TRANSNEURONAL EFFECTS ON AMPHIBTAN LIMB REGENERATTON Thesis for the Degree of Ph. D. MTCHTGAN STATE UNIVERSITY CHARLES D. TWEEDLE 1970 LIBRARY MTG! Egan State. University This is to certify that the thesis entitled TRAN SN L1} UR ONAL EFFE CT 3 ON Al'vIPiIIBIAI‘J LIIZ'IB REGEHQRATIOI‘é presented by C {ABLE 5 D . Tu‘v’EJDLE has been accepted towards fulfillment of the requirements for Ph.D. degree in 20010?” W Major professor I Date 6/10/ 70 0-169 T 1 ! $.57 “91% - ABSTRACT TRANSNEURONAL EFFECTS ON AMPHIBIAN LIMB REGENERATION BY Charles D. Tweedle There has been controversy in the regeneration litera— ture concerning the effect that removal of more than one limb has on regeneration rate. Series 1 of this study was designed to assess the effect of the removal of a second limb on regeneration rate in Ambystoma mexicanum larvae. It was found that the removal of a second limb brought about a significantly slower regen- eration rate both in length and onset of morphogenesis but only if the second limb removed was contralateral to the first. Series 2 of this study demonstrated cytochemically that there existed transneuronal changes following single forelimb amputation so that chromatolytic neurons could be seen on both sides of the Spinal cord. Following double forelimb removal, it was found that chromatolysis in the brachial neurons was both of a greater degree and more prolonged. Series 5 of this study demonstrated the same phenomenon with the use of 3H uridine uptake as an index of chromatolysis. Charles D. Tweedle It was found that single forelimb removal brought about sig— nificantly increased uridine uptake in brachial neurons on both sides of the spinal cord and that double forelimb remov- al brought about even greater uridine uptake. In the brachial spinal cord cells it was also seen that the increased uridine uptake was for a longer period of time following double limb amputation than following single limb amputation. This gave further evidence for the idea that increased neural disruption following double, contralateral, limb removal might be the reason for a slower regeneration rate. Series 4 was an investigation of the effect of double and single limb removal on regeneration rate in aneurogenic ~Ambystoma larvae. Here, without any intervening nervous influ- ence, no effect was seen with the removal of a second forelimb. Series 5 demonstrated that the removal of a second limb in the newt brought about slower linear regeneration rate but only if the second limb removed were contralateral to the first. It was also found that just the severence of the brachial innervation of one forelimb brought about a signifi- cant slow-down in the regeneration rate of the contralateral forelimb. Series 6 was designed to investigate a possible mechanism for the growth inhibition obtained with contralateral forelimb removal. It was found that at 7 days after amputation, the amputation or denervation of one forelimb would bring about Charles D. Tweedle significantly decreased DNA synthesis in the regenerating contralateral forelimb. The results of this investigation led to the following conclusions: 1. The removal of a second limb in either the axolotl or newt brings about a slower regeneration rate but only if the two limbs removed are contralateral to one another. Single forelimb removal brings about contralateral neural changes in the brachial spinal cord region and double forelimb removal brings about even more profound and prolonged contralateral neural disrup- tion. It is felt that the increased neural disruption brought about by the removal of two contralateral limbs is the mechanism by which the decreased regen- eration rate of the limbs is effected. Either through slower regrowth of nerve fibers or slower production of trophic substances by the neurons, the trOphic effect of the nerve on blastemal prolifera- tion is lessened and this slows down the regeneration rate. TRANSNEURONAL EFFECTS ON AMPHIBIAN LIMB REGENERATION BY \ 1’ Charles DJ Tweedle A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1970 ACKNOWLEDGMENTS The author wishes to express thanks to Dr. C. S. Thornton for his interest, encouragement, and excellent guidance during the course of the investigation. Gratitude is also extended to Dr. S. K. Aggarwal and Dr. B. E. Walker for their helpful suggestions concerning the experimentation and aid in preparation of the thesis and to Dr. Martin Balaban for teaching me what questions to ask. Special appreciation goes to Thos. Connelly for his valuable technical assistance and spiritual guidance and to Dr. Roy Tassava and Dr. Charles Taban for their encouragement and many critical discussions. Special thanks are extended to Dr. N. C. Shuraleff and Dr. W. E. Cooper for their interest and assistance in the statistical analyses, to Mrs. Mary Thornton for providing aneurogenic larvae, and to Mrs. B. Henderson for her help with supplies. I am grateful for financial support from an NIH Trainee- ship during the major portion of this investigation. ii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . . . . . . . . . LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . INTRODUCTION MATERIALS, METHODS, AND RESULTS. . . . . . . . . . . . GENERAL METHODS . . . . . . . . . . . . . . . . . SERIES SERIES SERIES SERIES SERIES SERIES DISCUSSION SUMMARY. . 1--Growth Relationships Between Single and Double Limb Amputations . . . . . . . . 2-—Cytochemical Investigation of Trans- neuronal Effects in Ambystgma . . . . . 5—-Autoradiographic Analysis of Contra- lateral Effects Following Limb Amputa- tion. . . . . . . . . . . . . . . . . . 4--Growth Relationships Between Double and Single Limb Amputations in Aneurogenic Ambystoma Larvae. . . . . . . . . . . . 5-—Investigation of Contralateral Effects on Limb Regeneration in the Newt. . . . 6--Autoradiographic Analysis of Contra- lateral Effects at an Early Phase of Regeneration. . . . . . . . . . . . . . LITERATURE CITED . . . . . . . . . . . . . . . . . . . iii Page iv 14 24 SO 52 59 45 52 55 LIST OF TABLES TABLE Page 1. A comparison of the time of completion of com— plete digit formation in Ambystoma mexicanum larvae following various amputation procedures. It may be seen that the removal of a second limb slows down the speed of complete digit formation, but only if the second limb removed is contra- lateral to the first one removed. It may also be seen that the hindlimb takes longer to com- plete digit formation than the forelimb. This may be either due to the fact that the hindlimb is larger in volume or that the hindlimb has more digits to reform. . . . . . . . . . . . . . . 15 A comparison of the uptake of tritiated uridine into brachial nerve cells of Ambystoma mexicanum larvae following various amputations at 1 and 5 days after amputatiOn. At both day 1 and 5 after amputation grain counts were done on 5 ani- mals in each amputation group. The averages given each represent grain counts on 20 nerve cells for the ventral Spinal cord cells and 25 nerve cells for the ganglion cells. Comparison of uptake of label was done statistically through the use of a Mann-Whitney U test. The results are given in Series 5. . . . . . . . . . . . . . . 27 A comparison of number of cells labeled with tritiated thymidine in the newt limb at 7 days after removal of: A. 1 forelimb plus sham denervation of opposite forelimb (control) B. 1 forelimb plus denervation of opposite fore— limb C. both forelimbs The sampling technique is given in Series 6. . . . 42 iv LIST OF FIGURES FIGURE Page 1. A comparison of the mean linear regeneration length of limbs of Ambystoma mexicanum larvae following 1 of 5 amputation procedures: A. 1 forelimb removed B. 1 hindlimb and 1 forelimb removed C. 1 hindlimb removed D. both hindlimbs removed E. both forelimbs removed A one—way analysis of variance and new multiple range test run at 55 days post-amputation showed that the animals in groups A, B, and C displayed significantly (P'< 0.01) greater growth than the other 2 groups. For the statis- tical analysis only 1 limb was measured in each animal of groups D and E and only the forelimb was measured in group B. . . . . . . . . . . . . . 11 Light micrograph of a Feulgen preparation of normal ventral spinal cord nuclei in an unampu- tated animal. Note the relatively diffuse state of the chromatin. (5000K). . . . . . . . . . 18 Light micrograph of a similar Feulgen prepara- tion of ventral Spinal cord nuclei at 9 days after limb amputation. The chromatin is seen to change from the more diffuse appearance of the normal nuclei to a more aggregated appearance in the chromatolytic neuron. The nuclear changes were seen on both sides of the Spinal cord after Single limb removal. (SOOOX) . . . . . . . . . . . 18 Light micrograph of a cross section of a ventral spinal cord cell showing a degenerating nerve fiber ending (F) and bouton (B). .Degenerating fibers were seen on both sides of the spinal cord following single limb amputation, giving evidence. for transneuronal degeneration . . . . . . . . . . 23 LIST OF FIGURES—-continued FIGURE Page 5. Light micrograph of a cross section of the ventral Spinal cord region at 15 days after single forelimb removal, showing a number of degenerating nerve fiber endings (arrows) and a degenerating bouton (B). (1250X). . . . . . . 25 A comparison between the growth rates in length of aneurogenic Ambvstoma maculatum larvae following amputation of either 1 or 2 forelimbs. -A Mann-Whitney U test run at day 14 after amputation Showed no Significant difference in amount of regeneration . . . . . . . . . . . . . 34 A comparison of the mean linear regeneration length of limbs of adult newts following 1 of the following 4 amputation procedures: A. 1 forelimb removed, opposite forelimb sham denervated B. 1 forelimb and 1 hindlimb removed C. 1 forelimb removed, opposite forelimb denervated D. both forelimbs removed A one-way analysis of variance and new multiple range test run at 55 days after amputation Showed that groups A and B diSplayed Signifi- cantly greater linear growth than groups C and D (P <10.05). 1 micrometer unit = 0.125 mm. For the statistical analysis only the fore- limb was measured in group B and only 1 fore- limb was measured in group D . . . . . . . . . . 58 vi INTRODUCTION Students of regeneration too often make the tacit assump- tion that a regenerating limb in an animal is not influenced by other regenerating limbs in the same animal. Experiments have commonly been designed so that one limb of the animal is used as a control while another limb of the same animal receives some experimental treatment. This practice makes no allowance for any factor of interaction that might exist with the removal of more than one limb. T. H. Morgan (1906) reported from unaided visual observa- tions that the removal of one, two, or three limbs from adult Diemyctylus viridescens did not seem to alter the rate of regeneration. A more elaborate experiment focused on this problem was carried out by Zeleny in 1909. In a series of studies on both vertebrates and invertebrates, Zeleny set out to determine the effect of the "degree of injury" on regenera- tion rate. As part of this study he used Ambystoma gpacum larvae with different numbers of limbs removed and sometimes also the tail. rAlthough he concluded that the larvae with the greater degree of injury (more appendages removed) had an increased regeneration rate, modern statistical analysis of his data does not bear out his conclusions. Not only is there great overlap between regeneration rates of the various groups but the small number of animals used precludes statis- tical Significance. There have also been a number of studies done on the effect of hindlimb removal on tail regeneration. Zeleny (1909) studied the effect of the removal of one or both hind— limbs on the rate of tail regeneration in g, climitans tadpoles and found that, while the amputation of one hindlimb had the effect of accelerating tail regeneration, the removal of both hindlimbs brought about a weaker acceleration of the regeneration rate. Zeleny concluded from this that there was an optimal amount of injury to stimulate regeneration; more than this optimum brought about a lessened stimulation. However, in 1926 Hiller repeated these eXperiments with axolotls and reported slower tail regeneration with the simultaneous removal of limbs as well as the tail. In an attempt to settle this problem, Blacher §£_§l, (1932) carried out extensive studies with tadpoles on the effect of limb amputation on the tail regenerate in order to determine whether there was any time relationship with the effects of the additional amputation. They found that the removal of the hindlimbs stimulated tail regeneration, but only if the tail was amputated not more than two days before the limbs were removed. There are a number of reports of regeneration promoters that may be active in the growth stages of regeneration. Issekutz-WOlsky and Fogarty (1962) demonstrated that a tail blastema implanted beneath the skin of the lateral body sur- face stimulated tail regeneration in newts. The authors also reported that neither implantation of amputated normal tail ends nor infliction of wounds brought about accelerated re- generation. Skowron g£‘_l. (1963) found acceleration of regenerative growth in axolotls with not only the implanta- tion of whole regeneration blastemata beneath the Skin but also with the implantation of muscles, injections of dissocir ated blastemal cells or of homogenates from such cells. Weber and Maron (1965) have also reported increased regenera— tion after the implantation of stump muscle under the abdominal skin in axolotls where it dedifferentiated into blastema-like cells. They localized the promoter activity mainly in the microsomal fraction which contained soluble proteins; when this was injected into the body wall of axolotls with amputated tails, an accelerated regeneration rate was noted. It remained to be determined whether the presence of such a circulating factor might be of importance with the existence of more than one regeneration blastema in the same animal. Work by Twitty and Delanney (1959) has Shown that competition for circulating nutrients did not effect the Speed of Size regulation of eyes transplanted from younger to older Ambystoma larvae with regenerating tails. From these and other experiments the authors concluded that there is a moderate abundance of nutritive materials in circulation even when the animals are starved. These are gradually depleted but apparently not to the point where competition becomes a vital factor in their distribution. Thus, if one may extrapo- late somewhat, it seems unlikely that this should be an important consideration in a well-fed animal with more than one regenerating limb. The growth and maintenance of each organ depends to a considerable extent on its innervation (Wyburn-Mason, 1950). If a growing limb is denervated its growth is seriously retarded, particularly if reinnervation is prevented (Huxley and DeBeer, 1934). On the other hand, an augmentation of the normal nerve supply to a limb may cause great hypertrophy or the production of supernumerary limbs. The trophic effect of the peripheral nerve is also seen in regenerating limbs (Singer, 1952, 1959, 1960) so much that the limbs of adult Anura which do not regenerate normally will do so with extra nerves added. If regenerating limbs are denervated their growth ceases until they become reinnervated. Thus, it is seen that any change in the nerve supply can also be a factor in the rate of regeneration. It was not clear which of these factors, if any, might be of importance in altering the rate of limb regeneration when more than one limb in an animal is removed. Therefore the experiments described herein were designed to determine the effect of amputation of an extra limb and then to access the cause of any effect found. It was found that removal of a second limb brought about a Slower regeneration rate, but only if the two limbs removed were directly opposite one another. Evidence is presented that a contralateral neural injury is responsible for this effect. MATERIALS, METHODS, AND RESULTS GENERAL METHODS Larval axolotls, Ambystoma mexicanum, used in these experiments were obtained through the generosity of Dr. R. R. Humphreys at Indiana University. The Ambystoma maculatum used in the aneurogenic experiment were obtained from Glen Gentry, Donelson, Tennessee. Adult newts, Notgphthalmus (Triturus) viridescens, were obtained from Lewis Babbitt, Petersham, Massachusetts. (All experimental animals were fed every other day on Sliced beef liver. The animals were main- tained at a uniform laboratory temperature of 22‘: 2°C. All amputations were made through the mid-humerus following anesthesia in 1:2000 MS 222 (Sandoz). In all amputations the bone was carefully trimmed with iridectomy scissors to avoid interference with regeneration. Care was taken to make certain that the amputations were as nearly as possible at the same level on the limb, since level of amputation has been reported to have an effect on the rate of regeneration in a number of regenerating systems. In amphibia in general the absolute rate of regeneration increases with the percentage of limb removed (Needham, 1952). To check this factor, the length of limb stump remaining after amputation was measured in each case and plotted against amount of linear regeneration at various time intervals. No correlation was found with the amputation levels used. Length measurements were used as an index of regenera— tive growth. It was felt that this was a valid index of regeneration, eSpecially for Ambystoma, as Karczmar (1946) determined that length changes are directly correlated with volume changes in regressing Ambystoma limbs. Length changes were also measured in newts, but these measurements were sup- plemented with autoradiographic data. Length measurements were made with a dissecting microscope fitted with an optical micrometer. Following anesthesia, the animals were positioned on their backs on moist gauze on the microsc0pe stage so as to achieve accurate and reproducible linear measurements. Repetition of these measurements Showed the results to be very consistent. Each measurement was made at least twice and the average used. Amount of linear regeneration was determined as the amount of growth in length beyond the length of the amputation stump. For measurements of linear growth the total length of the regenerating limb was taken and the length of the amputation stump subtracted to give the amount of new growth. Since the stump was heavily pigmented compared to the Sparsely pigmented blastema, the plane of amputation could easily be seen at the proximal border of the regenerate. Observations were also made on morphogenesis to supplement the length measurements. SERIES 1 Growth Relationships Between Single and Double Limb Amputations Procedure Fifty-five laboratory—raised Ambystoma mexicanum larvae of 60 mm. in length were used. These were maintained in individual small plastic dishes. The animals were divided into 5 groups on the basis of amputation procedure. These groups were as follows: A. one forelimb removed B. one hindlimb and one forelimb removed C. one hindlimb removed D. both hindlimbs removed E. both forelimbs removed. In order to satisfy the independence requirements in statistics, in the groups with two hindlimbs or two forelimbs removed only one of the limbs was arbitrarily chosen for measurements. In the groups with one hindlimb and one fore- limb removed only the forelimb regenerate was measured for the same reason. Length determinations were made only until shortly after the last of the experimental animals had re- formed all digits (about 45 dayS post-amputation). After this time, the growth of the digits made both measurements and interpretation difficult. Results Figure 1 Shows a plot of the mean regenerate length of each of the 5 groups plotted against time after limb ampu- tation. In the graph length is given in micrometer units; each micrometer unit is equal to 0.15 mm. About day 20 there develops a difference among the experimental classes whereby the animals with both hindlimbs or both forelimbs amputated show a slower linear regeneration rate than the other 5 groups. After ascertaining compliance with assumptions of the statis- tical tests, a one-way analysis of variance and new multiple range test were run at 55 days post-amputation and found to Show, indeed, that the 2 Single limb amputations and the single hindlimb plus Single forelimb groups display statis- tically greater (P <.0.01) growth by 55 days after amputation than the other 2 groups. The morphogenetic stages closely paralleled the growth results. Table 1 presents the time of complete digit formation in the 5 groups. Just as the animals with Single forelimb or single hind- limb amputation undergo greater regeneration by 55 days than those with double forelimb and double hindlimb amputations, it is also seen that the latter 2 groups Show a delayed onset of morphogenesis. The forelimb of the group with both a hind— limb and forelimb removed, however, Showed a morphogenetic rate similar to the single forelimb amputation. It is inter- esting that the hindlimb, which undergoes as rapid growth in length as the forelimb, lags behind it in morphogenesis. 10 .m msonm CH pwusmmma mm3 QEHHmHom map >Hco new m pew Q mmsoum mo HmEHcm some CH pmusmmoe mm3 QEHH a waco mflmwamcm HMUHumHumum may Mom .mmsoum N Hmnuo on» swap £u3oum Hmumoum Aao.o V_mv ManamUAMHcmflm commammfic U 0cm .m .< mmsoum CH managed on» umnu noBonm coflumusmEMIumom m>m© mm um can ummu mmcmu mamwuase 3o: cam mocmHHm> mo mamaamcm >m31wco d Uo>oaou mQEHHouom buon .m ©o>oamn mnaflaocfin buon .Q Uw>oEmH Deflapcfln a .0 ©o>oewu QEHHoHom a can QEHHGCAS a .m pw>oEoH QEflHouom a .d "mouspmooum coHumusmfim m no a mcHBOHHom om>uma Escmowxmfi mEoummnE¢ mo mQEwH mo numcma coflumuocmmmu Hmmcfla some may «0 conflummaoo ¢ .H wusmflm 11 nV ov mm a wusmam ZO;<.—Dm<<< amiss m> SH Hmmuma we QEHHocHE mnu menu uomm map ou map Hmnuflm mg was mane .Qaflamuom mnu amen coflum IEHom uHmHU mumamaou ou nmmcoa mmxmu Qawapcfin mnu umnu :mmm ma omHm mme uH .nm>oamn mco Umufim may on Hmumumamuucou ma Um>oamn QEAH Ucoomm mzu we >aco usn .coflumauom uHmHU mumHmEoo mo Ummmm may c306 mBOHm QEHH pcoumm m mo Hm>OEmH mnu umnu cmmm mn mmE pH .mmuspmo loud coflumvsmam m50wum> mSABOHHom mm>uma Escmowxme meoumNnE< SH :oflumanom uflmwp mumHmEoo mo :oHumHmEoo mo mafia may mo comHHmmEoo m .H magma 15 H magma OOH 00H OCH 00H 05 Ole macaw mSRAMMOh + mzHAQZH: LO mZHAHMOM 00H No 0: 0H mHIz mmSHAQZHm maom 00H OOH 00H on 04 OHuz mEHAQZHm mzo OCH «0 On On mm «:2 mmEHAMMOh meom OOH OCH OCH 00H onz mZHAanh n20 m4 mm on 20H9OEmH QESH mamaym Hmymm UHOU Hmaymm may mo mmpflm ayoa co ammm mum3 mmmamau Homausa mas .cousma oHy>H0ymEouao may CH muamymmmmm amymmmnmmm mHOE m oy Hmaosc HmEHoa may no moam tummmmm mmsmmwp mHoE may Sony mmamao 0y ammm we awymEOHao maB .cowymySmEm QEHH ymymm mmmo m ym Hmaosa oyoo Hmcflmm Hmuyam> mo GOHymummmHm ammasmh HMHHEHm m we ammanHUHE yamyn. .m mysmflh Axooomv .cyymaonaU may no mymym mmsmmap >Hm>flymamy may myoz .ymeyam UmyMysmEm Is: no CH ymausa Unoo Hmaflmm Hmyyam> HmEHo: mo aofiymummmum ammasmm M NO ammumOHUHE yamyq .N muzmflm 18 1" i V 4-!“ .9 m mHDmHm N musmflm 19 amputation. He reported that within the nucleus the granular and fibrillar material becomes more aggregated, occurring in clumps. The great reproducibility of these nuclear changes following different fixations (Carnoy's, formalin, Bouins, and Zenker's) precludes the possibility of it being a fixation artifact. After single limb amputation, these nuclear changes were seen in neurons on both Sides of the spinal cord, although less on the side Opposite the transection. This occurred both in Ambystoma and Triturus. In the 2 u thick methacrylate sections and in Ambystoma in particular chromatin clumping was also seen in the ganglia on bg£h_sides of the animal following unilateral limb removal, although the degree of nuclear change was much less in the ganglia contralateral to the limb amputation. This indicated some sort of contra- lateral change following the transection of just the brachial nerves supplying one limb. The neurons most obviously af- fected by unilateral limb removal were the cells of the dorsal and ventral portions of the Spinal cord and ganglia of the side of limb removal; however, there were detectable effects on just about every neuron in the spinal cord. It was difficult to ascribe all the changes seen to known neural pathways along which transneuronal degeneration might pass. There were neurons showing definite nuclear changes on the Side of the animal opposite the amputation that would have no direct synaptic connections to the operated Side--at least according to the scheme laid down by van Gehuchten (1897). This same 20 phenomenon has been reported by others such as Ochs gt 3;. (1960) who found chromatolysis in lateral nuclear neurons contralateral to the side of unilateral sciatic nerve crush in the cat. Here, also, no known neural connections could explain the results. In Ambystoma these nuclear changes were seen in the spinal cord and ganglion cells by one day after nerve section and lasted about 10-12 days in the case of Single forelimb amputation and 15-18 days in the case of the double forelimb amputation. It is thought that the lengthier disruption seen in the animals with double forelimb amputa- tion is due to the fact that not only must the nerve cells recover from the direct injury of having their axons severed with limb removal but also from contralateral changes carried from the injured neurons of the opposite side of the spinal cord. The phenomenon of the chromatin aggregation is especially interesting in the light of a report by Causey and Strattman (1959) who presented microspectrophotometric evidence that there is a loss of Feulgen staining material (DNA) during chromatolysis in the cell bodies of the cervical ganglion of the rabbit. Preliminary studies (Tweedle, unpub— lished) Show a similar loss of Feulgen positive material in the case of chromatolytic brachial ganglia of Ambystoma. However, it is debatable whether this represents a loss in DNA. Noeske (1969) has recently presented evidence that, as a consequence of chemical and biophysical alterations in chromatin during different functional states, variations in Feulgen 21 values may be found in cell nuclei that really contain the same amount of DNA. Another index of neural injury is the presence of de- generating nerve fiber endings or synapses (see Gray and Guillery, 1966, for review). The technique of Fink and Heimer (1967) was used to stain for degenerating axonal endings in Ambystoma at 7 and 15 days after unilateral limb removal in order to detect the presence of neural pathways that might be involved in the contralateral changes seen. Sections from unamputated animals were also run through the stain as a control. It was found that unilateral limb removal brought about degenerating fiber endings in both the dorsal and ventral areas of the spinal cord and occasionally in the ventral area of the unamputated side (see Figures 4 and 5). The degenerating fiber endings were mostly noticeable at the level of the brachial ganglia, which supports the idea that they are indicative of reflex pathways between the forelimbs. The appearance of degenerating axonal endings in the ventral portion of the unamputated Side of the spinal cord would argue for the involvement of transneuronal upset across one extra neuron-—the commissural fibers from the dorsal area of the amputated side to the ventral area of the contralateral Side. This type of transneuronal disruption is not rare in young animals and in neural pathways with few collaterals (Bleier, 1969). 2 2 Axommav .Amv SOysoa msyymumammmn m can Am3ouumv mmcflpam Hmnwm m>nma mad lymnmcmmmp mo Hmnasa m mcfl3oam .coflymysmEm aeflamyom mamaam Hmyym mwma ma ym aofimmu CHOU Hosamm Hmnyam> may mo aoayomm mmouu m yo ammymoyofla yamflq .m musmflm ANommdvn.coHymnmammmU Hmaousmcmamuy How moamofl>m maH>Hm .aoflymysmfim Dada mamaym mSHBOHHOM UHOU Hmchm may yo mmoym ayoa co ammm mumB mumaflm maflymumcmmmn .Amv aoyson Uam Amv mcflpcm Hmaflm m>uma mew lymumammmo m mSABOam Hamo UHOU Hmaflmm Hmuyam> m yo COHyumm mmOHU m mo ammumouuaa yamflq .e mysmflm 25 Figure 5 Figure 4 24 It is interesting that although degenerating fibers are seen, there is no great evidence of nerve cell death follow- ing limb amputation. This may be related to the fact that there is known to be some degree of regeneration in the amphibian CNS (Clemente, 1964) or the fact that long-term observations on the brachial neurons were not made following limb amputation. In summary, the results of Series 2 demon- strate that following unilateral limb amputation there can be seen bilateral changes such that brachial neurons on both Sides of the spinal cord are affected. With double limb removal the chromatolytic appearance of the brachial neurons (both in the spinal cord and the ganglia) is prolonged for a greater period time than with Single limb removal; this is apparently due in part to transneuronal degeneration. SERIES 5 Autoradioqraphic Analysis of Contralateral Effects Following Limb Amputation The results of Series 2 gives morphological evidence for transneuronal disruption following limb amputation. Series 5 is designed to give a more quantitative evaluation of this disruption. Advantage was taken of the fact that chromato— lytic neurons have been found to increase RNA production during the earlier stages of the nerve reaction (Haddad §£_§l,, 1969; Watson, 1965; Lison, 1962). 25 Procedure Uridine-S—SH (S.a. greater than 20 c/mM, New England Nuclear) was used as a precursor of RNA and the uptake of the label into the spinal cord and brachial ganglia of Ambystoma was taken as an index of neural disruption. AS there is no definite motor horn in Ambystoma, the label was counted in the large neurons of the ventral part of the spinal cord and thus included both commissural and motor neurons (Herrick and Coghill, 1915). It should be noted also that there are two main types of motor cells in the salamander-- primary and secondary. The primary motor neurons are somewhat mediolateral in the spinal cord, but were also included in grain counts. At various times after single or double limb amputation grain counts were done on ventral spinal neurons and cells of the brachial ganglia. These were statistically evaluated in reference to control sections from unamputated animals. In each case there were 5 animals per experimental group. In each animal grain counts of 25 ganglion cells were randomly made from the 5 brachial ganglia to arrive at a mean number of silver grains per nerve cell. At the level of the ganglia grain counts were also done on 20 ventral Spinal cord cells to obtain a mean value. Mann—Whitney U tests (Siegel, 1956) were run to compare the medians for animals in each group with those in other groups or with controls. Table 2 gives the data from these grain counts, corrected for back- ground. 26 .m mmHHmm CH am>Hm mum myazmmu mas .ymmy D mmcyflazwaamz m «0 mm: may amsoyay maamoflymflymym maoa mm3 HQOH mo mamyms mo comflymmEoo .maamu COHHmcmm may you maamo m>uma mm Ucm mHHmo Unoo Hmaflmm Hmuyam> may you mHHmU m>yma om co mycsoo samum yammmnmmu aumm am>am mmmmym>m maB .msoym c0aymysmem aomm CH mHmEHcm m so meow mHmB myasoo aymym aoflymysmem Hmymm m Ucm H wma ayon yd .coHymysmEm Hmymm mwmo m ace 6 yo mcoflyMmeEm msoHHm> mGHBOHHom mm>uma,85amoflxm8 mEoym>AE< mo mHHmo m>umc Hmyaomua oyCy maapflns UmyMyyflHy mo mamym: may mo consummaoo « .N mHQMB N manna *COAyMySmEmtymom.Nmmlmao .HMEHGM mamaflm M GA maamo sofiamamm mm no maamo puou Hmaflmm om Hmayym so meow mycsoo Sony mafimym mo amass: mmmum>m may mysmmmummu amass: aomma .GOHymuomuoocH ysoa m.N “mama ma How ammome "maoflyomm 1 m “molmtmawvflns 01 m+ .sowymyomuooaa Hsoa m “mamm3 w you ammomxm “mSOAyumm 1 m “mmtmlmaflpflus Q: a * ma.mawdm.om dm.ddem.mm m>.oa«m¢.>m «v.5 Hem.m ma.maamm.wm mw.m Hm>.¢N nm.oaamm.>m mm.w HNN.HN 7.mm.>dwd¢.mm >m.mdymd.¢m mo.dNHmo.N¢ em.m Hmm.m em.maama.mm om.m “mm.mm H¢.demm.mm md.> Hm¢.mm $.mo.mmfimm.mm >>.>awam.o¢ wa.maaa¢.mm mm.m Hom.m wa.dmam>.mm mm.mayow.mm >m.m H¢¢.dm mo.m www.mm mm.mdwmm.mm ¢H.¢HHOm.mm om.maumm.>¢ w¢.m Hmm.m mm.mHHNe.mm mm.m Hmm.>m mm.mdymm.¢m mm.m Hom.mm Na.mayom.¢m O>.m Hum.>m d>.mdyoo.oe Ho.m He¢.> mm.omumm.0m ah.m Hmd.mm mo.mHHN>.mN am.m www.mm +aoflyMmeEmemom m>mn m>Hm a¢.wmwoo.>> >>.mmahe.dm mm.NNHN¢.mm m¢.mHHao.Hm am.mHHN¢.mm mm.omyom.m¢ em.mdwam.om mo.mawdm.em >N.>NH®H.Nm m>.mmnme.mm em.maw~m.0¢ am.mfiyma.om oe.onwhd.m> em.mafimm.mm aa.amymfi.oodma.mmwom.m> Ha.>dnem.>m pm.maymm.em mm.mdyme.>> pm.mmyma.oh mm.NNHmm.om No.0NHmw.m> mm.mfiao¢.mm H¢.mayom.m¢ ooa A OOH A m¢.omwom.m> >0.mmwwa.mm mm.emym>.mm a¢.mmwmm.m> m>.m www.mm md.haydm.am gilalmflmo om flmcmo 348 um dHesse mflmo om seam Hmumumamuycoo seam vmymysmea a0wymy5ma¢ mansoa aoflyMyDQad mammflm Houyaou deZDOU 2H¢m0 Mm QHBflUHGZH m4.mAANU m>mmz OBZH ZOHBdflOmMOUZH NZHQHmD QEBfiHBHmB 28 All injections of label were intraperitoneal and made through the tail musculature to prevent the loss of label by leakage. Ribonuclease digestion (1% for 4 hrs. at 40°C.) was done on Slides from all groups to ascertain that the label was going into just the nucleic acid. The dipping method of autoradiography was used according to the technique of Kopriwa and Leblond (1962) with NTB-5 emulsion diluted 1:1. After development in Dektol the autoradiographs were thoroughly rinsed and stained with hemotoxylin or methylene blue (1% at pH 7.0). As different amounts of label and exposure time were used at the 5 time intervals tested, which were at 1, 5, and 10 days following amputation, these are also noted on Table 2. To avoid counting the same cell twice only sections at 20 u intervals were used for grain counts. Results One day after amputation: In the group of animals with a Single forelimb removed both the ganglion cells and ventral Spinal cord cells on both Sides of the Spinal cord showed statistically greater label incorporation (P = 0.004) than their respective controls when evaluated by the Mann-Whitney U test. The animals of the group with both forelimbs ampu— tated Showed such great incorporation of label into ganglion and ventral spinal cord cells that it was impossible to do meaningful grain counts on them. Five days after amputation: The ventral Spinal cord cells of the group of animals with one limb amputated had returned 29 to normal levels. However, in this group it was found that the ganglion cells on both the side of limb transection as well as the contralateral Side still Showed increased uridine uptake (P = 0.004) as compared to their reSpective controls. Likewise the ganglion cells of the group with both forelimbs removed showed increased label (P = 0.004) as compared to their controls. It was interesting to note that the ventral Spinal cord cells of the double amputation group still Showed increased uptake of label (P = 0.004) when compared to either the ventral spinal cord cells of the control or of the group with one forelimb removed. By 10 days after amputation all label incorporation in the experimental animals had returned to control levels. Thus, it was found that, using increased uridine uptake as an index of neural disruption, single limb removal quickly brought about increased labelling on both Sides of the spinal cord and also in the ganglia of both the side of limb removal and the contralateral Side. The most unexpected result was that the ganglion cells of the side of the animal contra- lateral to the limb amputation showed increased uridine uptake. It may be remembered that there was also Slight indication of nuclear changes in the contralateral ganglia following single forelimb removal. However, the increase in uridine uptake by these cells was of an overall increase in number of grains per cell and not due to the presence of a few cells Showing greatly increased uridine uptake, as if they had been injured 50 only to a slight degree. The ganglion cells of the animals with one or both forelimbs removed also Show continued in- crease of uridine uptake at 5 days after limb removal. The much larger amount of cytoplasm and Nissl material (RNA) in the ganglion cells as compared to Spinal cord neurons is thought to be responsible for this more prolonged reaction. It should be noted that the ventral spinal cord neurons of the group of animals with both forelimbs removed do Show more prolonged uptake of label than the group with one forelimb removed. This is thought to be the results of the combined effects of direct neural disruption on each side plus contra— lateral effects adding together to produce a chromatolytic reSponse of longer duration. This is Similar to the results obtained from the morphological study in Series 2. Ribonu- clease digestion was found to remove virtually all label from the preparations. SERIES 4 Growth Relationships Between Double and Single Limb Amputations in Aneurogenic Amhygggmghggggag Interestingly enough, under certain conditions nerves are not necessary for regeneration. In 1959 Yntema produced aneurogenic limbs by removal of the apprOpriate portion of the spinal cord and neural crest at an early stage in develOp- ment. Amputation of the nerveless limbs was followed by typical regeneration. Whether regeneration of aneurogenic 51 limbs results from persistence of embryonic growth factors in the non-neural tissues and/or from diminished threshold to normally present factors is unknown. This offers a unique system for the evaluation of agents in the inhibition of regeneration by the removal of a contralateral limb. If the neural connections between the 2 limbs are the determining factor, then one would not expect a growth inhibition with the removal of both forelimbs in the aneurogenic larvae. Procedure Ambystoma maculatum eggs obtained from Glenn Gentry were used. At the tail bud stage of embryonic deveIOpment the technique of Thornton and Steen (1962) was used for pro- ducing aneurogenic limbs. After the completion of limb development, 16 animals had one forelimb removed and 12 had both forelimbs removed. These operations were made with fine iridectomy scissors through the humerus. The animals were kept in sterile Steinberg's solution with sodium sulfadiazine added to prevent bacterial infection. Throughout the experi- ment they were maintained in an incubator at 180C. Both growth in length and morphogenetic stages were used to gauge the rate of regeneration. Measurements were made in this case without the use of anesthesia as the larvae are quite immobile. For statistical reasons only one of the limbs of the double forelimb amputation group was measured in analyz— ing the growth rate. 52 Results Figure 6 gives the mean regenerate length of the limbs of the 2 groups beyond the original limb stump throughout limb regrowth. It can be seen that there appears to be no difference in the growth rate of the 2 groups. Indeed, statis— tical analysis by a t test at day 14 after amputation shows no statistical difference at the 5% level between the amount of linear growth achieved by the 2 groups at this time. The completion of digit formation was achieved by all animals by 18 days; no difference was seen between the two groups in Speed of morphogenesis. The fact that there is no growth inhibition with double forelimb removal in the aneurogenic larvae supports the idea that general body (systemic) effects are not involved. SERIES 5 Investigation of Contralateral Effects on Limb Regeneration in the Newt The foregoing series of experiments have presented evidence of contralateral effects on the trOphic role of the nerve in Ambystroma limb regeneration. Ambystoma has a large number of fibers crossing the spinal cord via the ventral commissure (Herrick and Coghill, 1915) that might be Signifi- cant in effecting these contralateral changes. It was there- fore of some interest to determine whether such pronounced contralateral effects on growth are also present in the newt, 55 .EE mmH.o u yHc: HmymaoyoHE H .coHymumcmmmu mo yaSOEm :H mocmumHMHp ycmoHMHcmHm o: pm3oam aoHy Imysmfim nmymm HH amp ym can ymmy D mmsyHaB cam: é .maEHHmnom N no H umayHm yo coHymysmfim maHBOHHom mm>HmH ESyMHSUmE maoywwaad oHcmmousmcm mo aymamH :H mmymy ayBOHm may amm3ym£ comHymmEoo < .m mHDmHm 54 m muomHh zo_._.<.—=._8¢ cur: m >2— Op I an? N 3923 O o 0. mp .z = or 55225.! 2. nuhczuzmoux 2523 ON so“ 55 NotOphthalmus (Triturus) viridescens, which is used widely in regeneration studies. Therefore, a growth study was designed to determine the rate of regeneration with the re- moval of 1 or 2 forelimbs in various combinations in the newt. It was also designed to test whether or not the transection of only the brachial nerves on one Side of the animal could bring about a Slower regeneration of the fore- limb of the Opposite Side. Procedure Forty adult newts were randomly divided into 4 grOUps. These were: A. Those with only 1 forelimb amputated and the Opposite forelimb Sham denervated by cutting through the skin over the brachial plexus. B. Those with 1 forelimb and 1 hindlimb amputated. C. Those with only 1 forelimb amputated and the fore- limb of the opposite Side of the animal denervated by cutting through the 5 brachial nerves at the brachial plexus. D. Those with both forelimbs amputated. All amputations were made as closely as possible at the same level of the humerus or femur. The rate of regeneration was by measurement of the growth of the regenerate in length beyond the original stump length and supplemented by morpho- genetic observations. For statistical purposes only one of the limbs of the double amputation group was measured. Only the forelimb of the group with both 1 forelimb and 1 hindlimb removed was measured. During the course of the experiment all 56 animals were fed every other day on sliced beef liver and maintained at laboratory temperature of 22 i_2°C. A few of the animals deveIOped mold infestation and were discarded from the experiment. Results Figure 7 Shows the mean regeneration in length of the limbs of the 4 groups of animals. As regeneration proceeds there appears a difference in growth rate whereby the animals in groups A and B Show faster linear regeneration than the animals in groups C and D. At 45 days after amputa- tion statistical analysis by a one-way analysis of variance and new multiple range test Shows that the apparent differences are significant (P < 0.05). Morphogenesis in the animals of groups C and D was found to lag behind those of the other 2 groups by about 5-4 days. Neither this difference in morpho- genesis nor the differences in linear growth were so clear- cut as was found in Ambystoma larvae. A part of the reason for this probably lies in the greater variation normally seen in the regeneration rates of a group of newts as compared to a group of axolotls. Thus, in the newt as well as in the axolotl contralateral double forelimb removal brings about a Slower linear regenera- tion rate than single forelimb removal. The fact that neural connections may be responsible for this phenomenon is strongly supported by the fact that Single forelimb amputation on the Side of an animal opposite a severence«of the brachial 57 .Q msoum CH amusmmme mmB QEHHmHOm H >Hco cam m mmsoum CH Umysmmma mm3 QEHHmHOy may cho memeam HmoHymHymym may you. .88 mNH.o u yHcs HmymEOHUHE H Jamo.o V.mv a cam U mmsoym away ayBOym ymmsHH ymymmym waycmoHyHamym Um>MHmmH© m 0cm d mmsoym ymay UmBOam coHymysmEm ymyym mwmp mm yo soy ymmy mmcmu mHmHstE 3ma can muamHHm> mo mHmSHmcm mm3tmco a pm>oamu maEHHmHOy ayoa .Q pmym>nmcmp QEHHmHom myHmommo .pm>OEmH QEHHmuOm H .0 pm>OEmH aEHHcha H cam QEHHmHoy H .m pmym>umcmp Emam QEHHmyom myHmommO .Um>OEmH QEHHmHOm H .< "mmyspmooum coHymysmEm e mcH3oHHom may mo H mcH3oHHom my3mc stpm yo maEHH mo aymamH coHymHmcmmmH HmmaHH cmma may yo OOmHymmEoo 4 .h mHDmHm a mysmym onHHm mH mquaaumy mcHHmEMm mas maEHHmuoy ayOQ .0 QEHHmHOm myHmOmmO yo aOHym>Hmcmp mDHm QEHHmHOH H .m AHOHySOUV QEHHmHom myHmommo yo aOHym>nmcmp Ewan msHm QEHHmuom H .d "no Hm>OEmH Hmymm mwmp 5 ya QEHH mec may CH mchHemay pmymHyHHy ayHB pmHmamH mHHmo yo ymaasa mo aomHymmEoo 4 .m magma 45 m. mHQMB No.0a u 24m: NH.H© a 24m: 45.0“ . zam: o m < F rrr - :31 ..... 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