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Retrograde degeneration of primary optic fibers in the cat
EXPERIMENTAL
44, 21-34 (1974)
NEUROLOGY
Retrograde
Degeneration HOMIN
Departlnerrts
of Anatomy
of Primary
Optic
LIN AND W. R. INGRAM and Iowa
Received
Fibers 1
Ophthalmology, The University City, Iowa 52242 February
in the Cat
of Iowa,
6,1974
Unilateral electrolytic lesions were placed in the optic tract of adult cats, midway between the optic chiasm and the ventral lateral geniculate nucleus, or in the terminal portions of the tract. Transection of one optic nerve was performed in other cases. The animals were maintained postoperatively for 2-21 days. A few cats with long-standing optic-nerve incision were also available. The brain and nerve sections were processed with three current silver methods for the study of axonal disintegration in the primary optic pathways distal (retinal) to the lesions. In all cases retrogression could be seen in those parts of the optic tract or nerve near the lesions (traumatic degeneration). The amount and extent of decomposition distally from the lesion site was related to the length of the postoperative survival and to the proximity of the lesion to the retina. These findings favor an interpretation involving retrograde deterioration of afferent (centripetal or retinofugal) optic fibers, and fail to confirm the often suggested existence of efferent (centrifugal or retinopetal) fibers in the cat.
INTRODUCTION In previous studies, all presumed origins of efferent (centrifugal or retinopetal) optic fibers proposed for mammals have been systematically tested in the cat (18, 19, 21) . Despite extensive lesions and relatively long postoperative durations (up to 21 days) no retinopetal Wallerian degeneration of optic efferents could be demonstrated in the subcortical visual pathways distal (peripheral or retinal) to the lesions. The only evidence of optic-fiber devolution was found immediately adjacent to the lesions in the tract or nerve. Thus, retrograde or traumatic decomposition, or both, of afferent (centripetal or retinofugal) optic fibers was thought to be responsible for the observed axonal changes (18, 21). To test this possibility further, a follow-up investigation was carried out with optic tract lesions and optic nerve sections, and the resultant axonal 1 Supported by NINDS
Grants NS08166, NS05249, and NS03354. 21
Copyright All rights
@ 1974 by of reproduction
AcademicPrw. Inc. in any
form
wwwd.
22
LIN
AND
INGRAM
degradation in the primary optic pathways distal to the lesions was examined after various survival times. These experiments have confirmed the occurrence of traumatic and retrograde degeneration of centripetal fibers but, by failing to produce evidence of Wallerian axonal disintegration rostra1 to lesions, offer no support for the notion of centrifugal fibers in the optic nerve. MATERIALS
AND
METHODS
Altogether 38 adult cats were operated on under sodium pentobarbital anesthesia. All operations were unilateral and on the left side. For optic tract surgery, unipolar electrodes were inserted vertically with stereotaxic localization. Lesions were produced in the optic tract midway between the optic chiasm and the ventral lateral geniculate in 12 cats with 7, 10, 12, or 14 days of survival. In five other cats, allowed to survive for 12, 14, or 21 days, the lesions were made in the central (proximal or terminal) portions of the tract at the level of the ventral lateral geniculate nucleus. In most cases several lesions were placed at adjacent loci and the electrodes were moved up and down to ensure extensive damage to the tract. An intraorbital optic nerve transection (18) was performed upon 1S cats which were autopsied 2, 4, 7, 9, 10, 12, 14, or 21 days after surgery. Three additional cats with optic nerve transection were allowed to survive for 2, 3, or 4 mo. At autopsy the brains were perfused with saline, followed by fixation with 10% cacodylate-buffered formalin-sucrose solution (18). The optic nerves were separated from the brain just distal to the chiasm, or at the incision site. Frozen sections of the brains were cut at 30pm thickness frontally, parasagittally, or horizontally. The nerves, along with the posterior portions of the eyeball, were cut at the same thickness parallel to their long axis. Most of these sections were processed with the de Olmos-Ingram and the Nauta methods, but some were impregnated according to the Fink-Heimer technique (18). For each staining procedure, efforts were made to bring out maximal amounts of degeneration, or subjecting alternating sections to different degrees of the “oxidation” called for by this category of techniques, in the hope that comparison of variously prepared specimens might be more objective. The primary optic pathways referred to in this report include the optic tract, chiasm, and nerve peripheral to the lesions. Those parts central (cerebral) to the optic axotomy will not be considered. The optic nerves contralateral to those operated upon were used as normal controls, since previous experiments with the same silver techniques failed to disclose direct interretinal connections (22).
RETROGRADE
23
DEGENERATION
FIG. 1. Un’operated optic nerve controls. De Olmos-Ingram preparations, except (D) which is a Nauta preparation. X 250. Only connective tissue fibers are stained while axons are not. Random silver granules are present. See text for details. All these and subsequent pictures of the optic nerve have been oriented so that the retinal end is at the top. (A) The middle portion of the optic nerve. (B) The distal portion just central (cerebral) to the lamina cribrosa. (C) The upper half shows the lamina cribrosa while the lower portion is the optic nerve proper. (D) The upper third of the field shows axons of the retina which are unstained. Lower third is the optic nerve
proper
containing
silver
deposits.
Horizontal
middle third of the field (lamina cribrosa).
connective
tissue
fibers
appear
in
the
24
LIN
AND
INGRAM
FIG. 2. Examples of the lesions. Nissl preparations. X 8. (A) Coronal section of a cat brain with lesions in the ventral lateral geniculate nucleus. (B) Horizontal section of a cat brain with lesions in the middle portion of the left optic tract. Rostra1 end to the right and the midline below. CP, cerebral peduncle; LGHd, dorsal lateral geniculate nucleus ; OT, optic tract.
RESULTS Control Optic Nerves. The general picture varied with the cat, section, and the impregnation technique. Such preparations were essential in order to minimize possible misidentification of normal as well as degenerated axons and their terminals (also seeRef. 12). A brief account follows. The connective tissue fibers associated with blood vessels and interfascicular septa were stained selectively, and darkly when stained, particularly with the de Olmos-Ingram and the Fink-Heimer methods (Fig. lA-C). They could be distinguished from nerve fibers by their often larger caliber, wavier course, granular appearance, and occasional branching (Fig. lA-C) . Their special locations and often transverse or oblique directions across the course of retinal fibers supplied additional criteria for their identification (Fig. 1). The background was generally clear in de Olmos-Ingram material, with only sporadic heavily impregnated normal axons (one or two per section at most), while a few to numerous fibers were stained in FinkHeimer or Nauta specimens. Random silver deposits, present with any of the three techniques, were associated in particular with cut edges, tissue cracks, and in and around the lamina cribrosa (Fig. lC, D). In the latter, connective tissue fibers often mimicked axonal disintegration (Fig. lC, D). They could usually be differentiated by their perpendicular relationships to optic fibers or by their congruence with blood vessels. No axons or their terminal boutons were impregnated in retinal layers or the lamina cribrosa except
RETROGRADE
DEGENERATION
25
FIG. 3. De Olmos-Ingram preparations from the same cat (Z-day survival) as in Fig. ZA, with the same orientation. X 190. (A) The optic tract just distal (retinal) to the lesion, showing profuse axonal degeneration which decreases drastically peripheral to this point. (B) Some sparse disintegration in the proximal part of the contralateral optic nerve, which is absent in the more distal portion of the nerve (not shown).
for sporadic short fragments. Random argyrophilic granules often were seenin the retina without apparent relationship to any nervous elements or layers. Connective tissue fibers were also stained occasionally, especially those affiliated with blood vessels which were interposed among retinal neurons or layers. Cats with Lesions at the Ventral Lateral Geniculate. The lesions varied in size and location slightly from one cat to another. The ventral lateral geniculate and portions of the optic tract were partially or totally destroyed (Fig. 2A). Encroachments upon other areas and electrode tracks should pose no complication in the following discussion, since such damage could cause degradation of optic fibers only in a limited region of the tract near the lesions (21). This area was largely covered by the present electrocoagulations. All de Olmos-Ingram preparations showed intensive axonal breakdown in the proximal portion of the optic tract near the lesions (Fig. 3A). In two cats (14 days of survival), little or no degeneration was observed in the distal part of the tract and farther peripherally, although massive fiber degeneration appeared in the supraoptic commissures. In two others (1214 days), in which lesions were more distally placed, some fiber disintegration was also seen in the more retinal aspects of optic pathways, excluding the distal moieties of optic nerves. In the Zl-day animal, the decomposition
26
LIN
AND
INGRAM
FIG. 4. De Olmos-Ingram specimen from the same cat (E-day survival) as in Fig. ZB, with the same orientation. (A) A section taken from the dorsalmost level of the optic chiasm on the side contralateral to the lesion. The chiasm appears in the central and upper-right portions of the field, and contains profuse degeneration as well as “normal” fibers. The contralateral optic tract (OT) and hypothalamus (H) are free of stained axons of any kind at this level. X 30. (B) Higher magnification of the same optic chiasm. X 190.
in the optic pathways was quite profuse, with a diminution toward, and absence at, the ocular end of the nerves (Fig. 3B). Nauta-stained material of these cats revealed little or no regression in more distal pathways, while that in the tract next to lesions was rather immense. Neither normal nor disintegrated fibers or terminal boutons were ever detected with certainty in the lamina cribrosa and beyond in the retina. The same was true for the following experimental cats, and will not be mentioned repetitively. Hence, the optic nerve in the subsequent presentation does not include the lamina cribrosa. Cats with Lesions in the Middle Portion of the Optic Tract. Brain injuries varied somewhat from cat to cat with respect to size and location. The tract was totally or nearly transected, judging by serial Nissl sections (Fig. 2B). Coincidental damage outside the classical optic pathways included such regions as the hypothalamus and the anterior accessory optic tract (19). These effects could be ignored since lesions involving these structures could not produce demonstrable disintegration in the distal visual pathways with the same silver-impregnation techniques as here employed (18, 19).
FIG. 5. Nauta preparations from the brain of cat 37, which underwent left opti: tract lesions 12 days before autopsy. Coronal section.(A) A fragment of the tract isolated by lesions from the hypothalamus (H) and optic chiasm (OC). X 30. (B) Higher magnification of a portion of the isolated tract fragment to show “preserved” fibers. X 170.
Silver preparations of each brain showed profuse fiber degeneration in the ipsilateral optic tract just distal to the lesions, as well as in the supraoptic commissures. Various amounts could be traced from lesions in
FIG. 6. The optic nerve of a cat 4 days after its transection. preparation. (A) The portion just distal (peripheral) to the cut, axonal degradation and its debris. X 250. (B) The same nerve distal to (A), without apparent difference from controls (Fig. 1).
De Olmos-Ingram depicting traumatic section immediately X 100.
28
LIN
AND
INGRAM
FIG. 7. Profuseretrogradedeteriorationin the optic nerve of a cat 21 daysafter its axotomy. Taken just central to the laminacribrosa. De Olmos-Ingrampreparation. x 250.
the visual pathways for some distance. In de Olmos-Ingram specimenswith 7-10 days of survival, a moderate number of apparently degenerating fibers could be followed to the optic chiasm. In the optic nerve, such fibers were either absent or confined to the proximal portion. For cats living 12 days or longer, the deterioration patterns in the corresponding regions became profuse (Fig. 4). The devolution in the optic nerve could be demonstrated either throughout the specimen, with concentration near the proximal end, or restricted to the central portions of the nerves. Nauta preparations revealed little or no retrogression in the optic nerves, except in those of cat 37 (12-day survival) in which the lesions also damaged the lateral aspect of the chiasm (Fig. 5A). Part of the optic tract in this cat was completely isolated by electrocoagulations from the rest of the brain (Fig. 5A), and contained many normal-appearing fibers (Fig. 5B). These were likely “preserved” fibers as discussedelsewhere (20). In seven cats killed 2-7 days after Cats with Optic Nerve Transection. the operation axonal decomposition and debris were observed near the lesions (Fig. 6A), the amount of which could not be correlated with postoperative life durations. The remainder of the optic nerve in such cases was similar to the control (Fig. 6B). This was also true for one of the two animals which survived 9 days. In the other, a little degradation extended
RETROGRADE
DEGENERATION
29
slightly toward the eye as stained in de Olmos-Ingram and Fink-Heimer preparations, but did not appear in the Nauta material. Profuse devolution throughout the distal segment of the optic nerve began with the tenth postoperative day. Such deterioration in general intensified with increasing degeneration times and tapered toward, and disappeared in, the retinal end of the nerve. Only in animals surviving for 14 days or longer was the disintegration evenly distributed throughout the nerve (Fig. 7). The degeneration attained a maximum quantitatively at the twenty-first day and leveled off thereafter (Fig. 7). DISCUSSION The chief advantages of the de Olmos-Ingram method (19, 22) could be substantiated by the present results. The near-total suppression of normal axons (Figs. 1, 4A, 6B) and the fine sensitivity for degeneration were invariably observed. The Fink-Heimer technique seemed to be sensitive to a similar degree. However, positive identification of degraded fibers was often difficult in Fink-Heimer preparations especially when their number was small, because of the background normal fibers. The Nauta procedure was less sensitive (19, 22, 24) in that it required longer survival times to reveal degeneration, except for regions near lesions, and the disintegration when impregnated was often less conspicuous. Another merit of the de Olmos-Ingram method is that, with due experience and controls, slightly hypertrophic, darkly stained “normal” axons could be taken to indicate developing Wallerian disintegration (19, 22). The same holds true for the retrograde decomposition of centripetal optic fibers which should be obvious by consulting the present illustrations, particularly Fig. 4. Traumatic degeneration has been defined and discussed before (20-22). To reiterate briefly, this is a type of degeneration which could occur soon on either side of axonal injury for a short distance. Restricted distribution of such disturbance was noted in the peripheral optic nerve segments of cats up to 9 days after optic nerve transection (Fig. 6)) and in the optic tract of cats with optic tract or lateral geniculate lesions up to 10 and 14 days, respectively. A comparable condition has been observed in the central segment of the optic nerve some time before the characteristic anterograde changes of optic afferents could be demonstrated by the silver methods here used (22). Therefore, such early and limited degeneration must be traumatic according to definition. Whether Wallerian degradation in general commences near the nerve injury, or proceeds from the terminal toward the cut, or uniformly throughout the nerve segment is in dispute (5, 7, 10, 13, 22). Nevertheless, it is highly questionable that Wallerian degeneration of optic efferents could begin with the second day, be confined to a limited area, and stabilized over the next week or longer in quantity and distribution.
30
LIN
AND
INGRAM
Whether traumatic disturbance as here discussed represents a prelude to retrograde disintegration of centripetal optic fibers or a distinct variety of deterioration is a matter of definition. We did see degeneration spreading toward the eye from the lesion in other cats with longer survivals. Despite variables affecting the impregnation of axonal degradation (12, 19, 24), several generalizations can be derived from our experimental results. First, degeneration was present in all casesin that part of the optic tract or nerve near the lesionsdue to traumatic effects. Second, the observed disintegration tapered from the lesions rostrad in the visual pathways of cats with longer survivals. Third, this retrogression varied with postoperative intervals and the locations of lesions; viz., the closer the lesions to the retina and the longer the survival the more intensive and wider the distribution of the degeneration became. This was evident not only when comparing the three experimental groups, but also among animals in the same group. Such differences could be interpreted as evidence of retrograde changes (3, 5, 14) of optic afferents, but seem incompatible with a Wallerian degeneration of centrifugal fibers in the optic nerve. Finally, orthograde degradation of cat afferent axons shown with the same impregnation procedures starts near optic centers and propagates toward the cut (22). One would expect efferent fibers in the optic nerve to possessa similar property. However, the present findings suggest a centrifugal spread of the degenerative process, making the occurrence of efferents more unlikely. The problem of retrograde axonal degeneration, or the lack of it, has been the subject of recent reviews (3, 5). “There is no consistent model or law applicable to all portions of the nervous system” (5). In addition to numerous factors shaping the fate of this part of axons, traumatic degeneration has contributed to the confusion (3, 5). It is reasonably well establishedthat silver impregnation of retrograde degeneration of immature CNS fibers progresses somatofugally (9, 19). This generality does not apply to adult animals however. The existing literature shows that retrograde axonal decomposition may proceed somatofugally or somatopetally (3, 5, 14). Possible explanation for this disparity between newborn and adult animals is that rapid cell death after axotomy is the prerequisite for the also rapid retrograde fiber reaction in the former (9) whereas in adult animals retrograde neuronal changes may have a wide range of manifestations, and some perikarya may even achieve morphological and functional recovery (3, 5, 17). B eresford (3) formulated an interesting hypothesis to account for both cellulipetal and cellulifugal progression of retrograde axonal breakdown based on relative pressure and nutritional deficiency among cell bodies and different portions of the transected fibers. In this context our observation of retinopetal spread of disintegration should not be surprising. There is ample evidence that retinal ganglion cells and their
RETROGRADE
DEGENERATION
31
connecting axons may persist for a long time after optic nerve injury, provided that the lesi,on is not too close to the retina and that the retinal blood supply is not impaired (20). It is granted that Wallerian breakdown of mammalian centrifugal optic fibers might take a little longer than the time ordinarily optimal for the Wallerian devolution of other CNS fibers (4, 6). However, such a suggestion fails to explain the retinopetal progression of disintegration in our cats up to 14 days after optic nerve transection and the continuing increase in the amount of degeneration up to 21 days in all three operation groups. Neither could it account for the late onset and confined occurrence of degeneration in the optic nerves of cats with lesions of the optic tract, and the complete absence of such degradation after all presumed origins were destroyed in cats with survivals up to 21 days (18, 19, 21). All these could, nonetheless, be accounted for by a retrograde reaction (3, 5, 14) of retinofugal optic fibers. In most cats with optic nerve lesions, the two segments of the cut nerve were connected by scar tissue, and thus could be processed simultaneously. Hence, the effects due to staining variations ( 12, 24) could be minimized and a more objective comparison of the degenerative changes was possible. As noted previously (22) the degradation central (cerebral) to the incision reached a plateau in 9-10 days, and gradually decreased beginning with the third postoperative month. The amount of argyrophilic debris was insignificant after 6 mo. On the other hand, the degeneration in the distal segment as here described arrived at a maximum in 3 wk without apparent diminution throughout the period of observation. There were times when the degradation visible in the distal segment nearly equaled (from 3 wk to 3 mo) or actually exceeded (after 3 mo) the amount present in the proximal segment. This difference in evolution of the degenerative process, likewise, suggests a retrograde disintegration of optic afferent fibers rather than a Wallerian degeneration of centrifugal axons in the optic nerve. Since mammalian optic efferents, if they exist at all, should constitute only a small percentage of the fiber population of the optic nerve, one can hardly expect their Wallerian degeneration to match or exceed the combined Wallerian disintegration of afferents and retrograde changes of efferents. Furthermore, we know of no evidence that Wallerian degeneration takes place later than, and outlasts, retrograde alteration of the same fibers. Our results are in essential agreement with those of others who found no retinopetal degeneration of optic efferents in the cat 14 days after extensive damage of the dorsal lateral geniculate and adjacent optic tract (23), or up to 10 days after intracranial optic nerve transection (4, 11). of Leinfelder (15, 16) noted different spatial and temporal distributions optic fiber degradation in the cat and monkey distal to optic nerve incision and tract lesions at two loci. He also found that bilateral tract injuries
32
LIN
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INGRAM
produced much less degeneration in the nerve than nerve axotomy in animals with corresponding survival times. None of these findings lends any support for the idea of centrifugal optic fibers. The argument that some such fibers might come from the hypothalamus or mesencephalic tegmentum without coursing through the optic tract also lacks a sound basis (18, 19). Cragg (6) crushed the rabbit optic nerve near the optic foramen and described Wallerian disintegration of efferents in the nerve and retina with the use of the Nauta and Glees techniques. Such degeneration first appeared in 8-10 days. As discussed before, his results were probably artifactual, or represented a retrograde degradation of optic afferents (21). We could not detect any significant normal or degenerated fibers or boutons in either the lamina cribrosa or the retina in any of the normal controls or experimental specimens. Hedreen (11) also failed to find evidence for centrifugal axons in the cat retina in experiments by the methods of Nauta and Fink-Heimer. We call attention to possible sources of misinterpretation of degeneration as exemplified by our control optic nerves (Fig. 1) . Granit (8) believed that if optic efferents were present in mammals at all, the ventral lateral geniculate nucleus would likely be their site of cellular origin, since the latter is closest to the retina among all visual centers. Astruc (1, 2) reported orthograde dissolution of cat centrifugal fibers in distal visual pathways with the Nauta technique after this nucleus was damaged. However, strangely enough, he noted that such disintegrated fibers were very difficult to stain in the optic nerve peripheral to its transection. No postoperative intervals were given by Astruc to warrant meaningful comments on his results. Our own experiments produced no evidence of such efferent degradation. This article concludes our present investigations on the presumed centrifugal optic fibers of the cat (18-22). The most important findings of the series are briefly recapitulated below. With a “full-impregnation” silver method (Bodian’s technique), overt axonal breakdown in the distal segment of the optic nerve could not be seen 3 mo after its axotomy, in contrast to the degeneration in the proximal segment which started at the seventh day (20). Fifteen months after surgery numerous normal fibers still persisted in the distal segment while those in the proximal had long vanished (20). The observations made by the aid of current silver techniques suggest that the degree of axonal degeneration in the peripheral pathways is a function of postoperative duration and of the distance of the lesion from the retina (this study). None of these observations can substantiate the concept of centrifugal fibers in the optic nerve. Even though plausible hints seemed present as to their possible origins (22), all pertinent neural entities have been tested with negative results (18, 19, 21, present report). Thus, it must be concluded that the currently available fiber-
RETROGRADE
degeneration the retina.
methods
DEGENERATION
are unable to demonstrate
33 fibers from the brain to
REFERENCES 1. ASTRUC, J. 1964. Fibres centrifuges de la r&tine. C. R. Ass. Anat. 49: 212-213. 2. ASTRUC, J. 1968. Interconnections of the lateral geniculate bodies through the ventral supraoptic commissure. Anat. Rec. 160 : 309. 3. BERESFORD,W. A. 1965. A discussion on retrograde changes in nerve fibers. Progr. Bra& Res. 14 : 33-56. 4. BRINDLEY, G. S., and D. I. HAMASAKI. 1966. Histological evidence against the view that the cat’s optic nerve contains centrifugal fibers. J. Physiol. 184: 444449. 5. COLE, M. 1968. Retrograde degeneration of axon and soma in the nervous system, pp. 269-300. In “Structure and Function of Nervous Tissue.” G. H. Bourne [Ed.]. Academic Press, New York. 6. CRAGG, B. G. 1962. Centrifugal fibers to the retina and olfactory bulb, and composition of the supraoptic commissures in the rabbit. Exp. Neztrol. 5: 46 427. 7. DONAT, J. R., and H. M. WISNIEWSKI. 1973. The spatio-temporal pattern of Wallerian degeneration in mammalian peripheral nerves. Brain, Res. 53: 41-54. 8. GRANIT, R. 1962. Retina and optic nerve, pp. 541-574. In “The Eye.” Vol. 2. The Visual Process. H. Davson [Ed.]. Academic Press, New York. 9. GRANT, G. 1970. Neuronal changes central to the site of axonal transection. A method for the identification of retrograde changes in perikarya, dendrites and axons by silver impregnation, pp. 173-185. In “Contemporary Research Methods in Neuroanatomy.” W. J. H. Nauta and S. 0. E. Ebbesson [Ed.]. SpringerVerlag, New York. 10. GUILLERY, R. W. 1970, Light- and electron-microscopical studies of normal and degenerating axons, pp. 77-105. In. “Contemporary Research Methods in Neuroanatomy.” W. J. H. Nauta and S. 0. E. Ebbesson [Eds.]. Springer-Verlag, New York. 11. HEDREEN, J. 1969. Patterns of axon terminal degeneration after optic nerve section in cats. Avtat. Rec. 163: 198. 12. HEIMER, L. 1970. Selective silver-impregnation of degenerating axoplasm, pp. 106-131. In “Contemporary Research Methods in Neuroanatomy.” W. J. H. Nauta and S. 0. E. Ebbesson [Eds.]. Springer-Verlag, New York. 13. JOSEPH, B. S., and D. G. WHITLOCK. 1972. The spatio-temporal course of Wallerian degeneration within the CNS of toads (Bufo marinzcs) as defined by the Nauta silver method. Brain Behaz~. Evol. 5: l-17. 14. KALIL, K. 1973. Retrograde axon degeneration in the pyramidal tract of the hamster. Anat. Rec. 175 : 352. 1.5. LEINFELDER, P. J. 1938. Retrograde degeneration in the optic nerves and retinal ganglion cells. Trans. Amer. Ophthalmol. Sot. 36: 307-316. 16. LEINFELDER, P. J. 1940. Retrograde degeneration in the optic nerves and tracts. An experimental study of changes in the axis cylinders. Amer. .I. Ophthalmol. 23: 796-802. 17. LIEBERMAN, A. R. 1971. The axon reaction: a review of the principal features of perikaryal responses to axon injury. 1st. Rev. Neurobiol. 14: 50-124. 18. LIN, H., and W. R. INGRAM. 1972. Probable absence of connections between the retina and the hypothalamus in the cat. Exp. Nezcrol. 37: 23-36.
34
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19. LIN,
20. 21. 22. 23. 24.
AND
INGRAM
H., and W. R. INGRAM. 1972. An anterior component of the accessory optic system of the cat, with evidence for the absence of reticuloretinal fibers. Exp. Neurol. 37: 37-49. LIN, H., and W. R. INGRAM. 1973. Axonal degeneration in the peripheral optic pathway of the cat. Exp. Newel. 39: 234-248. LIN, H., and W. R. INGRAM. 1973. A search for centrifugal optic fibers in the cat. Bull. Imt. Zool. Acad. Sin. 12 : 51-57. LIN, H., and W. R. INGRAM. 1974. Spatial and temporal distribution of axonal degeneration in the primary optic system of the cat. Exp. Neural. 44: l&20. MAGOUN, H. W., and M. RANSON. 1942. The supraoptic decussations in the cat and monkey. J. Co)np. Newel. 76: 435-459. POWELL, E. W., and R. SCHNURR. 1972. Silver impregnation of degenerating axons ; comparisons of postoperative intervals, fixatives and staining methods. Stain Technol. 47 : 95-100.
44, 21-34 (1974)
NEUROLOGY
Retrograde
Degeneration HOMIN
Departlnerrts
of Anatomy
of Primary
Optic
LIN AND W. R. INGRAM and Iowa
Received
Fibers 1
Ophthalmology, The University City, Iowa 52242 February
in the Cat
of Iowa,
6,1974
Unilateral electrolytic lesions were placed in the optic tract of adult cats, midway between the optic chiasm and the ventral lateral geniculate nucleus, or in the terminal portions of the tract. Transection of one optic nerve was performed in other cases. The animals were maintained postoperatively for 2-21 days. A few cats with long-standing optic-nerve incision were also available. The brain and nerve sections were processed with three current silver methods for the study of axonal disintegration in the primary optic pathways distal (retinal) to the lesions. In all cases retrogression could be seen in those parts of the optic tract or nerve near the lesions (traumatic degeneration). The amount and extent of decomposition distally from the lesion site was related to the length of the postoperative survival and to the proximity of the lesion to the retina. These findings favor an interpretation involving retrograde deterioration of afferent (centripetal or retinofugal) optic fibers, and fail to confirm the often suggested existence of efferent (centrifugal or retinopetal) fibers in the cat.
INTRODUCTION In previous studies, all presumed origins of efferent (centrifugal or retinopetal) optic fibers proposed for mammals have been systematically tested in the cat (18, 19, 21) . Despite extensive lesions and relatively long postoperative durations (up to 21 days) no retinopetal Wallerian degeneration of optic efferents could be demonstrated in the subcortical visual pathways distal (peripheral or retinal) to the lesions. The only evidence of optic-fiber devolution was found immediately adjacent to the lesions in the tract or nerve. Thus, retrograde or traumatic decomposition, or both, of afferent (centripetal or retinofugal) optic fibers was thought to be responsible for the observed axonal changes (18, 21). To test this possibility further, a follow-up investigation was carried out with optic tract lesions and optic nerve sections, and the resultant axonal 1 Supported by NINDS
Grants NS08166, NS05249, and NS03354. 21
Copyright All rights
@ 1974 by of reproduction
AcademicPrw. Inc. in any
form
wwwd.
22
LIN
AND
INGRAM
degradation in the primary optic pathways distal to the lesions was examined after various survival times. These experiments have confirmed the occurrence of traumatic and retrograde degeneration of centripetal fibers but, by failing to produce evidence of Wallerian axonal disintegration rostra1 to lesions, offer no support for the notion of centrifugal fibers in the optic nerve. MATERIALS
AND
METHODS
Altogether 38 adult cats were operated on under sodium pentobarbital anesthesia. All operations were unilateral and on the left side. For optic tract surgery, unipolar electrodes were inserted vertically with stereotaxic localization. Lesions were produced in the optic tract midway between the optic chiasm and the ventral lateral geniculate in 12 cats with 7, 10, 12, or 14 days of survival. In five other cats, allowed to survive for 12, 14, or 21 days, the lesions were made in the central (proximal or terminal) portions of the tract at the level of the ventral lateral geniculate nucleus. In most cases several lesions were placed at adjacent loci and the electrodes were moved up and down to ensure extensive damage to the tract. An intraorbital optic nerve transection (18) was performed upon 1S cats which were autopsied 2, 4, 7, 9, 10, 12, 14, or 21 days after surgery. Three additional cats with optic nerve transection were allowed to survive for 2, 3, or 4 mo. At autopsy the brains were perfused with saline, followed by fixation with 10% cacodylate-buffered formalin-sucrose solution (18). The optic nerves were separated from the brain just distal to the chiasm, or at the incision site. Frozen sections of the brains were cut at 30pm thickness frontally, parasagittally, or horizontally. The nerves, along with the posterior portions of the eyeball, were cut at the same thickness parallel to their long axis. Most of these sections were processed with the de Olmos-Ingram and the Nauta methods, but some were impregnated according to the Fink-Heimer technique (18). For each staining procedure, efforts were made to bring out maximal amounts of degeneration, or subjecting alternating sections to different degrees of the “oxidation” called for by this category of techniques, in the hope that comparison of variously prepared specimens might be more objective. The primary optic pathways referred to in this report include the optic tract, chiasm, and nerve peripheral to the lesions. Those parts central (cerebral) to the optic axotomy will not be considered. The optic nerves contralateral to those operated upon were used as normal controls, since previous experiments with the same silver techniques failed to disclose direct interretinal connections (22).
RETROGRADE
23
DEGENERATION
FIG. 1. Un’operated optic nerve controls. De Olmos-Ingram preparations, except (D) which is a Nauta preparation. X 250. Only connective tissue fibers are stained while axons are not. Random silver granules are present. See text for details. All these and subsequent pictures of the optic nerve have been oriented so that the retinal end is at the top. (A) The middle portion of the optic nerve. (B) The distal portion just central (cerebral) to the lamina cribrosa. (C) The upper half shows the lamina cribrosa while the lower portion is the optic nerve proper. (D) The upper third of the field shows axons of the retina which are unstained. Lower third is the optic nerve
proper
containing
silver
deposits.
Horizontal
middle third of the field (lamina cribrosa).
connective
tissue
fibers
appear
in
the
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FIG. 2. Examples of the lesions. Nissl preparations. X 8. (A) Coronal section of a cat brain with lesions in the ventral lateral geniculate nucleus. (B) Horizontal section of a cat brain with lesions in the middle portion of the left optic tract. Rostra1 end to the right and the midline below. CP, cerebral peduncle; LGHd, dorsal lateral geniculate nucleus ; OT, optic tract.
RESULTS Control Optic Nerves. The general picture varied with the cat, section, and the impregnation technique. Such preparations were essential in order to minimize possible misidentification of normal as well as degenerated axons and their terminals (also seeRef. 12). A brief account follows. The connective tissue fibers associated with blood vessels and interfascicular septa were stained selectively, and darkly when stained, particularly with the de Olmos-Ingram and the Fink-Heimer methods (Fig. lA-C). They could be distinguished from nerve fibers by their often larger caliber, wavier course, granular appearance, and occasional branching (Fig. lA-C) . Their special locations and often transverse or oblique directions across the course of retinal fibers supplied additional criteria for their identification (Fig. 1). The background was generally clear in de Olmos-Ingram material, with only sporadic heavily impregnated normal axons (one or two per section at most), while a few to numerous fibers were stained in FinkHeimer or Nauta specimens. Random silver deposits, present with any of the three techniques, were associated in particular with cut edges, tissue cracks, and in and around the lamina cribrosa (Fig. lC, D). In the latter, connective tissue fibers often mimicked axonal disintegration (Fig. lC, D). They could usually be differentiated by their perpendicular relationships to optic fibers or by their congruence with blood vessels. No axons or their terminal boutons were impregnated in retinal layers or the lamina cribrosa except
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25
FIG. 3. De Olmos-Ingram preparations from the same cat (Z-day survival) as in Fig. ZA, with the same orientation. X 190. (A) The optic tract just distal (retinal) to the lesion, showing profuse axonal degeneration which decreases drastically peripheral to this point. (B) Some sparse disintegration in the proximal part of the contralateral optic nerve, which is absent in the more distal portion of the nerve (not shown).
for sporadic short fragments. Random argyrophilic granules often were seenin the retina without apparent relationship to any nervous elements or layers. Connective tissue fibers were also stained occasionally, especially those affiliated with blood vessels which were interposed among retinal neurons or layers. Cats with Lesions at the Ventral Lateral Geniculate. The lesions varied in size and location slightly from one cat to another. The ventral lateral geniculate and portions of the optic tract were partially or totally destroyed (Fig. 2A). Encroachments upon other areas and electrode tracks should pose no complication in the following discussion, since such damage could cause degradation of optic fibers only in a limited region of the tract near the lesions (21). This area was largely covered by the present electrocoagulations. All de Olmos-Ingram preparations showed intensive axonal breakdown in the proximal portion of the optic tract near the lesions (Fig. 3A). In two cats (14 days of survival), little or no degeneration was observed in the distal part of the tract and farther peripherally, although massive fiber degeneration appeared in the supraoptic commissures. In two others (1214 days), in which lesions were more distally placed, some fiber disintegration was also seen in the more retinal aspects of optic pathways, excluding the distal moieties of optic nerves. In the Zl-day animal, the decomposition
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FIG. 4. De Olmos-Ingram specimen from the same cat (E-day survival) as in Fig. ZB, with the same orientation. (A) A section taken from the dorsalmost level of the optic chiasm on the side contralateral to the lesion. The chiasm appears in the central and upper-right portions of the field, and contains profuse degeneration as well as “normal” fibers. The contralateral optic tract (OT) and hypothalamus (H) are free of stained axons of any kind at this level. X 30. (B) Higher magnification of the same optic chiasm. X 190.
in the optic pathways was quite profuse, with a diminution toward, and absence at, the ocular end of the nerves (Fig. 3B). Nauta-stained material of these cats revealed little or no regression in more distal pathways, while that in the tract next to lesions was rather immense. Neither normal nor disintegrated fibers or terminal boutons were ever detected with certainty in the lamina cribrosa and beyond in the retina. The same was true for the following experimental cats, and will not be mentioned repetitively. Hence, the optic nerve in the subsequent presentation does not include the lamina cribrosa. Cats with Lesions in the Middle Portion of the Optic Tract. Brain injuries varied somewhat from cat to cat with respect to size and location. The tract was totally or nearly transected, judging by serial Nissl sections (Fig. 2B). Coincidental damage outside the classical optic pathways included such regions as the hypothalamus and the anterior accessory optic tract (19). These effects could be ignored since lesions involving these structures could not produce demonstrable disintegration in the distal visual pathways with the same silver-impregnation techniques as here employed (18, 19).
FIG. 5. Nauta preparations from the brain of cat 37, which underwent left opti: tract lesions 12 days before autopsy. Coronal section.(A) A fragment of the tract isolated by lesions from the hypothalamus (H) and optic chiasm (OC). X 30. (B) Higher magnification of a portion of the isolated tract fragment to show “preserved” fibers. X 170.
Silver preparations of each brain showed profuse fiber degeneration in the ipsilateral optic tract just distal to the lesions, as well as in the supraoptic commissures. Various amounts could be traced from lesions in
FIG. 6. The optic nerve of a cat 4 days after its transection. preparation. (A) The portion just distal (peripheral) to the cut, axonal degradation and its debris. X 250. (B) The same nerve distal to (A), without apparent difference from controls (Fig. 1).
De Olmos-Ingram depicting traumatic section immediately X 100.
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FIG. 7. Profuseretrogradedeteriorationin the optic nerve of a cat 21 daysafter its axotomy. Taken just central to the laminacribrosa. De Olmos-Ingrampreparation. x 250.
the visual pathways for some distance. In de Olmos-Ingram specimenswith 7-10 days of survival, a moderate number of apparently degenerating fibers could be followed to the optic chiasm. In the optic nerve, such fibers were either absent or confined to the proximal portion. For cats living 12 days or longer, the deterioration patterns in the corresponding regions became profuse (Fig. 4). The devolution in the optic nerve could be demonstrated either throughout the specimen, with concentration near the proximal end, or restricted to the central portions of the nerves. Nauta preparations revealed little or no retrogression in the optic nerves, except in those of cat 37 (12-day survival) in which the lesions also damaged the lateral aspect of the chiasm (Fig. 5A). Part of the optic tract in this cat was completely isolated by electrocoagulations from the rest of the brain (Fig. 5A), and contained many normal-appearing fibers (Fig. 5B). These were likely “preserved” fibers as discussedelsewhere (20). In seven cats killed 2-7 days after Cats with Optic Nerve Transection. the operation axonal decomposition and debris were observed near the lesions (Fig. 6A), the amount of which could not be correlated with postoperative life durations. The remainder of the optic nerve in such cases was similar to the control (Fig. 6B). This was also true for one of the two animals which survived 9 days. In the other, a little degradation extended
RETROGRADE
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29
slightly toward the eye as stained in de Olmos-Ingram and Fink-Heimer preparations, but did not appear in the Nauta material. Profuse devolution throughout the distal segment of the optic nerve began with the tenth postoperative day. Such deterioration in general intensified with increasing degeneration times and tapered toward, and disappeared in, the retinal end of the nerve. Only in animals surviving for 14 days or longer was the disintegration evenly distributed throughout the nerve (Fig. 7). The degeneration attained a maximum quantitatively at the twenty-first day and leveled off thereafter (Fig. 7). DISCUSSION The chief advantages of the de Olmos-Ingram method (19, 22) could be substantiated by the present results. The near-total suppression of normal axons (Figs. 1, 4A, 6B) and the fine sensitivity for degeneration were invariably observed. The Fink-Heimer technique seemed to be sensitive to a similar degree. However, positive identification of degraded fibers was often difficult in Fink-Heimer preparations especially when their number was small, because of the background normal fibers. The Nauta procedure was less sensitive (19, 22, 24) in that it required longer survival times to reveal degeneration, except for regions near lesions, and the disintegration when impregnated was often less conspicuous. Another merit of the de Olmos-Ingram method is that, with due experience and controls, slightly hypertrophic, darkly stained “normal” axons could be taken to indicate developing Wallerian disintegration (19, 22). The same holds true for the retrograde decomposition of centripetal optic fibers which should be obvious by consulting the present illustrations, particularly Fig. 4. Traumatic degeneration has been defined and discussed before (20-22). To reiterate briefly, this is a type of degeneration which could occur soon on either side of axonal injury for a short distance. Restricted distribution of such disturbance was noted in the peripheral optic nerve segments of cats up to 9 days after optic nerve transection (Fig. 6)) and in the optic tract of cats with optic tract or lateral geniculate lesions up to 10 and 14 days, respectively. A comparable condition has been observed in the central segment of the optic nerve some time before the characteristic anterograde changes of optic afferents could be demonstrated by the silver methods here used (22). Therefore, such early and limited degeneration must be traumatic according to definition. Whether Wallerian degradation in general commences near the nerve injury, or proceeds from the terminal toward the cut, or uniformly throughout the nerve segment is in dispute (5, 7, 10, 13, 22). Nevertheless, it is highly questionable that Wallerian degeneration of optic efferents could begin with the second day, be confined to a limited area, and stabilized over the next week or longer in quantity and distribution.
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Whether traumatic disturbance as here discussed represents a prelude to retrograde disintegration of centripetal optic fibers or a distinct variety of deterioration is a matter of definition. We did see degeneration spreading toward the eye from the lesion in other cats with longer survivals. Despite variables affecting the impregnation of axonal degradation (12, 19, 24), several generalizations can be derived from our experimental results. First, degeneration was present in all casesin that part of the optic tract or nerve near the lesionsdue to traumatic effects. Second, the observed disintegration tapered from the lesions rostrad in the visual pathways of cats with longer survivals. Third, this retrogression varied with postoperative intervals and the locations of lesions; viz., the closer the lesions to the retina and the longer the survival the more intensive and wider the distribution of the degeneration became. This was evident not only when comparing the three experimental groups, but also among animals in the same group. Such differences could be interpreted as evidence of retrograde changes (3, 5, 14) of optic afferents, but seem incompatible with a Wallerian degeneration of centrifugal fibers in the optic nerve. Finally, orthograde degradation of cat afferent axons shown with the same impregnation procedures starts near optic centers and propagates toward the cut (22). One would expect efferent fibers in the optic nerve to possessa similar property. However, the present findings suggest a centrifugal spread of the degenerative process, making the occurrence of efferents more unlikely. The problem of retrograde axonal degeneration, or the lack of it, has been the subject of recent reviews (3, 5). “There is no consistent model or law applicable to all portions of the nervous system” (5). In addition to numerous factors shaping the fate of this part of axons, traumatic degeneration has contributed to the confusion (3, 5). It is reasonably well establishedthat silver impregnation of retrograde degeneration of immature CNS fibers progresses somatofugally (9, 19). This generality does not apply to adult animals however. The existing literature shows that retrograde axonal decomposition may proceed somatofugally or somatopetally (3, 5, 14). Possible explanation for this disparity between newborn and adult animals is that rapid cell death after axotomy is the prerequisite for the also rapid retrograde fiber reaction in the former (9) whereas in adult animals retrograde neuronal changes may have a wide range of manifestations, and some perikarya may even achieve morphological and functional recovery (3, 5, 17). B eresford (3) formulated an interesting hypothesis to account for both cellulipetal and cellulifugal progression of retrograde axonal breakdown based on relative pressure and nutritional deficiency among cell bodies and different portions of the transected fibers. In this context our observation of retinopetal spread of disintegration should not be surprising. There is ample evidence that retinal ganglion cells and their
RETROGRADE
DEGENERATION
31
connecting axons may persist for a long time after optic nerve injury, provided that the lesi,on is not too close to the retina and that the retinal blood supply is not impaired (20). It is granted that Wallerian breakdown of mammalian centrifugal optic fibers might take a little longer than the time ordinarily optimal for the Wallerian devolution of other CNS fibers (4, 6). However, such a suggestion fails to explain the retinopetal progression of disintegration in our cats up to 14 days after optic nerve transection and the continuing increase in the amount of degeneration up to 21 days in all three operation groups. Neither could it account for the late onset and confined occurrence of degeneration in the optic nerves of cats with lesions of the optic tract, and the complete absence of such degradation after all presumed origins were destroyed in cats with survivals up to 21 days (18, 19, 21). All these could, nonetheless, be accounted for by a retrograde reaction (3, 5, 14) of retinofugal optic fibers. In most cats with optic nerve lesions, the two segments of the cut nerve were connected by scar tissue, and thus could be processed simultaneously. Hence, the effects due to staining variations ( 12, 24) could be minimized and a more objective comparison of the degenerative changes was possible. As noted previously (22) the degradation central (cerebral) to the incision reached a plateau in 9-10 days, and gradually decreased beginning with the third postoperative month. The amount of argyrophilic debris was insignificant after 6 mo. On the other hand, the degeneration in the distal segment as here described arrived at a maximum in 3 wk without apparent diminution throughout the period of observation. There were times when the degradation visible in the distal segment nearly equaled (from 3 wk to 3 mo) or actually exceeded (after 3 mo) the amount present in the proximal segment. This difference in evolution of the degenerative process, likewise, suggests a retrograde disintegration of optic afferent fibers rather than a Wallerian degeneration of centrifugal axons in the optic nerve. Since mammalian optic efferents, if they exist at all, should constitute only a small percentage of the fiber population of the optic nerve, one can hardly expect their Wallerian degeneration to match or exceed the combined Wallerian disintegration of afferents and retrograde changes of efferents. Furthermore, we know of no evidence that Wallerian degeneration takes place later than, and outlasts, retrograde alteration of the same fibers. Our results are in essential agreement with those of others who found no retinopetal degeneration of optic efferents in the cat 14 days after extensive damage of the dorsal lateral geniculate and adjacent optic tract (23), or up to 10 days after intracranial optic nerve transection (4, 11). of Leinfelder (15, 16) noted different spatial and temporal distributions optic fiber degradation in the cat and monkey distal to optic nerve incision and tract lesions at two loci. He also found that bilateral tract injuries
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produced much less degeneration in the nerve than nerve axotomy in animals with corresponding survival times. None of these findings lends any support for the idea of centrifugal optic fibers. The argument that some such fibers might come from the hypothalamus or mesencephalic tegmentum without coursing through the optic tract also lacks a sound basis (18, 19). Cragg (6) crushed the rabbit optic nerve near the optic foramen and described Wallerian disintegration of efferents in the nerve and retina with the use of the Nauta and Glees techniques. Such degeneration first appeared in 8-10 days. As discussed before, his results were probably artifactual, or represented a retrograde degradation of optic afferents (21). We could not detect any significant normal or degenerated fibers or boutons in either the lamina cribrosa or the retina in any of the normal controls or experimental specimens. Hedreen (11) also failed to find evidence for centrifugal axons in the cat retina in experiments by the methods of Nauta and Fink-Heimer. We call attention to possible sources of misinterpretation of degeneration as exemplified by our control optic nerves (Fig. 1) . Granit (8) believed that if optic efferents were present in mammals at all, the ventral lateral geniculate nucleus would likely be their site of cellular origin, since the latter is closest to the retina among all visual centers. Astruc (1, 2) reported orthograde dissolution of cat centrifugal fibers in distal visual pathways with the Nauta technique after this nucleus was damaged. However, strangely enough, he noted that such disintegrated fibers were very difficult to stain in the optic nerve peripheral to its transection. No postoperative intervals were given by Astruc to warrant meaningful comments on his results. Our own experiments produced no evidence of such efferent degradation. This article concludes our present investigations on the presumed centrifugal optic fibers of the cat (18-22). The most important findings of the series are briefly recapitulated below. With a “full-impregnation” silver method (Bodian’s technique), overt axonal breakdown in the distal segment of the optic nerve could not be seen 3 mo after its axotomy, in contrast to the degeneration in the proximal segment which started at the seventh day (20). Fifteen months after surgery numerous normal fibers still persisted in the distal segment while those in the proximal had long vanished (20). The observations made by the aid of current silver techniques suggest that the degree of axonal degeneration in the peripheral pathways is a function of postoperative duration and of the distance of the lesion from the retina (this study). None of these observations can substantiate the concept of centrifugal fibers in the optic nerve. Even though plausible hints seemed present as to their possible origins (22), all pertinent neural entities have been tested with negative results (18, 19, 21, present report). Thus, it must be concluded that the currently available fiber-
RETROGRADE
degeneration the retina.
methods
DEGENERATION
are unable to demonstrate
33 fibers from the brain to
REFERENCES 1. ASTRUC, J. 1964. Fibres centrifuges de la r&tine. C. R. Ass. Anat. 49: 212-213. 2. ASTRUC, J. 1968. Interconnections of the lateral geniculate bodies through the ventral supraoptic commissure. Anat. Rec. 160 : 309. 3. BERESFORD,W. A. 1965. A discussion on retrograde changes in nerve fibers. Progr. Bra& Res. 14 : 33-56. 4. BRINDLEY, G. S., and D. I. HAMASAKI. 1966. Histological evidence against the view that the cat’s optic nerve contains centrifugal fibers. J. Physiol. 184: 444449. 5. COLE, M. 1968. Retrograde degeneration of axon and soma in the nervous system, pp. 269-300. In “Structure and Function of Nervous Tissue.” G. H. Bourne [Ed.]. Academic Press, New York. 6. CRAGG, B. G. 1962. Centrifugal fibers to the retina and olfactory bulb, and composition of the supraoptic commissures in the rabbit. Exp. Neztrol. 5: 46 427. 7. DONAT, J. R., and H. M. WISNIEWSKI. 1973. The spatio-temporal pattern of Wallerian degeneration in mammalian peripheral nerves. Brain, Res. 53: 41-54. 8. GRANIT, R. 1962. Retina and optic nerve, pp. 541-574. In “The Eye.” Vol. 2. The Visual Process. H. Davson [Ed.]. Academic Press, New York. 9. GRANT, G. 1970. Neuronal changes central to the site of axonal transection. A method for the identification of retrograde changes in perikarya, dendrites and axons by silver impregnation, pp. 173-185. In “Contemporary Research Methods in Neuroanatomy.” W. J. H. Nauta and S. 0. E. Ebbesson [Ed.]. SpringerVerlag, New York. 10. GUILLERY, R. W. 1970, Light- and electron-microscopical studies of normal and degenerating axons, pp. 77-105. In. “Contemporary Research Methods in Neuroanatomy.” W. J. H. Nauta and S. 0. E. Ebbesson [Eds.]. Springer-Verlag, New York. 11. HEDREEN, J. 1969. Patterns of axon terminal degeneration after optic nerve section in cats. Avtat. Rec. 163: 198. 12. HEIMER, L. 1970. Selective silver-impregnation of degenerating axoplasm, pp. 106-131. In “Contemporary Research Methods in Neuroanatomy.” W. J. H. Nauta and S. 0. E. Ebbesson [Eds.]. Springer-Verlag, New York. 13. JOSEPH, B. S., and D. G. WHITLOCK. 1972. The spatio-temporal course of Wallerian degeneration within the CNS of toads (Bufo marinzcs) as defined by the Nauta silver method. Brain Behaz~. Evol. 5: l-17. 14. KALIL, K. 1973. Retrograde axon degeneration in the pyramidal tract of the hamster. Anat. Rec. 175 : 352. 1.5. LEINFELDER, P. J. 1938. Retrograde degeneration in the optic nerves and retinal ganglion cells. Trans. Amer. Ophthalmol. Sot. 36: 307-316. 16. LEINFELDER, P. J. 1940. Retrograde degeneration in the optic nerves and tracts. An experimental study of changes in the axis cylinders. Amer. .I. Ophthalmol. 23: 796-802. 17. LIEBERMAN, A. R. 1971. The axon reaction: a review of the principal features of perikaryal responses to axon injury. 1st. Rev. Neurobiol. 14: 50-124. 18. LIN, H., and W. R. INGRAM. 1972. Probable absence of connections between the retina and the hypothalamus in the cat. Exp. Nezcrol. 37: 23-36.
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19. LIN,
20. 21. 22. 23. 24.
AND
INGRAM
H., and W. R. INGRAM. 1972. An anterior component of the accessory optic system of the cat, with evidence for the absence of reticuloretinal fibers. Exp. Neurol. 37: 37-49. LIN, H., and W. R. INGRAM. 1973. Axonal degeneration in the peripheral optic pathway of the cat. Exp. Newel. 39: 234-248. LIN, H., and W. R. INGRAM. 1973. A search for centrifugal optic fibers in the cat. Bull. Imt. Zool. Acad. Sin. 12 : 51-57. LIN, H., and W. R. INGRAM. 1974. Spatial and temporal distribution of axonal degeneration in the primary optic system of the cat. Exp. Neural. 44: l&20. MAGOUN, H. W., and M. RANSON. 1942. The supraoptic decussations in the cat and monkey. J. Co)np. Newel. 76: 435-459. POWELL, E. W., and R. SCHNURR. 1972. Silver impregnation of degenerating axons ; comparisons of postoperative intervals, fixatives and staining methods. Stain Technol. 47 : 95-100.