Methane-Rich Saline Alleviates CA/CPR Brain Injury by Inhibiting Oxidative Stress, Microglial Activation-Induced Inflammatory Responses, and ER Stress-Mediated Apoptosis

Abstract

Brain injury induced by cardiac arrest/cardiopulmonary resuscitation (CA/CPR) is the leading cause of death among patients who have recovery of spontaneous circulation (ROSC). Inflammatory response, apoptosis, and oxidative stress are proven pathological mechanisms implicated in neuronal damage. Methane-rich saline (MRS) has been proven that exerts a beneficial protectiveness impact in several models of ischemia-reperfusion injury. The goal of this paper is to ascertain the role of MRS in CA/CPR-induced brain injury and its potential mechanisms. The tracheal intubation of Sprague-Dawley (SD) rats was clamped for 6 min to establish an asphyxiating cardiac arrest model. After that, chest compressions were applied; then, MRS or saline was administered immediately post-ROSC, the rats were sacrificed, and brain tissue was collected at the end of 6 hours. We observed that MRS treatment attenuated neuronal damage in the hippocampal CA1 region by inhibiting microglial activation, leading to a decrease in the overexpression of proinflammatory cytokines such as TNF-α, IL-6, and iNOS. The results also illustrated that MRS treatment diminished apoptosis in the hippocampal CA1 region , reduced the expression of apoptosis-associated proteins Bax and cleaved caspase9, and increased Bcl-2 expression, as well as inhibited the expression of endoplasmic reticulum (ER) stress pathway-related proteins GRP78, ATF4, and CHOP. Further findings showed that MRS treatment significantly attenuated hippocampal ROS and MDA levels and increased GSH and SOD antioxidant factor levels, which indicated that MRS treatment could inhibit oxidative stress. Our results suggest that MRS exerts a protective effect against CA/CPR brain injury, by inhibiting oxidative stress, microglial activation-induced inflammatory responses, and ER stress-mediated apoptosis.

Figure 1

Experiment procedure flowchart. CA: cardiac arrest; CPR: cardiopulmonary resuscitation; ROSC: recovery of spontaneous circulation.

Figure 2

Changes in mean arterial pressure and heart rate during ROSC after CA: (a) mean arterial pressure (MAP) and (b) heart rate (HR). Bars represent the mean and SEM. BL indicates baseline. The statistical significance was assessed by the one-way ANOVA test (^δp < 0.05, and ^∗p < 0.01 vs. sham control group; ^ξp < 0.05 and ^#p < 0.01 vs. MRS control group; ^&p < 0.05 and ^$p < 0.01 vs. CPR+NS group; ns: no statistically significant difference).

Figure 3

MRS rescued hippocampal neuronal damage after CA/CPR. (a) Representative photomicrographs of Nissl staining showing neurons. (b) Immunohistochemical staining of antineuronal nucleus antibody NeuN to detect neuronal survival status. (c) Surviving neurons counting per high-power field (×400). (d) Quantification of the NeuN-positive relative intensity area (%). Scale bars indicated 25 μm. Data were reported as mean ± SD. The statistical significance was assessed by the one-way ANOVA test (^δp < 0.05, and ^∗p < 0.01 vs. sham control group; ^ξp < 0.05 and ^#p < 0.01 vs. MRS control group; ^&p < 0.05 and ^$p < 0.01 vs. CPR+NS group).

Figure 4

MRS inhibited microglial activation in the hippocampus after CA/CPR: (a) TMEM119 immunohistochemical staining, (b) CD68 immunohistochemical staining, and (c) Iba1 immunofluorescence to assess microglial activation status; (d) quantification of TMEM119 relative intensity area (%); (e) quantification of CD68 relative intensity area (%); (f) activated microglial relative intensity area (%). Data were reported as the mean ± SD. The statistical significance was assessed by the one-way ANOVA test (^δp < 0.05, and ^∗p < 0.01 vs. sham control group;^ξp < 0.05 and ^#p < 0.01 vs. MRS control group; ^&p < 0.05 and ^$p < 0.01 vs. CPR+NS group).

Figure 5

MRS alleviated the inflammatory response in the hippocampus after CA/CPR. The levels of (a) TNF-α, (b) IL-6, and (c) IL-10 in hippocampal tissues were measured 6 h after CA/CPR using commercial ELISA kits. (d) The expression levels of TNF-α, IL-6, and IL-10 in hippocampal tissue protein at 6 h after CA/CPR. (e) Relative band intensity of TNF-α, IL-6, and IL-10. (f) iNOS immunohistochemistry staining to detect hippocampal CA1 region inflammation response. (g) Quantification of iNOS relative intensity area (%). Scale bars indicated 25 μm. Data were reported as mean ± SD. The statistical significance was assessed by the one-way ANOVA test (^δp < 0.05, and ^∗p < 0.01 vs. sham control group; ^ξp < 0.05 and ^#p < 0.01 vs. MRS control group; ^&p < 0.05 and ^$p < 0.01 vs. CPR+NS group).

Figure 6

MRS reduced the hippocampal neuron apoptosis after CA/CPR. (a) TUNEL assay. (b) TUNEL-positive cells per high-power field (×400). (c) The expression levels of Bax, Bcl₂, caspase9, and cleaved caspase9 in the hippocampus. (d) The relative band intensities (fold of the sham control group). Scale bars indicated 25 μm. Data were reported as mean ± SD. The statistical significance was assessed by the one-way ANOVA test (^δp < 0.05, and ^∗p < 0.01 vs. sham control group; ^ξp < 0.05, and ^#p < 0.01 vs. MRS control group; ^&p < 0.05 and ^$p < 0.01 vs. CPR+NS group).

Figure 7

MRS ameliorated the ER stress and related apoptosis process in the hippocampus after CA/CPR. (a) ER stress-associated apoptosis signaling pathway protein GRP78, ATF4, and CHOP expression levels. (b) The relative band intensities (fold of the sham control group). Data were reported as mean ± SD. The statistical significance was assessed by the one-way ANOVA test (^δp < 0.05, and ^∗p < 0.01 vs. sham control group; ^ξp < 0.05 and ^#p < 0.01 vs. MRS control group; ^&p < 0.05 and ^$p < 0.01 vs. CPR+NS group).

Figure 8

MRS attenuated oxidative stress in the hippocampus after CA/CPR. (a) The DHE fluorescence staining of brain tissue cryosection. (b) The ROS relative fluorescence intensity area (fold of sham control group). The levels of (c) SOD, (d) GSH, and (e) MDA in hippocampal tissue were assessed. Scale bars indicated 25 μm. Data were reported as mean ± SD. The statistical significance was assessed by the one-way ANOVA test (^δp < 0.05, and ^∗p < 0.01 vs. sham control group; ^ξp < 0.05, and ^#p < 0.01 vs. MRS control group; ^&p < 0.05 and ^$p < 0.01 vs. CPR+NS group).

Figure 9

The underlying mechanism for MRS protection against CA/CPR brain injury.