Evidence suggests that the activation of the transcription factor hypoxia-inducible factor 1α (HIF-1α) may promote cell survival in hypoxic or ischemic brain. To help understand the role of HIF-1α in neonatal hypoxic-ischemic brain injury, mice with conditional neuron-specific inactivation of HIF-1α underwent hypoxia-ischemia (HI). Mice heterozygous for Cre recombinase under the control of the calcium/calmodulin-dependent kinase II promoter were bred with homozygous ‘floxed’ HIF-1α transgenic mice. The resulting litters produced mice with a forebrain predominant neuronal deletion of HIF-1α (HIF-1α^Δ/Δ), as well as littermates without the deletion. In order to verify reduction of HIF-1α at postnatal day 7, HIF-1α^Δ/Δ and wild-type mice were exposed to a hypoxic stimulus (8% oxygen) or room air for 1 h, followed by immediate collection of brain cortices for determination of HIF-1α expression. Results of Western blotting of mouse cortices exposed to hypoxia stimulus or room air confirmed that HIF-1α^Δ/Δ cortex expressed a minimal amount of HIF-1α protein compared to wild-type cortex with the same hypoxic stimulus. Subsequently, pups underwent the Vannucci procedure of HI at postnatal day 7: unilateral ligation of the right common carotid artery followed by 30 min of hypoxia (8% oxygen). Immunofluorescent staining of brains 24 h after HI confirmed a relative lack of HIF-1α in the HIF-1α^Δ/Δ cortex compared to the wild type, and that HIF-1α in the wild type is located in neurons. HIF-1α expression was determined in mouse cortex 24 h after HI. Histological analysis for the degree of injury was performed 5 days after HI. HIF-1α protein expression 24 h after HI showed a large increase of HIF-1α in the hypoxic-ischemic cortex of the wild-type compared to the hypoxic only cortex. Histological analysis revealed that HI injury was increased in the neuronally deficient HIF-1α^Δ/Δ mouse brain (p < 0.05) and was more severe in the cortex. Genetic reduction of neuronal HIF-1α results in a worsening of injury after neonatal HI, with a region-specific role for HIF-1α in the setting of neonatal brain injury.

BACKGROUND Hypoxic preconditioning (HPc) protects the neonatal brain in the setting of hypoxia-ischemia (HI). The mechanisms of protection may depend on activation of hypoxia inducible factor (HIF-1α). The present study sought to clarify the role of HIF-1α after HPc and HI. METHODS To induce HPc, HIF-1α knockout and wildtype mice were exposed to hypoxia at postnatal day 6. At day 7, the mice underwent HI. Brain injury was determined by histology. HIF-1α, downstream targets, and markers of cell death were measured by Western blot. RESULTS HPc protected the wildtype brain compared to wildtype without HPc, but did not protect the HIF-1α knockout brain. In wildtype, HIF-1α increased after hypoxia and after HI, but not with HPc. The HIF-1α knockout showed no change in HIF-1α after hypoxia, HI, or HPc/HI. After HI, spectrin 145/150 was higher in HIF-1α knockout, but after HPc/HI, it was higher in wildtype. LAMP2 was higher in wildtype early after HI, but not later. After HPc/HI, LAMP2 was higher in HIF-1α knockout. CONCLUSION These results indicate that HIF-1α is necessary for HPc protection in the neonatal brain, and may affect cell death after HI. Different death and repair mechanisms depend on the timing of HPc.

ABSTRACT:The effect of hypoxic preconditioning (PC) on hypoxic-ischemic (HI) injury was explored in glutathione peroxidase (GPx)-overexpressing mice (human GPx-transgenic [hGPx-tg]) mice. Six-day-old hGPx-tg mice and wild-type (Wt) littermates were preconditioned with hypoxia for 30 min and subjected to the Vannucci procedure of HI 24 h after the PC stimulus. Histopathological injury was determined 5 d later (P12). Additional animals were killed 2 h or 24 h after HI and ipsilateral cerebral cortices assayed for GPx activity, glutathione (GSH), and hydrogen peroxide (H 2 O 2 ). In line with previous studies, hypoxic PC reduced injury in the Wt brain. Preconditioned Wt brain had increased GPx activity, but reduced GSH, relative to naive 24 h after HI. Hypoxic PC did not reduce injury to hGPx-tg brain and even reversed the protection previously reported in the hGPx-tg. GPx activity and GSH in hGPx-tg cortices did not change. Without PC, hGPx-tg cortex had less H 2 O 2 accumulation than Wt at both 2 h and 24 h. With PC, H 2 O 2 remained low in hGPx-tg compared with Wt at 2 h, but at 24 h, there was no longer a difference between hGPx-tg and Wt cortices. T he developing brain is particularly susceptible to oxidative stress, more so than the mature brain (1). One reason for this susceptibility may be the different developmental profiles of antioxidant enzymes in the newborn brain compared with the mature brain. For example, total GPx activity increases sharply between E18 and P1, declines in the early postnatal period, then stabilizes through P21 (2). GSH levels also increase between E18 and P1, but remain lower than P21 (2). One consequence of this difference is that the developing brain accumulates H 2 O 2 after HI, whereas the mature brain does not (3). H 2 O 2 accumulation has also been associated with increased injury in superoxide dismutase-overexpressing neonatal murine brain (4), and greater cell death is seen when immature neurons are exposed to H 2 O 2 compared with mature neurons (5). Increased H 2 O 2 accumulation may be the result of relative insufficiency of the endogenous enzyme GPx. Under physiologic circumstances, the brain has efficient antioxidant defense mechanisms, including GPx, which converts potentially harmful H 2 O 2 to oxygen and water at the expense of reduced GSH. Under oxidative stress, in the immature brain, endogenous levels of GPx may be inadequate for converting excess H 2 O 2 . Transgenic mice that overexpress GPx (hGPx-tg), when subjected to HI have less histologic brain injury than their Wt littermates (6). In addition, the cortex exhibits increased GPx enzyme activity at 24 h, whereas GPx activity remains unaltered in the Wt brain. In addition, neurons cultured from GPx-tg brain are resistant to injury from exogenously applied H 2 O 2 (7). Neurons cultured from hippocampus and cortex that are transfected with genes for catalase and GPx also show protection from neurotoxic insults and a corresponding decrease in H 2 O 2 accumulation (8). These findings indicate that adequate GPx ac...

No abstract