Using quantitative anatomical techniques, we show that after intraorbital optic nerve transection in adult rats, virtually all retinal ganglion cells (RGCs) survive for 5 d and then die abruptly in large numbers, reducing the RGC population to approximately 50% of normal by day 7 and to less than 10% on day 14. During this period of rapid cell loss, some RGCs show cytochemical alterations indicative of apoptosis ("programmed cell death"), a change not previously categorized after axotomy in adult mammals. With intracranial lesions 8-9 mm from the eye, the onset of cell death is delayed until day 8 and is greater with cut than crush. The demonstration that axotomy results in apoptosis, the long interval between axonal injury and RGC death, and the different time of onset of the massive RGC loss with optic nerve lesions near or far from the eye suggest that axonal interruption triggers a cascade of molecular events whose outcome may be critically dependent on the availability of neuronal trophic support from endogenous or exogenous sources. The role of such molecules in RGC survival and the reversible nature of these injury-induced changes is underscored by the temporary rescue of most RGCs by a single intravitreal injection of brain-derived neurotrophic factor during the first 5 d after intraorbital optic nerve injury (Mansour-Robaey et al., 1994). The delayed pattern of RGC loss observed in the present experiments likely explains such a critical period for effective neurotrophin administration.

Optic nerve tansection in adult rats results in the death of -50% of the axotomized retinal on cells (RGCs) by 1 week and nearly 90% by 2 weeks after injury. RGC Lang. ROCs were retrogradely labeled with Fluorogold (Fluorochrome, Englewood, CO; 2% in 0.9% NaCl containing 10%o dimethyl sulfoxide) applied to the surface of both SC, as described for 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (diI) (11,12). For the experiments in which a correlation between RGC survival and axonal regrowth was investigated (as described below), horseradish peroxidase (HRP; Boehringer Mannheim) was applied to the distal tip of the PN graft, "1 cm from the eye, 6 weeks after the graft was attached to the ocular stump ofthe ON (13).ON Transection. One week after Fluorogold application, the left ON was transected 0.5 mm from the eye (1). In some animals, a segment of autologous sciatic nerve was attached to the ocular stump of the transected ON to test the capacity of the RGCs to regenerate and extend their axons (13).Injection Procedure. Anesthetized animals received single injections 3 or 6 days before or 0-10 days after ON transection. Multiple injections were given on postoperative days 0, 3, 7, and 10 for the animals without PN grafts and on days 0, 3, and 7 for the animals with PN grafts. Intraocular injections were made with a 10-A4 Hamilton syringe fitted with a 26-gauge needle whose tip was inserted into the vitreous space by an anterior or posterior approach. For the anterior approach, a drop of 2% lidocaine (Xylocaine) was applied to the conjunctiva, and the needle was inserted through the cornea-sclerajunction and advanced into the vitreous chamber, avoiding direct contact with the retina. By this approach, the needle usually pierced the margins of the iris and could damage the surface of the lens. After injection, Polysporin ointment was applied to the puncture site. For the posterior approach, the needle was inserted through the sclera and retina at the time of ON transection; this route avoided direct injury to the iris or lens. All experiments to determine the range of effective times for BDNF and control injections, as

To investigate the short- and long-term effects of axotomy on the survival of central nervous system (CNS) neurons in adult rats, retinal ganglion cells (RGCs) were labelled retrogradely with the persistent marker diI and their axons interrupted in the optic nerve (ON) by intracranial crush 8 or 10 mm from the eye or intraorbital cut 0.5 or 3 mm from the eye. Labelled RGCs were counted in flat-mounted retinas at intervals from 2 weeks to 20 months after axotomy. Two major patterns of RGC loss were observed: (1) an initial abrupt loss that was confined to the first 2 weeks after injury and was more severe when the ON was cut close to the eye; (2) a slower, persistent decline in RGC densities with one-half survival times that ranged from approximately 1 month after intraorbital ON cut to 6 months after intracranial ON crush. A small population of RGCs (approximately 5%) survived for as long as 20 months after intraorbital axotomy. The initial loss of axotomized RGCs presumably results from time-limited perturbations related to the position of the ON injury. A persistent lack of terminal connectivity between RGCs and their targets in the brain may contribute to the subsequent, more protracted RGC loss, but the differences between intraorbital cut and intracranial crush suggest that additional mechanisms are involved. It is unclear whether the various injury-related processes set in motion in both the ON and the retina exert random effects on all RGCs or act preferentially on subpopulations of these neurons.

In adult rats, one optic nerve was transected and replaced by a 4 cm segment of autologous peripheral nerve (PN) that linked one eye and the superior colliculus (SC) along a predominantly extracranial course. Retrograde and orthograde studies with the tracers HRP or rhodamine-B- isothiocyanate (RITC), as well as immunocytochemical neuronal labels, indicated the following: (1) Regenerating axons from the axotomized retinal ganglion cells extended along the entire PN grafts, covering a distance nearly twice that of the normal retinotectal projection of intact rats. (2) Some of these axons penetrated the SC and formed terminal arborizations up to 500 microns from the end of the graft. (3) By electron microscopy, the arborizations of these regenerated axons in the SC were seen as small HRP-labeled axonal profiles that contacted neuronal processes in the SC; some of these contacts showed pre- and postsynaptic membrane specializations. These findings indicate that injured retinal ganglion cells in the adult rat are not only able to regrow lengthy axons, but may also form synapses in the SC.