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S. J. McKinnon, L. T. Kasmala, A. L. Dixon; Severe B- and T-Lymphocyte Immunodeficiency Caused by Rag1 Knockout Prevents Optic Nerve Axon Loss in a Mouse Glaucoma Model. Invest. Ophthalmol. Vis. Sci. 2010;51(13):2523.
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Interactions of T-lymphocytes and receptors of the TNF superfamily have been implicated in immune-related RGC death. We have begun to investigate the role of lymphocytes in murine glaucoma using immune deficient Rag1 knockout mouse which lack mature B- and T-lymphocytes.
Mice lacking the Rag1 gene show early arrest of B- and T- lymphocyte differentiation correlating with the inability to perform V(D)J recombination, similar to that seen in severe combined immunodeficiency (SCID) mice. Rag1 KO mice display normal behavior, have intact neuroanatomy, and are viable and fertile. We obtained Rag1 KO mice and age-matched C57BL/6J mice from Jackson Labs. 12 Rag1 KO and 9 C57BL/6J were treated in the right eye to induce IOP elevation by the Morrison method of limbal injection of hypertonic (1.3 M) saline. IOP was recorded weekly for 22 weeks by TonoLab tonometry (Colonial Medical Supply). After sedation and enucleation, optic nerve cross-sections were stained with toluidine blue. Axon counts were obtained in a masked fashion using the KS400 imaging system (Zeiss), and % axon survival (treated/control axon counts) for each mouse was calculated. % axon survivals for the two groups were tested for significance with Student’s t-test.
9 Rag1 KO and 6 C57BL/6J mice met inclusion criteria, yielding a conversion rate of 71% (15/21). Mean IOP integrals for the Rag1 KO and C57BL/6J groups were 440 and 523 mm-days, respectively. 2-tailed Student’s t-test showed no significant difference between the two groups in IOP integral distribution (P=0.77), indicating equivalent levels of IOP exposure between the two groups. The two groups showed average axon counts of: Rag1 KO: 25,745 ± 8,293 (right eye; mean ± SD) and 34,496 ± 10,433 (left eye); C57BL/6J: 8,912 ± 9,662 (right eye) and 31,514 ± 6,028 (left eye). The two groups showed average % axon survivals (treated/control axon counts) of: Rag1 KO: 79.6% ± 31.0% (mean ± SD); C57BL/6J: 27.8% ± 25.4%. 2-tailed Student’s t-test showed a significant difference between the two groups in % axon survival (P=0.02).
Rag1 KO with resulting deletion of mature B- and T-lymphocytes provided robust neuroprotection when compared to wild-type controls in a chronic mouse glaucoma model. We hypothesize that lymphocytes are required for RGC cell death in glaucoma. Rag1 mice represent a powerful tool for selective hematopoietic stem cell reconstitution experiments designed to identify specific B- or T-lymphocyte sub-populations that may be critical to RGC death in glaucoma.
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