T and B Lymphocyte Deficiency in Rag1-/- Mice Reduces Retinal Ganglion Cell Loss in Experimental Glaucoma

Abstract

Purpose: We previously demonstrated that passive transfer of lymphocytes from glaucomatous mice induces retinal ganglion cell (RGC) damage in recipient animals, suggesting a role for immune responses in the multifactorial pathophysiology of glaucoma. Here we evaluate whether absence of an adaptive immune response reduces RGC loss in glaucoma.

Methods: Elevated intraocular pressure (IOP) was induced in one eye of C57BL/6J (B6) or T- and B-cell-deficient Rag1-/- knockout mice. After 16 weeks RGC density was determined in both the induced and the normotensive contralateral eyes. Data were compared to mice having received injections of "empty" vector (controls). The number of extravascular CD3+ cells in the retinas was determined using FACS.

Results: Retinas of eyes with elevated IOP contain significantly more extravasated CD3+ cells than control retinas (46.0 vs. 27.1, P = 0.025). After 16 weeks of elevated IOP the average RGC density in B6 mice decreased by 20.7% (P = 1.9 × 10-4). In contrast, RGC loss in Rag1-/- eyes with elevated IOP was significantly lower (10.3%, P = 0.006 vs. B6). RGC loss was also observed in the contralateral eyes of B6 mice, despite the absence of elevated IOP in those eyes (10.1%; P = 0.008). In RAG1-/- loss in the contralateral eyes was minimal (3.1%) and significantly below that detected in B6 (P = 0.02).

Conclusions: Our findings demonstrate that T Rag1-/- mice are significantly protected from glaucomatous RGC loss. In this model, lymphocyte activity contributes to approximately half of all RGC loss in eyes with elevated IOP and to essentially all loss observed in normotensive contralateral eyes.

Figure 1.

IOP (mm Hg) in mice following injection of Ad5myoc into the anterior chamber of the left eye (OS). (A) Elevated IOP was only observed in injected eyes. (B) Summation of IOP over the experimental period was similar in injected eyes between the two groups of mice (area under the curve: AUC).

Figure 2.

RGC density in age-matched control eyes (hashed bars) in comparison to B6 eyes (white bars) and Rag1−/− eyes (gray bars) after 16 weeks of elevated IOP. Each dot represents one eye. Values are obtained in the retinal periphery, mid-periphery (Mid) and Centrally (Central). *: P < 0.05, **P < 0.01, ns = not significant, n = 15/group.

Figure 3.

RGC density in contralateral eyes in control mice (hashed bars), B6 (white bars), and Rag1−/− (gray bars). Each dot represents one eye. Values are obtained in the retinal periphery, mid-periphery (Mid) and centrally (Central). *: P < 0.05, **P < 0.01, ns = not significant, n = 15/group.

Figure 4.

Extravasation of T cells in the retina of C57BL/6J eight weeks after induction of elevated IOP. (A) The IOP profile of the mouse whose retinas are depicted in B and C demonstrates a modest elevation of IOP throughout the eight-week period. (B) Retinal wholemount of the injected eye (OS) indicating Brn3a positive RGC (red) and T-cell receptor–positive T-cells (TCR, green, arrows) in the retinal parenchyma. The unlabeled retinal vasculature is faintly visible. (C) Retinal wholemount of the contralateral eye (OD). In this particular eye no T-cell extravasation was apparent although, in the absence of heparin perfusion, TCR positive cells are present within the larger retinal vessels.