Hypoxic Preconditioning Reverses Protection After Neonatal Hypoxia-Ischemia in Glutathione Peroxidase Transgenic Murine Brain

Abstract

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...

Vulnerability of white matter towards antenatal hypoxia is linked to a species-dependent regulation of glutamate receptor subunits

“…In various animal models, a short period of hypoxia (preconditioning) may prevent ischemia-or stroke-related brain injury (33)(34)(35). To our knowledge, we provide the first evidence prenatal hypoxic preconditioning induces down-regulation of specific glutamate-receptor subunits, leading to a neuroprotective effect also detectable after pharmacological inhibition of NR2B subunit.…”

Mechanisms of Perinatal Arterial Ischemic Stroke

“…86 Several other distinct characteristics of the immature brain, such as high oxygen consumption, higher iron levels, and lower expression of several endogenous antioxidant enzymes, contribute to the higher vulnerability of the immature brain due to the deleterious actions of free radicals. 87 However, overexpression of antioxidant enzymes does not necessarily protect the neonatal brain against H-I, as was shown for superoxide dismutase overexpressing pups, and is dependent on the balance of free radical scavenging pathways 88,89 and cell types. 90 Inflammation, the third major contributor to neonatal brain injury can induce neuronal death by itself as well as by enhancing excitotoxicity and oxidative stress through the release of cytokines, free radicals, and other toxic products or trigger release of excitotoxic molecules, including glutamate.…”

Exogenous low dose hydrogen peroxide increases hypoxia-inducible factor-1alpha protein expression and induces preconditioning protection against ischemia in primary cortical neurons

“…In the setting of H/I preconditioning, there is complete loss of ischemic preconditioning-induced cardioprotection in mice heterozygous for deficiency of HIF-1 α, which is associated with a 50% decrease in H 2 O 2 production in these mice [9]. In neurons, we previously showed that hypoxic preconditioning reverses protection after neonatal H/I in GPx 1 transgenic murine brain, indicating the importance of H 2 O 2 in mechanisms of hypoxic preconditioning [39]. Furthermore, generation of H 2 O 2, but not superoxide, during brief oxygen-glucose deprivation (OGD, an in vitro model of ischemia) induces preconditioning neuronal protection in mature primary cortical neurons [15].…”

“…Preconditioning the neonatal brain with H/I before a severe H/I insult results in preservation of neurons and brain volume [16,40]. Recent studies suggest that preconditioning protection in vitro and in vivo is dependent on H 2 O 2 production [15,39]. From this study, preconditioning protection appears to be tightly correlated to the levels of H 2 O 2 present in the neurons at the time of preconditioning, suggesting that H 2 O 2 may regulate whether downstream regulators will be pro or anti-apoptotic (i.e., high levels are necrotic and pro-apoptotic, whereas low levels induce an anti-apoptotic program).…”