Astrocyte Reactivity: A Biomarker for Retinal Ganglion Cell Health in Retinal Neurodegeneration

Abstract

Retinal ganglion cell (RGC) loss in glaucoma is sectorial in nature and preceded by deficits in axonal transport. Neuroinflammation plays an important role in the pathophysiology of glaucoma in the retina, optic nerve and visual centers of the brain, where it similarly appears to be regulated spatially. In a murine model, we examined the spatial characteristics of astrocyte reactivity (migration/proliferation, hypertrophy and GFAP expression) in healthy retina, retina with two glaucoma-related risk factors (aging and genetic predisposition) and glaucomatous retina and established relationships between these reactivity indices and the spatial organization of astrocytes as well as RGC health. Astrocyte reactivity was quantified by morphological techniques and RGC health was determined by uptake and transport of the neural tracer cholera toxin beta subunit (CTB). We found that: (1) astrocyte reactivity occurs in microdomains throughout glaucomatous retina as well as retina with risk factors for glaucoma, (2) these astrocyte microdomains are primarily differentiated by the degree of retinal area covered by the astrocytes within them and (3) percent retinal area covered by astrocytes is highly predictive of RGC health. Our findings suggest that microdomains of astrocyte reactivity are biomarkers for functional decline of RGCs. Based on current and emerging imaging technologies, diagnostic assessment of astrocytes in the nerve fiber layer could succeed in translating axonal transport deficits to a feasible clinical application.

Probing electrical signals in the retina via graphene-integrated microfluidic platforms

“…31 Briefly, anesthetized (2.5% isoflurane) adult mice (2 – 4 months) received a 1.5–2 μl intravitreal injection of CTB (10ug/μl in sterile PBS; Life Technologies) using a 33 gauge needle attached to a Hamilton syringe. Three days after CTB injection, mice were sacrificed by cervical dislocation and decapitation for live or dehydrated retina recordings or by transcardial perfusion with 4% paraformaldehyde for fixed retina recordings.…”

Glia–neuron interactions in the mammalian retina

“…Moreover, morphologically undistinguishable astrocytes can exhibit different physiological behaviour (Matyash and Kettenmann, 2010). The existence of different astrocyte populations is reinforced by the finding that astrocyte reactivity takes place in selected areas of the glaucomatous retina, these "microdomains" possibly serving as biomarkers and predictors of RGC health (Formichella et al, 2014).…”

Assessment of retinal ganglion cell damage in glaucomatous optic neuropathy: Axon transport, injury and soma loss

“…Application of CTB fluorescent conjugates clearly labels individual RGC axons regardless of transport direction (Dengler-Crish et al, 2014, Fig. 1D, E and H), and have been used in studies of axonal regrowth in the ON (Liu et al, 2009;Sun et al, 2011;Yang and Cho, 2005) and transport disruption in experimental GON Chidlow et al, 2011;Dengler-Crish et al, 2014;Formichella et al, 2014;Fujishiro et al, 2014;Lambert et al, 2011;Ward et al, 2014).…”

“…Conjugating CTB to a fluorophore thus allows visualization of endocytotic vesicles for tracking of AT . The CTB molecule 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 itself, in combination with in vitro anti-CTB IHC, has been used extensively as a neuronal tracer since the 1980's (Angelucci et al, 1996;Flink and Westman, 1986;Formichella et al, 2014;Luppi et al, 1990;Matteau et al, 2003). However, the development of CTB fluorescent conjugates (Dederen et al, 1994) eliminated the need for secondary labeling, reducing immuno-associated error and simplifying its use.…”