Rat Model Of Cerebral Hypoxia-ischemia Research Articles

GSK-3β/mTORC1 Couples Synaptogenesis and Axonal Repair to Reduce Hypoxia Ischemia-Mediated Brain Injury in Neonatal Rats.

Glycogen synthase kinase 3 beta (GSK-3β) plays an important role in neurological outcomes after brain injury. However, its roles and mechanisms in hypoxia-ischemia (HI) are unclear. Activation of mTOR complex 1 (mTORC1) has been proven to induce the synthesis of proteins associated with regeneration. We hypothesized that GSK-3β inhibition could activate the mTORC1 signaling pathway, which may reduce axonal injury and induce synaptic protein synthesis and functional recovery of synapses after HI. By analyzing a P7 rat model of cerebral HI and an in vitro ischemic (oxygen glucose deprivation) model, we found that GSK-3β inhibitors (GSK-3β siRNA or lithium chloride) activated mTORC1 signaling, leading to increased expression of synaptic proteins, including synapsin 1, PSD95, and GluR1, and the microtubule-associated protein Tau and decreased expression of the axonal injury-associated protein amyloid precursor protein. These changes contributed to attenuated axonal injury (decreased amyloid precursor protein staining and axonal loss by silver staining), improved electrophysiological properties of synapses, and enhanced spatial memory performance in the Morris water maze. However, inhibition of mTORC1 by rapamycin blocked the benefits induced by GSK-3β inhibition, suggesting that GSK-3β inhibition induces synaptogenesis and axonal repair via mTORC1 signaling, which may benefit neonatal rats subjected to HI.

MiR-30d-5p Plays an Important Role in Autophagy and Apoptosis in Developing Rat Brains After Hypoxic-Ischemic Injury.

Increasing evidence has demonstrated a vital role of microRNAs (miRNAs) in diverse biological processes. However, their functions in developing brain with hypoxia-ischemia (HI) remain largely unknown. Through a miRNA microarray analysis in a P10 rat model of cerebral HI, we found that miR-30d-5p was one of the most deregulated miRNAs in neonatal brains in response to HI. MiR-30d-5p was downregulated in a time-dependent manner in brain cortex after HI, which was accompanied by increased expression of Beclin1 both at transcript and protein levels. Increase of miR-30d-5p by agomir (AG) resulted in reduction of autophagy and increase of apoptosis, whereas inhibition of miR-30d-5p by antagomir (AT) enhanced autophagy and inhibited apoptosis in rat brains after HI. Moreover, miR-30d-5p AG increased infarct volume, delayed recovery of neurological function, and impaired improvement of spatial memory ability. MiR-30d-5p AT decreased infarct volume, promoted neurological recovery, and improved behavior performance of rats subjected to HI. Collectively, these results indicated that miR-30d-5p modulated survival programs of neural cell by regulating autophagy and apoptosis.

Prophylactic Subacute Administration of Zinc Increases CCL2, CCR2, FGF2, and IGF-1 Expression and Prevents the Long-Term Memory Loss in a Rat Model of Cerebral Hypoxia-Ischemia.

Prophylactic subacute administration of zinc decreases lipoperoxidation and cell death following a transient cerebral hypoxia-ischemia, thus suggesting neuroprotective and preconditioning effects. Chemokines and growth factors are also involved in the neuroprotective effect in hypoxia-ischemia. We explored whether zinc prevents the cerebral cortex-hippocampus injury through regulation of CCL2, CCR2, FGF2, and IGF-1 expression following a 10 min of common carotid artery occlusion (CCAO). Male rats were grouped as follows: (1) Zn96h, rats injected with ZnCl2 (one dose every 24 h during four days); (2) Zn96h + CCAO, rats treated with ZnCl2 before CCAO; (3) CCAO, rats with CCAO only; (4) Sham group, rats with mock CCAO; and (5) untreated rats. The cerebral cortex-hippocampus was dissected at different times before and after CCAO. CCL2/CCR2, FGF2, and IGF-1 expression was assessed by RT-PCR and ELISA. Learning in Morris Water Maze was achieved by daily training during 5 days. Long-term memory was evaluated on day 7 after learning. Subacute administration of zinc increased expression of CCL2, CCR2, FGF2, and IGF-1 in the early and late phases of postreperfusion and prevented the CCAO-induced memory loss in the rat. These results might be explained by the induction of neural plasticity because of the expression of CCL2 and growth factors.

Intranasal administration of aTf protects and repairs the neonatal white matter after a cerebral hypoxic–ischemic event

Our previous studies showed that the intracerebral injection of apotransferrin (aTf) attenuates white matter damage and accelerates the remyelination process in a neonatal rat model of cerebral hypoxia-ischemia (HI) injury. However, the intracerebral injection of aTf might not be practical for clinical treatments. Therefore, the development of less invasive techniques capable of delivering aTf to the central nervous system would clearly aid in its effective clinical use. In this work, we have determined whether intranasal (iN) administration of human aTf provides neuroprotection to the neonatal mouse brain following a cerebral hypoxic-ischemic event. Apotransferrin was infused into the naris of neonatal mice and the HI insult was induced by right common carotid artery ligation followed by exposure to low oxygen concentration. Our results showed that aTf was successfully delivered into the neonatal HI brain and detected in the olfactory bulb, forebrain and posterior brain 30 min after inhalation. This treatment successfully reduced white matter damage, neuronal loss and astrogliosis in different brain regions and enhanced the proliferation and survival of oligodendroglial progenitor cells (OPCs) in the subventricular zone and corpus callosum (CC). Additionally, using an in vitro hypoxic model, we demonstrated that aTf prevents oligodendrocyte progenitor cell death by promoting their differentiation. In summary, these data suggest that iN administration of aTf has the potential to be used for clinical treatment to protect myelin and to induce remyelination in demyelinating hypoxic-ischemic events in the neonatal brain.

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

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. Laboratory investigation. University research laboratory. Postnatal day ten Sprague-Dawley rats. 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. 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. Selective cyclooxygenase-2 inhibition protected neonatal rats against death, progression of brain injury, growth retardation, and neurobehavioral deficits after a hypoxic-ischemic insult.

Limited role of the c‐Jun N‐terminal kinase pathway in a neonatal rat model of cerebral hypoxia–ischemia

D-JNKI1, a cell-permeable peptide inhibitor of the c-Jun N-terminal kinase (JNK) pathway, has been shown to be a powerful neuroprotective agent after focal cerebral ischemia in adult mice and young rats. We have investigated the potential neuroprotective effect of D-JNKI1 and the involvement of the JNK pathway in a neonatal rat model of cerebral hypoxia-ischemia (HI). Seven-day-old rats underwent a permanent ligation of the right common carotid artery followed by 2 h of hypoxia (8% oxygen). Treatment with D-JNKI1 (0.3 mg/kg intraperitoneally) significantly reduced early calpain activation, late caspase 3 activation and, in the thalamus, autophagosome formation, indicating an involvement of JNK in different types of cell death: necrotic, apoptotic, and autophagic. However, the size of the lesion was unchanged. Further analysis showed that neonatal HI induced an immediate decrease in JNK phosphorylation (reflecting mainly JNK1 phosphorylation) followed by a slow progressive increase (including JNK3 phosphorylation 54 kDa), whereas c-jun and c-fos expression were both strongly activated immediately after HI. In conclusion, unlike in adult ischemic models, JNK is only moderately activated after severe cerebral HI in neonatal rats and the observed positive effects of D-JNKI1 are insufficient to give neuroprotection. Thus, for perinatal asphyxia, D-JNKI1 can only be considered in association with other therapies.

Therapeutic Window for Cycloheximide Treatment after Hypoxic-Ischemic Brain Injury in Neonatal Rats

We have previously shown that cycloheximide significantly inhibited apoptosis, and reduced ensuing cerebral infarction in a newborn rat model of cerebral hypoxia-ischemia. This study was performed to determine the therapeutic window for cycloheximide therapy. Seven day-old newborn rat pups were subjected to 100 min of 8% oxygen following a unilateral carotid artery ligation, and cycloheximide was given at 0, 6, 12 and 24 hr after hypoxia-ischemia (HI). Apoptosis or necrosis was identified by performing flow cytometry with a combination of fluorescinated annexin V and propidium iodide, and the extent of cerebral infarction was evaluated with triphenyl tetrazolium chloride (TTC) at 48 hr and 72 hr after HI, respectively. With cycloheximide treatment at 0 hr after HI, both apoptotic and necrotic cells by flow cytometry were significantly reduced, only necrotic cells were significantly reduced at 6 and 12 hr, and no protective effect was seen if administration was delayed until 24 hr after HI compared to the HI control group. Infarct volume, measured by TTC, was significantly reduced by 92% and 61% when cycloheximide was given at 0 or 6 hr after HI respectively; however, there was an insignificant trend in infarct reduction if cycloheximide was administered 12 hr after HI, and no protective effect was observed when administration was delayed until 24 hr after HI. In summary, cycloheximide was neuroprotective when given within 6 hr after HI in the developing newborn rat brain.

Neuroprotective effects of N-acetylaspartylglutamate in a neonatal rat model of hypoxia-ischemia

Neuroprotective effects of N-acetylaspartylglutamate (NAAG), the precursor of glutamate and a selective agonist at the Group II metabotropic glutamate (mGlu) receptor, against hypoxic-ischemic brain injury were examined in a neonatal rat model of cerebral hypoxia-ischemia. The neonatal hypoxia-ischemia procedure (unilateral carotid artery ligation followed by exposure to an 8% oxygen hypoxic condition for 1.5 h) was performed in 7-day-old rat pups. Following unilateral carotid artery ligation, NAAG (0.5 to 20 mg/kg, i.p.) was administered before or after the hypoxic exposure. Brain injury was examined 1-week later by weight reduction in the ipsilateral brain and by neuron density in the hippocampal CA1 area. In the saline-treated rat, neonatal hypoxia-ischemia resulted in severe brain injury as indicated by a 24% reduction in the ipsilateral brain weight. Low doses of NAAG (2–10 mg/kg, but not 0.5 mg/kg), administered before or even if 1 h after the hypoxic exposure, greatly reduced hypoxia-ischemia-induced brain injury (3.8–14.2% reduction in the ipsilateral brain weight). A high dose of NAAG (20 mg/kg) was ineffective. While l(+)-2-Amino-4-phosphonobutyric acid ( l-AP4) and trans-[1S,3R]-1-Amino-cyclopentane-1, 3-dicarboxylic acid ( t-ACPD) were unable to provide protection against hypoxic-ischemic brain injury, 2-(phosphonomethyl) pentanedioic acid (2-PMPA), an inhibitor of N-acetylated alpha-linked acidic dipeptidase (NAALADase), which hydrolyzes endogenous NAAG into N-acetyl-aspartate and glutamate, significantly reduced neonatal hypoxia-ischemia-induced brain injury. (αS)-α-Amino-α-[(1S, 2S)-2-carboxycyclopropyl]-9H-xanthine-9-propanoic acid (LY341495), a selective antagonist at the mGlu 2/3 receptor, prevented the neuroprotective effect of NAAG. Neuron density data measured in the hippocampal CA1 area confirmed that ipsilateral brain weight reduction was a valid measure for hypoxic-ischemic brain injury. Neonatal hypoxia-ischemia stimulated an elevation of cyclic AMP (cAMP) concentration in the saline-treated rat brain. NAAG, l-AP4 and t-ACPD all significantly decreased hypoxia-ischemia-induced elevation of cAMP. LY341495 blocked the effect of NAAG, but not of l-AP4 or t-ACPD, on hypoxia-ischemia-stimulated cAMP elevation. The overall results suggest that the neuroprotective effect of NAAG is largely associated with activation of mGlu 2/3 receptor.

Apoptosis in a neonatal rat model of cerebral hypoxia-ischemia.

The mechanisms of excitotoxic cell death in cerebral ischemia are poorly understood. In addition to necrosis, apoptotic cell death may occur. The purpose of this study was to determine whether an established model of cerebral hypoxia-ischemia in the neonatal rat demonstrates any features of apoptosis. Seven-day-old neonatal rats underwent bilateral, permanent carotid ligation followed by 1 hour of hypoxia, and their brains were examined 1, 3, and 4 days after hypoxia-ischemia. The severity of ischemic damage was assessed in the dentate gyrus and frontotemporal cortex by light microscopy. Immunocytochemistry was performed to detect the cleavage of actin by caspases, a family of enzymes activated in apoptosis. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) reactivity was examined in the cortical infarction bed and dentate gyrus. Neonatal rat brain DNA was run on agarose gel electrophoresis to detect DNA fragmentation. Ethidium bromide-staining and electron microscopy were used to determine whether apoptotic bodies, 1 of the hallmarks of apoptosis, were present. The frontotemporal cortex displayed evidence of infarction, and in most rats the dentate gyrus showed selective, delayed neuronal death. Immunocytochemistry demonstrated caspase-related cleavage of actin. TUNEL and DNA electrophoresis provided evidence of DNA fragmentation. Ethidium bromide-staining and electron microscopy confirmed the presence of chromatin condensation and apoptotic bodies. Features of apoptosis are present in the described model of cerebral hypoxia-ischemia. Apoptosis may represent a mode of ischemic cell death that could be the target of novel treatments that could potentially expand the therapeutic window for stroke.