Combined Cyclosporin A and Hypothermia Treatment Inhibits Activation of BV-2 Microglia but Induces an Inflammatory Response in an Ischemia/Reperfusion Hippocampal Slice Culture Model

Abstract

Introduction: Hypothermia attenuates cerebral ischemia-induced neuronal cell death associated with neuroinflammation. The calcineurin inhibitor cyclosporin A (CsA) has been shown to be neuroprotective by minimizing activation of inflammatory pathways. Therefore, we investigated whether the combination of hypothermia and treatment with CsA has neuroprotective effects in an oxygen-glucose deprivation/reperfusion (OGD/R) injury model in neuronal and BV-2 microglia monocultures, as well as in an organotypic hippocampal slice culture (OHSC).

Methods: Murine primary neurons, BV-2 microglia, and OHSC were pretreated with CsA and exposed to 1 h OGD (0.2% O₂) followed by reperfusion at normothermia (37°C) or hypothermia (33.5°C). Cytotoxicity was measured by lactate dehydrogenase and glutamate releases. Damage-associated molecular patterns (DAMPs) high mobility group box 1 (HMGB1), heat shock protein 70 (Hsp70), and cold-inducible RNA-binding protein (CIRBP) were detected in cultured supernatant by western blot analysis. Interleukin-6 (IL-6), Interleukin-1α and -1β (IL-1α/IL1-β), tumor necrosis factor-α (TNF-α), monocyte chemotactic protein 1 (MCP1), inducible nitric oxide synthase (iNOS), glia activation factors ionized calcium-binding adapter molecule 1 (Iba1), and transforming growth factor β1 (TGF-β1) gene expressions were analyzed by RT-qPCR.

Results: Exposure to OGD plus 10 μM CsA was sufficient to induce necrotic cell death and subsequent release of DAMPs in neurons but not BV-2 microglia. Moreover, OGD/R-induced secondary injury was also observed only in the neurons, which was not attenuated by cooling and no increased toxicity by CsA was observed. BV-2 microglia were not sensitive to OGD/R-induced injury but were susceptible to CsA-induced toxicity in a dose dependent manner, which was minimized by hypothermia. CsA attenuated IL-1β and Iba1 expressions in BV-2 microglia exposed to OGD/R. Hypothermia reduced IL-1β and iNOS expressions but induced TNF-α and Iba1 expressions in the microglia. However, these observations did not translate to the ex vivo OHCS model, as general high expressions of most cytokines investigated were observed.

Conclusion: Treatment with CsA has neurotoxic effects on primary neurons exposed to OGD but could inhibit BV-2 microglia activation. However, CsA and hypothermia treatment after ischemia/reperfusion injury results in cytotoxic neuroinflammation in the complex ex vivo OHSC.

Keywords: BV-2 microglia; DAMPs; cyclosporin A; hypothermia; inflammation; organotypic hippocampal slice culture; oxygen-glucose deprivation/reperfusion; primary neuron.

FIGURE 1

Experimental time-temperature protocol. Cyclosporin A (CsA) was applied 1 h before the simulated ischemia phase by oxygen-glucose-deprivation (OGD at 0.2% O₂ in glucose-depleted medium) and maintained after the reperfusion phase for the duration of experiment. Exposure to OGD for 1 h was performed at 37°C, followed by reperfusion (OGD/R) for 24 h at 37°C (normothermia) or 33.5°C (hypothermia).

FIGURE 2

Lactate dehydrogenase releases were measured from primary neurons (A) and BV-2 microglia cells (B) in the experimental control group exposed to 1 h normoxia (21% O₂ in glucose-containing medium) and simulated ischemia group exposed to 1 h oxygen-glucose deprivation (OGD at 0.2% O₂ in glucose-depleted medium) at 37°C, and also after reperfusion from primary neurons (C) and BV-2 microglia cells (D) exposed to 1 h normoxia + 24 h reperfusion with complete medium (Normoxia/R) or 1 h OGD + 24 h reperfusion (OGD/R) at either 37 or 33.5°C. All CsA groups were pre-treated 1 h before experimental start with either 1 or 10 μM CsA at 37°C. Data from 4 to 6 individual experiments are presented as box-and-whiskers plot (box from 25th to 75th percentile and whisker min to max). Statistical analysis were conducted using one-way ANOVA followed by the Tukey post hoc test; ^∗∗p < 0.01, ^∗∗∗p < 0.001 compared to Normoxia at 37°C, and ^#p < 0.05 for group comparison were considered significant.

FIGURE 3

(A) LDH and (B) glutamate releases from OHSCs were assessed from the cultured supernatants of the experimental control group exposed to 1 h normoxia (21% O₂ in glucose-containing medium) and simulated ischemia group exposed to 1 h oxygen-glucose deprivation (OGD at 0.2% O₂ in glucose-depleted medium) at 37°C. All CsA containing groups were pre-treated 1 h before experimental start with 10 μM CsA at 37°C. Data from 4 to 5 individual experiments are presented as box-and-whiskers plot (box from 25th to 75th percentile and whisker min to max). Statistical analysis were conducted using one-way ANOVA followed by the Tukey post hoc test; ^∗∗p < 0.01, ^∗∗∗p < 0.001 compared to Normoxia at 37°C, and ^#p < 0.05 for group comparison were considered significant.

FIGURE 4

Propidium iodide (PI) staining; OHSCs in the experimental control group (Normoxia/R) were exposed to 1 h normoxia (21% O₂ in glucose-containing medium) + 24 h reperfusion (21% O₂ in glucose-containing medium) and simulated ischemia groups were exposed to 1 h oxygen-glucose deprivation (OGD at 0.2% O₂ in glucose-depleted medium) + 24 h reperfusion (OGD/R at 21% O₂ in glucose-containing medium) at either 37 or 33.5°C. All cyclosporin A (CsA) containing groups were pre-treated 1 h before experimental start with 10 μM CsA at 37°C and maintained throughout the duration of the experiment. Images are shown at 40× magnification. CA, Cornu Ammonis; DG, Dentate Gyrus.

FIGURE 5

Western Blot analysis was used to assess extracellular DAMPs, (A,D) heat shock protein 70 (Hsp70), (B,E) high mobility group box 1 (HMGB1), and (C) cold-inducible RNA-binding protein (CIRBP) released into the cultured supernatant from primary neurons and BV-2 microglial cells. The experimental control group was exposed to 1 h normoxia (21% O₂ in glucose-containing medium) and simulated ischemia group was exposed to 1 h oxygen-glucose deprivation (OGD at 0.2% O₂ in glucose-depleted medium) at 37°C. Quantitative densitometric analysis from 5 to 6 individual experiments is presented as box-and-whiskers plot (box from 25th to 75th percentile and whisker min to max), along with the representative immunoblots. Statistical analysis were conducted using one-way ANOVA followed by the Tukey post hoc test; ^∗∗p < 0.01, ^∗∗∗p < 0.001 compared to normoxia at 37°C, and ^#p < 0.05 for group comparison were considered significant.

FIGURE 6

RT-qPCR was used to assess gene expressions in BV-2 microglia, OHSCs, and primary neurons for inflammatory (A,E,I) IL-6, (B,F) TNF-α, (C,G) IL-1β, and (D,H) IL-1α after exposure to Normoxia/R (1 h normoxia + 24 h reperfusion at 21% O₂ in glucose-containing medium) or OGD/R (1 h OGD + 24 h reperfusion at 0.2% O₂ in glucose-depleted medium) at either 37 or 33.5°C. All CsA containing groups were pre-treated with 10 μM CsA at 37°C. Data from 4 to 5 individual experiments are presented as box-and-whiskers plot (box from 25th to 75th percentile and whisker min to max). Statistical analysis were conducted using one-way ANOVA followed by the Tukey post hoc test; ^∗p < 0.01, ^∗∗p < 0.01, ^∗∗∗p < 0.001 compared to Normoxia/R at 37°C, and ^#p < 0.05 for group comparison were considered significant.

FIGURE 7

RT-qPCR was used to assess gene expressions in the BV-2 microglia, OHSCs, and primary neurons for (A,E,I) MCP1, (B,F) Iba1, (C,G) TGF-β1, and (D,H) iNOS after exposure to Normoxia/R (1 h normoxia + 24 h reperfusion at 21% O₂ in glucose-containing medium) or OGD/R (1 h OGD + 24 h reperfusion at 0.2% O₂ in glucose-depleted medium) at either 37 or 33.5°C. All CsA containing groups were pre-treated with 10 μM CsA at 37°C. Data from 4 to 5 individual experiments are presented as box-and-whiskers plot (box from 25th to 75th percentile and whisker min to max). Statistical analysis were conducted using one-way ANOVA followed by the Tukey post hoc test; ^∗p < 0.01, ^∗∗p < 0.01, ^∗∗∗p < 0.001 compared to Normoxia/R at 37°C, and ^#p < 0.05 for group comparison were considered significant.