Hypoxia inhibits cardiomyocyte proliferation in fetal rat hearts via upregulating TIMP-4.

Maternal hypoxia inhibits cardiomyocyte proliferation in the heart of fetal and neonatal rats. The present study tested the hypothesis that hypoxia has a direct effect inhibiting cardiomyocyte proliferation via upregulating tissue inhibitors of metalloproteinases (TIMP) in fetal rat hearts. Isolated fetal rat hearts and rat embryonic ventricular myocyte H9c2 cells were treated ex vivo with 20% or 1% O(2) for 48 or 24 h, respectively. Hypoxia caused a significant reduction in cardiomyocyte Ki-67 expression and bromodeoxyuridine incorporation in fetal hearts and H9c2 cells. In both fetal hearts and H9c2 cells, hypoxia resulted in a significant decrease in a cell division marker cyclin D2 but an increase in a cell division inhibitor p27. Additionally, hypoxia caused an upregulation of TIMP-3 and TIMP-4 in fetal hearts and H9c2 cells. Knockdown of TIMP-3 in H9c2 cells significantly increased cyclin D2 and Ki-67 and partially blocked the hypoxia-induced inhibition of cyclin D2 and Ki-67 in H9c2 cells. Unlike TIMP-3, TIMP-4 knockdown had no significant effects on the basal levels of cell proliferation but completely abrogated the hypoxia-mediated effects. These findings provide evidence of a novel causal role of TIMP-4 and TIMP-3 in the direct inhibitory effect of hypoxia on cardiomyocyte proliferation in the developing heart.

Fetal hypoxia results in programming of aberrant angiotensin ii receptor expression patterns and kidney development.

AIMS: The present study tested the hypothesis that fetal hypoxia adversely affects kidney development in fetal and offspring rats and alter the expression patterns of angiotensin II type 1 (AT1R) and type 2 (AT2R) receptors. METHODS: Time-dated pregnant rats were divided between normoxic and hypoxic (10.5% O2 last period of gestation) groups. Protein expression, in the offspring, was determined using western blot. RESULTS: Hypoxic treatment significantly decreased body and kidney weight in 21-day fetuses (E21) and 7-day neonates (P7). In 3-month-old offspring there were no significant differences in body and kidney weight between hypoxic and control animals. Fetal hypoxia had no effect on kidney AT1R density in E21 or P7, but significantly decreased kidney AT1R protein and mRNA abundance in both male and female adults. In contrast, kidney AT2R density was not affected by fetal hypoxia throughout the developmental stages studied. The hypoxia-mediated reduction of nephron numbers was progressively from P7 worsened into the adulthood with females affected more than males. CONCLUSION: The results suggest that fetal hypoxia causes programming of aberrant kidney development and accelerates the aging process of the kidney during the postnatal development, which may contribute to an increased risk of cardiovascular disease.

Perinatal nicotine exposure increases vulnerability of hypoxic-ischemic brain injury in neonatal rats: role of angiotensin II receptors.

BACKGROUND AND PURPOSE: Maternal cigarette smoking increases the risk of neonatal morbidity. We tested the hypothesis that perinatal nicotine exposure causes heightened brain vulnerability to hypoxic-ischemic (HI) injury in neonatal rats through aberrant expression patterns of angiotensin II type 1 (AT(1)R) and type 2 (AT(2)R) receptors in the developing brain. METHODS: Nicotine was administered to pregnant rats through subcutaneous osmotic minipumps. HI brain injury was determined in 10-day-old pups. AT(1)R and AT(2)R expression patterns were assessed through Western blotting, quantitative polymerase chain reaction, immunofluorescence, and confocal imaging. RESULTS: Perinatal nicotine exposure significantly increased HI brain infarct size in male, but not female, pups. In fetal brains, nicotine caused a decrease in mRNA and protein abundance of AT(2)R but not AT(1)R. The downregulation of AT(2)R persisted in brains of male pups, and nicotine treatment resulted in a significant increase in methylation of CpG locus 3 bases upstream of TATA-box at the AT(2)R gene promoter. In female brains, there was an increase in AT(2)R but a decrease in AT(1)R expression. Both AT(1)R and AT(2)R expressed in neurons but not in astrocytes in the cortex and hippocampus. Central application of AT(1)R antagonist losartan or AT(2)R antagonist PD123319 increased HI brain infarct size in both male and female pups. In male pups, AT(2)R agonist CGP42112 abrogated nicotine-induced increase in HI brain infarction. In females, PD123319 uncovered the nicotine's effect on HI brain infarction. CONCLUSIONS: Perinatal nicotine exposure causes epigenetic repression of the AT(2)R gene in the developing brain resulting in heightened brain vulnerability to HI injury in neonatal male rats in a sex-dependent manner.

Maternal hypoxia alters matrix metalloproteinase expression patterns and causes cardiac remodeling in fetal and neonatal rats.

Fetal hypoxia leads to progressive cardiac remodeling in rat offspring. The present study tested the hypothesis that maternal hypoxia results in reprogramming of matrix metalloproteinase (MMP) expression patterns and fibrillar collagen matrix in the developing heart. Pregnant rats were treated with normoxia or hypoxia (10.5% O(2)) from day 15 to 21 of gestation. Hearts were isolated from 21-day fetuses (E21) and postnatal day 7 pups (PD7). Maternal hypoxia caused a decrease in the body weight of both E21 and PD7. The heart-to-body weight ratio was increased in E21 but not in PD7. Left ventricular myocardium wall thickness and cardiomyocyte proliferation were significantly decreased in both fetal and neonatal hearts. Hypoxia had no effect on fibrillar collagen content in the fetal heart, but significantly increased the collagen content in the neonatal heart. Western blotting revealed that maternal hypoxia significantly increased collagen I, but not collagen III, levels in the neonatal heart. Maternal hypoxia decreased MMP-1 but increased MMP-13 and membrane type (MT)1-MMP in the fetal heart. In the neonatal heart, MMP-1 and MMP-13 were significantly increased. Active MMP-2 and MMP-9 levels and activities were not altered in either fetal or neonatal hearts. Hypoxia significantly increased tissue inhibitors of metalloproteinase (TIMP)-3 and TIMP-4 in both fetal and neonatal hearts. In contrast, TIMP-1 and TIMP-2 were not affected. The results demonstrate that in utero hypoxia reprograms the expression patterns of MMPs and TIMPs and causes cardiac tissue remodeling with the increased collagen deposition in the developing heart.

Cyclooxygenase-2 inhibition provides lasting protection against neonatal hypoxic-ischemic brain injury.

OBJECTIVE: To investigate whether inhibition of cyclooxygenase-2, a critical component of the inflammatory pathway, is neuroprotective in a neonatal rat model of cerebral hypoxia-ischemia. The development of brain inflammation largely contributes to neonatal brain injury that may lead to a lifetime of neurologic deficits. DESIGN: Laboratory investigation. SETTING: University research laboratory. SUBJECTS: Postnatal day ten Sprague-Dawley rats. INTERVENTIONS: Neonatal hypoxia-ischemia was induced by ligation of the right common carotid artery followed by 2 hrs of hypoxia (8% oxygen). The pups in treatment groups were administered 10 mg/kg (low dose) or 30 mg/kg (high dose) of a known selective cyclooxygenase-2 inhibitor (NS398). Animals were euthanized at three time points: 72 hrs, 2 wks, or 6 wks. Inflammation outcomes were assessed at 72 hrs; brain damage was assessed at 2 wks and 6 wks along with other organs (heart, spleen). Detailed neurobehavioral examination was performed at 6 wks. MEASUREMENTS AND MAIN RESULTS: Pharmacologic inhibition of cyclooxygenase-2 markedly increased survivability within the first 72 hrs compared with untreated rats (100% vs. 72%). Low- and high-dose NS398 significantly attenuated the loss of brain and body weights observed after hypoxia-ischemia. Neurobehavioral outcomes were significantly improved in some parameters with low-dose treatment, whereas high-dose treatment consistently improved all neurologic deficits. Immunohistochemical results showed a marked decrease in macrophage, microglial, and neutrophil abundance in ipsilateral hemisphere of the NS398-treated group along with a reduction in interleukin-6 expression. CONCLUSIONS: Selective cyclooxygenase-2 inhibition protected neonatal rats against death, progression of brain injury, growth retardation, and neurobehavioral deficits after a hypoxic-ischemic insult.

Hyperbaric oxygen preconditioning reduces postoperative brain edema and improves neurological outcomes after surgical brain injury.

The present study was designed to examine if hyperbaric oxygen preconditioning (HBO-PC) is neuroprotective in a mouse model of surgical brain injury (SBI). C57BL mice were administered 100% oxygen for 1 h at 2.5 ATA for 5 consecutive days and subjected to SBI on the following day. The HBO-PC + SBI animals were compared to sham and normoxia + SBI groups for brain water content in different brain regions at 24 and 72 h after surgery. Blood-brain barrier (BBB) permeability was evaluated using Evan's blue dye extravasation at 24 h. Neurological assessment of the animals was done by a blinded observer at 24 and 72 h. The results showed that brain water content was significantly increased in the right (ipsilateral) frontal lobe surrounding the site of resection. This was attenuated by HBO-PC at 24 and 72 h. However, HBO-PC did not have any effect on the increased BBB permeability observed after SBI. Significant neurological deficits were observed after SBI. HBO-PC improved neurological deficits at 72 h on the 21-point sensorimotor scale and at 24 and 72 h on the wire hang and beam balance scoring. In conclusion, HBO-PC attenuates post-operative brain edema and improves neurological outcomes following SBI.

Cyclo-oxygenase-2 mediates hyperbaric oxygen preconditioning-induced neuroprotection in the mouse model of surgical brain injury.

BACKGROUND AND PURPOSE: We investigated the role of cyclo-oxygenase-2 (COX-2) in mechanisms of hyperbaric oxygen preconditioning (HBO-PC) in the mouse model of surgical brain injury (SBI). METHODS: C57BL mice were administered 100% oxygen for 1 hour at 2.5 atmosphere absolute for 5 consecutive days and subjected to SBI. Neurological status and brain edema were evaluated at 24 hours and 72 hours after the brain insult. Fluorescent immunostaining and Western blotting were performed to study hypoxia-inducible factor-1alpha and COX-2, respectively. Two doses of COX-2 inhibitor, NS398 (3 mg/kg and 10 mg/kg) were used to verify the role of COX-2 signaling pathway in the mechanism of HBO-PC. RESULTS: HBO-PC improved neurological status and decreased brain edema at 24 hours and 72 hours after SBI. HBO-PC by itself and SBI independently increased COX-2 levels by 2-fold and 4-fold, respectively. HBO-PC, however, reduced increase in hypoxia-inducible factor-1alpha and COX-2 expression after SBI. The HBO-PC-induced improvement in neurological status and brain edema was reversed by a suboptimal dose of the COX-2 inhibitor, NS398 (10 mg/kg intraperitoneally; 1/4th of dose shown to provide neuroprotection), which itself had no effect on investigated end points. CONCLUSIONS: HBO-PC attenuates postoperative brain edema and improves neurological outcomes after SBI. The HBO-PC-induced neuroprotection is mediated through COX-2 signaling pathways.

Reduced brain injury in CD18-deficient mice after experimental intracerebral hemorrhage.

Many studies have indicated leukocytes are a major contributor to brain injuries caused by intracerebral hemorrhage (ICH). Leukocyte-expressed CD18 is important for neutrophil-endothelial interactions in the vasculature, and CD18 deficiency protects against ischemia-reperfusion injury. We investigated whether CD18 deficiency provides protection against ICH-induced brain injury. Male wild-type (WT) CD18(+/+) mice and CD18(-/-) -knockout mice were used in this study. ICH was induced by a collagenase injection. Mortality, neurological function, brain edema, and myeloperoxidase (MPO) activity as well as tissue expression of nitrotyrosine and MPO were evaluated 24 hr after ICH. We discovered significantly reduced brain edema and diminished mortality with a concomitant decrease in MPO and nitrotyrosine immunoreactivity in brains of CD18-knockout mice.

Rosiglitazone, a PPAR gamma agonist, attenuates inflammation after surgical brain injury in rodents.

INTRODUCTION: Surgical brain injury (SBI) is unavoidable during many neurosurgical procedures. This inevitable brain injury can result in post-operative complications including brain edema, blood-brain barrier disruption (BBB) and cell death in susceptible areas. Rosiglitazone (RSG), a PPAR-gamma agonist, has been shown to reduce inflammation and provide neuroprotection in experimental models of ischemia and intracerebral hemorrhage. This study was designed to evaluate the neuroprotective effects of RSG in a rodent model of SBI. METHODS: 65 adult male Sprague-Dawley rats were randomly divided into sham, vehicle and treatment groups. RSG was administered intraperitoneally in two dosages (1 mg/kg/dose, 6 mg/kg/dose) 30 min before surgery, and 30 min and 4 h after surgery. Animals were euthanized 24 h following neurological evaluation to assess brain edema and BBB permeability by IgG staining. Inflammation was examined using myeloperoxidase (MPO) assay and double-labeling fluorescent immunohistochemical analysis of IL-1beta and TNF-alpha. RESULTS: Localized brain edema was observed in tissue surrounding the surgical injury. This brain edema was significantly higher in rats subjected to SBI than sham animals. Increased IgG staining was present in affected brain tissue; however, RSG reduced neither IgG staining nor brain edema. RSG also did not improve neurological status observed after SBI. RSG, however, significantly attenuated MPO activity and qualitatively decreased IL-1beta and TNF-alpha expression compared to vehicle-treated group. CONCLUSION: SBI causes increased brain edema, BBB disruption and inflammation localized along the periphery of the site of surgical resection. RSG attenuated inflammatory changes, however, did not improve brain edema, BBB disruption and neurological outcomes after SBI.

Melatonin decreases mortality following severe subarachnoid hemorrhage.

Aneurysmal subarachnoid hemorrhage (SAH) is a devastating disease that is associated with significant morbidity and mortality. There is substantial evidence to suggest that oxidative stress is significant in the development of acute brain injury following SAH. Melatonin is a strong antioxidant that has low toxicity and easily passes through the blood-brain barrier. Previous studies have shown that melatonin provides neuroprotection in animal models of ischemic stroke. This study hypothesizes that melatonin will provide neuroprotection when administered 2 hr after SAH. The filament perforation model of SAH was performed in male Sprague-Dawley rats weighing between 300 and 380 g. Melatonin (15 or 150 mg/kg), or vehicle was given via intraperitoneal injection 2 hr after SAH. Mortality and neurologic deficits were assessed 24 hr after SAH. A significant reduction in 24-hr mortality was seen following treatment with high dose melatonin. There was no improvement in neurologic scores with treatment. Brain water content and lipid peroxidation were measured following the administration of high dose melatonin to identify a mechanism for the increased survival. High dose melatonin tended to reduce brain water content following SAH, but had no effect on the lipid peroxidation of brain samples. Large doses of melatonin significantly reduces mortality and brain water content in rats following SAH through a mechanism unrelated to oxidative stress.

Fetal hypoxia and programming of matrix metalloproteinases.

Fetal hypoxia adversely affects the brain and heart development, yet the mechanisms responsible remain elusive. Recent studies indicate an important role of the extracellular matrix in fetal development and tissue remodeling. The matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMPs) have been implicated in a variety of physiological and pathological processes in the cardiovascular and central nervous systems. This review summarizes current knowledge of the mechanisms by which fetal hypoxia induces the imbalance of MMPs, TIMPs and collagen expression patterns, resulting in growth restriction and aberrant tissue remodeling in the developing heart and brain. Collectively, this information could lead to the development of preventive diagnoses and therapeutic strategies in the fetal programming of cardiovascular and neurological disorders.

Maternal hypoxia increases the activity of MMPs and decreases the expression of TIMPs in the brain of neonatal rats.

A recent study has shown that increased activity of matrix metalloproteinases-2 and metalloproteinases-9 (MMP-2 and MMP-9) has detrimental effect on the brain after neonatal hypoxia. The present study determined the effect of maternal hypoxia on neuronal survivability and the activity of MMP-2 and MMP-9, as well as the expression of tissue inhibitors of metalloproteinase 1 and 2 (TIMP-1 and TIMP-2) in the brain of neonatal rats. Pregnant rats were exposed to 10.5% oxygen for 6 days from the gestation day 15 to day 21. Pups were sacrificed at day 0, 4, 7, 14, and 21 after birth. Body weight and brain weight of the pups were measured at each time point. The activity of MMP-2 and MMP-9 and the protein abundance of TIMP-1 and TIMP-2 were determined by zymography and Western blotting, respectively. The tissue distribution of MMPs was examined by immunofluorescence staining. The neuronal death was detected by Nissl staining. Maternal hypoxia caused significant decreases in body and brain size, increased activity of MMP-2 at day 0, and increased MMP-9 at day 0 and 4. The increased activity of the MMPs was accompanied by an overall tendency towards a reduced expression of TIMPs at all ages with the significance observed for TIMPs at day 0, 4, and 7. Immunofluorescence analysis showed an increased expression of MMP-2, MMP-9 in the hippocampus at day 0 and 4. Nissl staining revealed significant cell death in the hippocampus at day 0, 4, and 7. Functional tests showed worse neurobehavioral outcomes in the hypoxic animals.