Hypoglossal nerve stimulation in a pre-clinical anesthetized rabbit model relevant to OSA.

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.

Gender differences in hypoxic acclimatization in cyclooxygenase-2-deficient mice.

The aim of this study was to determine the effect of cyclooxygenase-2 (COX-2) gene deletion on the adaptive responses during prolonged moderate hypobaric hypoxia. Wild-type (WT) and COX-2 knockout (KO) mice of both genders (3 months old) were exposed to hypobaric hypoxia (~0.4 ATM) or normoxia for 21 days and brain capillary densities were determined. Hematocrit was measured at different time intervals; brain hypoxia-inducible factor -1α (HIF-1α), angiopoietin 2 (Ang-2), brain erythropoietin (EPO), and kidney EPO were measured under normoxic and hypoxic conditions. There were no gender differences in hypoxic acclimatization in the WT mice and similar adaptive responses were observed in the female KO mice. However, the male KO mice exhibited progressive vulnerability to prolonged hypoxia. Compared to the WT and female KO mice, the male COX-2 KO mice had significantly lower survival rate and decreased erythropoietic and polycythemic responses, diminished cerebral angiogenesis, decreased brain accumulation of HIF-1α, and attenuated upregulation of VEGF, EPO, and Ang-2 during hypoxia. Our data suggest that there are physiologically important gender differences in hypoxic acclimatization in COX-2-deficient mice. The COX-2 signaling pathway appears to be required for acclimatization in oxygen-limiting environments only in males, whereas female COX-2-deficient mice may be able to access COX-2-independent mechanisms to achieve hypoxic acclimatization.

HIF-1α/COX-2 expression and mouse brain capillary remodeling during prolonged moderate hypoxia and subsequent re-oxygenation.

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 micro-vessels regress, identifies these proteins as vascular remodeling molecules crucial for angioplasticity.

Defining the role of HIF and its downstream mediators in hypoxic-induced cerebral angiogenesis.

Now that some of the basic mechanisms that underlie hypoxia-induced cerebral angiogenesis have been described, it has become clear that the hypoxia-inducible transcription factors, HIF-1 and HIF-2, play an important role in the process by causing the upregulation of vascular endothelial growth factor (VEGF). The heterogeneity of the brain parenchyma means that further progress in understanding capillary pathophysiology requires techniques that allow determination of the roles of individual components of the neurovascular unit. Multi-stain fluorescence co-localization techniques provide one such approach.

Increased HIF-1α and HIF-2α accumulation, but decreased microvascular density, in chronic hyperoxia and hypercapnia in the mouse cerebral cortex.

The partial pressure of oxygen in the brain parenchyma is tightly controlled, and normal brain function is delicately sensitive to continuous and controlled oxygen delivery. The objective of this study was to determine brain angiogenic adaptive changes during chronic normobaric hyperoxia and hypercapnia in mice. Four-month-old C56BL/6 J mice were kept in a normobaric chamber at 50 % O2 and 2.5 % CO2 for up to 3 weeks. Normoxic littermates were kept adjacent to the chamber and maintained on the same schedule. Physiological variables were measured at time points throughout the 3 weeks or when the mice were sacrificed. Freshly collected or fixed brain specimens were analyzed by Western blot analysis and immunohistochemistry (IHC). We found significant accumulation of hypoxia-inducible factors 1α and 2α (HIF-1α and HIF-2α) and increased expression of erythropoietin (EPO), cyclooxygenase-2 (COX-2), and angiopoietin-2 (Ang-2) in hyperoxia and hypercapnia. Conversely, vascular endothelial growth factor (VEGF), and VEGF receptor-2 (KDR/Flk-1), peroxisome proliferator-activated receptor gamma coactivator 1-α (PGC-1α), and prolyl hydroxylase-2 (PHD-2) expressions were decreased in hyperoxia and hypercapnia. Capillary density was significantly diminished by the end of the 3rd week of hyperoxia and hypercapnia as compared to control. We conclude that HIF-independent mechanisms contribute to brain capillary density modulation that is continuously adjusted in accordance with tissue oxygen tension.

Kidney EPO expression during chronic hypoxia in aged mice.

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.

Decreased VEGF expression and microvascular density, but increased HIF-1 and 2α accumulation and EPO expression in chronic moderate hyperoxia in the mouse brain.

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% O(2) 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.

Hypoxia-induced angiogenesis is delayed in aging mouse brain.

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 4month old ("young") C57BL/6 mice and 24month old ("aged") C57BL/6 mice to chronic hypobaric hypoxia of 0.4atm (290torr). 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 3weeks 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.