Brimonidine prevents axonal and somatic degeneration of retinal ganglion cell neurons

Abstract

Background: Brimonidine is a common drug for lowering ocular pressure and may directly protect retinal ganglion cells in glaucoma. The disease involves early loss of retinal ganglion cell transport to brain targets followed by axonal and somatic degeneration. We examined whether brimonidine preserves ganglion cell axonal transport and abates degeneration in rats with elevated ocular pressure induced by laser cauterization of the episcleral veins.

Results: Ocular pressure was elevated unilaterally by 90% for a period of 8 weeks post- cauterization. During this time, brimonidine (1mg/kg/day) or vehicle (phosphate-buffered saline) was delivered systemically and continuously via subcutaneous pump. Animals received bilateral intravitreal injections of fluorescent cholera toxin subunit β (CTB) two days before sacrifice to assess anterograde transport. In retinas from the vehicle group, elevated pressure induced a 44% decrease in the fraction of ganglion cells with intact uptake of CTB and a 14-42% reduction in the number of immuno-labelled ganglion cell bodies, with the worst loss occurring nasally. Elevated pressure also caused a 33% loss of ganglion cell axons in vehicle optic nerves and a 70% decrease in CTB transport to the superior colliculus. Each of these components of ganglion cell degeneration was either prevented or significantly reduced in the brimonidine treatment group.

Conclusions: Continuous and systemic treatment with brimonidine by subcutaneous injection significantly improved retinal ganglion cell survival with exposure to elevated ocular pressure. This effect was most striking in the nasal region of the retina. Brimonidine treatment also preserved ganglion cell axon morphology, sampling density and total number in the optic nerve with elevated pressure. Consistent with improved outcome in the optic projection, brimonidine also significantly reduced the deficits in axonal transport to the superior colliculus associated with elevated ocular pressure. As transport deficits to and from retinal ganglion cell projection targets in the brain are relevant to the progression of glaucoma, the ability of brimonidine to preserve optic nerve axons and active transport suggests its neuroprotective effects are relevant not only at the cell body, but throughout the entire optic projection.

Figure 1

Systemic BMD does not affect IOP. Ocular pressure in mmHg as measured using TonoLab following laser photocoagulation of episcleral veins at two time points (arrows). Systemic BMD (1 mg/kg/day) delivered via subcutaneous osmotic pump did not affect IOP for control or OHT eyes compared to vehicle treatment (p ≥ 0.6; mean ± SEM; n = 16 eyes per group).

Figure 2

BMD preserves RGC morphology in retinas exposed to OHT. (A-C) Representative confocal images of whole-mounted naïve rat retina demonstrating co-localization of CTB (green) with phosphorylated heavy-chain neurofilament (SMI31; red) in RGC axons (dashed lines) and somas (open arrowheads). Similar co-localization was observed in vehicle (D) and BMD (J) control retinas. Vehicle OHT retinas (E-I) demonstrated fewer CTB + RGC somas and axons, dystrophic axons (arrowheads), and swollen RGCs with dendrites accumulating SMI31 (arrows). In BMD OHT retinas (K, L), co-localization of CTB and SMI31 was similar to that in naïve and control retinas. Distance from optic disc is 1 mm (A, D, E, G, J, K), 2 mm (B, H, I), or 4 mm (C, F, L).

Figure 3

BMD preserves CTB uptake by RGCs during OHT. Bar chart shows CTB+ and SMI31+ RGCs as the ratio of OHT to control retina for the vehicle- and BMD-treated rats (mean ± SEM; n = 6 retina each). The naïve group is represented simply as the ratio of the right to left retina. In the vehicle group, OHT decreased the number of CTB+ RGCs so that the ratio was significantly less than one (*, p = 0.01). Treatment with BMD improved CTB uptake in OHT retinas compared to vehicle (**, p < 0.001). The number of SMI31+ RGCs in vehicle-treated retinas was also decreased by OHT, but this result was not significant. The ratio of OHT to control eye for both CTB+ and SMI31+ RGCs was identical for the naïve and BMD-treated rats (p ≥ 0.11).

Figure 4

BMD prevents modest RGC somatic loss with OHT. RGC density is shown for control, vehicle- and BMD-treated groups across eccentricity (mean ± SEM; n = 6 retina each). RGC density decreased with OHT in vehicle retinas compared to control at all locations in each quadrant, with significant decreases indicated (*). In BMD-treated rats, RGC density with OHT was the same as control retinas except at 3 locations in the nasal quadrant (†, p ≤ 0.02) and was significantly higher than vehicle OHT retinas at locations in both temporal and nasal quadrants (§, p ≤ 0.05). Inset: Diagram showing locations in retinal quadrants 1-4 mm eccentric from the optic disc (black circle) for which SMI31+ RGCs were quantified. Size of each field is 0.101mm².

Figure 5

BMD preserves RGC axon morphology, density and total number following OHT. High-magnification light photomicrographs demonstrating degenerating axon profiles (arrowheads) and gliotic scars (asterisks) in cross-sections of vehicle OHT optic nerves (A and B). Like vehicle control nerve (C), nerves from BMD OHT rats (D) had significantly fewer degenerating profiles. (E) Axon density and total axon number expressed as the ratio of OHT to control nerve for vehicle and BMD groups; naïve group is the ratio of the right to left nerve (mean ± SD; n = 16 each). Density and axon number decreased in vehicle treated rats so that the ratio for each differed significantly from one (*, p ≤ 0.02). BMD treatment improved both outcome measures compared to vehicle (**, p = 0.011).

Figure 6

BMD partially preserves anterograde transport following acute IOP elevation. (A) Representative cross-section through medial superficial superior colliculus (left, outlined) from naïve rat showing normal RGC anterograde transport of CTB (green). Corresponding retinotopic map reconstructed from serial cross-sections through the colliculus (right) shows representation of optic disc gap (circle). Colorimetric scale indicates levels of transport from 100% (red) to 50% (green) to 0% (blue). (B) Comparable colliculus section (left) and retinotopic map (right) corresponding to vehicle control eye also shows intact transport. (C) Sections of colliculus corresponding to OHT eye in vehicle group show deficits ranging from sectorial (arrowheads on left) to complete loss (right). Corresponding retinotopic maps were 58% (left) and 7% (right) intact, respectively. (D) Colliculus from OHT eye in BMD-treated rats show range of rescued transport from complete (left) to modest (85% intact, right). (E) CTB transport in colliculus calculated as the fraction of the retinotopic map with ≥70% maximum signal for three groups (mean ± SEM; n = 11 each). Naïve group represents transport to the left and right colliculi. OHT decreased CTB transport 70% in vehicle-treated rats (*, p = 0.001) but only 35% in BMD-treated animals (*, p = 0.004). BMD significantly improved transport with OHT when compared to vehicle eyes (**, p = 0.002).

Figure 7

Deficits in anterograde transport begin in the nasal retina. Retinotopic maps of CTB transport transformed into retinal quadrant and eccentricity coordinates following Siminoff et al. (1966) [27] and Drager and Hubel (1976) [28]. (A) Colliculus maps for naïve (left), vehicle-group control (middle) and BMD-group control (right) eyes are complete (98-99%) and show location of optic disk (*). (B) Moderate transport deficits (shaded regions) in colliculi from vehicle OHT eyes with 58%, 50% and 43% intact transport (left to right respectively) appear to spread from the nasal retinal representation to the optic disk. (C) Moderate to severe transport deficits in vehicle OHT colliculi with 39%, 19% and 2% intact transport (left to right respectively) continue to spread from the optic disk to other retinal quadrants. (D) BMD treatment ameliorates most transport deficits (96% intact, left) but the same spatial pattern of progression applies for even modest deficits (84% and 80% intact, middle and right respectively). Abbreviations: I, N, S, T indicate inferior, nasal, superior and temporal quadrants of the retina, respectively.