The present study revealed that the combination of C+P (4 mg/kg) and DHC (0.5 mg/kg) induced an effective hypothermic state, and that the combination induced an enhanced neuroprotection as compared to each alone. This neuroprotection was evidenced by reduction in infarct volumes, neurological deficits, NOX activity and its subunit gp91, ROS, cell death, glucose, and lactate, with simultaneous increase in ATP levels and improved overall glucose metabolism, with consideration for both hyperglycolysis and gluconeogenesis. The improvement of glucose metabolism with C+P+DHC was evidenced by decreased levels of glycolytic enzymes (GLUT-1, GLUT-3, PFK and LDH), through inhibition of NOX level, and gluconeogenic enzymes (PCK1 and PCK2). These findings support our hypothesis that PH, especially by combination of C+P and DHC, reversed dysfunctional hyperglycolysis and gluconeogenesis induced by stroke, resulting in reduced brain damage.
Systemic hypothermia is a traditional therapeutic hypothermia method to prevent brain cell death from acute ischemic stroke. Early attempts of harnessing hypothermia to prevent brain tissue death from acute ischemic stroke frequently utilized physical means of decreasing the systemic temperature. Clinical limitations of physical hypothermia induction included delays in cooling initiation and the onset of target temperature. The late start necessitated a prolonged hypothermia duration, which in turn required intensive medical support and caused secondary complications such as pneumonia . The only means of physically inducing hypothermia that found significant success was selective hypothermia (or regional cooling), achieved through injecting cool saline in midst of a mechanical thrombectomy procedure [35, 36]. While highly effective, this procedure has a stringent inclusion criteria, limiting the patient population that it benefits. Pharmacologic cooling is attractive because of its ease of use. Although it induces systemic hypothermia, pharmacological hypothermia (PH) is a strong stroke therapy candidate that may be able to provide the benefits of cooling without the intense labor, unintended consequences, and limited patient population of physical hypothermia. Furthermore, it also counteracts the physiological resistance to cooling by inhibiting shivering [37, 13]. While single drugs require toxic doses to achieve therapeutic levels of hypothermia, combinations of lower doses avoid toxicity and work synergistically to achieve efficient cooling . There are eight classes of pharmacological agents that can induce hypothermia, which are the cannabinoid system, Transient Receptor Potential Vanilloid channel 1 (TRPV1) receptor, opioid receptor, neurotensin, thyroxine derivatives, dopamine receptor activators, gaseous hypothermia, and adenosine/adenine nucleotides.
DHC, an analog and congener of capsaicin in chili peppers (Capsicum), is a TRPV1 agonist [38, 39]. TRPV1 is a nonspecific cation channel and confers neuroprotection through its ability to induce hypothermia . Activation of TRPV1 resets thermoregulation into the hypothermic range, which permits effective physiological hypothermia while eliminating concerns for natural rewarming responses such as shivering . The thermoregulation reset is achieved at several brain regions, including the preoptic area of the hypothalamus . However, given the toxicity and complications associated with high doses of DHC that are required to achieve effective hypothermia, its use as monotherapy is limited [42, 43]. Previously, we have demonstrated that a low dose (0.5 mg/kg) DHC combined with physical hypothermia (ice pad) could provide enhanced hypothermia and neuroprotection . Additionally, DHC was seen to act synergistically at low, non-toxic doses with phenothiazine class drugs to induce effective PH .
Chlorpromazine and promethazine (C+P), two members of the phenothiazine class of neuroleptic drugs, have been widely used for their antipsychotic and sedative effects . Previously, we have reported that C+P could confer neuroprotection in stroke via the induction of an “artificial hibernation”-like state, achieved by decreasing brain activity and glucose metabolism . The depressive effect on glucose utilization is similar to those of local anesthetics . Combining physical hypothermia with C+P significantly enhanced the neuroprotective effects of mild hypothermia . Most importantly, C+P, as members of the phenothiazine class, act synergistically with DHC, inducing hypothermia without concern for toxic doses.
The mechanism of ischemic damage has been well described. In sufficiently perfused brain tissue, ATP is primarily generated by oxidative phosphorylation in mitochondria . In hypoxic conditions, such as AIS, mitochondrial oxidative phosphorylation is no longer possible. Instead, the ischemic brain tissue depends on glycolysis, a considerably inefficient method compared to the perfused state . Thus, the brain attempts to compensate with excessive glucose uptake through GLUT-1 and GLUT-3 transporters, leading to neuronal injury . This state of increased glucose uptake and metabolism is described as hyperglycemia and hyperglycolysis, which is frequently seen in ischemia/reperfusion injuries and associated with poor outcomes [49, 26, 25]. Pharmacological and physical hypothermia have been seen to target this pathway effectively. Pharmacological hypothermia with DHC and C+P suppressed ROS and lactic acid accumulation and prevented ATP depletion in achieving neuroprotection .
In the present study, hyperglycolysis inhibition attenuated brain damage, as observed by the relationship between improved amelioration and decreased glucose, lactate, and PFK. Recent studies indicate that hyperglycolysis-exacerbated injury, especially during reperfusion, is due to activation of NOX, independent of lactic acidosis . NOX is a multi-component (p47phox, p67phox, p40phox and Rac2) membrane-bound enzyme complex located in both the cytosol and plasma membrane . When NOX is phosphorylated at its p47phox subunit, it forms a complex and translocates to the plasma membrane to dock with specific plasma membrane subunits such as gp91phox . The catalytic core of the enzyme is composed of gp91phox . NOX is dependent on glucose metabolism, specifically the hexose monophosphate shunt, which supplies the NADPH necessary for enzymatic activity . NADPH is a necessary cofactor for NOX as it transfers its electron to O2 to create superoxide (O2−) . Hence, the presence of glucose during reperfusion increases neuronal NOX activity [56, 57] by functioning as the requisite electron donor for neuronal superoxide production through the generation of NADPH . Moreover, activity of the catalytic subunit, gp91phox, is dependent on the presence of NADPH produced by glycolysis . After ischemia/reperfusion injury, there may be a time window in which hyperglycolysis-induced NOX activation enhances ROS generation. The present study found that stroke exacerbates NOX activation, ROS generation, and hyperglycolysis, which were reduced by C+P and DHC, suggesting that PH induced neuroprotection via improved glucose metabolism with NOX inhibition.
Inhibiting hyperglycolysis has proven to be therapeutic in contexts other than AIS. In peritoneal dialysis, peritoneal fibrosis is prevented through hyperglycolysis inhibition . In thromboembolic cerebral ischemia, neuroprotection was induced with ethanol and therapeutic hypothermia through hyperglycolysis attenuation . In AIS, various ameliorative therapies were observed to confer benefit while inhibiting hyperglycolysis. Ethanol and modafinil, each of which are known to be neuroprotective, in combination inhibited hyperglycolysis in attenuating AIS damage . Normobaric O2  and ischemic pre-conditioning  modulated signaling pathways to subdue hyperglycolysis in postischemic states. Most relevant to this study, C+P also attenuated hyperglycolysis .
More recently, it has been observed that the brain also undergoes gluconeogenesis. One of the crucial means of maintaining glucose levels in humans is gluconeogenesis. The common perception is that gluconeogenic activities are only present in the liver, kidneys, intestines, and muscle tissue. However, many studies have now proven that the brain also undergoes gluconeogenesis in significant levels. While the organs that are classically associated with gluconeogenesis usually overshadow it, the brain is definitely capable of and undergoes gluconeogenesis . This may be the missing link to our understanding of the mechanism of damage induced by AIS. In an attempt to compensate for ATP depletion, the ischemic brain tissue initiates gluconeogenesis. However, due to ATP depletion, gluconeogenesis is incomplete and results in undesirable byproduct of lactic acid instead. The consequence of dysfunctional gluconeogenesis of the ischemic brain tissue is an excessively active phosphoenolpyruvate carboxy kinase (PCK) enzyme, which contributes to the neurotoxic pathways of lactic acid and ROS accumulation . In ischemia, when mitochondrial oxidative phosphorylation and ATP production are disrupted, anaerobic glycolysis becomes the primary source of ATP. Anaerobic glycolysis alone cannot produce sufficient ATP to maintain brain function and produces lactate, leading to acidosis and ROS. In an attempt to compensate for insufficient energy, we see a full circle where gluconeogenesis is increasingly attempted after ischemia to provide additional substrate for energy production, which may not function correctly due to lack of ATP , leading to excess lactic acidosis. We must note that gluconeogenesis is anabolic, which increases the amount of glucose, while oxidative phosphorylation is catabolic and decreases the amount of glucose. In our previous study, cerebral gluconeogenesis was actively found after stroke . In the present study, we further found C+P and DHC conferred neuroprotection partially through inhibition of cerebral gluconeogenesis evidenced by PCK enzymes.
In conclusion, the present study found that PH was effective in conferring therapy to rats undergoing AIS through a pathway involving prevention of dysfunctional gluconeogenesis and hyperglycolysis. These findings justify the exploration of new therapies that target gluconeogenesis, not only in stroke patients, but also in other forms of brain injuries such as trauma and epilepsy.