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Jillian Foltz, Lisa Schildmeyer, Woojin Han, Dillon Brown, Andrés García, C Ross Ethier; Morphometric analysis of rat optic nerve head (ONH) astrocytes grown in a 3D cell culture system. Invest. Ophthalmol. Vis. Sci. 2021;62(8):2374.
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© ARVO (1962-2015); The Authors (2016-present)
ONH astrocyte reactivity is an early response to elevated IOP, a key risk factor in glaucomatous optic neuropathy. To better understand astrocyte mechanobiology, we have developed a 3D hydrogel cell culture system to mimic the in situ ONH environment. Here our goals were: (i) to quantify morphology of astrocytes cultured in this system, as assessed by semi-automated image analysis, and (ii) compare to in situ morphology.
Primary astrocytes isolated from rat ONH were cultured in a synthetic hydrogel based on 4-arm polyethylene glycol norbornene (PEG-4NB) macromer functionalized with 1.0 mM RGD for 2 to 14 days. Astrocytes within hydrogels were stained with DAPI and phalloidin and imaged using a Leica DM6 fluorescent microscope at post-seeding day 5. Images were skeletonized (AnalyzeSkeleton plugin in ImageJ/FIJI), and individual astrocytes were isolated (n=11). The number and length of astrocytic processes were then quantified from each astrocyte and used to calculate branching frequency (number of processes emanating directly from the cell body divided by total number of processes), a measure of branching complexity. This morphometric quantity was then compared to data in the literature (Butt et al., J Neurocytology, 1994).
We successfully identified, segmented, and analyzed astrocytes from hydrogel cultures. Branching patterns were qualitatively similar to astrocytes in situ; however, quantitative analysis showed that cultured astrocytes had higher branch frequency than cells in situ (Fig. 2, chi-squared test, Χ2 (df=8) = 116.68, p <0.001).
Qualitative morphological similarities of astrocytes in situ vs. those cultured in 3D hydrogels suggest that the PEG-4NB hydrogel system is a viable model for studying ONH astrocyte mechanobiology. However, astrocytes grown in 3D hydrogels are less elongated and branch more frequently than in situ, possibly explained by the additional constraints imposed in situ on the astrocytes by the cylindrical optic nerve shape. Ongoing efforts seek to develop this 3D hydrogel cell culture system, along with image analysis tools, as a novel platform for studying the mechanobiology of astrocytes in glaucoma.
This is a 2021 ARVO Annual Meeting abstract.
Steps of image processing in ImageJ/FIJI.
Histogram of astrocyte branching frequency of cells in 3D hydrogel culture vs. in situ.
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