Background
Ischemic stroke is the most common form of stroke and a major cause of disability and death in adults worldwide. The obstruction of cerebral blood flow initiates the acute phase of cerebral injury, including neuroinflammation and cerebral edema [
1]. Neuroinflammation is a crucial contributor to neuronal death after ischemic stroke, and activated microglia have been the object of studies concentrating on neuroinflammation [
2‐
5]. Cerebral edema, mainly resulting from the increase of cerebral vascular permeability, is the most common cause of neurologic deficiency and mortality during acute ischemic stroke, highlighting the importance of maintaining blood-brain barrier (BBB) integrity [
6‐
9]. Despite enormous efforts in both preclinical and clinical research, treatments to protect against ischemic stroke are still insufficient.
A novel approach to protecting against ischemic stroke injury is the administration of low levels of exogenous CO [
10]. Endogenous carbon monoxide (CO) is a product of heme catabolism by heme oxygenase (HO) [
11‐
14]. Exogenous CO is commonly regarded as poisonous because its high affinity for hemoglobin can cause rapid elevation of carboxyhemoglobin (COHb) to toxic levels that compromise oxygen delivery to the tissues [
11]. However, accumulating evidence suggests that low levels can offer protection through potential anti-inflammatory, anti-apoptotic, and anti-proliferative effects [
15‐
17]. Carbon monoxide-releasing molecules (CORMs), a group of compounds capable of carrying and liberating controlled quantities of CO, have shown promise for delivering exogenous CO without altering COHb to toxic levels [
11,
18‐
20]. Although the mechanisms by which CORM-derived CO might offer its beneficial effects have not yet been thoroughly investigated, many studies substantiate the protective role of CORM-derived CO against cellular and tissue damage in numerous models of injury, such as renal ischemia-reperfusion injury, hemorrhagic stroke, traumatic brain injury, transplantation, sepsis, hypertension, and cardiovascular disorders [
21‐
28].
Currently, the effect of water-soluble CORM-3 [
11,
22,
24] on ischemic stroke remains unclear. In this study, we investigated the therapeutic potential of CORM-3 for ischemic stroke using a mouse model of transient middle cerebral artery occlusion (tMCAO). We found that CORM-3 treatment may contribute to better outcomes after cerebral injury by attenuating brain infarct and edema, lessening BBB disruption, and improving neurologic functions. Further investigation into mechanisms underlying the beneficial effects of CORM-3 on ischemic stroke revealed that CORM-3 increased the protein levels of neuronal nuclear antigen (NeuN) and microtubule-associated protein 2 (MAP2), and suppressed microglial activation, which reduced the quantities of reactive microglia and downregulated the expression of ionized calcium-binding adapter molecule (Iba)-1, tumor necrosis factor (TNF)-α, and interleukin (IL) 1β. CORM-3 also elevated the expression of the pericyte marker platelet-derived growth factor receptor (PDGFR)-β, tight junction protein ZO-1, and matrix protein laminin, which reduced BBB leakage. Finally, CORM-3 reduced the protein level of matrix metalloproteinase (MMP)-9. Taken together, our findings demonstrate clear benefits of CORM-3 on ischemic stroke through suppression of neuroinflammation and alleviation of BBB disruption.
Discussion
Our findings suggest that CORM-3 has a protective effect in a mouse model of ischemic stroke injury. We found that CORM-3 treatment reduced infarct volume and brain water content, alleviated BBB disruption, and promoted neurologic recovery after ischemic stroke. Furthermore, CORM-3 increased the protein levels of NeuN and MAP2, reduced microglial activation, decreased the expression of pro-inflammatory cytokines TNF-α and IL1β, increased the expression of PDGFRβ, ZO-1, and laminin, and downregulated the protein level of MMP-9 compared with corresponding levels in saline-treated mice. Our study suggests that CORM-3 ameliorates ischemic stroke injury by suppressing neuroinflammation and alleviating BBB disruption.
Against general dogma that CO is poisonous, particularly to the brain, accumulating evidence suggests that it might be associated with cytoprotection and maintenance of homeostasis in several organs and tissues [
10,
23,
27,
28,
42‐
44]. Therefore, exogenous administration of CO at low levels (as inhaled CO gas or as CORMs) could be explored as a potential therapeutic method. It has been shown that low levels of inhaled CO provide benefit against ischemia-reperfusion brain injury by attenuating ischemia-induced infarct volume, reducing edema formation, and restoring cerebral blood flow [
45]. A study that used a model of permanent ischemic stroke indicated that a low concentration of inhaled CO provided neuroprotective effect by activating the Nrf2 pathway [
46]. However, the application of CO gas presents several limitations, such as the potential for partial systemic hypoxia and toxicity and the need for CO inhalation facilities and monitoring of blood oxygen levels [
11]. Given these barriers, transition metal carbonyls are good candidates for delivering CO, as they function as CORMs in biological systems [
11]. Water-soluble CORM-3 has beneficial effects in many models of injury, such as hemorrhagic stroke, pulmonary hypertension, and ischemia-reperfusion injury during kidney transplantation [
21,
22,
47]. However, the effect of CORM-3 on ischemic stroke and its mechanism of protection are still unclear. In this study, we treated tMCAO mice with CORM-3 at the time of reperfusion and investigated the therapeutic effects. We used a CORM-3 dose of 4 mg/kg because of studies showing that this dose releases an amount of CO in mice that may be sufficient to exert protective effect [
22,
24]. We did not monitor the effects of CORM-3 on the level of COHb because studies have already indicated that various doses of CORM-3 have no effects on COHb in mice [
19,
20]. We found that the administration of CORM-3 significantly decreased cerebral infarct volume and water content after ischemic stroke, similar to results from a previous study that used inhaled CO [
45]. Additionally, the protein levels of NeuN and MAP2 in the ischemic brain of CORM-3-treated tMCAO mice were significantly increased compared with those of saline-treated controls. NeuN and MAP2 are well recognized as markers of mature neurons. It is likely that CORM-3 increases the levels of these two proteins by decreasing neuronal death or apoptosis, rather promoting neurogenesis, because 3 days is not long enough for neural stem cells in the subventricular zone to proliferate, migrate to the peri-infarction zone, and differentiate into mature neurons [
40,
48,
49]. In addition, CORM-3 treatment markedly reduced BBB permeability and promoted neurologic recovery after ischemic stroke. These results suggest that CORM-3 might provide protection against ischemic stroke. Our study focused on the acute phase after ischemia, but it will be important to determine the long-term effects of CORM-3 on post-stroke neurogenesis, angiogenesis, and recovery of ischemic white matter injury.
Upregulation of endogenous neuroinflammatory processes is a crucial secondary cell death mechanism that follows ischemic stroke and initiates a feedback loop of inflammatory cascades that can expand the region of brain damage [
1]. Evidence has shown that inflammation not only affects the infarct tissue in the near-term but also causes long-term damage in the ischemic penumbra [
2,
4]. Microglia, the brain’s resident immune cells, are rapidly activated from their resting state after ischemic stroke and are considered to be major cellular contributors to neuroinflammation [
50,
51]. Activated microglia initiate neuroinflammation by producing cytotoxic and inflammatory factors, including the cytokines TNF-α and IL-1β, thereby aggravating brain damage [
5,
52]. At high concentrations, TNF-α and IL1β have direct toxic effects on neurons and neural cells [
53,
54]. Consequently, activated microglia have been the target of studies focusing on neuroinflammation. CORMs possess anti-inflammatory properties, and CORM-3 has been shown to reduce microglial activation in BV-2 microglia and in a rat model of hemorrhagic stroke [
22,
55]. To investigate whether CORM-3 regulates neuroinflammation after ischemic stroke, we characterized activated microglia as Iba1
+ cells with special morphology as previously described [
14,
40,
56]. We found that the administration of CORM-3 decreased the number of activated microglia in the peri-infarction zone and reduced the expression of Iba-1, TNF-α, and IL1β in ipsilateral tissue at 72 h after reperfusion. Consistent with the anti-inflammatory effect shown in previous studies, our results indicate that CORM-3 suppressed neuroinflammation after ischemic stroke. We hypothesize that CORM-3 might reduce the release of damage-associated molecular pattern molecules (DAMPs), such as high-mobility group box 1 (HMGB1) and galectin-3 (Gal3) [
57], which have been shown to stimulate microglia after ischemic stroke. HMGB1 is released from necrotic neurons and mediates the activation of microglia via Toll-like receptors (TLRs) or the receptor of advanced glycation end-products (RAGE) on microglia [
58]. Gal3 is released from microglia, and Gal3-dependent TLR4 activation promotes microglial activation [
59]. CORM-3 might directly modulate microglia phenotype by inhibiting microglial nucleotide-binding domain (NOD)-like receptor protein 3 (NLRP3) signaling, but this possibility needs to be further investigated. In addition to the resident microglial activation, the infiltration of circulating monocytes/macrophages is considered to play a vital role in post-stroke neuroinflammation [
57]. Details regarding the mechanisms that underlie the effects of CORM-3 on microglia and macrophages after ischemic stroke require additional exploration.
Normally, the BBB protects the brain from toxic substances. BBB disruption is a crucial event in the pathogenesis of acute ischemic stroke that leads to cerebral edema, brain hemorrhage, and neuronal death [
7]. Previous data suggest that overexpression of metalloproteinases, especially MMP-9, after ischemic stroke disrupts the BBB by degrading the tight junctions and basal lamina proteins [
24,
60‐
63]. Furthermore, a recent study showed that pericytes, a key component of the BBB, contribute to BBB disruption in the capillary bed during acute ischemic stroke, with rapid MMP-9 activation at pericyte somata before capillary leakage [
64]. In a mouse model of traumatic brain injury, CORM-3 was shown to reduce pericyte death and MMP-9 expression at pericyte somata [
24]. However, the effect of CORM-3 on BBB disruption after ischemic stroke remains unknown. In this study, we showed that CORM-3 treatment reduced Evans blue leakage and brain edema, indicating that it can reduce BBB permeability in mice after tMCAO. The administration of CORM-3 increased the expression of PDGFRβ, ZO-1, and laminin and decreased the expression of MMP-9, indicating that it can protect pericytes, tight junction proteins, and matrix proteins after tMCAO. These results suggest that CORM-3 can potentially protect BBB integrity from ischemic stroke injury. Although we showed that CORM-3 decreased MMP-9 expression after ischemia, its effect on MMP-9 function would be better supported by gelatin gel zymography assessment [
65]. Considering the peak concentration of reactive oxygen species (ROS) during reperfusion [
66], we hypothesize that CORM-3 may rescue pericytes after tMCAO by stabilizing the ROS/hypoxia-inducible factor-1α axis, leading to microvascular stabilization. Although studies have shown that CORM-3-derived CO may be a potent enhancer of angiogenesis in vitro [
67], and pericytes may contribute to post-stroke angiogenesis [
68‐
70], it would be interesting to identify the direct effect of CORM-3 on endothelial cells after ischemic stroke, and the indirect effect of CORM-3 on angiogenesis, for example whether CORM-3 affects pericyte-endothelial cell crosstalk after stroke.
One limitation of our study is that we administered CORM-3 only at the time of reperfusion by way of retro-orbital injection. The effects of CORM-3 administrations at different time points and by different routes should be further investigated. Additionally, microscopic analysis of the brains was made only on day 3 after tMCAO, which may be sufficient to support our conclusion, but longitudinal observations, such as on days 7, 14, and 21, are needed to clarify the mechanism by which CORM-3 promotes post-stroke recovery. It would be interesting to study the mechanism by which CORM-3 treatment suppresses microglial activation in the ischemic brain and to determine whether CORM-3 affects newly generated microvessels. Identification of the long-term effects of CORM-3 on ischemic stroke will provide better support for the potential therapeutic value of the CORMs.