Abstract
Purpose :
To measure the effect of short-term exposure to dissociation enzyme on the mechanical response of the optic nerve head (ONH) in explanted mouse eyes.
Methods :
The ONH was imaged with two-photon laser scanning microscopy (Zeiss LSM 710) in explanted mouse eyes using previously-published inflation testing methods (FVB/N-Tg(GFAP-GFP)14Mes mice, both sexes, 3-8 months of age). Inflation tests were performed before and again after 60 minutes exposure to TrypLE Express (Gibco 12604013), a non-animal-derived recombinant enzyme and a purified trypsin alternative. These (n=8) were compared to sequential inflation of explants exposed only to buffer (n=8). Deformation of the astrocytic lamina (AL) and of connective tissues of the peripapillary sclera (PPS) was analyzed by digital volume correlation (Korneva et al., 2020). After inflation-testing, treated specimens were prepared either for transmission electron microscopy or for fluorescent immunolabeling with phalloidin to quantify changes in the actin network (Ling et al., 2020).
Results :
AL nasal-temporal strain (Exx) increased significantly post TrypLE treatment (post: 3.59±1.10%, pre: 2.52±0.73%, p=0.01). AL superior-inferior strain (Eyy) also significantly increased post TrypLE treatment (post: 2.50±0.64%, pre: 0.86±0.75%, p=002). The change in AL strain after treatment with buffer was insignificant (Exx post: 2.77±1.17%, pre: 2.92±1.41%, p=0.64). The effect of TrypLE on PPS strain response was insignificant (Exx post: 0.00±0.34%, pre: 0.06±0.41%, p=0.93). The differences in PPS strain after TrypLE and buffer were similar (ΔExx p=0.99, ΔEyy p=0.81). The PPS maximum principal strain after TrypLe significantly increased from before treatment (p=0.01) but not compared to after buffer (p=0.67). Quantitative morphological analysis of astrocyte processes in these eyes will be presented.
Conclusions :
TrypLe treatment led to greater strain of the AL but similar strain of the PPS in inflation testing of explanted mouse eyes. Morphological studies suggest that astrocyte junctional complexes to the PPS are disrupted in experimental mouse glaucoma eyes (Quillen et al., 2020). Comparisons of experimental alterations of mouse model tissue may elucidate the underlying mechanisms of optic nerve head remodeling.
This is a 2021 ARVO Annual Meeting abstract.