MQRF‘HOLQGY 0F Ez‘éléfii'E-‘oMQBTEM EI’EGENERAWON: Wé‘SEE‘EEJLG’EQURuUiAEE E’RACT AE'N'EE‘ EMS HAEEE'EU‘LAR HUGEE! EN THE ECG manna Ea»? {Em Dawes a? M. 5. 3 MECEEEGRR SEME UNEVERSHY Dug-me E. Haimez ages? masts LIBRARY MghEgan State mversity MORPHOLOGY 0F POST-MORTEM DEGENERATION: HABENULOPEDUNCULAR TRACT AND THE HABENUIAR NUCLEI ‘IN THE DOG by Duane E. Haines A THESIS Submitted to Michigan State university in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Anatomy 1967 < I /../'."/4.1 1" . A " ‘ \x‘ / J I (u \4 x/igup",g .’ ’ V x k, ,I ‘\ ACKNOWLEDGEMENTS The author wishes to express his thanks and sincere appreciation to his major Professor, Dr. Thomas W. Jenkins, and to the members of his committee, Dr. D. R. Swindler and Dr. Al W. Stinson for their valued criticism and patient guidance. Thanks is also extended to Dr. M. L. Calhoun, Chairman of Anatomy, for unhampered use of departmental facilities. Special gratitude is due Dr. William Adam for his critical evaluation of this manuscript and photographic advice, to Dr. Clyde F. Cairy, and members of the cardiovascular research unit, Department of Physiology, for their assistance in the collection of specimens. The author is deeply indebted to the following individuals for their considerate contributions to the production of this manuscript: Dr. Arlene R. Seamen for her technical advice and German translation, Dr. Hiroshi Minaguchi for assistance in Japanese translation, Mr. John Seamen, Attorney, Lansing, Michigan, for Italian translation, Mrs. Edith Jones for technical advice, German and French translation, Mrs. Barbara Wheaton for her technical advice, To the personnel of the Interlibrary Loan Service of the Main Library: without their cooperation it would ii have been impossible to obtain many of the articles used in this research, Mrs. Lester L. Haines, my mother, for her patient and laborous typing of this manuscript. No man works alone, therefore I am very grateful to my colleagues whose criticism and advice have undoubtedly added a great deal to this manuscript. iii TABLE OF CONTENTS page Introduction ---------------- , ------- 1 Review Of the Literature ----------------- 3 Material & Methods -------------------- 9 Tables -------------------------- 11 Results and Discussion ------------------ 14 Anatomical Note ------------------- 14 Cell Bodies --------------------- 15 0-hour post-mortem -------------- 15 6-hour post-mortem -------------- l6 12-hour post-mortem -------------- l7 18-hour post-mortem -------------- 18 24-hour post-mortem -------------- 19 36-hour post-mortem -------------- 20 48-hour post-mortem -------------- 21 Anatomical Note ------------------- 23 Fiber Tract --------------------- 23 0-hour post-mortem -------------- 23 6-hour post-mortem -------------- 24 12-hour post-mortem -------------- 25 18-hour post-mortem -------------- 25 24-hour post-mortem -------------- 26 36-hour post-mortem -------------- 27 48-hour post-mortem -------------- 28 iv Summary and Conclusion ------------------ 29 Literature Cited --------------------- 32 Figures """"""""""""""" 35 TABLE II III LIST OF TABLES page Specimen Date, Temperature Ranges and Degeneration Time Intervals --------- 11 Generalized Dehydration Procedure -------- 12 Osmic Acid Staining Procedure ---------- 13 Vi LIST OF FIGURES FIGURES 1. Diagrammatic Synopsis of Post-Mortem Degeneration. 2. Overall Architecture of the Habenular Nucleus. 3. Medial Habenular Nucleus - 0-hour post-mortem. 4. Lateral Habenular Nucleus - 0-hour post-mortem. 5. Lateral Habenular Hucleus showing Nuclear-Cytoplasmic ratio. 6. Cells of Dorsomedial Nucleus of the Thalamus to show Nuclear- Cytoplasmic ratio. 7. Medial Habenular Nucleus - 6-hour post-mortem. 8. Lateral Habenular Nucleus - 6-hour post-mortem. 9. Medial Habenular Nucleus - 12-hour post-mortem. 10. Lateral Habenular Hucleus - 12-hour post-mortem. 11. Medial Habenular Nucleus - 18-hour post-mortem. 12. Medial Habenular Nucleus - 18-hour post-mortem. 13. Lateral Habenular Nucleus - 18-hour post-mortem. 14. Medial Habenular Hucleus - 24-hour post-mortem. 15. Medial Habenular Nucleus - 24-hour post-mortem. 16. Lateral Habenular Nucleus - 24-hour post-mortem. 17. Lateral Habenular Nucleus - 24-hour post-mortem. 18. Medial Habenular Nucleus - 36-hour post—mortem. 19. Medial Habenular Nucleus - 36-hour post-mortem. 20. Lateral Habenular Nucleus - 36-hour post-mortem. 21. Medial Habenular Nucleus - 48-hour post-mortem. 22. Lateral Habenular Nucleus - 48-hour post-mortem. vii 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35. 36. Habenulopeduncular Habenulopeduncular Habenulopeduncular Habenulopeduncular Habenulopeduncular Habenulopeduncular Habenulopeduncular Habenulopeduncular Habenulopeduncular Habenulopeduncular Habenulopeduncular Habenulopeduncular Habenulopeduncular Habenulopeduncular Tract Tract Tract Tract Tract Tract Tract Tract Tract Tract Tract Tract Tract Tract 0-hour 0-hour 6-hour 6-hour 12-hour 12-hour 18-hour 18-hour 24-hour - 24-hour - 36-hour - 36-hour 48-hour 48-hour viii post-mortem of the Myelin. post-mortem of the Axis Cylinder. post-mortem of the Myelin. post-mortem of the Axis Cylinder. post-mortem post-mortem post-mortem post-mortem post-mortem post-mortem post-mortem post-mortem post-mortem post-mortem of of of of of of of of of of the the the the the the the the the the Myelin. Axis Cylinder. Myelin. Axis Cylinder. Myelin. Axis Cylinder. Myelin. Axis Cylinder. Myelin. Axis Cylinder. INTRODUCTION Since the time of Galen (Riese, 1959), who first observed that total transection of the spinal cord would deprive all regions below the transection of movement and sensation, periodic studies of the degeneration phenomena of nerves have been carefully conducted. After the first scientific exploits of the Greeks and Romans, almost every type of anatomical investigation fell under the shadows of superstition for over 1200 years. When the Fabrica by Andreas Vesalius appeared in the 16th Century (Riese, 1959), the stage was set for gross advancements in the science of Anatomy. Degeneration studies of the nervous system were limited to descriptions of lesions or descriptions of transections until the classical works of Santiago Ramon y Cajal (Riese, 1959, Ramon y Cajal, 1928). His detailed descriptions of degeneration and regeneration in the nervous system were amazingly accurate, and many of his observations are still authoritative. However, studies on the structural changes of cell bodies, fibers, and tracts of the nervous system under post-mortem conditions are rarely found in the English literature. Limited work in this area has been reported by Italian, Japanese, German and French investigators during the early decades of this century. Neppi (1897) described the post-mortem change of the Nissl substance. Barbacci and Campacci (1897) also conducted studies on the Nissl substance. Post-mortem studies of the entire nerve cell were conducted by Levi in 1898 (Cammermeyer, 1962). Since the turn of the century only limited work has been done on the structural changes that occur under poSt-mortem conditions. Morphologic studies were conducted on both the pheripheral and central nervous systems, with supplementary information obtained by histochemical studies on nerve tissue and cerebrospinal fluid. It is the purpose of this study to note the structural changes occurring in myelin, axis cylinder, Nissl substance, nucleus and its membrane, and general cell structures at specific post-mortem time intervals. The neuropathologist must contend with post-mortem auto- lytic changes; however, very little information exists as to the nature of these changes. The desirability of fixing nerve tissue immediately after death is often questioned. Does nerve tissue undergo definite structural change if not immediately fixed, and if so, what is the nature of these changes? When studying histologic sections of nerve tissue what can be interpreted as post-mortem arti- fact versus any type of pathology? These and related questions are at this time unanswered. It is the desire of the author to briefly summarize the work done in post-mortem degeneration as well as to contribute to the existing knowledge of the post-mortem changes of the central nervous system. REVIEW OF LITERATURE A careful search of the literature revealed only sparse, and many times inconclusive reports of studies on the post-mortem alterations of the peripheral and central nervous systems.. These reports, in a variety of languages are diffusely spread over the past 70 years. Tirelli in 1896 (Ugo, 1927), using methylene blue and the Golgi method, noted an early dissolution of the peripheral chromatic substance in the cell body, and a clouding over of the achromatic residue. At later stages he noted gross protoplasmic changes. Neppi (1897), using immersion fixed material, studied the ventral horn cells in dogs. He reported no change at 24 hours, but noted a progressive agglutination and fragmentation in the Nissl substance and the appearance of cyto- plasmic vacuoles from 48 to 96 hours post-mortem. The nucleolus remained intact even after 96 hours. The results of Neppi (1897) were directly contradicted by Barbacci and Campacci (1897). Utilizing the central nervous system of rabbits they observed a fragmentation and pallidation of the Nissl substance, and the appearance of cytoplasmic vacuoles as early as 3 hours post-mortem. An early swelling and atrophy of the nucleus was noted, and the nucleolus appeared normal at 72 hours post- mortem. Levi (1898), using the Nissl method, noted post-mortem changes beginning to appear from 18 - 24 hours after death. He reported the early changes to consist of protoplasmic augmentation and peripheral chromatolysis of the cell body. Alessi in 1899 observed that general post-mortem changes in nerve cells occur sooner in animals that are high on the zoological scale (e.g. dog and cat), and later in those low members, (e.g. frog) (Ugo, 1927). 3 Faworsky (1900) studied the post-mortem changes in spinal cord cells of various animals. He noted a Nissl substance pallidation and border obliteration, and a tendency for the Nissl fragments to agglut- inate. Alterations occurred later in posterior horn cells than in anterior horn cells. He was one of the first to contest the difference between post-mortem and pathologic chromatolysis of nerve cells. Ugo (1927), using the Bielschowsky method, conducted a detailed study of the post-mortem alterations of neurofibrils in human and rabbit brains. No change was noted until 24 hours, and by 30 - 36 hours the intracellular fibrils appeared swollen and segmented. Dendritic and axonal fibrils became irregular and in some areas mimicked fragmented cords. By 48 hours the aforementioned changes had advanced, the nucleus appeared hypertrophied, and a perinuclear halo was observed, resulting from the disappearance of neurofibrils in this area. From this work the following points should be noted: 1.) neurofibrils are highly resistant to post-mortem change, 2.) alterations occur slightly faster in small cells than in large cells, 3.) the appearance of a perinuclear halo that enlarges toward the peripheral area of the cell body as post- mortem time lengthens, 4.) intracellular fibrils are more resistant to alteration than dendritic or axonal fibrils. Hamada (1927), using a modification of the Cajal method, studied post-mortem changes in nerve endings of the rat tongue. The nerve endings became swollen after 30 minutes, underwent rapid fragmentation, and appeared as fine diffuse granules by 7 - 8 hours post-mortem. These fine granules were interpreted as indicative of complete destruction of the nerve ending. These changes were initiated more slowly under conditions of low temperature, but complete dissolution of the endings still occurred by 8 hours post-mortem. Sannomiya (1927) reported the post-mortem alterations in Schmidt- Lantermann incisures of peripheral nerves fixed in formalin or sprayed igflziyg with cocain or chlorethyl solutions. In untreated nerves in- cision width is constant for a considerable time, however, there is a tendency for the incisures to become narrow and irregular with undulating borders as post-mortem time increases. Incisures of formalin fixed nerves were of constant structure for 4 hours, but after this time they became irregular and completely disappeared by 23_- 30 hours. In nerves treated EEHXEXE with cocain and chlorethyl solutions, incisures were not seen or only slightly visible. Maintenance of post-mortem nerve samples in low temperature also produced narrow, faintly visible incisures. In studies of the post-mortem change of myelin in brains and spinal cords, Kreiner (1939-1940) noted a progressive change from.an irregular tube-like structure of the myelin immediately after death to an irregular pattern of spindle-like swellings and constrictions by 36 hours to 3 days post-mortem. In advanced stages (4 days) the myelin became arranged into rosary-like rows of balls held together by thin thread- like structures that were possibly myelin extensions. The threads eventually broke leaving rows of loose globules of different sizes (6 days). An attempt was made by Camerer (1943) to correlate certain post- mortem alterations of nerve cells with the mode of organism death, be it sudden or slow, natural or pathologic. He observed only vague correlations and his reports are self-contradictory at points. This work was discussed and its fallacies pointed out by Ferrero (1947). Koenig and Koenig (1952), using the central nervous system of adult guinea pigs and a perfusion method, noted slight fragmentation of the Nissl substance at 30 minutes. This fragmentation was progressive and the substance was almost completely dispersed by 23-1/2 hours post-mortem. Cytoplasmic vacuoles were present at 3 hours in some cells with a steady increase until vacuoles were noted in most cells by 10 hours post-mortem. The large cells of the nucleus ruber and the large ventral horn cells demonstrated the most rapid progression of change Maros and Lazar (1959) studied the post-mortem change of nerves after maintenance in springwater, physiological salt solution, blood serum, and cerebrospinal fluid. The myelin sheath and axis cylinder of nerves kept in blood serum retained a relatively normal configuration even after extended periods of time, but nerves kept in other media under- went relatively rapid characteristic post-mortem alterations. The initial change was a shriveling and distortion of the axis cylinder due to dehy- dration, and by 24 hours general alterations were well advanced. Shingaki and Nakayama (1960) reported on the post-mortem alterations of the Golgi apparatus and Nissl corpuscles in pyramidal cells, Purkinje cells, and spinal ganglion cells. At 8 hours the Golgi apparatus was swollen with a tendency to granulate, and the Nissl corpuscles appeared agglutinated (literal translation - melted together). The Nissl sub- stance continued to fragment (literal translation - melt) until only a small amount was observed in small spinal ganglion cells at 24 hours. The Golgi apparatus continued to split and granulate until only dust- like granules were present at 30 - 40 hours. Cerebellar Purkinje cells were most susceptible to change and small spinal ganglion cells were most resistant because cell boundries and a granular Golgi apparatus could be observed at 32 hours in these cells. Smythies and Inman (1960) noted that post-mortem degeneration is essential for a good demonstration of boutons termineaux in the cerebral cortex of the dog. At 1 hour post-mortem few boutons were observed around Betz cells, however, at 24 - 48 hours boutons were plainly visible as regular masses or globular-like structures closely associated with the cell body. It was concluded in this study that the post-mortem chemical change in the boutons are essential for their effective retention of the stain. Cammermeyer (1960, 1961, 1962) has extensively studied the signi- ficance of post-mortem trauma on nerve tissue. Trauma in the form of manipulation of the tissue, pull of an artery when removing the specimen, or instrument compression will produce "dark" neurons in the immediate area. A "dark" neuron is defined as a neuron having closely packed Nissl substance lending a uniform dark color to the cell body and nucleus. Although this thesis does not deal with histochemical observations, these works will be briefly reviewed. Mayer (1928) reported a rapid increase in brain tissue lactic acid in the grey matter, and a less rapid increase in the white matter. He also noted that experimental anemia would bring about a rapid post-mortem increase in lactic acid. Those areas of the brain most often affected by cerebrovascular accidents (grey matter) also exhibited the greatest degree of chemical change. He felt that there could be a correlation drawn between these occurrences. Friede (1958) reported extremely inconsistent results in guinea pig brains when an attempt was made to correlate phosphorylase activity with postmortal neuron change. Robins fig 51. (1958) studied 11 enzymes in post-mortem brains to ascertain the effects of fever and uremia. They noted no appreciable change in enzyme stability after 6 hours post-mortem. In the histochemical observations of Lazarus 25 El: (1962) marked cytochemical changes were noted at 24 hours in acid phosphatase and adenosine triphosphatase, but no change was observed in glucose ~6- phosphate dehydrogenase and lactic acid. Cell granules underwent swelling, clumping and a slow disappearance. Sumegi and Findeisen (1931) in studies of post-mortem cerebrospinal fluid noted an elevation of albumin, decrease in carbohydrate, lowering of the albumin-globulin quotient, and a pH shift to acidity shortly after death. The cerebrospinal fluid studies by Dito (1964) showed a progressive increase in glutamic-oxaloacetic tranaminase activity up to 70 hours. He correlated this activity with the autolysis of central nervous system tissue. MATERIALS AND METHODS The habenulopeduncular tract and habenular nuclei of 18 adult dogs were used in this study. Since this is a bilateral tract, a total of 36 specimens was collected. The animals used consisted of 10 males and 8 females, and included various breeds and sizes of dogs (Table I). The dogs were obtained from the Department of Physiology, Michigan State University, in cooperation with Dr. Clyde Cairy who collected the pituitary from each specimen, and with the assistance of members of the cardiovascular research unit. The heads and brains in £2£g_were kept at room temperature for varying lengths of time (Table I). The post-mortem degeneration temperature range of all specimens was 24.300. - 26.0°C., with an average temperature of 24.940C. At 0, 6, 12, 18, 24, 36, and 48 hour intervals after death, the brains were removed from the heads, and the habenulopeduncular tract and nuclei were removed in a piece of tissue approximately 15mm X 15mm X 8mm. These pieces were immediately fixed by immersion in 10% formalin buffered with sodium.acetate (Guyer 1941). All specimens were kept in 10% buffered formalin until dehydration. Prior to dehydration each specimen block was trimmed so that the tract and nuclei were parallel to the intended cutting surface of the block. All blocks (except those stained with osmic acid) were rinsed in running tap water and then dehydrated through a graded series of ethyl alcohols to n-butyl alcohol, infiltrated with 3 changes of paraffin and 10 embedded in fresh paraffin (Table II). Paraplast1 Omelting point 56-5700.) was used as the infiltration and embedding medium. The tissues in this study were stained for myelin, axis cylinders, and Nissl substance. Osmium tetroxide2 in a 2% solution was used as the myelin stain (Baker 1958, Baker 1960). Preparatory to osmic staining, the tissues were rinsed for twenty-four hours in running tap water, then stained for forty-eight hours in the fumes of a 2% solution of osmium tetroxide (Guyer 1941). (Prior to staining, the solution was prepared and allowed to equalize for twenty-four hours.) The tissue blocks were rinsed in running H20 for six hours after the osmic procedure (Table III), then dehydrated and embedded using a standard procedure (Table II). Axis cylinders were stained by the two hour Protargol method of Davenport, McArthur, and Bruesch (Conn, Darrow, Emmel, 1962). ' The standard luxol fast blue-cresyl echt violet staining procedure was employed to demonstrate fibers and Nissl substance (Gridley and Ambrogi, 19573). Sections in this study were cut at 6n, 8u, lOu, lZu, and 14q on a Spencer "820" rotatory microtome.4 1. Fisher Scientific Company, Chicago, Ill. 2. Fisher Scientific Company, New York, N. Y. 3. Manual of Histological and Special Staining Technics, 1957. Armed Forces Institute of Pathology, washington, D. C. 4. American Optical Company, St. Louis, Mo. 11 TABLE I. SPECIMEN DATA, TEMPERATURE RANGES AND DEGENERATION TIME INTERVALS. Animal Breed Sex Approx. Degeneration Degeneration No. (not Wt. Temperature Time .__ pure bred), 4gij== Range*# Interval CF-l Beagle Female 9 Kg. 24.8% 12 hr. CF-2 Collie Male 12 Kg. 25.0°C 6 hr. CF-3 Beagle Female 10 Kg. 24.500 24 hr. CF-4 Boxer Female 22 Kg. 24.9°c 24 hr. CF-S Unknown Male 12 Kg. 24.8°C 48 hr. CF-6 Unknown Male 12 Kg. 24.9°c 48 hr. CF-7 Cocker Male 10 Kg. 24.5°C 18 hr. CF-8 3:33:18“ Female 9 Kg. 26.0°C 6 hr. CF-9 Unknown Female 16.5 Kg. 26.0% 6 hr. CF-lO Beagle Male 11 Kg. 24.3% 12 hr. CF-ll Unknown Male 15 Kg. 24.3°C 12 hr. CF-12 Terrier Female 10 Kg. 25.0°C 36 hr. CF-13 Labrador Female 15 Kg. 24.9°c 36 hrs. (IF-14 Cocker Male 10 Kg. 25.3°C 18 hr. CF-15 Labrador Male 15 Kg. 25.0°C 6 hr. CF-16 Labrador Male 12 Kg. 24.8% 12 hr. CF-l7 Unknown Female 13 Kg. none 0 hr. CF-18 unknown Male 15 Kg. none 0 hr. *Average degeneration temperature - 24.94°C. #Each temperature fluctuated approximately tloc. 12 TABLE II. GENERALIZED DEHYDRATION PROCEDURE. Solution Hours/Solution Rinse in running H20 24 hrs. 60% Ethyl Alcohol I 3 hrs. 60% Ethyl Alcohol II 3 hrs. 70% Ethyl Alcohol I 3 hrs. 70% Ethyl Alcohol II 3 hrs. 80% Ethyl Alcohol I 3 hrs. 80% Ethyl Alcohol II 3 hrs. 95% Ethyl Alcohol I 3 hrs. 95% Ethyl Alcohol II 3 hrs. 100% Ethyl Alcohol I 3 hrs. 100% Ethyl Alcohol II 3 hrs. N-Butyl Alcohol I 3 hrs. N-Butyl Alcohol II 3 hrs. Paraffin I (paraplast) 4 hrs. Paraffin II " 6-8 hrs. Paraffin III " 4-6 hrs. Vacuum (paraplast) 1/2 hr. Embed (paraplast) 13 TABLE III. OSMIC ACID STAINING PROCEDURE. Procedure Steps Hours/Step Solution prepared and allowed to equalize 24 hrs. Nerve sample suspended over a 2% solution 48 hrs. Rinse in Running H20 6 hrs. Standard Dehydration Procedure* *At this point tissues were placed in 60% Alcohol I and carried through the standard dehydration procedure as noted in Table II. RESULTS AND DISCUSSION Anatomical Note. The habenular nucleus in the dog is composed of medial and lateral groups of cell bodies. The medial group consists of a dense population of closely packed cells with intensely staining granulations. The lateral group consists of slightly larger cells with lightly staining granulations. The lateral group also has a considerable less dense population of cell bodies. This observation on general architecture closely corresponds with previous reports on the structure of the medial and lateral habenular nuclei (Crosby, Humphrey and Lauer, 1962). Using this criterion, all further references to the habenular nucleus will be to either the medial or lateral habenular nucleus (figure 2). As the habenulopeduncular tract (and other contributing tracts) appear in the medial and lateral nuclei, they are separated into three main bundles, a large lateral bundle and smaller intermediate and medial bundles (figure 2). The lateral bundle passes laterally and dorsally around the lateral habenular nucleus, with its fibers extending as far as the stria medullaris thalami (figure 2). The intermediate bundle passes between the medial and lateral habenular nuclei and sends branches to both of these areas. The medial bundle passes between the medial habenular nucleus and the ependymal lining of the third ventricle. Fibers of the intermediate and medial bundles also reach as far dorsally as the stria medullaris thalami (figure 2). 14 CELL BODIES 0-hour post-mortem. The medial habenular nucleus consists of densely packed cells with intensely staining granulations. The nucleus of the neuron is round and the demarcation between nucleus and surrounding cytoplasm is distinct but delicate. There is a tendency for the nucleus to retain some of the stain even when nuclei of the surrounding pulvinar cells are clear. The nucleolus can be distinctly defined in most cells. and is sharply delimited from adjacent karyoplasm. The cell membrane is quite distinct in most cells, and Nissl bodies can be demonstrated in larger cells (figure 3). The lateral habenular nucleus has a sparse population of cells with light staining granulations. The nucleus of the neuron retains a slight amount of the stain, and the nucleolus can be sharply defined from surrounding karyoplasm. The nuclear membrane is distinct but delicate, and gives the nucleus a uniform round appearance. The cell borders of most cells can be made out with relative accuracy. Nissl bodies can be demonstrated in the larger cells (figure 4). The prominence of glial cell nuclei indicate that this cell type is evenly dispersed throughout both the medial and lateral habenular nuclei. Even though the cell bodies of the medial and lateral nuclei are of the multipolar type, the amount of cytoplasm in relationship to the size of the nucleus is greatly reduced when compared to the surrounding thalamic (dorsomedial nucleus) cells (figures 5 and 6). The habenular 15 16 multipolar neurons have a medium sized nucleus surrounded by a thin layer of cytoplasm (figure 4). Therefore, the nuclei of the cells are the most prominent structures seen when scanning the field. This characteristic nuclear cytoplasmic ratio is normal in the medial and lateral nuclei even though medial cells have a tendency to stain more intensely. It is difficult to analyze discrete structural changes in cell bodies when the cytoplasm occupies such a limited portion in the cell. The individuality of neuropil fibers in normal sections is indis- tinct, and the neuropil is closely applied to the cell bodies. It can be noted that in normal tissue sections there are no spaces between in- dividual elements of the neuropil or between neuropil and cell bodies. 6-hour post-mortem. Some of the cell bodies of the medial habenular nucleus appear slightly swollen. The cell membrane can be determined in most cells, however, there are distinct spaces around most cell bodies and among fibers of the neuropil. The spaces in the neuropil accentuate each fiber as an individual unit. The neuropil fibers appear more prominent not only because of the interfiber spaces, but because they appear to stain more intensely than normal neuropil. The nuclear membrane is distinct and delicate, but is assuming an irregular pattern in some cells, giving the nuclei of these cells a bizarre shape (figure 7). The Nissl substance is clumped in dark staining areas toward the periphery of the larger cells, but cannot be clearly defined in smaller cells. These observations on Nissl substance correspond to the reports of Shingaki and Nakayama (1960). The nucleolus is clear in most cells and sharply delimited from the karyoplasm. Some cells are beginning 17 to show spaces in the cytoplasm that may be interpreted as forming vacuoles according to previous observers (Barbacci and Campacci 1897, Koenig and Koenig 1952). The changes in the cell bodies of the lateral nucleus are essentially the same as those changes observed in cell bodies of the medial nucleus. Some of the lateral nucleus cell bodies are beginning to have irregularities in the cell membrane, and the appearance of cytoplasmic vacuoles in this area (figures 7 and 8). Immediate post-mortem changes are the appearance of spaces in the tissue sections and vacuoles in the cytoplasm, and irregularities of the cell membrane in some cells. Irregularities in the appearance of the nucleus are caused by structural changes in the nuclear membrane (figures 7 and 8). 12-hour post-mortem. Cell bodies of the medial nucleus show only slight changes from the 6 hour sample. More cells have vacuoles and the previously existing vacuoles have become enlarged. The cytoplasm has become slightly granular and the Nissl substance can be seen only in larger cells as dark clumps in the peripheral area of the cell body. In cells with such limited amounts of cytoplasm, Nissl substance can be observed only in the largest cells. The nuclear membrane is eccentric, giving the entire nucleus a bizarre shape. The irregularities of the cell membrane appear as vacuoles in or near the periphery of the cell, or as invaginations of the cell membrane (figure 9). Progressive changes in the cell bodies of the lateral nucleus mimic those changes in the medial nucleus. These changes are noted on figure 10. 18 Spaces around the cell bodies (pericellular) and in the neuropil (interfiber) are becoming more evident. The nucleolus is distinct in most cells and remains well defined from the karyoplasm (figure 10). Pericellular and interfiber spaces, Nissl substance agglutination, and presence of cytoplasmic vacuoles are noted. These changes as well as cell and nuclear membrane irregularities are artifacts interpreted as post-mortem structural change of the cell body. l8-hour post-mortem. The nuclear membrane of cells in the medial nucleus is irregular and appears broken in a few cells, although this is not the rule. The nuclear membrane in a few cells is beginning to show alternating light and dark areas (figure 11). Most cells have a limited number of dark granules present in the nucleus. The cell border is indistinct in most cells, and the pericellular spaces are prominent (figure 11). Vacuoles are present in the cytoplasm of most cells and light spaces are appearing in the perinuclear area (figure 12). The nucleolus in some cells is staining lighter and it is sometimes difficult to sharply distinguish it from surrounding karyoplasm. In some nuclei the nucleolus appears to be sending out processes or fragments. According to earlier reports (Koenig and Koenig, 1952) this indicates the onset of nucleolar fragmentation (figure 12). Changes of the cell bodies in the lateral nucleus follow a parallel course with medial nucleus cell bodies. The general appearance of these changes can be noted on figure 13. The light granular appearance of the periphery of the cell body is probably the fragmented Nissl bodies. It has been reported previously 19 (Koenig and Koenig 1952, Shingaki and Nakayama 1960) that this fragmentation takes place from 18-23 hours post-mortem and that the fragmented Nissl substance is located in the periphery of the cell. However, the fragmented Nissl bodies can only be located in large cells, because of the limited amount of cytoplasmic area in smaller cells. 24-hour post-mortem. The cell membrane of cells in the medial nucleus is indistinct for the most part. Vacuoles that are present in the cytoplasm and in the area of the cell membrane are becoming especially prominent in the perinuclear area. The nuclear membrane stains intensely, and shows alternating light and dark areas in many cells (figure 14). A few cells have nuclei with membranes that appear granular, or seem to be made up of rows of granules giving the membrane the appearance of a string of beads (figure 15). ‘Most nuclei contain some coarse granules, and the nucleolus has not changed significantly from the 18 hour stage. In the lateral nucleus the cell bodies appear much the same as those of the medial nucleus. Changes in the nuclear membrane are similar to the changes observed in the medial nucleus. In some of the large cells the nuclear membrane has broken into segments separated by areas where no membrane can be distinguished. The obscurity of the cell membrane in many cells is shown on figure 16. In a few cells the cell membrane is almost completely gone (figure 17). Nissl substance can be seen only in the large cells, and it appears as fine granules in the periphery of the cell. Previous authors reported that the Nissl bodies have undergone almost complete autolysis 20 by this time (Koenig and Koenig 1952, Shingaki and Nakayama 1960). The neuropil of both the medial and lateral nuclei stains rather intensely and shows a highly irregular configuration. The intense staining property and fiber irregularity are progressive post-mortem changes. 36-hour post-mortem. Distinct structural changes have occurred between 24 and 36 hours post-mortem. Most cell nuclei of the medial nucleus appear shrunken (pyknotic) and have a finely granular complexion. They seem shrunken because of the relatively large amount of cytoplasm that can be determined around the nucleus when compared to earlier cells. A distinct nuclear membrane can be delineated in only a very few cells. In most cells, the nuclear membrane is completely gone, or only irregular sections remain (figure 18). In large cells not only are the nuclei shrunken and pyknotic, but the cell bodies are slightly swollen and the cytoplasm contains extremely coarse granulations. Cell membranes are highly irregular and in some cells appear almost ruptured (figure 19). Vacuoles are present in all cells, and most large cells have a distinct light area around the nucleus, the perinuclear halo. This has been described by Ugo (1927) as indicative of advanced post- mortem change of nerve cell bodies. It has been noted previously that this halo appears at 18 - 24 hours post-mortem in large cells and becomes progressively more distinct by 36 hours post-mortem (figure 19). The nucleolus has lost some of its staining ability and is undergoing slow but definite fragmentation (figure 19). Spaces in 21 the neuropil and around the cell bodies are prominent, and the neuropil has also lost some of its staining ability. Structural changes in the cell bodies of the lateral nucleus are the same as those in the medial. A higher percentage of cells appears to be without a nuclear membrane. This is believed to stem from the fact that since there is normally a sparce population of cell bodies in the lateral nucleus, it is only an illusion that a higher percentage of cells do not have a nuclear membrane (figure 20). 48-hour pOSt-mortem. Few cells of the medial nucleus have nuclei that even slightly resemble the normal structure. Most cells Bhow no nucleus at all, or only very irregular segments that are apparently remnants of the nuclear membrane, enclosing a slightly lighter area of the cell body. The cytoplasm and karyoplasm are filled with coarse granules and are of equal complexion in most cells. 'Nuclei that are still relatively intact appear shrunken and pyknotic similar to 36 hour cells. The cell border can be seen in some cells, but is highly irregular in others (figure 21). Vacuoles are noted in all cells, and a perinuclear halo is evident in most of the larger cells. The nucleolus is difficult to distinguish in some cells, and is undergoing progressive fragmentation in all cells. The neuropil is fragmented and stains lightly. In the lateral nucleus the large cells are swollen and any intact nuclei are markedly shrunken and pyknotic. Almost all cells display no nuclear membrane or only fragments. In some cells the area of the nucleus is light, but no membrane can be demonstrated (figure 22). Cytoplasmic vacuoles are present in all cells, and a perinuclear halo is noted in cells which still contain remnants of the nucleus. Cell 22 borders are distinct in some cells, but highly irregular in most (figure 22). Nuclei of glial cells are not reduced considerably in number, but have lost some of their staining ability. The nucleolus is faint and undergoing fragmentation. Nissl bodies cannot be defined in either 36 or 48 hour post- mortem cell bodies. 23 Anatomical Note. The habenulopeduncular tract pursues a medially concave course from the habenular nucleus of the epithalamus to the peduncular nucleus of the mesencephalon. It is a grossly dissectible tract, but is easily destroyed if care is not taken. Random measure- ment of the diameter of the tracts show a great deal of variation. The smallest diameter is 440 microns (u) and the largest diameter is 710m, Average diameter of individual tracts ranges from 500u,to 612m. It is impossible to state the exact diameter of the tract, but it is the opinion of the author that a reasonable range is from 650q_to 700m, The smaller measurements result from off-center cuts through the tract. A micrometer was used to determine the relative sizes of the tracts. The habenulopeduncular tract is composed of fibers with varying diameters. In histological section the large fibers appear to be located mainly in the peripheral area of the tract, and the more delicate fibers located mainly in the central area of the tract. Almost all fibers, regardless of axon diameter, are lightly myelinated. A very few heavily myelinated fibers are noted in the periphery of the tract. The configuration of the tract as it appears in the habenular nucleus has been previously noted (page 14). FIBER TRACT 0-hour post-mortem. Most fibers are lightly myelinated with a few heavily myelinated fibers in the peripheral area of the tract. Individual fibers are defined as two black myelin layers surrounding the light unstained area of the axis cylinder. The myelin surrounding 24 the axis cylinder area has only a few slight irregularities and generally shows a smooth contour (figure 23). It is impossible to discern individual fibers in the central area of the tract, although large individual fibers in the periphery of the tract can be demonstrated (figure 23). The axis cylinder is slightly irregular in most areas, but maintains a relatively constant diameter. A space around the axon (periaxonal space) indicates the position of the myelin since it does not impregnate with silver preparations. The small axons in the central area of the tract are extremely difficult if not impossible to distinguish, and only large axons in the periphery of the tract can be demonstrated (figure 24). In all areas it is difficult to clearly define individual axons, and glial cell nuclei are indistinct. 6-hour post-mortem. Individual myelinated fibers are easier to define, because of the appearance of interfiber spaces in the tract and in the adjacent tissue. There is little or no change in the myelin sheath, although some fibers show discrete light areas within the myelin. The general contour of the myelin tube is unaltered at this stage (figure 25). The appearance of spaces in the tract and in the surrounding tissue help to accentuate individual axons. Axons centrally located in the tract can be defined at this stage. The axons have lost some of their irregular or wavy pattern, but still maintain a constant diameter. A few axis cylinders have light areas within the axolemma (figure 26). Glial cell nuclei are prominent, and in many areas 25 form distinct rows. Due to previous reports on glial cells in the central nervous system (Crosby, Humphrey and Lauer, 1962) it is reasonable to assume these nuclei belong to oligodendrocytes (figure 26). 12-hour post-mortem. The structure of the myelin tube around the axon has not changed significantly from the 6 hour sample. The light central area of the myelin tube that represents the axon is shaded in some fibers. A few fibers have occasional slight dilations of the axon area. Light irregular areas can be noted in the myelin sheath (figure 27). Interfiber spaces are evident and help to accentuate the individual fibers of the tract. No progressive change in the axis cylinder can be noted. A few axons of large diameter have areas of slight dilation and constriction accompanied by occasional light areas within the axolemma (figure 28). The increasing amount of tissue spaces as well as the increased staining ability of the axon help to accentuate each axon as an individual unit. Oligodendrocyte nuclei are prominent and often found in rows parallel to the axis cylinders (figure 28). 18-hour post-mortem. Little progressive change is noted in the myelin tube structure from the 12 hour sample. A few fibers show a slight swelling of the myelin in some areas, and the axis cylinder area is shaded over in some fibers (figure 29). Only slight changes are noted in the large diameter axons. There is a tendency for them to become irregular and have alternating dark and light areas. Segments of dilation and constriction are also evident 26 in some axons. Oligodendrocyte nuclei are prominent, and even the small diameter axons in the central area of the tract can be defined as individual units (figure 30). 24-hour post-mortem. The myelin tube is slightly swollen and irregular, and light areas can be seen in the substance of the myelin. The axis cylinder core of the myelin tube is shaded over in most fibers, and showing segments of dilations and constrictions. It is becoming progressively difficult to distinguish individual fiber units of the tract, and in a few fibers the myelin tube appears jagged and irregular (figure 31). The overall architecture of the tract has not changed significantly since 18 hours post-mortem. Only random fibers show distinct change. It is interesting to note that cell bodies of the surrounding thalamus show coarse granules at 24 hours post-mortem. These granules are assumed to be lipoid in nature because they are clearly seen in osmic stained sections. No progressive changes can be noted in the axis cylinder that differ from those discussed for 18 hour samples. Oligodendrocyte nuclei are clear and each individual axon is extremely prominent. The occasional dilations and constrictions previously observed are present, and the alternating light and dark areas are also noted (figure 32). Previous reports (Ugo, 1927) of the relatively slow post-mortem alterations of the axis cylinder and neurofibrils are verified by this study. It is observed that as post-mortem time increases it becomes progressively easier to demonstrate glial cell nuclei and axis cylinders 27 when using a silver method. Based on the work of Smythies and Inman (1960), it is evident that neural tissue stained by a silver method will show more discrete structural components if it is allowed to undergo post-mortem decomposition prior to staining. The distinctive- ness of the glial cell nuclei and individual axis cylinders stem not only from the spacing in the tissue, but also from the increased ability to retain a silver stain. If it is the investigators purpose to demonstrate only axis cylinders and neurofibrils, the tissue should be allowed to decompose for 18 - 24 hours prior to fixation and staining for best results. This is justifiable from two aspects. First, the axon undergoes slow post-mortem alteration, and shows almost no post-mortem artifact at these times. Secondly, silver impregnation methods produce better results if applied to neural tissue that has undergone a certain amount of post-mortem decomposition. 36-hour post-mortem. Gross structural changes in the myelin tube are still not observed. Previously observed alternating light and dark areas are noted, and the axis cylinder area is slightly shaded over in most fibers. The shaded axonal area also shows areas of dilation and constriction (figure 33). It is noted that the myelin sheath undergoes slow post-mortem alteration. Kreiner (1939-1940) has reported that only an irregular pattern of swellings and constrictions can be observed from 36 hours to 3 days post-mortem. A strikingly characteristic structural change is noted in the axis cylinder at this stage. Almost every axon, regardless of diameter size, demonstrates short discrete loops (figure 34). Discrete loops 28 and irregularities of the smaller axons are not as distinct as large axons, however, all axons show a looping configuration. Most axons also have alternating light and dark areas as well as areas of dilation and constriction of the axon (figure 34). 48-hour post-mortem. The myelin tube is swollen almost to the point of completely obliterating the axonal area of some fibers. Even though the myelin tube is considerably swollen the general config- uration is relatively unaltered, supporting the previous report of Kreiner (1939-1940). The previously noted light areas in the myelin are still recognizable. In a few fibers, dilated areas can be noted in the boundaries of the axis cylinder (figure 35). A few axons have lost the loops so characteristic of 36 hour post-mortem samples, but the loops are still present in most fibers to some degree. The alternating light and dark areas are present, and occasional dilations can be noted in some axons. The internal area of the larger axis cylinders appears finely granular. Based on previous work (Ugo, 1927) these fine granules can be interpreted as the product of neurofibril fragmentation (figure 36). The integrity of the axolemma remains unaltered in all stages studied. The entire gamut of post-mortem structural alterations of nerve cell bodies and fibers observed in this research is diagrammatically summarized on figure 1. SUMMARY AND CONCLUSION In conclusion, the progressive changes in the cell bodies of the medial and lateral habenular nucleus can be noted (figure 1). The smooth overall complexion of the sections are interrupted at 6 hours post-mortem by the appearance of interfiber and pericellular spaces. From 6 - 24 hours three main areas of change can be noted. Firstly, there is an agglutination, fragmentation and obliteration of the Nissl bodies, secondly a progressive increase in the irregularity of the nuclear membrane and cell border, and thirdly a progressive increase in vacuoles and in the perinuclear halo. From 24 - 36 hours the cytoplasm becomes extremely granular with the granular appearance extending into the nucleus. At 36 hours many cells appear swollen and demonstrate an indistinct nucleus, or no nucleus at all. This nuclear disappearance stems from a progressive breakdown of the nuclear membrane. The nucleolus begins fragmentation at about 24 hours and progresses to 48 hours. From 36 - 48 hours, the cytoplasm becomes increasingly granular, and by 48 hours very few cells demonstrate a nucleus with most cells having a uniform color in both cytoplasm and in the area of the nucleus. Any nuclei remaining from 36 - 48 hours post-mortem appear pyknotic, shrunken and have a finely granular complexion. Initial changes in the fiber tract consist of the appearance of interfiber spaces in the sections. These spaces help to accentuate each fiber as an individual unit. The myelin and axis cylinder show limited structural change over the time span of this study. The myelin 29 30 tube becomes slightly swollen and shows light and dark areas, however, it retains a relatively normal configuration even up to 48 hours post- mortem. Axonal changes are also relatively minor. After 6 hours there is a tendency for the oligodendrocyte nuclei and axons to stain with a progressive degree of clarity and intensity. At 36 hours all axons show a variable degree of irregularity and looping, and the axoplasm of the axon has become finely granular by 48 hours post- mortem. The axolemma, as well as the borders of the myelin tube, retain their integrity throughout the time span of this study. The slow post-mortem alteration of fibers reported in this research have been previously observed by Ugo (1927) and Kreiner (1939-1940). Post-mortem alterations of cell bodies can be recognized as discrete structural changes, however, changes in the fiber tract up to 48 hours are very limited. Most nuclei have lost their integrity from 36 - 48 hours post-mortem. This change is accompanied by cell border nucleolar and cyloplasmic change. It would be advisable in future investigations to utilize areas that have larger multipolar neurons, thus providing a more favorable nuclear-cytoplasm ratio. In larger cells with more cytoplasmic area, more exacting observations could be made. The observations of Smythies and Inman (1960) for effective demonstration of fine neural detail using a silver method, are also noted in this project. As post-mortem time progresses the axis cylinder stains with increasing clarity. From 18 - 36 hours post- mortem individual axis cylinders can be easily demonstrated even in 31 the central areas of the tract where the more delicate axons are located. In comparison with previous reports (Smythies and Inman, 1960) it is the opinion of the author that fine structural details can be more efficiently demonstrated with silver methods only after the tissue has undergone post-mortem decomposition for a certain length of time. The post-mortem structural change of nerve cell bodies in the central nervous system is a research area where inadequate work has been done. In view of the results obtained in this present project more extensive work in this area is certainly merited. LITERATURE CITED Baker, John R. 1958. Principles of biological microtechnique. John Wiley and Sons, Inc., New York. Baker, John R. 1960. Cytological Technique. John Wiley and Sons, Inc., New York. Barbacci, O. and G. Campacci 1897. Sulle lesioni cadaveriche delle cellule nervose. Rivista di Patologia nervosa e mentale 2:337-347. Camerer, Joachim 1943. Untersuchungen fiber die postmortalen veranderungen am zentralneversystem, insbesondere an den ganglienzellen. Ztschrft.f. ges. Neurol. u. Psychiat. 176:596-635. Cammermeyer, Jan 1960. The post-mortem origin and mechanism of neuronal hyperchromatosis and nuclear pyknosis. Exp. Neurol. 2:379-405. Cammermeyer, Jan 1961. The importance of avoiding "dark" neurons in experimental neuropathology. Acta Neuropathologica 1:245-270. Cammermeyer, Jan 1962. An evaluation of the significance of the "dark" neuron. Ergebnisse der Anatomie und Entwicklungs- geSChiChte B. 36:8. 1-610 Conn, H. J., Mary A. Darrow and Victor M. Emmel 1962. Staining procedures used by the Biological Stain Commission. The Williams and Wilkins Co., Baltimore. Crosby, Elizabeth, Tryphena Humphrey, and Edward W. Lauer 1962. Correlative Anatomy of the Nervous System. The MacMillan Co., New York, New York. Dito, William R. 1964. Transaminase in postmortem cerebrospinal fluid. Amer. J. Clin. Pathol. 42:360-363. Faworsky, A. 1900. Die postmortalen veranderungen der ganglienzellen des Ruckenmarks beim gesunden Tier. Monatsschrift f. Psychiatr. u. Neurol. 8:294-296. Ferrero, C. 1947. Alterations du systeme nerveux central apres la mort. Monatsschrift f. Psychiatr. u. Neurol. 113:118-125. Friede, Reinhard L. 1958. Histochemical demonstration of phosphorylase in brain tissue: association of postmortal neuron changes with phosphorylase activity. J. Histochem. Cytochem. 7:34-38. 32 33 Guyer, M. F. 1941. Animal Micrology. University of Chicago Press, Chicago. Hamada, Inazumi 1927. (Experimental studies on the post-mortem changes of nerve endings.)* Trans. Jap. Path. Soc. Koenig, R. S. and H. Koenig 1952. An experimental study of post- mortem alterations in neurons of the central nervous system. Jour. Neuropath. and Exptl. Neurol. 11:69-78. Kreiner, J. 1939-40. Uber postmortale veranderung der Markscheide im Bilde Weigerts and Spielmeyer. Beitraege zur Pathol. Anat. und zur allegemeinen Pathologica 103-104:l69-l72. Lazarus, Sydney 8., Barbara J. Wallace, George W. F. Edgar, and Bruno W. Volk 1962. Enzyme localization in rabbit cerebellum and effect of post mortem autolysis. J. Neurochem. 9:227-232. Levi, Giulio 1898. Alterazioni cadaveriche della cellula nervosa studiate col metodo di Nissl. Rivista di Patologica, nervosa e mentale 3:18-20. Lillie, R. D. 1965. Histopathologic Technic and Practical Histo- chemistry. McGraw Hill Book Co., New York. Manual of Histologic and Special Staining Technics. 1957. Armed Forces Institute of Pathology, Washington D. C. Maros, Tibor and Laszlo Lazar 1959. Zur frage der postmortalen veranderungen in peripheren nerven. Acta Anat. 38:34-55. Mayer, M. E. 1928. Uber postmortale Milchsaurezunahme in der Gehirnsubstanz von versuch stieren. Arch. Exp. Pathol. und Pharmakol. 134:218-224. Neppi, A. 1897. Sulle alterazioni cadaveriche delle cellule nervosa rilevabili col metodo di Nissl. Rivista di Patologia nervosa e mentale 2:152-155. Ramon y Cajal, S. 1928 (Translated and Edited by Raoul M. May, 1959) Degeneration and Regeneration of the Nervous System. Vol. I & II. Hafner Publishing Co., New York. Riese, Walther 1959. A History of Neurology. MD Publications, Inc. New York. Robins, E., David E. Smith, Geraldine E. Daesch and Kathryn E. Payne 1958. The validation of the quantitative histochemical method for use on post mortem material 11. The effects of fever and uraemia. J. Neurochem. 3:19-27. *Japanese 34 Sannomiya, Nobuhiko 1927 (Post mortem change of peripheral myelinated nerve fiber Schmidt-Lantermann incisures and effect of various pre and post mortem actions on nerve fibers.)* Okayama Igakkwai Zasshi 444:104-111. Seaman, Arlene R. Personal communication. Department of Anatomy, Michigan State University, East Lansing, Michigan. Shingaki, Y. and M. Nakayama 1960. (Study on the post mortem changes of Golgi apparatus in nerve cells.)* Igaku Kenkyu 30:499-507. Smythies, J. R. and O. R. Inman 1960. The effect of post-mortem autolysis on synaptic terminals in cerebral cortex of dog. Jour. Anat. 94:241-243. Sumegi, St. and L. Findeisen 1931. Uber veranderungen der Ruckenmarksflussigkeit nach dem Tode. Frankfurter Zeitschr. Path. 41:431-434. Ugo, Geacanelli V. 1927. Le alterazioni cadaveriche delle neurofibrille studiate col metodo di Bielschowsky. Pathologica 19:328-335. *Japanese FIGURES 35 36 Figure I. Diagrammatic Synopsis of Post-mortem Degeneration. ......... ooooooo ,.. a ... a E Ea E 7» \a \\ Ea Ea REE aa 7? E EEE a a E E E E E aa a E aux-Ema g m RIF—bun g E kg a. EN 2:. 3.2.23 nausea. see: no a 2.22.2 ggggg cozonocoooo Soto: non. ac 382$ ozoeeoeogo ._ 22m 38 Figure 2. Overall architecture of the habenular nucleus. medial fiber bundle medial habenular nucleus intermediate fiber bundle lateral habenular nucleus lateral fiber bundle stria medullaris thalmi . habenulopeduncular tract \lO‘U‘IJ—‘WNH Luxol fast blue - cresyl echt violet x29 Figure 3. Medial habenular nucleus - O-hour post-mortem. 1. cell membrane 2. nuclear membrane 3. Nissl substance 4. nucleolus Luxol fast blue - cresyl echt violet x756 39 40 Figure 4. Lateral habenular nucleus — O-hour post-mortem. . cell membrane nuclear membrane nucleolus Nissl substance DWNH Luxol fast blue - cresyl echt violet x756 Figure 5. Lateral habenular nucleus - O—hour post-mortem. Note the nuclear-cytOplasmic ratio of most cells of the habenular nuclei (compare with Figure 6.) Luxol fast blue - cresyl echt violet x756 42 Figure 6. Thalamic cells - O-hour post-mortem. Note the nuclear-cytoplasmic ratio and size of cells of the surrounding dorsal medial nucleus of the thalamus. (Compare with figure 5) Luxol fast blue — cresyl echt violet x756 Figure 7. Medial habenular nucleus - 6—hour post-mortem. 1. clumped Nissl substance 2. irregular nuclear membrane 3. vacuole Luxol fast blue - cresyl echt violet x756 43 44 Figure 8. Lateral habenular nucleus - 6-hour post—mortem. clumped Nissl substance vacuoles nuclear membrane irregular cell membrane «L‘LAJNH Luxol fast blue - cresyl echt violet x756 Figure 9. Medial habenular nucleus - 12-hour post-mortem. l. Nissl substance 2. vacuoles 3. irregular nuclear membrane Note nucleolus and cell membrane irregularities Luxol fast blue — cresyl echt violet x756 46 Figure 10. Lateral habenular nucleus - lZ-hour post-mortem. l. clumped Nissl substance 2. vacuole 3. nucleolus 4. irregular cell membrane Luxol fast blue - cresyl echt violet x756 Figure 11. Medial habenular nucleus - l8-hour post-mortem. light and dark areas of the nuclear membrane broken cell membrane pericellular space light perinuclear area . vacuole mwal-J Luxol fast blue - cresyl echt violet x756 47 48 Figure 12. Medial habenular nucleus - l8-hour post—mortem. l. clumped Nissl substance 2. light perinuclear area 3. nucleolar fragmentation Luxol fast blue - cresyl echt violet x756 Figure 13. Lateral habenular nucleus - 18—hour post-mortem. l. clumped Nissl substance 2. light perinuclear area 3. light and dark areas of nuclear membrane 4. nucleolar fragmentation Luxol fast blue - cresyl echt violet x1150 Figure 14. Figure 15. 50 Medial habenular nucleus - 24—hour post-mortem. l. 2 3. light and dark areas of nuclear membrane light perinUClear area irregular cell membrane Luxol fast blue — cresyl echt violet x1150 Medial habenular nucleus - 24-hour post-mortem. l. 2. 3. 4. finely granulated Nissl substance nuclear membrane - exhibiting a string-of-beads appearance cytoplasmic granulations nucleolar fragmentation Luxol fast blue - cresyl echt violet x1150 51 52 Figure 16. Lateral habenular nucleus - 24-hour post-mortem. 1. light and dark areas of nuclear membrane 2. vacuolated cytoplasm and granulations 3. cell border irregularities Luxol fast blue - cresyl echt violet x756 Figure 17. Lateral habenular nucleus - 24-hour post-mortem. Note the rupture of the nuclear membrane of some cells. (see arrows) Luxol fast blue - cresyl echt violet x756 53 54 Figure 18. Medial habenular nucleus - 36-hour post-mortem. pyknotic nucleus cytoplasmic granulations ruptured nuclear membrane nuclear membrane partially absent J-‘LAJNH Luxol fast blue - cresyl echt violet x756 Figure 19. Medial habenular nucleus - 36—hour post—mortem. irregular cell membrane vacuolated cytoplasm light perinuclear area . nucleolar fragmentation buNH Luxol fast blue - cresyl echt violet x756 56 Figure 20. Lateral habenular nucleus - 36-hour post-mortem. 1. obliterated nuclear membrane 2. pyknotic nucleus 3. vacuoles in granulated cytoplasm Luxol fast blue - cresyl echt violet x756 Figure 21. Medial habenular nucleus - 48-hour post-mortem. fragmenting nuclear membrane partially obliterated nuclear membrane pyknotic nucleus irregular areas of the cell membrane DWNH Luxol fast blue - cresyl echt violet x1150 57 58 Figure 22. Lateral habenular nucleus - 48-hour post-mortem. l. pyknotic nucleus nuclear membrane fragments 3. note the over-all light area of the nucleus even though the membrane is fragmented 4. irregular cell border 5. nucleolar fragmentation N Luxol fast blue - cresyl echt violet x1150 Figure 23. Habenulopeduncular tract - O—hour post-mortem of the myelin. 1. black myelin bisected by light axonal area 2. even contour of axonal and myelin areas Note other fibers of various diameters Osmium tetroxide x756 59 60 Figure 24. Habenulopeduncular tract - O-hour post-mortem of the axon. l. axon 2. myelin space Note the over-all unclear pattern of the tract Two hour Protargol method x580 Figure 25. Habenulopeduncular tract - 6-hour post-mortem of the myelin. 1. individual fiber: note irregular myelin with light spaces and slight clouding of axonal area 2. distinct axonal space Osmium tetroxide x1150 61 62 Figure 26. Habenulopeduncular tract - 6—hour post-mortem of the axon. l. axon: note the relatively constant diameter 2. myelin space 3. glial cell nuclei Two hour Protargol method x1150 Figure 27. Habenulopeduncular tract - 12-hour post-mortem of the myelin. 1. shaded axonal area of a large fiber 2. light area in myelin 3. areas of dilation and constriction of axonal area Osmium tetroxide x1150 63 64 Figure 28. Habenulopeduncular tract - lZ-hour post-mortem of the axon. 1. axon 2. areas of dilation and constriction 3. glial cell nuclei Two hour Protargol method x675 Figure 29. Habenulopeduncular tract - l8—hour post-mortem of the myelin. Note how the myelin has become irregular and the axonal area is clouded over in most fibers Osmium tetroxide x675 65 66 Figure 30. Habenulopeduncular tract - 18-hour post-mortem of the axon. axon light area of the axon neuron cell body constricted area of axon dilated area of axon WDWNH Two hour Protargol method x1150 Figure 31. Habenulopeduncular tract - 24-hour post-mortem of ' the myelin. Note that the myelin appears swollen and shows light areas. Axonal area is shaded over in some fibers. Osmium tetroxide x1150 67 68 Figure 32. Habenulopeduncular tract - 24-hour post—mortem of the axon. l. axon 2. light area in axon 3 dilated area of two axons Two hour Protargol method x1150 Figure 33. Habenulopeduncular tract - 36-hour post-mortem of the myelin. 1. Note the irregular axon and spaces in the myelin Note that other fibers show similar patterns. Osmium tetroxide x1150 70 Figure 34. Habenulopeduncular tract - 36-hour post—mortem of the axon. l. axonal 100ps 2. dilation and constriction 3. light axonal area 4. dark axonal area Two hour Protargol method x1150 Figure 35. Habenulopeduncular tract - 48-hour post-mortem of the myelin. Note the swollen myelin and reduced axonal area. Light spaces are seen in the myelin. Osmium tetroxide x725 71 72 Figure 36. Habenulopeduncular tract - 48-hour post-mortem of the axon. l. axonal loops 2. dilation of the axon 3. light and dark areas of axon Two hour Protargol method x1150