Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2021 Jun 1;62(7):5.
doi: 10.1167/iovs.62.7.5.

Sigma 1 Receptor Modulates Optic Nerve Head Astrocyte Reactivity

Jing Zhao  1   2 , Graydon Gonsalvez  3 , Manuela Bartoli  3   2 , Barbara A Mysona  1   3   2 , Sylvia B Smith  1   3   2 , Kathryn E Bollinger  1   3   2
Affiliations

Sigma 1 Receptor Modulates Optic Nerve Head Astrocyte Reactivity

Jing Zhao et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: Stimulation of Sigma 1 Receptor (S1R) is neuroprotective in retina and optic nerve. S1R is expressed in both neurons and glia. The purpose of this work is to evaluate the ability of S1R to modulate reactivity responses of optic nerve head astrocytes (ONHAs) by investigating the extent to which S1R activation alters ONHA reactivity under conditions of ischemic cellular stress.

Methods: Wild type (WT) and S1R knockout (KO) ONHAs were derived and treated with vehicle or S1R agonist, (+)-pentazocine ((+)-PTZ). Cells were subjected to six hours of oxygen glucose deprivation (OGD) followed by 18 hours of re-oxygenation (OGD/R). Astrocyte reactivity responses were measured. Molecules that regulate ONHA reactivity, signal transducer and activator of transcription 3 (STAT3) and nuclear factor kappa B (NF-kB), were evaluated.

Results: Baseline glial fibrillary acidic protein (GFAP) levels were increased in nonstressed KO ONHAs compared with WT cultures. Baseline cellular migration was also increased in nonstressed KO ONHAs compared with WT. Treatment with (+)-PTZ increased cellular migration in nonstressed WT ONHAs but not in KO ONHAs. Exposure of both WT and KO ONHAs to ischemia (OGD/R), increased GFAP levels and cellular proliferation. However, (+)-PTZ treatment of OGD/R-exposed ONHAs enhanced GFAP levels, cellular proliferation, and cellular migration in WT but not KO cultures. The (+)-PTZ treatment of WT ONHAs also enhanced the OGD/R-induced increase in cellular pSTAT3 levels. However, treatment of WT ONHAs with (+)-PTZ abrogated the OGD/R-induced rise in NF-kB(p65) activation.

Conclusions: Under ischemic stress conditions, S1R activation enhanced ONHA reactivity characteristics. Future studies should address effects of these responses on RGC survival.

PubMed Disclaimer

Conflict of interest statement

Disclosure: J. Zhao, None; G. Gonsalvez, None; M. Bartoli, None; B.A. Mysona, None; S.B. Smith, None; K.E. Bollinger, None

Figures

Figure 1.
Figure 1.
Characterization of cultured primary mouse ONHAs. (A) ONHAs were fixed and probed with antibodies against GFAP (red), S1R (red), OSP (red), and Iba-1 (red). The cells were counterstained with DAPI to label DNA (blue) as a marker for nuclei. Scale bar: 50 µm. (B) Quantitative analysis shows that more than 95% of the cells in culture express GFAP. (C) The cell lysates from ONHAs (lane 2) were positive for GFAP, a marker for astrocytes and S1R, but negative for Iba-1, a marker for microglial cells, and OSP, a marker for oligodendrocytes. The protein extract from mouse brain (lane 1) was used as positive controls. Analyses were repeated in duplicate with cells isolated from different dates, different animals and treated on different days. For each immunofluorescence evaluation, three coverslips were quantified per group. Iba-1 is a marker for microglial cells.
Figure 2.
Figure 2.
Effect of (+)-PTZ on WT ONHA viability, GFAP and S1R expression. WT ONHAs were treated with (+)-PTZ at varying concentrations (1, 3, 10, 20, or 50 µM) for 24 hours. (A) Representative images of GFAP expression detected by immunocytochemistry using GFAP antibody. Scale bar: 50 µm. (B) Quantification of GFAP staining intensity showed no significant differences with varying (+)-PTZ concentrations. (C) MTT assay showed no significant differences in ONHA viability with varying (+)-PTZ concentrations. (D) S1R expression was detected by Western blot and (E) quantified relative to GAPDH. No significant differences in S1R expression with varying (+)-PTZ concentrations were detected. Analyses were repeated in triplicate with cells isolated from different dates, different animals and treated on different days. For GFAP expression experiments, three coverslips were quantified from each group of each isolation. Eight microscopic fields were quantified per coverslip.
Figure 3.
Figure 3.
GFAP expression in Sigma 1 Receptor knock out (S1R KO) ONHAs. (A) Western analysis showed that ONHAs isolated from S1R KO mice lacked S1R. (B) Immunocytochemistry showed that ONHAs isolated from S1R KO mice lacked S1R. Both WT and S1R KO ONHAs were plated on coverslips and incubated for 24 hours. (C) Representative images show cells stained with GFAP antibody. Scale bar: 50 µm. (D) S1R KO ONHAs expressed higher GFAP than WT ONHAs under the same cell culture conditions. These results were repeated five times with cells isolated from different dates, different animals and treated on different days. For each group of each isolation, three coverslips were quantified. Significantly different from control ****P < 0.0001. Data were analyzed using Student's t-test.
Figure 4.
Figure 4.
Effect of (+)-PTZ on WT and S1R KO ONHA migration. WT and S1R KO ONHAs were pretreated with 10 µM (+)-PTZ or vehicle for 1h before scratching. After scratching, the cells were incubated with 10 µM (+)-PTZ or vehicle for 24 hours. (A) Representative images show the original scratch (day 0), and migration of WT ONHAs into the wound area after 24 hours of incubation with (+)-PTZ treatment or vehicle control. Scale bar: 200 µm. (B) Quantification of the number of WT ONHAs migrated into the wounded (scratched) area. Cells in wounded area were counted using ImageJ. For each group, four coverslips were quantified. WT ONHA migration increased significantly when cells were treated with (+)-PTZ. Significantly different from control ****P < 0.0001. (C) Representative images show the original scratch (day 0) and migration of KO ONHAs into the wound area after 24 hours of incubation with (+)-PTZ treatment or vehicle control. Scale bar: 200 µm. (D) Quantification of the number of KO ONHAs migrated into the wounded (scratched) area. Cells in wounded area were counted by ImageJ. For each group, three coverslips were quantified. (+)-PTZ did not affect S1R KO ONHA migration. (E) S1R KO control ONHAs had significantly more migrated cells than WT control ONHAs. Significantly different from control *P < 0.05. Data were analyzed using Student's t-test. These results were repeated in triplicate with cells isolated from different dates, different animals and treated on different days. For each group of each isolation, three coverslips were quantified.
Figure 5.
Figure 5.
Effect of (+)-PTZ on WT ONHAs migration after five hours of treatment and on lamellipodia marker expression. WT ONHAs were pretreated with 10 µM (+)-PTZ or vehicle for one hour before scratching. After scratching, the cells were incubated with 10 µM (+)-PTZ or vehicle for five hours. (A) Representative images show the original scratch (day 0), and migration of WT ONHAs into the wound area after five hours of incubation with (+)-PTZ treatment or vehicle control. Scale bar: 200 µm. (B) Wound distance was measured using ZEN software. The wound distance significantly decreased when WT ONHAs were treated with (+)-PTZ. Significantly different from control **P < 0.01. The results were repeated in triplicate with cells isolated from different animals, on different dates, and treated on different days. (C) Representative images show cells stained with Arp3 antibody (red), phalloidin (green), and DAPI (blue). Scale bar: 50 µm. (D) Quantification of the number of lamellipodia for each cell. For each group, 100 cells, located at the wound edge, were quantified. (+)-PTZ treated cells had significantly more lamellipodia than control ONHAs. Significantly different from control **P < 0.01. Data were analyzed using Student's t-test. The results were repeated in triplicate with cell cultures isolated from different animals, on different dates, and treated on different days.
Figure 6.
Figure 6.
Effect of (+)-PTZ treatment on OGD/R-exposed WT ONHAs. (A) Representative images show GFAP expression in WT ONHAs under normal and OGD/R-exposed conditions, with or without (+)-PTZ treatment. Scale bar: 50 µm. (B) Quantitative analysis shows that OGD/R increases GFAP expression, and that (+)-PTZ treatment enhances the OGD/R-induced increase in GFAP expression. Significantly different from control ****P < 0.0001, **P < 0.01. (C) Cell proliferation was detected by MTT assay. Under conditions of OGD/R exposure, (+)-PTZ treated cells showed a higher proliferation rate than non-(+)-PTZ treated cells. Significantly different from control **P < 0.01. Data were analyzed using two-way ANOVA followed by Tukey-Kramer post-hoc test for multiple comparisons. These experiments were repeated in triplicate with cells isolated from different dates, different animals and treated on different days. For each group of each isolation, three coverslips were quantified.
Figure 7.
Figure 7.
Effect of (+)-PTZ treatment on OGD/R-exposed S1R KO ONHAs. (A) Representative images show GFAP expression in S1R KO ONHAs under normal and OGD/R-exposed conditions with or without (+)-PTZ treatment. Scale bar: 50 µm. (B) Quantitative analysis shows that OGD/R increases GFAP expression, and that (+)-PTZ treatment does not significantly enhance the OGD/R-induced increase in GFAP expression. Significantly different from control ****P < 0.0001, ***P < 0.001, *P < 0.05. (C) Cell proliferation was detected by MTT assay. Under conditions of OGD/R exposure, (+)-PTZ treatment did not significantly affect the S1R KO ONHA proliferation rate. Data were analyzed using two-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. These experiments were repeated in triplicate with cells isolated from different dates, different animals and treated on different days. For each group of each isolation, three coverslips were quantified.
Figure 8.
Figure 8.
Effect of (+)-PTZ on migration of OGD/R-exposed ONHAs. Both WT and S1R KO ONHAs were pretreated with 10 µM (+)-PTZ for one hour before scratching. After scratching, the cells were incubated with 10 µM (+)-PTZ under OGD for six hours, followed by reoxygenation for 18 hours. (A) Representative images show migration of WT versus S1R KO normoxic (control) and OGD/R-exposed cells into the wounded area with or without (+)-PTZ treatment. Scale bar: 200 µm. (B) Cells in wounded area were counted by ImageJ. For each group, four coverslips were quantified. WT ONHA migration increased when cells were treated with (+)-PTZ after OGD/R. Significantly different from control ****P < 0.0001. S1R KO ONHA migration was not affected by (+)-PTZ treatment. Data were analyzed using one-way ANOVA followed by Tukey-Kramer post hoc test for multiple comparisons. Experiments were repeated in triplicates with cells isolated from different dates, different animals and treated on different days. For each group of each isolation, three coverslips were quantified.
Figure 9.
Figure 9.
Effect of (+)-PTZ on phosphorylation of STAT3 and NFkB p-65 in WT ONHAs. (A) Representative Western blot showing increased phosphorylation of both STAT3 and NFkB p-65 after OGD/R in WT ONHAs. (+)-PTZ treatment enhanced OGD/R-induced STAT-3 phosphorylation, whereas (+)-PTZ treatment abrogated OGD/R-induced phosphorylation of p-65. In addition, S1R expression was increased under OGD/R exposure. (B–D) Quantitative analysis by ImageJ. Significantly different from control **P < 0.01, *P < 0.05. Data were analyzed using one-way ANOVA followed by Tukey-Kramer for multiple comparisons. These experiments were repeated in triplicate with cells isolated from different dates, different animals and treated on different days.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Quigley HA, Addicks EM, Green WR, Maumenee AE.. Optic nerve damage in human glaucoma. II. The site of injury and susceptibility to damage. Arch Ophthalmol. 1981; 99: 635–649. - PubMed
    1. Quigley HA, Hohman RM, Addicks EM, Massof RW, Green WR.. Morphologic changes in the lamina cribrosa correlated with neural loss in open-angle glaucoma. Am J Ophthalmol. 1983; 95: 673–691. - PubMed
    1. Tham YC, Li X, Wong TY, Quigley HA, Aung T, Cheng CY.. Global prevalence of glaucoma and projections of glaucoma burden through 2040: a systematic review and meta-analysis. Ophthalmology. 2014; 121: 2081–2090. - PubMed
    1. Wang R, Seifert P, Jakobs TC.. Astrocytes in the optic nerve head of glaucomatous mice display a characteristic reactive phenotype. Invest Ophthalmol Vis Sci. 2017; 58: 924–932. - PMC - PubMed
    1. Tehrani S, Davis L, Cepurna WO, et al. .. Astrocyte structural and molecular response to elevated intraocular pressure occurs rapidly and precedes axonal tubulin rearrangement within the optic nerve head in a rat model. Plos One. 2016; 11: e0167364. - PMC - PubMed

Publication types