Impact of cerebral hypoperfusion-reperfusion on optic nerve integrity and visual function in the DBA/2J mouse model of glaucoma

Abstract

Objective: One of the most important risk factors for developing a glaucomatous optic neuropathy is elevated intraocular pressure. Moreover, mechanisms such as altered perfusion have been postulated to injure the optical path. In a mouse model, we compare first negative effects of cerebral perfusion/reperfusion on the optic nerve structure versus alterations by elevated intraocular pressure. Second, we compare the alterations by isolated hypoperfusion-reperfusion and isolated intraocular pressure to the combination of both.

Methods and analysis: Mice were divided in four groups: (1) controls; (2) perfusion altered mice that underwent transient bi-common carotid artery occlusion (BCCAO) for 40 min; (3) glaucoma group (DBA/2J mice); (4) combined glaucoma and altered perfusion (DBA/2J mice with transient BCCAO). Optic nerve sections were stereologically examined 10-12 weeks after intervention.

Results: All experimental groups showed a decreased total axon number per optic nerve compared with controls. In DBA/2J and combined DBA/2J & BCCAO mice the significant decrease was roughly 50%, while BCCAO leaded to a 23% reduction of axon number, however reaching significance only in the direct t-test. The difference in axon number between BCCAO and both DBA/2J mice was almost 30%, lacking statistical significance due to a remarkably high variation in both DBA/2J groups.

Conclusion: Elevated intraocular pressure in the DBA/2J mouse model of glaucoma leads to a much more pronounced optic nerve atrophy compared with transient forebrain hypoperfusion and reperfusion by BCCAO. A supposed worsening effect of an altered perfusion added to the pressure-related damage could not be detected.

Keywords: Anatomy; Experimental & animal models; Glaucoma; Optic Nerve.

Figure 1

(A) Visualisation of the patent posterior communicating arteries within the circle of Willis, (B) assessment of visual function by optokinetic response to a rotating whole field stimulus, (C) fundus photo of a mouse during bicommon carotid artery occlusion. No signs for ischaemia, no bleedings were observed.

Figure 2

(A) Description of the fractionator tool, applied to imaging (EM cross-section, optic nerve of DBA/2J). For each systematically sampled micrograph four unbiased counting frames are virtually applied with the NewCAST programme. Axons laying within the rectangular frames, not touching the red lines (‘exclusion lines’) are counted; exemplary estimation in the top right frame (yellow ticks). (B) Description of the nucleator tool. The NewCAST programme allows integrating the nucleator tool directly on the virtual counting frames. The blue colour lines are computed isotropically from an arbitrarily chosen point in the centre of the axon, and the intersections of these lines with the boundary are then marked (yellow crosses). The four different obtained lengths from the centre are then directly integrated and calculated in the programme. Scale bar for (A, B)=5 µm (magnification ×3400). EM, electron microscopy.

Figure 3

(A) Intraocular pressure (IOP) of the four study groups. There was no difference in IOP levels between non glaucoma mice (SV-129) with or without (sham operated) bicommon carotid artery occlusion (BCCAO). There was no significant difference in IOP between DBA/2J mice with and without BCCAO as well. The graph shows the situation at 14 months of life. ** indicates p<0.001. (B) optokinetic head response at 0.066 cycle per degree as frequency of extinction (FE) (cycle/second). FE was significantly reduced in experimental animals 8 weeks after BCCAO at a spatial frequency of 0.066 cycle/degree. ** indicates p < 0.01, *** indicates p < 0.001.

Figure 4

(A) Absolute number of axons per entire optic nerve (ON) cross section. Bar indicates group mean, whiskers±1 SD and symbols indicate individual animal values. Symbols at the baseline indicate nerves with complete atrophy. Asterisk indicates significant differences with p<0.05, (B) Mean axon profile area on the ON cross sections. Bar indicates group mean, whiskers±1 SD and symbols indicate individual animal values. Symbols at the baseline indicate nerves with complete axonemal atrophy. BCCAO, bicommon carotid artery occlusion.