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.