Intraocular colchicine inhibits competition between resident and foreign optic axons for functional connections in the doubly innervated goldfish optic tectum

After unilateral optic tectum ablation in the goldfish, regenerating optic axons grow into the optic layers of the remaining ipsilateral tectal lobe and regain visual function. The terminal arbors of the foreign fibers are initially diffusely distributed among the resident optic axons, but within two months the axon terminals from each retina are seen to segregate into irregular ocular dominance patches. Visual recovery is delayed until after segregation. This suggests that the foreign fibers compete with the residents for tectal targets and that the segregation of axon terminations is an anatomical characteristic of the process. Here we investigate whether inhibiting axonal transport in the resident fibers inhibits competition with foreign fibers. The eye contralateral to the intact tectal lobe received a single injection of 0.1 μg colchicine, which does not block vision with the intact eye. We measured visual function using a classical conditioning technique. Segregation of axon terminations was examined shortly following visual recovery by autoradiography. The no-drug control fish showed reappearance of vision with the experimental eye at 9 weeks postoperatively and ocular dominance patches were well developed. Colchicine administered to the intact eye (resident fibers) several weeks postsurgery decreased the time to reappearance of vision with the experimental eye by several weeks. Autoradiography revealed some signs of axonal segregation but the labeled foreign axons were mainly continuously distributed. Administration of colchicine at the time of tectum ablation, or of lumicolchicine at two weeks postoperatively produced normal visual recovery times. Fast axonal transport of³H-labeled protein was inhibited by 1.0 and 0.5 μg but not by 0.1 μg of colchicine or by 1.0 μg of lumicolchicine. Previous studies showed that while 0.1 μg of colchicine does not block vision it is sufficient to inhibit axonal regeneration following optic nerve crush. We conclude that two retinas can functionally innervate one tectum without forming conspicuous ocular dominance columns, and that the ability of residents to compete with the in-growing foreign axons is very sensitive to inhibition of axoplasmic transport or other processes that are inhibited by intraocular colchicine.     

Effect of conditioning lesions on regeneration of goldfish optic axons: time course of the cell body reaction to axotomy

The time course of the cell body reaction to axotomy was determined in goldfish retinal ganglion cells by measuring cell body size and the amount of labelled protein conveyed by fast axonal transport to the optic tectum, both of which increase during regeneration of the optic axons. Following a single testing lesion of the optic nerve, the regenerating axons began to innervate the tectum at about 14 days after the lesion and the cell body reaction began to decline 2-3 weeks thereafter. If the testing lesion had been preceded by a conditioning lesion 2 weeks earlier, the time for the regenerating axons to arrive in the tectum was reduced by a week, because of the faster rate of axonal outgrowth, but the interval between their arrival and the beginning of the decline of the cell body reaction was unchanged. Electrophysiological measurements showed that synaptic transmission was initiated earlier when the axons reached the tectum faster. These results indicate that the mechanisms initiating the recovery of cell body metabolism are independent of those governing the rate of axonal outgrowth. The recovery of the cell body may begin shortly after synapses are established, regardless of whether they are correctly or incorrectly targetted. The correctness of the target may be a separate factor in determining how rapidly and completely the cell body recovers.     

Effect of target removal on goldfish optic nerve regeneration: analysis of fast axonally transported proteins.

How is axonal transport in regenerating neurons affected by contact with their synaptic target? We investigated whether removing the target (homotopic) lobe of the goldfish optic tectum altered the incorporation of 3H-proline into fast axonally transported proteins in the regenerating optic nerve. Regeneration was induced either by an optic tract lesion (to reveal the changes in the original axon segment that remained connected to the cell body) or by an optic nerve lesion (to reveal the changes in the newly formed axon segment). Of 26 proteins analyzed by 2-dimensional gel electrophoresis and fluorography, all but one showed increased labeling as a result of tectal lobe ablation. By 2 d after the lesion, significantly increased labeling of some proteins was seen with a 6-hr labeling interval, but not with a 24-hr labeling interval. This is probably indicative of an increased velocity of transport, which may have been a nonspecific consequence of the surgery. Otherwise, tectal lobe removal had relatively little effect until 3 weeks, when there was a transitory increase in labeling of transported proteins in the new axon segments of the tectum-ablated animals. Beginning at 5 weeks, tectal lobe ablation caused considerably higher labeling of many of the proteins in the original axon segments. Because this was seen with both 6-hr and 24-hr labeling intervals, it is probably indicative of increased protein synthesis. The increased synthesis lasted until at least 12 weeks, though some proteins were beginning to show a diminished effect at this time. In the late stages of regeneration (8-12 weeks), there was also increased labeling of proteins in the new axon segments as a result of the absence of the target tectal lobe. This included a disproportionately large increase in the relative contribution of cytoskeletal proteins and of protein 4, which is the goldfish equivalent of the growth-associated protein GAP-43 (neuromodulin). We conclude that, after the regenerating axons begin to innervate the tectum, the expression of most of the proteins in fast axonal transport is down-regulated by interaction between the axons and their target. However, the changes in expression may be preceded by a modulation of the turnover and/or deposition of proteins in the newly formed axon segment.     

Effect of optic nerve lesions and intraocular colchicine on cell proliferation in the germinal zone of the optic tectum and in the torus longitudinalis in the goldfish

Postembryonic development of the optic tectum occurs in part through proliferation of cells in the germinal zone located at the caudal edges of each lobe. Autoradiography experiments by others have shown that [3H]thymidine labeling in the germinal zone is decreased following optic nerve crush or enucleation and restored above normal levels during optic nerve regeneration. The present autoradiography experiments examined the relationship between retinal innervation and the rate of mitotic activity in the tectum germinal zone and in the torus longitudinalis. The fish received optic nerve crush to temporarily deafferent the tectum, enucleation for permanent deafferentation, or an intraocular injection of 0.01-1.0 microgram of colchicine to reversibly inhibit axonal transport in the optic nerve. Thymidine labeling in the tectum germinal zone showed that nerve crush resulted in decreased mitotic activity in most fish within 6 days followed by recovery by 21 days; enucleation decreased mitotic activity more uniformly and for more than 42 days with recovery by 84 days postaxotomy; colchicine produced a dose-dependent inhibition of mitotic activity which was reversed by 42 days postinjection. Axonal transport was restored by 42 days postinjection. In the torus longitudinalis, nerve crush produced a brief increase in mitotic activity followed by a return to normal; enucleation and colchicine resulted in a lasting decrease in mitotic activity and atrophy indicating a loss of cells or neuropil. The data are consistent with the proposal that cell proliferation in the tectum germinal zone is stimulated by the accretion of fibers from developing retinal ganglion cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recovery of regenerating goldfish retinal ganglion cells is slowed in the absence of the topographically correct synaptic target

When the axons of goldfish retinal ganglion cells are severed the cell bodies undergo a series of changes as the axons regenerate. These changes begin to reverse when the axons start to innervate the tectum and by 3 months after the lesion the cell bodies have nearly returned to normal. When the axons projecting to the caudal tectum were severed by a mediolateral transection of the tectum, only retinal ganglion cells in the nasal portion of the contralateral retina underwent the changes normally associated with regeneration, followed by a speedy return to normal. Because the injured fibers probably did not fully retract from the tectum, these results indicated that: (1) the complete removal of the axons from the tectal milieu was not essential for initiating the cell body changes, and (2) close proximity to the target sites would speed the recovery of the cells. When the caudal portion of the tectum was ablated the retinal ganglion cells of the nasal retina remained enlarged significantly longer than after tectal transection. During the time the cells remained enlarged the electrophysiological projection onto the remaining rostral part of the tectum revealed no significant 'compression' of the visual field. Compression of the visual field onto the rostral portion of the tectum can be accelerated if the caudal tectal ablation is accompanied by an optic nerve crush. However, under this condition the recovery of ganglion cells in the nasal retina was significantly slower than the recovery of cells in the temporal retina. This may reflect an element of topographical specificity in the regulation of the recovery of the cell body from axonal injury.     

Effect of different optic nerve lesions on retinal ganglion cell death in the frog Rana pipiens

Following optic nerve crush in various species of frog, a proportion of the retinal ganglion cells re-establishes functional contact with the optic tectum. However, as much as 50% of the retinal ganglion cells die during this process. The determinants of an individual ganglion cell's fate have not been established. In this study of Rana pipiens, cell survival after optic nerve crush was compared with that after nerve cut followed by stump separation, a procedure that considerably delayed entry of optic axons to the brain. It was also ascertained, in the case of delayed ingrowth, whether application of nerve growth factor immediately after lesion influenced the cell death process. This study confirmed that retinal ganglion cell death is a relatively late event in regeneration, because in several animals where anterograde HRP labeling demonstrated regenerating axons within the tectum, no cell death had occurred. There was no statistically significant difference in cell death at 75 days after lesion between animals receiving nerve crush and those receiving nerve cut with stump separation, even though most crush animals had regenerated a complete visual projection, whereas most nerve cut animals had not. The application of NGF did not influence the level of cell death at 75 days after lesion. These results suggest that contact of optic axons with the optic tract or tectum is not necessary for retinal ganglion cell death to occur. However, this does not necessarily mean that contact with the brain is not involved with cell death during regeneration following nerve crush because it is possible that the mechanisms of cell death are different when axons are prevented from regenerating. Further investigations are therefore required to establish the reasons for this cell death.     

Bidirectional axonal transport of glycoproteins in goldfish optic nerve

The goldfish visual system was used to study the relationships between anterograde and retrograde transport of axonal glycoproteins. After intraocular injection of radioactive glucosamine, determinations were made of the normal time course of appearance of labeled glycoproteins in the optic nerve and tectum and of their time course of accumulation on both sides of an optic nerve crush. The labeled glycoproteins, transported at a maximum velocity of about 80 mm/day, continued to pass through the optic nerve in significant amounts for as long as 24 h after the injection, with a maximum at about 6 to 14 h. Retrograde transport of labeled materials back from the optic tectum to the same point in the nerve began about 5 h later, indicating a minimum possible retrograde velocity of about 36 mm/day and a maximum possible lag time in the axon terminals (with the assumption of equal retrograde and anterograde velocities) of 1 to 2 h. When delivery of glycoproteins from the retina to the optic tectum was interrupted by a nerve crush 8 h after injection, a component with rapid turnover in the tectum was revealed having a half-life of not more than 6 h. At least 40% of this turnover could be attributed to retrograde transport. The amount of labeled glycoprotein transported in a retrograde direction 8 to 10 h after injection was greatly elevated in regenerating axons 2 weeks after the optic tract was cut.     

Axons added to the regenerated visual pathway of goldfish establish a normal fiber topography along the age-axis

Throughout a goldfish's life, new generations of ganglion cells are added on the retinal margin and their axons extend centrally to occupy predictable positions in the retinotectal pathway, adjacent to their predecessors and subjacent to the pia. The stacking of successive generations of axons defines the age-axis of the pathway. This study examined whether an ordered array of predecessor axons is a prerequisite for the patterned growth of new axons. One optic nerve was crushed intraorbitally and the fish was injected with 3H-thymidine to label the proliferating cells on the retinal margin. The ring of 3H-thymidine-labeled cells separated retina that was present at the time of nerve crush (inside the ring) from new retina added afterward (outside). After a period of 14-16 months postcrush, both tectal lobes received two punctate applications of horseradish peroxidase (HRP), one in the central and the other in peripheral tectum, to retrogradely label contralateral retinal ganglion cell bodies and their axons. The pattern of HRP labeling from the control tectum confirmed earlier work: axons on the central tectum had somata in the central retina, and axons on the peripheral tectum had somata in the peripheral retina. The labeled cells and axons were both in predictable patterns. The somata that were backfilled from applications to the center of the experimental tectum lay inside the radioactive ring and had therefore regenerated their axons. The patterns of their labeled axons in the optic pathway and of their somata in the retina were typical of the regenerated condition as described in earlier studies. The somata backfilled from the periphery of the experimental tectum were outside the radioactive ring and had been added after the optic nerve crush. The patterns of their labeled axons and somata were comparable to the normal pattern. These observations indicate that new axons do not depend on an ordered array of predecessors to reestablish normal order along the age-axis of the pathway.     

Expansion of the ipsilateral retinal projection in the frog brain during optic nerve regeneration: Sequence of reinnervation and retinotopic organization

The sequential reinnervation and distribution of optics axons in diencephalic and mesencephalic targets was studied after crushing the optic nerve in 37 adult Rana pipiens. Following intravitreal injection of ³H-proline at various times after nerve crush the distribution of regenerating optic axons was traced using autoradiographic methods. The maximal distribution of regenerating axons was reached between 6 and 8 weeks after optic nerve crush. On the contralateral side of the brain, the distribution of fibers was similar to the normal projection. Ipsilaterally, silver grain density was greater than normal in the optic tract and projections were expanded to all optic targets on this side of the brain. These abnormal projections were sustained for a least 6 months after nerve crush. The sequence of reinnervation of targets on both sides of the brain differed from normal development. Unlike development, regenerating optic axons were found in the ipsilateral optic tract prior to the time they were found on the contralateral side of the brain. Also unlike development, regenerating axons did not begin to reinnervate targets in the anterior thalamus until several weeks after reinnervation of the posterior thalamus and tectum had begun. The expanded distribution of regenerating axons within optic targets on the ipsilateral side of the brain became evident at the time optic axons first invaded each area. Most optic axons appeared to regenerate from the point of nerve crush. The retinal stump of the crushed nerve was filled with labeled axons in all six frogs given intravitreal ³H-proline injections between 1 and 7 days after nerve crush. In addition, using a modified Fink-Heimer method, few degenerating axons were found in the retinal stump of four frogs sacrificed between 2 and 8 days after optic nerve crush. In ten frogs studied between 3 and 6 months after nerve crush, horseradish peroxidase (HRP) was placed on different portions of the tectum ipsilateral to the crush. In each case HRP was retrogradely transported to ganglion cells in both retinae. The cells labeled in the ipsilateral retina corresponded in position to the same region of the contralateral retina although many fewer cells were labeled ipsilaterally. Cutting the optic tract on the side opposite the HRP placement did not affect the results. No ganglion cells were labeled in the ipsilateral retina of two frogs not receiving optic nerve crush. These results show that axons from all parts of the retina regenerate to the ipsilateral side of the brain during optic nerve regeneration and the distribution of these misrouted axons, at least to the tectum, overlaps the intact distribution from the other eye. Differences between development and regeneration in the patterns of growth of optic axons may be related to this anomaly.     

The re-establishment of synaptic transmission by regenerating optic axons in goldfish: Time course and effects of blocking activity by intraocular injection of tetrodotoxin

Intraocular injections of tetrodotoxin were used to block activity for 27 days in normal fish and for the first 27 or 31 days of regeneration in fish with one optic nerve crushed. Synaptic activity was then assessed by a current source-density analysis of field potentials evoked by optic nerve shock at different times following the TTX treatment. In normal fish, the lack of activity for 4 weeks had no significant effect on the maintenance of synaptic strength. Likewise, in fish with nerve crush, lack of activity did not prevent the regenerating optic fibers from forming synapses that were nearly as effective as those formed in controls injected with the citrate buffer vehicle. The earliest synapses were formed at the rostromedial corner of the tectum (where the tract enters) at 20 days after nerve crush, when fibers had not yet reached the caudal areas. By 28 days synaptic potentials could be recorded everywhere on the surface of the tectum in both controls and TTX injected fish. However, the latency of the responses with TTX were longer, suggesting a smaller caliber of fiber, which is consistent with an earlier finding of decreased axonal transport in TTX fish. Maturation of the regenerating fibers proceeded slowly in both TTX and control fish. After more than 5 months, the projections were nearly normal but still not completely normal.     

Growing and regenerating axons in the visual system of teleosts are recognized with the antibody RT97

We have analyzed the immunolabeling with the antibody RT97, a good marker for ganglion cell axons in several species, in the normal and regenerating visual pathways of teleosts. We have demonstrated that RT97 antibody recognizes several proteins in the tench visual system tissues (105, 115, 160, 200, 325 and 335 kDa approximately). By using immunoprecipitation and Western blot we have found that after crushing the optic nerve the immunoreactivity to anti RT97 increased markedly in the optic nerve. In immunohistochemical analysis we also found a different pattern of labeling in normal and regenerating visual pathways. In normal tench RT97 is a good marker for the horizontal cells in the retina, for growing ganglion cell axons which run along the optic nerve from the retina to the optic tectum and of the axon terminals in the stratum opticum and stratum fibrosum and griseum superficiale in the optic tectum. After optic nerve crush, no immunohistochemistry modifications were observed in the retina. However, in accordance with Western blot experiments, in the optic nerve intensely stained groups of regenerating axons appeared progressively throughout the optic nerve as far as the optic tectum. We conclude that the antibody RT97 is an excellent marker of growing and regenerating axons of the optic nerve of fish.     

Plasticin,a newly identified neurofilament protein,is preferentially expressed in young retinal ganglion cells of adult goldfish

The adult goldfish retina and optic nerve display continuous growth, plasticity, and the capacity to regenerate throughout the animal's life. The intermediate filament proteins in this pathway are different from those in adult mammalian nerves, which do not continuously grow or normally regenerate. One novel intermediate filament protein of the goldfish visual pathway is plasticin, which is synthesized in ganglion cells and transported into the optic nerve. Using specific polyclonal antibodies raised against a plasticin fusion protein, we investigated the distribution of this protein in the normal retina and nerve and in the retina and nerve following optic nerve crush. In the normal pathway, plasticin was localized predominantly to the axons of very young ganglion cells; however, there was considerable immunoreactivity in older axons as they approach the chiasm. In addition, following optic nerve crush, all ganglion cell somata and their axons proximal to the crush site became equally immunoreactive. The results suggest that plasticin may contribute to axonal growth, plasticity, and regeneration. © 1994 Wiley-Liss, Inc.     

A quantitative analysis of frog optic nerve regeneration: Is retrograde ganglion cell death or collateral axonal loss related to selective reinnervation?

The present study was designed to assess whether axon collateral formation and loss or retrograde cell death contribute to selective reinnervation during optic nerve regeneration in the frog, Rana pipiens. The right optic nerve was crushed in 18 frogs, and samples were taken near the optic disc (retinal segment) and near the optic chiasm (brain segment). These samples were studied quantitatively with the electron microscope at various postoperative survival times (1, 2, 3, 4, 6, 12 weeks, 6 months, 1 year; N = 2). The number and size of axons in each segment were estimated from a series of electron micrographs taken at intervals across the transverse extent of each nerve and compared with normal nerves (N = 4). Results show that there are 5.3 ± 1.8 × 10⁵ (S.D.) unmyelinated and 2.3 ± .5 × 104 myelinated axons in the normal nerve. One week post-crush (p.c.) there is a 27% decrease in the number of axons in the retinal segment (4.1 ± 1.4 × 10⁵), indicating early retrograde axonal loss. As expected, there is a greater loss of axons at this time in the brain segment (3.0 ± 1.3 × 105). Between 2 and 6 weeks p.c. the number of axons increases in the retinal segment to over twice the normal number (12.3 ± 3.8 × 105) and to over four times this number in the brain segment (20.0 ± 3.0 × 10⁵), showing collateral axon formation results from this injury. A large loss in the number of axons occurs in both nerve segments between 6 and 12 weeks p.c. (4.3 ± 1.5 × 10⁵) and an additional loss at 20 weeks p.c. (2.2 ± .98 × 10⁵). Subsequently, the number remains constant, approximately 40% of normal. Visual recovery was seen in the two frogs tested one year after optic nerve crush that were used for optic axon counts. Autoradiography in these same animals showed the optic nerve projections normally seen after regeneration. Besides axonal loss, our results also indicate that the size of both myelinated and unmyelinated axons is significantly above normal at chronic postoperative periods. This increase in axonal size is interpreted to be related to the increased territory each remaining optic axon must fill to restore the optic projections. The number and density of ganglion cells in the retina were estimated at various periods after nerve crush injury (Normal, 3 weeks, 6–8 weeks, 10–12 weeks, 6 months or more, N = 5) using diamidino yellow dihydrochloride (DY) as a retrograde label. Approximately 4.2 ± 1.3 × 10⁵ ganglion cells were estimated in normal retinae, 1 week after applying DY to the nerve. Ganglion cell loss is rapid so that by 6 weeks p.c., when collateral axon estimates are greatest, only 2.4 ± 8 × 10⁵ ganglion cells remain. Six months or more p.c., only 1.2 ± .7 × 10⁵ ganglion cells remain, 28% of the number estimated in normal retinae. In another experiment the formation of retinoretinal axons was prevented during optic nerve regeneration by ligating the other optic nerve at the optic chiasm (N = 3) at the same time one nerve was crushed. The same amount of axonal loss was found in these crushed nerves as in frogs receiving only optic nerve crush, Thus, misrouted retinoretinal axons do not appear to be a significant factor in the axonal or retrograde cell loss. We also estimated the number of ganglion cells having axons projecting into the opposite nerve during axonal regeneration. Ganglion cells were retrogradely labeled by placing DY or rhodamine in the opposite nerve 6 (N = 3), 7 (N = 4), or 8 weeks p.c. (N = 6). The number of these cells having a collateral projecting to a normal target was assessed by placing true blue on the tectal hemi-sphere contralateral to the injury 3 days prior to the time rhodamine was placed in the nerve (N = 10). Our estimates indicated that 26,000 ± 2,000 (S.E.) ganglion cells have a collateral projecting into the contralateral nerve between 6 and 7 weeks p.c. This number decreased to 18,200 ± 1,100 by 8 weeks p.c. These cells are in all portions of the retina and many were doubly labeled by placing true blue (TB) in the contralateral tectum (range 2%- 70%). The percentage of doubly labeled neurons increased as the tectum became more fully reinnervated. Our results suggest that selective reinnervation correlates with axon collateral formation and that loss of collaterals occurs as reinnervation takes place. Although ganglion cell loss also occurs as a result of nerve injury, the timing of this loss suggests that it is not directly related to selective reinnervation.     

Protein synthesis and fast axonal transport in regenerating goldfish retinal ganglion cells

To characterize the fast component of axonal transport in regenerating goldfish optic axons, the incorporation of l-2,3-[³H]proline into newly-synthetized proteins in the cell bodies of the retinal ganglion cells and the amount of transported labeled protein were determined at 2–36 days after cutting the optic tract. Both the incorporation and the amount of transported protein had doubled by 10 days after the lesion and continued to increase to about 5 times normal at 15 days, a time when a large proportion of the regenerating axon population had reached the optic tectum. Near-normal levels were recovered by 36 days. In contralateral control neurons, the incorporation of l-2,3-[³H]proline was unchanged from normal throughout, whereas the amount of labeled transported protein entering control axons was decreased by 55% at 2 and 10 days after the testing lesion, returning to normal by 15 days. An increase in fast transport velocity was seen in the regenerating axons beginning at 10 days after the lesion. However, a similar velocity increase was also seen in the contralateral control axons and in undamaged axons following removal of the cerebral hemispheres. Therefore, the velocity increase was not a specific consequence of axotomy.     

Axonal regeneration is associated with glial migration: Comparison between the injured optic nerves of fish and rats

The central nervous systems of mammals and fish differ significantly in their ability to regenerate. Central nervous system axons in the fish readily regenerate after injury, while in mammals they begin to elongate but their growth is aborted a the site of injury, an area previously shown to contain no glial cells. In the present study we compared the ability of glial cells to migrate and thus to repopulate the injured area in fish and rats, and used light and electron microscopy in an attempt to correlate such migration with the ability of axons to traverse this area. One week after the optic nerve was crushed, both axonal and glial responses to injury were similar in fish and rat. In both species glial cells were absent in the injured area (indicated by the disappearance of glial fibrillary acidic protein and vimentin immunoreactive cells from the site of injury in rat and fish, respectively), while at the same time axonal growth, indicated by expression of the growth-associated protein GAP-43, was restricted to the proximal part of the nerve. In fish, 2 weeks after the crush, GAP-43 staining (i.e., growing axons) was seen at the site of injury, in association with migrating vimentin-positive glial cells. One week later the site of injury in the fish optic nerve was repopulated by vimentin-positive glial cells, and GAP-43-positive axons had already traversed the site of injury and reached the distal part of the nerve. In contrast, the site of injury in the rat remained devoid of glial fibrillary acidic protein immunoreactive cells, and the expression of GAP-43 by growing axons was still restricted to the proximal part of the nerve. Double-labeling experiments and transmission electron microscopy performed 2 weeks after crush injury of the fish optic nerve revealed that the frontier of axonal growth (i.e., the leading growth cones) appeared to be 200–300 μm ahead of the nearest vimentin-positive glial cells. The leading growth cones were associated with other cells, presumably glial precursors, that seemed to have a high migratory potential. We suggest that the ability of fish glial precursor cells to migrate into the injured area may contribute to the potential of growing axons to traverse this area. The failure of glia and glial precursor cells to migrate into the injured area in the rat may partially account for the failure of rat axons to enter and traverse the injured area.     

Growth of retinal ganglion cell axons following optic nerve crush in adult hamsters

Regrowth of retinal ganglion cell axons was examined 2 to 60 days after intraorbital optic nerve crush lesions in adult hamsters. Anterograde axonal transport of intraocularly injected wheat germ agglutinin-horseradish peroxidase conjugate was used to label the axons after specific postinjury time periods. Labeled axons were present in the region of the optic nerve lying between the eye and the crush site at all times, but their numbers appeared to decrease with increasing survival time. Labeled axons were first detected in the segment of optic nerve lying distal to the crush site 1 week after injury and had extended as far as 2.3 mm beyond the crush site by 60 days postinjury, growing at a rate similar to that at which the collateral branches of developing ganglion cell axons extend into their targets. Although most axons failed to regrow after these lesions, the slow reextention exhibited by members of a small population of axons indicates that the degenerating adult mammalian optic nerve provides an adequate environment for a particular mode of regrowth by injured axons of the central nervous system.     

Immunohistochemical localization of CNTFRalpha in adult mouse retina and optic nerve following intraorbital nerve crush: evidence for the axonal loss of a trophic factor receptor after injury

Ciliary neurotrophic factor (CNTF) is important for the survival and outgrowth of retinal ganglion cells (RGCs) in vitro. However, in vivo adult RGCs fail to regenerate and subsequently die following axotomy, even though there are high levels of CNTF in the optic nerve. To address this discrepancy, we used immunohistochemistry to analyze the expression of CNTF receptor alpha (CNTFRalpha) in mouse retina and optic nerve following intraorbital nerve crush. In normal mice, RGC perikarya and axons were intensely labeled for CNTFRalpha. At 24 hours after crush, the immunoreactivity normally seen on axons in the nerve was lost near the lesion. This loss radiated from the crush site with time. At 2 days postlesion, labeled axons were not detected in the proximal nerve, and at 2 weeks were barely detectable in the retina. In the distal nerve, loss of axonal staining progressed to the optic chiasm by 7 days and remained undetectable at 2 weeks. Interfascicular glia in the normal optic nerve were faintly labeled, but by 24 hours after crush they became intensely labeled near the lesion. Double labeling showed these to be both astrocytes and oligodendrocytes. At 7 days postlesion, darkly labeled glia were seen throughout the optic nerve, but at 14 days labeling returned to normal. It is suggested that the loss of CNTFRalpha from axons renders RGCs unresponsive to CNTF, thereby contributing to regenerative failure and death, while its appearance on glia may promote glial scarring.     

α-Crystallin Protected Axons from Optic Nerve Degeneration After Crushing in Rats

In mature mammals, optic nerve injury results in apoptosis of retinal ganglion cells. The literature confirms that lens injury enhances retinal ganglion cells survival, but the mechanism is not very clear. Using silver staining method and computer image analysis techniques, the effect of alpha-crystallin, a major component of the lens in the survival of retinal ganglion cell axons, was investigated in vivo after intravitreal injections. The results showed that enhanced survival of axotomized axons was observed beyond the crush site after a single intravitreal administration of alpha-crystallin at the time of axotomy. Axonal density of the retinal ganglion cell was significantly greater than in the untreated controls until 2 weeks after injection. This effect declined by 4 weeks after injection but survival of axons remained greater than controls. These findings indicate that alpha-crystallin plays a key role in protecting axons after optic nerve injury.     

Axonal transport of phospholipid in goldfish optic system.

After injection of [³H] glycerol into the eye of goldfish, labeled lipid was conveyed by axonal transport in the optic axons to the optic tectum. The transported material was found to consist almost entirely of phosphoglycerides, including both zwitterionic and acidic species. The time course of appearance of the labeled phospholipid in the optic tectum suggested that it might be axonally transported at a rate intermediate between the rates of the fast and slow components in the axonal transport of protein. This possibility, however, was contradicted by the following evidence: a) the rate of transport of the phospholipid that first appeared in the tectum was the same as that of the fast protein component; b) during the first few days after the precursor injection the phase difference between the rates of bulid-up of labeled phospholipid in the optic nerve and tectum such as would be expected with an intermediate rate of transport was not seen; c) the accumulation of phospholipid in the tectum was rapidly terminated after the optic axons were separated from their cell bodies, as would be expected from a fast rather than intermediate transport rate in the isolated axons. The results suggest that the axonal transport of most of the phospholipid had the same rate as the fast component of protein transport, and that the prolonged period of accumulation of the phospholipid in the tectum reflected a prolonged period of release from the cell body into the axon. Only a small proportion of the phospholipid might have been transported at a slower rate. Inhibition of protein synthesis in the retina reduced the amount of transported phospholipid appearing in the tectum almost as much as the transported protein. It is probable, therefore, that the transport of phospholipid occurs in association with protein, possibly in the form of an assembled membrane structure. Whereas the protein that is transported is newly synthesized, the transported lipid need not be.     

Cooperative effects of bcl-2 and AAV-mediated expression of CNTF on retinal ganglion cell survival and axonal regeneration in adult transgenic mice

We used a gene therapy approach in transgenic mice to assess the cooperative effects of combining anti-apoptotic and growth-promoting stimuli on adult retinal ganglion cell (RGC) survival and axonal regeneration following intraorbital optic nerve injury. Bi-cistronic adeno-associated viral vectors encoding a secretable form of ciliary neurotrophic factor and green fluorescent protein (AAV-CNTF-GFP) were injected into eyes of mice that had been engineered to over-express the anti-apoptotic protein bcl-2. For comparison this vector was also injected into wildtype (wt) mice, and both mouse strains were injected with control AAV encoding GFP. Five weeks after optic nerve injury we confirmed that bcl-2 over-expression by itself promoted the survival of axotomized RGCs, but in contrast to previous reports we also saw regeneration of some mature RGC axons beyond the optic nerve crush. AAV-mediated expression of CNTF in adult retinas significantly increased the survival and axonal regeneration of RGCs following axotomy in wt and bcl-2 transgenic mice; however, the effects were greatest in the transgenic strain. Compared with AAV-GFP-injected bcl-2 mice, RGC viability was increased by about 50% (mean, 36 738 RGCs per retina), and over 1000 axons per optic nerve regenerated 1-1.5 mm beyond the crush. These findings exemplify the importance of using a multifactorial therapeutic approach that enhances both neuroprotection and regeneration after central nervous system injury.