Cell Death During Development of the Nervous System

To investigate the role of extrinsic influences on the survival and growth of axotomized retinal ganglion cells (RGCs) in the mature mammalian CNS, both optic nerves (ONs) of adult rats were transected intraorbitally and, on one side, replaced by an autologous segment of peripheral nerve (PN) that had been left unconnected distally. The survival of RGCs and the regrowth of their cut axons into the PN grafts were assessed using morphometric techniques, neuroanatomic tracers, and immunologic cell markers to identify and count RGCs at times ranging from 15 d to 9 months. It was observed that (1) in the absence of a PN graft, more than 90% of the RGCs died by 1 month after axotomy; (2) between 1 and 3 months after axotomy, survival of RGCs in the PN-grafted retinas was enhanced 2-4-fold; (3) nearly 20% of the surviving RGCs regrew lengthy axons into the grafts; and (4) although the density of surviving RGCs in PN-grafted retinas decreased significantly between 1 and 3 months after axotomy, the densities of RGCs with axons extending into the graft remained relatively stable. These results confirm that in the adult rat retina, neuronal death is a major effect of axotomy near the cell soma. Although such lesions lead to the degeneration of many RGCs, we show that extrinsic influences introduced by the apposition of a PN segment at the time of severing the ON can rescue a substantial number of these neurons. Because the enhanced survival of many axotomized RGCs in the PN-grafted retinas appears to be limited to the first few weeks after injury, while those of RGCs that regenerate axons into the grafts do not show a parallel decline, it is possible that, in these experiments, neuronal viability depends on a spectrum of differently timed influences that may include the early diffusion of critical molecules arising from the graft and the subsequent establishment of more complex interactions with graft components.

https://www.jneurosci.org/content/jneuro/8/1/265.full.pdf

Influences of peripheral nerve grafts on the survival and regrowth of axotomized retinal ganglion cells in adult rats

This review will describe the unique advantages that are offered by the visual system of mammals and other vertebrates for studying the regenerative responses of the central nervous system (CNS) to injury, and recent insights provided by such studies. In the mouse and rat visual system a variety of experimental paradigms promote survival of retinal ganglion cells (RGC) and optic nerve regeneration, probably through stimulation by neurotrophic factors (NTF) either directly, or indirectly through retinal astrocyte/Muller cell intermediary activation. NTF induce disinhibition of axon growth through regulated intramembranous proteolysis of p75NTR, and the inactivation of RhoA and EGFR signalling. The concomitant release of metalloproteinases (MMP) and plasminogen activators from RGC axons, and tissue inhibitors of metalloproteinases from optic nerve glia repress scarring and thereby reduce titres of scar-derived inhibitory ligands expressed in the wound. MMP also degrade myelin-derived inhibitory ligands along regenerating axon trajectories after regulated release from glia at the growing front of regenerating RGC axons. Optic nerve transection induces apoptosis of RGC which is blocked by anti-apoptotic regimes and thus, in combination with blockers of axon-growth inhibitory signalling and promoters of axon growth may be a therapeutic formula for promoting sustained axon regeneration. All these findings in the visual system are translatable to the CNS as a whole and thus strategies that successfully promote visual axon regeneration will be equally effective elsewhere in the CNS. Future developments likely to advance the field of regenerative research include a greater understanding of phylogenetic differences in the response of the CNS to injury, the role of NTF, cAMP, EGFR, glia/neuron interactions in disinhibiting and promoting axon growth, the control of neuron death, and effective drug delivery.

Regeneration of axons in the visual system.

The vertebrate retina is an extension of the brain, a hemisphere of neural tissue upon which is mapped an image of a particular species visual environment. Each point in visual space is subtended by a corresponding point on the neural retina which in turn is retinotopically mapped onto the visual centres of the brain. Light energy or the ‘optical image’ is transformed into electrical energy or a ‘neural image’ by the photoreceptors and, via a number of interneurons (bipolar, amacrine and horizontal cells), these signals reach the ganglion cells each of which possess an axon carrying information to the central nervous system via the optic nerve. Although processing at the level of the photoreceptors may not necessarily change the neural image, due to the over abundance of photoreceptors relative to the number of ganglion cells, it is the ganglion cells which ultimately define the perception of a species’ environment received by the central nervous system.

Behavioural ecology and retinal cell topography

Individual optic arbors, normal and regenerated, were stained via anterograde transport of HRP and viewed in tectal whole mounts. Camera lucida drawings were made of 119 normal optic arbors and of 242 regenerated arbors from fish 2 weeks to 14 months postcrush. These arbors were analyzed for axonal trajectory, spatial extent in the horizontal plane, degree of branching, number of branch endings, average depth, and degree of stratification. Normal optic arbors ranged in size from roughly 100 to 400 microns across in a continuous distribution, had an average of 20 branch endings with average of fifth-order branching, and were highly stratified into one of three planes within the major optic lamina (SO-SFGS). Small arbors arising from fine-caliber axons terminated in the most superficial plane of SO-SFGS; large arbors from coarse axons terminated in the superficial and middle planes; and medium arbors from medium-caliber axons terminated in the middle and deep planes of SO-SFGS, as well as deeper in the central gray and deep white layers. Arbors from central tectum tended to be much more tightly stratified than those in the periphery. No other differences between central and peripheral arbors were noted. Mature regenerated arbors (five months or more postcrush) were normal in their number of branch endings, order of branching, and depth of termination. Their branches covered a wider area of tectum, partially because of their early branching and abnormal trajectories of branches. Axonal trajectories were often abnormal with U-turns and tortuos paths. Fine-, medium-, and coarse-caliber axons were again present and gave rise to small, medium, and large arbors at roughly the same depths as in the normals. There was frequently a lack of stratification in the medium and large arbors, which spanned much greater depths than normal. Overall, however, regenerates reestablished nearly normal morphology except for axonal trajectory and stratification. Early in regeneration, the arbors went through a series of changes. At 2 weeks postcrush, regenerated axons had grown branches over a wider-than-normal extent of tectum, though they were sparsely branched and often tipped with growth cones. At 3 weeks, the branches were more numerous and covered a still wider extent (average of five times normal), many covering more than half the tectal length or width. At 4-5 weeks smaller arbors predominated, although a few enlarged arbors were present for up to 8 weeks. Additional small changes occurred beyond 8 weeks as the arbors became progressively more normal in appearance.(ABSTRACT TRUNCATED AT 400 WORDS)

Staining of regenerated optic arbors in goldfish tectum: progressive changes in immature arbors and a comparison of mature regenerated arbors with normal arbors.

In the frog Hyla moorei we have estimated there to be between approximately 450,000 and 750,000 cells in the retinal ganglion cell layer. Optic axon counts and retrograde transport of horseradish peroxidase (HRP) indicated that 72–76% of these were ganglion cells. Cells of this type were distributed as a temporally situated area centralis within a horizontal visual streak.



Cell and optic axon counts showed that there was an approximately 40% loss of ganglion cells during optic nerve regeneration. Ganglion cells appeared chromatolysed by 6–8 days after an extracranial nerve crush but there was no indication of cell death until 15 days. By this stage anterograde transport of HRP indicated that axons had reached the chiasma. Death was first seen in the area centralis, extended along the streak, and finally was observed in the periphery by 65 days; cell counts demonstrated that at this time the wave of death was almost complete. We have previously shown by electrophysiological visual mapping (Humphrey and Beazley, 1982) and confirmed in this study that visuotectal projections were retinotopically organized during regeneration. Multiunit receptive fields were initially large but progressively refined starting in nasal field (temporal retina) to restore a normal projection. The similar sequences whereby the visuotectal projection became refined and death took place in the retinal ganglion cell layer suggested that death may be related to a process of organization within the regenerating projection.



In normal animals primary visual pathways revealed by anterograde transport of HRP were essentially similar to those of Rana pipiens and R. esculenta. Regenerating axons generally remained within optic pathways. Exceptions were a retinoretinal projection which was not completely withdrawn even after 1,028 days and a direct projection to the ipsilateral tectum via an inappropriate part of the optic tract.

Retinal ganglion cell death during optic nerve regeneration in the frog Hyla moorei.

We have previously reported that a proportion of ganglion cells die during optic nerve regeneration in the adult frog Hyla moorei (Humphrey and Beazley, '85). Here we assess the effect of preventing optic nerve regeneration on this cell loss. The optic nerve was crushed unilaterally and regeneration was allowed to progress unimpeded in one experimental series but was prevented by ligating or capping the nerve in another.



We estimated total cell numbers in the ganglion cell layer from cresylstained wholemounts, comparing each experimental retina with its unoperated partner. At 70–78 days postcrush, mean cell numbers had fallen by 31.5% for frogs with unimpeded regeneration (N = 9), a significantly greater reduction than the 21.5% (N = 8) loss for the impeded regeneration series (p < 0.001). Thereafter, cell numbers were stable for frogs with unimpeded regeneration. Cell death continued in the series with impeded regeneration, and losses exceeded those of frogs with unimpeded regeneration from 110 days postcrush. When regeneration was impeded, ganglion cell somas underwent an intense cell soma reaction and became arranged in rows radiating from the optic nerve head.



Our findings indicate that some ganglion cells are transiently spared when regeneration of their axons is prevented. The abnormally extensive contacts formed between somas may delay ganglion cell loss. However, the eventual death of most ganglion cells shows them to be target-independent in the long term.

Prevention of optic nerve regeneration in the frog Hyla moorei transiently delays the death of some ganglion cells

Extensive regeneration of the optic nerve takes place in adult Amphibia. In this study, we have determined whether one aspect of retinotectal organisation, namely immunoreactive laminae in the retinorecipient layers of the optic tectum, is restored after optic nerve regeneration. To do so, the distributions of substance-P, bombesin, and leucine-enkephalin immunoreactivities were examined in the optic tectum of the frog Litoria (Hyla) moorei. Results of a normal series were compared with those at intervals up to 84 days and at 196 days after either unilateral deafferentation or optic nerve crush. In the normal series, distinct neuropeptide immunoreactive laminae were located within the retinorecipient tectal layers. There were two major laminae with substance-P, two with bombesin, and one with leucine-enkephalin immunoreactivities. Additional faint laminae of both substance-P and bombesin immunoreactivity were present in the tectal region that receives input from the visual streak. In addition, labelling of cell bodies and dendrites was seen elsewhere in the tectum. All except one immunoreactive lamina changed after deafferentation. The deeper of those with substance-P immunoreactivity, along with both bombesin laminae, were eventually lost; the lamina with leucine-enkephalin immunoreactivity was halved in intensity. We assume that these laminae are wholely or, in the case of the leucine-enkephalin lamina, partially associated with primary optic input. By contrast, the more superficial lamina with substance-P immunoreactivity remained unchanged and is presumably not directly related to visual input. During nerve regeneration, the intensity of all laminae associated with optic input initially fell as in the deafferentation series but, in the long term, recovered to approximately 80% of normal intensities. We conclude that ganglion cells associated with each of the immunoreactivities tested had successfully regenerated. The reduced intensity of immunoreactivities after regeneration is due presumably in part to the cell loss from the ganglion cell population. Furthermore, we discuss the findings of similar studies for Rana pipiens (Kuljis and Karten [1983] J. Comp. Neurol. 217:239-251 and [1985] 240:1-15) in light of the present findings. We argue that some of the previous observations can be reinterpreted to indicate that regeneration was not limited to ganglion cells associated with substance-P immunoreactivity as first thought.

M. F. Humphrey

Papers

Retinal ganglion cell death during optic nerve regeneration in the frog Hyla moorei.

Prevention of optic nerve regeneration in the frog Hyla moorei transiently delays the death of some ganglion cells

Substance P, bombesin, and leucine-enkephalin immunoreactivities are restored in the frog tectum after optic nerve regeneration.