Hypobaric hypoxia preconditioning protects against hypothalamic neuron apoptosis in heat-exposed rats by reversing hypothalamic overexpression of matrix metalloproteinase-9 and ischemia

Background: Hypoxia-inducible factor-1α (HIF-1α), heat shock protein-72 (HSP-72), hemeoxygenase-1 (HO-1), and matrix metalloproteinase-9 (MMP-9) have been identified as potential therapeutic targets in the brain for cerebral ischemia. To elucidate their underlying mechanisms, we first aimed to ascertain whether these proteins participate in the pathogenesis of heat-induced ischemic damage to the hypothalamus of rats. Second, we investigated whether hypobaric hypoxia preconditioning (HHP) attenuates heat-induced hypothalamic ischemic/hypoxic injury by modulating these proteins in situ. Methods: Anesthetized rats treated with or without HHP were subjected to heat stress. Hypothalamic ischemic/hypoxic damage was evaluated by measuring hypothalamic levels of cerebral blood flow (CBF), partial oxygen pressure (PO2), and hypothalamic temperature via an implanted probe. Hypothalamic apoptotic neurons were counted by measuring the number of NeuN/caspase-3/DAPI triple-stained cells. Hypothalamic protein expression of HIF-1α, HSP-72, HO-1, and MMP-9 was determined biochemically. Results: Before the start of the thermal experiments, rats were subjected to 5 hours of HHP (0.66 ATA or 18.3% O2) daily for 5 consecutive days per week for 2 weeks, which led to significant loss of body weight, reduced brown adipose tissue (BAT) wet weight and decreased body temperature. The animals were then subjected to thermal studies. Twenty minutes after heat stress, heat-exposed rats not treated with HHP displayed significantly higher core and hypothalamic temperatures, hypothalamic MMP-9 levels, and numbers of hypothalamic apoptotic neurons but significantly lower mean blood pressure, hypothalamic blood flow, and PO2 values than control rats not exposed to heat. In heat-exposed rats, HHP significantly increased the hypothalamic levels of HIF-1α, HSP-72, and HO-1 but significantly alleviated body and hypothalamic hyperthermia, hypotension, hypothalamic ischemia, hypoxia, neuronal apoptosis and degeneration. Conclusions: HHP may protect against hypothalamic ischemic/hypoxic injury and overexpression of MMP-9 by upregulating the hypothalamic expression of HIF-1α, HSP-72, and HO-1 in rats subjected to heatstroke.

In rodents, heat-induced thermoregulatory deficits and mortality may result from ischemic and oxidative damage to the hypothalamus (the essential thermoregulatory center in the mammalian brain). Compared to normothermic controls, mice subjected to heatstroke exhibit significantly higher levels of cellular ischemia markers (e.g., glutamate and the lactate-to-pyruvate ratio) and damage markers (e.g., glycerol) in the hypothalamus [5,6]. In cerebral ischemia, enhanced expression of matrix metalloproteinase-9 (MMP-9) is associated with various complications, including neuronal damage, apoptosis, and blood-brain barrier (BBB) disruption [7]. Hemeoxygenase-1 (HO-1) protects cells and tissues by exerting anti-inflammatory, antiapoptotic, antiproliferative, and antioxidative effects in response to noxious stimuli [8]. Hypoxia-inducible factor-1α (HIF-1α) acts as a potential therapeutic target for cerebral ischemia [9]. Heat shock protein-72 (HSP-72) overexpression appears to be critical for the development of thermotolerance and protection against heat-induced hypothalamic ischemic and oxidative damage in rodents. However, the interrelationships among these proteins in the hypothalamus during the pathogenesis of heatstroke remain unclear.
Changes associated with acclimation to hypoxia, including carotid body-mediated minute ventilation, which can improve oxygen saturation [10], diuresis, which can effectively increase hemoglobin and hematocrit concentrations, and stimulation of erythropoiesis, which can increase the number and oxygen-carrying capacity of red blood cells; occur within 1-3 weeks of high-altitude living (1500-3000 meters) or intermittent exposure to hypoxia [11]. These systemic responses have been experimentally linked to several downstream effects of HIF-1α, HSP-72 and other related proteins in hypothalamic tissues.
Hypobaric hypoxia preconditioning (HHP) is a transient phenomenon caused by brief episodes of sublethal hypoxia that induce various endogenous atrophic signals and robustly protect against subsequent lethal insults [12,13]. Although HHP has been shown to exert neuroprotective effects against ischemic brain injury [13,14], it is still unknown whether HHP protects against heat-induced hypo-thalamic injury [15] by modulating hypothalamic proteins including HIF-α, MMP-9, HSP-72, and HO-1.
To address this question, the present study aimed to assess the effects of HHP on heat-induced ischemic and hypoxic damage to the rat hypothalamus. In addition, the temporal profiles of HIF-1α, HSP-72, HO-1, and MMP-9 in the hypothalamus were assessed in rats subjected to heatstroke and treated with or without HHP.

Animal care
All animal experiments were conducted on adult male Sprague-Dawley rats weighing 248-306 g. All experimental and animal care protocols were approved by the Institutional Animal Care and Use Committee of Chi Mei Medical Center, Tainan, Taiwan (approval no.: 106121110) in accordance with the guidelines of Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health [16], and care was taken to minimize pain and suffering. The rats were housed in a temperature-controlled room (24±1.0 °C) with a relative humidity of 50±10% on a 12/12 hour-light/ dark cycle, and food pellets and water were provided ad libitum.

Physiological variable monitoring
Each animal was anesthetized with sodium pentobarbital (40 mg/kg body weight, i.p.), and the femoral artery was cannulated with polyethylene tubing to measure mean arterial blood pressure (MABP) and heart rate (HR). Body core temperature (Tco) was monitored continuously by a thermocouple inserted into the rectum, and MABP and HR were continuously monitored with a pressure transducer. The head of the animal was mounted on a stereotaxic apparatus (David Kopf Instruments, Tujunga, CA, USA) with the nose bar positioned 3.3 mm below the horizontal line. Following a midline incision, the skull was exposed, and a burr hole was made in the skull to allow insertion of a 100-μm-diameter thermocouple and two 230-μm fibers attached to an oxygen probe. This combined probe was used to measure oxygen, temperature, and microvascular blood flow. OxyLite and OxyFlo instruments were used for the measurements. The OxyLite 2000 instrument (Oxford Optronix, Oxford, UK) is a two-channel device (measures partial oxygen pressure [PO2] and temperature at 2 locations simultaneously), whereas the OxyFlo 2000 instrument is a two-channel laser Doppler perfusion monitoring instrument. Together, these 2 instruments simultaneously provided tissue blood flow, oxygenation, and temperature data [17].

HHP simulation
Animals in the HHP groups were exposed to 5 hours of simulated HHP of 4000 m in a hypobaric chamber (0.66 ATA or 18.3% O 2 ) daily for 5 consecutive days per week for 2 weeks before the start of heat exposure [18]. All animals in the non-HPP groups were exposed to 1.0 ATA (21% O 2 ) for the equivalent period of time.

Induction of heatstroke
The Tco of the normothermic control group was maintained at about 37 °C with a folded heating pad at a room temperature of 26 °C throughout the entire experiment. For the heatstroke group, the heating pad was removed before the start of heat stress (ambient temperature [Ta] of 42 °C with a relative humidity of 60%). In the heatstroke group, an MABP value of 25 mmHg below the peak accompanied by excessive body hyperthermia (i.e., ~41.5 °C Tco and ~40.0 °C hypothalamic temperature) was observed approximately 70 min after the initiation of heat stress; this time point was arbitrarily used as the onset of heatstroke [19]. Heat was withdrawn from the heat-exposed animals 70 min after heat stress, and the animals were allowed to recover at room temperature (26 °C).

Experimental groups
The animals were randomly assigned randomly to one of the following four groups: the non-HHP + non-heat-exposed group, HHP + non-heat-exposed group, non-HP + heat-exposed group, and HHP + heat-exposed group. In the non-heat-exposed groups, the Tco was maintained at approximately 37 °C with a heating pad. The animals in the heat-exposed groups were exposed to heat stress (40 °C) for exactly 70 min.

Western blot analysis
The rats were anesthetized and underwent intracardiac perfusion with 0.1 M PBS (pH 7.4) [20,21]. The hypothalamic region of the brain was rapidly isolated, and the brain tissues were homogenized using a homogenizer. Total protein was extracted using protein extraction reagent (Bio-Rad Laboratories, CA, USA), and the protein concentration was quantified by using the Bradford method (Bio-Rad protein assay kit, Bio-Rad Laboratories). The protein samples (50 µg) were separated by 20% sodium dodecyl sulfatepolyacrylamide gel electrophoresis and were subsequently transferred onto polyvinylidene fluoride membranes. The blots were blocked and incubated with a primary antibody specific for HSP-72, HIF-1α or HO-1. The membranes were then incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G and anti-mouse IgG. Following secondary antibody incubation, the immunoblots were visualized with an enhanced chemiluminescence detection system. The expression of HSP-72, HIF-1α, HO-1 and β-actin was semiquantified using a gel densitometry scanning program.

Gelatin zymography
The levels of MMP-9 in the brain homogenates were measured by gelatin zymogram following a previously described technique [22]. The brains were quickly removed from the rats under general anesthesia, and the tissues were frozen immediately in liquid nitrogen and stored at -80 °C. Total protein concentrations were determined using the bicinchoninic acid protein assay. Protein samples (50 μg) were prepared, loaded on 10% tris-glycine gels with 0.1% gelatin as a substrate and separated. After electrophoresis and incubation, the gels were stained with 3% methanol, 10% acetic acid, and 0.5% wt/vol Coomassie brilliant blue for 2 hours and then destained. Gelatinolytic activity was manifested as horizontal white bands on a blue background. MMP bands were identified after standard techniques using molecular weight criteria and a human MMP-9 control, which was purchased from Chemicon [23].

Hematoxylin and Eosin (H&E) staining
We evaluated brain tissue damage following a previously described method [24]. At the end of the experiments, the rats were anesthetized as described above and intracardially perfused with isotonic chloride solution followed by 4% (w/v) paraformaldehyde (Bio-Rad Laboratories) in 0.1 M sodium phosphate buffer (pH 7.4; Bio-Rad Laboratories). The brains were subsequently removed, fixed for 48 hours in 4% (w/v) paraformaldehyde, and embedded in paraffin (Bio-Rad Laboratories), and brain tissues were sliced into 4-μm coronal sections and stained with hematoxylin-eosin (H&E) (Sigma-Aldrich, MA, USA).
Semiquantitative histological analysis was performed after H&E staining to evaluate morphological changes in neural cells and the severity of damage to the brain following heat stress. Brain sections (corresponding to coronal coordinates of Paxinos and Watson [25] at bregma -2.2~-3.6 mm, lateral 0~1 mm, and depth 8.0~10 mm) were semiquantitatively scored [26] from 0 to 4 (0= no detectable lesion, 1 = lesion involving <25% of the field, 2 = lesion involving 25%-50% of the field, 3 = lesion involving 50%-75% of the field, and 4 = lesion involving >75% of the field). Each hypothalamus region was evaluated for morphological changes as follows [27]: 0 = normal morphology, 1 =minor damage (edema and few pyknotic cells), 2 = moderate damage (structural disorganization, edema, many pyknotic cells, and vacuolization), and 3 = intense damage (structural disorganization, edema, many pyknotic cells, and vacuolization). Brain damage was scored and evaluated, and the scores were multiplied to yield final scores, which ranged from 0 to 12, for each animal. Representative images were collected.

Determination of hypothalamic glutamate levels, glycerol levels and lactate-to-pyruvate ratio
To measure hypothalamic glutamate concentrations, glycerol concentrations, and lactateto-pyruvate ratio, aliquots of samples were injected into a CMA600 microdialysis analyzer (Carnegie Medicine, Stockholm, Sweden) [28].

Data presentation and statistical analysis
The data are presented as the means±SDs ANOVA was used to analyze factorial experiments, and Duncan's multiple range test was used for post hoc comparisons among means. GraphPad Prism (version 7.01 for Windows; GraphPad Software, San Diego, CA, USA) was used for these analyses. A p-value of less than 0.05 was considered statistically significant.

HHP reduces body weight, brown adipose tissue (BAT) weight and temperature
As shown in Table 1, after 2 weeks, the average body weight of HHP-treated rats was significantly lower than that of untreated rats, which remained unchanged. Both the BAT temperature and weight values of the HHP group were significantly lower than those of the non-HHP group (Table 1).
(E) The hypothalamic damage scores of the different groups are presented as the means±SDs; n=10 per group. *P<0.05 for the non-HHP + heat-exposed group versus the non-HHP + non-heat-exposed group; + P<0.05 for the HHP + heat-exposed group versus the non-HHP + heat-exposed group. 3V= third ventricle; ME= median eminence.

Figure 4.
Photomicrographs of immunofluorescence staining of hypothalamic apoptotic neurons (NeuN+caspase-3+DAPI-positive cells) in a non-HHP + non-heat-exposed rat (A), an HHP + non-heat-exposed rat (B), a non-HHP+heat-exposed rat (C), and an HHP+heat-exposed rat (D). The animals were killed 90 min after heat stress or at the equivalent time for the non-heat-exposed groups. Scale bar=50 µm. (E) The values are presented as the means±SDs; n=10 per group. *P<0.05 for the non-HHP + heat-exposed group versus the non-HHP + non-heat-exposed group; + P<0.05 for the HHP + heat-exposed group versus the non-HHP + heat-exposed group.

Figure 5.
Photographs of immunofluorescence staining of hypothalamic apoptotic neurons (NeuN+TUNEL+DAPI-positive cells) in a non-HHP+non-heat-exposed rat (A), an HHP+non-heat-exposed rat (B), a non-HHP+ heat-exposed rat (C), and an HHP+ heat-exposed rat (D). The animals were killed 90 min after heat stress or at the equivalent time for the non-heat-exposed groups. Scale bar= 50 µm). (E) The values are presented as the mean±SDs; n=10 per group. *P<0.05 for the non-HHP+heat-exposed group versus the non-HHP+non-heat-exposed group; +P<0.05 for the HHP+heat-exposed group versus the non-HHP+heat-exposed group.

HHP decreases heat-induced MMP-9 overexpression and activity in heat-exposed rats
Gelatin zymography analysis revealed that the non-HHP+heat-exposed group exhibited significantly higher levels of MMP-9 activity in the hypothalamus than the non-HHP+non-heat-exposed group and the HHP+non-heat-exposed group (Fig. 6C and Fig. 6D). However, the HHP+heat-exposed rats exhibited significantly lower hypothalamic MMP-9 activity than the non-HHP+heat-exposed rats (Fig. 6C and Fig. 6D).

Discussion
In the present study, which used an experimental rat model of heatstroke, heatstroke-exposed animals displayed hyperthermia (i.e., Tco of 42 °C), arterial hypotension (i.e., MABP of 20 mmHg), bradycardia (i.e., HR of ~200 beats/min), hypothalamic ischemia (i.e., CBF of 40% of the original level), hypoxia (i.e., PO2 of ~5 mmHg), neuronal degeneration (i.e., increased neuronal damage scores) and apoptosis (i.e., increased numbers of Neu-N/caspase-3/DAPI-positive cells and NeuN+TUNEL+DAPI-positive cells). In addition, heat-induced hypothalamic ischemic/hypoxic injury were associated with increased hypothalamic levels of MMP-9 activity. The most striking finding of the present study was that HHP protected against heat-induced hypotension (e.g., increased MABP to ~60 mmHg), bradycardia (e.g., increased HR to ~350 beats/min), hypothalamic ischemia (e.g., increased CBF to ~100%), hypoxia (increased the PO 2 to 17 mmHg), hypothalamic neuronal degeneration (e.g., decreased neuronal damage scores) and apoptosis (e.g., reduced the number of apoptotic neurons) and increased hypothalamic levels of MMP-9 activity. Simultaneously, HHP increased the hypothalamic expression of HSP-72, HIF-1α, and HO-1 in heat-exposed animals. Although HHP did not affect heat-induced hyperthermia, it attenuated heatinduced hypothalamic overexpression of MMP-9 as well as hypothalamic ischemia/hypoxia and its complications (e.g., hypothalamic neuronal degeneration and apoptosis). HIF-1α maintains oxygen homeostasis and facilitates cellular adaptation to low oxygen conditions by regulating many downstream genes that participate in angiogenesis, erythropoiesis, energy metabolism, apoptosis and neuronal stem/progenitor cell proliferation [9]. Heat acclimation (HA) confers protection against acute heat stress and delays thermal injury [29,30]. Increased levels of HIF-1α as well as HSP72 and its metabolic targeted genes are among the cytoprotective changes that occur during HA [31]. HIF-1α, the master regulator of oxygen homeostasis, participates in the Figure 6. (A) Western blot analysis of HSP72, HIF-1α and HO-1 in hypothalamic tissues from a non-HHP + non-heat-exposed rat, an HHP + non-heat-exposed rat, a non-HHP + heat-exposed rat, and an HHP + heat-exposed rat. (B) The fold-change values represent the mean of 10 samples (n=10) divided by the mean of the 10 controls (n=10). (C) Gelatin zymography analysis of MMP-9 in hypothalamic tissues from a non-HHP + non-heat-exposed rat, an HHP + non-heat-exposed rat, a non-HHP + heat-exposed rat, and an HHP + heat-exposed rat. (D) The percent expression values represent the mean of 10 samples (n=10). The results are presented as the means±SDs *P<0.01 compared with the non-HHP and non-heat-exposed groups; + P<0.01 compared with the non-HHP + heat-exposed group.
process of HA [32,33]. Our present data show that HHP exerts the same cytoprotective effects as HA. HHP may protect against heat-induced hypothalamic injury by upregulating HIF-1α. In contrast, our present results are not consistent with several previous findings. Neuron-specific HIF-1α-deficient mice display reductions in both the infarct volume and expression of proapoptotic genes following bilateral common carotid artery occlusion [34]. In addition, early inhibition of HIF-1α with small interfering RNA reduces ischemia-reperfusioninduced brain injury in rats [35]. Inhibition of HIF-1α by HIF-1α inhibitors such as 2-methoxyestradiol [35], mir-335 [9] and hyperbaric oxygen [36] in the acute phase of ischemic stroke exerts beneficial effects via inhibition of apoptotic genes. Thus, downregulating HIF-α expression in the early period of cerebral ischemia could be efficacious in reducing cerebral ischemic damage. The reason for the discrepancy between our results and the results of these previous studies is currently unclear.
Transgenic mice have been used to examine the role of HSP72 in experimental heatstroke [6]. Compared to transgene-negative littermate controls ([-]HSP72 mice), transgenic mice heterozygous for the porcine HSP-72 β gene ([+]HSP 72 mice) exhibit lower levels of cellular ischemia, inflammatory and oxidative damage markers in the hypothalamus and lower neuronal damage scores 4 hours after heat stress.
[-]HSP72 mice were subjected to heat stress, and 4 hours after exposure, none of the 12 mice were still living, and the Tco of the mice was 34.2±0.4 °C. When [+]HSP-72 mice were exposed to the same heat treatment, the fraction survival and Tco values were significantly increased to 12/12 and 37.4±0.3 °C, respectively. This study indicates that HSP72 overexpression appears to be critical for the development of thermotolerance and protection from heat-induced hypothalamic ischemic and oxidative damage. In the present study, we further confirmed that HHP-induced HSP72 overexpression in the hypothalamus protects against ischemic/hypoxic injury in the hypothalamus in heat-exposed rats. In studies of cerebral ischemia, neurodegenerative disease, epilepsy, and trauma, HSP72 has been shown to be neuroprotective [37]. Proteins that accumulate in Alzheimer's disease models are able to be degraded in HSP72-overexpressing transgenic mice, leading to neurological improvements [38].
Hemeoxygenase-1 (HO-1) is an inducible isoform of the first and rate-limiting enzyme of the degradation of heme into iron, carbon monoxide, and biliverdin. It possesses anti-inflammatory, antiapoptotic, angiogenic, and cytoprotective functions, suggesting that it plays potential protective and defensive role in several pathophysiological states, including ischemic stroke [39]. HO-1 knockout mice exhibit greater ischemic damage than wild-type mice [40]. Induction of HO-1 expression with acetyl-11keto-β-boswellic acid [41] or saponins [42] effectively reduces stroke-or reperfusion-induced injury. These observations provide compelling information that HO-1 activation contributes to neuroprotection under many conditions. HO-1 exerts its anti-inflammatory effects by upregulating the expression of interleukin-10 (an anti-inflammatory cytokine) and interleukin-1 receptor antagonist [43]. In the present study, HHP may have attenuated hypothalamic inflammation and ischemic/hypoxic injury by enhancing HO-1 expression in the hypothalamus.
MMP-9 plays several important roles associated with the actions of neutrophils, such as degradation of extracellular matrix, activation of IL-1β, and cleavage of several chemokines [44]. In a mouse model, MMP-9 deficiency results in resistance to endotoxin shock, suggesting that MMP-9 is important in sepsis [45]. Knockout of MMP 9 results in delayed apoptosis, vascularization, and ossification of hypertrophic chondrocytes [46]. Enhanced MMP-9 expression and activity have been observed in many neuropsychiatric disorders and central nervous system disorders, including ischemic stroke [28,47,48]. In cerebral ischemia, enhanced expression of MMP 9, which is associated with various complications, including excitotoxicity, neuronal damage, apoptosis, and BBB opening, which leads to cerebral edema and hemorrhagic transformation, is observed [7]. It is evident that brain ischemia, inflammation, and oxidative damage rather than hyperthermia of the body are the main causes of heatstroke [1,4]. In addition, heatstroke resembles septic shock in many ways [1,4]. In the present study, HHP may have inhibited the overexpression of MMP-9 in the hypothalami of rats subjected to heatstroke, resulting in a reduction in hypothalamic ischemia, hypoxia, neuronal degeneration and apoptosis.
Hypoxia-inducible factors are key molecules that regulate the cellular response to hypoxia and inflammation and play a major role in normal cell function and survival [49]. Current evidence also indicates that the HIF-1α pathway in the hypothalamus may be involved in the regulation of food intake and energy expenditure [49]. Previous studies have shown that 30 cycles of intermittent hypoxia per hour for 8 hours per day for 21 days reduces body weight and BAT wet weight in rats [50]. In our present study, we further confirmed that five hours of HHP (0.66 ATA or 18.3% O2) daily for 5 consecutive days per week for 2 weeks before the start of heat exposure caused both weight loss and reduced BAT wet weight in rats. BAT activation occurs at temperatures between 4 and16 °C [51]. In our present study, all HHP experiments were conducted at 26 °C. It is, therefore, improbable that procedure-induced BAT behavior or uncoupling protein 1 function influenced the protective effects of HHP in heatstroke. Fig. 7A depicts core the molecular networks identified by ingenuity pathway analysis (IPA) [5]. The altered proteins, including HSP72, HIF-1α, HO-1, MMP-9, and other ischemia-related mediators, are Figure 7. Core molecular net words identified by IPA. The altered genes are involved in biological functions associated with HIF-1α signaling (A). The red nodes and lines indicate the heat shock protein (HSP)70/HIF-1α/HMOX1 (or HO-1)/MMP-9 cell death pathway might be involved in the present results. (B) A flowchart of the proposed pathogenic mechanism of heatstroke in rats. Rodents subjected to heatstroke (evidenced by excessive hyperthermia and hypotension) exhibit higher levels of cellular ischemia and damage markers (e.g., MMP-9) in the hypothalamus, which leads to hypothalamic neuronal degeneration and apoptosis (I). Based on the core molecular net words identified by IPA shown in (B), HHP may attenuate heat-induced hypothalamic degeneration and apoptosis via the HSP70 (or HSP72)/HIF-1α/HO-1/MMP-9 pathways (II). (+), upregulation; (-) downregulation. HHP might ameliorate hypothalamic overexpression of MMP-9 in heat-exposed animals by reducing hypotension (or increasing CBF) and/or by enhancing HSP-72/HIF-1α/HO-1 signaling.
involved in significant biological functions associated with HIF-1α. Severe heat stress may cause both hypotension and hyperthermia and result in hypothalamic ischemic/hypoxia and overexpression of MMP-9, leading to hypothalamic degeneration and apoptosis (Fig. 7B). HHP may attenuate heat-induced hypothalamic degeneration and apoptosis by inhibiting the pathways involved in hypotension, hypothalamic ischemic and hypothalamic MMP-9 overexpression or enhancing the HSP-72/HIF-α/ HO-1/MMP-9 pathway.

Limitations
This study used adult male rats and therefore cannot reflect the complicated human clinical situation, which may involve different comorbidities. Male rats were used to avoid the hormonal fluctuations that occur in female rats. Additionally, in this study, thermal experiments were performed in rats under general anesthesia.

Conclusions
Based on our present data, we conclude that severe heat stress causes hypothalamic ischemia/ hypoxia, upregulation of hypothalamic MMP-9, and hypothalamic neuronal degeneration and apoptosis. HHP upregulates the hypothalamic protein expression of HSP-72, HIF-1α, and HO-1 and downregulates hypothalamic MMP-9 activity to attenuate hypothalamic neuronal degeneration and apoptosis in heat-exposed animals.