Neuroinflammation is a key contributor to the pathogenic cascades induced by hypoxic-ischemic (HI) insult in the neonatal brain. AD-16 is a novel anti-inflammatory compound, recently found to exert potent inhibition of the lipopolysaccharide-induced production of pro-inflammatory and neurotoxic mediators. In this study, we evaluated the effect of AD-16 on primary astrocytes and neurons under oxygen-glucose deprivation (OGD) in vitro and in mice with neonatal HI brain injury in vivo. We demonstrated that AD-16 protected against OGD-induced astrocytic and neuronal cell injury. Single dose post-treatment with AD-16 (1 mg/kg) improved the neurobehavioral outcome and reduced the infarct volume with a therapeutic window of up to 6 h. Chronic administration reduced the mortality rate and preserved whole-brain morphology following neonatal HI. The in vitro and in vivo effects suggest that AD-16 offers promising therapeutic efficacy in attenuating the progression of HI brain injury and protecting against the associated mortality and morbidity.
Animals ; Animals, Newborn ; Astrocytes/pathology* ; Brain/pathology* ; Brain Injuries/pathology* ; Glucose ; Hypoxia ; Hypoxia-Ischemia, Brain/drug therapy* ; Mice ; Neuroinflammatory Diseases ; Neuroprotective Agents/therapeutic use* ; Oxygen/therapeutic use*

BACKGROUNDStudies showed that propentofylline enhances the action of adenosine and protects hippocampal neuronal damage against transient global cerebral ischaemia. Our study was to investigate the effect of propentofylline on hypoxic-ischaemic brain damage in neonatal rat.

METHODSSeven-day-old Wistar rats were subjected to unilateral common carotid artery ligation and hypoxia in oxygen 8 kPa for two hours at 37 degrees C. Propentofylline (10 mg/kg) was administered intraperitoneally one hour after hypoxia-ischaemia (treated group). Control group rats were received an equivalent volume of saline. The effects of propentofylline were assessed by observing the body mass gain, behavioural alteration and neurohistological changes. The rats were sacrificed at 72 hours after hypoxia-ischaemia, and the brain sections were examined after haematoxylin and eosin staining.

RESULTSThe propentofylline-treated rats had better body mass gain and better behavioural response than the paired saline-controls did. In the control group, the rats either lost body mass or had little mass gain after the insult, their average body mass gain was 97.3% at 24 h, 100.3% at 48 h, and 114.1% at 72 h of recovery. In propentofylline-treated group, there was a significant improvement of body mass gain at 24 h (100.2%, P < 0.05) and 48 h (110.3%, P < 0.01) of recovery; the percentage of rats that performed well on behavioural test was significantly higher from 48 h to 72 h of recovery (P < 0.05); the incidence of severe brain damage to the cerebral cortex and dentate gyrus was significantly reduced in propentofylline-treated rats (cortex, 93% - 70.8%, P < 0.01; dentate gyrus 95% - 66.7%, P < 0.01) as compared with control rats.

CONCLUSIONSAdministration of propentofylline 1 hour after hypoxia-ischaemia significantly attenuates brain damage in both the cerebral cortex and dentate gyrus, and also improves the body mass gain as well as behavioural disturbance in 7-day-old rats.

Animals ; Animals, Newborn ; Brain ; pathology ; Female ; Hypoxia-Ischemia, Brain ; drug therapy ; pathology ; Male ; Neuroprotective Agents ; therapeutic use ; Rats ; Rats, Wistar ; Weight Gain ; drug effects ; Xanthines ; therapeutic use

OBJECTIVETo evaluate the long-term effects of delayed hyperbaric oxygen (HBO) therapy on neonatal rats with hypoxic-ischemic brain injury (HIBD).

METHODPostnatal 7 days newborn rats (n = 52) were randomly set to three groups: control (n = 18, sham operation), HIBD (n = 17), or HBO (n = 17). Pups in the HBO group were subjected to hyperbaric oxygen treatment with 2 atmosphaera absolutus, 5 x 30 min at a 24 h intervals since 48-72 h after the HIBD model. All the animals were tested for the spatial learning and memory ability in the Morris water maze from postnatal days 37 to 41. At day-42, rats were decapitated and the brains were analyzed for morphological and histological changes, including brain shapes and weights, survival neurons, percentage of AchE positive area and NOS positive neurons in hippocampal CA1 region.

RESULTSRats in HBO and HIBD groups displayed significant morphological and histological damages, as well as severe spatial learning and memory disability. The average escape latency of Morris water maze in HBO group [(56 +/- 23) s] and HIBD group [(56 +/- 22) s] were longer than the control [(23 +/- 16) s] (P < 0.05). The swimming time in HBO group [(30 +/- 5) s] and HIBD group [(29 +/- 6) s] were shorter than the control [(51 +/- 5) s] (P < 0.05). The swimming length in HBO group [(572 +/- 92) cm] and HIBD group [(548 +/- 92) cm] were shorter than the control [(989 +/- 101) cm] (P < 0.05). The weight of left brains in HBO group [(598 +/- 46) mg] and HIBD group [(601 +/- 59) mg] were lighter than the control [(984 +/- 18) mg] (P < 0.05). The survival neurons of hippocamal CA1 region in HBO group [(97 +/- 27)/mm] and HIBD group [(100 +/- 27)/mm] were less than the control [(183 +/- 8)/mm] (P < 0.05). The percentage of AchE-positive fibers in HBO group [(18.4 +/- 2.2)%] and HIBD group [(18.5 +/- 2.2)%] were less than the control [(27.5 +/- 2.2)%,] (P < 0.05). NOS-positive neurons in HBO group [(21 +/- 5)/mm(2)] and HIBD group [(19 +/- 4)/mm(2)] were also less than the control [(34 +/- 6)/mm(2)] (P < 0.05).

CONCLUSIONDelayed HBO therapy resulted in no protection against either HIBD-induced brain morphological and histological deficits or spatial learning and memory disability.

Acetylcholinesterase ; analysis ; Animals ; Animals, Newborn ; Brain ; pathology ; Female ; Hippocampus ; pathology ; Hyperbaric Oxygenation ; Hypoxia-Ischemia, Brain ; drug therapy ; pathology ; Male ; Maze Learning ; Nitric Oxide Synthase ; analysis ; Rats ; Time

OBJECTIVEImmunosuppressant tacrolimus (FK506) has shown neuroprotective effects on hypoxic-ischemic brain damage (HIBD) in the adult animal model. This study investigated whether FK506 has a protection against HIBD in neonatal rats by examining growthjassociated protein-43 (GAP-43) expression in the hippocampus.

METHODSNinety-six seven-day-old Sprague-Dawley rats were randomly divided into three groups: sham-operation, HIBD and FK506 intervention group. HIBD was induced in the later two groups. The FK506 intervention group was intraperitoneally injected with FK506 immediately after HIBD, at a dosage of 1 mg/kg daily, for three days. The HIBD group was injected with normal saline. Immunohistochemical technical was applied to examine GAP-43 expression in the hippocampus 24 and 72 hrs and 7 and 14 days after HIBD.

RESULTSCompared with the HIBD group, hematoxylin-eosin staining showed attenuated neuronal necrosis in the FK506 intervention group. In the HIBD group, the expression of GAP-43 increased significantly 72 hrs, and 7 and 14 days after HIBD compared with that in the sham-operation group. The GAP-43 expression in the FK506 intervention group was significantly higher than that in the HIBD group 72 hrs and 7 days after HIBD.

CONCLUSIONSFK506 might have neuroprotective effects against HIBD in neonatal rats.

Animals ; Animals, Newborn ; GAP-43 Protein ; analysis ; Hippocampus ; chemistry ; drug effects ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; Immunosuppressive Agents ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Tacrolimus ; pharmacology

Animals ; Animals, Newborn ; Antioxidants ; administration & dosage ; pharmacology ; Apoptosis ; drug effects ; Brain ; drug effects ; pathology ; Disease Models, Animal ; Female ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; Immunohistochemistry ; Male ; Melatonin ; administration & dosage ; pharmacology ; Neuroprotective Agents ; administration & dosage ; pharmacology ; Rats ; Rats, Sprague-Dawley

This study was done to determine the neuroprotective effect of cycloheximide on neonatal hypoxic-ischemic brain injury. Seven day-old newborn rat pups were subjected to 90 min of 8% oxygen following a unilateral carotid artery ligation. The extent of cerebral infarction was evaluated at 1 and 4 week of recovery. Apoptosis was identified by performing terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and flow cytometry with a combination of fluoresceinated annexin V and propidium iodide. Brain infarction area was significantly increased at 4 week compared to 1 week after hypoxia-ischemia in the control group. With cycloheximide treatment, the number of TUNEL positive cells in the ipsilateral cerebral cortex at 48 hr and peri-infarct area at 1 and 4 week of recovery was significantly reduced, both apoptotic and necrotic cells by flow cytometry 48 hr after the injury were significantly reduced, and the extent of cerebral infarction at 1 and 4 week of recovery was also significantly attenuated compared to the hypoxia-ischemia control group. In summary, our data suggest that apoptosis plays an important role in the development of delayed infarction, and inhibition of apoptosis with cycloheximide significantly reduces the ensuing cerebral infarction in a newborn rat pup model of cerebral hypoxia-ischemia.
Time Factors ; Rats, Sprague-Dawley ; Rats ; Propidium ; Neuroprotective Agents/*pharmacology ; In Situ Nick-End Labeling ; Hypoxia-Ischemia, Brain/*drug therapy/metabolism/pathology ; Cycloheximide/*pharmacology ; Brain Infarction/pathology/prevention & control ; Apoptosis/drug effects ; Annexin A5/metabolism ; Animals, Newborn ; Animals

OBJECTIVEPrevious research suggests that dexamethasone (Dex) pretreatment protects neonatal rats against hypoxic-ischemic brain damage (HIBD). Some of the pharmacological effects of baicalin (a traditional Chinese medicine extracted from Scutellaria baicalensis Georgi) are similar to Dex. This study was designed to explore the effect of baicalin on the neuronal apoptosis following HIBD in neonatal rats.

METHODSSix-day-old Sprague-Dawley rats were randomly assigned into Control (without HI), HIBD, Dex-pretreatment and post-treatment, Baicalin-pretreatment and -post-treatment groups. HIBD was induced by ligating the left common carotid artery, followed by exposure to hypoxia. In the pretreatment groups either baicalin (16 mg/kg) or Dex (0.1 mg/kg) was administered to the rats 24 hrs before HIBD; in the post-treatment groups baicalin or Dex was given 30 minutes after HIBD. The rat pups were sacrificed on postnatal day 10, and brain tissues were harvested. Brain water content was determined, morphological changes were observed under a light microscope, and neuronal apoptosis was measured by terminal deoxynucleotidyl transferase mediated dUTP biotin nick end labeling (TUNEL) staining.

RESULTSThe brain water content and the number of apoptotic cells were significantly higher in the HIBD group than those of the Control group (P < 0.05). Both baicalin and Dex pretreatment decreased the brain water content from 88.9 +/- 1.7 % (HIBD group) to 87.4 +/- 0.7% (baicalin) or 87.3 +/- 0.6% (Dex) (P < 0.05) and the number of apoptotic cells were reduced from 251 +/- 28 (HIBD group) to 102 +/- 47 (baicalin) or 75 +/- 26 (Dex) (P < 0.05). Baicalin and Dex post-treatment had no effects on the brain water content and the number of apoptotic cells. Loss and degeneration of neurons could be observed in the HIBD group. Baicalin and Dex pretreatment significantly alleviated neuronal injury, but post-treatment did not.

CONCLUSIONSPretreatment with baicalin, as with Dex, has a protective effect against HIBD in neonatal rats, but baicalin or Dex post-treatment do not reverse the neuronal injuries.

Animals ; Apoptosis ; drug effects ; Body Water ; metabolism ; Brain ; metabolism ; pathology ; Female ; Flavonoids ; therapeutic use ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; In Situ Nick-End Labeling ; Male ; Neuroprotective Agents ; therapeutic use ; Rats ; Rats, Sprague-Dawley

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Brain ; drug effects ; pathology ; Disease Models, Animal ; Estrogens ; pharmacology ; Female ; Hypoxia-Ischemia, Brain ; drug therapy ; In Situ Nick-End Labeling ; Male ; Rats ; Rats, Wistar

The aim of this study was to investigate the effect of erythropoietin (EPO) on histological brain injury, subventricular zone (SVZ) expansion, and sensorimotor function deficits induced by hypoxia-ischemia (HI) in newborn rat pups. Seven-day-old male rat pups were divided into six groups: normoxia control, normoxia EPO, hypoxia control, hypoxia EPO, HI control, and HI EPO group. Sham surgery or HI was performed in all animals. HI was induced by ligation of the right common carotid artery followed by 90 min of hypoxia with 8% oxygen. Recombinant human EPO 3 U/g or saline was administered intraperitoneally, immediately, at 24- and 48-hr after insult. At two weeks after insult, animals were challenged with cylinder-rearing test for evaluating forelimb asymmetry to determine sensorimotor function. All animals were then sacrificed for volumetric analysis of the cerebral hemispheres and the SVZ. The saline-treated HI rats showed marked asymmetry by preferential use of the non-impaired, ipsilateral paw in the cylinder-rearing test. Volumetric analysis of brains revealed significantly decreased preserved ipsilateral hemispheric volume and increased ipsilateral SVZ volume compared with the sham-operated animals. Treatment of EPO significantly improved forelimb asymmetry and preserved ipsilateral hemispheric volume along with decreased expansion of ipsilateral SVZ following HI compared to the saline-treated HI rats. These results support the use of EPO as a candidate drug for treatment of neonatal hypoxic-ischemic brain damage.
Animals ; Animals, Newborn ; Carotid Artery, Common ; Cerebral Ventricles/*pathology ; Erythropoietin, Recombinant/*pharmacology ; Female ; Hypoxia-Ischemia, Brain/*drug therapy/*pathology ; Ligation ; Male ; Motor Activity/drug effects ; Pregnancy ; Rats ; Rats, Sprague-Dawley ; Recovery of Function/drug effects

OBJECTIVETo examine the expression of calreticulin (CRT) and the changes of intracellular free calcium and neuronal apoptosis in the cerebral cortex of neonatal rats with hypoxic-ischemic brain damage (HIBD), and to investigate the intervention effects of Shenfu injection.

METHODSSeven-day-old rats were randomly assigned to three groups: control, hypoxic-ischemia (HI) and Shenfu-treated. Each group (n=50) was subdivided into 5 groups sacrificed at 3, 6, 12, 24 and 72 hours. Rat models of HIBD were prepared according to the Rice's method. Rats in the control group only underwent the separation of right common carotidartery. Shenfu injection was administered by intraperitoneal injections right after HI insults and then once daily at a dosage of 10 mL/kg for 3 days in the Shenfu-treated group. The expression of CRT in the cerebral cortex was detected by RT-PCR and Western blot. The free calcium concentrations were determined under a fluorescent microscope. The apoptosis rate was measured by the flow cytometry.

RESULTSCompared with the control group, the expression levels of CRT in the HI and the Shenfu-treated groups were obviously up-regulated (P<0.05), and the expression levels of CRT in the Shenfu-treated group were notably higher than those in the HI group (P<0.05) at all time points. The concentrations of intracellular free calcium and the apoptosis rate of neurons in the cerebral cortex in the Shenfu-treated group were significantly reduced compared with those in the HI group (P<0.05), but increased significantly compared with those in the control group at all time points (P<0.05).

CONCLUSIONSShenfu injection may have neuroprotective effects against HIBD by up-regulation of CRT expression and relief of calcium overload.

Animals ; Animals, Newborn ; Apoptosis ; drug effects ; Calcium ; metabolism ; Calreticulin ; analysis ; Cerebral Cortex ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hypoxia-Ischemia, Brain ; drug therapy ; metabolism ; pathology ; Injections ; Male ; Neurons ; drug effects ; Rats, Sprague-Dawley