Stabilization of hypoxia-inducible factor-1α in buffer containing cobalt chloride for Western blot analysis.

Hypoxia-inducible factor-1α (HIF-1α) is a widely studied protein with significant biomedical impact. Care is needed to stabilize HIF-1α protein during sample preparation for Western blot analysis due to its rapid degradation in the presence of oxygen. Enzyme inhibitor cocktails can be complex and expensive. We present a protease inhibitor-free buffer, containing cobalt chloride, which is effective at stabilizing HIF-1α, while being inexpensive, straightforward, and convenient, and has potential for widespread application.

A method for measuring brain partial pressure of oxygen in unanesthetized unrestrained subjects: the effect of acute and chronic hypoxia on brain tissue PO(2).

The level of tissue oxygenation provides information related to the balance between oxygen delivery, oxygen utilization, tissue reactivity and morphology during physiological conditions. Tissue partial pressure of oxygen (PtO(2)) is influenced by the use of anesthesia or restraint. These factors may impact the absolute level of PtO(2). In this study we present a novel fiber optic method to measure brain PtO(2). This method can be used in unanesthetized, unrestrained animals, provides absolute values for PO(2), has a stable calibration, does not consume oxygen and is MRI compatible. Brain PtO(2) was studied during acute hypoxia, as well as before and after 28 days of high altitude acclimatization. A sensor was chronically implanted in the frontal cortex of eight Wistar rats. It is comprised of a fiber optic probe with a tip containing material that fluoresces with an oxygen dependent lifetime. Brain PtO(2) declines by 80% and 76% pre- and post-acclimatization, respectively, when the fraction of inspired oxygen declines from 0.21 to 0.08. In addition, a linear relationship between brain PtO(2) and inspired O(2) levels was demonstrated r(2)=0.98 and r(2)=0.99 (pre- and post-acclimatization). Hypoxia acclimatization resulted in an increase in the overall brain PtO(2) by approximately 35%. This paper demonstrates the use of a novel chronically implanted fiber optic based sensor for measuring absolute PtO(2). It shows a very strong linear relationship in awake animals between inspired O(2) and tissue O(2), and shows that there is a proportional increase in PtO(2) over a range of inspired values after exposure to chronic hypoxia.

A near-infrared calibration method suitable for quantification of broadband data in humans.

Broadband near-infrared spectroscopy (bNIRS) is a powerful non-invasive technique for the measurement of hemoglobin. bNIRS systems are relatively simple to construct compared with many near-infrared instruments since they operate on the principle of continuous wave. The advantage of the broadband method is the capacity to model the spectra and to use "the second differential method" to quantify deoxyhemoglobin (HHb). An "anoxia pulse" method can be applied to quantify total haemoglobin (tHb) and tissue oxygen saturation (S(t)O(2)). A disadvantage is that this calibration method is not suitable for application in humans. In this study, we compared the "anoxia pulse" method with "graded hypoxia" method, which can be applied for human studies, to quantify tHb and S(t)O(2). The values obtained with the two methods were respectively (tHb=47.8+/-2.8 and 49.4+/-7.7 microM, mean+/-S.D., n=8) and (S(t)O(2)=72.8+/-3.7% and 73.2+/-5.7%, mean+/-S.D., n=8). There was no significant difference (p<0.05) between the two methods, indicating that the graded hypoxia method could be used for quantification of tHb and S(t)O(2) in human subjects.

Monitoring angiogenesis noninvasively with near-infrared spectroscopy.

Near-infrared (NIR) spectroscopy is used to quantify cerebral blood volume (CBV) as a marker of angiogenesis (formation of new blood vessels). Rats are exposed to chronic hypoxia for 3 weeks at half atmospheric pressure to stimulate angiogenesis, and second-differential NIR spectroscopy is used to quantify total cerebral hemoglobin before and after angiogenesis. The cerebral hemoglobin (from broadband NIR spectroscopy), and the large vessel hemoglobin and hematocrit (from blood samples), are used to derive values for the calculation of CBV. The total hemoglobin in brain is 46.6+/-1.9 micromoll (mean+/-SD, n=5) preacclimation and increases by 72% postacclimation. CBV is initially 3.26+/-0.41% v/v and increases by 31% with acclimation. Each individual animal shows a measureable increase in CBV. This study indicates that NIR broadband spectroscopy can be used for repeated measurements of CBV and can be applied as a noninvasive method to study angiogenesis.

CRYAB and HSPB2 deficiency alters cardiac metabolism and paradoxically confers protection against myocardial ischemia in aging mice.

The abundantly expressed small molecular weight proteins, CRYAB and HSPB2, have been implicated in cardioprotection ex vivo. However, the biological roles of CRYAB/HSPB2 coexpression for either ischemic preconditioning and/or protection in situ remain poorly defined. Wild-type (WT) and age-matched ( approximately 5-9 mo) CRYAB/HSPB2 double knockout (DKO) mice were subjected either to 30 min of coronary occlusion and 24 h of reperfusion in situ or preconditioned with a 4-min coronary occlusion/4-min reperfusion x 6, before similar ischemic challenge (ischemic preconditioning). Additionally, WT and DKO mice were subjected to 30 min of global ischemia in isolated hearts ex vivo. All experimental groups were assessed for area at risk and infarct size. Mitochondrial respiration was analyzed in isolated permeabilized cardiac skinned fibers. As a result, DKO mice modestly altered heat shock protein expression. Surprisingly, infarct size in situ was reduced by 35% in hearts of DKO compared with WT mice (38.8 +/- 17.9 vs. 59.8 +/- 10.6% area at risk, P < 0.05). In DKO mice, ischemic preconditioning was additive to its infarct-sparing phenotype. Similarly, infarct size after ischemia and reperfusion ex vivo was decreased and the production of superoxide and creatine kinase release was decreased in DKO compared with WT mice (P < 0.05). In permeabilized fibers, ADP-stimulated respiration rates were modestly reduced and calcium-dependent ATP synthesis was abrogated in DKO compared with WT mice. In conclusion, contrary to expectation, our findings demonstrate that CRYAB and HSPB2 deficiency induces profound adaptations that are related to 1) a reduction in calcium-dependent metabolism/respiration, including ATP production, and 2) decreased superoxide production during reperfusion. We discuss the implications of these disparate results in the context of phenotypic responses reported for CRYAB/HSPB2-deficient mice to different ischemic challenges.