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

Abstract

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.

Keywords: heatstroke; hypobaric hypoxia; hypothalamus; ischemic/hypoxic injury.

Figure 1

Changes in Ta (A), Tco (B), MABP (C), HR (D), hypothalamic temperature (E), CBF (F) and PO₂ (G) in non-HHP + non-heat-exposed rats (⚫), HHP + non-heat-exposed rats (■), non-HHP + heat-exposed rats (▲) and HHP + heat-exposed rats (▼) over time. The values are the means±SDs; n=10 animals per group. *P<0.05 vs non-HHP + non-heat-exposed rats; +P<0.05 vs non-HHP + heat-exposed rats. Twenty minutes after heat exposure concluded, the heat-exposed rats not treated with HHP (non-HHP) displayed hyperthermia (~42 °C vs. ~37 °C), hypotension (~20 mmHg vs. ~90 mmHg), bradycardia (~300 beats/min vs. ~450 beats/min), hypothalamic ischemia (~50% vs. ~100%), and hypothalamic hypoxia (~15 mmHg vs. 22 mmHg).

Figure 2

Changes in hypothalamic (A) glutamate levels, (B) glycerol levels, and (C) lactate/pyruvate ratio in non-HHP+non-heat-exposed rats (⚫), HHP + non-heat-exposed rats (■), non-HHP + heat-exposed rats (▲) and HHP + heat-exposed rats (▼) over time. The values are the means± SDs; n=10 rats per group. *P<0.05 vs. non-HHP+non-heat-exposed group; +P<0.05 vs. non-HHP+heat-exposed group.

Figure 3

(A) Photomicrographs of hypothalamic hematoxylin-eosin staining of 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). Images of brain morphology are shown at 50x, 100x, 200x, 400x and 1000x magnification. Scale bars= 500 μm, 200 μm, 100 μm, 50 μm, and 20 μm. Rats in the heat-exposed groups were killed 90 min after heat stress, and rats in the non-heat-exposed groups were skilled at the equivalent time. (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.

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.

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.