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Regeneration of ganglion cell axons in the adult mouse retina
362
Brain Research, 241 (1982) 362-365
Elsevier Biomedical Press
Regeneration of ganglion cell axons in the adult mouse retina P. McCONNELL and M. BERRY Department of Anatomy, Medical School, University of Birmingham, Birmingham, B15 2TJ ( U.K.)
(Accepted February 25th, 1982) Key words: regeneration - - retina - - stratum opticarum - - ganglion cell - - unmyelinated nerve fibers
The hypothesis that regenerative failure of axons in the adult mammalian CNS is due to release of a growth inhibitor from injured oligodendrocytes and/or myelin2, predicts that regeneration of injured fibers would proceed unchecked in unmyelinated CNS regions. This prediction was borne out by observations on the stratum opticarum of the mouse retina. Axonal sprouts, first seen 14-16 h post-lesion (pl), continued growing until at least 100 days pl, well beyond the time at which regeneration fails in myelihated CNS regions. It has long been accepted that injured axons in the mature avian and mammalian CNS exhibit only a transitory regenerative response. Cajal's seminal studies 6 of mammalian spinal cord and optic nerve revealed that damaged fibers were capable of sprouting from 2 to 3 days post-lesion (pl), but that growth ceased by I0 14 days pl. Regenerated axons subsequently atrophied and were resorbed. These early observations were confined, however, to myelinated CNS regions. More recently, work on nonmyelinated fiber systems has provided evidence of a number of exceptions to this generally held view that post-injury growth is abortive. Thus, successful regeneration has been observed in the adult neurohypophyseal system s, olfactory nerve 14, monoaminergic system 4 and unmyelinated cholinergic nerves 2z, as well as in the immature unmyelinated CNS 16. These findings, coupled with the well-documented regenerative capacity of myelinated axons within the peripheral nervous system of mammals1, 6 and the CNS offish and amphibia 7, have led to the formulation of a hypothesis proposing that failure of central axonal regeneration, in mature mammals and birds, is due to the release of an active axonal-growth inhibitory factor (AGIF) from degenerating oligodendrocytes and/or myelin in the immediate postinjury period 2. Release of A G I F s from an astrocyte or mesodermal source was excluded because reactive astrocytes, fibroblasts and macrophages are features 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
of all CNS wounds, independent of the type of axon injured, or the vigour of outgrowth. The A G I F hypothesis has been tested by investigating regeneration in the stratum opticarum of the retina, an area of the CNS where, in most species, axons are entirely unmyelinated 2°. Ganglion cell fibers are tightly fasciculated, running radially over the inner retinal surface to the optic papilla. Hence, fiber paths are planar and unidirectional, providing a unique model in which to study post-injury axonal growth. Moreover, the retina may be prepared as a whole-mount, thus avoiding errors otherwise introduced by serial sectioning 1°,~3. The experiment was designed with the aim of quantifying the extent of the growth-response of damaged retinal ganglion cell axons, predicting that growth would proceed unchecked in the absence of oligodendrocyte myelin. Thus, using applantation ophthalmoscopy 2~, small scratch lesions were placed between radial blood vessels in the ganglion cell fibre layer of the retina of adult female albino mice (MF 1 strain) by the technique of Goldberg and Frank 11. Groups of 10 animals were killed at 14, 16, 24, 36 and 48 h pl and 4, 6, 8, 10, 20, 30, 45 and 100 days pl, their eyes silver-stained 17 and retinal whole-mounts prepared by carefully dissecting away the lens, cornea, sclera and pigment epithelium, incising the retina radially, and flat-mounting in glycerol. The typical pattern of fiber outgrowth at various
363
Fig. 1. Photomontages of the pattern of fiber outgrowth at various intervals post-lesion, seen in retinal whole-mounts, a: fibers proximal to the lesion site 16 h pl, the majority of terminals have developed swollen end bulbs and some sprouts (arrows) are already present, b: by 36 h pl, the majority of injured proximal fibers had developed sprouts which either joined pre-existing axon fascicles or else showed random unfasciculated growth, occasionally by-passing or growing into the substance or the lesion (star). c: at 8 days pl, the regenerating axonal arborization has increased in extent and complexity, d: at 10 days pl, the proximal fiber plexus persists although, in many cases, the severed axons distal to the lesion (asterisk) have degenerated by this time. e: by 45 days pl, randomly oriented axons persist deep to the fiber layer (the lesion site is off the lower edge of the photograph), f: at 100 days pl, regenerating fibers are still present proximal to the lesion site. In all cases 'L' indicates the lesion site, and the optic papilla lies beyond the bottom edge of the photograph. Marker = 10/~m. intervals pl is illustrated in Fig. 1. Up until 24 h pl, the majority of fibers, both proximal and distal to the lesion site, showed terminal swellings or 'end
bulbs' similar to those described by Cajal 6. However, in contrast to previous reports2,12, there appeared to be sprouting of proximal fibers at even the earliest post-lesion intervals (Fig. la). Sprouts initially grew towards the lesion but, by 36-48 h pl, the majority had looped through 180 ° and either joined a pre-existing axon fascicle or else remained unfasciculated, growing in an apparently random manner just deep to the fiber layer. A few axons failed to loop, continuing to grow either into the substance of the lesion or around its edge (Fig. lb). Since it was impossible to follow the fate of the fibers which grew into fascicles, we describe here only non-fasciculated growth. The extent and complexity of fiber outgrowth increased with time (Fig. lc) and, by 10 days pl, a rich plexus of axons was present. By this stage, the severed distal portions of axons had degenerated in most cases, but the proximal sprouts showed no sign of atrophy (Fig. ld). Indeed, although a proportion of proximal fibers did begin to degenerate from about 15 days pl, many sprouts persisted and continued to grow until 100 days pl - - well beyond the time at which Cajal a noted the disappearance of regenerated axonal arbors (Fig. le, f). The precise extent and direction of axonal outgrowth was determined by tracing regenerating fibers using a camera lucida ( × 1250) in conjunction with a Kontron M O P / A M O 2 on line to a Hewlett Packard 9825A computer. At all but the earliest survival times (14, 16 and 24 h pl), 30 regenerating fibers were measured. Only 20 fibers could be traced from each of the 3 early groups. The measurements of fiber length (Fig. 2) revealed a phase of rapid (20 /~m/day) growth until 10 days pl, followed by a 50C
400
8 300 D)
~ 2oo tL 1 0 0
0
I
I
5
10
_1
I
I
I
I
15 20 25 30 35 Days post lesion
I
I
40 45
.~
I
100
Fig. 2. The extent of axonal outgrowth with increasing time post-injury.
364
OPTIC DISC
14hpl
24hpl
2dpl
4dpl
6dpl
lOdpl
30dpl
45dpl
1 O0 dpl
Fig. 3. Mean orientation of regenerating retinal axons The 360 ° histograms show the relative proportion of 2 mm fiber 'steps' per 10° of arc at the various post-lesion intervals indicated. Initially (14-24 h pl) most fibers grew towards the optic disc, but 36-48 h pl the majority looped through 180 ° producing a bipolar pattern of fiber outgrowth (4-6 days pl). This orientation was progressively lost, however, as the proportion of random growth increased. Thus, at the later survival times fibers showed no sign of any preferred direction of growth.
decline in overall g r o w t h rate, p r e s u m a b l y as regener a t e d sprouts b e g a n to degenerate. Nevertheless, the actual length o f the persisting regenerating fibers c o n t i n u e d to increase t h r o u g h o u t the experiment. The o r i e n t a t i o n o f successive 2 m m 'steps' o f the fiber tracings was d e t e r m i n e d with respect to the optic disc, a n d 360 ° h i s t o g r a m s c o n s t r u c t e d showing the relative p r o p o r t i o n o f 'steps' per 10° o f arc (Fig. 3). These i n d i c a t e d t h a t the direction o f o u t g r o w t h o f the regenerating nerve fibers was entirely r a n d o m . Thus, the lesioned optic axons in the retina o f the a d u l t m o u s e fail to rc-establish their original connections, possibly due to the absence o f a p p r o p r i a t e guidance m e c h a n i s m s ~2. However, their capacity for r e g r o w t h following injury is far greater t h a n anticip a t e d on the basis o f Cajal's 6 descriptions o f the a b o r t i v e g r o w t h response. One possible e x p l a n a t i o n for this d i c h o t o m y between the present observations in the retina a n d the results o f studies in the spinal c o r d a n d optic nerve, m a y lie in the fact t h a t fibers in the latter sites are
1 Aguayo, A. J., Bray, G. M., Perkins, C. S. and Duncan, I. D., Axon-sheath cell interactions in peripheral and central nervous system transplants. In J. A. Ferrendelli (Ed.), Aspects of Developmental Neurobiology, Society for Neuroscience Symposia, Vol. IV, Society for Neuroscience,
Bethesda, MD, 1979, pp. 361-383. 2 Berry, M., Post-injury myelin-breakdown products inhibit axonal growth: an hypothesis to explain the failure of axonal regeneration in the mammalian central nervous system, BibL anat., in press. 3 Bignami, A. and Dahl, D., The radial glia of MOiler in the rat retina and their response to injury. An immuno-fluorescence study with antibodies to the glial fibrillary acidic (GFA) protein, Exp. Eye Res., 28 (1979) 63-69. 4 Bj6rklund, A. and Stenevi, V., Regeneration of monoaminergic and cholinergic neurons in the mammalian central nervous system, PhyioL Rev., 59 (1979) 62-100. 5 Bfissow, H., The astrocytes in the retina and optic nerve head of mammals: a special glia for the ganglion cell axons, Cell Tiss. Res., 206 (1980) 367-378. 6 Cajal, S. Ram6n y, Degeneration and Regeneration of the Nervous System, (translation R. M. May), Hafner, London, 1968. 7 Clemente, C. D., Regeneration in the vertebrate central nervous system, Int. Rev. NeurobioL, 6 (1964) 251-301. 8 Dellman, H. D., Degeneration and regeneration of neurosecretory systems, Int. Rev. CytoL, 36 (1973) 215-315. 9 Dupouey, P., Jacque, C., Bourre, J. M., Cesselin, F., Privat, A. and Baumann, N., Immunochemical studies of myelin basic protein in shiverer mouse devoid of major dense line of myelin, Neurosci. Lett., 12 (1979) 113-118. 10 Goldberg, S., Silver staining, featuring rapid reduction, for whole mounts of retina and optic pathways in chick embryos, Stain TechnoL, 47 (1972) 65-69. 11 Goldberg, S. and Frank, B., The guidance of optic axons in the developing and adult mouse retina, Anat. Rec., 193 (1979) 763-774.
myelinated. Thus, the results s u p p o r t the hypothesis t h a t b r e a k d o w n p r o d u c t s o f m a m m a l i a n oligodendrocytes or C N S myelin m a y inhibit a x o n a l growth, a l t h o u g h the evidence is circumstantial. W e are further testing the hypothesis m o r e directly by stud y i n g the injury response o f axons in the m y e l i n a t e d segment o f the r a b b i t retina I9 a n d in the C N S o f the shiverer m u t a n t mouse, in which there is severe l e u c o d y s t r o p h y with a specific absence o f myelin basic p r o t e i n 9,18. The results also vindicate the a s s u m p t i o n t h a t astrocytes are n o t involved in axon a l - g r o w t h inhibition. A l t h o u g h the presence o f astrocytes in the m o u s e retina has n o t been specifically d e t e r m i n e d , these cells are plentiful in the s t r a t u m o p t i c a r u m o f o t h e r species 5,15,2~, b e c o m i n g reactive after injury z~ t o g e t h e r with the astrocyte-like retinal Mtiller cells 3. The w o r k was s u p p o r t e d by grants f r o m the MRC, U.K.
12 Goldberg, S. and Frank, B., Will central nervous systems in the adult mammal regenerate after bypassing a lesion? A study in the mouse and chick visual systems, Exp. Neurol., 70 (1980) 675-689. 13 Goldberg, S. and Galin, M. A., Response of retinal ganglion cell axons to lesions in the adult mouse retina, lnvestOphthalmol., 21 (1973) 382-385. 14 Graziadei, P. P. C. and Monti Graziadei, G. A., The olfactory system: a model for the study of neurogenesis and axon regeneration in mammals. In C. W. Cotman (Ed.), Neuronal Plasticity, Raven Press, New York, 1978, pp. 131-153. 15 Hogan, M. J. and Feeney, L., The ultrastructure of retinal vessels III. Vascular-glial relationships, J. Ultrastruct. Res., 9 (1963) 47-64. 16 Kalil, K. and Reh, T., Regrowth of severed axons in the neonatal central nervous system: establishment of normal connections, Science, 205 (1979) 1158-1161. 17 McConnell, P. and Berry, M., Regeneration of axons in the mouse retina after injury, Bibl. anat., in press. 18 Mikoshiba, K., Aoki, E. and Tsukada, Y., 2',3'-Cyclic nucleotide 3'-phosphohydrolase activity in the central nervous system of a myelin deficient mutant (shiverer), Brain Research, 192 (1980) 195-204. 19 Prince, J. H. and McConnell, D. G., Retina and optic nerve. In J. H. Prince (Ed.), The Rabbit in Eye Research, Thomas, Springfield, IL, 1964, pp. 385-415. 20 Rodieck, R. W., The Vertebrate Retina, W. H. Freeman, San Francisco, CA, 1973. 21 Shakib, M. and Ashton, N., Focal retinal ischaemia. Part II. Ultrastructural changes in focal retinal ischaemia, Brit. J. Ophthalmol., 50 (1966) 325-359. 22 Svendgaard, N. A., BjSrklund, A. and Stenevi, U., Regeneration of central cholinergic neurones in the adult rat, Brain Research, 102 (1976) 1-22. 23 Wise, G., Dollery, C. and Henkind, P., The Retinal Circulation, Harper and Row, New York, 1971.
Brain Research, 241 (1982) 362-365
Elsevier Biomedical Press
Regeneration of ganglion cell axons in the adult mouse retina P. McCONNELL and M. BERRY Department of Anatomy, Medical School, University of Birmingham, Birmingham, B15 2TJ ( U.K.)
(Accepted February 25th, 1982) Key words: regeneration - - retina - - stratum opticarum - - ganglion cell - - unmyelinated nerve fibers
The hypothesis that regenerative failure of axons in the adult mammalian CNS is due to release of a growth inhibitor from injured oligodendrocytes and/or myelin2, predicts that regeneration of injured fibers would proceed unchecked in unmyelinated CNS regions. This prediction was borne out by observations on the stratum opticarum of the mouse retina. Axonal sprouts, first seen 14-16 h post-lesion (pl), continued growing until at least 100 days pl, well beyond the time at which regeneration fails in myelihated CNS regions. It has long been accepted that injured axons in the mature avian and mammalian CNS exhibit only a transitory regenerative response. Cajal's seminal studies 6 of mammalian spinal cord and optic nerve revealed that damaged fibers were capable of sprouting from 2 to 3 days post-lesion (pl), but that growth ceased by I0 14 days pl. Regenerated axons subsequently atrophied and were resorbed. These early observations were confined, however, to myelinated CNS regions. More recently, work on nonmyelinated fiber systems has provided evidence of a number of exceptions to this generally held view that post-injury growth is abortive. Thus, successful regeneration has been observed in the adult neurohypophyseal system s, olfactory nerve 14, monoaminergic system 4 and unmyelinated cholinergic nerves 2z, as well as in the immature unmyelinated CNS 16. These findings, coupled with the well-documented regenerative capacity of myelinated axons within the peripheral nervous system of mammals1, 6 and the CNS offish and amphibia 7, have led to the formulation of a hypothesis proposing that failure of central axonal regeneration, in mature mammals and birds, is due to the release of an active axonal-growth inhibitory factor (AGIF) from degenerating oligodendrocytes and/or myelin in the immediate postinjury period 2. Release of A G I F s from an astrocyte or mesodermal source was excluded because reactive astrocytes, fibroblasts and macrophages are features 0006-8993/82/0000-0000/$02.75 © Elsevier Biomedical Press
of all CNS wounds, independent of the type of axon injured, or the vigour of outgrowth. The A G I F hypothesis has been tested by investigating regeneration in the stratum opticarum of the retina, an area of the CNS where, in most species, axons are entirely unmyelinated 2°. Ganglion cell fibers are tightly fasciculated, running radially over the inner retinal surface to the optic papilla. Hence, fiber paths are planar and unidirectional, providing a unique model in which to study post-injury axonal growth. Moreover, the retina may be prepared as a whole-mount, thus avoiding errors otherwise introduced by serial sectioning 1°,~3. The experiment was designed with the aim of quantifying the extent of the growth-response of damaged retinal ganglion cell axons, predicting that growth would proceed unchecked in the absence of oligodendrocyte myelin. Thus, using applantation ophthalmoscopy 2~, small scratch lesions were placed between radial blood vessels in the ganglion cell fibre layer of the retina of adult female albino mice (MF 1 strain) by the technique of Goldberg and Frank 11. Groups of 10 animals were killed at 14, 16, 24, 36 and 48 h pl and 4, 6, 8, 10, 20, 30, 45 and 100 days pl, their eyes silver-stained 17 and retinal whole-mounts prepared by carefully dissecting away the lens, cornea, sclera and pigment epithelium, incising the retina radially, and flat-mounting in glycerol. The typical pattern of fiber outgrowth at various
363
Fig. 1. Photomontages of the pattern of fiber outgrowth at various intervals post-lesion, seen in retinal whole-mounts, a: fibers proximal to the lesion site 16 h pl, the majority of terminals have developed swollen end bulbs and some sprouts (arrows) are already present, b: by 36 h pl, the majority of injured proximal fibers had developed sprouts which either joined pre-existing axon fascicles or else showed random unfasciculated growth, occasionally by-passing or growing into the substance or the lesion (star). c: at 8 days pl, the regenerating axonal arborization has increased in extent and complexity, d: at 10 days pl, the proximal fiber plexus persists although, in many cases, the severed axons distal to the lesion (asterisk) have degenerated by this time. e: by 45 days pl, randomly oriented axons persist deep to the fiber layer (the lesion site is off the lower edge of the photograph), f: at 100 days pl, regenerating fibers are still present proximal to the lesion site. In all cases 'L' indicates the lesion site, and the optic papilla lies beyond the bottom edge of the photograph. Marker = 10/~m. intervals pl is illustrated in Fig. 1. Up until 24 h pl, the majority of fibers, both proximal and distal to the lesion site, showed terminal swellings or 'end
bulbs' similar to those described by Cajal 6. However, in contrast to previous reports2,12, there appeared to be sprouting of proximal fibers at even the earliest post-lesion intervals (Fig. la). Sprouts initially grew towards the lesion but, by 36-48 h pl, the majority had looped through 180 ° and either joined a pre-existing axon fascicle or else remained unfasciculated, growing in an apparently random manner just deep to the fiber layer. A few axons failed to loop, continuing to grow either into the substance of the lesion or around its edge (Fig. lb). Since it was impossible to follow the fate of the fibers which grew into fascicles, we describe here only non-fasciculated growth. The extent and complexity of fiber outgrowth increased with time (Fig. lc) and, by 10 days pl, a rich plexus of axons was present. By this stage, the severed distal portions of axons had degenerated in most cases, but the proximal sprouts showed no sign of atrophy (Fig. ld). Indeed, although a proportion of proximal fibers did begin to degenerate from about 15 days pl, many sprouts persisted and continued to grow until 100 days pl - - well beyond the time at which Cajal a noted the disappearance of regenerated axonal arbors (Fig. le, f). The precise extent and direction of axonal outgrowth was determined by tracing regenerating fibers using a camera lucida ( × 1250) in conjunction with a Kontron M O P / A M O 2 on line to a Hewlett Packard 9825A computer. At all but the earliest survival times (14, 16 and 24 h pl), 30 regenerating fibers were measured. Only 20 fibers could be traced from each of the 3 early groups. The measurements of fiber length (Fig. 2) revealed a phase of rapid (20 /~m/day) growth until 10 days pl, followed by a 50C
400
8 300 D)
~ 2oo tL 1 0 0
0
I
I
5
10
_1
I
I
I
I
15 20 25 30 35 Days post lesion
I
I
40 45
.~
I
100
Fig. 2. The extent of axonal outgrowth with increasing time post-injury.
364
OPTIC DISC
14hpl
24hpl
2dpl
4dpl
6dpl
lOdpl
30dpl
45dpl
1 O0 dpl
Fig. 3. Mean orientation of regenerating retinal axons The 360 ° histograms show the relative proportion of 2 mm fiber 'steps' per 10° of arc at the various post-lesion intervals indicated. Initially (14-24 h pl) most fibers grew towards the optic disc, but 36-48 h pl the majority looped through 180 ° producing a bipolar pattern of fiber outgrowth (4-6 days pl). This orientation was progressively lost, however, as the proportion of random growth increased. Thus, at the later survival times fibers showed no sign of any preferred direction of growth.
decline in overall g r o w t h rate, p r e s u m a b l y as regener a t e d sprouts b e g a n to degenerate. Nevertheless, the actual length o f the persisting regenerating fibers c o n t i n u e d to increase t h r o u g h o u t the experiment. The o r i e n t a t i o n o f successive 2 m m 'steps' o f the fiber tracings was d e t e r m i n e d with respect to the optic disc, a n d 360 ° h i s t o g r a m s c o n s t r u c t e d showing the relative p r o p o r t i o n o f 'steps' per 10° o f arc (Fig. 3). These i n d i c a t e d t h a t the direction o f o u t g r o w t h o f the regenerating nerve fibers was entirely r a n d o m . Thus, the lesioned optic axons in the retina o f the a d u l t m o u s e fail to rc-establish their original connections, possibly due to the absence o f a p p r o p r i a t e guidance m e c h a n i s m s ~2. However, their capacity for r e g r o w t h following injury is far greater t h a n anticip a t e d on the basis o f Cajal's 6 descriptions o f the a b o r t i v e g r o w t h response. One possible e x p l a n a t i o n for this d i c h o t o m y between the present observations in the retina a n d the results o f studies in the spinal c o r d a n d optic nerve, m a y lie in the fact t h a t fibers in the latter sites are
1 Aguayo, A. J., Bray, G. M., Perkins, C. S. and Duncan, I. D., Axon-sheath cell interactions in peripheral and central nervous system transplants. In J. A. Ferrendelli (Ed.), Aspects of Developmental Neurobiology, Society for Neuroscience Symposia, Vol. IV, Society for Neuroscience,
Bethesda, MD, 1979, pp. 361-383. 2 Berry, M., Post-injury myelin-breakdown products inhibit axonal growth: an hypothesis to explain the failure of axonal regeneration in the mammalian central nervous system, BibL anat., in press. 3 Bignami, A. and Dahl, D., The radial glia of MOiler in the rat retina and their response to injury. An immuno-fluorescence study with antibodies to the glial fibrillary acidic (GFA) protein, Exp. Eye Res., 28 (1979) 63-69. 4 Bj6rklund, A. and Stenevi, V., Regeneration of monoaminergic and cholinergic neurons in the mammalian central nervous system, PhyioL Rev., 59 (1979) 62-100. 5 Bfissow, H., The astrocytes in the retina and optic nerve head of mammals: a special glia for the ganglion cell axons, Cell Tiss. Res., 206 (1980) 367-378. 6 Cajal, S. Ram6n y, Degeneration and Regeneration of the Nervous System, (translation R. M. May), Hafner, London, 1968. 7 Clemente, C. D., Regeneration in the vertebrate central nervous system, Int. Rev. NeurobioL, 6 (1964) 251-301. 8 Dellman, H. D., Degeneration and regeneration of neurosecretory systems, Int. Rev. CytoL, 36 (1973) 215-315. 9 Dupouey, P., Jacque, C., Bourre, J. M., Cesselin, F., Privat, A. and Baumann, N., Immunochemical studies of myelin basic protein in shiverer mouse devoid of major dense line of myelin, Neurosci. Lett., 12 (1979) 113-118. 10 Goldberg, S., Silver staining, featuring rapid reduction, for whole mounts of retina and optic pathways in chick embryos, Stain TechnoL, 47 (1972) 65-69. 11 Goldberg, S. and Frank, B., The guidance of optic axons in the developing and adult mouse retina, Anat. Rec., 193 (1979) 763-774.
myelinated. Thus, the results s u p p o r t the hypothesis t h a t b r e a k d o w n p r o d u c t s o f m a m m a l i a n oligodendrocytes or C N S myelin m a y inhibit a x o n a l growth, a l t h o u g h the evidence is circumstantial. W e are further testing the hypothesis m o r e directly by stud y i n g the injury response o f axons in the m y e l i n a t e d segment o f the r a b b i t retina I9 a n d in the C N S o f the shiverer m u t a n t mouse, in which there is severe l e u c o d y s t r o p h y with a specific absence o f myelin basic p r o t e i n 9,18. The results also vindicate the a s s u m p t i o n t h a t astrocytes are n o t involved in axon a l - g r o w t h inhibition. A l t h o u g h the presence o f astrocytes in the m o u s e retina has n o t been specifically d e t e r m i n e d , these cells are plentiful in the s t r a t u m o p t i c a r u m o f o t h e r species 5,15,2~, b e c o m i n g reactive after injury z~ t o g e t h e r with the astrocyte-like retinal Mtiller cells 3. The w o r k was s u p p o r t e d by grants f r o m the MRC, U.K.
12 Goldberg, S. and Frank, B., Will central nervous systems in the adult mammal regenerate after bypassing a lesion? A study in the mouse and chick visual systems, Exp. Neurol., 70 (1980) 675-689. 13 Goldberg, S. and Galin, M. A., Response of retinal ganglion cell axons to lesions in the adult mouse retina, lnvestOphthalmol., 21 (1973) 382-385. 14 Graziadei, P. P. C. and Monti Graziadei, G. A., The olfactory system: a model for the study of neurogenesis and axon regeneration in mammals. In C. W. Cotman (Ed.), Neuronal Plasticity, Raven Press, New York, 1978, pp. 131-153. 15 Hogan, M. J. and Feeney, L., The ultrastructure of retinal vessels III. Vascular-glial relationships, J. Ultrastruct. Res., 9 (1963) 47-64. 16 Kalil, K. and Reh, T., Regrowth of severed axons in the neonatal central nervous system: establishment of normal connections, Science, 205 (1979) 1158-1161. 17 McConnell, P. and Berry, M., Regeneration of axons in the mouse retina after injury, Bibl. anat., in press. 18 Mikoshiba, K., Aoki, E. and Tsukada, Y., 2',3'-Cyclic nucleotide 3'-phosphohydrolase activity in the central nervous system of a myelin deficient mutant (shiverer), Brain Research, 192 (1980) 195-204. 19 Prince, J. H. and McConnell, D. G., Retina and optic nerve. In J. H. Prince (Ed.), The Rabbit in Eye Research, Thomas, Springfield, IL, 1964, pp. 385-415. 20 Rodieck, R. W., The Vertebrate Retina, W. H. Freeman, San Francisco, CA, 1973. 21 Shakib, M. and Ashton, N., Focal retinal ischaemia. Part II. Ultrastructural changes in focal retinal ischaemia, Brit. J. Ophthalmol., 50 (1966) 325-359. 22 Svendgaard, N. A., BjSrklund, A. and Stenevi, U., Regeneration of central cholinergic neurones in the adult rat, Brain Research, 102 (1976) 1-22. 23 Wise, G., Dollery, C. and Henkind, P., The Retinal Circulation, Harper and Row, New York, 1971.