Neuroprotection of NSC Therapy is Superior to Glibenclamide in Cardiac Arrest-Induced Brain Injury via Neuroinflammation Regulation

Abstract

Cardiac arrest (CA) is common and devastating, and neuroprotective therapies for brain injury after CA remain limited. Neuroinflammation has been a target for two promising but underdeveloped post-CA therapies: neural stem cell (NSC) engrafting and glibenclamide (GBC). It is critical to understand whether one therapy has superior efficacy over the other and to further understand their immunomodulatory mechanisms. In this study, we aimed to evaluate and compare the therapeutic effects of NSC and GBC therapies post-CA. In in vitro studies, BV2 cells underwent oxygen-glucose deprivation (OGD) for three hours and were then treated with GBC or co-cultured with human NSCs (hNSCs). Microglial polarization phenotype and TLR4/NLRP3 inflammatory pathway proteins were detected by immunofluorescence staining. Twenty-four Wistar rats were randomly assigned to three groups (control, GBC, and hNSCs, N = 8/group). After 8 min of asphyxial CA, GBC was injected intraperitoneally or hNSCs were administered intranasally in the treatment groups. Neurological-deficit scores (NDSs) were assessed at 24, 48, and 72 h after return of spontaneous circulation (ROSC). Immunofluorescence was used to track hNSCs and quantitatively evaluate microglial activation subtype and polarization. The expression of TLR4/NLRP3 pathway-related proteins was quantified via Western blot. The in vitro studies showed the highest proportion of activated BV2 cells with an increased expression of TLR4/NLRP3 signaling proteins were found in the OGD group compared to OGD + GBC and OGD + hNSCs groups. NDS showed significant improvement after CA in hNSC and GBC groups compared to controls, and hNSC treatment was superior to GBC treatment. The hNSC group had more inactive morphology and anti-inflammatory phenotype of microglia. The quantified expression of TLR4/NLRP3 pathway-related proteins was significantly suppressed by both treatments, and the suppression was more significant in the hNSC group compared to the GBC group. hNSC and GBC therapy regulate microglial activation and the neuroinflammatory response in the brain after CA through TLR4/NLRP3 signaling and exert multiple neuroprotective effects, including improved neurological function and shortened time of severe neurological deficit. In addition, hNSCs displayed superior inflammatory regulation over GBC.

Keywords: Cardiac arrest; Inflammasome; Neural stem cells (NSCs); Neuroinflammation; Neurology outcome; TLR4.

Figure 1.

Proliferation and activation of BV2 cells after OGD. Cell viability decreased in three OGD-treated groups compared to the normal group (A). More activated BV2 cells with enlarged cellular bodies were found in the control group (A, red arrows). Further quantification showed that hNSCs treatment markedly protected the cells’ ability to proliferate (B) and inhibited the cells’ activation (C) after OGD. The TLR4 / NLRP3 signaling pathway was activated in BV2 cells after OGD. Immunofluorescence quantification of TLR4, NLRP3, and caspase-1 showed that TLR4/NLRP3-related inflammation was maximally up-regulated in the control group (D). hNSCs and GBC treatment downregulated the expression of inflammatory molecules, and the downregulatory effect of hNSCs treatment was more profound compared to GBC treatment (E, F, G). hNSCs or GBC treatment induced changes of phenotype related to the inflammatory process in BV2 cells. After OGD, either pro-inflammation (M1, marked with iNOS) or anti-inflammation (M2, marked with CD206) phenotype BV2 cells increased in the three OGD-treated groups (H). hNSCs and GBC- treated BV2 cells showed significant inhibition of M1 polarization and promotion of M2 polarization. The co-culture of hNSCs had a more significant effect than GBC on the above regulatory process (I, J). *: p < 0.05; **: p < 0.01; ***: p < 0.001. 200/400X, Scale bar = 75μm/50μm.

Figure 2.

Quantitative analysis of neurological function and outcome between groups. The aggregate analysis of surviving NDS shows a significant functional improvement in hNSCs and GBC groups compared to the control group (NSCs VS. Control: p < 0.01, GBC VS. Control: p < 0.05). Further comparison between the two treatment groups showed that the hNSCs group had better functional improvement than the GBC group on aggregate surviving NDS (hNSCs VS. GBC: p < 0.01). Of note, hNSCs therapy displayed better functional recovery than the GBC group at all assessment points (A). Both therapeutic interventions significantly shortened the length of time under severe neurological deficit (SND) (B). The cumulative survival analysis exhibited a trend of improved survival after hNSCs or GBC therapy (C). *: p < 0.05; **: p < 0.01.

Figure 3.

Ischemic neuron assessment by cresyl violet staining. The damaged neurons in the hippocampus were evaluated by region (CA1, CA2, CA3). Neurons in the CA1 region underwent the most serious injury after ischemia, as this region had the highest proportion of damaged neurons (A). Quantitative analysis (HDS, %) among the three groups showed that the percentage of injured neurons in the hippocampus of rats transplanted with hNSCs was significantly lower than that of the control group in CA1, CA2, and CA3 (B). In the ischemia-sensitive region, CA1, hNSCs therapy exhibited a stronger protective effect than GBC (B). *: p < 0.05, **: p < 0.01; 50/200X, Scale bar = 500 /100μm.

Figure 4.

The recruitment of microglia in the hippocampus and tracking of transplanted hNSCs after CA. Immunofluorescence-labeled microglia showing differences in microglial recruitment and activation between groups after ischemia-reperfusion injury of CA (A). Quantitative results showed that the GBC and hNSCs therapy suppressed the recruitment and activation of microglia (B). **: p < 0.01; 50X, Scale bar = 50/250μm.

Figure 5.

The definition and comparison of microglial resting-state vs. activated state based on morphology in the cortex and hippocampus. In the hippocampus, the hNSCs group retained the maximum resting-state microglia (type 1) and minimum activated, non-phagocytic microglia (type 3) compared to GBC or control groups (A, B). In the cortex, the hNSCs group and GBC group had significantly higher proportions of resting-state microglia (type 1) compared to the control group. The hNSCs treatment group had the smallest proportion of activated microglial cells (type 3 and type 4) in the cortex (C, D). Microglia classification according to morphology (E). *: p < 0.05, **: p < 0.01; 400X, Scale bar = 25μm.

Figure 6.

hNSCs inhibit the polarization of pro-inflammatory phenotype of microglia (M1) in the hippocampus. (A) In the control group, Iba-1 positive cells were dense, and more iNOS positive cells were found compared to the treatment groups. (B) Quantitative analysis showed that the proportion of M1 polarization microglia in the control group was significantly higher than that in the hNSCs group and GBC group. (C) hNSCs treatment promoted polarization of the anti-inflammatory phenotype of microglia (M2). Immunofluorescence staining revealed more CD206 positive cells (M2) were co-located with Iba-1 positive cells. (D) The effects of GBC and hNSCs were similar in promoting M2 phenotype polarization in microglia compared to the control group. **: p < 0.01; ***: p < 0.001; 200/400X, Scale bar = 100/50μm.

Figure 7.

TLR4 expression in microglia was downregulated by hNSCs and GBC treatment. Representative double immunofluorescence staining with TLR4 and Iba-1 showed that TLR4 was expressed in a variety of central nervous system cells, including microglia (A). Both TLR4 and Iba-1 expression had an increasing trend in the control group (A). The quantitative results showed that after treatment, the percentages of TLR4+ / Iba-1+ cells in microglia were significantly decreased in the GBC and hNSCs groups (B). Therapeutic intervention inhibited the expression of the NLRP3 inflammasome. Using co-staining with Iba-1, microglia with an expression of NLRP3 were double marked in 10X and 40X microscopic fields. All groups had the expression of NLRP3 at different levels (C). Further quantitative analysis based on the proportion of NLRP3 positive cells showed that hNSCs therapy had a significant and stronger regulatory effect on the expression of this key protein compared to GBC (D). Evaluation of the expression of Caspase-1 in microglia showed the proportion of Caspase-1 positive microglia (marked as both Caspase-1 and Iba-1 positive) in all microglia (marked as Iba-1 positive) decreased after treatments (E). There was a significant decrease in this proportion in the two treatment groups compared to the Control group. In addition, the comparison between the two treatment groups suggested that the hNSCs group had a stronger inhibition of Caspase-1 expression in microglia (F). Localization and quantification of IL-1β expressed in the three groups showed IL-1β was expressed in a small amount in the extracellular area (G). The levels of IL-1β in GBC and hNSCs treated groups were similar and significantly lower than in the control group (H). *: p < 0.05, **: p < 0.01, ***: p < 0.001; 50 100/200/400X, Scale bar = 200/100/50μm.

Figure 8.

The neuroinflammatory regulation of hNSCs therapy is driven by the TLR4 / NLRP3 pathway. Three rats in each group were included for western blot (WB) quantitative analysis. (A) Representative Western Blots for four key proteins involved in receptor expression, signal transduction, assembly of the inflammasome, and expression of inflammatory factors in the TLR4 / NLRP3 pathway. (B-E) The quantitative analysis between the three groups showed that GBC treatment and hNSCs treatment significantly reduced the expression of these proteins compared to the control group (***: p < 0.001) (arbitrary units, A. U.). The comparison between the two treatment groups showed that the downregulatory effect of hNSCs treatment on TLR4/NLRP3 pathway-related proteins was greater than the GBC group. (*: p < 0.05, **: p < 0.01).

Figure 9

(A) Proposed two-way model of TLR4/NLRP3 signal pathways. TLR4 senses multiple compounds (signal 1) including lipopolysaccharide (LPS) and induces activation of the transcription factor NF-κB. This causes the expression of NLRP3 and proIL-1β. NLRP3 senses different pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs) (signal 2), which drives the assembly of the NLRP3 inflammasome (NLRP3, ASC, and pro-caspase-1). Caspase-1 is activated by pro-caspase-1 and cleaves pro-IL-1β (pro-inflammatory cytokine). Finally, the mature cytokines induce inflammation. (B) Possible mechanism of TLR4 / NLRP3 signaling pathway affecting microglia polarization. ASC: apoptosis-associated speck-like protein containing a CARD (Caspase activation and recruitment domain). DAMPs: danger-associated molecular patterns. IL: interleukin. NFκB: nuclear factor κ-light-chain-enhancer of activated B-cells. PAMPs: pathogen-associated molecular patterns.