Neuroinflammation and gliosis in the injured and contralateral retinas after unilateral optic nerve crush.

The main purpose of this study is to analyze the effects of unilateral optic nerve crush in the gene expression of pro- and anti-inflammatory mediators, and gliosis markers in injured and contralateral retinas. Retinas from intact, unilaterally optic nerve injured or sham-operated C57BL/6J mice were analyzed 1, 3, 9 and 30 days after the surgery (n = 5/group and time point) and the relative expression of TGF-ß1, IL-1ß, TNF-α, Iba1, AQP4, GFAP, MHCII, and TSPO was analyzed in injured and contralateral using qPCR. The results indicated that compared with intact retinas, sham-operated animals showed an early (day 1) upregulation of IL-1ß, TNF-α and TSPO and a late (day 30) upregulation of TNF-α. In sham-contralateral retinas, TNF-α and TSPO mRNA expression were upregulated and day 30 while GFAP, Iba1, AQP4 and MHCII downregulated at day 9. Compared with sham-operated animals, in retinas affected by optic nerve crush GFAP and TSPO upregulated at day 1 and TNF-α, Iba1, AQP4 and MHCII at day 3. In the crushed-contralateral retinas, TGF-ß1, TNF-α, Iba1 and MHCII were upregulated at day 1. TSPO was upregulated up to day 30 whereas TGF-ß1 and Iba1 downregulated after day 9. In conclusion, both sham surgery and optic nerve crush changed the profile of inflammatory and gliosis markers in the injured and contralateral retinas, changes that were more pronounced for optic nerve crush when compared to sham.

Comparative Analysis of Retinal Organotypic Cultures and In Vivo Axotomized Retinas.

Retinal organotypic cultures (ROCs) are used as an in vivo surrogate to study retinal ganglion cell (RGC) loss and neuroprotection. In vivo, the gold standard to study RGC degeneration and neuroprotection is optic nerve lesion. We propose here to compare the course of RGC death and glial activation between both models. The left optic nerve of C57BL/6 male mice was crushed, and retinas analyzed from 1 to 9 days after the injury. ROCs were analyzed at the same time points. As a control, intact retinas were used. Retinas were studied anatomically to assess RGC survival, microglial, and macroglial activation. Macroglial and microglial cells showed different morphological activation between models and were activated earlier in ROCs. Furthermore, microglial cell density in the ganglion cell layer was always lower in ROCs than in vivo. RGC loss after axotomy and in vitro followed the same trend up to 5 days. Thereafter, there was an abrupt decrease in viable RGCs in ROCs. However, RGC somas were still immuno-identified by several molecular markers. ROCs are useful for proof-of-concept studies on neuroprotection, but long-term experiments should be carried out in vivo. Importantly, the differential glial activation observed between models and the concomitant death of photoreceptors that occurs in vitro may alter the efficacy of RGC neuroprotective therapies when tested in in vivo models of optic nerve injury.

Axonal Injuries Cast Long Shadows: Long Term Glial Activation in Injured and Contralateral Retinas after Unilateral Axotomy.

BACKGROUND: To analyze the course of microglial and macroglial activation in injured and contralateral retinas after unilateral optic nerve crush (ONC). METHODS: The left optic nerve of adult pigmented C57Bl/6 female mice was intraorbitally crushed and injured, and contralateral retinas were analyzed from 1 to 45 days post-lesion (dpl) in cross-sections and flat mounts. As controls, intact retinas were studied. Iba1+ microglial cells (MCs), activated phagocytic CD68+MCs and M2 CD206+MCs were quantified. Macroglial cell changes were analyzed by GFAP and vimentin signal intensity. RESULTS: After ONC, MC density increased significantly from 5 to 21 dpl in the inner layers of injured retinas, remaining within intact values in the contralateral ones. However, in both retinas there was a significant and long-lasting increase of CD68+MCs. Constitutive CD206+MCs were rare and mostly found in the ciliary body and around the optic-nerve head. While in the injured retinas their number increased in the retina and ciliary body, in the contralateral retinas decreased. Astrocytes and Müller cells transiently hypertrophied in the injured retinas and to a lesser extent in the contralateral ones. CONCLUSIONS: Unilateral ONC triggers a bilateral and persistent activation of MCs and an opposed response of M2 MCs between both retinas. Macroglial hypertrophy is transient.

Ly6c as a New Marker of Mouse Blood Vessels: Qualitative and Quantitative Analyses on Intact and Ischemic Retinas.

Ly6c is an antigen commonly used to differentiate between classical and non-classical monocytes/macrophages. Here we show its potential as a marker of the mouse vasculature, particularly of the retinal vascular plexuses. Ly6c was immunodetected in several tissues of C57BL/6 mice using isolectin IB4 as the control of vasculature staining. In the retina, Ly6c expression was analyzed qualitatively and quantitatively in intact, ischemic, and contralateral retinas from 0 to 30 days after the insult. Ly6c expression was observed in all organs and tissues tested, with a brighter signal and more homogeneous staining than the IB4. In the retinas, Ly6c was well expressed, allowing a detailed study of their anatomy. The three retinal plexuses were morphologically different, and from the superficial to the deep one occupied 15 ± 2, 24 ± 7, and 38 ± 1.4 percent of the retinal surface, respectively. In the injured retinas, there was extravasation of the classically activated monocyte/macrophages (Ly6chigh) and the formation of new vessels in the superficial plexus, increasing the area occupied by it to 25 ± 1%. In the contralateral retinas, the superficial plexus area decreased gradually, reaching significance at 30 days, and Ly6c expression progressively disappeared in the intermediate and deep plexuses. Although the role of Ly6c in vascular endothelial cell function is still not completely understood, we demonstrate here that Ly6c can be used as a new specific marker of the mouse vasculature and to assess, qualitatively and quantitatively, vascular changes in health and disease.