Stroke is the leading cause of disability and the third leading cause of death in adults worldwide 1 . In human stroke, there exists a highly variable clinical state; in the development of animal models of focal ischemia, however, achieving reproducibility of experimentally induced infarct volume is essential. The rat is a widely used animal model for stroke due to its relatively low animal husbandry costs and to the similarity of its cranial circulation to that of humans 2,3 . In humans, the middle cerebral artery (MCA) is most commonly affected in stroke syndromes and multiple methods of MCA occlusion (MCAO) have been described to mimic this clinical syndrome in animal models. Because recanalization commonly occurs following an acute stroke in the human, reperfusion after a period of occlusion has been included in many of these models. In this video, we demonstrate the transient endovascular suture MCAO model in the spontaneously hypertensive rat (SHR). A filament with a silicon tip coating is placed intraluminally at the MCA origin for 60 minutes, followed by reperfusion. Note that the optimal occlusion period may vary in other rat strains, such as Wistar or Sprague-Dawley. Several behavioral indicators of stroke in the rat are shown. Focal ischemia is confirmed using T2-weighted magnetic resonance images and by staining brain sections with 2,3,5-triphenyltetrazolium chloride (TTC) 24 hours after MCAO. Video LinkThe video component of this article can be found at https://www.jove.com/video/1978/ Protocol MCAO Rat Model Presurgical PreparationsAseptic technique should be used for all survival surgical procedures. Disinfect the surgical work surface with commercial disinfectant and prepare sterile surgical packs of instruments, drapes, gauze, swabs, sutures, and scalpel blades by autoclaving. A surgical mask, hair bonnet and sterile gloves should be worn. A Germinator dry bead sterilizer is also used to resterilize surgical instruments between procedures if multiple rat surgeries will be done during one session. Prewarm a water-jacketed homeothermic blanket and place under an absorbent pad in order to prevent hypothermia of the rat during surgery.1. Place spontaneously hypertensive rat (or other rat strain of choice) into an induction chamber and induce anesthesia with 5% isoflurane (anesthesia machine should be set to 1.0 L/min O 2 and 1.0 L/min N 2 O). Lower to 1-2% isoflurane to maintain anesthesia. 2. Apply Artificial Tears ointment to both eyes. 3. Shave the throat and left neck region beyond the prospective incision site using clippers (Oster A5 with #10 blade). 4. Apply Betadine to a gauze pad and disinfect the skin starting from the center of the surgical region, spiraling outward. Rinse with sterile gauze pad containing 70% ethanol, moving pad in a similar pattern. Repeat both steps for a total of three cycles. 5. Inject 0.2 mL of 0.5% bupivacaine subcutaneously along the prospective incision site. 6. Place rat in sterile stockinette or cover with a sterile surgical drape.

We investigated the role of Na(+)/H(+) exchanger isoform 1 (NHE-1) in neonatal hypoxia/ischemia (HI). HI was induced by unilateral ligation of the left common carotid artery in postnatal day 9 (P9) mice, and subsequent exposure of animals to 8% O(2) for 55 min. A pre/posttreatment group received a selective and potent NHE-1 inhibitor HOE 642 (0.5 mg/kg, intraperitoneally) 5 min before HI, then at 24 and 48 h after HI. A posttreatment group received HOE 642 (0.5 mg/kg) at 10 min, 24 h, and 48 h after HI. Saline injections were used as vehicle controls. The vehicle-control brains at 72 h after HI exhibited neuronal degeneration in the ipsilateral hippocampus, striatum, and thalamus, as identified with Fluoro-Jade C positive staining and loss of microtubule-associated protein 2 (MAP2) expression. NHE-1 protein was upregulated in glial fibrillary acidic protein-positive reactive astrocytes. In HOE 642-treated brains, the morphologic hippocampal structures were better preserved and displayed less neurodegeneration and a higher level of MAP2 expression. Motor-learning deficit was detected at 4 weeks of age after HI in the vehicle control group. Inhibition of NHE-1 in P9 mice not only reduced neurodegeneration during the acute stage of HI but also improved the striatum-dependent motor learning and spatial learning at 8 weeks of age after HI. These findings suggest that NHE-1-mediated disruption of ionic homeostasis contributes to striatal and CA1 pyramidal neuronal injury after neonatal HI.

In the present study, we tested whether the ongoing differentiation of microglia in the immature brain results in more robust microglial activation and pro-inflammatory responses than juvenile brains following hypoxia-ischemia (HI). Under normoxic conditions, microglial activation profiles were assessed in postnatal day 9 and postnatal day 30 mice (P9 and P30) by analyzing relative expression levels of CD45 in CD11b+/CD45+ microglia/macrophages. Flow cytometry analysis revealed that the hippocampi of P9 and P30 brains exhibited higher levels of CD45 expression in CD11b+/CD45+ cells than in the cortex and striatum. In response to HI, there was an early increase in number of CD11b+/CD45+ microglia/macrophages in the ipsilateral hippocampus of P9 mice. These cells transformed from a “ramified” to an “amoeboid” morphology in the CA1 region, which was accompanied by a loss of microtubule-associated protein 2 immunostaining in this brain region. The peak response of microglial activation in the ipsilateral hippocampus of P9 mice occurred on day 2 post-HI, which was in contrast to a delayed and persistent microglial activation in the cortex and striatum (peak on day 9 post-HI). P9 brains demonstrated a 2–3 fold greater increase in microglia counts than P30 brains in each region (hippocampus, cortex, and striatum) during day 1–17 post-HI. P9 brains also showed more robust expression of pro-inflammatory cytokines (tumor necrosis factor-alpha, interleukin-1β) than P30 brains. Taken together, compared to P30 mice, P9 mice demonstrated differences in microglial activation and pro-inflammatory responses after HI, which may be important in brain damage and tissue repair.

The OC and JT are the main bony prominences obstructing the anterolateral surface of the brainstem. Neurosurgeons should be familiar with variations of the structures surrounding the FM in order to perform the safest and widest exposure possible.

In this study, we investigated the effects of a bioactive high-affinity TrkB receptor agonist 7,8-dihydroxyflavone (7,8 DHF) on neonatal brain injury in female and male mice after hypoxia ischemia (HI). HI was induced by exposure of postnatal day 9 (P9) mice to 10% O2 for 50 minutes at 37°C after unilateral ligation of the left common carotid artery. Animals were randomly assigned to HI-vehicle control group [phosphate buffered saline (PBS), intraperitoneally (i.p.)] or HI + 7,8 DHF-treated groups (5 mg/kg in PBS, i.p at 10 min, 24 h, or with subsequent daily injections up to 7 days after HI). The HI-vehicle control mice exhibited neuronal degeneration in the ipsilateral hippocampus and cortex with increased Fluoro-Jade C positive staining and loss of microtubule associated protein 2 expression. In contrast, the 7,8 DHF-treated mice showed less hippocampal neurodegeneration and astrogliosis, with more profound effects in female than in male mice. Moreover, 7,8 DHF-treated mice improved motor learning and spatial learning at P30-60 compared to the HI-vehicle control mice. Diffusion tensor imaging of ex-vivo brain tissues at P90 after HI revealed less reduction of fractional anisotropy values in the ipsilateral corpus callosum of 7,8 DHF-treated brains, which was accompanied with better preserved myelin basic protein expression and CA1 hippocampal structure. Taken together, these findings strongly suggest that TrkB agonist 7,8 DHF is protective against HI-mediated hippocampal neuronal death, white matter injury, and improves neurological function, with a more profound response in female than in male mice.

We investigated the effects of perinatal hypoxia-ischemia (HI) on brain injury and neurological functional outcome at postnatal day (P)30 through P90. HI was induced by exposing P9 mice to 8% O₂ for 55 min using the Vannucci HI model. Following HI, mice were treated with either vehicle control or Na⁺/H⁺ exchanger isoform 1 (NHE1) inhibitor HOE 642. The animals were examined by the accelerating rotarod test at P30 and the Morris water maze (MWM) test at P60. T₂-weighted MRI was conducted at P90. Diffusion tensor imaging (DTI) was subsequently performed in ex vivo brains, followed by immunohistochemical staining for changes in myelin basic protein (MBP) and neurofilament protein expression in the corpus callosum (CC). Animals at P30 after HI showed deficits in motor and spatial learning. T₂ MRI detected a wide spectrum of brain injury in these animals. A positive linear correlation was observed between learning deficits and the degree of tissue loss in the ipsilateral hemisphere and hippocampus. Additionally, CC DTI fractional anisotropy (FA) values correlated with MBP expression. Both FA and MBP values correlated with performance on the MWM test. HOE 642-treated mice exhibited improved spatial learning and memory, and less white matter injury in the CC. These findings suggest that HI-induced cerebral atrophy and CC injury contribute to the development of deficits in learning and memory, and that inhibition of NHE1 is neuroprotective in part by reducing white matter injury. T₂-weighted MRI and DTI are useful indicators of functional outcome after perinatal HI.

Hypoxia ischemia (HI)-related brain injury is the major cause of long-term morbidity in neonates. One characteristic hallmark of neonatal HI is the development of reactive astrogliosis in the hippocampus. However, the impact of reactive astrogliosis in hippocampal damage after neonatal HI is not fully understood. In the current study, we investigated the role of Na+/H+ exchanger isoform 1 (NHE1) protein in mouse reactive hippocampal astrocyte function in an in vitro ischemia model (oxygen/glucose deprivation and reoxygenation, OGD/REOX). 2 h OGD significantly increased NHE1 protein expression and NHE1-mediated H+ efflux in hippocampal astrocytes. NHE1 activity remained stimulated during 1–5 h REOX and returned to the basal level at 24 h REOX. NHE1 activation in hippocampal astrocytes resulted in intracellular Na+ and Ca2+ overload. The latter was mediated by reversal of Na+/Ca2+ exchange. Hippocampal astrocytes also exhibited a robust release of gliotransmitters (glutamate and pro-inflammatory cytokines IL-6 and TNFα) during 1–24 h REOX. Interestingly, inhibition of NHE1 activity with its potent inhibitor HOE 642 not only reduced Na+ overload but also gliotransmitter release from hippocampal astrocytes. The noncompetitive excitatory amino acid transporter inhibitor TBOA showed a similar effect on blocking the glutamate release. Taken together, we concluded that NHE1 plays an essential role in maintaining H+ homeostasis in hippocampal astrocytes. Over-stimulation of NHE1 activity following in vitro ischemia disrupts Na+ and Ca2+ homeostasis, which reduces Na+-dependent glutamate uptake and promotes release of glutamate and cytokines from reactive astrocytes. Therefore, blocking sustained NHE1 activation in reactive astrocytes may provide neuroprotection following HI.

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