Hypoxia-ischemia In Neonatal Rats Research Articles

112 G-Csf Prevents Long-Term Behavioral Deficits and Brain Injury Following Neonatal Hypoxia-Ischemia in Rats

112 G-Csf Prevents Long-Term Behavioral Deficits and Brain Injury Following Neonatal Hypoxia-Ischemia in Rats

951 Tacrolimus and Leu-Ile Inhibit Hypoxic-Ischemic Encephalopathy in Newborn Rats

We used a neonatal rat hypoxia ischemia (HI)/ reperfusion brain damage model and investigated the neuroprotective effects of the immunosuppressant tacrolimus (FK506) and the hydrophobic peptide Leu-Ile, which has an active site similar to that of FK506 but is not an immunosuppressant. Method: HI was induced in 7- or 8-day-old Wister rat pups by transient right carotid artery occlusion, followed by exposure to 8% O. After HI insult, the rats were intraperitoneally administered FK506, Leu-Ile, a combination of the 2 drugs, or vehicle. Twenty-four hours or 7 d after HI, we analyzed the decreasing ratio of cerebral hemispheric weights and then histologically examined the brain tissue by HE, Iba-1, and caspase-3 staining. Body weight gain was also monitored. Result: The decreasing ratio of cerebral hemispheric weights in the FK506, Leu-Ile, and combination groups was lower than that in the vehicle group, but this ratio was significantly different only between the vehicle and FK506 groups. Pathological analysis showed that neurocytic injury had a strong suppressive effect in the FK506 and combination groups and a mild effect in the Leu-Ile group. During the 7-d experiment, the FK506 group showed lower body weight gain than the other groups. Conclusion: Combination treatment with Leu-Ile and FK506 had a stronger neuroprotective effect and lesser side effects; thus, we concluded that combination therapy might have potential for clinical application in neonatal HIE.

PP-33. Possible neuroprotective effect of magnesium sulfate administered alone or in combination with melatonin in neonatal hypoxic–ischemic rat model

PP-33. Possible neuroprotective effect of magnesium sulfate administered alone or in combination with melatonin in neonatal hypoxic–ischemic rat model

Short-Term Effect of Erythropoietin on Brain Lesions and Aquaporin-4 Expression in a Hypoxic-Ischemic Neonatal Rat Model Assessed by Magnetic Resonance Diffusion Weighted Imaging and Immunohistochemistry

Erythropoietin (Epo) is an endogenous cytokine that regulates hematopoiesis and is widely used to treat anemia. In addition, it has recently increased interest in the neurosciences since the new concept of Epo as a neuroprotective agent has emerged. The potential protective effect of human recombinant Epo (r-hu-Epo) on a hypoxic-ischemic (HI) pup rat model was studied. Cerebral HI was obtained by permanent left carotid artery ligature of pups followed by a 2-h hypoxia. Three hours after carotid occlusion, brain lesions were assessed by magnetic resonance diffusion weighted imaging. Intraperitoneal administration of r-hu-Epo (30,000 U/kg dose) limited both the HI-induced brain lesion area and the decrease in apparent diffusion coefficient (ADC) in the lesion. To identify potential mechanisms underlying the effects of Epo, immunohistochemical detection of caspase-3 and water channel protein aquaporin-4 (AQP4) were performed. No early apoptosis was detected, but up-regulation of AQP4 expression was observed in HI pups that received r-hu-Epo compared with HI animals without treatment. This study demonstrates an early neuroprotective effect of Epo with regard to brain lesion area and ADC values. One possible mechanism of Epo for decreasing brain edema and cellular swelling could be a better clearance of water excess in brain tissue, a process possibly mediated by AQP4.

Sulforaphane protects brains against hypoxic–ischemic injury through induction of Nrf2-dependent phase 2 enzyme

Neonatal hypoxia–ischemia (HI) brain injury involves reactive oxygen species (ROS) and inflammatory responses. Sulforaphane (SFN), an isothiocyanate found in cruciferous vegetables, has cytoprotective effects against oxidative stress and its effect was mediated by NF-E2-related factor-2 (Nrf2), a transcription factor, and heme oxygenase 1 (HO-1) which is one of Nrf2 downstream target genes. This study was undertaken to investigate the neuroprotective mechanisms of SFN in a neonatal HI rat model. Seven-day-old rat pups were subjected to left common carotid artery ligation and hypoxia (8% oxygen at 37 °C) for 90 min. SFN (5 mg/kg) was systemically administered 30 min before HI insult. Brain injury was assessed by 2,3,5-triphenyltetrazoliumchloride (TTC), Nissl, TUNEL staining, malondialdehyde (MDA), 8OH-dG level, and caspase-3 activity in the cortex and hippocampus. SFN pretreatment increased the expression of Nrf2 and HO-1 in the brain and reduced infarct ratio at 24 h after HI. The number of TUNEL-positive neurons as well as activated macroglia and the amount of 8OH-dG, were markedly reduced after SFN treatment, accompanied by suppressed caspase-3 activity and reduced lipid peroxidation (MDA) level. These results demonstrated that SFN could exert neuroprotective effects through increasing Nrf2 and HO-1 expression.

Expressions of matrix metalloproteinase-9 (MMP-9) and occludin in brain tissue of hypoxic-ischemic neonatal rats

Objective To investigate the effect of MMP-9 mRNA and occludin mRNA expressions after hypoxic-ischemic brain damage( HIBD). Methods Neonatal 7-day-old Sprague Dawley rats were ran-domly divided into control group,HIBD 3 h,6 h,24 h,48 h and 72 h groups (n =6 per group). The ligated cerebral hemisphere was observed for the morphological changes. The expression of MMP-9 mRNA and oc-cludin mRNA were measured by Real-time Q-PCR. Results (1)The ligated cerebral hemisphere of neonatal rats showed obvious edema from 24 h to 48 h after hypoxia-ischemia. (2) As compared to HIBD groups,the expression of MMP-9 mRNA was lower in control group (0. 7933 ± 0.0859) (P＜ 0.05). In the group of HIBD,it began to increase at 3h(l.1533 ±0.1291) ,and peaked at 24 h(3.4883 ±0.1759) .maintained the high level at 72 h(1.625 0±0. 1157). (3) The expression of occludin mRNA was higher in the control group(2.1780 ±0.0773) (P＜0.05), while in the group of HIBD, it began to decrease at 3 h(1.718 6 ± 0.1168) ,was lowest at 24 h(0.9066 ±0.0602) ,and maintained the low level at 72 h( 1.5966 ±0.0799). (4) The expression of MMP-9 mRNA negatively correlated with the expression of occludin mRNA (r = -0.815, P ＜0.05). Conclusion The increased expression of MMP-9 mRNA and the decreased expression of occlu-din mRNA after HIBD may play a role in the pathogenesis of HIBD in neonatal rats. Key words: Hypoxic-ischemia, brain; Matrix metalloproteinase-9; Tight junction protein; Occludin; Neonatal rats

Hemispheric Brain Injury and Behavioral Deficits Induced by Severe Neonatal Hypoxia-Ischemia in Rats Are Not Attenuated by Intravenous Administration of Human Umbilical Cord Blood Cells

Neonatal hypoxia-ischemia (HI) is an important cause of mortality and morbidity in infants. Human umbilical cord blood (HUCB) is a potential source of cellular therapy in perinatology. We investigated the effects of HUCB cells on spatial memory, motor performance, and brain morphologic changes in neonate rats submitted to HI. Seven-day-old rats underwent right carotid artery occlusion followed by exposure to 8% O(2) inhalation for 2 h. Twenty-four hours after HI, rats received either saline solution or HUCB cells i.v. After 3 wk, rats were assessed using a Morris Water Maze and four motor tests. Subsequently, rats were killed for histologic, immunohistochemical, and polymerase chain reaction (PCR) analyses. HI rats showed significant spatial memory deficits and a volumetric decrease in the hemisphere ipsilateral to arterial occlusion. These deficits and decreases were not significantly attenuated by the injection of HUCB cells. Moreover, immunofluorescence and PCR analysis revealed few HUCB cells located in rat brain. Intravenous administration of HUCB cells requires optimization to achieve improved therapeutic outcomes in neonatal hypoxic-ischemic injury.

Docosahexaenoic acid pretreatment confers neuroprotection in a rat model of perinatal cerebral hypoxia-ischemia

We hypothesized that pretreatment with docosahexaenoic acid (DHA), a potentially neuroprotective polyunsaturated fatty acid, would improve function and reduce brain damage in a rat model of perinatal hypoxia-ischemia. Seven-day-old rats were divided into 3 treatment groups that received intraperitoneal injections of DHA 1, 2.5, or 5 mg/kg as DHA-albumin complex and 3 controls that received 25% albumin, saline, or no injection. Subsequently, rats underwent right carotid ligation followed by 90 minutes of 8% oxygen. Rats underwent sensorimotor testing (vibrissae-stimulated forepaw placing) and morphometric assessment of right-sided tissue loss on postnatal day 14. DHA pretreatment improved forepaw placing response to near-normal levels (9.5 +/- 0.9 treatment vs 7.1 +/- 2.2 controls; normal = 10; P < .0001). DHA attenuated hemisphere damage compared with controls (P = .0155), with particular benefit in the hippocampus with 1 mg/kg (38% protection vs albumin controls). DHA pretreatment improves functional outcome and reduces volume loss after hypoxia-ischemia in neonatal rats.

Cooling Combined with Immediate or Delayed Xenon Inhalation Provides Equivalent Long-Term Neuroprotection after Neonatal Hypoxia—Ischemia

Hypothermia (HT) improves outcome after neonatal hypoxia-ischemia. Combination therapy may extend neuroprotection. The noble anesthetic gas xenon (Xe) has an excellent safety profile. We have shown earlier that 3 h of 50% Xe plus HT (32 degrees C) additively gives more protection (72%) than either alone (HT=31.1%, Xe=10.2%). Factors limiting clinical use include high-cost and specialist administration requirements. Thus, combinations of 1 h of 50% Xe were administered concurrently for either the first (1 h(Immediate)Xe) or last (1 h(Delayed)Xe) of 3 h of posthypoxic-ischemic HT as compared with 3 h of 50%Xe/HT to investigate how brief Xe exposure with a delay would affect efficacy. An established neonatal rat hypoxia-ischemia model was used. Serial functional neurologic testing into adulthood was performed, followed by neuropathological examination. Xenon with HT was more effective with longer Xe duration (3 h versus 1 h) (P=0.015). However, 1 h Xe/3 h HT resulted in better neuroprotection than 3 h HT alone (P=0.03), this significant effect was also present with 1 h Xe after a 2-h delay. One (immediate or with a delay) or 3 h Xe also significantly improved motor function (P=0.024). Females had significantly better motor scores than males, but no sex-dependent difference in pathology results. The neuroprotection of short, delayed Xe treatment would allow transport to specialist facilities to receive Xe.

Hydrogen gas is ineffective in moderate and severe neonatal hypoxia–ischemia rat models

Hydrogen gas (H 2) has been shown to ameliorate brain injury in experimental adult rat focal ischemia and in a mild neonatal hypoxia–ischemia (HI, 90 min hypoxia) rat model. In this study we tested H 2 in moderate (120 min hypoxia) and severe (150 min hypoxia) neonatal HI rat models. We hypothesized that H 2 would improve outcomes after neonatal HI by scavenging free radicals. Two hundred (200) unsexed Sprague–Dawley rats at day 10 of life (p10) underwent neonatal HI with the Rice–Vannucci model. Multiple treatment protocols were studied, including pre-ischemic treatment, intra-ischemic treatment, and post-ischemic treatment (Sham n = 32, HI n = 82, HI + H 2 n = 86). We also tested H 2 in middle cerebral artery occlusion (MCAO) in adult rats (MCAO n = 9, MCAO + H 2 n = 7) for comparison. Analysis at 24 h included infarction volume, measurement of brain concentration of malondialdehyde (MDA) (an end-product of lipid peroxidation), daily weight, Nissl histology, and mortality. In moderate and severe neonatal HI models, hydrogen gas therapy (2.9% concentration H 2) was not associated with decreased volume of infarction or decreased concentration of MDA. H 2 gas pretreatment (2.9%) was associated with increased infarction volume in neonatal HI. In MCAO in adult rats, H 2 gas therapy demonstrated a trend of beneficial effect. Exposure of H 2 gas to non-ischemic neonates resulted in a significant increase in brain concentration of MDA. We conclude that 2.9% H 2 gas therapy does not ameliorate moderate to severe ischemic damage in neonatal hypoxia–ischemia.

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.

Neuroprotective effects of hydrogen saline in neonatal hypoxia–ischemia rat model

Cerebral hypoxia–ischemia (HI) represents a major cause of brain damage in the term newborn. This study aimed to examine the short and long-term neuroprotective effect of hydrogen saline (H 2 saline) using an established neonatal HI rat pup model. Seven-day-old rat pups were subjected to left common carotid artery ligation and then 90 min hypoxia (8% oxygen at 37 °C). H 2 saturated saline was administered by peritoneal injection (5 ml/kg) immediately and again at 8 h after HI insult. At 24 h after HI, the pups were decapitated and brain morphological injury was assessed by 2,3,5-triphenyltetrazolium chloride (TTC), Nissl, and TUNEL staining. Acute cell death, inflammation and oxidative stress were evaluated at 24 h by studying caspase-3 activity, MDA measurement as well as Iba-1 immunochemistry in the brain. At 5 weeks after HI, spontaneous activity test and Morris water maze test were conducted. We observed that H 2 saline treatment reduced the caspase activity, MDA, Iba-1 levels, the infarct ratio, and improved the long-term neurological and neurobehavioral functions. H 2 saline has potentials in the clinical treatment of HI and other ischemia-related cerebral diseases.

Intravenous Grafts Recapitulate the Neurorestoration Afforded by Intracerebrally Delivered Multipotent Adult Progenitor Cells in Neonatal Hypoxic-Ischemic Rats

Once hypoxic-ischemic (HI) injury ensues in the human neonate at birth, the resulting brain damage lasts throughout the individual's lifetime, as no ameliorative treatments are currently available. We have recently shown that intracerebral transplantation of multipotent adult progenitor cells (MAPCs) results in behavioral improvement and reduction in ischemic cell loss in neonatal rat HI-injury model. In an attempt to advance this cellular therapy to the clinic, we explored the more practical and less invasive intravenous administration of MAPCs. Seven-day-old Sprague-Dawley rats were initially subjected to unilateral HI injury, then 7 days later received intracerebral or intravenous injections of allogeneic rat MAPCs. On post-transplantation days 7 and 14, the animals that received MAPCs via the intracerebral or intravenous route exhibited improved motor and neurologic scores compared with those that received vehicle infusion alone. Immunohistochemical evaluations at day 14 after transplantation revealed that both intracerebrally and intravenously transplanted MAPCs were detected in the ischemic hippocampal area. The degree of hippocampal cell preservation was almost the same in the two treatment groups and greater than that in the vehicle group. These results show that intravenous delivery of MAPCs is a feasible and efficacious cell therapy with potential for clinical use.

Reduction of Brain Injury in Neonatal Hypoxic—Ischemic Rats by Intracerebroventricular Injection of Neural Stem/Progenitor Cells Together With Chondroitinase ABC

Perinatal hypoxia-ischemia (HI) remains a critical issue. Cell transplantation therapy could be a potent treatment for many neurodegenerative diseases, but limited works on this kind of therapy have been reported for perinatal HI. In this study, the therapeutic effect of transplantation with neural stem/ progenitor cells (NSPCs) and chondrotinase ABC (ChABC) in a neonatal HI rat model is evaluated. Histological studies showed that the unaffected area of the brain in animals treated with NSPCs together with ChABC was significantly larger than that in the animals treated with vehicle or NSPCs alone. The wet weight of the brain that received the combined treatment was also significantly higher than those of the vehicle and their individual treatments. These results indicate that intracerebroventricular injection of NSPCs with ChABC reduces brain injury in a rat neonatal HI model.

Characterization of White Matter Injury in a Hypoxic-Ischemic Neonatal Rat Model by Diffusion Tensor MRI

We evaluate white matter (WM) injury after hypoxic-ischemic (HI) insult in a neonatal rat model using diffusion tensor imaging (DTI) to determine whether lambda(parallel) and lambda(perpendicular) are able to characterize type and severity of brain damage. Eighteen 7-day-old Sprague-Dawley rats underwent unilateral ligation of left common carotid artery followed by 50 minutes (n=9) or 90 minutes (n=9) of hypoxia at 37 degrees C. Rats were divided into 2 groups, according to absence (group A, n=11) or presence (group B, n=7) of cystic lesions on D7 post-HI T2-weighted imaging. DTI was performed for all rats at D1 and for group A rats at D7 post-HI. Signal intensity of ipsilateral and contralateral external capsule (EC) on D1 was compared by paired t test, with histological correlation. Group A rats had significantly reduced FA, elevated trace, elevated lambda(perpendicular), and similar lambda(parallel) on D1 in the ipsilateral compared to contralateral EC, whereas group B rats had significant reduction in all parameters in the ipsilateral EC. Elevated trace normalized on D7 in group A rats. Histopathologic results demonstrated reduced myelination in group A noncystic HI and severe necrosis in group B cystic HI. Increased lambda(perpendicular) with no significant change in lambda(parallel) appears to characterize noncystic WM injury with reduced myelination, whereas reduction in both lambda(parallel) and lambda(perpendicular) characterize severe damage with loss of structural integrity and necrosis. Combining with FA and trace, lambda(parallel) and lambda(perpendicular) provide additional information which reflects type and severity of HI injury.

Hydrogen therapy reduces apoptosis in neonatal hypoxia–ischemia rat model

Hypoxia-ischemia (HI) brain injury is a major cause of neuronal cell death especially apoptosis in the perinatal period. This study was designated to examine the effect of hydrogen therapy on apoptosis in an established neonatal HI rat pup model. Seven-day-old rat pups were subjected to left common carotid artery ligation and then 90 min hypoxia (8% oxygen at 37 degrees C). Immediately after HI insult, pups were placed into a chamber filled with 2% H2 for 30 min, 60 min, or 120 min, respectively. 24 h after 2% H2 therapy, the pups were decapitated and brain injury was assessed by 2,3,5-triphenyltetrazoliumchloride (TTC), Nissl, and TUNEL staining, as well as caspase-3, caspase-12 activities in the cortex and hippocampus. H2 treatment in a duration-dependent manner significantly reduced the number of positive TUNEL cells and suppressed caspase-3 and -12 activities. These results indicated H2 administration after HI appeared to provide brain protection via inhibition of neuronal apoptosis.

Mechanism of hyperbaric oxygen preconditioning in neonatal hypoxia–ischemia rat model

Hypoxic ischemic (HI) injury in neonates damages brain tissues. We examined the mechanism of hyperbaric oxygen preconditioning (HBO-PC) in neonatal HI rat model. Seven-day-old rat pups were subjected to left common carotid artery ligation and hypoxia (8% oxygen at 37 °C) for 90 min. HBO (100% O 2, 2.5 atmospheres absolute for 2.5 h) were administered by placing pups in a chamber 24 h before HI insult. Brain injury was assessed by the survival rate, 2,3,5-triphenyltetrazolium chloride (TTC), Nissl, TUNEL straining and caspase-3,caspase-9 activities after HI. In HBO preconditioned animals, survival rate was increased, infarct ratio was decreased, and the positive stained TUNEL cells were reduced, accompanied by the suppression of caspase-3 and -9 activities. These results indicate that a single HBO-PC appears to provide brain protection against HI insult via inhibition of neuronal apoptosis pathways.

Transplantation of human umbilical cord blood (hUCB) mononuclear cells leads to extensive restoration of gross motor function after cerebral hypoxia-ischemia in neonatal rats

Transplantation of human umbilical cord blood (hUCB) mononuclear cells leads to extensive restoration of gross motor function after cerebral hypoxia-ischemia in neonatal rats

Expression of Nogo-A and NgR in the developing rat brain after hypoxia–ischemia

Nogo-A and its receptor, NgR, have been shown to inhibit neurite growth in the adult rat. Therefore, we hypothesized that Nogo-A and NgR will be upregulated and thus play a similar role in the damage in developing rat brain following hypoxia–ischemia (HI). To test this hypothesis, we subjected postnatal day 7 (P7) rats to HI by permanently ligating the right common carotid artery, followed by exposure to 8%O 2/92% N 2 for 3 h. Rat brains at 0 h, 6 h, 12 h, 24 h and 72 h after HI, as well as from sham controls, were collected to determine histopathological damage and expression levels of Nogo-A and NgR using hematoxylin and eosin (H&E) staining, immunohistochemistry, fluorescence immunolabeling, Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR). We found neuronal degeneration and edema in the ischemic cortex, becoming most prominent at 24 h following HI in this model. Accordingly, the expression of Nogo-A and NgR protein was significantly upregulated at 24 h compared with the sham controls ( p < 0.01). The upregulated Nogo-A and NgR immunoreactive cells were mainly located in the core of the ischemic cortex and colocalized to neurons. Meanwhile, we found the expression of both Nogo-A and NgR mRNA was increased at 6 h and peaked at 12 h in the ischemic cortex after HI, compared with sham controls. Our findings of upregulation of neurite growth inhibitor Nogo-A and its receptor NgR in ischemic cortex suggest that Nogo-A and NgR may participate in the pathology seen after HI in neonatal rats.

Oxygen treatment after experimental hypoxia-ischemia in neonatal rats alters the expression of HIF-1α and its downstream target genes

Recently, mounting evidence has emerged to suggest that hyperbaric oxygenation (HBOT)-induced neuroprotection after experimental global ischemia and subarachnoid hemorrhage entails a decrease in the expression of hypoxia-inducible factor-1alpha (HIF-1alpha). Therefore, the purpose of this study was to test the hypothesis that oxygen-induced neuroprotection after neonatal hypoxia-ischemia involves alterations in the expression of HIF-1alpha. Seven-day-old rat pups were subjected to unilateral carotid artery ligation followed by 2 h of hypoxia (8% O(2) at 37 degrees C). Pups were then treated with HBOT (2.5 ATA) or normobaric oxygenation treatment (NBOT) for 2 h. The expression and phosphorylation status of HIF-1alpha was evaluated at intervals up to 24 h after the insult, as was the expression of glucose transporter (GLUT)-1, GLUT-3, lactate dehydrogenase (LDH), aldolase (Ald), and p53. The protein-protein interaction of HIF-1alpha and p53 was also examined. An elevated expression of HIF-1alpha, GLUT-1, GLUT-3, Ald, and LDH was observed after the insult. An increase in the dephosphorylated form of HIF-1alpha was followed by an increase in the association of HIF-1alpha with p53 and an increase in p53 levels. Both HBOT and NBOT reduced the elevated expression of HIF-1alpha and decreased its dephosphorylated form. Furthermore, both treatments promoted a transient increase in the expression of GLUT-1, GLUT-3, LDH, and Ald, while decreasing the HIF-1alpha-p53 interaction and decreasing the expression of p53. Therefore, the alteration of the HIF-1alpha phenotype by a single oxygen treatment may be one of the underlying mechanisms for the observed oxygen-induced neuroprotection seen when oxygen is administered after a neonatal hypoxic-ischemic insult.