The Role of Aquaporin-4 in Optic Nerve Head Astrocytes in Experimental Glaucoma

Abstract

PurposeTo study aquaporin channel expression in astrocytes of the mouse optic nerve (ON) and the response to IOP elevation in mice lacking aquaporin 4 (AQP4 null).MethodsC57BL/6 (B6) and AQP4 null mice were exposed to bead-induced IOP elevation for 3 days (3D-IOP), 1 and 6 weeks. Mouse ocular tissue sections were immunolabeled against aquaporins 1(AQP1), 4(AQP4), and 9(AQP9). Ocular tissue was imaged to identify normal AQP distribution, ON changes, and axon loss after IOP elevation. Ultrastructure examination, cell proliferation, gene expression & transport block was also analyzed.ResultsB6 mice presented abundant AQP4 in Müller cells, astrocytes of retina and myelinated ON (MON), but minimal expression in prelaminar and unmyelinated ON (UON). MON of AQP4 nulls had smaller ON area, smaller axon diameter, higher axon density, and larger proportionate axon area than B6 (all p≤0.05). Bead-injection led to comparable 3D-IOP elevation (p=0.42) and axonal transport blockade in both strains. In B6, AQP4 distribution was unchanged after 3D-IOP. At baseline, AQP1 and AQP9 were present in retina, but not in UON and this was unaffected after IOP elevation in both strains. In 3D-IOP mice, ON astrocytes and microglia proliferated, more in B6 than AQP4 null. After 6 week IOP elevation, axon loss occurred equally in the two mouse types (24.6%, AQP4 null vs. 23.3%, B6).ConclusionLack of AQP4 was neither protective nor detrimental to the effects of IOP elevation. The minimal presence of AQP4 in UON may be a vital aspect of the regionally specific phenotype of astrocytes in the mouse optic nerve head.

Regional Gene Expression in the Retina, Optic Nerve Head, and Optic Nerve of Mice with Experimental Glaucoma and Optic Nerve Crush

“…An important pathway in common between UON and MON in our analysis was axon guidance. In the mouse UON, the vast majority of cells locally generating RNA are astrocytes [24]. The major cellular content difference between UON and MON is the presence of oligodendrocytes in the latter region.…”

“…The injection of microbeads into the anterior chamber produces IOP elevation known to cause RGC death that is maximal by 6 weeks [23] (Figure 4A, Figure S4, Table S1). Mice exposed to elevated IOP followed prior experience with bead injection [23,24], having significant mean IOP elevation at three days, decreasing at two weeks and with minimal difference from baseline at six weeks (Figure S4, Table S1). Micro-dissected UON, MON, and retinal tissue was collected at three days (early, 3D), two weeks (middle, 2W), and six weeks (late, 6W) post-injection to characterize gene expression changes spanning the time course of this model (Figure 4A).…”

“…Dissected tissue was immediately placed into individual, pre-chilled 1.5 mL Eppendorf tubes and flash frozen in liquid nitrogen before long-term storage at -80°C. Tissue taken from naïve WT GFAP-GFP animals was immediately placed into QIAzol Lysis Reagent (QIAgen) and processed using the manufacturer's protocol as previously reported [24]. For qPCR, six biological replicates were collected for each tissue region.…”

“…The MON had more increases in cell cycle genes. Both astrocytes and microglia proliferate in the mouse glaucoma model [24]. In both mouse and rat ocular hypertension models, there was loss of oligodendrocytes in the MON and oligodendrocyte precursor cells proliferated, while activation of microglia was detected only in advanced damage nerves [36].…”