Assessment of Brain-Derived Neurotrophic Factor on Retinal Structure and Visual Function in Rodent Models of Optic Nerve Crush

Abstract

The effects of brain-derived neurotrophic factor (BDNF) on retinal ganglion cell (RGC) survival and visual function were assessed in rat and mouse models of optic nerve (ON) crush. ONs were crushed on Day 1, followed by intravitreal injections of a vehicle or BDNF on Days 1 and 8. The spatial frequency threshold was measured using optokinetic tracking on Days 7 and 14. On Day 15, ganglion cell complex (GCC) thickness was quantified using optical coherence tomography. Furthermore, all eyes were enucleated for immunohistochemical analysis of the surviving RGC somas and axons. BDNF significantly reduced the RGC soma in mice and increased GCC thickness in intact eyes, with apparent axonal swelling in both species. It displayed significantly greater RGC soma survival in eyes with ON injury, with moderately thicker axonal bundles in both species and a thicker GCC in rats. Visual function was significantly reduced in all ON-crushed animals, regardless of BDNF treatment. Thus, we obtained a comprehensive analysis of the structural and functional impact of BDNF in intact and ON-crushed eyes in two rodent models. Our results provide a foundation for further BDNF evaluation and the design of preclinical studies on neuroprotectants using BDNF as a reference positive control.

Keywords: brain-derived neurotrophic factor; glaucoma; neuroprotection; optic nerve crush; retinal ganglion cells.

Figure 1

An outline of optic nerve crush experiments in rats and mice. The optic nerve was crushed just before the intravitreal injection of the vehicle, or BDNF, on Day 1. A second intravitreal injection was performed on Day 8. Optokinetic tracking was performed to assess visual function on Days 7 and 14. Optical coherence tomography was performed to quantify the ganglion cell complex (GCC) thickness on Day 15, followed by animal euthanasia, after which eyeballs were enucleated for flatmount immunohistochemistry to label retinal ganglion cells (RGCs) with RBPMS and TUJ1.

Figure 2

Effects of BDNF on retinal ganglion cell survival in rats. RGC soma count (a), as measured in uncrushed and crushed eyes administered a vehicle or BDNF injection. Vehicle, closed column; BDNF, diagonal column. Each value represents the mean ± standard error of the mean (SEM) for n = 8 (a). *** p < 0.001, as determined using Student’s t-test. Representative images from retinal flatmounts stained with anti-RBPMS to label surviving RGCs in peripheral regions (b) and with anti-TUJ1 to label RGC axons close to the optic nerve head (c) in rats. No difference in RGC soma count was observed between the uncrushed eyes, regardless of treatment. Crushed eyes receiving BDNF displayed significantly greater RGC survival compared to those injected with the vehicle. Axonal density and integrity were decreased in the crushed eyes. However, axon bundles appeared moderately thicker in BDNF-injected eyes compared to vehicle-injected controls. Note: The bright spot in the top left represents the optic nerve head (c).

Figure 3

Effects of BDNF on retinal ganglion cell survival in mice. RGC soma number, as measured in uncrushed and crushed eyes administered a vehicle or BDNF injection. Vehicle, closed column; BDNF, diagonal column. Each value represents the mean ± SEM for n = 8 (a). ** p < 0.01, *** p < 0.001, as determined using Student’s t-test. Representative images from retinal flatmounts stained with anti-RBPMS to label surviving RGCs in peripheral regions (b) and with anti-TUJ1 to label RGC axons close to the optic nerve head (c) in mice. BDNF slightly decreased RGC soma survival in uncrushed eyes compared to vehicle-injected eyes. Crushed eyes receiving BDNF displayed greater RGC survival compared to those injected with the vehicle. Axonal density and integrity were decreased in crushed eyes. However, axon bundles appeared thicker in eyes administered BDNF compared to vehicle-injected controls. Note: The bright spot in the top left represents the optic nerve head (c).

Figure 4

Effects of BDNF on ganglion cell complex (GCC) thickness in rats. GCC thickness (a), as measured in uncrushed and crushed eyes administered a vehicle or BDNF injection. Vehicle, closed column; BDNF, diagonal column. Each value represents the mean ± standard error of the mean (SEM) for n = 4–8 (a). ** p < 0.01, *** p < 0.001, as determined using Student’s t-test. Representative peripapillary circle scans from optical coherence tomography measurements in rats (b). The GCC thickness, which consists of the retinal nerve fiber layer (RNFL), ganglion cell layer (GCL), and inner plexiform layer (IPL), was measured. ONC reduced GCC thickness in vehicle-injected eyes. Increased thickness was evident in the RNFL of BDNF-injected eyes without optic nerve crush. A slight RNFL increase was also qualitatively observed in BDNF-injected crushed eyes (c). Scale bar = 50 mm.

Figure 5

Effects of BDNF on ganglion cell complex (GCC) thickness in mice. GCC thickness (a), as measured in uncrushed and crushed eyes administered a vehicle or BDNF injection. Vehicle, closed column; BDNF, diagonal column. Each value represents the mean ± SEM for n = 7–8 (a). *** p < 0.001, as determined using Student’s t-test. Representative peripapillary circle scans from optical coherence tomography measurements in mice (b). The GCC thickness was measured. ONC reduced GCC thickness in vehicle-injected eyes. Increased thickness was only evident in the RNFL of uncrushed eyes administered BDNF (c). Scale bar = 100 mm.

Figure 6

Effects of BDNF on optokinetic tracking in rats. Spatial frequency threshold, as measured in uncrushed and crushed eyes administered a vehicle or BDNF injection. Vehicle, closed column; BDNF, diagonal column. Each value represents the mean ± standard error of the mean (SEM) for n = 8–10.

Figure 7

Effects of BDNF on optokinetic tracking in mice. Spatial frequency threshold, as measured in uncrushed and crushed eyes administered a vehicle or BDNF injection. Vehicle, closed column; BDNF, diagonal column. Each value represents the mean ± SEM for n = 5–8.