Chronic moderate hypoxia results in systemic and central nervous system adaptations that allow acclimatization. Long-term responses to hypoxia involve systemic physiological changes, metabolic regulation, and vascular remodeling. To investigate whether aging affects systemic and cerebral angiogenic adaptational changes in response to prolonged hypoxia, the present study assessed the responses of 4 month old (“young”) C57BL/6 mice and 24 month old (“aged”) C57BL/6 mice to chronic hypobaric hypoxia of 0.4 ATM (290 torr). Compared to young mice, delayed body weight-loss recovery and a lag in polycythemic response were observed in aged mice. As previously shown, Hypoxia Inducible Factor-1α (HIF-1α) accumulation was attenuated and vascular endothelial growth factor (VEGF) expression was decreased in the cerebral cortex of aged mice. Conversely, Cyclooxygenase-2 (COX-2), angiopoietin-2 (Ang-2) and Peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α) protein upregulation were not affected in the aged mice. Despite an initial delay in cerebral angiogenic response in aged mice in the first week of hypoxia, no significant differences were observed in microvascular density between young and aged mice in normoxia and at 2 and 3 weeks of hypoxia. Taken together, these observations indicate that even though the HIF-1 response to hypoxia is greatly attenuated, HIF-1 independent compensatory pathways are eventually able to maintain baseline and cerebral angiogenic adaptational changes to chronic hypoxia in aged mice. The delayed adaptive response, however, may result in decreased survival in the aged cohort.

Normal brain function is dependent on continuous and controlled oxygen delivery. Chronic moderate hypoxia leads to angiogenesis, suggesting a modulatory role for oxygen in determining capillary density. The objective of this study was to determine physiologic and brain angiogenic adaptational changes during chronic moderate normobaric hyperoxia in mice. Four-month old C56BL/6J mice were kept in a normobaric chamber at 50% O2 for up to 3 weeks. Normoxic littermates were kept in the same room outside the chamber. Freshly collected or fixed brain specimens were analyzed by RT-PCR, Western blot analysis and immunohistochemistry. Results show accumulation of hypoxia inducible factors 1 and 2α (HIF-1 and 2α), and increased expression of erythropoietin (EPO), cyclooxygenase-2 (COX-2) and angiopoietin-2 (Ang-2). Conversely, vascular endothelial growth factor (VEGF), and VEGF receptor-2 (KDR/Flk-1), Peroxisome proliferator-activated receptor gamma coactivator 1-α(PGC-1α) and prolylhydroxylase-2 (PHD-2) expressions were decreased. VEGF mRNA level was diminished but there was no change in HIF-1α mRNA and von Hippel Lindau E3 ubiquitin ligase (VHL) protein expression. Microvascular density was significantly diminished by the end of the 3rd week of hyperoxia. Overall, our results are: 1) increased expression of the potent neuroprotective molecule, EPO; 2) diminished expression of the potent angiogenic factor, VEGF; and 3) decreased microvascular density. We can, therefore, conclude that brain microvascular density can be controlled by HIF-independent mechanisms, and that brain capillary density is a continuously adjusted variable with tissue oxygen availability as one of the controlling modulators.

Dynamic microvascular remodeling maintains an optimal continuous supply of oxygen and nutrients to the brain to account for prolonged environmental variations. The objective of this study was to determine the relative time course of capillary regression during re-oxygenation after exposure to prolonged moderate hypoxia and expression of the primary signaling factors involved in the process. Four-month old male C57BL/6 mice were housed and maintained in a hypobaric chamber at 290 torr (0.4 ATM) for 21 days and allowed to recover at normoxia (room air) for up to 21 days. The mice were either decapitated or perfused in-situ and brain samples collected were either homogenized for Western blot analysis or fixed and embedded in paraffin for immunohistochemistry. Hypoxia inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and erythropoietin (EPO) expression were increased during hypoxic exposure and diminished during subsequent re-oxygenation. However, cyclooxygenase-2 (COX-2) and angiopoietin-2 (Ang-2) were both elevated during hypoxia as well as subsequent re-oxygenation. Significantly increased capillary density at the end of the 3rd week of hypoxia regressed back toward normoxic baseline as the duration of re-oxygenation continued. In conclusion, elevated COX-2 and Ang-2 expression during hypoxia where angiogenesis occurs and re-oxygenation, when microvessels regress, identifies these proteins as vascular remodeling molecules crucial for angioplasticity.

In order to maintain normal cellular function, mammalian tissue oxygen concentrations must be tightly regulated within a narrow physiological range. The hormone erythropoietin (EPO) is essential for maintenance of tissue oxygen supply by stimulating red blood cell production and promoting their survival. In this study we compared the effects of 290 Torr atmospheric pressure on the kidney EPO protein levels in young (4-month-old) and aged (24-month-old) C57BL/6 mice. The mice were sacrificed after being anesthetized, and kidney samples were collected and processed by Western blot analysis. Relatively low basal expression of EPO during normoxia in young mice showed significant upregulation in hypoxia and stayed upregulated throughout the hypoxic period (threefold compared to normoxic control), showing a slight decline toward the third week. Whereas, a relatively higher normoxic basal EPO protein level in aged mice did not show significant increase until seventh day of hypoxia, but showed significant upregulation in prolonged hypoxia. Hence, we confirmed that there is a progressively increased accumulation of EPO during chronic hypoxia in young and aged mouse kidney, and the EPO upregulation during hypoxia showed a similarity with the pattern of increase in hematocrit, which we have reported previously.

We tested the functional effects of hypoglossal (CNXII) stimulation in the anesthetized rabbit before and after injections of saline into the tongue base to obstruct the airway. Data (n = 6) show little or no effect of CN XII trunk stimulation; however, medial branch stimulation (20-100 Hz; 50-500 μs pulse width, and incremental increases from 10 μA) reduced upper airway resistance. Medial branch stimulation was less effective in reducing resistance than anterior advancement of the hyoid. Endoscopic viewing (n-3) of the retropalate showed this region as the narrowest and dynamically changed by anterior hyoid displacement, with less evident effects than CNXII stimulation. We conclude that under these conditions CNXII medial branch stimulation reduces airway resistance, especially after induced obstruction.