Mechanical strain in the mouse astrocytic lamina increases after exposure to recombinant trypsin

Astrocytic glial cells support the retinal ganglion cell axons in the optic nerve head (ONH). In a mouse eye, the tissue in the center of the ONH is comprised largely of astrocytes and is thus sometimes called the astrocytic lamina (AL). In addition, there are capillaries, microglia, and axons of retinal ganglion cells. In larger mammals, astrocytes envelop connective tissue beams to provide mechanical support to axons exiting the eye. Intraocular pressure (IOP) applies stress circumferentially to the AL via the expansion of the scleral canal, and the difference between the IOP and the optic nerve (ON) pressure applies a pressure gradient across the ONH. The astrocytes of this unmyelinated portion of the ON are unique in the central nervous system in that they are subjected to translaminar mechanical stress. In the mouse eye, some individual astrocytes in the unmyelinated region of the ONH can span the entire AL in the coronal plane [1]. The processes of the astrocytes are anchored by dense junctional complexes to the basement membrane of the peripapillary sclera (PPS) [2]. The astrocytes in the mouse AL occupy as much as 80% of tissue volume. Their processes are composed primarily of F-actin and of intermediate filaments, such as glial fibrillary acidic protein (GFAP), vimentin, and nestin [3].

In human glaucoma and in animal models of glaucoma, long-term IOP elevation produces axon damage initially at the ONH [4], [5], [6]. We hypothesize that this injury to axons occurs in part from the mechanical strains affecting ONH astrocytes. Studies of the ONH astrocytes in experimental glaucoma have reported changes to astrocyte microstructure, biochemical signaling, gene expression, and function [3,[7], [8], [9], [10], [11], [12], [13], [14]]. We developed an ocular explant model to measure the change in the mechanical behavior of the mouse ONH resulting from acute and chronic IOP elevation [7]. Measurement of the mechanical response of the ONH after chronic IOP elevation demonstrated that changes to the strain response evolve over time, with an initial increase in the compliance of the strain response to IOP increase followed by a transition to lower compliance at a later timepoint [15].  Astrocyte processes also separated from the ONH basement membrane early in the time course of chronic IOP elevation, but were again attached to the basement membrane six weeks after induction of the IOP elevation [2]. To simulate this important aspect of glaucomatous remodeling in a non-IOP dependent model, we considered treating mouse eyes with trypsin, which is widely used for dissociation of cultured cells from the extracellular matrix [16]. In one study, a collagen gel containing cells was treated with trypsin, which led to the dissociation of all cells from the gel without a change in the collagen content [17].

Trypsin is extracted from animal tissue, usually from porcine pancreas, and therefore may contain additional proteases. TrypLE is a trypsin-like protease prepared from recombinant bacteria expressing a microbial protease. TrypLE proteolytic activity is similar to, but not identical to that of trypsin [18,19]. In two studies, TrypLE and trypsin dissociated comparable numbers of cells and did not affect cell viability [20,21]. Therefore, TrypLE was selected in this study in order to simulate astrocyte separation from the ONH basement membrane and the sclera. Astrocyte separation from the ONH basement membrane has been reported in experimental glaucoma, although we recognize that this was not caused by trypsin or TrypLE in vivo [2]. We hypothesized that reproducing a feature of glaucoma, albeit artificially by TrypLE, would allow examination of the induced changes in the mechanical response of the ONH and PPS when performing inflation testing of explanted mouse eyes. We treated explanted mouse eyes with TrypLE and buffer for one hour and compared the effect on the astrocyte microstructure using both laser scanning and electron microscopy. We hypothesized that the reduction of astrocyte attachment to the ONH basement membrane and the PPS would increase the strain response of the AL in a manner similar to that observed in a mouse model of glaucoma.

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