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% O 2 ) 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.

Cooling and Sterile Inflammation in an Oxygen-Glucose-Deprivation/Reperfusion Injury Model in BV-2 Microglia

“…Our results indicate that cooling, whilst increasing cell viability and expression of cold shock proteins in a model of OGD/R-induced injury, also activates BV-2 microglial cells and is accompanied by an upregulation of both pro-and anti-inflammatory cytokine gene expressions and the release of TNF-α. Moreover, we have previously reported similar results in BV-2 microglial cells with Iba-1 and MCP-1 gene expressions being upregulated after 24 h of cooling [30]. Furthermore, TNF-α release was increased due to cooling alone in the reperfusion phase 12 h after experimental start (Figure 9).…”

“…Immortalized murine BV-2 microglial cells [29] were kindly provided by Prof. Ullrich (Zurich, Switzerland). BV-2 cells were cultured as previously described in high-glucose Dulbecco's modified Eagle's medium (DMEM) supplemented with 1% natrium-pyruvate (Biochrom), 10% heat inactivated fetal bovine serum (FBS, Biochrom), and incubated at 37 °C, 21% O 2, and 5% CO 2 [30]. Both cultivating media as well as experimental media were supplemented with 100 U/ml penicillin and 100 μg/ml streptomycin (Merck Milipore).…”

“…The extinction was measured at 490 nm minus 630 nm using a microtiter plate reader (Thermo Fisher Multiskan Ascent). Cytotoxicity is presented as a percentage in relation to maximum LDH content assessed by a lysed normoxic control group as previously described by our research group [30].…”

“…Protein concentration was assessed via Pierce Bicinchoninic Acid (BCA) Protein Assay (Thermo Scientific). Extracellular proteins were precipitated using a trichloroacetic acid protocol as previously described [30]. 3 Mediators of Inflammation room temperature.…”

Bone Marrow-Derived Mononuclear Cell Transplants Decrease Retinal Gliosis in Two Animal Models of Inherited Photoreceptor Degeneration

“…We did not find significant differences in the numbers of Iba-1 + cells between the left (PBS or hBM-MSCs) injected eyes of the animals that had received IVI or SRI ( Table A1 ). Thus, we failed to document differences in microglial cell activation between the PBS or hBM-MSCs left injected eyes, either for the IVI or the SRI, possibly due to the effect of immunosuppression on these cells [ 62 , 63 , 64 ].…”