Effects of ocular injury and administration ofbrain-derived neurotrophic factor on survival and regrowth of axotomized retinalganglion cells.

Abstract

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

Lens Injury Stimulates Axon Regeneration in the Mature Rat Optic Nerve

“…Quantitation revealed a cell density of 1806 Ϯ 54 RGCs/mm 2 (n ϭ 14 cases; Fig. 9a,d), similar to previously reported results Mansour-Robaey et al, 1994;Clarke et al, 1998;Koeberle and Ball, 1998). Very few RGCs remained alive 3 Figure 3.…”

“…Both cell death and regenerative failure can be altered by manipulating extracellular conditions. Intravitreal injections of brain-derived neurotrophic factor (BDNF), neurotrophin-(NT-) 4/5, ciliary neurotrophic factor (CNTF), glial-derived neurotrophic factor (GDNF), and other polypeptide growth factors enhance RGC survival after nerve crush, although this effect is transient (Carmignoto et al, 1989;Mey and Thanos, 1993;Cohen et al, 1994;Mansour-Robaey et al, 1994;Rabacchi et al, 1994;Di Polo et al, 1998;Koeberle and Ball, 1998). In culture, isolated RGCs require a combination of insulin (or IGF-2), CNTF (or LIF), BDNF (or NT-4/5), and elevated intracellular cAMP for their survival (Meyer-Franke et al, 1995).…”

Cellular and Molecular Pathways of Ischemic Neuronal Death

“…The neuroprotective effects of neurotrophins have been demonstrated under various pathological conditions. For example, neurotrophins protect various populations of neurons from axotomy (Hefti, 1986;Yan et al, 1992;Mey and Thanos, 1993;Chiu et al, 1994;Cohen et al, 1994;Mansour-Robaey et al, 1994;Friedman et al, 1995). Neurotrophins can attenuate neuronal death following global or focal cerebral ischemia (Shigeno et al, 1991;Beck et al, 1994;Chan, 1996).…”

Methods for Experimental Manipulations after Optic Nerve Transection in the Mammalian CNS

“…This procedure requires some practice, as it is critical not to injure the lens with the tip of the glass pipette. Lens damage has been shown to cause the release of growth factors, altering cell survival and regeneration 20,21 . It is also important to carefully insert and withdraw the glass pipette parallel to the direction of the tip.…”