Inflammation and NFkappaB activation is decreased by hypothermia following global cerebral ischemia

Abstract

We previously showed that hypothermia attenuates inflammation in focal cerebral ischemia (FCI) by suppressing activating kinases of nuclear factor-kappa B (NFkappaB). Here we characterize the inflammatory response in global cerebral ischemia (GCI), and the influence of mild hypothermia. Rodents were subjected to GCI by bilateral carotid artery occlusion. The inflammatory response was accompanied by microglial activation, but not neutrophil infiltration, or blood brain barrier disruption. Mild hypothermia reduced CA1 damage, decreased microglial activation and decreased nuclear NFkappaB translocation and activation. Similar anti-inflammatory effects of hypothermia were observed in a model of pure brain inflammation that does not cause brain cell death. Primary microglial cultures subjected to oxygen glucose deprivation (OGD) or stimulated with LPS under hypothermic conditions also experienced less activation and less NFkappaB translocation. However, NFkappaB regulatory proteins were not affected by hypothermia. The inflammatory response following GCI and hypothermia's anti-inflammatory mechanism is different from that observed in FCI.

Fig. 1. Inflammatory reaction after global cerebral ischemia

Increased OX42 staining in microglia is observed in the region between the blades of the dentate (A) and within hippocampal CA1 (B) 72 h post GCI in the rat, compared to a sham animal where little staining is seen (F). OX42 cells usually possessed processes (C), consistent with a microglial morphology. In contrast, rare cells near the dentate (D), and CA1 (E) stained for a neutrophil marker (myeloperoxidase, MPO), but the morphology of these cells was not consistent with them being neutrophils (arrows).

Fig. 2. Mild hypothermia reduced ischemic damage in the CA1 hippocampus region 72h following GCI treatment

Hematoxylin & eosin staining revealed less cell death in the 33°C and sham operated rats (n=4–6 per group) quantified as the percent of ischemic damage after 72h (A) (*P< 0.05). Images (B) are representative of H&E stained CA1 sections 72 h post GCI shown at both 100 × and 400 × magnification. Scale bars=100 μm 100x, 20 μm.

Fig. 3. Mild hypothermia decreases microglial staining following GCI

Histological scores (see text) from the isolectin B4 (IB4) stained sections were assessed for each section and quantified at 72 h. Staining scores were significantly lower in hypothermic ischemic rats (GCI-33) than normothermic (GCI-37, *P<0.05 vs. GCI37, N =6/group). IB4 staining was nearly undetectable among sham animals, and scores were no different between hypothermic (SHAM-33) and normothermic (SHAM-37) groups. Representative histochemical stains (B) of the regions between and around the blades of the dentate gyrus 72 h following GCI are shown. Scale bars=100 μm 100×, 20 μm.

Fig. 4. Hypothermia reduces microglial cell death due to ischemia-like insults

Less cell death is observed when microglia are subjected to glucose deprivation (BSS0) or combined oxygen glucose deprivation (OGD) under hypothermic (33C) conditions compared to normothermic (37C). Control cultures (BSS5.5) suffered low levels of cell death, and this was not affected by temperature. (**P<0.001 vs. cultures studied at 37C)

Fig. 5. Microglia suffer less cell death and are less activated following ischemia-like insults under hypothermic conditions

Less cell death is observed when microglia are subjected to oxygen glucose (OGD) or glucose deprivation (GD) at 33°C (B, D) compared to 37°C (A, C). Arrows indicate representative dead cells. Fewer amoeboid (and thus, activated) microglia were observed at 33°C compared to 37°C as well (arrowheads), indicating that hypothermia might also prevent microglial activation.

Fig. 6. Mild hypothermia reduces NFκB translocation following GCI

Normothermic, hypothermic, and sham operated rat brain sections were stained for the p65 subunit of NFκB and counterstained with hematoxylin to identify nuclei. Once NFκB is activated, it translocates into the nucleus. Images (A) are representative of the hippocampal CA1 region 72h after GCI. In the normothermic GCI group (37°C GCI), NFκB staining was seen in the nucleus of most cells (arrowheads) and cytosol. In hypothermic brains (33°C GCI), staining was mostly confined to the cytosol (arrowheads) with very faint staining in the nucleus. Among sham animals (Sham), cytosolic staining was observed, but no nuclear staining was seen as evidenced by the bluish appearance of nuclei (arrowheads) from the hematoxylin stain (Scale bar=20μm). The extent of NFκB expression and translocation was quantified in figures B and C. All positive cells in the CA1 region were counted and quantified per high power field. Ischemia led to higher numbers of positive cells, and fewer positive cells were observed in hypothermic brains (GCI-33) compared to normothermic ischemic brains (GCI-33, *P<0.001 vs. shams, **P<0.01 vs. GCI-33). Furthermore, numbers of cells that exhibited nuclear NFκB staining per field were much fewer among GCI-33 compared to GCI-37 (C) (*P<.001 vs. shams, **P<0.001 vs. GCI-37).

Fig. 7. Mild hypothermia decreases NFκB activation

A NFκB p65 DNA binding assay was used to measure NFκB activity in nuclear extracts of brain samples taken from mice exposed to LPS injection or GCI. Data represent the % increase in optical density (OD) relative to the activity detected in sham samples. Following both GCI and LPS, lower temperature led to decreased DNA binding (*P<0.05, **P<0.01 vs. 37C, n=4–8 samples/group).

Fig. 8. Hypothermia inhibits nuclear NFκB translocation in primary microglia following 2h OGD and 4h reperfusion

Primary microglia subjected to OGD and reperfusion were stained for NFκB’s p65 subunit. In the absence of injury, microglia appeared elongated with processes (BSS5.5, phase), but OGD at normothermic conditions caused microglia to assume a phagocytic phenotype (37C) which was not observed under hypothermic conditions (33C). Under normothermic conditions, OGD led to increased nuclear NFκB staining (37C, nuclei delineated by DAPI staining), compared to hypothermic and control conditions (33C and BSS5.5, respectively). Under hypothermic conditions, NFκB was primarily cytosolic, and less intense than microglia exposed to OGD at normothermia. Both morphology and NFκB staining patterns were similar between control and hypothermic cultures. (scale bar = 10 μm)

Fig. 9. The inhibition of NFκB translocation by hypothermia is not due to differences in the expression of NFκB modulatory proteins

Western blots of IKKγ following GCI (A, E) and LPS injection (B, F) showed increased expression compared to sham, but expression was no different between normothermic and hypothermic brains. Total levels of NFκB’s inhibitory protein, IκB-α was unchanged by GCI or LPS, and levels were no different with hypothermia (C, G). GCI led to increases in IκB-α phosphorylation (D, upper gel), but hypothermia failed to alter phosphorylated IκB-α levels (D, lower gel, H). (*P<0.05, β-actin shown as a housekeeping control, n=6/group)

Fig. 10. Mild hypothermia decreases inflammatory mediator secretion by cultured microglia

Primary microglial cultures were stimulated with LPS (A–C) at either 37°C or 33°C for 24 h. Measurement of glutamate (A), IL-1β(B) and TNF-α (C) showed decreased levels in culture supernatants at lower temperatures. Similarly, microglia exposed to 2 h OGD followed by 24 h reperfusion at 33°C led to less TNF- α secretion compared to 37°C (D). (*P<0.05, **P<0.01)