Approximately half of the retinal ganglion cells (RGCs) present in the rodent retina at birth normally die during early development. Overexpression of the proto-oncogene bcl-2 recently has been shown to rescue some neuronal populations from natural cell death and from degeneration induced by axotomy of nerves within the peripheral nervous system. Here we study in vivo the role of the overexpression of bcl-2 in the natural cell death of RGCs and in the degenerative process induced in these cells by transection of the optic nerve. We find that in newborn bcl-2 transgenic mice, the number of RGCs undergoing natural cell death is considerably lower than in wild-type pups. Consistently, a vast majority (90%) of the ganglion cells found in the retina of neonatal transgenics are maintained in adulthood, whereas only 40% survive in wild-type mice. After transection of the optic nerve, the number of degenerating ganglion cells, determined by counting pyknotic nuclei or nuclei with fragmented DNA, is substantially reduced in transgenic mice. In wild-type animals, almost 50% of ganglion cells degenerate in the 24 hr after the lesion, whereas almost the entire ganglion cell population survives axotomy in transgenic mice. Therefore, overexpression of bcl-2 is effective in preventing degeneration of this neuronal population, raising the possibility that ganglion cells are dependent on the endogenous expression of bcl-2 for survival. The remarkable rescue capacity of bcl-2 overexpression in these neurons makes it an interesting model for studying natural cell death and responses to injury in the CNS.

The expression of the nerve growth factor-inducible gene VGF has been examined by in situ hybridization. Western blot and immunohistochemical studies in the developing and adult rat central nervous system, with particular emphasis on the visual system. Both the messenger RNA and the protein are particularly abundant in the developing dorsal lateral geniculate nucleus, appearing, respectively, at embryonal day 16 and 18. After its onset at E16, VGF messenger RNA expression increases progressively in the dorsal lateral geniculate nucleus and remains high during the first two post-natal weeks; afterwards, it gradually decreases and, at the offset of the plasticity period, it reaches very low levels maintained in adulthood. A similar time course has been observed for VGF protein in the dorsal lateral geniculate nucleus area, by semi-quantitative Western blots. In addition to the presence of the protein in the geniculate neurons, a strong, transient immunoreactivity has been found at the embryonic cortical subplate at E18, reflecting the presence of the antigen in axonal terminals originating from thalamic neurons. Interestingly, we found that the blockade of afferent electrical activity by intraocular injection of tetrodotoxin strongly reduces the level of VGF messenger RNA in the dorsal lateral geniculate nucleus. Although the function of the VGF protein is not known, it had been previously proposed that VGF could be a precursor for neuropeptide/s. The spatiotemporal expression of VGF, together with the observation of a regulation by electrical activity, suggest that this protein may be relevant in the process of synaptogenesis and/or synaptic stabilization in the developing geniculocortical connections.

It has recently been reported that the degeneration of retinal ganglion cells induced by transection of the optic nerve in the neonatal rat is due to an active process of apoptosis, as opposed to passive necrosis. Here we tested whether the administration of the trophic factor nerve growth factor could prevent the apoptotic death of the axotomized cells. We administered nerve growth factor by two intraocular injections, one immediately after the lesion and the second 12 h later. The retinas were taken at 24 h post-lesion and stained as whole mounts with Cresyl Violet. Pyknotic as well as surviving cells were counted in the retinal ganglion cell layer. In this layer at least 95% of the total cell population is composed by ganglion cells, as revealed by retrogradely labelling these cells with horseradish peroxidase injected in the superior colliculi. We found that intraocular administration of nerve growth factor diminishes the degeneration induced by optic nerve transection in the neonatal rat. After nerve growth factor injection, in fact, the number of pyknotic cells is reduced by 39% compared with controls (lesioned, injected with saline); in addition, nerve growth factor also increases the survival of retinal ganglion cells by 30% at 24 h post-lesion.

Cell death can be ascribed to one of two distinct modes of degeneration: apoptosis (programmed or active cell death) or necrosis (passive degeneration). While apoptosis is generally assumed to occur in physiological conditions such as normal development or tissue turnover, necrotic cell degeneration is induced in pathological situations. Here we report that also in a pathological situation, such as after axotomy in the CNS, apoptotic type of cell death comes into play: following intracranial transection of the optic nerve in the neonatal rat in vivo, retinal ganglion cells undergo an active, apoptotic cell death. In fact, the administration of protein synthesis inhibitors (actinomycin D and cycloheximide) prevents the appearance of pyknotic nuclei as well as of fragmented DNA of ganglion cells at 24 hr postlesion. Correspondingly, the number of surviving cells after actinomycin D and cycloheximide treatment is comparable to normal, unlesioned retinas. In addition, cycloheximide decreases the number of pyknotic ganglion cells during spontaneous cell death.