Neonatal hypoxic-ischemic brain injury (HIBI) and hypoxic-ischemic encephalopathy (HIE) continue to produce significant morbidity and mortality despite implementation of therapeutic hypothermia (TE) as a standard of care. There is consequently an urgent need for alternative therapeutics. The Rice-Vannucci method is an accepted approach for modelling neonatal HIBI in rodents. These rodent models have facilitated discovery of numerous therapeutic targets and analysis of the structural and functional consequences of modulating activity at these targets. Owing to their key roles in diverse physiological and pathophysiological processes, several such targets are ion channels. 4 promising ion channel targets are highlighted in this mini-review, with a focus on key rodent model-based experiments which have helped identify them as such. These are transient receptor potential (TRP) melastatin (M) 7 (TRPM7) and 2 (TRPM2) channels, ATP- sensitive potassium (KATP) channels, and volume- regulated anion channels (VRACs). These and other foundational experiments create hope that future work in rodent models and beyond will enable development of novel therapeutics to reduce the burden of neonatal HIBI and HIE.

• Neonatal hypoxic-ischemic brain injury
• Hypoxic-ischemic encephalopathy
• Therapeutic hypothermia
• Rodent Models

Cross E, Feng ZP, Sun HS (2024) Utility of Rodent Models of Neonatal HIBI in Identifying Therapeutic Targets, with a Focus on Ion Channels. J Vet Med Res 11(3): 1273.

Neonatal hypoxic-ischemic brain injury (HIBI) is a state of insufficient blood and oxygen supply to the newborn brain. The associated clinical syndrome, hypoxic-ischemic encephalopathy (HIE), is life-threatening and leaves survivors with long-term neurodevelopmental consequences, including cognitive, motor, and memory impairments, attentional deficits, and cerebral palsy [1-3]. The standard of care for HIE is currently therapeutic hypothermia (TE), which involves cooling the brain, via either targeted head cooling or whole-body cooling, to disrupt pathological processes [2-5]. Although TE has substantially improved overall outcomes, it is not always effective, and a significant proportion of affected neonates continue to suffer mortality and disability [2,3]. This is likely owing in part to the narrow window of opportunity to initiate it: within 6 hours of HIBI [2]. Additionally, TE is only recommended for neonates who meet certain gestational age, weight, and umbilical cord pH criteria [2,4]. Alternative therapeutics are therefore urgently required to help combat neonatal HIBI and HIE.

The Rice-Vannucci method is a well-established approach to studying neonatal HIBI in rodents. The method, as described originally, involves subjecting 7-day-postnatal rats to unilateral common carotid artery ligation, followed, 4 to 8 hours later, by hypoxic conditions, defined as 8% oxygen at 37°C for 3.5 hours [6]. This induces moderate-to-severe ischemic injury in ipsilateral brain regions [6], a finding which has been extensively reproduced [7-9]. The method represents a breakthrough in research on neonatal HIBI, the mechanisms of which are significantly influenced by brain immaturity, as HIBI was previously only modelled in adult rodents [6,10]. In the over 40 years since its development, the Rice-Vannucci method has been adapted for use in mice in addition to rats [11-13]. Although some groups have adjusted factors such as the recovery time between ischemia and hypoxia and the nature of hypoxic conditions, this continues to be the preferred method for studying neonatal HIBI in rodents [14]. These rodent models have enabled identification of numerous promising therapeutic targets to advance treatment of neonatal HIBI and HIE. Several such targets are ion channels; transmembrane proteins which mediate selective transport of ions across cell membranes. This ion transport plays essential roles in diverse physiological and pathophysiological processes. Ion channels are therefore the third most common family of drug targets, surpassed only by receptors and enzymes [15]. Ion channel agonists and antagonists have been developed for treatment of various conditions [16]. This mini-review highlights 4 ion channels and the key rodent model-based experiments which have contributed to their discovery as promising therapeutic targets for neonatal HIBI.

Transient receptor potential (TRP) channels are a group of cation channels which, upon activation by diverse stimuli, play essential roles in both physiological and pathophysiological signaling [17]. 8 of 28 TRP channels expressed in mammals belong to the melastatin (M) subgroup (TRPM1-8). TRPM channels are expressed in various tissues and participate in processes such as proliferation of cells, detection of temperature changes, and angiogenesis, as well as cancer and neurological diseases [17]. TRPM7 is a calcium (Ca2+)- and magnesium (Mg2+)-regulated channel which conducts most divalent cations [17]. It is activated by changing cellular energy requirements and regulates cation movement to accommodate these. It was thus implicated as a plausible mediator of the pathological influx of divalent cations that occurs during hypoxia [18], and subsequently identified to be necessary for anoxia-induced neuron death [19]. The latter study showed that, under conditions of oxygen and glucose deprivation (OGD), TRPM7 is necessary for creation of a fatal reactive oxygen species (ROS)-induced current which promotes excessive Ca2+ influx and further ROS release in a positive feedback loop [19]. Several subsequent studies in adult rodents have shown that TRPM7 expression is upregulated in response to HIBI, and validated the observed neuroprotective effects of TRPM7 suppression [20-24].

The first evidence of these phenomena in neonatal rodents was published by our group [12]. HIBI was induced in postnatal day 7 mice using the Rice-Vannucci method with slightly modified hypoxic conditions (7.5% oxygen at 37°C for 100 minutes after a 1.5-hour recovery period post- ligation). 24 hours later, TRPM7 protein expression was significantly elevated in the ipsilateral, compared to the contralateral, hemisphere. Treating the mice, 30 minutes before HIBI, with carvacrol, a TRPM7 inhibitor which previously demonstrated neuroprotective efficacy against HIBI in adult rats [25], similarly conferred neuroprotection [1]. The pre-treated mice had reduced infarct volumes 24 hours and 7 days post-HIBI, as measured through 2, 3, 5-triphenyltetrazolium chloride (TTC) and Nissl staining, respectively, as well as improved scores on neurobehavioural tests. Carvacrol also reduced caspase-3 protein expression and the quantity of cells staining positive for TUNEL, while restoring pre-HIBI Bcl/Bax3 and p-Akt/t-Akt ratios. These findings collectively suggest anti- apoptotic effects [12].

Notable limitations of this study were the fact that carvacrol lacks strong specificity for TRPM7 and that its effects were only studied with pre-HIBI administration [12,26]. These were addressed in a follow-up study testing the effects of waixenicin A, a TRPM7-specific inhibitor [27], in postnatal day 7 mice subjected to the Rice-Vannucci method [26]. Both pre-treatment (30 minutes before ischemia) and post-treatment (30 minutes before hypoxia onset, immediately after hypoxia, or 1 hour after hypoxia) substantially reduced infarct volumes 24 hours later. Administration of naltriben, a TRPM7 activator, significantly increased infarct volumes measured at the same timepoint, but waixenicin A overpowered this effect. Pre- and post-HIBI waixenicin A treatment also protected against loss of brain mass and improved performance on neurobehavioural tests up to 7 days post-HIBI. Pre-treatment continued to improve neurobehavioural performance for 4 weeks [26]. Potential downstream mediators of TRPM7-induced neuronal death in neonatal HIBI were also identified as targets for future study: calmodulin-dependent protein kinase II (CaMKII), phosphatase calcineurin, calmodulin, p38, and the cofilin cascade [26]. These experiments implicate TRPM7 as a powerful mediator of neonatal HIBI in rodents at the structural and functional levels, thus supporting the potential therapeutic utility of TRPM7 inhibitors in human neonates.

Rodent models have also facilitated identification of another TRPM channel, TRPM2, as a promising therapeutic target for neonatal HIBI. TRPM2 is a cation channel, expressed in several cell types including neurons, which is non-selectively permeable to Ca2+ [28]. In addition to its roles in physiological cellular processes, TRPM2 sensitizes the cell to oxidative stress as it is activated by hydrogen peroxide (H2O2) and its effector molecules and promotes Ca2+ influx which induces cell death [28-30]. These channels have therefore been shown to mediate several diseases, including neurological ones [31]. This, coupled with their extensive expression in the brain, their sequence similarity to TRPM7, and their apparent inhibition by TRPM7- targeted siRNA, led TRPM2 channels to be implicated as another potential mediator of fatal OGD-induced currents [19]. Evidence has since been published to support the role of TRPM2 channels as a mediator of ischemia-induced brain damage in adult rodents [32-34].

TRPM2 channels were expected to play an even more pronounced role in HIBI in neonatal rodents because immature brains are hypersensitive to oxidative stress and therefore endure significantly greater H2O2 accumulation in response to HIBI [4]. A study by our group provided support for this. Postnatal day 7 TRPM2-deficient (TRPM2+/- and TRPM2-/-) mice [35], along with wild-types, were subjected to HIBI via the Rice- Vannucci method [36]. The knockout mice had significantly reduced infarct volumes on TTC-stained coronal sections 24 hours post-HIBI and Nissl-stained sections 7 days post-HIBI. They also performed better on tests of sensorimotor capabilities 7 days post-HIBI. Anti-apoptotic signaling through the Akt/GSK- 3β/caspase-3 pathway was implicated as a likely mechanism as the HIBI-induced reductions in ratios of phosphorylated to unphosphorylated Akt and glycogen synthase kinase 3β (GSK- 3β) were overcome in knockout mice [36]. Akt is a pro-survival kinase which is active in its phosphorylated form, while GSK-3β is a pro-apoptotic one which is inactive in its phosphorylated form. Further, significantly less astrocytes and microglia were activated in the ischemic penumbra of knockout mice post-HIBI. Overall, this study showed that genetic TRPM2 deficiency is neuroprotective for neonatal HIBI [36].

In a follow-up study, the effects of pharmacological, rather than genetic, TRPM2 antagonism were investigated using AG490, an inhibitor of TRPM2 activation by endogenous H2O2 [37]. A similar set of experiments were again performed in postnatal day 7 mice using the Rice-Vannucci method [6,37]. When administered 20 minutes pre-ischemia, immediately post- ischemia, or immediately post-hypoxia, AG490 significantly decreased infarct volumes on TTC-stained coronal sections 24 hours post-HIBI and Nissl-stained coronal sections 7 and 32 days post-HIBI. It also promoted significantly more weight gain and improved performance on tests of functional status in the short- and long-term. The Akt signaling pathway was again implicated as a mediator of these effects [37]. A limitation of this study was the fact that AG490 also inhibits the JAK2 signaling pathway, although there is evidence that these effects are isolated from those on TRPM2 [37]. Testing a specific pharmacological inhibitor of TRPM2 using a similar methodology may help further clarify its putative role in neonatal HIBI.

ATP-sensitive potassium (KATP) channels are activated by reductions in ATP levels, often induced by pathological processes, and promote efflux of potassium (K+) from cells, thus decreasing their excitability [38]. Such channels were first described in neurons by Ashford et al. (1988), following evidence of their role in regulating the activity of other types of excitable cells, such as pancreatic β-cells and cardiac muscle cells [1,39,40]. These channels were shown to help establish connections between metabolism and excitability in states of metabolic compromise, including ischemia, hypoxia, and anoxia [41,42].

The neuroprotective functions of KATP channels have been studied in both adult [13,43-46], and neonatal [7-9,47], rodent models of HIBI. An early study in postnatal day 7 rats showed that electro-acupuncture (EA) performed before Rice-Vannucci- induced HIBI was neuroprotective, and that these effects may be mediated by KATP channels. Through analysis of c- Fos and c-Jun expression, the authors found that both EA and diazoxide, a KATP channel agonist, reduced pathological HIBI signaling, while glibencalmide, a KATP antagonist, enhanced it [7]. In a subsequent study using the same model, they showed that diazoxide-mediated KATP activation decreases proteolysis of A-calcium-dependent neutral protease (A-calpain), a mediator of apoptosis, necrosis, and cytotoxicity, along with c-Fos and c-Jun expression. This provides insight into a potential mechanism of the neuroprotective effects of KATP agonists against HIBI [8].

Additionally, our group investigated the role of KATP channels in neonatal HIBI using a similar experimental paradigm as described for the TRP channels in postnatal day 7 mice [47]. Brief exposure to hypoxic conditions before true hypoxic insult, known as hypoxic preconditioning, was associated with significantly reduced infarct volumes and quantities of TUNEL positive cells, as well as improved performance on neurobehavioural tests. Administration of tolbutamide, a KATP channel blocker, eliminated these pre-conditioning effects, while diazoxide mimicked them. This indicates that KATP channels contribute to the neuroprotective effects of hypoxic preconditioning in neonatal HIBI [47]. These rodent model-based studies have thus implicated KATP channels as another promising therapeutic target.

Alongside the cation channels discussed above and others, anion channels have been identified as important therapeutic targets in rodent models of neonatal HIBI. Chloride (Cl-) is the dominant anion present under physiological conditions [11]. Dysregulation of chloride channels is associated with various pathologies, including epilepsy, lung fibrosis, neurodegeneration, and blindness [48]. Volume-regulated anion channels (VRACs) are a class of chloride channels extensively expressed in the brain which promote Cl- efflux in response to cell swelling, thus contributing to osmotic regulation [11]. They have been shown to contribute to HIBI in adult rodents [49,50]. Ischemia- induced excitotoxicity induces extreme depolarization which promotes water influx and cell swelling. This dysregulates VRACs, causing them to mediate Cl- influx, rather than efflux, thus further enhancing swelling via a positive feedback mechanism. This ultimately leads to cell death [51].

Our group investigated the role of VRACs in neonatal HIBI using the Rice-Vannucci method, with unilateral common carotid artery occlusion being induced by bipolar electrical coagulation in this case. Pre-ischemia treatment with 4-(2-butyl-6,7-dichloro-2- cyclopentyl-indan-1-on5-yl) oxobutyric acid (DCPIB), a selective VRAC antagonist, significantly reduced infarct volumes 24 hours later and improved functional recovery both 4 and 24 hours later, as evidenced by increased participation in activity compared to vehicle-treated controls. These results corroborated those elucidated in vitro and implicated VRACS as mediators of osmotic disturbances and consequential cell death under ischemic conditions [11]. In a follow-up study, the role of the swelling- induced Cl- current (ICl, swell) in this mechanism was further elucidated. The neonatal mice were again treated pre-ischemia with DCPIB, which significantly decreased infarct volumes on TTC and Nissl staining, 24 hours and 7 days post-HIBI, respectively. DCPIB pre-treatment was also associated with significantly improved post-HIBI performance on neurobehavioural tests. This provided greater support for the role of ICl, swell as a mediator of the neuroprotective effects of DCPIB [52-54]. Both VRACs and ICl, swell appear to be promising therapeutic targets for neonatal HIBI.

Neonatal HIBI is modelled in rodents using the Rice-Vannucci method, which involves unilateral carotid artery ligation followed by exposure to hypoxic conditions. These models have proven to be instrumental in translating findings from the better-studied and markedly distinct process of adult HIBI to identify promising therapeutic targets in neonates. They allow for multiple experiments to be conducted in parallel to analyze structural and functional changes, as well as the effects of target agonism and antagonism, with greater ease, accessibility, and cost-effectiveness than more advanced animal models. This mini-review summarizes rodent model-based evidence of the therapeutic potential of agents targeting 4 classes of ion channels: TRPM7, TRPM2, KATP channels, and VRACs, which represent just a subset of the promising ion channel targets that have been identified. The hope is that future studies in these rodent models and beyond will promote development of highly efficacious, more universal agents to treat neonatal HIBI and circumvent HIE.

1. American College of Obstetricians and Gynecologists. Executive summary: Neonatal encephalopathy and neurologic outcome. Obstet Gynecol. 2014; 123: 896-901.
2. Lemyre B, Chau V. Hypothermia for newborns with hypoxic-ischemic encephalopathy. Paediatric Child Health. 2018; 23: 285-291.
3. Natarajan G, Pappas A, Shankaran S. Outcomes in childhood following therapeutic hypothermia for neonatal hypoxic-ischemic encephalopathy (HIE). Semin Perinatol. 2016; 40: 549-555.
4. Korf JM, McCullough LD, Caretti V. A narrative review on treatment strategies for neonatal hypoxic ischemic encephalopathy. Transl Pediatr. 2023; 12: 1552-1571.
5. Shankaran S, Laptook AR, Ehrenkranz RA, Tyson JE, McDonald SA, Donovan EF, et al. Whole-body hypothermia for neonates with hypoxic–ischemic encephalopathy. N Engl J Med. 2005; 353: 1574-1584.
6. Rice JE, Vannucci RC, Brierley JB. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Ann Neurol. 1981; 9: 131- 141.
7. Jiang K, Zhao Z, Shui Q, Xia Z. Electro-acupuncture preconditioning abrogates the elevation of c-Fos and c-Jun expression in neonatal hypoxic-ischemic rat brains induced by glibenclamide, an ATP- sensitive potassium channel blocker. Brain Res. 2004; 998: 13-19.
8. Jiang KW, Yu ZS, Shui QX, Xia ZZ. Activation of ATP-sensitive potassium channels prevents the cleavage of cytosolic mu-calpain and abrogates the elevation of nuclear c- Fos and c-Jun expressions after hypoxic-ischemia in neonatal rat brain. Brain Res Mol Brain Res. 2005; 133: 87-94.
9. Ren X, Wang Z, Ma H, Zuo Z. Sevoflurane postconditioning provides neuroprotection against brain hypoxia-ischemia in neonatal rats. Neurol Sci. 2014; 35: 1401-1404.
10. Huang BY, Castillo M. Hypoxic-ischemic brain injury: Imaging findings from birth to adulthood. RadioGraphics. 2008; 28: 417-439.
11. Alibrahim A, Zhao LY, Bae CY, Barszczyk A, Sun CL, Wang GL, Sun HS. Neuroprotective effects of volume-regulated anion channel blocker DCPIB on neonatal hypoxic-ischemic injury. Acta Pharmacol Sin. 2013; 34: 113-118.
12. Chen W, Xu B, Xiao A, Liu L, Fang X, Liu R, et al. TRPM7 inhibitor Carvacrol protects brain from neonatal hypoxic-ischemic injury. Molecular Brain. 2015; 8.
13. Takaba H, Nagao T, Yao H, Kitazono T, Ibayashi S, Fujishima M. An ATP-sensitive potassium channel activator reduces infarct volume in focal cerebral ischemia in rats. Am J Physiol. 1997; 273: R583-586.
14. Vannucci SJ, Back SA. The Vannucci Model of Hypoxic-Ischemic Injury in the Neonatal Rodent: 40 years Later. Dev Neurosci. 2022; 44: 186-193.
15. Dabrowski MA, Dekermendjian K, Lund PE, Krupp JJ, Sinclair J, Larsson O. Ion channel screening technology. CNS Neurol Disord Drug Targets. 2008; 7: 122-128.
16. Bagal SK, Brown AD, Cox PJ, Omoto K, Owen RM, Pryde DC, et al. Ion channels as therapeutic targets: A drug discovery perspective. J Med Chem. 2012; 56: 593-624.
17. Zhang M, Ma Y, Ye X, Zhang N, Pan L, Wang B. Trp (transient receptor potential) ion channel family: Structures, biological functions and therapeutic interventions for diseases. Signal Transduction and Targeted Therapy. 2023; 8.
18. Nadler MJ, Hermosura MC, Inabe K, Perraud AL, Zhu Q, Stokes AJ, et al. LTRPC7 is a MG·ATP-regulated divalent cation channel required for cell viability. Nature. 2001; 411: 590-595.
19. Aarts M, Iihara K, Wei WL, Xiong ZG, Arundine M, Cerwinski W, et al. A key role for TRPM7 channels in anoxic neuronal death. Cell. 2003; 115: 863-877.
20. Jiang H, Tian SL, Zeng Y, Li LL, Shi J. TrkA pathway(s) is involved in regulation of TRPM7 expression in hippocampal neurons subjected to ischemic-reperfusion and oxygen–glucose deprivation. Brain Res Bull. 2008; 76: 124-130.
21. Sun H-S, Jackson MF, Martin LJ, Jansen K, Teves L, Cui H, et al. Suppression of hippocampal TRPM7 protein prevents delayed neuronal death in brain ischemia. Nat Neurosci. 2009; 12: 1300-1307.
22. Zhan HQ, Li PF, Li SY, Yan FL, Yang J, Liu RL, Zhang HF. Influence of lactuside B on the expression of AQP4 and TRPM7 mRNAs in the cerebral cortex after cerebral ischemia injury. Eur Rev Med Pharmacol Sci. 2014; 18: 1151-1157.
23. Zhang Y, Zhou L, Zhang X, Bai J, Shi M, Zhao G. Ginsenoside-RD attenuates TRPM7 and ASIC1A but promotes ASIC2A expression in rats after focal cerebral ischemia. Neurol Sci. 2012; 33: 1125-1131.
24. Zhao L, Wang Y, Sun N, Liu X, Li L, Shi J. Electroacupuncture regulates TRPM7 expression through the trka/PI3K pathway after cerebral ischemia–reperfusion in rats. Life Sci. 2007; 81: 1211-1222.
25. Yu H, Zhang Z-L, Chen J, Pei A, Hua F, Qian X, et al. Carvacrol, a food- additive, provides neuroprotection on focal cerebral ischemia/ reperfusion injury in mice. PLoS ONE. 2012; 7.
26. Turlova E, Wong R, Xu B, Li F, Du L, Habbous S, et al. TRPM7 Mediates Neuronal Cell Death Upstream of Calcium/Calmodulin-Dependent Protein Kinase II and Calcineurin Mechanism in Neonatal Hypoxic- Ischemic Brain Injury. Transl Stroke Res. 2021; 12: 164-184.
27. Zierler S, Yao G, Zhang Z, Kuo WC, Porzgen P, Penner R, et al. Waixenicin A inhibits cell proliferation through magnesium dependent block of transient receptor potential melastatin 7 (TRPM7) channels. J Biol Chem. 2011; 286: 39328-39335.
28. Lange I, Yamamoto S, Partida-Sanchez S, Mori Y, Fleig A, Penner R. TRPM2 functions as a lysosomal Ca2+-release channel in beta cells. Sci Signal. 2009; 2: ra23.
29. Hara Y, Wakamori M, Ishii M, Maeno E, Nishida M, Yoshida T, et al. LTRPC2 ca2+- permeable channel activated by changes in redox status confers susceptibility to cell death. Mol Cell. 2002; 9: 163-173.
30. Jiang LH, Yang W, Zou J, Beech DJ. TRPM2 channel properties, functions and therapeutic potentials. Expert Opin Ther Targets. 2010; 14: 973-988.
31. Naz?ro?lu M. TRPM2 cation channels, oxidative stress and neurological diseases: Where are we now? Neurochem Res. 2010; 36: 355-366.
32. Alim I, Teves L, Li R, Mori Y, Tymianski M. Modulation of NMDAR subunit expression by TRPM2 channels regulates neuronal vulnerability to ischemic cell death. J Neurosci. 2013; 33: 17264- 17277.
33. Fonfria E, Mattei C, Hill K, Brown JT, Randall A, Benham CD, et al. TRPM2 is elevated in the tMCAO stroke model, transcriptionally regulated, and functionally expressed in C13 microglia. J Recept Signal Transduct Res. 2006; 26: 179- 198.
34. Verma S, Quillinan N, Yang YF, Nakayama S, Cheng J, Kelley MH, et al. TRPM2 channel activation following in vitro ischemia contributes to male hippocampal cell death. Neurosci Lett. 2012; 530: 41-46.
35. Yamamoto S, Shimizu S, Kiyonaka S, Takahashi N, Wajima T, Hara Y, et al. TRPM2-mediated Ca2+influx induces chemokine production in monocytes that aggravates inflammatory neutrophil infiltration. Nat Med. 2008; 14: 738-747.
36. Huang S, Turlova E, Li F, Bao M, Szeto V, Wong R, et al. Transient receptor potential melastatin 2 channels (TRPM2) mediate neonatal hypoxic-ischemic brain injury in mice. Exp Neurol. 2017; 296: 32-40.
37. Shimizu S, Yonezawa R, Hagiwara T, Yoshida T, Takahashi N, Hamano S, et al. Inhibitory effects of AG490 on H2O2-induced TRPM2- mediated Ca(2+) entry. Eur J Pharmacol. 2014; 742: 22-30.
38. Bavencoffe A, Chen SR, Pan HL. Regulation of nociceptive transduction and transmission by nitric oxide. Vitam Horm. 2014; 1-18.
39. Cook DL, Hales CN. Intracellular ATP directly blocks K+ channels in pancreatic B-cells. Nature. 1984; 311: 271-273.
40. Noma A. ATP-regulated K+ channels in cardiac muscle. Nature. 1983; 305: 147-148.
41. Ashford ML, Sturgess NC, Trout NJ, Gardner NJ, Hales CN. Adenosine- 5’-triphosphate- sensitive ion channels in neonatal rat cultured central neurones. Pflugers Arch. 1988; 412: 297-304.
42. Hansen AJ. Effect of anoxia on ion distribution in the brain. Physiol Rev. 1985; 65: 101-148.
43. Héron-Milhavet L, Xue-Jun Y, Vannucci SJ, Wood TL, Willing LB, Stannard B, et al. Protection against hypoxic- ischemic injury in transgenic mice overexpressing Kir6.2 channel pore in forebrain. Mol Cell Neurosci. 2004; 25: 585-593.
44. Liu R, Wang H, Xu B, Chen W, Turlova E, Dong N, et al. Cerebrovascular Safety of Sulfonylureas: The Role of KATP Channels in Neuroprotection and the Risk of Stroke in Patients with Type 2 Diabetes. Diabetes. 2016; 65: 2795-2809.
45. Sun HS, Feng ZP, Miki T, Seino S, French RJ. Enhanced neuronal damage after ischemic insults in mice lacking Kir6.2-containing ATP- sensitive K+ channels. J Neurophysiol. 2006; 95: 2590-2601.
46. Sun H-S, Feng ZP, Barber PA, Buchan AM, French RJ. Kir6.2-containing ATP-sensitive potassium channels protect cortical neurons from ischemic/anoxic injury in vitro and in vivo. Neurosci. 2007; 144: 1509-1515.
47. Sun HS, Xu B, Chen W, Xiao A, Turlova E, Alibraham A, et al. Neuronal K (ATP) channels mediate hypoxic preconditioning and reduce subsequent neonatal hypoxic-ischemic brain injury. Exp Neurol. 2015; 263: 161-171.
48. Planells-Cases R, Jentsch TJ. Chloride channelopathies. Biochim Biophysica Acta (BBA) - Molecular Basis of Disease. 2009; 1792: 173- 189.
49. Kimelberg HK, Jin Y, Charniga C, Feustel PJ. Neuroprotective activity of tamoxifen in permanent focal ischemia. J Neurosurg. 2003; 99: 138-142.
50. Zhang Y, Zhang H, Feustel PJ, Kimelberg HK. DCPIB, a specific inhibitor of volume regulated anion channels (VRACs), reduces infarct size in MCAo and the release of glutamate in the ischemic cortical penumbra. Exp Neurol. 2008; 210: 514-520.
51. Inoue H, Okada Y. Roles of volume-sensitive chloride channel in excitotoxic neuronal injury. J Neurosci. 2007; 27: 1445-1455.
52. Wong R, Abussaud A, Leung JW, Xu B, Li F, Huang S, et al. Blockade of the swelling- induced chloride current attenuates the mouse neonatal hypoxic-ischemic brain injury in vivo. Acta Pharmacologica Sinica. 2018; 39: 858-865.
53. Lafemina MJ, Sheldon RA, Ferriero DM. Acute hypoxia-ischemia results in hydrogen peroxide accumulation in neonatal but not adult mouse brain. Pediatr Res. 2006; 59: 680-683.
54. Tinker A, Aziz Q, Thomas A. The role of ATP-sensitive potassium channels in cellular function and protection in the cardiovascular system. British J Pharmacol. 2013; 171: 12-23.