T UBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before due due. | DATE DUE DATE DUE DATE DUE MSU Is An Nflrmetlve ActiorVEqual Opportunlty Institution ammo“ NEUROPATHOLOGIC AND ELECTROPHYSIOLOGIC EFFECTS OF ORGAN OPHOSPHORUS DELAYED NEUROT OXICANT S ON THE CENTRAL NERVOUS SYSTEM OF THE RAT by Ellen J . Lehning A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Animal Science and Institute for Environmental Toxicology 1992 ABSTRACT NEUROPATHOLOGIC AND ELECTROPHYSIOLOGIC EFFECTS OF ORGANOPHOSPHORUS DELAYED NEUROTOXICANTS ON THE CENTRAL NERVOUS SYSTEM OF THE RAT by Ellen J. Lehning Certain organophosphorus chemicals (OPS) cause organophosphoruS-induced delayed neurotoxicity (OPIDN). In order to enhance the rat model of Type I and Type II OPIDN, the Fink-Heimer silver impregnation technique was used to determine the extent of degeneration in the central nervous system (CNS) of adult male Long-Evans rats after exposure to a single dose of diisopropyl phosphorofluoridate (DFP) (4 mg/kg body weight, sc), a Type I OP, or to three doses of triphenyl phosphite (TPPQ (1184 mg/kg body weight, sc), 3 Type II OP, at three day intervals. DFP rats did not display clinical Signs, and at twenty-eight days after dosing, CNS degeneration was confined to the rostral gracile fasciculus and nucleus. The results provide evidence the rat may not be suited for study of Type I OPS relative to species which exhibit clinical signs and more extensive CNS degeneration such as the chicken or ferret. TPP, rats exhibited hindlimb ataxia, circling, and backward movement. CNS degeneration, examined after onset of clinical signs, consisted of widespread degeneration in the hindbrain, midbrain, and forebrain. The results indicate the rat is suitable for study of the effects of Type II OPS. A second study evaluated the effects of 'I'PPi on function of CNS sensory systems. Adult male Long-Evans rats were dosed dermally on two successive days with corn oil (5 rats) or with TPP at 450 (10 rats) or 600 (2 rats) mg/kg body weight and were evaluated by observation of movement, an evoked potential (EP) battery, and a neuropathological assessment. Most low dose rats became slightly ataxic. Auditory brainstem, flash, and somatosensory evoked potential results indicated Slight effects. High dose rats developed hindlimb ataxia, circling, and backward movement. EPs indicated moderate brainstem effects and severe cerebral cortical effects. Vacuoles which contained degenerating axons occurred in deeper layers of the cerebral cortex. The results indicate that Type II OPS interfere with the function of CNS sensory systems. ACKNOWLEDGEMENTS I would like to thank the members of my guidance committee, Dr. Steven Bursian, Dr. Duke Tanaka, Jr., Dr. Karen Chou, Dr. Robert Bowker, and Dr. Joel Mattsson, for their valuable assistance during the preparation of this manuscript. Sincere appreciation is extended to my major professor, Dr. Steven Bursian, for introducing me to neurotoxicology, for the encouragement and thoughtful guidance which has made possible the attainment of this degree, and for the friendship we have shared during our collaboration. I am genuinely grateful to Dr. Duke Tanaka, Jr. for providing me with a solid foundation in neuroanatom . I would also like to thank Dr. Chou, Dr. Bowker, and Dr. Mattsson for the advice ey have provided throughout my graduate training. I am grateful to the Department of Animal Science for its long-lasting support of my graduate career and for providing me with numerous research and teaching opportunities. I would like to thank my fellow graduate students and the faculty and staff of the Department of Animal Sc1ence for making graduate school an enjoyable and rewarding experience. Special thanks are extended to Dennis Bush, Julie Cameron, Carolyn Daniel, Kim Howard, Scott Kramer, Tadd Dawson, Margaret Benson, and Li Chen for the many experiences we have shared. Special acknowledgement belongs to Dr. Joel Mattsson, Pam Spencer, Ken Stebbins, Dr. Jan Wilmer, Julie Redmond, Ralph Albee, Malena Lewis, Dr. Keith Johnson, and everyone in the Departments of N eurotoxicology and Pathology at Dow Chemical Company. Their generous and unending support has contributed immeasurably to my personal and career growth and has made possible the completion of this degree. Additional thanks go to Carolyn Daniel for typing the more troublesome tables and figures. I would especial-121? like to thank gay parents, brother, Sister, and brother-in—law. Their help and mo support suppli the foundation that made this achievement possible. iv TABLE OF CONTENTS LIST OF FIGURES ....................................... ix LIST OF ABBREVIATIONS ................................ xiv INTRODUCTION ........................................ 1 LITERATURE REVIEW ................................... 6 Organophosphorus Chemicals .............................. 6 Chemical Structure .................................... 7 Type 1 OPIDN .................................... 7 Type H OPIDN ................................... 7 Incidence of OPIDN ................................... 9 I OPIDN .................................... 9 Type II OPIDN ................................... 10 Clinical Signs ........................................ 10 I OPIDN .................................... 10 Type II OPIDN ................................... l2 Neuropathological Features ............................... 13 I OPIDN - Sensitive Species ........................ 13 Type I OPIDN - Less Sensitive Species ..................... 15 H OPIDN ................................... 17 Electrophysiological Features .............................. 18 Type I OPIDN - Sensitive Species ........................ 18 Type I OPIDN - Less Sensitive Species ..................... 20 II OPIDN ................................... 20 Inhibition of Neuropathy Target Esterase ....................... 20 Type I OPIDN - Sensitive Species ........................ 20 Type I OPIDN - Less Sensitive Species ..................... 23 Type II OPIDN ................................... 24 Summary ........................................... 25 EXPERIMENT I ........................................ 27 Objectives .......................................... 27 Rationale .......................................... 27 Methods .................................... . ....... 29 Test Species and Husbandry ............................ 29 Test Materials .................................... 29 ExperimentalDesign........................... ..... 29 Evaluation of Body Weight and NTE Activity ................. 30 vi Neuropathological Analysis ............................ g1 Results ............................................ 32 DFP Trial ....................................... 32 Body Weight Gain ............................... 32 NTE Activity .................................. 32 Clinical Signs ................................ A. . 33 Central Nervous System Degeneration ................... 33 TPP, Trial ....................................... 40 Animal Condition ................................ 40 Body Weight Gain ............................... 40 Clinical Signs .................................. 40 Central Nervous System Degeneration ................... 43 Discussion ......................................... 7O DFP Trial ....................................... 70 NTE Activity .................................. 70 Central Nervous System Degeneration and Clinical Signs ........ 70 TPP, Trial ....................................... 72 Clinical Signs .................................. 72 Correlation of CNS Degeneration with Clinical Signs .......... 75 Species Comparisons .............................. 79 Comparison to Type I OPIDN ........................ 80 EXPERIMENT II ........................................ 83 Objectives .......................................... 83 Rationale .......................................... 83 Methods ........................................... 85 Test Material ..................................... 85 Test Species and Husbandry ............................ 85 Experimental Design ................................. 85 Dosing Regimen ................................... 86 Hindlimb Landing Foot Splay ........................... 88 Surgery ......................................... 88 Electrophysiological System and Test Battery .................. 89 Flash Evoked Potentials ............................ 89 Auditory Brainstem Responses ........................ 89 Somatosensory Evoked Potentials ...................... 90 Caudal Nerve Action Potentials ....................... 90 Digital Filtering ................................. 91 Waveform Analysis ................................. 91 Waveform Composites ............................. 91 Auditory Brainstem Responses ........................ 91 Somatosensory Evoked Potentials ...................... 93 Flash Evoked Potentials ............................ 93 Caudal Nerve Action Potentials ....................... 93 Data Analysis ..................................... 94 Neuropathological Analysis ............................. 95 Results ............................................ 96 Application Site .................................... 96 Clinical Signs ................. * .................... 96 Hindlimb landing Foot Splay ........................... 97 vii Body Weights ..................................... 57 Body Temperature ................................. 100 Tail Temperature .................................. 100 Auditory Brainstem Responses .......................... 100 ABRlo and ABRw ............................... 100 ABRc ...................................... l 10 Somatosensory Evoked Potentials ........................ 119 Somatosensory Cortex ............................ 119 Cerebellar Cortex ............................... 119 Flash Evoked Potentials .............................. 134 Visual Cortex ................................. 134 Cerebellar Cortex ............................... 134 Caudal Nerve Action Potentials ......................... 150 Neuropathology ................................... 150 Discussion ........................................ 166 Body Temperature ................................. 166 Clinical Signs .................................... 167 Evoked Potentials ................................. 167 Auditory Brainstem Responses ....................... 167 Somatosensory Evoked Potentials - Somatosensory Cortex ...... 168 Flash Evoked Potentials - Visual Cortex ................. 168 Cerebellar Potentials - Somatosensory and Visual . . . .‘ ....... 168 Caudal Nerve Action Potentials ...................... 169 Neuropathology ................................... 169 Summary ....................................... 170 SUMMARY .......................................... 171 BIBLIOGRAPHY ....................................... 175 LIST OF TABLES Table Bass 1. The effect of DFP on body weight gain in the rat ............. I ..... 32 2. The effect of DFP on whole-brain neuropathy target esterase (NTE) activity in t3; 3. The effect of TPP, on body weight gain in the rat .................. 40 4. The effect of TPP, on development of clinical Signs in the rat ........... 44 5. Rat central nervous system regions which contained axonal, preterminal axonal, and/or terminal degeneration after administration of TPPi .............. 45 6. Summary of experimental design for rats dosed dermally with 450 mg TPPi/kg bogg weight ............................................. 7. Summary of experimental design for rats dosed dermally with 600 mg TPP/ kg 8; weight ............................................. viii LIST OF FIGURES figure Base 1. Structure of Type I and Type II organophosphorus chemicals .......... 8 2. Inhibition and aging of neuropathy target esterase (NTE) ............. 22 3. Line drawings illustrating approximate levels of the brain and Spinal cord used to depict degeneration in subsequent figures ....................... 34 4. Line drawings of cross-sections depicting the location of degeneration in the , caudal hindbrain and rostral spinal cord of the rat after exposure to DFP ............................................. 36 5 . Photomicrographs illustrating control and 28 day post-DFP Fink-Heimer silver impregnated cross-sections through the gracile nucleus of the rat ........ 38 6. Line drawings of sagittal sections depicting the location of axonal degeneration in fiber tracts and preterminal axonal and terminal degeneration in nuclear regions of the brain of rat 25 .............................. 48 7. Line drawings of sagittal sections which are a lateral continuation of Fig. 6 and depict the location of preterminal axonal and terminal degeneration in gray matter of the brain of rat 25 .............................. 50 8. Line drawings of cross-sections depicting the location of preterminal axonal and tzerminal degeneration in gray matter and nuclear regions of the forebrain of rat52 2 .............................................. 9. Line drawings of cross-sections which are a caudal continuation of Fig. 8 and Show the extent of preterminal axonal and terminal degeneration in the gray matter and nuclear regions of the forebrain of rat 22 ............... 54 10. Line drawings of cross-sections which are a caudal continuation of Fig. 9 and Show the extent of preterminal axonal and terminal degeneration in the gray matter and nuclear regions of the midbrain of rat 22 ................ 56 ll. Photomicrographs illustrating Fink-Heimer silver impregnated sections through the somatosensory cortex of rats 25 and 24 ..................... 58 12. l3. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. Base Photomicrographs illustrating control and TPP Fink-Heimer silver impregnated sections through the medial mamillary nucleus, medial part and the medial vestibular nucleus of the rat ............................... 60 Line drawings of cross-sections illustrating the location of degeneration 1n the cerebellum of rat 22 ................................... 64 Line drawings of cross-sections which are a caudal continuation of Fig. 10 and Show the extent of axonal degeneration 1n fiber tracts and preterminal axonal and terminal degeneration 1n nuclei of the hindbrain of rat 22 ............. 66 Line drawings of cross- -sections which are a caudal continuation of Fig. 14 and Show the extent of axonal degeneration 1n fiber tracts and preterminal axonal degeneration 1n the hindbrain of rat 22 ........................ 68 Summary schematics 1n the sagittal plane which compare qualitatively the extent of degeneration 1n the CNS of the rat, chicken, and ferret after a Single exposure to DFP ........................................... 73 Summary schematics 1n the sagittal plane which co mam qualitatively the extent of degeneration 1n the CNS of the rat, chicken, and t after acute (chicken and ferret) or subacute (rat) exposure to TPP .................... 81 Plot of contrast differences estimated for hindlimb landing foot splay data ............................................. 98 Plot of contrast differences estimated for body weight data ............ 99 Plot of contrast differences estimated for body temperature data ........ 101 Composites of auditory brainstem responses collected in response to 10 kHz tone pips (ABRIO). Rats were dosed dermally with 450 mg TPP/kg body weight on days one and two .............................. 102 Composites of auditory brainstem responses collected in response to 10 kHz tone pips (ABRIO). Rats were dosed dermally with 600 mg TPP/kg body weight on days one and two .................................... 1 Composites of auditory brainstem responses collected in response to 30 kHz tone pips (ABR30). Rats were dosed dermally with 450 mg TPP/kg body weight on days one and two .............................. 106 Composites of auditory brainstem responses collected in response to 30 kHz tone pips (ABRJO). Rats were dosed dermally with 600 mg TPP/ kg body weight on days one and two .................................... 108 Plot of contrast differences estimated for ABR, IPL data ............ 111 Plot of contrast differences estimated for ABRc IPL data corrected for body temperature (CIPL) ................................... 112 38. 39. 41. 42. 43. 45. 46. 47. 48. 238: Composites of somatosensory evoked potentials recorded from the cerebellar electrode (SEP-C) with a 200 msec data sweep (long latency components). Rats were dosed dermally with 600 mg TPP/kg body weight on days one and two ............................................. 135 Plot of contrast differences estimated for PEP-V peak N 1 power ....... 137 Composites of flash evoked potentials collected from the visual electrode (FEP- V) in response to low or medium intensity flashes with a 150 msec data sweep (early latency components). Rats were dosed dermally with 450 mg TPPi/kg1 body weight on days one and two .......................... Composites of flash evoked potentials collected from the visual electrode (FEP- V) in response to low or medium intensity flashes with a 150 msec data sweep (early latency components). Rats were dosed dermally with 600 mg TPP/kgl body weight on days one and two .......................... Composites of flash evoked potentials collected from the visual electrode (FEP- V) in response to low or medium intensity flashes with a 600 msec data sweep (long latency components). Rats were dosed dermally with 450 mg TPPi/ kg body weight on days one and two .......................... 142 Composites of flash evoked potentials collected from the visual electrode (FEP— V) in response to low or medium intensity flashes with a 600 msec data sweep (long latency components). Rats were dosed dermally with 600 mg TPP/kg body weight on days one and two .......................... 144 Composites of flash evoked potentials recorded from the cerebellar electrode (FEP-C) 1n response to low or medium intensity flashes and with a 150 msec data sweep (early latency components). Rats were dosed dermally with 450 mg TPP/kg body weight on days one and two ..................... 146 Composites of flash evoked potentials recorded from the cerebellar electrode (FEP- C) 1n response to low or medium intensity flashes and with a 150 msec data sweep (early latency components). Rats were dosed dermally with 600 mg TPP/kg body weight on days one and two ..................... 148 Composites of flash evoked potentials recorded from the cerebellar electrode (PEP-C) in response to low or medium intensity flashes and with a 600 msec data sweep (long latency components). Rats were dosed dermally with 450 mg TPP/kg body weight on days one and two ..................... 151 Composites of flash evoked potentials recorded from the cerebellar electrode (PEP-C) in response to low or medium intensity flashes and with a 600 msec data sweep (long latency components). Rats were dosed dermally with 600 mg TPPilkg body weight on days one and two ..................... 153 Composites of caudal nerve action potentials collected in response to sin 1e (CNAP,) or paired (CNAPz) stimuli. Rats were dosed dermally with 45 mg TPP/kg body weight on days one and two ..................... 155 49. 50. 51. 52. Composites of caudal nerve action tentials collected in response to sin 1e (CNAP,) or paired (CNAPZ) stimuli. Rats were dosed dermally with mg TPP/kg body weight on days one and two ..................... 157 Photomicrographs illustrating control and TPPi H & E stained cross—sections through the somatosensory cortex at the level of the optic chiasm ....... 159 Higher power photomicrographs illustrating control and TPPi H & E stained cross-sections through layer V of somatosensory cortex at the level of the optirl:62 chiasm ........................................... Electron micrograph of a vacuole in the neuropil of layer V of somatosensory cortex of a TPPi-treated rat .............................. 164 LIST OF ABBREVIATIONS Spinal cord laminae Cerebellar lobules Oculomotor nucleus Facial nucleus Hypoglossal nucleus Third ventricle Fourth ventricle anterior commissure, anterior part Area postrema Anterior pretectal nucleus Aqueduct (Sylvius) brachium of the inferior colliculus Fields CAI-CA3 of Ammon’s horn Corpus callosum Central (periaqueductal) gray Central nucleus of the inferior colliculus Centrolateral thalamic nucleus Central medial thalamic nucleus Copula of the pyramis Cerebral peduncle, basal part Caudate putamen (striatum) Crusl of the ansiform lobule Crus2 of the ansiform lobule Cuneate nucleus Cuneate fasciculus Cortex Dorsal cochlear nucleus Dentate gyrus Dorm-intermediate posterior thalamic nucleus Dorsal raphe nucleus External cuneate nucleus External cortex of the inferior colliculus Fomix Flocculus Forceps minor of the corpus callosum Gigantocellular reticular nucleus Gracile nucleus Gracile fasciculus Hippocampal region Nucleus of the horizontal limb of the diagonal band Inferior colliculus xiv Internal eapsule Internal medullary lamina Intermediate gray layer of the superior colliculus Intermediate whrte layer of the superior colliculus Inferior oliv nucleus Lateral funicu us of the spinal cord Lateral cervical nucleus Lateral geniculate nucleus Lateral reticular nucleus Lateral ventricle Lateral vestibular nucleus Mamillary body Magnocellular preoptic nucleus Mediodorsal thalamic nucleus Medullary reticular nucleus Mesencephalic trigeminal nucleus Medial geniculate nucleus Medial mamillary nucleus, lateral part Medial lemniscus Lateral mesencephalic nucleus, pars dorsalis Medial longitudinal fasciculus Medial mamillary nucleus, medial part Medial mamillary nucleus, median part Medial mamillary nucleus, posterior part Medial vestibular nucleus Medial vestibular nucleus, ventral part Nucleus of the tractus solitarius Optic tectum Optic chiasm Paracentral thalamic nucleus Predorsal bundle Paraflocculus Pontine gray matter Lateral paragigantocellular nucleus Paramedian lobule Pontine nuclei Postsubiculum Paleostriatum primitivum Paramedian reticular nucleus Parasolitary nucleus Pyramidal tract Red nucleus Agranular retrosplenial cortex Sensory root of the trigeminal nerve Superior colliculus Simple lobule Somatosensory cortex Substantia nigra, compact part Substantia nigra, reticular part Superior olivary nucleus Nucleus of the solitary tract XV Spinal trigeminal nucleus Spinal trigeminal nucleus, caudal part Spinal trigeminal nucleus, interpolar part Spinal trigeminal nucleus, oral part lateral spiriform nucleus Spinal vestibular nucleus Subthalamic nucleus Superior vestibular nucleus Pedunculopontine tegmental nucleus, pars compacta Ventral cochlear nucleus, posterior part Nucleus of the vertical limb of the diagonal band Descending vestibular nucleus Ventral funiculus of the spinal cord Ventrolateral thalamic nucleus Ventromedial thalamic nucleus Vestibular nuclear complex Ventral posterior thalamic nucleus Ventral posterolateral thalamic nucleus Ventral posteromedial thalamic nucleus xvi INTRODUCTION OrganOphosphorus compounds (OPS) are used extensively in agriculture as pesticides and in industry as petroleum additives and plasticizers. Benefits derived from these uses of organophosphorus chemicals have been offset by the ability of certain of them to cause neurotoxicity in animals and humans. Two kinds of nervous system effects can result from exposure to OPS, those which are apparent immediately (acute effects) and those which develop a number of days after exposure (delayed effects). Neurotoxic OPS may produce one or both kinds of effects (Davis and Richardson, 1980). Acute effects are due to inhibition of acetylcholinesterase (Fikes, 1990). Delayed effects are distinct from acute effects and are known collectively as organophosphorus-induced delayed neurotoxicity (OPIDN) (Davis and Richardson, 1980). OPIDN was first described as a human syndrome in 1899. Since that time, an estimated 40,000 human cases have been reported (Chemiak, 1988). Because of its widespread occurrence, OPIDN is of concern. OPIDN was first studied nearly six decades ago by Smith and his colleagues at the National Institutes of Health (Smith et al., 1930a, 1930b, 1932, 1933; Smith and Lillie, 1931; Lillie and Smith, 1932). As a result of their work, two types of OPIDN were identified: OPIDN eaused by organic esters of phosphoric acid and OPIDN caused by organic esters of phosphorus acid (Smith et al. , 1932). The categorization of OPIDN into two types has been substantiated by recent research. OPIDN produced by organic esters of phosphoric acid is now designated Type I while OPIDN produced by organic esters of phosphorus 2 acid is designated Type II (Abou-Donia and Lapadula, 1990). Even though two types of OPIDN have been identified, research has focused on Type 1 because all known human eases have been characterized as such. More recently, research on Type II has increased Since it has been shown that potential exists for human exposure to an organic ester of phosphorus acid, triphenyl phosphite, which can precipitate OPIDN in experimental animals (U .8. Environmental Protection Agency, 1986). Type I and Type II OPIDN are distinguished, not only by chemical structure of the causative OP, but also by a combination of biochemical, clinical, and neuropathological features. These include the degree of inhibition of certain esterases, the nature of clinieal signs and their latency to onset, and the pattern of degeneration in the nervous system, respectively. Characteristics of Type I OPIDN are inhibition of the nervous system enzyme neuropathy target esterase (NTE) in excess of 70% , a relatively long delay period of 10 to 14 days before onset of hindlimb ataxia, and restriction of accompanying nervous system degeneration to hindbrain, Spinal cord, and peripheral nerves. Type I OPS, organic esters of phosphoric acid, contain a central pentavalent phosphorus atom. Examples include tri-ortho—cresyl phosphate, diisopropyl phosphorofluoridate (DFP), mipafox (MN-diisopropyl phosphorodiamidofluoridate) , and leptophos (0-2,5-dichloro—4- bromophenyl 0-methyl phenylphosphonothionate). Type H OPIDN ean occur, in some species, when NTE inhibition is less than 65-70% . Other characteristics of Type II OPIDN include a Shortened delay period of four to seven days before onset of hindlimb ataxia, additional clinical signs in some species of hyperexcitability, circling, and extensor rigidity, and presence of lesions in the forebrain and midbrain as well as in the hindbrain, Spinal cord, and peripheral nerves. Type H OPS, organic esters of phosphorus acid, contain a central trivalent phosphorus atom. Examples include triphenyl phosphite (TPPi) and t1i-ortho-, tri-meta-, and tri-para-cresyl phosphite (Abou-Donia and Lapadula, 1990). 3 Using the characteristics described above, several avian and mammalian species have been screened for the ability to serve as a Type I er Type II OPIDN animal model. Species tested for suitability include the chicken, cat, ferret, mouse, and rat (Smith at al. , 1933; Abou-Donia, 1981; Abou-Donia er al., 1983; Veronesi and Dvergsten, 1987; Carrington and Abou-Donia, 1988a; Padilla and Veronesi, 1988; Tanaka et al., 1990a, 1991; Veronesi er al. , 1991). The chicken has long been considered the most appropriate OPIDN animal model and has been used to study OPIDN Since the 19303 (Abou-Donia, 1981). Although OPIDN has been studied for more than five decades, it has been suggested that limited progress has been made in understanding this neurological syndrome. The lack of progress is thought to have resulted, at least in part, from the almost exclusive use of the chicken, a species with a relatively small scientific database. Consequently, it has been recommended that understanding of OPIDN could be enhanced by using a species with a larger scientific database, such as the rat, as the animal model (Padilla and Veronesi, 1988). The pattern of degeneration in the central nervous system (CNS) is an important consideration in determining the suitability of a species as an OPIDN model. Several studies utilizing Type I (Veronesi and Abou-Donia, 1982; Veronesi, 1984a, 1984b; Padilla and Veronesi, 1985; Veronesi er al., 1986a; Carboni er al., 1991; Jortner er al., 1991) and Type II (Veronesi er al., 1986b; Veronesi and Dvergsten, 1987) OP compounds have included a histopathological examination of nervous system tissue in the rat. In these studies, histopathological examination of the CNS was confined to the hindbrain and spinal cord and Staining methods employed were designed to evaluate the nature of the pathological change rather than the distribution of CNS lesions. Therefore, the first objective of the present study was to more completely describe the pattern of degeneration in the CNS of the rat after exposure to a Type I or a Type II organophosphorus delayed neurotoxicant. DFP and TPPi were used as the prototype 4 Type I and Type H OPS, respectively. CNS degeneration was mapped using a Pink- Heimer silver impregnation procedure, a technique which has been Shown to be sensitive for studying the distribution of OP-induced CNS damage (Cavanagh and Patangia, 1965; Tanaka et al. , 1992b). Exposure to TPP, produces a complex set of clinical signs in the rat (Veronesi and Dvergsten, 1987). Thus, a second objective of this study was to assess TPPi-induced clinical signs so that they could be correlated to CNS degeneration. The Fink—Heimer procedure has also been used to map Type I and Type H OP-induced degeneration in the CNS of the chicken and ferret (Tanaka and Bursian, 1989; Tanaka et al., 1990a, 1990b, 1991, 1992a). Accordingly, a third objective of this study was to compare rat degeneration patterns to those reported for the chicken, the most commonly used OPIDN model, and the ferret, a mammalian OPIDN model, to determine the suitability of the rat as a model for Type I or Type H OPHDN. The neuropathological studies Showed that TPPi induces widespread degeneration in the auditory, the somatosensory, and the visual pathways in the CNS of the rat. This suggests that Type H neurotoxicants interfere with central processing of sensory information. CNS sensory processing is readily evaluated using evoked potential electrophysiological techniques (Mattsson and Albee, 1988). Consequently, a third objective of this study was to use an evoked potential test battery to survey the function of CNS sensory pathways in the rat after exposure to the Type H OP delayed neurotoxicant TPPi. The final objective of this study was to correlate TPPi-induced CNS degeneration with effects on CNS sensory processing. Thus, specific aims of this study were: 1. To use aFink-Heimer Silver impregnation method to map the extent of degeneration in the CNS of the rat after exposure to the Type I or Type II organophosphorus delayed neurotoxicants, DFP and TPP,, respectlvely. 2. To assess TPPi-induced clinical signs so that they can be correlated to CNS degeneration in the rat. 5 To compare CNS degeneration patterns in the rat to those reported for the chicken and the ferret to determine, based on neuropathological considerations, the suitability of the rat as a model for Type I or Type H OPIDN. To use an evoked potential test battery (auditory brainstem responses, flash evoked potentials, somatosensory evoked potentials, and caudal nerve action potentials) to assess the ability of the CNS to process sensory information in the rat after exposure to the Type H organophosphorus delayed neurotoxicant TPPi. To correlate TPPi-induced neuropathological lesions with effects on central processing of sensory information in the rat. LITERATURE REVIEW Organophosphorus Chemicals Organophosphorus chemicals are used in agriculture as pesticides to kill undesired insects, worms, fungi, and weeds and in industry as plasticizers, antioxidants, stabilizers, plastic extenders, and oil and gasoline additives (Etc, 1979; Davis and Richardson, 1980; Cherniak, 1988). Numerous benefits have resulted from these uses including increases in agricultural productivity and protection of human health through control of vector- bome disease (Ecobichon, 1991). Unfortunately, many OPS also cause reproductive, teratogenic, and/or nervous system effects (Proctor et al. , 1975; Davis and Richardson, 1980; Chapin er al., 1991). Of these, nervous system effects are the most prevalent. Two kinds of nervous system effects result from exposure to OPS, those which are apparent immediately (acute effects) and those which develop a number of days after exposure (delayed effects). N eurotoxic OPS may produce one or both kinds of effects (Davis and Richardson, 1980). Acute effects are the desired response when OPS are applied as insecticides, but are undesirable when non-target organisms are affected. Acute effects usually develop within minutes to hours of exposure and are due to inhibition of acetylcholinesterase, a nervous system enzyme responsible for inactivating the neurotransmitter acetylcholine (Fikes, 1990). Delayed effects are undesirable effects which result from exposure to certain OPS. They are distinct from acute effects and become apparent several days after exposure. Delayed effects are known collectively as 7 organophosphorus-induced delayed neurotoxicity (OPIDN). Two types of OPIDN have been identified, Type I and Type H (Abou-Donia and Lapadula, 1990). The purpose of this review is to compare and contrast the characteristics of Type I and Type H OPIDN. Characteristics which will be described are chemical Structure of causative OPS, incidence, clinieal Signs, neuropathological features, electrophysiological features, and inhibition of neuropathy target esterase. Olendcal Structure Type I OPIDN. Type I OPS contain a central pentavalent phosphorus atom and are derivatives of phosphorus-containing acids including phosphoric, phosphonic, phosphoroamidic, and phosphorofluoridic acids and their thiono analogues. The general structural formula of a Type I OP is given in Fig. 1A. In this figure, X is an oxygen or sulfur atom, RI and R2 are highly variable alkyl and aryl groups which are either directly bonded to P or are linked to P through an oxygen (ester), nitrogen (amido), or sulfur (thiolo) atom, and R3 is any acid residue and includes halide, cyanide, thiocyanate, phenoxy, thiophenoxy, and carboxylate groups (Stuart and Oehme, 1982). The structure of diisopropyl phosphorofluoridate (DFP), the prototypical Type I OP used in the present Study, is given in Fig. 1A. ' Type II OPmN. Relatively few OPS have been tested for Type H delayed neurotoxic potential. Of the OPS examined, four have been determined to produce Type H OPIDN. They are triphenyl phosphite (TPPi) and tri-ortho-, tri-meta-, and tri-para- cresyl phosphite. They contain a central trivalent phosphorus atom and are tri-aryl derivatives of phosphorus acid (Abou-Donia and Lapadula, 1990). The structure of TPP,, the prototypical Type H OP used in the present studies, represents the general structure of the known Type H OPS and is presented in Fig. 1B. P-—R3 R2 / GENERAL STRUCTURE OF A TYPE I 0P C113 tug—CH—o\fi P—F CH3—Cll—0/ CH3 DIISOPROPYL PHOSPHOROFLUORIDATE . H°~7-° , ‘ * 0 TRIPHENYL PHOSPHITE Fig. 1. Structure of Type I (A) and Type II (B) organOphosphorus chemicals. The structure of triphenyl phosphite represents the general structure of a Type II organophosphorus chemical. Incidence of OPEN Type I OPEN. Type I OPIDN was first identified as a human syndrome in 1899 when the neuropathy developed in tuberculosis patients who were treated with phospho- creosote, a mixture of phosphoric acid esters and phenols (Davis and Richardson, 1980). Since then, most occurrences of Type I OPHDN have been due to ingestion of beverages or foods contaminated with tri-ortho—cresyl phosphate ('TOCP.). For example, during prohibition several thousand persons in the Midwestern and Southwestern United States developed Type I OPHDN after drinking a ginger extract which had been adulterated with TOCP,. In 1959 an estimated 10,000 people in Morocco were afflicted after cooking with cresyl phosphate-adulterated oil (Metcalf, 1982). Less frequently, human Type I OPIDN has been related to OP insecticides. Documented exposures have been primarily occupational, accidental, or intentional in cases of attempted suicide. Implicated OP insecticides include mipafox, leptophos, EPN, methamidophos, trichlorphon, isophenphos, fenthion, and chlorpyrifos (Lotti er al. , 1986; Cherniak, 1988). Incidences of Type I OPIDN in domestic animals have been relatively rare. The most widespread exposure occurred in 1971 in Egypt. The OP leptophos was sprayed to control cotton leafworm, and 1300 water buffalo which came in contact with the insecticide developed Type I OPHDN (Metcalf, 1982). Because OP insecticides have been linked to human Type I delayed neurotoxicity, the US Environmental Protection Agency introduced federal screening guidelines for OPS in 1978 and updated them recently (U .8. Environmental Protection Agency, 1991). Following implementation of the original guidelines, occurrence of Type I OPIDN has been uncommon. However, more than 40 OP insecticides currently marketed were registered for use prior to adoption of the guidelines, and thus, have not been screened for Type I delayed neurotoxic potential. Of these, 11 have structures which suggest that 10 they are Type I delayed neurotoxins. Also, because the recommended testing protocol does not use routes and durations of exposure that the human population typieally encounters, the screening procedure may not adequately detect Type I OPIDN (Cherniak, 1988). Therefore, although risk of Type I OPIDN has been minimized, potential exists for additional incidences. Iypc II OPEN. Occurrence of Type H OPIDN in humans has not been reported. The syndrome was identified in studies using experimental animals. However, at least one Type H OP, TPP,, is used commercially. TPPi is used primarily as an intermediate in the synthesis of phosphite derivatives employed as antioxidants and stabilizers in vinyl plastics. This creates potential for occupational and consumer exposure. In the worlquace, long-term, low-level dermal or inhalation exposure to TPP, could occur during manufacturing while consumer exposure could result from contact with TPPi-Stabilized plastics or from release of TPPi into the environment. Occupational exposure is thought to represent the most probable TPPi exposure risk (U .8. Environmental Protection Agency, 1986). Clinical Signs Type I OPEN. Clinical signs of Type I OPEN in humans first become apparent 5 to 21 days after a Single exposure. Initial signs are sharp, cramp-like pains in calf muscles and occasional numbness and tingling in toes and fingers. Then, in about 7 to 10 days, distal muscles in the lower and upper limbs become weak. Weakness is followed by foot and wrist drop and ataxia. Ataxia can worsen and the lower and upper limbs may become paralyzed. The paralysis is flaccid, bilateral, and symmetrical. Lower limb deficits usually occur earlier, are more severe, and extend farther up the limb than upper limb deficits (Smith at al. , 1930a; Davis and Richardson, 1980). Ataxia 11 and subsequent paralysis are the main Signs of Type I OPEN, and they may or may not be associated with other features. For example, Smith et al. (1930a) reported that tactile, pain, and temperature sensation and vision and cranial nerve function are not affected while Davis and Richardson (1980) stated that sensory loss can occur. Type I OPEN has also been associated with higher order dysfunction such as nervousness, depression, concentration problems, and psychiatric disturbances (Tabershaw and Cooper, 1966; Amr, 1989). Recovery from Type I OPEN is Slow and limited. Weakness and partial paralysis have been reported to continue for up to 30 years after exposure (Metcalf, 1982). The probability of clinical expression of Type I OPEN increases with age. In general, clinical signs appear consistently in adults and infrequently in preadolescents (Smith et al., 1930a; Smith and Elvove, 1931; Davis and Richardson, 1980). Clinieal signs of Type I OPEN in animals are very Similar to those exhibited by humans (Davis and Richardson, 1980). However, degrees of species susceptibility have been described in adult animals. Sensitive Species display clinical Signs after a single exposure and include cats, dogs, and some non-human primates as well as farm animals such as cows, pigs, horses, Sheep, chickens, and water buffalo. Less sensitive Species do not display clinical signs after a single exposure but may display clinical Signs after repeated exposure. Less sensitive species include typical laboratory Species such as rabbits, rats, mice, and guinea pigs (Davis and Richardson, 1980; Lapadula er al., 1985; Abou-Donia and Lapadula, 1990). Young of sensitive Species respond like adults of less sensitive species. Clinieal Signs are not exhibited after a single exposure but may be displayed after repeated exposure (Taylor, 1967; Johnson and Barnes, 1970). Clinieal Signs have not been examined in young of less sensitive Species. Also, sex does not influence susceptibility to Type I OPEN in animals or humans (Cavanagh, 1964; Abou- Donia and Lapadula, 1990). Gill: W81 CON are YOU per 111V: 011 Bur Ho' a ii for and arm and of] not 12 type II OPEN. Clinical manifestation of Type H OPEN has been studied in chickens, cats, ferrets, and rats. Acute and delayed Stages of clinical Signs are evident. The acute stage develops within a few hours of exposure, is characterized by tremors, weakness, and lethargy, and subsides within two to three days. Since Type H OPS can contain phenol impurities or hydrolyze to phenol in sin; (Smith et al. , 1933; Veronesi er al. , 1986b), acute effects may be due to phenol poisoning. The acute and delayed stages are separated by a latent period which lasts from a few to several days depending upon route of administration, dose, Species, and individual variation. In general, the latent period to onset of delayed effects is shorter for Type H than for Type I OPEN. The late Stage consists of hindlimb ataxia and subsequent paresis with some forelimb involvement possible. In addition, cats can develop extensor rigidity, ferrets may circle or have extensive forelimb involvement, and rats may circle or develop tail kinks (Smith et al., 1933; Veronesi and Dvergsten, 1987; Carrington and Abou-Donia, 1988a; Bursian, perS. comm.). Degrees of species sensitivity are not as apparent for Type H as for Type I OPEN. However, as for Type I OPEN, chickens, cats, and ferrets display Type H signs after a single dose while rats require repeated dosing although not as frequently as is required for the display of Type I clinical Signs (Smith et al., 1933; Veronesi, 1984a; Veronesi and Dvergsten, 1987; Carrington and Abou-Donia, 1988a; Tanaka et al., 1990a). In contrast to Type I OPEN, Type H clinical signs can be expressed in young animals after a single exposure. One-week old chicks developed clinical Signs after a Single oral exposure to a high dose of TPPi (Abou-Donia and Brown, 1990), and chicks and ferret kits first consistently showed clinical Signs when injected with a single dose of TPP, at four weeks of age (Bursian, pers. comm.). As for Type I OPEN, sex does not influence susceptibility to Type H OPEN (Bursian, pers. comm.). Ni 1C! 53' fir. all 011; ‘31 13 In summary, the clinical expression of Type H OPEN is Similar to, but is easily distinguished, from Type I OPEN. Both syndromes are characterized by delayed and progressive development of hindlimb ataxia and paralysis with some forelimb involvement. However, earlier onset of late effects, presence of additional clinical signs, susceptibility of young animals to a single exposure at an early age, and susceptibility of rats to less frequent repeated dosing serve to differentiate the clinical expression of ’lype H from Type I OPEN. Neumparhological Features Iype I OPEN - Sensitive Species. Neuropathological features of Type I OPEN in adults of sensitive species have been assessed at the system, cellular, and subcellular level. Neuropathological changes have not been detected in young animals of sensitive species after a Single exposure to a Type 1 OP (Tanaka, pers. comm.). Degeneration occurs in both the peripheral (PNS) and central (CNS) nervous systems after a single exposure to a Type I OP and is usually apparent at about the same time as clinical signs. However, degenerative changes have been detected in advance of clinieal signs (Wilson et al., 1988; El-Fawal et al., 1990a). Peripheral nerves typieally affected innervate distal muscles of hind- or lower limbs and fore- or upper limbs and include the common peroneal and tibial branches of the sciatic nerve, the nerve to the lateral head of the gastrocnemius muscle, and the ulnar and radial nerves. Fore— or upper limb nerves are less affected than those in hind- or lower limbs (Barnes and Denz, 1953; Cavanagh, 1954, 1964; Bouldin and Cavanagh, 1979; Krinke er al., 1979). Depending upon the nerve examined, the dose, the time point after dosing, and the clinical state of the animal, from 1 to 40% of fibers in a nerve may be affected, and various stages of degeneration may be exhibited at any point in time (J ortner, 1984). de V: be ch pn ge: 1111 dB; 191 the Hi: mg 14 Regeneration of peripheral nerves can occur several weeks after onset of clinical signs and is correlated with improvement in clinical deficits (Cavanagh, 1964; Glazer er al. , 1978; Jortner et al., 1989). The primary CNS targets are the long ascending and descending pathways of the spinal cord. No main ascending pathways, the gracile fasciculus and the dorsal Spinocerebellar tract, are lesioned. These pathways develop axonal degeneration which is heaviest at cervical levels and which can be traced to preterminal axonal and terminal degeneration in the gracile nucleus (the gracile-cuneate nucleus in birds) in the medulla and the anterior lobe of the cerebellum, respectively. In general, the dorsal Spinocerebellar pathway exhibits more degeneration than the gracile fasciculus. In addition, the ascending ventral Spinocerebellar, spinovestibular, spinoolivary, and Spinoreticular pathways may exhibit axonal and terminal degeneration. One main descending spinal cord pathway, the corticospinal tract, is affected. The lateral and ventral corticospinal tracts may contain axonal degeneration in mammals while the avian homologue of the corticospinal tract, the medial pontine-Spinal tract, is affected in chickens. Degeneration in this system is heaviest at lumbar levels and can be traced to preterminal and terminal degeneration in lamina VH of thoracic and lumbar cord. In general, the descending pathways are less involved than the ascending pathways (Barnes and Denz, 1953; Cavanagh, 1954; Cavanagh and Patangia, 1965; Tanaka and Bursian, 1989; Tanaka et al., 1990b, 1991). As for the PNS, not all fibers in a CNS pathway degenerate and various stages of degeneration may exist at any one point in time (J ortner, 1984). At'the cellular level, Type I OPS initially produce a focal ‘chemical transection’ of the distal, but not terminal, axon mainly in longer, larger diameter myelinated fibers. The ‘chemical transection’ then precipitates Wallerian degeneration of the entire distal region of the neuron including sensory and motor nerve terminals (Cavanagh, 1954, 196 of ll deg: haw 197i Car: lgll neun OPII 35868 111 hi Pefipl arena cord. 31111 in [Etta 15 1964; Glazer et al., 1978; Bouldin and Cavanagh, 1979). Although the distal portion of the axon is usually affected first, terminal degeneration ean be apparent before axonal degeneration (Illis et al. , 1966; Tanaka and Bursian, 1989). Also, although some authors have reported neuronal somatic degeneration (J anzik and Glees, 1966; LeVay et a1 . , 1971), most studies report a lack of an effect on cell bodies (Barnes and Denz, 1953; Cavanagh, 1954, 1964; Tanaka and Bursian, 1989). Ultrastructurally, axonal degeneration is characterized by initial hypertrophy of agranular endoplasmic reticulum (AER) in the axoplasm. The AER then aggregates to form stacks of tubulovesicular arrays, and as degeneration progresses, neurotubules and neurofilaments are lost and mitochondria degenerate (J ortner, 1984). Iype I OPEN - Less Sensitive Species. Neuropathologieal features of Type I OPEN in less sensitive species have been studied in adult rats and mice and have been assessed mainly at the system and subcellular level. N europathological assessment in the rat has been limited to peripheral nerves and the spinal cord. After a single exposure, peripheral nerves feature regenerating axonal Sprouts instead of axonal degeneration, but axonal degeneration is present in the gracile fasciculus at cervical levels of the spinal cord. If exposure is repeated, the distal regions of tibial and plantar nerves are affected, and in the spinal cord, in addition to the gracile fasciculus, the dorso- and ventro—lateral columns at cervical and lumbar levels and the ventral columns and dorsal corticospinal tract at lumbar levels contain axonal degeneration. Ultrastructurally, axonal degeneration in the rat is characterized by early and late changes. Early changes consist of tubulovesicular profiles interspersed with intraaxonal vacuoles and groups of intact mitochondria or intraaxonal and intramyelinic vacuolation. Later changes are characterized by swollen axons packed with accumulations of vesicular profiles, dense amorphous bodies, and mitochondria (V eronesi and Abou-Donia, 1982; Veronesi, ‘ 1984a, 1984b; Padilla and Veronesi, 1985; Veronesi et al., 1986a). for : axor cord 3X01 hm Adu 35cc 00ml Imp 00ml a Its is lit affec am 3110" Elaci 16 Neuropathological assessment in the mouse has been limited to peripheral nerves, the Spinal cord, and the caudal medulla. In mice, the pattern of degeneration is Similar for Single and repeated exposure. Peripheral nerves feature regenerating axonal sprouts, axonal degeneration is found in the lateral and ventral columns at all levels of the spinal cord, and the gracile fasciculus is spared. Although, the gracile fasciculus is spared, axonal degeneration in the gracile nucleus, the medullary nucleus which receives projections from the gracile fasciculus, has been documented after a single exposure, and degeneration has also been noted in the inferior olivary nucleus. Ultrastructurally, tubulovesicular profiles are rarely seen in mice. The main ultrastructural manifestations consist of swollen axons filled with neurofilaments or floccular axoplasm and frequent intraaxonal vacuolation (Lapadula et al., 1985; Veronesi et al., 1991). In summary, neuropathological features of Type I OPEN depend on the species. Adults of sensitive species exhibit degeneration after a Single exposure. Degeneration occurs in the distal branches of peripheral nerves and in the distal regions of the ascending dorsal spinocerebellar and gracile fasciculus pathways and in the descending corticospinal tract in the CNS. The ascending systems carry exteroceptive and proprioceptive somatosensory information while the descending system regulates activity of a-motor neurons which innervate distal muscles. Degeneration in these. systems, in combination with peripheral nerve lesions, would be expected to cause ataxia. In the rat, a less sensitive species, nervous system damage is apparent after a single exposure, but is limited in its extent. As the extent of exposure increases, nervous system regions affected are Similar to those affected in sensitive species and clinical signs may become apparent. Neuropathological features in the mouse, also a less sensitive species, are anomalous. The extent of nervous system damage is qualitatively Similar after single and repeated exposure, but clinical signs are apparent only after repeated exposure, and the gracile fasciculus is spared. For all species, damage is restricted to the hindbrain, spinal ves mic Sen Die the Systi CXhil “Ides Cord ; 17 cord, and peripheral nerves, the initial Site of degeneration occurs in the distal region of longer, larger diameter axons, and cell bodies are not affected. Type II OPEN. Neuropathological features of Type H OPEN have been assessed mainly at the system level in the CNS of adult chickens, cats, ferrets, and rats. Chickens, cats, and ferrets develop nervous system injury after a single dose while rats require repeated dosing. Although the specific pattern of CNS degeneration varies, it is generally analogous among the species. In the Spinal cord and hindbrain, the pattern of degeneration is very Similar to that associated with Type I OPEN. The long ascending (dorsospinocerebellar tract and gracile fasciculus) and descending (corticospinal tract) pathways and their terminations in the hindbrain and in the ventral horn of the spinal cord, respectively, are affected. However, Spinal cord and cerebellar degeneration are more extensive after exposure to a Type H OP, and neuronal somatic degeneration may occur in the ventral hem of the spinal cord, especially at lumbar levels, and in the vestibular nuclei in the medulla. Unlike Type I OPEN, degeneration also occurs in the midbrain and the forebrain of the chicken, cat, and ferret after exposure to a Type H OP. Sensorimotor, auditory, and visual pathways are targeted, and axonal, terminal, and cell body degeneration occur (Lillie and Smith, 1932; Veronesi et al. , 1986b; Veronesi and Dvergsten, 1987; Tanaka et al., 1990a, 1992a). Degeneration has not been assessed in the midbrain and forebrain of the rat after exposure to a Type H OP. The nervous system of young animals is also susceptible to Type H OPS. Chicks and ferret kits exhibit adult-like patterns of CNS degeneration when injected with a Single dose of a Type H OP at four weeks of age (Tanaka, pers. comm.). ' Thus, the neuropathological expression of Type H OPEN is similar to, but is more widespread than that of Type I OPEN. Degeneration is more extensive in the Spinal cord and hindbrain and is also found in the midbrain and forebrain. Also, cell bodies CI lib PIC Va]. Der dur 18 are affected in addition to the distal axon and terminal. The more extensive injury explains why additional clinical signs are associated with Type H OPEN. Electmphysiological Features Iype I OPEN - Sensitive Species. Electrophysiologieal assessment of Type I OPEN in sensitive species has been limited to the PNS. Electromyographic, sciatic and tibial‘nerve compound action potential, and nerve-muscle preparation studies have been conducted. ' Electromyographic studies have been conducted in humans, cats, and chickens after exposure to TOCP,, trichlorphon (dimethyl 1-hydroxy-2-trichloroethylphosphonate), or phenyl saligen phosphate (PSP) at a time point when the subjects were either ataxic or paralyzed. Abnormal spontaneous muscle activity consisting of fibrillation potentials and positive waves were consistently present in the gastrocnemius and anterior tibialis muscles of hindlimbs (humans and cats) and abductor pollicis brevis of the right hand (humans). Fibrillation potentials and positive waves are the electromyographic manifestation of muscle denervation (Hierons and Johnson, 197 8; Abou—Donia et al. , 1986). In contrast, fibrillation potentials were detected in only two of twenty ataxic chickens which had been previously exposed to PSP or TOCP, (Shell et al. , 1988). Anderson and her colleagues (Robertson et al. , 1987, 1988; Anderson et al., 1988) studied the effects of TOCP,, di-n-butyl-2,2-dichlorovinyl phosphate, and DFP on the properties of sciatic and tibial nerve compound action potentials in chickens. Conduction velocity, duration, and amplitude were minimally affected, but refractoriness in the tibial nerve was decreased, refractoriness in the sciatic nerve was increased, and the Strength- duration curves of sciatic and tibial nerves were elevated. These changes were apparent pril WC} dur dur wer incr Stud mus hinc gent tetal mus p051 clini mus but I 5mg dual dry-'31 19 prior to manifestation of clinical signs and were markedly affected after clinical signs were evident. , , El-Fawal and colleagues (El-Fawal et al., 1988, 1990a, 1990b) Studied strength- duration curves in biventer cervicis nerve-muscle preparations obtained from the neck region 'of chickens at various time points after exposure to TOCP, or PSP. Strength- duration curves were elevated, and rheobase and chronaxie calculated from the curves were increased and Shortened, respectively. This combination of effects indicated an increase in excitability threshold and muscle denervation. Lowndes and colleagues (Lowndes et al. , 1974, 1975; Baker and Lowndes, 1980) studied motor and sensory nerve terminal function in the cat in an in viva soleus nerve- muscle preparation at various time points after intraarterial injection of DFP into the hindlimb. In the affected hindlimb, the capacity of soleus al.-motor nerve terminals to generate stimulus—evoked repetitive discharge (SBR), SBR-dependant soleus muscle post- tetanic potentiation (PTP), and position sensitivities of secondary, but not primary, soleus muscle Spindle endings were attenuated. SBR, PTP, and secondary muscle spindle position sensitivities were lost as clinical Signs became evident and were regained as clinical Signs improved. Thus, both sensory and motor nerve terminals were affected. PTP was also studied in chicken plantaris muscle, the avian homolog of the cat soleus muscle, after exposure to TOCP,. TOCP, treatment reduced PTP in plantaris muscle, but the effect was not statistically significant (Durham and Ecobichon, 1984). In summary, electrophysiological assessment of Type I OPEN in sensitive Species has been limited to the PNS and has indicated that Type I OPEN consists of a mixed sensory and motor denervation of distal muscles of hind- or lower limbs and fore- or upper limbs. Of the electrophysiological properties assessed, changes in strength- duration curves and refractoriness of peripheral nerves are early indicators of subsequent development of clinical signs and nervous system damage. 20 Type I OPEN - Less Sensitive Species. Averbrook and Anderson (1983) and Anderson and Dunham (1985) studied the time-course of electrophysiological changes in the sciatic nerve compound action potential of male Sprague-Dawley rats after subchronic exposure to trichlorphon or DFP. The treatment increased nerve excitability (decreased duration and peak conduction velocity, increased rise time, and reduced refractoriness) but did not produce clinical signs or histopathological changes. Nerve excitability returned to normal with continued exposure suggesting nerve membrane compensatory changes. Thus, nerve excitability is an early indicator of exposure in less sensitive species, but changes occur in a direction opposite to that observed in sensitive species. Veronesi et al. (1987) evaluated somatosensory evoked potentials in the CNS of adult Long-Evans rats given a single subcutaneous dose of DFP (2 mg/kg body weight). Activity transmitted from the hindlimb to somatosensory cortex was depressed coincident with spinal cord neuropathic damage. Type II OPEN. Electrophysiological techniques have not been used to assess Type H OPEN. Inhibition of Neuropathy Target Esterase Type I OPEN - Sensitive Species. Although the exact mechanism of action of organophosphorus delayed neurotoxicants is unknown, inhibition and aging of the nervous system enzyme neuropathy target esterase (NTE) has been proposed as the initiating event (Johnson, 1982). NTE is a membrane-bound protein with no known physiological function and iS found in all regions of the nervous system. In addition, NTE activity has been measured in lymphocytes, the human placenta, and the testes of the chicken (Gurba and Richardson, 1983; Lotti et al., 1985; Johnson, 1990). 21 Neurotoxic OPS, or their active metabolites, phosphorylate the active Site of NTE and inhibit its eatalytic activity. All ester or amido bond which links an alkyl group to the central phosphorus atom is then cleaved in a process known as aging. The alkyl group is released and binds to an adjacent macromolecular site and a negative charge is left on the phosphorylated enzyme (Fig. 2). If aging occurs, NTE is permanently inhibited, and replacement of activity requires synthesis of new enzyme. Biochemical events between aging and expression of Type I OPEN are unknown. Two classes of compounds interact with the active Site of NTE: chemicals which inhibit and age (phosphates, phosphonates, and phosphoramidates) and chemicals which inhibit, but, because they do not contain a labile ester or amide bond, do not age (phosphinates, sulfonates, and carbamates). Administration of a non-aging inhibitor, such as phenyl methanesulfonyl flouride (PMSF), prior to exposure to a neuropathic OP prevents occupation of the active Site of NTE by a compound capable of aging and thus, prevents the ensuing neuropathy (Johnson, 1982). The degree of early inhibition of NTE can predict subsequent clinical and pathological expression of Type I OPEN. Exposure to a single dose of a neuropathic OP that produces a threshold value of at least 70 to 80% inhibition of brain and spinal cord NTE within one to 40 hours after dosing results in expression of Type 1 OPEN. Inhibition less than 60% appears to have no clinical or pathological correlate. The relationship between inhibition of NTE and repeated exposure is not as clear, but repeated exposure which raises inhibition of NTE to plateau values of 45 to 60% results in neuropathy (Johnson and Richardson, 1983; Johnson, 1990). A There are exceptions to the relationship between NTE inhibition and aging and the subsequent development of clinical and neuropathologic changes characteristic of Type I OPEN. Whole-brain NTE was inhibited greater than 70% in Japanese quail, bobwhites, pheasants, and young chickens after exposure to TOCP, or PSP but clinical ‘19. 2. 22 l) Characterised lirst lay selective p _ labelling with [32 -DFP. TARGET 21 Further characterised in vitro as an esterase PROTEIN OH with selective response to progressive inhibitors: called neurotoxic esterase (NTE). 3) Function in vivo a) Not known b) Not vitally essential 63 IN VIVO ORGANOPHOSPHORYLATION (or PHOSPHINYIATION, eicl or ACTIVE SITE or THE TARGET INHIIBITS NTE ACTIVITY. MODIFIED QR TARGET o-P.R PROTEIN ' BIOLOGICAL RESPONSES IN VIVO DEFEND ON THE NEXT CHEMICAL CHANGE - IF lor2 R-P bonds are C-O-P IF 80th R-P. bonds are C-P I Phosphate or Phosphonatel l Phosphinatel THEN THEN . 'Aging’ is possible Aging is impossmle (Usually rapid on NTE) 9.0- NO CHANGE O-P..R N0 NEUROPATHY also INHIBITED NTE IS PROTECTED _ AGAINST NEUROPATHIC O. P. ESTERS SO ANIMAL IS PROTECTED AGAINST THEIR NEUROPATHIC ETFECTSX. FORMATION OF AGED INHIBITED NTE INITIATES NEUROPATHY I/ Fig. 2. Inhibition and aging of neurOpathy target esterase (NTE). NTE is also called neurotoxic esterase (from Johnson, 1982). ex“ (Vex T0C 23 signs were not expressed (Bursian er al., 1983; olson and Bursian, 1988). Johnson (1990) suggests that damage is initiated, but these species and young animals either repair or adapt to the damage. Also, the half-life for recovery of NTE activity after inhibition is four to six days. Thus, NTE activity is recovering to control values when clinical and pathological expression occurs. Johnson (1990) ' states that recovery of NTE catalytic activity is not related to the neuropathy and that it is the initial formation of a critical amount of inhibited, aged NTE which precipitates the syndrome. Finally, if PMSF is administered. after a neuropathic OP, instead of before, the syndrome is potentiated rather than prevented (Pope and Padilla, 1990; Lotti er al., 1991). Authors of these studies concluded, however, that .potentiation of Type I OPIDN by PMSF probably occurs at a site distinct from NTE. , Iype I OPEN - Less Sensitive Species. Inhibition and aging of NTE are also thought to be the initiating events in expression of Type I OPIDN in rats (Johnson, 1990). NTE is thought to be the initiating event because: 1)the biochemical process of inhibition and aging in rats appears to be very similar to that in sensitive species (Richardson, 1984; Novak and Padilla, 1986§ Veronesi er al., 1991), 2)the degree of early inhibition of NTE after exposure to a single dose predicts subsequent spinal cord damage (Padilla and Veronesi, 1985; Veronesi et al., 1986a), and 3)administration of PMSF prior to a single dose of a neuropathic OP prevents development of spinal cord damage (Veronesi and Padilla, 1985). The relationship between inhibition of NTE and Type I OPIDN in mice is not as clear. The degree of early inhibition of NTE after exposure to a single dose does not predict subsequent spinal cord damage in mice (Veronesi er al., 1991), but mice which were gavaged with daily oral doses of 225 mg TOCPJkg body weight were ataxic and exhibited greater than 90% NTE inhibition after 270 days of exposure. 24 Iype II OPwN. The relationship between inhibition of NTE and development of Type II OPIDN has been assessed in the chicken, ferret, and rat. Carrington and Abou- Donia (1988a) injected adult female chickens with a single subcutaneous neuropathic dose of TPPi (1000 mg/kg body weight). NTE activity was inhibited more than 80% in whole brain and sciatic nerve 24 hours after dosing. Sciatic nerve NTE activity remained depressed throughout the 21 day study period, but whole-brain NTE activity recovered to 50% of control activity by 14 days after dosing and was still at this level at 21 days after dosing. The authors suggested that the long term depression of NTE activity was due to slow absorption of TPP, from the injection site. Long-term depression of NTE activity in chicken whole brain after exposure to a single subcutaneous dose of 1000 mg TPPi/kg body weight was also noted by Tanaka et al. (1992a). TPP, is not a potent in viva inhibitor of NTE in ferrets and rats. In an unpublished study (Bursian, pers. comm.), adult ferrets were injected with a single subcutaneous neuropathic dose of TPPi (1000 mg/ kg body weight). Whole-brain NTE activity was inhibited 46 and 35 % two and eight days after dosing, respectively. Veronesi et al. (1986b) and Padilla er al. (1987) injected adult male Long-Evans rats subcutaneously with 1184 mg TPP, body weight once per week for two weeks. Although the neuropathy developed, NTE inhibition was never greater than 39 96 on average in brain or spinal cord at any of the time points assessed (4 and 48 hours after the first dose and 4, 24, 48 and 72 hours after the second dose). The authors found, however, that TPP, is a potent in vitra inhibitor of rat brain NTE. The 1,0 for inhibition of whole-brain NTE by TPP. was 0.98 uM. This value is ten times less than that for mipafox and is equivalent to that of DFP, both of which are potent in viva and in vitra inhibitors of NTE. The authors suggested the disparity between in vivo and in vitra potency could be due to: 1)chemical reactivity of TPPi in viva, 2)metabolism of TPP, to compounds not eapable of inhibiting NTE, or 3)reversible inhibition of NTE allowing spontaneous reactivation. In summary, 25 inhibition and aging of NTE is thought to be the initial biochemical lesion in Type I OPIDN, but the relationship between NTE activity and Type II OPIDN is not as clear. NTE activity is depressed for long periods in chickens exposed to ’TPPi, and Type II OPIDN can manifest in the ferret and rat with subthreshold NTE inhibition. Summary Characteristics of Type I OPIDN in adults of sensitive species include a delay period of 5 to 21 days before onset of ataxia, confinement of lesions to the hindbrain, spinal cord, and peripheral nerves, and degeneration of the distal axon and terminal. Electrophysiological changes indicate denervation of distal muscles in hind- or lower and fore- or upper limbs, and the initiating event is believed to be inhibition and aging of NTE. Characteristics of Type I OPIDN in young of sensitive species and in the adult rat, a less sensitive species, is dependant on the extent of exposure. Exposure to a single dose does not produce clinical signs, but does result in threshold inhibition of NTE. Adult rats also develop a limited distribution of nervous system damage. As the extent of ' exposure increases, nervous system damage is comparable to that exhibited by adults of sensitive species and clinical signs can develop. The response of mice, also a less sensitive species, to Type I OPs is anomalous and includes uncharacteristic nervous system damage and an unclear relationship to NTE inhibition. Species variation in susceptibility has been attributed to pharmacokinetic disparities, qualitative and quantitative differences in NTE, and neuroanatomical differences (V eronesi, 1984a). Characteristics of Type II OPIDN in chickens, ferrets, cats, and rats include susceptiblity of young animals, a shortened delay period before onset of ataxia, additional clinical signs of extensor rigidity in cats and circling in ferrets and rats, presence of 26 lesions in the forebrain and midbrain as well as in the hindbrain, spinal cord, and peripheral nerves, and degeneration of cell bodies as well of the distal axon and terminal. The relationship between NTE and development of Type H OPIDN is not clear. Type II OPIDN can develop in rats and ferrets without threshold inhibition of NTE. EXPERINIENT I OBJECTIVES To map the extent of degeneration in the central nervous system (CNS) of the rat after exposure to the Type I or Type II organophosphorus delayed neurotoxicants diisoprocyl phosphorofluoridate (DFP) and triphenyl phosphite (TPPi), respectively, by use a a Fink-Heimer silver' impregnation method. To assess TPPi-induced clinical signs "so that they can be correlated to CNS degeneration in the rat. To compare CNS degeneration patterns in the rat to those reported for the chicken and ferret to determine, based on neuropathological considerations, the suitability of the rat as a model for Type I or Type H OPIDN. RATIONALE The CNS is organized anatomically and functionally. Anatomically, it is divided into two regions, the spinal cord and brain. The brain is rostral to the spinal cord and can be divided into three bilaterally paired anatomical regions which are, from caudal to rostral, the hindbrain, the midbrain, and the forebrain. The hindbrain consists of the medulla, pans, and cerebellum; the midbrain contains the inferior and superior colliculi; and the forebrain is comprised of the diencephalon (thalamus and hypothalamus) and the cerebral hemispheres (cerebral cortex, basal ganglia, hippocampus, and amygdala) (Kelly and Dodd, 1991). The CNS is divided into three major functional systems known as the sensory, motor, and motivational (limbic) systems. The sensory system detects environmental or 27 int: i I inf; by sys CV6 pic Sy: Ni: are fur (T: 361‘. of 28 internal stimuli and relays the information to the motor system which plans and executes a behavioral act. The motivational system impinges upon the motor system and influences the initiation and completion of a behavior. Specific behaviors are mediated by anatomically and functionally distinct systems which occur within the major functional systems, such as the somatosensory and visual sensory systems. These systems contain even more specialized divisions which are known as pathways. For example, the somatosensory system contains separate pathways for the perception of touch and pain. Pathways are comprised of functionally related and interconnected nuclei and fiber tracts that are found throughout the spinal cord and brain. Nuclei and fiber tracts in a pathway process and relay specific information within and between functional systems. Nuclei and fiber tracts are comprised of the fundamental units of the nervous system, neurons. A relay nucleus contains dendrites and cell bodies of neurons and the axon terminals which innervate the neurons while fiber tracts contain nerve cell axons which connect the relay nuclei in a pathway (Kelly and Dodd, 1991). Thus, the function of the nervous system is dependent on the anatomical organization of neurons within nuclei and fiber tracts in CNS pathways. The Fink-Heimer technique selectively stains degenerating cell bodies, axons, and axon terminals in nuclei and fiber tracts at all levels of the CNS (Tanaka et al. , 1992b). Nissl stains can be used to identify nuclei and fiber tracts. Therefore, when these stains are used in combination, pathways damaged by neurotoxicants ean be identified and their function can be correlated to neurotoxicant-induced behavioral deficits (clinical signs) (Tanaka et al. , 1992b). Because the Fink-Heimer technique has also been found to be sensitive for detecting organophosphorus-induced CNS degeneration (Cavanagh and Patangia, 1965; Tanaka et al., 1992b), it, in conjunction with a Nissl stain, is the method of choice for mapping Type I and Type II-induced CNS degeneration in the rat. CI m2 dix 29 TheFink—Heimerprocedurehasalsobeenusedto mapTypeIandTypeIIOP- induced degeneration in the CNS of the chicken and ferret (Tanaka and Bursian, 1989; Tanaka et al., 1990a, 1990b, 1991, 1992a). Therefore, CNS degeneration patterns among these species can be compared to determine, based on neuropathologieal considerations, the suitability of the rat as a model for Type I and Type II OPIDN. METHODS Test Species and Husbandry. Adult male Long-Evans rats (Harlan Sprague Dawley, Indianapolis, IN) were used for these trials. Rats were housed individually in polycarbonate boxes with aspen chip bedding. The holding room was maintained at approximately 22°C and 50% humidity with a 12 hour light-dark cycle. Rats were acclimated to the environment for at least two weeks prior to the start of a trial. Drinking water and Purina Rodent Lab Chow #5001 (Purina Mills Inc. , St. Louis, M0) were available ad libitum throughout the study. Rats which developed neuropathy had difficulty in obtaining water and food, and thus, were injected subcutaneously with 10 ml of lactated Ringer’s solution three times a day and had food placed in an accessible location. All aspects of this study were approved by the Michigan State University Committee for Animal Use and Care. Test Materials. Diisopropyl phosphorofluoridate (DFP) was purchased from Sigma Chemical Company (St. Louis, MO) and triphenyl phosphite (TPP9 from Aldrich Chemical Company (Milwaukee, WI). The purity of TPPi was 97% according to the manufacturer. Experimental Design. A DFP trial and a TPPi trial were conducted. In the DFP trial, eight rats, 17 weeks old and ranging in weight from 340 to 400 g, were randomly divided into two groups of four rats each. Rats in one group were injected Iva 0th of 15 rat. we 8,‘ 0f: 81'0 clin Var} lieu Eac On( gTOL Ofp 3O subcutaneously in the nape of the neck with 4 mg DFP/kg body weight on day one. DFP was delivered in dimethyl sulfoxide in a volume of 0.5 ml/kg body weight. Rats in the other group served as controls. In order to protect against the anticholinesterase action of DFP, treated rats were also injected with atropine sulfate (1.5 mg/kg body weight, ip) 15 minutes prior to and just after DFP injection. Twenty-four hours after dosing, two rats from each group were killed by decapitation. Brains were removed rapidly, weighed, frozen on dry ice, and subsequently assayed for neuropathy target esterase (NTE) activity according to the method of Johnson (1977). The remaining two rats in each group were observed daily in an open field for clinical signs of neurologic impairment. The open field was circular (diameter = 95 cm; height = 15 cm) and constructed of plastic-coated wire (1.4 x 1.4 cm mesh). On day 28, the remaining two rats in each group were weighed and perfused for neuropathological analysis. In the TPP, trial, eight rats, 16 weeks old and ranging in weight from 350 to 500 g, were randomly divided into a group of six rats and a group of two rats. The group of six rats was injected subcutaneously in the nape of the neck with 1184 mg TPP/kg body weight (undiluted, 1.0 ml/kg body weight) on days one, four, and seven. The group of two rats served as controls. All rats were observed daily in the open field for clinical signs of neurologic impairment. The latent period to onset of clinical signs was variable among TPPi-treated rats. Therefore, two TPPi-treated rats were perfused for neuropathological analysis on day 11, one on day 15, two on day 21, and one on day 25. Each TPP, rat was weighed prior to perfusion. Control rats were weighed and perfused on day 25. Evaluation of Body Weight and N112 Activity. For the DFP trial, the percent gain in average body weight from day one to day 28 was calculated for the control and treated groups. For the TPP, trial, the percent gain in body weight from day one until the day of perfusion was calculated for each rat. NTE activities obtained in the DFP trial were ph 5P CT. er. in f01 ph de mi We of dei 31 analyzed statistically with the Student’s t test (Gill, 1978), and percent inhibition of NTE activity was calculated using the control activity. Neuropathological Analysis. Rats were deeply anesthetized with two ml of sodium pentobarbital (40 mg/ml, ip) and perfused transcardially with a 10% formalin-saline solution. Just prior to perfusion with fixative, one ml of heparin (168 USP units/ml physiological saline) was injected into the left ventricle. Following perfusion, brains and spinal cords were removed and placed initially in a 10% formalin-saline solution for one to two days. Spinal cord blocks were obtained from the first cervical cord segment and the cervical and lumbar enlargements. Brains and spinal cord blocks were then cryoprotected for two to three days in a 30 % sucrose-formalin solution. After cryoprotection, frozen sections were cut from brains in the coronal or sagittal plane and from spinal cord blocks in the coronal plane at a thickness of 40 um. Every 5th or 10th section was processed using a variation of the Fink-Heimer silver impregnation method for staining degenerating cell bodies, axons, and terminals (Tanaka, 1976). Adjacent sections were stained with cresyl violet to delineate nuclear boundaries and to assess perikaryal effects. Selected adjacent Fink-Heimer and cresyl violet-stained sections were photographed at low power with a Wild M400 Photomacroskop. Areas containing degeneration were mapped directly onto the photographs with the aid of a compound microscope, and line drawings were traced from the photographs. Nuclei and fiber tracts were identified according to Zilles (1985) and Paxinos and Watson (1986). The postsubiculum was identified according to Van Groen and Wyss (1990). The severity of degeneration was described using the classification of no degeneration, light degeneration, moderate degeneration, or heavy degeneration. 32 RESULTS DFP Dial Body Weight Gain. The effect of DFP on body weight grain is presented in Table 1. Body weight gain was similar for control and DFP rats over the 28 day trial period. ME Activity. The effect of DFP on whole—brain NTE activity is presented in Table 2. Whole-brain NTE activity was inhibited significantly (p < 0.05) by administration of DFP. Inhibition was 87% 24 hours after dosing. TABLE 1 THE EFFECT OF DFP ON BODY WEIGHT GAIN IN THE RAT — % Gain Treatment‘ Day 1 to day 28 Control 11.6 :1; 2.6" DFP 12.7 i 5.5 ‘DFP rats were injected with 4 mg DFP/kg body weight (so) on day one. ”Data presented as mean :1; standard deviation. Sample size is 2. TABLE 2 THE EFFECT OF DF P ON WHOLE-BRAIN NEUROPATHY TARGET ESTERASE (NTE) ACTIVITY IN THE RAT _ Whole-brain Treatment' NTE activityb % Inhibition Control 1154 i 24" - DFP 150 j; 4" 87 'DFP rats were injected with 4 mg DFP/kg body weight (ac) on day one. ”Brains were removed for determination of NTE activity 24 hours after dosing. Units of activity are nmol phenyl valerate hydrolyzed/min/g brain. ‘Datapresentedasmean :1: standarderrorofthemean. Samplesize is 2. ‘Significantly different from control (p < 0.05). the DI hat SIU im} deg ant sill dc; Sl1\ loo 1x0 COD grai nor nor 33 Clinical Signs. Control rats did not exhibit any signs of neurologic deficit during the 28 day observation period. Within two to three hours after dosing, rats treated with DFP exhibited signs typical of anticholinesterase toxicosis including labored respiration, tremors, and reduced activity. Signs appeared diminished 24 hours after dosing, and rats had recovered by 48 hours after dosing. No further signs of neurologic impairment were observed in DFP rats during the 28 day observation period. ' Central Nervous System Degeneration. Silver-impregnated degeneration appeared as black debris against a light brown or yellow background. Electron microscopic studies (Guillery, 1970; Heimer, 1970) have shown that the silver preferentially impregnates degenerating axons and terminals. Axonal degeneration in fiber tracts consisted of linearly arranged black fragments. Preterminal axonal and terminal degeneration in nuclear regions appeared as short, irregularly arranged fiber fragments, and somewhat variably shaped punctate debris, respectively. Sources of artifact included silver impregnation of reticular fibers of blood vessels and neuronal somata, selective deposition of silver within the cytoplasm of specific neuronal groups, and nonspecific silver precipitation. Degeneration was distinguished from artifact based on appearance, location within known fiber tracts or nuclei, continuity in serial sections, and its bilateral occurrence. Degeneration was not detected in the CNS of control rats. Degeneration detected in the CNS of DFP-exposed rats was bilateral and was similar for both rats. Moderate axonal degeneration in the gracile fasciculus could be traced from the first cervical spinal cord segment to moderate preterminal axonal and terminal degeneration throughout the gracile nucleus of the medulla (Figs. 4 and 5). Axonal and terminal degeneration were not detected in any other region of the spinal cord or brain. Cell body degeneration was not detected in the CNS of DFP-exposed rats. 34 Fig. 3. Line drawings illustrating approximate levels of the brain and spinal card used to depict degeneration in subsequent figures. Horizontal lines at the top of (1) indicate cross-section levels selected for the DFP (short line) and TPPi (long line) trials. Solid vertical lines in (1) correspond to similarly lettered sections in Figs. 4 and 6-10 while dashed vertical lines in (1) correspond to similarly lettered sections in Fig. 11. Solid vertical lines in (2) indicate sagittal levels selected for the TPP, trial and correspond to similarly lettered sections in Figs. 12 and 13. -I-w, s__ l \ \ I\\ \ /- / I 36 Fig. 4. Line drawings of cross-sections depicting the location of degeneration in the caudal hindbrain (L) and rostral spinal cord (M) of the rat after exposure to DFP. Two rats were injected with a single dose of 4 mg DFP/kg body weight (sc) and were perfused on day 28. The brain levels from which sections L and M were obtained is depicted in Fig. 3. 38 Fig. 5. Photomicrographs illustrating control (A) and 28 day post-DFP (B) Fink-Heimer silver impregnated cross-sections through the gracile nucleus of the rat. DFP rats were injected with a single dose of 4 mg DFP/kg body weight (so). The gracile nucleus of DFP-injected rats contained irregularly and variably shaped punctate debris which probably represents a mixture of preterminal axonal and terminal degeneration (arrows). In the control plate (A), note the silver impregnation of reticular fibers of a blood vessel (star) and of cell bodies (arrowhead) and the absence of preterminal axonal and terminal degeneration (magnification x100). 39 Fig. 5 TPP, r A. develo probal times groom E 3. TI the tri weigh the 2s \ figns 4o TPP, Trial Animal Condition. Firm cutaneous masses approximately 1 cm "in diameter developed at some of the injection sites in all TPPi-treated rats. The masses were probably the result of an inflammatory reaction to TPPi. Rats with the longest survival times also developed hair loss, at the injection site. TPP, rats also appeared poorly groomed at the time they were perfused. Body Weight Gain. The effect of TPPi on body weight gain is presented in Table 3. The two control rats had an 8 to 12% gain in body weight over the 25 day period of the trial while TPPi rats lost from 5 to 28% in body weight. In general, the loss of body weight was greatest for TPPi rats with the shortest survival times. TABLE 3 THE EFFECT OF TPPI ON BODY WEIGHT GAIN IN THE RAT Day of Rat number Treatment' Icrfusion % Gain” 20 Control ' 25 12 21 Control 25 8 25 TPP, 1 1 -28 23 TPPi 1 l -22 22 TPPi 15 ~17 19 TPP, 21 -5 24 TPP, 21 -16 18 TPPi 25 -5 'TPP, rats were injected with 1184 mg TPP/kg body weight (so) on days one, four, and seven. |’Represents percent gain in body weight from day one to the day of perfusion. Clinical Signs. Control rats did not exhibit any signs of neurologic deficits during the 25 day observation period. All TPPi-exposed rats developed clinical signs. Clinical signs in general were similar among TPPi rats, but differences were apparent and the latencg provid genera on the increa behav side-ti and ti head I flaccit behav and ti 41 latency to onset and order of progression varied. Therefore, a clinical description is provided for each TPP, rat (rats 25, 23, 22, 24, 19, and 18). Clinical signs in rat 25 initially became apparent on day nine when the rat exhibited generalized weakness and had long periods of inactivity. When active, all movement was directed forward but the rat did not rear. Hindlimbs were slightly ataxic, the rat walked on the tip-toes of hindfeet, and the distal tail was flaccid. On day 10, activity was increased and consisted of repetitive movement of the forebody from side-to—side. The behavior was executed using forelimbs while hindlimbs remained mostly stationary. The side-to-side movement was unbalanced and the rat often fell backwards onto hindquarters and then backed up, using hind- and forelimbs, for several steps. The rat did not rear, head and forelimb support appeared weakened, and hindlimbs and the distal tail were flaccid. On day 11, movement was directed forward, and side-to-side and backing behaviors did not occur. However, head support was weak and the forelimbs, hindlimbs, and the tail were completely flaccid with the hindlimbs splayed to the side. Rat 25 was perfused on day 11. Clinical signs in rat 23 initially became apparent on day nine with the majority of movement directed side-to-side as described above. The rat never reared but occasionally moved forward for two to three steps displaying slight hindlimb ataxia. Also, the rat walked on the tip-toes of its hindfeet and the distal tail was flaccid. On day 10, when first placed in the open field, the rat circled counterclockwise. Circling was very similar to side-to-side behavior in that forelimbs were used to pull the animal in a circle while hindlimbs remained mostly stationary. Forward movement was very ataxic consisting of a repeating cycle of one to two steps forward followed by falling back onto lower hindlegs or sometimes to the side. The rat did not rear and the distal tail was flaccid. On day 11, rat 23 did not circle, but exhibited repetitive side-to-side movements. The movement was unbalanced and evolved into backing movement similar were I forwz follm the n were rat W: l‘het: 3PM andfi flaCCIl have alatltic Veil i prim, were f the tai: 42 to that described for rat 25. Also, the rat did not rear and hindlimbs and the distal tail were flaccid. Rat 23 was perfused on day 11. Clinical signs were first apparent in rat 22 on day 12. Movement was directed forward. Hindlimb gait was slightly ataxic and consisted of four to five steps forward followed by falling back onto the lower portion of the hindlimbs or to the side. Also, the rat did not rear and the distal tail was flaccid. On days 13 and 14, clinical signs were similar to those observed on day 12 except hindlimb ataxia had increased and the rat walked on the tip—toes of his hindfeet. On day 15, hindbody deficits had progressed. The tail appeared very rigid and was held in a semi-circle above the body, hindlimbs also appeared rigid, foot placement was very deliberate, and the rat again walked on the tip- toes of its hindfeet. Rat 22 was perfused on day 15 . Gait deficits in rat 19 were first observed on day 20. Direction of movement was unaffected and the animal could rear but moderate hindlimb ataxia was displayed and was characterized by dropping to lower legs or falling to the side. By day 21, deficits were more pronounced. The rat displayed repetitive side—to-side movement as previously described. Periodically, the animal also moved forward two to three steps. Side-to—side and forward movement was extremely ataxic, the rat did not rear, hindlimbs were flaccid, and the tail was rigid. Rat 19 also developed a tail kink about one inch from the base of the tail on day nine which persisted until the rat was perfused on day 21 . Gait deficits were first observed in rat 24 on day 20 when movement alternated between repetitive side-to-side activity and bidirectional circling. The animal was not ataxic but it did not rear and the distal tail was flaccid. On day 21, clinical signs were very similar to those observed on day 20, but were more severe. Movement was primarily side-to-side or circular. The rat did not rear and hindlimbs and the distal tail were flaccid. Also, on day nine, a kink was observed about one inch from the base of the tail which persisted until the rat was perfused on day 21. and wen held the I circl were rapic com] dege was : of C0 Axor TPP. SimiL Van's. CNS: deger. belov, the Si: betWel 43 Rat 18 initially displayed gait deficits on day 25 . Movement was directed in a circle and was interspersed with rearing or movement forward. All three types of movement were very ataxic, and the rat often dropped onto lower legs. Also, the tail was rigid and held in a semi-circle above the body. A kink approximately one inch from the base of the tail was observed on day 9 and was still apparent when the rat was perfused on day 25 . In summary, clinical signs were comprised mainly of hindlimb deficits (inability to rear and hindlimb ataxia and flaccidity) and directional deficits (side-to-side, backing, and circling movements) and in general were similar for all TPPi rats, although differences were apparent and the order of progression did vary. Clinical signs also progressed rapidly. Rats which had post-onset survival times of two or more days exhibited the full complement of deficits. Clinical signs are summarized in Table 4. Central Nervous System Degeneration. The appearance of silver-impregnated degenerating axons in fiber tracts and preterminal axons and terminals in nuclear regions was similar to that observed in the DFP trial. Degeneration was not detected in the CNS of control rats nor was cell body degeneration detected in the CNS of TPPi-exposed rats. Axonal, preterminal axonal, and terminal degeneration were detected in the CNS of all TPPi-exposed rats and were bilateral. The general pattern of degeneration observed was similar for the six TPPi-exposed rats. However, the extent and density of degeneration varied among rats. Differences are noted as necessary in the following description. The CNSs of rats 22 and 25 contained the most degeneration and were used to depict degeneration in the cross- (rat 22) and sagittal (rat 25) section line drawings referred to below. Table 5 provides a list of affected brain regions and degeneration densities for the six TPPi—exposed rats. In the forebrain, degeneration was detected in transitional cortex (cortex which lies between neocortex and allocortex), the neocortex, the septum and hypothalamus, the 44 TABLE 4 THE EFFECT OF TPPl ON DEVELOPMENT OF CLINICAL SIGNS IN THE RAT Rat No.‘ Predominant clinical signs" 1232.2 DILIQ 1291.11 25 Inability to rear Inability to rear Inability to rear Tip-toe walking Hindlimb ataxia Forelimb, hindlimb, Hindlimb ataxia Distal tail flaccidity and tail flaccidity Distal tail flaccidity Side-to-side movement Backing movement 1281.2 1231.19 M 23 Distal tail flaccidity Inability to rear Inability to rear Hindlimb ataxia Distal tail flaccidity Hindlimb and distal Side-to-side movement Hindlimb ataxia tail flaccidity Circling Side-to—side movement - Backing movement ’ M15 1291.1; 22 - Inability to rear Inability to rear Tip-toe walking Tip-toe walking Distal tail flaccidity Hindlimb and tail Hindlimb ataxia rigidity M29 12312.1 19 - Hrnd' limb ataxia Inability to rear Tail rigidity Hindlimb flaccidity Side-to-side movement Day 20 Day 21 . 24 - Inability to rear Inability to rear Distal tail flaccidity Hindlimb and distal Side-to-side movement tail flaccidity Circling Side-to-side movement Circling no.2; . 18 - - Tail rigidity Hindlimb ataxia Circl'flg 'Rats were injected with 1184 mg TPPilkg body weight on days one, four, and seven. ‘The first day listed for each rat represents the day of onset of gait deficits and the last day represents the day of perfusion. Rate 19, 24, and 18 also developed tailldnksondayninewhichpersisteduntiltheday ofperfusion. RAT A) FOREBRAIN intsitio lateral Ihdial lgranul corte lecortex Sensori Auditor Visual Motor c -£P0 HP HM 45 TABLE 5 RAT CENTRAL NERVOUS SYSTEM REGIONS WHICH CONTAINED AXONAL, PRETERMINAL AXONAL, AND/OR TERMINAL DEGENERATION AFTER ADMINISTRATION OF TPPi. Rat Rat Rat Rat Rat Rat 25a 23 22 19 24 18 FOREBRAIN Transitional Cortex Lateral orbital area +++b ++ +++ ++ + + Medial prefrontal cortex + + + 0 O 0 Agranular retrosplenial cortex + + + 0 0 Neocortex , Somatosensory cortex +++ +++ +++ + + + Sensorimotor cortex +++ +++ +++ + + + Auditory cortex + + + + 0 + Visual cortex + + + 0 0 + Motor cortex ++ ++ + 0 O 0 Septum and Hypothalamus HDB ++ ++ + 0 0 0 MCPO ++ ++ + 0 0 0 MP + + + + + + MM +++ ++ +++ +++ +++ +++ ML + + + + + + MMn - + + + + + + Hippocampal Region CAléCAB + + + 0 0 0 06 + + + ‘O O' 0 P05 + + + + 0 0 Thalamus MD + + ++ + + ++ .VM + ++ +++ ++ + +++ MG ++ ++ + ++ 0 + CM + + + + + + pc + + + + + + CL + 0 + + 0 + HIDBRAIN Basal Ganglia . _ , STh + + + .+ ’0 + SNC + + + + 0 + Superior Colliculus InG ++ + ++ + +. - + Inwh' ++ + ++ + + + Pretectum + .+ ++ + + ++ Inferior Colliculus c1c ++ + ++ ++ + ++ ECIC + + + + + + Miscellaneous 3 + + + + 0 + DR + + g + + 0 + All 5 (c IIIIDBRAIN the lll'eV Spi’e Su‘i’e PSoI :mh aranul ar "I“ I'll) Hill I a r 46 TABLE 5 (can‘t) Rat Rat Rat Rat . . Rat Rat 25a 23 22 19 . 24 18 HINDBRAIN Vestibular Nuclear Complex MVe +++ ++ +++ ++ ++ ++ MVeV -l- + + + + + SpVe ++ + ++ + ++ + SuVe . + + + + 0 + PSol + + + + + + Cerebellum Granular cell layerC + + + 0 0 0 Cochlear Nuclei DC + + + + + + VCP ‘ 0 + + + 0 0 Medullary Reticular Formation 0 0 + 0 0 0 Spinal Cord ' gr ' 0 O + + 0 0 Laminae v-x of c1d -e o + - - - a Rats listed were injected with 1184 mg TPPi/kg body weight (so) on days one, four, and seven and were perfused at various stages of progression of clinical signs (see Table 4 for a description of clinical signs). Control rats are not listed because degeneration was not detected in their CNS. “2 b Represents degeneration density: 0 = none; + = light; ++ = moderate; and +++ = heavy. ‘ , C Terminal degeneration was present in the granular cell layer of all cerebellar lobules. d Represents the first cervical spinal cord segment. e Spinal cord was not examined. hipl deg: path pref sens 9, l prin area deg: laye 47 hippocampal region, and the thalamus. In transitional cortex, light to heavy terminal degeneration was noted in the lateral orbital area of all TPPi-exposed rats while a mixed pattern of light preterminal axonal and terminal degeneration occurred in the medial prefrontal and agranular retrosplenial cortices of rats 25, 23, and 22 (Table 5, Figs. 6, 7, 8, 9). In the neocortex, degeneration was detected in the somatosensory, sensorimotor, auditory, visual, and motor cortices of rats 25 , 23, and 22 (Figs. 6, 7, 8, 9, 10). These rats showed heavy terminal degeneration throughout layer IV of the primary and supplementary somatosensory cortices and in the forelimb and hindlimb areas of sensorimotor cortex. They also contained light to moderate terminal degeneration in layer IV of the auditory, visual, and motor cortices. Degeneration in layer IV occasionally extended to layers 11, III, and V. Preterminal axonal degeneration in layer V appeared to project to layer IV in the more heavily affected neocortical areas. The deeper part of layer V of the primary somatosensory cortex of rat 25 contained particularly dense preterminal axonal degeneration (Fig. 7). Neocortical degeneration in rats 19, 24, and 18 was light and was confined to layer IV of somatosensory, sensorimotor, auditory, and visual cortices (Table 5, Fig. 11). In the septum and hypothalamus, degeneration was detected in three brain regions. Light to moderate terminal degeneration was found in the nucleus of the horizontal limb of the diagonal band and the magnocellular preoptic nucleus of rats 25 , 23, and 22 (Table 5 , Figs. 6, 8). Moderate to heavy terminal degeneration occurred in the medial part and light terminal degeneration was noted in the posterior, lateral, and median parts of the medial mamillary nucleus of all TPPi-treated rats (Table 5, Figs. 6, 10, 12). Degeneration in the hippocampal region consisted of light axonal degeneration seattered throughout the dentate gyrus and fields CAI-CA3 of Ammon’s horn (rats 25, 23, and 22) and a mixed pattern of light preterminal axonal and temtinal degeneration in the postsubiculum (rats 25, 23, 22, and 19) (Table 5, Figs. 6, 7, 9, 10). 48 Fig. 6. Line drawings of sagittal sections depicting the location of axonal degeneration in fiber tracts (dashed lines) and preterminal axonal (dashed lines) and terminal (dots) degeneration in nuclear regions of the brain of rat 25. Rat 25 was injected with 1184 mg TPPJkg body weight (so) on days one, four, and seven and was perfused on day 11. Drawings are arranged from medial (A) to lateral (C). The brain levels from which sections A-C were obtained are depicted in Fig. 3. 49 Emma manna“ ’ \ . 777 A" " ‘ "Ca‘ ¢,\/ (5"; w c Fig. 6 50 Fig. 7. Line drawings of sagittal sections which are a lateral continuation of Fig. 6 and depict the location of preterminal axonal (dashed lines) and terminal (dots) degeneration in gray matter of the brain of rat 25. Rat 25 was injected with 1184 mg TPPilkg body weight (sc) on days one, four, and seven and was perfused on day 11. Drawings are arranged from medial (D) to lateral (F). The brain levels from which sections D-F were obtained are depicted in Fig. 3. j 51 .r“ .3..q:.:~’..'. I‘.- “2......“ "°.. '.'0. a . . ”.32.”. ;~\};:;W- .. .FAUV‘s-og _;-..7..r.‘ . :bfl'.’” . I "7' r .. ‘ug "‘ .— ‘_}ééw Cruel Crud V "I Own 52 Fig. 8. Line drawings of cross-sections depicting the location of preterminal axonal (dashed lines) and terminal (dots) degeneration in gray matter and nuclear regions of the forebrain of rat 22. Rat 22 was injected with 1184 mg TPP/kg body weight (sc) on days one, four, and seven and was perfused on day 15 . Drawings are arranged from rostral (A) to caudal (C). The brain levels from which sections A-C were obtained are shown in Fig. 3. 54 Fig. 9. Line drawings of cross-sections which are a caudal continuation of Fig. 8 and show the extent of preterminal axonal (dashed lines) and terminal (dots) degeneration in the gray matter and nuclear regions of the forebrain of rat 22. Rat 22 was injected with 1184 mg TPP/kg body weight (sc) on days one, four, and seven and was perfused on day 15. Drawings are arranged from rostral (D) to caudal (F). The brain levels from which sections D-F were obtained are depicted in Fig. 3. 55 PRIMARY SOIATOSENSORY SUPPLEMENTARY somroaeusonv - . conrex it?“ \ ‘. -'.-' ~‘. 4.5:" ‘ 1435?. :1 .. 'I ‘ u \ s . \ I'ou‘ ‘ . . ,5($.' -& \‘ ‘ C ‘ "MARY SOMTOSENIOHV COME! SWLE‘NTAIW somromonv -- , comsx . . - Fig. 9 56 Fig. 10. Line drawings of cross-sections which are a caudal continuation of Fig. 9 and show the extent of preterminal axonal (dashed lines) and terminal (dots) degeneration in the gray matter and nuclear regions of the midbrain of rat 22. Rat 22 was injected with 1184 mg TPPJkg body weight (sc) on days one, four, and seven and was perfused on day 15. Drawings are arranged from rostral (G) to caudal (H). The brain levels from which sections G and H were obtained are depicted in Fig. 3. VIS COR 57 VISUAL CORTEX VISUAL COBTEX 58 Fig. 11. Photomicrographs illustrating Fink-Heimer silver impregnated sections through the somatosensory cortex of rats 25 (A and C) and 24 (B). Rats 25 and 24 were injected with 1184 mg TPPilkg body weight (sc) on days one, four, and seven and were perfused on days 11 and 21 , respectively. Plate A shows heavy terminal degeneration in layer IV of the somatosensory cortex of rat 25 while plate B shows that the same region of the brain in rat 24 contained light terminal degeneration. Plate C exhibits light preterminal axonal degeneration (arrows) in layer V of the somatosensory cortex of rat 25 . Plate-D is a control section through layers IV and V of somatosensory cortex (magnification x100). \x u .. n . '1 u‘ , . . . as. . - .4. \‘n.. b . ..\ . .. V. D. ‘ . . . 7.. x.‘\ . a . . .. _. . .......a...~....n. .. a... . .. ... Haze- ..\.£. ...f..w..»1_.rw. I . Fig. 11 Fig. 12. Photomicrographs illustrating control (left) and TPPi (right) Fink-Heimer silver impregnated sections through the medial mamillary nucleus, medial part (A, B) and the medial vestibular nucleus (C, D) of the rat. TPP, rats were injected with 1184 mg TPPi/kg body weight on days one, four, and seven and were perfused at various time points. Exposure to TPPi resulted in heavy terminal degeneration (variably shaped punctate debris) in the medial mamillary nucleus, medial part (B), while the medial vestibular nucleus (D) contained moderate preterminal axonal degeneration (arrows) interspersed with terminal degeneration. Also note the absence of degeneration in the control sections (A and C) (magnification x100). 12 Fig. the rats deg thal the deg app DUC app inf: det: sub Deg all lerr reg- Slip In t nuc dor sun alig Tap, terr 62 Several thalamic nuclei also contained degeneration (Figs 6, 7, 9, 10). In general, the pattern of degeneration in the thalamus was consistent among the six TPP,—exposed rats although some differences in density did exist (Fable 5). Light to heavy terminal degeneration was seen in the mediodorsal, the ventromedial, and the medial geniculate thalamic nuclei. In addition, degeneration was noted in the intralaminar thalamic nuclei, the central medial, paracentral and centrolateral nuclei. Light preterminal axonal degeneration coursed in a medial to lateral direction in the central medial nucleus and appeared to project to light terminal degeneration in the paracentral and the centrolateral nuclei. Preterminal axonal degeneration in the central medial thalamic nucleus also appeared to project to the ventromedial thalamic nucleus. In the midbrain, degeneration was detected in the basal ganglia, the superior and inferior colliculi, and in the oculomotor and dorsal raphae nuclei. Degeneration was detected in two basal ganglia nuclei associated with the midbrain tegmentum, the subthalamic nucleus and the substantia nigra, pars compacta (Figs. 6, 9, 10). Degeneration in these nuclei was moderate, was terminal in nature, and was detected in all TPPi-exposed rats except rat 24 (Table 5). In the superior colliculus, moderate terminal degeneration was found in the intermediate gray and white layers and in the region of the pretectum just ventral and lateral to the those layers. Degeneration in the superior colliculus occurred in all TPPi-treated rats except rat 24 (Table 5, Figs. 6, 10). In the inferior colliculus, light to moderate terminal degeneration was found in the central nucleus of all TPP, rats. Terminal degeneration in this nucleus was interspersed with dorso—ventrally oriented preterminal axonal degeneration. In addition, the external cortex surrounding the central nucleus contained light, scattered preterminal axonal degeneration aligned in a dorsoventral direction (Table 5 , Figs. 6, 10). The oculomotor and dorsal raphe nuclei of the midbrain also contained degeneration. The degeneration was light, terminal in nature, and present in all TPP, rats except rat 24 (Table 5, Figs. 6, 10). In comp]: matter contair (I-X an of the in the nuclei. axonal degent vestibt 14). 001mm 14), ] fatS, b in rats degent Could I 22 alsc and la] eXPOSe 63 In the hindbrain, degeneration was detected in the cerebellum, the vestibular nuclear complex, two cochlear nuclei, the reticular formation, the gracile fasciculus, and the gray matter of the first cervical spinal cord segment. The cerebellum of rats 25, 23, and 22 contained light, scattered terminal degeneration in the granular cell layer of all vermal (I-X and flocculus) and hemispheral (simple, crusl and 2 ansiform, paramedian, copula pyramis, and paraflocculus) lobules (Table 5, Figs. 6, 7, 13). The six TPPi—exposed rats exhibited a consistent pattern of degeneration in the nuclei of the vestibular complex (Table 5). Moderate to heavy axonal degeneration was present in the fiber tracts which are distributed throughout the medial and spinal vestibular nuclei. The fiber degeneration was interspersed with light to moderate preterrrrinal axonal and terminal degeneration in the gray matter of those nuclei. Light terminal degeneration was also located in the medial vestibular nucleus, ventral part, the superior vestibular nucleus, and the parasolitary nucleus of the vestibular complex (Figs. 6, 12, 14). The dorsal cochlear nucleus and the ventral cochlear nucleus, posterior part, contained light terminal and preterminal axonal degeneration, respectively (Figs. 6, 7, 14). Degeneration in the dorsal cochlear nucleus was similar among all TPPi-exposed rats, but degeneration in the ventral cochlear nucleus, posterior part, was observed only in rats 22, 23, and 19 (Table 5). Two of the six TPPi-exposed rats (rats 22 and 19) contained very light axonal degeneration in the gracile fasciculus of the first cervical spinal cord segment which could be traced just to its entry point in the gracile nucleus of the medulla (Fig. 15). Rat 22 also exhibited light scattered axonal degeneration in the medullary reticular formation and laminae V-X of the first cervical spinal cord segment (Fig. 15). No other TPP,- exposed rat contained degeneration in the reticular formation or the first cervical spinal Fig. 13. Line drawings of cross-sections illustrating the location of degeneration in the cerebellum of rat 22. Rat 22 was injected with 1184 mg TPPJkg body weight (sc) on days one, four, and seven and was perfused on day 15. Drawings are arranged from rostral (A) to caudal (C). The brain levels from which sections A-C were obtained are depicted in Fig. 3. Fig. 14. Line drawings of cross-sections which are a caudal continuation of Fig. 10 and show the extent of axonal degeneration in fiber tracts (dashed lines) and preterminal axonal (dashed lines) and terminal (dots) degeneration in nuclei of the hindbrain of rat 22. Rat 22 was injected with 1184 mg TPPilkg body weight (sc) on days one, four, and seven and was perfused on day 15 . Drawings are arranged from rostral (I) to caudal (K). The brain levels from which sections I-K were obtained are depicted in Fig. 3. 67 68 Fig. 15. Line drawings of cross-sections which are a caudal continuation of Fig. 14 and show the extent of axonal degeneration in fiber tracts (dashed lines) and preterminal axonal degeneration (dashed lines) in the hindbrain of rat 22. Rat 22 was injected with 1184 mg TPP/kg body weight (sc) on days one, four, and seven and was perfused on day 15 . Drawings are arranged from rostral (L) to caudal (M). The brain levels from which sections L and M were obtained are depicted in Fig. 3. 7O cord segment (Table 5). Cervical and lumbar spinal cord enlargements were examined in, rats 22 and 23. Degeneration was not detected in these spinal cord regions. DISCUSSION DFP Trial NTE Activity. At least 66% inhibition of whole-brain NTE activity within one to 44 hours of administration of a single dose of a Type I OP is required for subsequent expression of neuropathic damage in the rat (Padilla and Veronesi, 1985; Veronesi et al. , 1986a). The level of whole-brain NTE inhibition measured in this study exceeded the threshold for neuropathic damage and was comparable to that reported in other studies in which rats were exposed to a single subcutaneous neuropathic dose of DFP (V eronesi et al., 1987; Jortner et al., 1991). Central Nervous System Degeneration and Clinical Signs. Degeneration detected in the central nervous system of the rat 28 days after administration of a single subcutaneous dose of DFP consisted of axonal degeneration in the gracile fasciculus of the upper cervical spinal cord and preterminal axonal and terminal degeneration throughout the gracile nucleus of the medulla. The axonal degeneration in the rostral gracile fasciculus is consistent with that found in other studies in which rats were administered a single neuropathic dose of DFP (Ehrich et al. , 1991; Jortner et al., 1991) and is also similar to that reported for the rat after administration of a single neuropathic dose of the Type I OPs tri-ortho—cresyl phosphate (Padilla and Veronesi, 1985 ; Ehrich et al. 1991; Jortner et al. 1991), phenyl saligenin phosphate (Jortner et al. 1991), and mipafox (Veronesi and Padilla, 1985; Carboni et al., 1991). Because the gracile nucleus of the rat has not previously been examined after exposure to a Type 1 OP, this is the 71 first report of Type I OP-induced degeneration in the neuropil of that nucleus. The gracile fasciculus and nucleus of the rat constitute the first relay in the ascending dorsal column pathway which carries conscious proprioceptive somatosensory information from the hindlimb (Tracey, 1985a). This first relay consists of primary afferents and second order axons which enter or originate, respectively, at caudal levels of the spinal cord, ascend in the ipsilateral gracile fasciculus, and terminate throughout the gracile nucleus (Tracey, 1985a, 1985b). Therefore, in the rat, a single dose of DFP . preferentially affects distal axons and terminals of fibers which carry conscious proprioceptive somatosensory information from the hindlimb. A ‘ time course study will need to be conducted to determine if distal axons and terminals are affected simultaneously or in sequence. An appropriate Type I OPIDN animal model should express sequelae similar to those expressed by humans. Unfortunately, Type TOP-induced CNS degeneration has not been well-defined for humans. Neuropathological analysis of the human CNS was often done several years after exposure and/or was assessed with myelin stains ’which were not sensitive for detecting the full extent of degeneration (Smith and Lillie, 1931; Aring, 1942). The rat, however, may be assessed relative to other animal species. We have used the Fink-Heimer technique to map degeneration in the CNS of the chicken, a species thought to closely mimic OPIDN in humans (Abou-Donia, 1981), and the ferret, a mammalian OPIDN model, after adminisuation of a single subcutaneous dose of DFP at a time point when hindlimb ataxia was severe (Tanaka et al., 1990b; Tanaka et al., 1991). The pattern of degeneration in these two species is very similar and consists of, as it does in the rat, damage in the gracile fasciculus and nucleus. In addition, the chicken and ferret develop distal axonal and terminal degeneration in the ascending spinocerebellar, spinoolivary, spinovestibular, and spinoreticular pathways and in the 72 descending corticospinal tract (ferret) or in its avian homolog, the medial pontine-spinal tract (chicken) (Fig. 16). Thus, degeneration in the CNS of the rat is much less extensive than in the CNS of the chicken or ferret after a single exposure to a Type I OP. The less extensive injury to the rat CNS in combination with minimal effects in peripheral nerves (Padilla and Veronesi, 1985) may explain why rats do not become ataxic after a single exposure. In the CNS , sparing of the spinocerebellar and corticospinal tracts is probably related to the lack of functional effects. Degeneration in these pathways is critical for production of hindlimb deficits in the ferret (Tanaka et al. , 1991), and is associated withoataxia in rats after repeated exposure to tri-ortho—cresyl phosphate or after exposure to hexacarbons or acrylamide (Spencer and Schaumberg, 1977; Veronesi, 1984a). Because the rat does not express the full neuropathological and concomitant functional sequelae of Type I OPIDN after a single exposure, the rat’s utility as a paradigm for this condition is questionable. Nonetheless, if degeneration in the rat gracile fasciculus is predictive of human Type I OPIDN, then it could be useful in a screening capacity. The rat gracile fasciculus is affected by the human Type I delayed neurotoxicants tri-ortho—cresyl phosphate (Padilla and Veronesi, 1985) and mipafox (V eronesi and Padilla, 1985), but the response to mipafox is inconsistent (Jortner et al. , 1991). The model needs to be assessed with other known human delayed neurotoxicants. Also, study of why certain regions of the rat nervous system are refractory after a single exposure could help elucidatethe mechanism of Type I OPIDN. TPP, Trial Clinical Signs. Latency to onset of clinical signs varied among the TPP, rats. Carrington et al. (1988b) showed that the disappearance of TPP, from a subcutaneous 73 Fig. 16. Summary schematics in the sagittal plane which compare qualitatively the extent of degeneration in the CNS of the rat, chicken, and ferret after a single exposure to DFP. The location of degeneration was assessed using a modified Fink-Heimer silver impregnation technique. Solid lines and dashed lines indicate ascending and descending pathways, respectively, which contained axonal degeneration, and dots indicate nuclear or gray matter areas which contained terminal degeneration. The widths of the lines do not imply degeneration density. a t DFP ------ .. @‘fl C) 6‘5 é .3. Chicken Ferret Fig. 16 75 injection site in the chicken has a slow component with a half—life of two weeks. Thus, slow and possibly variable absorption of TPP, from the injection sites may account for latency differences. Clinieal deficits observed in this study are in general similar to those observed in other studies in which rats were exposed to TPPi (Smith et al., 1933; Veronesi et al. 1986b; Veronesi and Dvergsten, 1987). However, this is the first report of TPPi-induced side-to—side and backing movements and hindlimb rigidity in the rat. ' Correlation of CNS Degeneration with Clinical Signs. Subacute subcutaneous exposure to TPP, resulted in widespread loss of afferent input (preterminal axonal and terminal degeneration) to nuclei and gray matter areas in the forebrain, midbrain, and hindbrain of the rat. Although widespread, loss of afferents was associated with specific CNS systems and pathways. Inputs to layer IV in the somatosensory, sensorimotor, motor, auditory, and ’visual neocortices were extensively damaged. Since layer IV receives input from thalamic relay nuclei (Zilles, 1990) , thalamocortical afferents were most likely the source of the degeneration. The thalamus relays information in the sensory and motor systems to the neocortex (Faull and Mehler, 1985). Loss of thalamocortical afferents would be expected to interfere with sensory perception, impair sensory guided behaviors, and- hinder integration of sensory and motor information (Chapin and Lin, 1990), and thus, may mediate, in part, TPPi-induced behavioral deficits. Degeneration of thalamocortical afferents does not, however, correlate to clinical signs. Rats with minimal neocortical degeneration exhibited clinical signs similar to those with more extensive degeneration. It may be that deficits associated with neocortical sensory deprivation are not readily apparent in an open field. Neocortical degeneration does correlate to length of survival afier the onset of clinical signs. Rats allowed the longest survivals had the most neocortieal damage. These afferents, therefore, are one of the last CNS systems to be affected in the rat after subcutaneous exposure to NE. nuc rats aris coll post heat 198; sour and nucl affet 76 Afferent inputs to the ventromedial thalamic nucleus, the intralaminar thalamic nuclei, and the intermediate layers of the superior colliculus were damaged in all TPP, rats. These nuclei receive afferent input from a basal ganglia output pathway which arises in the substantia nigra, pars reticulata and projects to the thalamus and superior colliculus. The pathway is thought to be a means by which the basal ganglia influence posture and locomotion (thalamic and collicular projections) and orientation of eye and head movements (collicular projection) (Kilpatrick et al. , 1982; Starr and Summerhayes, 1983; Chevalier and Deniau, 1987). Interestingly, injury to neurons, which are the source of this pathway produces circling behavior in rats (Kilpatrick et al. , 1982; Starr and Summerhayes, 1983). Thus, effects on this pathway could be responsible, at least in part, for hindlimb and directional deficits produced by TPPi. Two other basal ganglia nuclei, the substantia nigra, pars compacta and the subthalamic nucleus, were minimally affected by TPP, and probably had little contribution to deficits. ‘ Afferent input to nuclei in the vestibular nuclear complex (the superior, spinal, and medial vestibular nuclei) and areas associated with the vestibular nuclei (the parasolitary and oculomotor nuclei and the cerebellum) were damaged by administration of TPP,. The pattern of degeneration in the vestibular nuclear complex was remarkably consistent among TPPi rats. Vestibular nuclei receive input from the vestibular portion of the eighth nerve, the cerebellum, and the spinal cord. Because the inputs overlap, it is not clear which were the source of the affected afferents. Three functional roles have been identified for the vestibular nuclear complex: coordination of eye and head movements, maintenance of balance, and maintenance of muscle tone (Baloh and Honrubia, 1990). Thus, degeneration in this nuclear region could be the major source of hindlimb ataxia, inability to rear, hindlimb and tail flaccidity, and directional deficits. Degeneration in areas assdciated with the vestibular nuclear complex was light and probably had a minimal contribution to deficits. Arn of: nun lam: ass: the hon" hnp duet nucl Collit deger andnl andtl damn 77 Preterminal axonal and axonal degeneration were noted in fields CAl-CA3 of Ammon’s horn, the dentate gyrus, the postsubiculum, the nucleus of the horizontal limb of the diagonal band, the magnocellular preoptic nucleus, the mamillary nuclei, the mediodorsal thalamic nuclei, and the retrosplenial agranular, medial prefrontal, and lateral orbital transitional cortical areas. These areas are considered part of, or - are associated with, the limbic system in the rat (Hamilton, 1976). The overall function of the limbic system in the rat is thought to be regulation of sensory, motor, and homeostatic systems with involvement in learning and memory as well (Hamilton, 1976). Impairment of limbic system regulatory functions could have contributed to hindlimb and directional deficits. Of affected limbic regions, the lateral orbital area, the mamillary nuclei, and the mediodorsal thalarrric nucleus were moderately to severely affected in all TPP, rats. The lateral orbital area is part of orbital frontal cortex. Functions of orbital frontal cortex have not been well-defined, but lesions in this area result in social and sexual behavioral, sequential behavioral, and behavioral flexibility effects (Kolb, 1990). Lesions in the mamillary nuclei and mediodorsal thalamic nucleus have been related to the Wernicke-Korsakoff amnesic syndrome in humans (Squire, 1987) and in the rat, lesion of the mamillary region results in long lasting spatial memory impairments (Saravis et al. , 1990). Thus, TPPi affected CNS regions which mediate complex behaviors and cognitive functions (memory) as well as regions which mediate balance and locomotion. The ventral cochlear nucleus, posterior part, the dorsal cochlear nucleus, the inferior colliculus, and the medial geniculate nucleus contained preterminal axonal and terminal degeneration afier exposure to TPPi. These nuclei are consecutive relays in an ascending auditory pathway which originates from the descending bifurcation of the auditory nerve and terminates in the auditory cortex (Webster, 1985). The heaviest and most consistent damage occurred in the inferior colliculus and the medial geniculate nucleus. The infe l0n( gen: Mel the hav sub VCS the but pre Spa deg isr Var hin (So Whi and We 78 inferior colliculus is a obligatory relay in the auditory pathway, is organized tonotopically, and mediates sound localization (Webster, 1985) while the medial geniculate nucleus is the source of afferents to layer IV of the auditory cortex (Faull and Mehler, 1985). Degeneration of afferents to these nuclei would be expected to interrupt the flow of tonotopic and sound localization information to the auditory cortex. Veronesi and colleagues (Veronesi et al., 1986b; Veronesi and Dvergsten, 1987) have examined the medulla and spinal cord in the Long-Evans rat after subacute subcutaneous exposure to TPP,. In the medulla, they noted axonal debris in the medial vestibular nucleus, the spinocerebellar tracts, and the medullary reticular formation. In the spinal cord, damage occurred in the ventrolateral and ventral columns at all levels but was not detected in the gracile fasciculus. These results differ from the results of the present study, especially in the spinal cord. The ventrolateral and ventral columns were spared in this study while degeneration was detected in the gracile fasciculus. The degeneration in the gracile fasciculus was very light and occurred in only two of the six TPP, rats. It appears, as Veronesi and colleagues suggested, that the gracile fasciculus is not a major target for Type II OPs in the rat. Dissimilarity between the two studies could be due to different staining techniques and survival periods or may represent variability in the response of the rat. In summary, this study shows that subacute subcutaneous exposure to TPPi resulted in loss of afferent input to nuclei in the midbrain and forebrain as well as in the hindbrain of the rat. Affected afferents were associated with three sensory systems (somatosensory, visual, and auditory), the motor system (motor cortex), two systems which modulate posture and locomotion (the basal ganglia and the vestibular nuclear complex), and a portion of the limbic system associated with memory (malnillary nuclei and mediodorsal thalamic nucleus). TPP, also damages peripheral nerves in the rat (Veronesi et al. , 1986b). Most behavior is a result of the coordination of multiple 79 peripheral and CNS sensory, motor, and limbic systems. Therefore, although TPP,- induced deficits ean be associated with specific systems and pathways, the deficits may also be a result of the combined system degeneration. This could be the case for deficits difficult to ascribe to a specific functional system such as the side-to—side and backing movements, hindlimb rigidity, and body weight loss. Also, differences in the relative contribution of the affected systems over time could explain why the order of progression of clinical signs varied among TPP, rats. A more sophisticated behavioral and/ or electrophysiological analysis than that used in this study would aid in better characterization of deficits, could reveal deficits which are not detectable in open field assessment, could help elucidate the relative role of the affected systems in TPPi-induced deficits, and could determine if and what type of memory is impaired. ‘ Although TPP,-induced degeneration was widespread, damage was selective within certain neuronal populations. It is unclear from the results of this study what determines susceptibility to degeneration. Further anatomical, physiological, and biochemical characterization of the involved systems is required to understand what traits confer susceptibility to degeneration. It is interesting that CNS systems affected by TPP, are somewhat similar to those affected by iminodiproprionitrile, a neurotoxicant which also produces a complex set of behavioral deficits (Cadet et al. , 1989). Comparison of these syndromes could help in understanding factors which confer susceptibility to degeneration. ' Species Comparisons. The Fink-Heimer technique has also been used to map degeneration in the CNS of the chicken and ferret after exposure to TPPi. Tanaka et al. (1992a) present a detailed comparison of the neuropathological effects of TPPi in the chicken, ferret, and rat. In summary, the pattern of TPPi-induced CNS degeneration is very similar among the species, especially in the midbrain and forebrain, but in this study, the rat exhibited less medullary brainstem and spinal cord degeneration and did 80 not develop neuronal somatic degeneration. In addition, the rat displayed damage in memory associated regions (Fig. 17). Thus, the rat model of Type II OPIDN should be used to study effects of TPP, on memory and could be used to study midbrain and forebrain CNS effects but would be less useful for study of hindbrain and spinal cord or neuronal somatic effects. ' . Comparison to Type I OPEN. Results of ' this study and others (Padilla and Veronesi, 1985 ; Veronesi et al. , 1986a) show that Type I OPIDN in the rat after a single exposure consists of a relationship between inhibition of NTE and subsequent expression of neuropathic damage, the lack of clinical signs, and confinement of CNS degeneration to the gracile fasciculus and nucleus in the spinal cord and hindbrain. Also, Type .I OPIDN in the rat consists of damage to the distal axon and its terminations. In contrast, Type II OPIDN in the rat is characterized by a lack of a relationship between in vivo inhibition of NTE and expression of neuropathic damage (Padilla et a1 . , 1987), delayed onset of a complex set of behavioral deficits, and widespread degeneration throughout the sensory, motor, and limbic systems in the hindbrain, midbrain, and forebrain. This study also showed that Type II OPs damage distal axons and terminals. Veronesi and colleagues (V eronesi et al. , 1986b, Veronesi and Dvergsten, 1987) provide evidence that Type II OPs produce neuronal necrosis as well. In summary, although Type I and Type II OPIDN are both organophosphorus-induced delayed neurotoxicities, each. exhibits distinct biochemical, clinical, and neuropathological traits in the rat. 81 Fig. 17. Summary schematics in the sagittal plane which compare qualitatively the extent of degeneration in the CNS of the rat, chicken, and ferret after acute (chicken and ferret) or subacute (rat) exposure to TPPi. The location of degeneration was assessed using a modified Fink-Heimer silver impregnation technique. Solid lines and dashed lines indicate ascending and descending pathways, respectively, which were affected, dots indicate nuclear or gray matter areas which contained terminal degeneration, and stars represent neuronal somatic degeneration. The widths of the lines do not imply degeneration density. 82 Chicken Ferret Fig. 17 EXPERIMENTII OBJECTIVES 1. To assess the ability of the rat's central nervous system (CNS) to process sensory information after exposure to the Type II organophosphorus delayed neurotoxicant triphenyl phosphite (TPPi) by use of an evoked potential test battery (auditory brainstem responses, somatosensory evoked potentials, flash evoked potentials, and caudal nerve action potentials) . 2. To assess clinical signs and CNS neuropathologic changes in the rat after exposure to TPPi so that they can be correlated to effects on CNS sensory processing. RATIONALE As a normal biological process, the nervous system generates background electrical activity resulting from the asynchronous firing of large groups of unrelated neurons. An electroencephalogram (EEG) is a record of this background electrical activity. If the nervous system is stimulated by a discrete event, such as a sound, flash of light, or a touch, neurons in a specific sensory system fire synchronously and produce electrical activity which is time-locked to the stimulus. This activity is known as an evoked potential. Evoked potentials can be distinguished from the ongoing EEG using computer averaging techniques which average the random EEG towards zero and enhance non-random sensory evoked potentials (signal to noise enhancement) (Dyer, 1985; Martin, 1991; Mattson et al., 1992). An electrical recording of a sensory evoked potential consists of many peaks and valleys. The peaks and valleys represent the sum of electrical activity which was generated as the sensory signal passed through the nuclei and fiber tracts of the stimulated system. The identity of nuclei and fiber tracts which are associated with specific peaks or valleys has been 83 84 elucidated in some cases but not in others. Also, since sensory processing occurs in serial and parallel, most peaks and valleys are generated by more than one nuclei or fiber tract and each of these may contribute to more than one wave. (Dyer, 1985; Mattsson et al., 1992). Because sensory evoked potentials reflect activity in a sensory system, they can be used to assess its functional integrity. For example, the amplitude or power (area under the curve) and latency of peaks and valleys in the sensory evoked waveform can be measured before and after exposure to a neurotoxicant. Changes in these variables indicate that the function of the sensory system has been altered. More specifically, decreases in peak amplitude (or power) indicate toxicant-induced loss of neurons, cell connections, or firing synchrony while increases in peak latency suggest myelin damage. Changes in amplitude (or power) and latency may also be mixed so that altered potentials can be small, slow, and poorly shaped. In addition, if the generator of an affected peak or valley is known, damage can be localized to a specific brain region (Mattsson et al., 1992). Results of the first experiment showed that TPPi induces widespread degeneration in the auditory, somatosensory, and visual systems in the CNS of the rat. Therefore, an evoked potential test battery was conducted to survey the function of CNS sensory systems in the rat after exposure to the Type 11 OF TPPi. Auditory brainstem responses (ABRs), somatosensory evoked potentials (SEPs), flash evoked potentials (FEPs), and caudal nerve action potentials (CNAPs) were measured in this study. ABRs assess frmction in the peripheral auditory apparatus (cochlea) and the hindbrain and midbrain regions of the auditory pathway. SEPs and FEPs consist of early and long latency components which measure function of subcortical and initial cortical processing and higher order cortical processing, respectively. CNAPs measure peripheral nerve function. Clinical signs and neuropathological changes were also assessed so they could be correlated to evoked potential alterations. 8 5 METHODS Test Material TPP; was obtained from Aldrich Chemical Company (Milwaukee, WI). Prior to the start of the study the identity of the test material was confirmed with infrared spectroscopy and its purity was determined to be 97.2%. Test Species and Husbandry Adult male Long-Evans rats (Charles River Laboratories, Portage, MI) were used for this study. Rats were housed individually in suspended stainless steel cages with wire-mesh floors. The holding room was maintained at approximately 22°C and 50% humidity with a 12 hour light- dark cycle. Rats were acclimated to their environment for at least two weeks prior to the start of a trial. Municipal drinking water and Certified Purina Rodent Chow #5002 (Purina Mills Inc., St. Louis, MO) were available ad libitum throughout the study. Rats which developed neuropathy had difficulty obtaining water and food, and thus, were injected subcutaneously with 10 ml of normal saline two times a day and had food placed in an accessible location. All aspects of this study were approved by the Dow Chemical Company Institutional Animal Use and Care Committee. Experimental Design Two trials were conducted. In the first trial (Table 6), 15 rats, nine weeks old and ranging in weight from 299 to 323 g, were utilized. Five rats were assigned to the control group and 10 rats were assigned to the treated group based on body weight distribution. One day prior to treatment, a preexposure electrophysiology test battery and hindlimb landing foot splay were measured. On days one and two, TPPi (450 mg / kg body weight) was applied dermally. Then, on days five, seven and nine, rats were weighed, observed in an open-field (50 cm x 50 cm clear plastic box) for clinical signs of neurologic impairment, and assessed by the electrophysiology test battery. On days six and eight, hindme landing foot splay was measured. 8 6 On day seven, a subsample of five treated animals was randomly chosen and perfused for neuropathological analysis. On day nine, the remaining treated animals and the control animals were perfused. TABLE 6 SUMMARY OF EXPERIMENTAL DESIGN NR RATS DCBED DERMALLY WITH 450 MC TPPi/KG ”DY WEIGHT Electro- Hindlimb TPPi Gait physiology landing foot Body Test day dosing observation battery splay weight Perfusion Preexposure X X X l X X X 2 X 3 4 5 X X X 6 X 7 X X X 5 treated 8 X 9 X X X 5 treated 5 control The TPPi dosing regimen used in the first trial was expected to produce widespread functional and neuropathological changes. However, deficits were mild. Therefore, to provide additional data, a second trial (Table 7) was concluded using a modified dosing regimen. In the second trial, three 13-week-old rats were used. One rat was designated as the control and two rats were subsequently dosed with mi. The electrophysiology test battery was performed twice prior to dosing. Then, on days one and two, TPPi (600 mg/ kg body weight) was applied dermally. Rats were observed daily in the open-field for clinical signs of neurologic impairment. On days five and six, rats were weighed and the electrophysiology test battery was conducted. On day six, the three rats were perfused for neuropathological analysis. Dosing Regimen For the first trial, treated rats were dosed dermally with TPPi at 450 mg/ kg body weight (undiluted, 0.375 ml / kg body weight). Control rats were dosed dermally with 0.375 ml corn oil/ kg body weight. Rats were dosed once per day on days one and two. One day prior to application of 87 the first dose, all rats were anesthetized with MetofaneO and the rostral two-thirds of the back was shaved. The first dose was applied on the rostral half of the shaved region. The second dose was placed further caudally in the shaved region. On each day, after the TPPi or corn oil was applied, the application site was covered with an absorbent gauze patch held in place with an elastic bandage. The elastic bandage consisted of a two-inch wide strip of VetrapO, cut to a length suitable for wrapping one to two times around the body of the animal. The Vetrapo was held in place with ElastikonO elastic tape (one inch wide) cut to lengths similar to the VetrapO . The bandages were removed approximately two hours after dosing. TABLE 7 SUMMARY OF EXPERIMENTAL DESIGN m RATS DOSED DERMALLY WITH 6m MG TPPi/ KG BODY WEIGHT Electro- ‘I'PPi Gait physiology Body Test day dosing observation battery weight Perfusion Preexposure X X 1 X X X 2 X X 3 X 4 X 5 X X X 6 X X X 1 control 2 treated For the second trial, treated rats were dosed dermally with TPPi at 600 mg/ kg body weight (undiluted, 0.504 ml/ kg body weight). The control rat was dosed with 0.504 ml corn oil/ kg body weight. Rats were dosed once per day on days one and two. Since the slight response observed in the first trial may have been due to loss of TPPi in newly regrown hair or in the materials used to wrap the application site, animals in the second trial were shaved just prior to dosing on the first day and the application site was not wrapped. Shaving and dermal application methods were the same as for the first trial. 88 Hindlimb Landing Foot Splay Hindlimb landing foot splay was assessed using the procedure of Edwards and Parker (1977). The tarsal joint pad of each hindfoot was marked with ink. After inking, the rat was grasped around the shoulders and flank and was held horizontally with its back up 30 cm above the floor of an open field box. The rat was dropped onto a recording sheet, and the distance, in cm, between the ink marks was measured. Each rat was dropped three times and joint pads were reinked between drops. The average of the three drops was used for statistical analysis. Surgery Epidural electrodes were surgically implanted when rats were approximately seven weeks old. Anesthesia was induced by methoxyflurane and maintained with isoflurane. Once an animal was anesthetized, the eyes were treated with an ophthalmic ointment and the eyelids were closed with skin clips to prevent corneal drying and retinal damage by lights. A rat was then placed in the stereotaxic instrument (#900, David Kopf Instruments, Tujunga, CA), and a rectal thermistor was used to monitor core temperature during surgery. Body temperature was supported with a warm water blanket. Epidural electrodes (seven mm long, No. 0-80, stainless-steel set screws) were inserted into the skull and supported with dental acrylic. The top three mm of the set screws remained exposed above the acrylic so recording leads could be directly attached to the screws. Four epidural electrodes were implanted: somatosensory cortex, visual cortex, cerebellar cortex, and reference. The somatosensory cortex electrode was placed 2.0 mm posterior and 2.0 mm lateral left of bregma, the visual cortex electrode was placed 1.5 mm anterior and 3.0 mm lateral right of lambda, the cerebellar electrode was located 3.0 mm posterior and 0.0 mm lateral of lambda, and the reference electrode was placed 8.0 mm anterior and 1.0 mm lateral left of bregma. 8 9 Electrophysiological System and Test Battery. The electrophysiological system was a Nicolet Pathfinder II (N icolet Biomedical Instruments, Madison, WI). Data sweeps (msec segments of EEG) were digitally sampled 512 times and averaged by an online computer. The following data were collected, in sequence, in the electrophysiology test battery: flash evoked potentials, auditory brainstem responses, somatosensory evoked potentials, and caudal nerve action potentials. Rats were physically restrained and were not anesthetized during the recording sessions. Animals were tested in opposing pairs and testing was counterbalanced for position. Tail temperature was recorded prior to the caudal nerve test, and body temperature was recorded with a rectal thermistor prior to all other electrophysiological tests. Flash Evoked Potentials. Flash evoked potentials (FEP) were recorded simultaneously from the visual cortex electrode (PEP-V) and the cerebellar electrode (PEP-C). Recording variables described below refer to both FEP-V and PEP-C. FEPs were collected in an isolation cubicle which was constructed of white plastic. Restrained rats were placed in the isolation cubicle facing the wall opposite a strobe light. The strobe light flashed at a rate of 0.7 flashes/ sec with a duration of 50 used flash. FEPs were collected at low and medium intensities of approximately 0.7 and 4.6 cd-sec/mz, respectively. Flash intensity was set with a Grass photic driver and was calibrated with a United Detector Technology 350 photodetector (plus 11 1 filter and lumilens 1153) placed in the same position as the rat facing the wall opposite the strobe. Amplifier filters collected EEG between 0.5 and 500 Hz with sensitivity set to 1 mV. Two sweep durations were collected using dual digitizers. Sweep duration was 150 msec for one digitizer and 600 msec for the other digitizer. Final waveforms were an average of 200 sweeps for each duration. In summary, FEPs were collected simultaneously from the visual (PEP-V) and cerebellar (PEP-C) cortex electrodes. Both the FEP-V and FEP-C were collected at two intensifies, and each intensity was collected at two sweep durations. Auditory Brainstem Responses. Auditory brainstem responses (ABRs) were collected in response to clicks (ABRC) and to tone pips of 10 (ABR1 o) and 30 (ABPeo) kHz representing middle and high frequency, respectively. ABRc, ABR1o, and ABR30 were collected in an isolation cubicle designed 90 for acoustic isolation and minimal sound reflections. Restrained rats were placed in the cubicle so that the distance from the speaker to the ears was about 17 cm. For all three ABRs, stimulus rate was 29.1 / sec with a duration of 100 usec/ stimulus. Stimulus intensity was 75 decibels (dB) (linear) for ABRc and ABR10 and was 80 dB (linear) for ABRao. Sound pressure level was calibrated with a Bruel and Kjar (N aerum, Denmark) sound level meter (model 2230) with a 1 / 4 inch condenser microphone (model 4135) placed in the cubicle at the location of the ears. Tone pip frequency was controlled with a peripheral system. For all three ABRs, amplifier filters collected EEG between 100-8000 Hz with sensitivity set to 200 uV. Sweep duration was 12 msec, and final waveforms were an average of 2000 sweeps. Somatosensory Evoked Potentials. Somatosensory evoked potentials (SEPs) were recorded simultaneously from the somatosensory cortex electrode (SEP-S) and cerebellar electrode (SEP-C). Recording variables described below refer to both SEP—S and SEP-C. SEPs were elicited in restrained rats by electrical stimulation of ventrolateral caudal nerves at the base of the tail. The stimulating electrodes, small needles separated by a distance of 0.5 cm, were placed subcutaneously on the ventral surface of the tail just in front of the hairline. The electrical stimulus consisted of a 3 mA pulse presented at a rate of 1.1 pulses/sec with a duration of 50 usec/pulse. Amplifier filters collected EEG between 1 and 1500 Hz with a sensitivity of 1 mV. Two sweep durations were collected using dual digitizers. Sweep duration was 35 msec for one digitizer and 200 msec for the other digitizer. Final waveforms were an average of 400 sweeps for each duration. In summary, SEPs were collected simultaneoulsy from the SEP-S and SEP-C electrodes. Both the SEP-S and SEP—C were collected at two sweep durations. Caudal Nerve Action Potentials. Caudal nerve action potentials (CNAPs) were recorded from the tail in response to a single stimulus (CNAP1) and paired stimuli (CNAPz). CNAPs were elicited by electrical stimulation of ventrolateral caudal nerves at the tip of the tail and were recorded at the base of the tail. The stimulating and recording electrodes were separated by 9 cm and were mounted in a plastic tray and attached to the tail according to Rebert (1983). For CNAPI, the electrical stimulus consisted of a 3 mA pulse presented at a rate of 10.1 pulses/sec with a pulse 9 1 duration of 20 usec. The physical parameters of the electrical stimulus were the same for CNAPz, except that the stimulus was presented as 10.1 paired pulses/secwithanmtervalof3msecbemeenthefirstandsecondsfimulus of a pair. For both CNAP1 and CNAPz, amplifier filters collected EEG between 1 and 3000 Hz with a sensitivity of ImV. Sweep duration was 20 msec, and final waveforms were an average of 200 sweeps. Digital Filtering. FEP, ABR, and SEP waveforms collected with the broad-band analog filter were digitally filtered using a computer routine (N icolet Biomedical Instruments, Madison, WI). Digital filter settings were: FEPs 1-250Hz;ABRs500-8000Hz;SEP-Srecorded witha35msecdata sweep 85-1500 Hz; SEP-C recorded with a 35 msec data sweep 1-750 Hz; SEP- SandSEP-CrecordedwithaZOOmsecdatasweep 1-500Hz. Waveform Analysis Waveform Composites. Using an automated computer routine, control and TPP; group waveform composites (grand averages of individual waveforms) were created for ABRs, SEPs, FEPs, and CNAPs collected in the first and second trials. Waveform composites were visually examined for treatment effects. In addition, automated computer routines were used to quantify peak latency and peak amplitude or power (RMS volts) of selected regions (described below) of ABR, SEP, FEP, and CNAP individual waveforms. These data were analyzed as described under data analysis. Body temperature of TPPi-treated rats in the second trial was greatly reduced (see results). The temperature decrease caused a sizable slowing and thus, large alteration in shape, of the waveforms collected on day five. To compensate for this effect, the treated rats were warmed on day six with a heating lamp prior to collection of waveforms and the day six waveforms were used for waveform composites and data analysis. Auditory Brainstem Responses. ABRs consist of seven peaks, I-VII. The probable neural generators are: I - the auditory nerve; II - the cochlear nuclei; III - the superior olive; IV - the lateral lemniscus; V - the inferior colliculus; VI - the medial geniculate nucleus; and VII - the acoustic radiation (Stockard et al., 1980). Alterations in peak I reflect changes in the peripheral auditory apparatus so this peak is used to screen for ototoxicity (Dyer, 1985). Peak I peak latency and power were measured in ABR1o and 9 2 ABR30 in order to screen for ototoxicity which might be associated with a specific frequency of sound. Since the neural generators of peaks II - VII are centrally located, alterations in these peaks suggest central dysfunction (Dyer, 1985). ABRcs were used to assess the effects of TPP; on central processing in the brainstem auditory pathway. Peak I to peak III, peak III to peak V, and peak I to peak V interpeak latencies (I-lII, III-V, I-V lPLs) were measured. These IPLs assess conduction in the lower, upper, and entire brainstem, respectively (Stockard et al., 1980). ABRc IPL was significantly increased by treatment with TPPi. Because wave latency and body temperature are inversely correlated (Hetzler et al., 1988; Sohmer et al., 1989), the effect of TPP; on body temperature was confounding the possible direct effect of TPP; on ABRc IPL. Therefore, a trial was conducted to develop an equation by which IPLs corrected for body temperature could be calculated. Four adult Fisher 344 male rats were used. The assumption was made that their response would be similar to that of the Long-Evans rat. The body temperature of the rats was decreased with isoflurane anesthesia. ABRcs were collected just after rats were anesthetized at a point before body temperature had dropped, and after a one, two, and three °C decrease in body temperature. I-III, III-V, and I-V IPLs were measured, and using the regression procedure of SAS (SAS Institute, Inc., Cary, NC), a linear and a quadratic regression model were fit to the relationship between the measured IPLs and body temperature decrease. For each IPL, the linear regression parameter was significant (p < 0.05), but the quadratic regression parameter was not (p > 0.05). The slopes (msec/°C) and correlations obtained from the linear regression models were -0.06, r=0.87; - 0.17, r=0.83; and -0.23, r=0.90 for LEI, III-V, and I-V IPL, respectively. The slopes were used in the following equation to calculate IPLs corrected for body temperature cooling (CIPL): CIPL = IPL + (38.8 — body temperature) at slope where 38.8 represents the average of all preexposure body temperatures and the body temperatures of control animals on days five, seven, and nine. Amplitudes of peaks I-VI were also measured in ABRcs. Peak VII was not well-defined so it was not assessed. Amplitudes were measured relative to a horizontal baseline which passed through the mid-point of the 93 descending limb of the second cochlear microphonic. Because the amplitude of peaks lI-VI are dependant upon the amplitude of peak I, the amplitude of peaks II-VI were normalized to peak I by calculating peak amplitude ratios. Somatosensory Evoked Potentials. SEP-Ss are comprised of early (SEP- Ss collected with a 35 msec data sweep) and long (SEP-Ss collected with a 200 msec data sweep) latency components. The neural generators of the early latency components have been identified by Wiederholt and Iragui- Madoz (1977). They consist of: 1)a complex of peaks which represents activity transmitted in the somatosensory system from the tail to the somatosensory cortex and 2)peak P1 which is thought to represent the first cortical response in the somatosensory pathway . The complex of peaks which represents activity transmitted from the tail to the somatosensory cortex was assessed qualitatively upon visual examination of SEP-S composites. The incidence of peak P1 was analyzed statistically using a Chi square statistic (Gill, 1978). The source of long latency SEP-S components is unknown, but these components probably represent higher order cortical processing in the somatosensory pathway (Mattsson et al., 1992). Power was measured in long latency SEP-5s. Little is known of the neural generators of peaks and valleys in SEP-Cs. However, alterations in SEP-Cs may indicate effects on cerebellar function related to somatic sensation (Mattsson, pers. comm.). Early (peaks N1, P1, and P2) and long latency components were evaluated qualitatively in SEP-C composites. Flash Evoked Potentials. FEP-Vs are also comprised of early (FEP-Vs collected with a 150 msec data sweep) and long (PEP-Vs collected with a 600 msec data sweep) latency components. Visual cortex and higher order visual processing appear to be the primary source of the early and long latency FEP-V components, respectively (Dyer et al., 1987). Peak latency and power of peak N1 were measured in the early latency FEP-V components, and power was measured in long latency PEP-Vs. Little is known of the neural generators of peaks and valleys in FEP-Cs, but alterations in FEP-Cs suggest that cerebellar function related to vision has been impaired (Mattsson, pers. comm.). Early (peak N1) and long latency components were evaluated qualitatively in FEP-C composites. Caudal Nerve Action Potentials. CNAP1 (single stimulus) and CNAPz (second of paired stimuli) are mixed motor and sensory nerve action 94 potentials used to assess function in ventrolateral peripheral tail nerves. Changes in power and latency. indicate axonal damage (loss of firing units) and myelin damage, respectively. When a second stimulus is presented to the nerves before the refractory period related to a single stimulus has ended, then the ability of axons in the nerves to recover and fire another action potential can be assessed. Recovery is assessed by calculating CNAPz/CNAP1 power ratios. If the ratio in the heated group is decreased relative to the control group, it indicates that fewer axons were able to respond to the second stimulus after heahnent. Power and latency were measured in CNAP1 and CNAPz, and CNAPz/CNAP1 power ratios were calculated for the day nine control and treated group means in the first trial and for the day six conhol and heated group means in the second trial. Data Analysis Data obtained from the first trial were analyzed statistically using a general linear models procedure for unbalanced data (SAS Institute, Inc., Cary, NC.). For each variable, the model was a repeated (measures ANOVA. Mean squares were calculated with Type III sums of squares, and the following conhast differences were estimated: 1. (TPPis - TPPiI) - (Conhols - Controll) 2. My - TPPil) - (Control7- Conholl) 3. (TPPi9 - TPPil) - (Conhol9 - Controli) where 1 is the preexposure average, 5 is the day five average, 7 is the day seven average, and 9 is the day nine average. For hindlimb splay data, contrasts estimated were the same ’as above except day six and day eight averages were used for the postexposure averages. The contrasts were designed to subdivide the heahnent by test day interaction to identify on which postexposure test day(s) the TPP; average had changed from its preexposure value. Because control values changed over time, the contrasts also included that change for the specified time period. That is, in order for the change in the TPP; average (from preexposure to a specific postexposure day) to be considered significant, the change had to be greater than the change in the conhol group over the same time period. All 95 conhasts were found to be estimable and were estimated using the estimate statement of the GLM procedure of SAS (SAS Institute, Inc., Cary, NC .). The significance of the conhasts was tested using the Bonferroni t test statistic (Gill, 1978). Contrast significance statements are based on p < 0.05. Similar contrasts were calculated for data collected in the second trial. However, because there was only one conhol rat, the conhast differences were not analyzed statistically. Neuropathological Analysis Rats were heparinized (200 units/ kg body weight, i.p.) and then anesthetized with methoxyflurane inhalation. While under anesthesia, the rats were hanscardially perfused with phosphate buffer (pH 7.4, 0.05M) followed by phosphate buffered 1 5% glutaraldehyde-4% formaldehyde (pH 7.4, 0.5M, 540 mOsM)- Brains were removed and stored in the 1.5% glutaraldehyde-4% formaldehyde fixative. From each brain, three forebrain, two midbrain, and three hindbrain tissue blocks, one or two mm thick, were obtained. Selected blocks were acquired in the hansverse plane using a zinc alloy brain mold (Harvard Apparatus, South Natick, MA) which had 0.3 mm wide channels precisely 1.0 mm apart. Sections obtained from the blocks corresponded approximately to the following figures in the stereotaxic rat atlas of Paxinos and Watson (1986): block 1 - figure 21; block 2 - figure 27; block 3 - figure 41; block 4 - figure 50; block 5 - figure 54; block 6 - figure 56; block 7 - figure 64; block 8 - figure 73. Blocks were processed through graded alcohols to xylene and embedded in paraffin. Sequential hansverse 8 um thick sections were cut from each block on a rotary microtome, spread on a 45°C distilled water bath, and mounted on elechostatically heated glass slides (Fischer Super Frost / Plus”, Fischer Scientific, Pittsburgh, PA). Mounted sections were air dried at room temperature for at least 24 hours and were stained, using a routine procedure, with ’hematoxylin and eosin. In addition, a 1 mm thick hansverse tissue block just rostral to block 1 was obtained from the brains of the high dose rats and was used for elechon microscopy. The tissue block was trimmed and post-fixed in buffered 1 % osmium tehoxide for one hour, and then dehydrated in increasing grades of ethanol, cleared in propylene glycol, and embedded in Mollenhauers EPON/ Araldite epoxy 9 6 resin. Sections (1.0 um) were cut with a glass knife and stained with toluidine blue. Areas of interest were then sectioned (60-80 nm) with a diamond knife and were stained with uranyl acetate and lead cihate and photographed with a Zeiss EM10 elechon microscope. RESULTS Application Site Within 12 to 24 hours after the first application of 450 or 600 mg TPPi/ kg body weight, the dermal application site appeared red. After the second application, redness increased and some fissures developed, but when the trials were terminated, redness appeared diminished and fissures were scabbed and appeared to be healing. Hair regrew in the application site of rats heated with 450 mg TPPi/ kg body weight, but did not regrow in the application site of rats heated with 600 mg TPPi / kg body weight. Clinical Signs Clinical signs of neurologic impairment were assessed on days five, seven, and nine in rats dosed with 450 mg TPPi/ kg body weight. Conhol rats did not exhibit any signs of neurologic impairment. on any day. However, one’of the conhol rats from the first hial appeared ill. The rat was listless and gaunt and did not gain weight at a rate equivalent to the other conhol rats during the course of the. study. Because of this, the rat was excluded from all waveform composites and statistical analyses. Clinical signs in treated rats were first apparent on day seven when seven of ten TPPi-treated rats were affected. The effects exhibited were not severe. Four of the seven rats showed tentativeness in placement of fore- and hindfeet while three of the seven displayed hindlimb ataxia. Although ataxic, they were able to rear and did not exhibit any other signs of neurologic impairment. The three unaffected rats and two of the three ataxic rats Were perfused on day seven. Two of the four rats which had tentative foot placement on day seven continued to exhibit that trait on day nine while two appeared recovered. The rat which was ataxic on day seven remained ataxic on day nine. 9 7 Clinical signs of neurologic impairment were assessed daily in rats dosed with 600 mg TPPi/ kg body weight. The conhol rat did not exhibit any sips of neurologic impairment during the course of the study. In the first TPP; rat, clinical sips were noted initially on day four at which time the rat directed movement in a forward direction with frequent stops to rear. Gait of hindlimbs was slightly ataxic during forward movement and rearing was unbalanced. On days five and six, the rat circled several times in both directions. Circling was interspersed with forward movement and rearing. During circling and forward movement, the rat displayed moderate to severe hindlimb ataxia, and rearing was extremely unsteady. In the second TPP; rat clinical sips were apparent initially on day four at which time the rat exhibited bidirectional circling. Circling Was occasionally interrupted as the rat moved forward or reared. Hindlimb gait during circling and forward movement was moderately ataxic. On day five, all movement was directed backwards in a circle. The backing behaviour was very unbalanced. Also on day five, hindlimbs and the tail were flaccid, the left hindleg was splayed to the side, and forelimb and head support was diminished. On day six, all movement was directed forward, the rat did not rear, and movement was exhemely unbalanced. As on day five, hindlimbs and the tail were flaccid, the left hindleg was splayed to the side, and forelimb and head support was weak. Hindlimb Landing Foot Splay Hindlimb splay in rats dosed with 450 mg TPPi/ kg body weight was not significantly affected on day six, but was sipificantly affected on day eight. On day eight, the conhast difference :I: the standard error was 2.17 :t: 0.63 cm (Fig. 18). Hindlimb splay was not assessed in rats dosed with 600 mg TPPi/ kg body weight. Body Weights Body weights of rats dosed with 450 mg TPPi/ kg body weight were sipificantly decreased below the preexposure average on days five, seven, and nine. The conhast difference :t: the standard error was -15 :t 5 g, -23 :I: 5 g and -22 :i: 6 g on days five, seven, and nine, respectively (Fig. 19). Body 98 3.5 3'0- 2.5-1 2.0-J V 1.5-t difference (cm) 1.04 3:. V e 6 8 Test day Hindlimb splay conhast 1 Fig. 18. Plot of conhast differences estimated for hindlimb landing foot splay data. Rats were dosed dermally with 450 mg TPPi/kg body weight on days one and two. Contrasts estimate the difference between the preexposure and day six or day eight TPP; group average and take into account the change in the conhol group average over the same time periods. The conhol mean :1: standard errorwas8.1 :t:0.9cmondayone, 6.510.7cmondaysix,and5.0i0.5cmon day eight. Error bars are the standard error of the contrast difference. Asterisks indicate conhast differences which are significant (p < 0.05). 99 ~15 . Body weight conhast difference (grams) 8 Test day Fig. 19. Plot of conhast differences estimated for body weight data. Rats were dosed dermally with 450 mg TPPi/kg body weight on days one and two. Conhasts estimate the difference between the preexposure and day five, seven,ornineTPPigroup averageand takeinto account thechange in the conhol group average over the same time periods. The conhol mean :1: standard errorwas311:t4gondayone,313:t5gondayfive,306:t4gonday seven,and321 :thondaynine. Errorbarsarethestandard errorofthe contrast difference. Asterisks indicate contrast differences which are significant (p < 0.05). 1 00 weights of rats dosed with 600 mg TPPi/ kg body weight were markedly reduced below the preexposure average on day six. On day six, the conhast difference was ~64 g. Body Temperature Mean body temperature of rats dosed with 450 mg TPPi/ kg body weight was significantly decreased below the preexposure average on days five and seven but had recovered to the preexposure mean on day nine. The conhast difference :i: the standard error was ~1.2 :t 0.4 °C and ~1.5 :t: 0.4 °C on days five and seven, respectively (Fig. 20). Mean body temperature of rats dosed with 600 mg TPPi/ kg body weight was greatly reduced below the preexposure average. On day five, the conhast difference was ~2.9 °C. Body temperature of TPP; rats was not measured prior to warming with the heating lamp on day six. Tail Temperature Tail temperature of rats dosed with 450 mg TPPi / kg body weight was not significantly affected on any test day. Average tail temperature of rats dosed with 600 mg TPPi/ kg body weight was not different from the preexposure mean on day five (data not presented). Auditory Brainstem Responses ABR10 and ABR30. ABRlo and ABRzo peak I average peak latency and power in rats dosed with 450 mg TPPi/ kg body weight were not sipificantly different from their preexposure means on any test day and were not altered by treatment with 600 mg TPPi/ kg body weight (data not presented, see Figs. 21-24 for waveform composites). Although no statistical differences were detected, visual examination of Fig. 23 suggests that the power of peak I in ABPeo was reduced in the day nine TPPi composite obtained from rats dosed with 450 mg TPPi/ kg. The power reduction apparent in the composite was a result of a poor response in one TPPi rat and was not apparent in the TPPi composite obtained from rats dosed with 600 mg TPPi/ kg body weight (Fig. 24). 0.0 r 7/ 8 a -1.0 . i3 _.L 41] T Test day Fig. 20. Plot of conhast differences estimated for body temperature data. Rats were dosed dermally with 450 mg TPPi/ kg body weight on days one and two. Conhasts estimate the difference between the preexposure and day five, seven, or nine TPP; group average and take into account the change in the conhol group average over the same fimeperiods.1heconuolmeantstandarderroroffliemeanwas38.7i0.l °Condayone, 39.1 i 0.2 °C on day five, 39.1 :t 0.1 °C on day seven, and 39.1 :1: 0.2 °C on day nine. Error bars are the standard error of the contrast difference. Asterisks indicate conhast differences which are significant (p < 0.05). 102 Fig. 21. Composites of auditory brainstem responses collected in response to 10 kHz tone pips (ABR1o). Rats were dosed dermally with 450 mg TPPi / kg body weight on days one and two. In the figure, the first nine msec of day one (preexposure) and day nine composites are presented, the dashed line is a vertical reference point for peak latency of peak I, the peak I power analysis window is the region between the shaded areas, and peak IV is labeled. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 500 ~ 8000 Hz. 103 Auditory Brainstem Response - 10 kHz 450 mg TPPi/kg body weight TPPi, day9 + TPPi, day 1 2 uV 0 1 2 3 4 5 6 7 8 9 Latency (msec) Conhol, day9 + Conhol, day 1 2 “v 0123456789 Latency(msec) Fig. 21 104 Fig. 22. Composites of auditory brainstem responses collected in response to 10 kHz tone pips (ABRlo). Rats were dosed dermally with 600 mg TPPi/ kg body weight on days one and two. In the figure, the first nine msec of day one (preexposure) and day six waveforms are presented, the dashed line is a vertical reference point for peak latency of peak I, the peak I power analysis window is the region between the shaded areas, and peak IV is labeled. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 500 ~ 8000 Hz. 105 Auditory Brainstem Response ~ 10 kHz 600 mg TPPi/kg body weight TPPi, day 6 + TPPi, day 1 2 uV Conhol, day 6 + Conhol, day 1 2 IN 106 Fig. 23. Composites of auditory brainstem responses collected in response to 30 kHz tone pips (ABR3o). Rats were dosed dermally with 450 mg TPPi/ kg body weight on days one and two. In the figure, the first nine msec of day one (preexposure) and day nine composites are presented, the dashed line is a vertical reference point for peak latency of peak I, the peak I power analysis window is the region between the shaded areas, and peak IV is labeled. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 500 ~ 8000 Hz. Auditory Brainstem Response - 30 kHz 450 mg TPPi/kg body weight TPPi, day 9 TPPi, day 1 0123456789 Latency(msec) Conhol, day 9 Conhol, day 1 0123456789 Latency(msec) Fig. 23 108 Fig. 24. Composites of auditory brainstem responses collected in response to 30 kHz tone pips (ABRao). Rats were dosed dermally with 600 mg TPPi/ kg body weight on days one and two. In the figure, the first nine msec of day one (preexposure) and day six waveforms are presented, the dashed line is a vertical reference point for peak latency of peak I, the peak 1 power analysis window is the region of waveform between the shaded areas, and peak IV is labeled. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 500 ~ 8000 Hz. 109 Auditory Brainstem Response - 30 kHz 600 mg TPPi/kg body weight TPPi,day6 + TPPi,day1 2uV 0 1 2 3 4 5 6 7 8 9 Latency (msec) Control,day6 Conhol, day 1 . 'a 2 uV 0123456789 Latency(msec) Fig. 24 1 10 ABRc. I~HI IPL of rats dosed with 450 mg TPPi/ kg body weight was significantly increased over the preexposure average on days five, seven, and nine. The conhast difference :1: the standard error was 0.14 :t: 0.05, 0.14 :t 0.05, and 0.16 :i: 0.05 msec on days five, seven, and nine, respectively. III-V and I~V IPLs were not significantly affected by heatment on any day (Fig. 25). I~III IPL corrected for body temperature (CIPL) was not significantly affected on days five and seven, but was significantly increased over the preexposure value on day nine. On day nine, the conhast difference :1: the standard error was 0.13 :l: 0.04 msec. III-V and I~V CIPLs were not significantly affected by heatment on any day (Fig. 26). Rats dosed with 600 mg TPPi/ kg body weight had increased I~III and I~V and shortened III-V IPLs and CIPLs on day six (Fig. 27). On day six, the MD IPL and CIPL conhast difference was 0.25 and 0.21 msec, respectively. The increase in HE IPL is not visibly apparent in ABRc composite waveforms obtained from rats treated with 450 mg TPPi/ kg body weight (Fig. 28) because the changes were slight, but the increase is readily apparent in the composite waveform obtained from rats dosed with 600 mg TPPi/ kg body weight (Fig. 29). Peak amplitude ratios were not significantly affected by treatment with 450 mg TPPi/ kg body weight on any test day, but there was a downward hend in the IV/ I peak amplitude ratio (Fig. 30). The conhast difference :1: the standard error was ~0.15 i 0.15, ~0.11 i 0.15 and ~0.28 i 0.16 on days five, seven, and nine, respectively. 111 / I and IV / 1 peak amplitude ratios were reduced below the preexposure mean after heatment with 600 mg TPPi/ kg body weight. On day six, the values of the III/ I and IV/ I peak amplitude ratio conhast differences were ~0.16 and ~0.70, respectively. The decrease in IV/ I peak amplitude ratio is not readily apparent in ABRc composite waveforms obtained from rats heated with 450 mg TPPi/ kg body weight because the change was slight (Fig. 28). The decrease in m/ I and IV/ I peak amplitude ratios, however, are easily visualized in waveforms obtained from rats heated with 600 mg TPPi/ kg body weight (Fig. 29). A decrease in the IV/ I ratio is also apparent in ABRlo and ABRso composite waveforms obtained from rats dosed with 450 or 600 TPPi/ kg body weight (Figs. 21-24). 111 0.3 0.2 q 8 g 11. +0 A a g 0.0 . ’3 g or . U g ‘02 I a I‘m ‘3 m-v -os . I I~V ~0.4 . 1 5 7 9 Test day Fig. 25. Plot of conhast differences estimated for ABRc IPL data. Rats were dosed dermally with 450 mg TPPi/kg body weight on days one and two. Conhasts estimate the difference between the preexposure and day five, seven, or nine TPPi group average and take into account the change in the conhol group average over the same time periods. For I~III IPL, the conhol rneanztstandarderrorwas 13210.03rnsecondayone, 1.23:0.01 mseconday five, 1.26:0.01 mseconday seven, and 1.22:t0.01 msec ondaynine. For III-V IPL, the control mean 1: standard error was 1.54 1: 011% msec on day one, 1.62 i: 0.01 msec on day five, 1.60 :t 003 msec on day seven, and 1.55 :t 0.04 msec on daynine. Forl-V IPLthecontrolmeanistandarderrorwas286i004msec on day one, 2.85 :t 0.02 rmec on day five, 2.86 :t 0.03 msec on day seven, and 2.78:0.04 msecondaynine. Errorbarsarethestandard erroroftheconhast difference. Asterisks indicate conhast differences which are significant (p < 0.05). 112 0.3 a I~IIICorrected 0.2-J - III-VCorrected 0.1 .1 0.0 q -0.1 1 ~02- ABRc CIPL conhast difference (mseC) 0.3-1 -OA 1 s 5 7 9 Test day Fig. 26. Plot of conhast differences estimated for ABRc IPL data corrected for body temperature (CIPL). Rats were dosed dermally with 450 mg TPPi/ kg body weight on days one and two. Conhasts estimate the difference between thepreexposureanddayfive,seven,ornine'IPPigroupmeanand takeinto accountthediangeintheconholgroupmeanoverthesamefimepefiods. For l~IIICIPL,theconholmean:tstandard errorwas 1.301001 msecondayone, 1.26 :t: 0.02 msec on day five, 1.26 :t 0.02 msec on day seven, and 1.24 i: 0.01 msecon day nine. For III-V CIPL, theconhol mean 1: standard error was 1.49 :t 0.07 msec on day one, 1.71 :t 0.02 msec on day five, 1.61 i: 0.06 msec on day seven, and 1.61 10.06 msec on day nine. For I~V CIPL, the control mean i standard error was 2.79 :t 0.07 msec on day one, 2.97 :t: 0.05 msec on day five, 2.87:1:0.07msecondayseven,and 2.85:0.06msecondaynine. Errorbarsare the standard error of the conhast difference. Asterisks indicate conhast differences which are significant (p < 015). 113 0.1-4 h\\\\\‘ . '//////l 0.0 a1 . a2 4 as 1 [2| IPL cm. 0.4 . e I-III III-V I-V Test day6 Fig. 27. Plot of conhast difference estimated for ABRc IPL and IPL data corrected for body temperature (CIPL). Rats were dosed dermally with 6(1) mg TPPi/kg body weight on days one and two. The conhasts estimate the difference between the preexposure and day six TPP; group average and take into account the change in the conhol rat value over the same time period. Because there was only one control rat, the conhasts were not analyzed statistically. For the conhol rat, I~III IPL was 1.22 and 1.22 msec on days one and six, respectively, III-V IPL was 1.56 and 1.66 msec on days one and six, respectively, and I~V IPL was 2.78 and 2.88 msec on days one and six, respectively. For the control rat, I~III CIPL was 1.22 and 120 msec on days one and six, respectively, III-V CIPL was 1.56 and 1.59 msec on days one and six, respectively, and I~V CIPL was2.78and2.79msecondaysoneandsix,respectively. 114 Fig. 28. Composites of auditory brainstem responses collected in response to clicks (ABRc). Rats were dosed dermally with 450 mg TPPi/ kg body weight on days one and two. In the figure, the first nine msec of day one (preexposure) and day nine composites are presented, dashed lines are vertical reference points for latency of peaks I and III, and peak IV is labeled. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 500 ~ 8000 Hz. 1 15 Auditory Brainstem Response ~ Click 450 mg TPPi/kg body weight I m I I ”IV I I TPPi,day9 I I I I l I IV I m’dayl I Mp; I + I I I I 29" I T1 I 11 I T fi I T fl 0 1 2 3 4 5 6 7 8 9 Latency(msec) I I.“ I I II” I I I ’ Conhol, day 9 l l I I I l I I Control,day1 : + I I 211V 116 Fig. 29. Composites of auditory brainstem responses collected in response to clicks (ABRc). Rats were closed dermally with 600 mg TPPi / kg body weight on days one and two. In the figure, the first nine msec of day one (preexposure) and day six composites are presented, dashed lines are vertical reference points for latency of peaks I and III, and peak IV is labeled. Waveforms were collected with a broad-band analog filter and then were digitally filtered with abandpassof500~8000Hz. 117 Auditory Brainstem nse ~ Click 600 mg TPPilkg y weight .1 III I I ‘ I I . N I I I I TPPi,day6 I I I l : “IV | l TPPi,dayl : I I 2+v , , I I: l I I I' 1‘ J; T f Y I U 1 0 1 2 3 4 5 6 7 8 9 Latency (msec) I III I I I I I I ' N I Conhol,day6 I I I I I j IV Conhol,da 1 ‘I' y l :1:va I I I 1' '1 U I I I I I 0 1 2 3 4 5 6 7 8 9 Latency (msec) 118 0.0 e ~02- -0.3 - -—L— ~0.4 a ABRc IV/ I conhast difference I | & ~05 . a 5 7 9 Test day Fig. 30. Plot of contrast differences estimated for ABRc IV/ I peak amplitude ratio data. Rats were dosed dermally with 450 mg TPPi/kg body weight on days one and two. Conhasts estimate the difference between the preexposure and day five, seven, or nine TPP; group average and take into account the change in the control group average over the same time periods. The conhol average :1: standard error was 0.97 :i: 0.15 on day one, 1.14 :t 0.15 on day five, 1.02 i 0.18 on day seven, and 1.14 :t 0.20 on day nine. Error bars are the standard error of the conhast difference. 1 l9 Somatosensory Evoked Potentials Somatosensory Cortex. Visual examination of SEP-S composites ( Figs. 31 and 32) shows that activity transmitted from the tail to the somatosensory cortex was not affected by heatment with 450 mg TPP/ kg body weight but was depressed by treatment with 600 mg TPP/ kg body weight. Visual examination of composite waveforms obtained from rats dosed with 450 mg TPP/ kg body weight ( Fig. 31) indicate that power of peak P1 was slightly reduced on day five, moderately reduced on day seven, and the peak was barely detectable on day nine. However, peak P1 was not detectable in all preexposure waveforms. Therefore, the effect of heatment on the incidence of peak P1, rather than on power, was analyzed statistically. Incidence of peak P1 decreased with postexposure time (Fig. 31), but the difference was not statistically significant (p > 0.05). The peak P1 response was present in all preexposure and in the control day six waveforms collected from rats used for the 600 mg hial. After heatment, peak P1 was barely detectable in the TPP; composite (Fig. 32). Power of long latency SEP-S components was not sipificantly affected by heatment with 450 mg TPP; / kg body weight on any test day (data not presented, see Fig. 33 for waveform composites). Power of long latency components was greatly reduced below the preexposure average in the day six TPPi composite obtained from rats dosed with 600 mg TPPi/ kg body weight (Fig. 34). On day six, the value of the long latency power conhast difference was ~28 uV. Cerebellar Cortex. Upon visual examination of SEP-C early latency composites, peaks N1, P1, and P2 do not appear to be affected by treatment with 450 mg TPPi/ kg body weight (Fig. 35). However, the day nine TPPi composite contains a large negative peak at 18 msec which is not present in the TPP; preexposure or the control composites (Fig. 35). This peak is not present in the day six composite obtained from rats dosed with 600 mg TPPi/ kg body weight, but peaks N1, P1, and P2 appear reduced in power (Fig. 36). Long latency SEP-C components in the day nine composite obtained from rats dosed with 450 mg TPPi/ kg body weight contain oscillations which are not present in the long latency components of the TPP; preexposure or the conhol composites (Fig. 37). Long latency components 120 Fig. 31. Composites of somatosensory evoked potentials recorded from the somatosensory electrode (SEP-S) with a 35 msec data sweep (early latency components). Rats were dosed dermally with 450 mg TPPi/ kg body weight on days one and two. The first 25 msec of day one (preexposure) and day nine composites are presented, the area between the dashed lines represents activity hansmitted from the tail to the somatosensory cortex, and peaks labeled with an arrow are peak P1. The number of waveforms which contained a detectable peak P1 out of the total number of waveforms forming the composite is presented in parentheses next to each composite. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 85-1500 Hz. 121 Somatosensory Evoked Potential ~ Sensory Cortex Early latency Components 450 mg TPPi/kg body weight TPPi, day 9 (1 of 5) TPPi, day 7 (3 of 10) TPPi, day 5 (5 of 10) TPPi, day 1 (9 of 10) Conhol, day 9 (2 of 4) Conhol, day7 (3 of 4) | Conhol, day 5 (3 of 4) Conhol ,day 1 (3 of 4) I =- _—_—a 1'0 {5 20 25 Latency (msec) Fig. 31 °1 (ll-I 122 Fig. 32. Composites of somatosensory evoked potentials recorded from the somatosensory elechode (SEP-S) with a 35 msec data sweep (early latency components). Rats were closed dermally with 600 mg TPPi / kg body weight on days one and two. The first 25 msec of day one (preexposure) and day six waveforms are presented, the area between the dashed lines represents activity hansmitted from the tail to the somatosensory cortex, and peaks labeled with an arrow are peak P1. Waveforms were collected with a broad~ band analog filter and then were digitally filtered with a bandpass of 85-1500 Hz. 123 Somatosensory Evoked Potential ~ Sensory Cortex Early Latency Components 600 mg TPPi/kg body weight TPPi, day 6 W TPPi,dayl Conhol, day 6 Conhol, day 1 0 “1 —-g ‘1 O _———- dd 0| N O N 01 124 Fig. 33. Composites of somatosensory evoked potentials recorded from the somatosensory electrode (SEP-S) with a 200 msec data sweep (long latency components). Rats were dosed dermally with 450 mg TPPi / kg body weight on days one and two. The first 100 msec of day one (preexposure) and day nine composites are presented, and the region between the shaded areas represents the power analysis window. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1~500 Hz. 125 Somatosensory Evoked Potential - Sensory Cortex Long Latency Components 450 mg TPPi/kg body weight TPPi, day 9 TPPi, day 1 Conhol, day 9 Control, day 1 126 Fig. 34. Composites of somatosensory evoked potentials recorded from the somatosensory elechode (SEP-S) with a 200 msec data sweep (long latency components). Rats were dosed dermally with 600 mg TPPi/ kg body weight on days one and two. The first 100 msec of day one (preexposure) and day six waveforms are presented, and the region between the shaded areas represents the power analysis window. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1~500 Hz. 127 Somatosensory Evoked Potential - Sensory Cortex Long Latency Components 600 mg TPPi/kg body weight TPPi, day 6 TPPi, day 1 Conhol, day 6 Conhol, day 1 128 Fig. 35. Composites of somatosensory evoked potentials recorded from the cerebellar elechode (SEP-C) with a 35 msec data sweep (early latency components). Rats were dosed dermally with 450 mg TPPi/ kg body weight on days one and two. The first 25 msec of day one (preexposure) and day nine composites are presented, and dashed vertical lines are reference points for peaks N1, P1, and P2. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1~750 Hz. Somatosensory Evoked Potential - Cerebellar Cortex Early latency Components 450 mg TPPi/kg body weight N1P1P2 I TPPi, day 9 I | I I I + TPPi, day 1 : : : 20 W I | - h I I I I I I I I o 5 1o 15 20 25 Latency (msec) Conhol, day 9 + Control, day 1 2° “V | i | I 10 130 Fig. 36. Composites of somatosensory evoked potentials recorded from the cerebellar electrode (SEP-C) with a 35 msec data sweep (early latency components). Rats were dosed dermally with 600 mg TPPi/ kg body weight on days one and two. The first 25 msec of day one (preexposure) and day nine composites are presented, and dashed vertical lines are reference points for peaks N1, P1, and P2. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1~750 Hz. Somatosensory Evoked Potential ~ Cerebellar Cortex Early Latency Components 600 mg TPPi/kg body weight N1 P1 P2 I I TPPi, day 6 W I I I I -—+ TPPi,dayl : : : 20 uV I I __ _ I I I I) 5 1'0 1'5 2'0 21 5 Latency (msec) N1 P1 P2 I I I I I Conhol, day 6 I : I I I I I l I I _ _ + Conhol, day 1 I I l | j ZOuV I I I I I I " "' " 0 5 lo 15 20 25 Latency (msec) Fig. 36 132 Fig. 37. Composites of somatosensory evoked potentials recorded from the cerebellar electrode (SEP-C) with a 200 msec data sweep (long latency components). Rats were dosed dermally with 450 mg TPPi/ kg body weight on days one and two. The dashed vertical line represents the onset of the long latency components. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1~750 Hz. 1 3 3 Somatosensory Evoked Potential - Cerebellar Cortex Long Latency Components 450 mg TPPi/kg body weight TPPi, day 9 8?? + . 40 p. V TPPi, day 1 (I 40 8‘0 120 160 200 Latency (msec) l | | | I | I Conhol, day 9 I I + l | 40 p. V I Control, day 1 I ~ I I I I I 40 so 120 160 200 latency (msec) 0! Fig. 37 l 3 4 in the day six composite obtained from rats dosed with 600 mg TPPi/ kg body weight do not contain oscillations but are much reduced in power relative to those same components in the TPPi preexposure and control composites (Fig. 38). Flash Evoked Potentials Visual Cortex. Peak latency and power of peak N1 were not sipificantly affected by heatment with 450 mg TPPi/ kg body weight on any day (p > 0.05) although there was a downward trend in the power of peak N1 over time post-heatment. The conhast difference :I: the standard error was ~11.6 :I: 10.1 IN, ~18.4 :i: 10.1 11V, and ~26.1 :I: 11.0 IN on days five, seven, and nine, respectively for low intensity FEP-V and was ~19.8 :i: 14.4 IN, ~24.0 :1: 14.4 uV, and ~26.4 :I: 15.7 IN on days five, seven, and nine, respectively for medium intensity FEP-V (Fig. 39). Because the change was slight, the hend is not readily apparent in the composites (Fig. 40). Treatment with 600 mg TPP; / kg body weight markedly increased peak latency and reduced power of peak N1 in both low and medium intensity FEP-Vs (Fig. 41). On day six, the values of the latency conhast differences were 6.3 and 6.8 msec for low and medium intensity FEP-Vs, respectively and the values of the power conhast differences were 40.9 and ~34.7 uV for low and medium intensity FEP-Vs, respectively. Power of the long latency FEP-V components was not significantly affected by heatment with 450 mg TPP; / kg body weight on any day (p > 0.05) in either low or medium intensity FEP-Vs (data not presented, see Fig. 42 for waveform composites). In conhast, treatment with 600 mg TPPi/ kg body weight affected power of this complex. At low intensity, TPP; rat 1 had a decrease in power while TPP; rat 2 showed a large increase in power. At high intensity, power was slightly and greatly increased in TPPi rat 1 and 2, respectively. In both rats, long latency components were also slow (Fig. 43). Cerebellar Cortex. Visual examination of composite waveforms shows that peak N1 in both low and medium intensity FEP-Cs did not appear to be affected by treatment with 450 mg TPPi/ kg body weight (Fig. 44) but the power was reduced and latency increased after heatment with 600 mg TPPi/ kg body weight (Fig. 45). Visual examination of composite 135 Fig. 38. Composites of somatosensory evoked potentials recorded from the cerebellar electrode (SEP-C) with a 200 msec data sweep (long latency components). Rats were dosed dermally with 600 mg TPPi/ kg body weight on days one and two. The dashed vertical line represents the onset of the long latency components. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bande of 1~750 Hz. 136 Somatosensory Evoked Potential ~ Cerebellar Cortex Long Latency Components 600 mg 'I'PPi/kg body weight I I I —— + I I TPPi,dayl I 40 ”V I I I I so 120 160 200 Latency (msec) °- ‘ 0 Control, day 6 Conhol, day 1 40 II V I I I I so 120 160 200 Latency (msec) O- «b 0 Fig. 38 137 FEP-V N1 power contrast difference (uV) '40 " a Low intensity I Medium intensity 5 7 9 Test day Fig. 39. Plot of contrast differences estimated for FEP-V peak N1 power. Rats were dosed dermally with450mg TPPi/kgbody weighton days oneand two. Contrasts estimate the difference between the preexposure and day five, seven, or nine TPPi group average and take into account the change in the control group average over the same time period. The control mean :I: standard error was 42.8 :t 5.9 LIV on day one, 51.5 :t 105 [IV on day five, 52.8 i: 113 [IV on day seven, and 65.2 :t: 16.1 11V on day nine for low intensity flash and 64.9 :t 6.9 11V on day one, 79.5 i 14.9 LIV on day five, 82.9 :t 13.4 11V on day seven, and 81.7 :I: 21.3 uV on day nine for medium intensity flash. Error bars are the standard error of the contrast difference. 138 Fig. 40. Composites of flash evoked potentials collected from the visual electrode (FEP-V) in response to low or medium intensity flashes with a 150 msec data sweep (early latency components). Rats were dosed dermally with 450 mg TPPi / kg body weight on days one and two. The first 90 msec of day one (preexposure) and day nine composites are presented, the dashed line is a vertical reference point for peak latency of peak N1, and the area between the shaded regions represents the power analysis window for peak N 1. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1-250 Hz. 139 Flash-Evoked Potential - Visual Cortex Early Latency Components 450 mg '1??ng body weight Low Intensity Medium Intensity 1s 36 54 72 Latency(msec) 140 Fig. 41. Composites of flash evoked potentials collected from the visual electrode (FEP-V) in response to low or medium intensity flashes with a 150 msec data sweep (early latency components). Rats were closed dermally with 600 mg TPPi/ kg body weight on days one and two. The first 90 msec of day one (preexposure) and day six composites are presented, the dashed line is a vertical reference point for peak latency of peak N1, and the area between the shaded regions represents the power analysis window for peak N1. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1-250 Hz. 141 Flash-Evoked Potential - Visual Cortex Early latency Components 600 mg TPPi/kg body weight Low Intensity Medium Intensity 1s 36 54 72 o 36 54 Latency(msec) Latency(msec) 142 Fig. 42. Composites of flash evoked potentials collected from the visual electrode (FEP—V) in response to low or medium intensity flashes with a 600 msec data sweep (long latency components). Rats were dosed dermally with 450 mg TPPi/ kg body weight on days one and two. Day one (preexposure) and day nine composites are presented, and the area between the shaded regions represents the power analysis window. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1- 250 Hz. 143 Flash-Evoked Potential - Visual Cortex Long Latency Components 450 mg TPPi/kg body weight Low Intensity Medium Intensity ’ : TPPi, day ’3 ; TPPi, day + 120 240 360 480 600 o 120 240 360 480 800 Latency(msec) Latency(msec) Control, day i: : :EOO uV + o 120 240 360 480 600 o 120 240 360 480 300 Latency(msec) Latency(msec) Fig. 42 144 Fig. 43. Composites of flash evoked potentials collected from the visual electrode (FEP-V) in response to low or medium intensity flashes with a 600 msec data sweep (long latency components). Rats were dosed dermally with 600 mg TPPi/ kg body weight on days one and two. Day one (preexposure) and day six composites are presented, and the area between the shaded regions represents the power analysis window. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1— 250 Hz. 145 Flash-Evoked Potential - Visual Cortex Long Latency Components 600 mg TPPiIkg Low Intensity Medium Intensity o 120 240 360 480 600 o 20 La24o cyéaso) 430 800 Latency(msec) 120 240 360 480 600 o 120 240 380 480 600 Latency (msec) Latency (msec) 119,0 n V o 120 240 360 480 600 o 120 240 380 480 600 latency (msec) Latency (msec) Fig. 43 146 Fig. 44. Composites of flash evoked potentials recorded from the cerebellar electrode (FEP-C) in response to low or medium intensity flashes and with a 150 msec data sweep (early latency components). Rats were dosed dermally with 450 mg TPPi/ kg body weight on days one and two. The first 90 msec of day one (preexposure) and day nine composites are presented, and the dashed vertical line is a reference point for peak N1. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1-250 Hz. More ”I 147 Flash-Evoked Potential - Cerebellar Cortex Early Latency Components 450 mg TPPilkg body weight Low Intensity Medium Intensity N1 N1 148 Fig. 45. Composites of flash evoked potentials recorded from the cerebellar electrode (FEP-C) in response to low and medium intensity flashes and with a 150 msec data sweep (early latency components). Rats were dosed dermally with 600 mg TPPi/ kg body weight on days one and two. The first 90 msec of day one (preexposure) and day six composites are presented, and the dashed vertical line is a reference point for peak N 1. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1-250 Hz. 149 Flash-Evoked Potential - Cerebellar Cortex Early Latency Components 600 mg TPPi/kg body weight Low Intensity Medium Intensity N1 N1 I I TPPi, day 6 I MINA I I I I r I I I 0 18 36 54 72 go o 18 36 54 72 Latency (msec) Latency (msec) Control, day 6 %. _I———-—— Control, day 1 : | I I I4 I l I *I l 54 72 90 0 I 18 36 8 36 54 Latency (msec) Latency (msec) Fig. 45 l 5 0 waveforms shows that long latency FEP-C components did not appear to be affected by treatment with 450 mg 'l'PPi/ kg body weight (Fig. 46) but in rats which were dosed with 600 mg TPPi/ kg body weight were slow, and in TPPi rat 2, were altered in shape (Fig. 47). Caudal Nerve Action Potentials CNAP1 and CNAP2 peak latency and power in rats dosed with 450 mg TPPi/ kg body weight were not significantly different from their preexposure values on any test day (data not presented, see Fig. 48 for composite waveforms). On day nine, the CNAle CNAP1 power ratio was 0.98 and 0.96 for the control and the treated group, respectively. Visual examination of CNAP1 and CNAPz composite waveforms obtained from rats dosed with 600 mg TPPi/ kg body weight (Fig. 49) indicate that peak latency was unaffected and that power of both CNAPs was reduced on day six. Power analysis showed that the reduction was similar for CNAP1 and CNAPz. On day six, the value of the power contrast difference was -1.8 and -1.9 [IV for CNAP1 and CNAPz, respectively. On day six, the CNAP2/CNAP1 power ratio was 0.95 and 1.04 for the control and TPPi rats, respectively. Neu ropathology No pathological changes were detected in the brain of rats dosed with 450 mg TPPi/ kg body weight. At the light microscopic level, both rats dosed with 600 mg TPPi / kg body weight exhibited bilaterally symmetrical discrete bands of clear, circular intercellular vacuoles in the neuropil of motor, somatosensory, and sensorimotor cortices at the level of the optic chiasm. Vacuoles in the somatosensory and sensorimotor cortices also were present at the level of the infundibulum. Vacuoles also occurred in visual and auditory cortices at the level of the mammillary nuclei and in the visual cortex at the level of the superior colliculus. Vacuoles were most prominent in cortical layer four and the deeper parts of layer five (Fig. 50). Vacuolization was moderately dense in somatosensory and sensorimotor cortices and was less dense in the other cortical areas. Also, the density of the vacuolization was greater in TPPi rat 2 than in TPPi rat 1 . 151 Fig. 46. Composites of flash evoked potentials recorded from the cerebellar electrode (FEP-C) in response to low or medium intensity flashes and with a 600 msec data sweep (long latency components). Rats were dosed dermally with 450 mg TPPi/ kg body weight on days one and two. Day one (preexposure) and day six composites are presented, and the dashed vertical line is a reference point for onset of long latency components. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1-250 Hz. 152 Flash-Evoked Potential - Cerebellar Cortex Long Latency Components 450 mg TPPi/kg body weight Low Intensity Medium Intensity TPPi, day 9 a TPPi, day 1 _ + I I I I fl I I I I 120 240 360 480 600 o 120 240 360 480 600 Latency (msec) Latency (msec) 3? 0! Control, day 9 'E’ i?) Control, day 1 I + I I I I I I I I l o 120 240 360 480 600 120 240 360 480 600 Latency (msec) Latency (msec) I 01 Fig. 46 153 Fig. 47. Composites of flash evoked potentials recorded from the cerebellar electrode (FEP-C) in response to low and medium intensity flashes and with a 600 msec data sweep (long latency components). Rats were dosed dermally with 600 mg TPPi/ kg body weight on days one and two. Day one (preexposure) and day six composites are presented, and the dashed vertical line is a reference point for onset of long latency components. Waveforms were collected with a broad-band analog filter and then were digitally filtered with a bandpass of 1-250 Hz. 154 Flash-Evoked Potential - Cerebellar Cortex Long Latency Components 600 mg TPPi/kg body weight Low Intensity Medium Intensity I I | TPPi rat 2, day 6 l I I l TPPi rat 2, day 1 _ I40 uV l + 5 120 210 aso 450 $0 0 120 2:0 3so 450 so'o Latency (msec) Latency (msec) I l l TPPi rat 1, day 6 TPPi rat 1, day 1 - I40 uV + 120 240 360 480 800 2:0 380 480 60'0 latency (msec) 0Latency (msec) Wat day 6% Control rat, day 1 - I40 uv I l I I I I I I I I I I 1 o 120 240 380 480 600 o 120 240 360 480 600 Latency (msec) latency (msec) Fig. 47 155 Fig. 48. Composites of caudal nerve action potentials collected in response to single (CNAP1) or paired (CNAPz) stimuli. Rats were dosed dermally with 450 mg TPPi/ kg body weight on days one and two. CNAP1 was subtracted from CNAPz to facilitate analysis of CNAPz. In the figure, the first 15 msec of day one (preexposure) and day nine composites are presented, the dashed line is a vertical reference point for peak latency, and the power analysis window is the region between the shaded areas. 156 Caudal Nerve Action Potential 450 mg TPPi/kg body weight Single stimulus Second of paired stimuli may : TPPi,dayl _. - 10 uV + 3 s s 12 15 o 3 s 9 12 15 latency (msec) latency (msec) I ' Control, day 9 Conhol,day 1 “ - 10uV o 3 s s 12 15 o 3 s s 12 15 latency (msec) latency (msec) 157 Fig. 49. Composites of caudal nerve action potentials collected in response to single (CNAP1) and paired (CNAP1)) stimuli. Rats were dosed dermally with 600 mg TPPi/ kg body weight on days one and two. CNAP1 was subtracted from CNAPz to facilitate analysis of CNAPZ In the figure, the first 15 msec of day one (preexposure) and day six waveforms are presented, the dashed line is a vertical reference point for peak latency, and the power analysis window is the region between the shaded areas. 158 Caudal Nerve Action Potential 600 mg TPPi/kg body weight Single stimulus Second of paired stimuli TPPi, day 6 TPPi, day 1 Control, da 6 _ ' “ Control, day 1 ' :[10uV . + 3utefincy(9 )12 15 1:18.43 3hte6ncy(9 )12 15 159 Fig. 50. Photomicrographs illustrating control (A) and TPPi (B) H & E stained cross— sections through the somatosensory cortex at the level of the optic chiasm. TPP, rats were dosed dermally with 600 mg TPPi/kg body weight on days one and two and were perfused on day six. Note the light vacuolization in layer IV and the more superficial parts of layer V, the extensive vacuolization in the deeper parts of layer V, and its absence in the control section (magnification x16.1). 160 Fig. 50 1 6 l TPPi rat 2 also exhibited bilaterally symmetrical clusters of vacuoles in the neuropil of the magnocellular preoptic nucleus and the nucleus of the horizontal limb of the diagonal band. Vacuoles ranged from approximately 2 to 15 microns in diameter, and either appeared empty or contained coarse, particulate matter in their lumens. Vacuolization was not accompanied by gliosis or inflammatory cell influx (Fig. 51). At the electron microscopic level, vacuoles were bordered by intact myelinated or unmyelinated axons, dendrites, and the cytoplasmic membranes of neuronal cell bodies. The vacuoles resulted in compression of the surrounding structures. Multiple circular to polygonal profiles of shrunken cytoplasmic processes occurred within the vacuoles. The processes were usually clustered together in the center of the vacuoles. The outer portion of the vacuoles were clear or contained small fragments of electron-dense material which probably originated from degenerative cytoplasmic processes. Some of the vacuolar components were recognizable as degenerating axons, with their mitochondria and myelin sheaths intact (Fig. 52). 162 Fig. 51. Higher power photomicrographs illustrating control (A) and TPPi (B) H & E stained cross-sections through layer V of somatosensory cortex at the level of the optic chiasm. TPPi rats were dosed dermally with 600 mg TPPi/kg body weight on days one and two and were perfused on day six. Note the variably sized intercellular vacuoles in the neuropil of the TPPi section. Endothelial cells (arrows in the control plate) distinguish blood vessels from vacuoles (magnification x102.4). 163 Fig. 51 164 Fig. 52. Electron micrograph of a vacuole in the neuropil of layer V of somatosensory cortex of a TPPi-treated rat. TPP, rats were dosed dermally with 600 mg TPP/kg body weight on days one and two and were perfused on day six. Vacuoles contained profiles of shrunken cytoplasmic processes clustered together in the center of the vacuole. Some of the processes could be identified as degenerating axons with their myelin sheaths intact (arrows) (magnification x3200). DISCUSSION Body Temperature Dermal exposure to TPP, produced a dose-dependant decrease in body temperature. Hypothermia interferes with temperature dependant biological processes and influences the effect of a toxicant on a biological system (Gordon et al. , 1988). Thus, TPPi-induced hypothermia may have enhanced the effects of TPP, on the CNS. Hypothermia results from failure of central or peripheral components of the thermoregulatory system or may arise from other toxicant-related effects such as a decrease in metabolic rate (Gordon et al., 1988). The medial preoptic/anterior hypothalamic region is the central thermoregulatory controller in mammals (Gordon et al., 1988). Damage was not detected in this region after exposure to TPPi. Exposure to TPP, did result in vacuolization of the magnocellular preoptic nucleus in the lateral part of the anterior hypothalamus, but this nucleus does not appear to be anatomically connected nor functionally related to the medial preoptic/anterior hypothalamic region (Simerly and Swanson, 1988; Vertes, 1988). Thus, TPP, could target the peripheral thermoregulatory systems or act in some other manner to lower body temperature. Body temperature in low dose animals recovered indicating that the effect is reversible or that thermoregulatory systems were able to compensate. Some Type I OPs also cause hypothermia (Gordon et al. , 1991). This is thought to be due to anticholinergic effects in the medial preoptic/anterior hypothalamic region (Gordon et al. , 1991). Because TPPi does not produce meaningful inhibition of acetylcholinesterase in rat brain (V eronesi et 166 167 al., 1986b) and does not target the central controller, Type I and Type II OPs appear to induce hypothermia through different mechanisms. Clinical Signs Clinieal signs observed depended on the dose of TPP,. Clinieal signs in the low dose animals were slight and ranged from no observable response to hindlimb ataxia and splay. Also, since two of the more slightly affected rats recovered, either the effects were reversible or compensation occurred. In contrast, both high dose rats exhibited severe gait and directional deficits similar to those previously reported for the rat after subacute subcutaneous exposure to TPPi (V eronesi et al. , 1986b; Veronesi and Dvergsten, 1987). Evoked Potentials Auditory Brainstem Responses. The lack of an effect on peak I of ABRs obtained in response to tones from both low and high dose rats indicates that TPPi is not ototoxic in the rat after subacute dermal application. However, this dosing regimen did result in alterations in central processing in the auditory pathway. I-III IPL, adjusted for the hypothermic state of the animals, was increased and the amplitude of peak IV decreased. The magnitude of the change was directly related to dose, and in the low dose group, also depended on time post-treatment. The I—III latency increase suggests that conduction in lower brainstem auditory tracts was affected while the peak IV amplitude reduction indicates that the lateral lemniscus was targeted (Stockard et al., 1980). Peak III also appeared to be reduced in the high dose rats. This peak corresponds to the superior 168 olivary nucleus (Stockard et al. , 1980). Thus, TPP, selectively affected the lower brainstem auditory pathway. Somatosensory Evoked Potentials - Somatosensory Cortex. In the low dose rats, subacute dermal exposure to TPP, produced a time-dependant and selective depression of peak P1, a peak thought to be associated with initial cortical processing in the somatosensory system (Wiederholt and lragui-Madoz, 1977). At the higher dose, long latency components were also greatly depressed indicating that higher order cortical processing of somatosensory information was impaired (Mattsson et al., 1992). The subcortical somatosensory pathway did not appear to be affected at either dose level. Thus, TPP, selectively targeted cortical and higher order processing of somatosensory information. Flash Evoked Potentials - Visual Cortex. In the low dose rats, power of peak N1 was reduced in both low and medium intensity PEP-Vs in a time-dependant manner. In the high dose rats, power of peak N1 was reduced in low and medium intensity PEP-Vs, but to a greater extent than in the low dose rats and long latency components at both intensities were depressed in one rat and slow and large in the other. Peaks in the FEP- V appear to be entirely of cortical origin (Dyer et al., 1987). Because of this, effects on the subcortical visual pathway cannot be assessed. Therefore, the depression of early and long latency components observed in this study could either be due to loss of subcortical input or to a direct cortieal effect. Whatever the source, it is clear that processing of visual information was impaired after exposure to TPPi. Cerebellar Potentials - Somatosensory and Visual. A negative peak and oscillations not typically present occurred in SEP—Cs collected from the low dose rats while SEP-Cs collected from the high dose rats were depressed. These changes suggest that cerebellar function associated with somatic sensation was impaired. PEP-Cs collected from the low dose rats did not appear to be affected by treatment while PEP-Cs collected from the 169 high dose rats were depressed, altered in shape, and slow suggesting that cerebellar function associated with visual processes was impaired. Caudal Nerve Action Potentials. CNAPs were not affected by exposure to the low dose of TPP,. CNAP, and CNAPz power were reduced without an apparent latency change after exposure to the high dose of TPP,. This combination of changes indicates axonal degeneration without myelin damage and suggests that in peripheral nerves the neuropathy produced by exposure to TPPi is an axonopathy. The CNAPJCNAP, power ratios were similar for the control and treated groups after exposure to either dose of TPP,. Thus, under the stimulus conditions used, TPP, did not affect refractoriness of the stimulated peripheral nerves. Neuropathology Pathologic damage was not detected with H & E at the light microscopic level in the brains of the low dose rats or in the auditory brainstem or cerebellum of the high dose rats. The functional effects observed in evoked potentials obtained from the low dose rats and the effects noted in ABRs and cerebellar potentials collected from the high dose rats could have arisen from subpathologieal, i.e. , biochemical or physiological, effects on the CNS. It also may be that if a more sensitive staining technique had been used or if an ultrastructural examination had been conducted, pathological changes would have been evident. Numerous vacuoles which contained degenerating axons were prevalent throughout the cerebral cortex in the brains of the high dose rats. The vacuoles were especially prominent in somatosensory and visual cortices, and thus, are the pathological correlates of the depression of cortical and higher order processing in the somatosensory and visual systems. Peripheral nerves in the tail were not examined for pathologic damage. 1 70 Summary This study indieates that subacute dermal exposure to TPP,, a Type II organophosphorus delayed neurotoxicant, results in notable alterations in central processing in the auditory, somatosensory, and visual systems in the rat. These effects would be expected to impair sensory guided behaviours and hinder integration of sensory and motor information (Chapin and Lin, 1990), and thus, are probably responsible for many of the clinieal signs associated with exposure to Type II delayed neurotoxicants. The magnitude of the effects observed was dependant on the dose. At the low dose, effects were slight and selective and involved the lower auditory brainstem and initial cortical processing in the somatosensory and visual systems. At the high dose, higher order cortical processing and peripheral nerve function were affected in addition to effects of greater magnitude on the previously mentioned parameters. In the low dose rats, alterations were also dependant on the time post-treatment. Evoked potential alterations in the CNS were greatest on day nine, a time point when body temperature and clinical signs showed recovery. This indicates that non-central effects may also play a role in TPPi-induced neurotoxicity or that repair or compensation occurred. If recovery was related to central compensation, it suggests that subsequent challenge with a non- neurotoxic dose of TPPi could reprecipitate clinical expression of the neuropathy. Beeause high dose rats were larger and older, their waveforms were more robust and more well-defined than those obtained from low dose rats (for an example, compare early latency SEP-S components in the preexposure composites in Figs. 31 and 32). Thus, the severe effects observed in that trial may not only be due to a higher dose, but could also be related to anatomical or functional maturation of the nervous system. Interestingly, Tanaka et al. (1992b) suggest that maturation plays a role in susceptibility of the ferret nervous system to TPPi-induced CNS degeneration. 1 71 Body weight loss causes a decrease in the amplitude of peak N1 in FEP-Vs (Albee et al. , 1987). Therefore, body weight loss could be partially responsible for this change in the present study. However, the magnitude of the change observed in the high dose rats and the presence of a verifying morphologic lesion suggest that the depression of peak N1 is neurological in origin. The sample size used in this study was small, especially in the high dose trial. Thus, normal biological variation could be responsible for some of the observed responses. However, presence of similar changes in both dose groups, the magnitude of the changes in the high dose evoked potentials, and the presence of verifying clinical signs and a morphological lesion provide evidence that the observed effects are related to TPPi-intoxication. SUMMARY The first experiment examined and correlated clinical and neuropathologic effects of organophosphorus delayed neurotoxicants on the central nervous system of the rat. Rats exposed to a single subcutaneous dose of DFP, a Type I OP, did not display clinical signs and CNS degeneration was confined to the rostral gracile fasciculus and gracilis. The lack of clinical signs and limited CNS degeneration indicate the rat may not be suited for study of Type I OPs relative to species such as the chicken or ferret which exhibit clinical signs and more extensive CNS degeneration. Rats which received subacute subcutaneous exposure to TPP,, a Type II OP, had widespread loss of afferent input to nuclei in the midbrain and forebrain as well as in the hindbrain of the rat. Affected afferents were associated with three sensory systems (somatosensory, sensorimotor, visual, and auditory), the motor system (motor cortex), and two systems which modulate posture and locomotion (the basal ganglia and the vestibular nuclear 1 72 complex). This combined system degeneration accounts for the complex set of clinical signs displayed by rats after exposure to TPPi and indicates the rat is suitable for study of many aspects of Type II OPIDN. This study is also the first report of TPPi-related damage in a region of the limbic system associated with memory (mamillary nuclei and mediodorsal thalamic nucleus). The second experiment examined and correlated electrophysiologic and neuropathologic effects of subacute dermal exposure to TPPi on the CNS of the rat. The results of the two experiments can also be correlated. As an example, two staining techniques verified the presence of degenerating axons in neocortical layers IV and V after either subcutaneous or dermal exposure to TPP,. The source of this degeneration was probably thalamocortieal afferents. Loss of thalamocortical afferents was directly associated with selective depression of cortical processing of somatosensory and visual information. Peak IV in ABRs was also selectively depressed. This peak is thought to arise mainly from activity in the lateral lemniscus. Fink-Heimer stained sections showed presence of preterminal axonal and terminal degeneration in the inferior colliculus, a mandatory relay in the auditory pathway. The lateral leminiscus provides the primary ascending input to the inferior colliculus and is probably the source of the degeneration in the inferior colliculus and the depression of peak IV in ABRs. The inability to process sensory information in combination with the presence of degeneration in motor and limbic systems would be expected to produce the severe gait and directional deficits observed. Although exposure to TPPi results in delayed onset neurotoxicity, some have questioned whether it should be categorized as a second type of OPIDN (V eronesi and Dvergsten, 1987). This judgement was based on the lack of similarity in hindbrain degeneration patterns produced by the two types of neurotoxicants in the rat. This study verifies those dissimilarities and also shows that TPP,, a Type II OP, induces widespread 1 73 degeneration in the midbrain and forebrain of the rat while DFP, a Type I OP, does not. In addition, electrophysiological evidence is provided which further differentiates the two types of neurotoxicity. Somatosensory evoked potentials have been measured in rats which were exposed to DFP (Veronesi et al. , 1987). Activity transmitted from the hindlimb to somatosensory cortex was depressed coincident with spinal cord neuropathic damage. This activity was spared after exposure to TPP,, and instead, somatosensory cortical activity was depressed and was related to cortieal degeneration. Thus, Type I OPs appear to selectively affect the initial part of the somatosensory pathway, and Type II OPs have preference for later stages in somatosensory processing. It is clear that the two syndromes do not share morphologic or electrophysiologic characteristics, and are instead, distinctly different in the rat. Mechanistic studies are required to further delineate these two syndromes. To date, TPPi-induced delayed neurotoxicity in the rat has been studied using only the subcutaneous route (Smith et al., 1933; Veronesi et al., 1986b; Veronesi and Dvergsten, 1987). The results of this study show that TPPi-related clinical and ' electrophysiologic neurotoxic effects occur after subacute dermal exposure at a dose (2 x 450 mg/kg body weight) lower than that required for production of clinical and neuropathological effects via the subcutaneous route (2 x 1184 mg/kg body weight) (Veronesi et al. , 1986b; Veronesi and Dvergsten, 1987). Chickens also express TPP,- related neurotoxic effects after dermal exposure at doses lower than in the rat. One of three hens exposed to a single dermal dose of 50 mgTPP/kg body weight displayed treatment-related neuropathological changes while hens exposed to multiple dermal doses of TPP, within a 24 hour period (1 x 100 mg/kg body weight + 2 x 200 mglkg body weight; total dose of 500 org/kg body weight) exhibited clinical signs and widespread neuropathological changes in the spinal cord and peripheral nerves (Borg-Warner Chemicals, 1982a as cited in U. S. Environmental Protection Agency, 1986). 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