Hyperbaric oxygen treatment promotes neural stem cell proliferation in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage

Hyperbaric oxygen therapy for the treatment of neonatal hypoxic-ischemic brain damage has been used clinically for many years, but its effectiveness remains controversial. In addition, the mechanism of this potential neuroprotective effect remains unclear. This study aimed to investigate the influence of hyperbaric oxygen on the proliferation of neural stem cells in the subventricular zone of neonatal Sprague-Dawley rats （7 days old） subjected to hypoxic-ischemic brain damage. Six hours after modeling, rats were treated with hyperbaric oxygen once daily for 7 days. Immunohistochemistry revealed that the number of 5-bromo-2＇-deoxyuridine positive and nestin positive cells in the subventricular zone of neonatal rats increased at day 3 after hypoxic-ischemic brain damage and peaked at day 5. After hyperbaric oxygen treatment, the number of 5-bromo-2＇- deoxyuddine positive and nestin positive cells began to increase at day 1, and was significantly higher than that in normal rats and model rats until day 21. Hematoxylin-eosin staining showed that hyperbaric oxygen treatment could attenuate pathological changes to brain tissue in neonatal rats, and reduce the number of degenerating and necrotic nerve cells. Our experimental findings indicate that hyperbaric oxygen treatment enhances the proliferation of neural stem cells in the subventricular zone of neonatal rats with hypoxic-ischemic brain damage, and has therapeutic potential for promoting neurological recovery following brain injury.

Ketamine induces tau hyperphosphorylation at serine 404 in the hippocampus of neonatal rats

Male Wistar 7-day-old rats were injected with 40 mg/kg ketamine intraperitoneally, followed by three additional injections of 20 mg/kg ketamine each upon restoration of the righting reflex. Neonatal rats injected with equivalent volumes of saline served as controls. Hippocampal samples were collected at 1,7 or 14 days following administration. Electron microscopy showed that neuronal structure changed noticeably following ketamine treatment. Specifically, microtubular structure became irregular and disorganized. Quantitative real time-PCR revealed that phosphorylated tau mRNA was upregulated after ketamine. Western blot analysis demonstrated that phosphorylated tau levels at serine 396 initially decreased at 1 day after ketamine injection, and then gradually returned to control values. At 14 days after injection, levels of phosphorylated tau were higher in the ketamine group than in the control group. Tau protein phosphorylated at serine 404 significantly increased after ketamine injection and then gradually decreased with time. However, the levels of tau protein at serine 404 were significantly greater in the ketamine group than in the control group until 14 days. The present results indicate that ketamine induces an increase of phosphorylated tau mRNA and excessive phosphorylation of tau protein at serine 404, causing disruption of microtubules in the neonatal rat hippocampus and potentially resulting in damage to hippocampal neurons.

Expression of Clock genes in the pineal glands of newborn rats with hypoxic-ischemic encephalopathy

Clock genes are involved in circadian rhythm regulation, and surviving newborns with hypoxic-ischemic encephalopathy may present with sleep-wake cycle reversal. This study aimed to determine the expression of the clock genes Clock and Bmall, in the pineal gland of rats with hypoxic-ischemic brain damage. Results showed that levels of Clock mRNA v^re not significantly changed within 48 hours after cerebral hypoxia and ischemia. Expression levels of CLOCK and BMAL1 protein were significantly higher after 48 hours. The levels of Bmall mRNA reached a peak at 36 hours, but were significantly reduced at 48 hours. Experimental findings indicate that Clock and Bmall genes were indeed expressed in the pineal glands of neonatal rats. At the initial stage （within 36 hours） of hypoxic-ischemic brain damage, only slight changes in the expression levels of these two genes were detected, followed by significant changes at 36-48 hours. These changes may be associated with circadian rhythm disorder induced by hypoxic-ischemic brain damage.

Oligodendrocyte transcription factor 1 overexpression promotes oligodendrocyte transcription factor 2 expression in the brains of neonatal rats exposed to hypoxia

To examine the expression profiles of oligodendrocyte transcription factors 1 and 2 （Oligl and Olig2） and the interaction between these two proteins, Oligl was transfected into the lateral ventricles of neonatal rats subjected to hypoxia. Immunohistochemistry demonstrated that Olig2 was expressed throughout the nuclei in the brain, and expression increased at 3 days following hypoxia and was higher than levels at 7 days following Ad5-Oligl transfection. Western blot revealed that Oligl and Olig2 expression increased in Oligl-transfected brain cells 3 days after hypoxia, but Oligl and Olig2 expression decreased at 7 days. These results indicate that Oligl overexpression enhances Olig2 expression in brain tissues of hypoxia rats.

Neurotoxicity of prenatal alcohol exposure on medullary pre-B?tzinger complex neurons in neonatal rats

Prenatal alcohol exposure disrupts the development of normal fetal respiratory function, but whether it perturbs respiratory rhythmical discharge activity is unclear. Furthermore, it is unknown whether the 5-hydroxytryptamine 2A receptor（5-HT2AR） is involved in the effects of prenatal alcohol exposure. In the present study, pregnant female rats received drinking water containing alcohol at concentrations of 0%, 1%, 2%, 4%, 8% or 10%（v/v） throughout the gestation period. Slices of the medulla from 2-day-old neonatal rats were obtained to record respiratory rhythmical discharge activity. 5-HT2 AR protein and m RNA levels in the pre-B？tzinger complex of the respiratory center were measured by western blot analysis and quantitative RT-PCR, respectively. Compared with the 0% alcohol group, respiratory rhythmical discharge activity in medullary slices in the 4%, 8% and 10% alcohol groups was decreased, and the reduction was greatest in the 8% alcohol group. Respiratory rhythmical discharge activity in the 10% alcohol group was irregular. Thus, 8% was the most effective alcohol concentration at attenuating respiratory rhythmical discharge activity. These findings suggest that prenatal alcohol exposure attenuates respiratory rhythmical discharge activity in neonatal rats by downregulating 5-HT2 AR protein and m RNA levels.

Estrogen inhibits lipid peroxidation after hypoxic-ischemic brain damage in neonatal rats

Sprague-Dawley neonatal rats within 7 days after birth were used in this study. The left common carotid artery was occluded and rats were housed in an 8% O2 environment for 2 hours to establish a hypoxic-ischemic brain damage model. 17β-estradiol （1 × 10-5 M） was injected into the rat abdominal cavity after the model was successfully established. The left hemisphere was obtained at 12, 24, 48, 72 hours after operation. Results showed that malondialdehyde content in the left brain of neonatal rats gradually increased as modeling time prolonged, while malondialdehyde content of 17β-estrodial-treated rats significantly declined by 24 hours, reached lowest levels at 48 hours, and then peaked at 72 hours after injury. Nicotinamide-adenine dinucleotide phosphate histochemical staining showed the nitric oxide synthase-positive cells and fibers dyed blue/violet and were mainly distributed in the cortex, hippocampus and medial septal nuclei. The number of nitric oxide synthase-positive cells peaked at 48 hours and significantly decreased after 17β-estrodial treatment. Our experimental findings indicate that estrogen plays a protective role following hypoxic-ischemic brain damage by alleviating lipid peroxidation through reducing the expression of nitric oxide synthase and the content of malondialdehyde.

Effects of dizocipine maleate on mitochondrial ultramicrostructure in neurons following traumatic brain injury in neonatal rats A quantitative time-course analysis

BACKGROUND： The effects of N-methyl-D-aspartic acid （NMDA） receptor antagonist on neurodegeneration in the immature brain following traumatic brain injury （TBI） are still widely unknown. OBJECTIVE： To study the effects of dizocipine maleate （MK-801）, a non-competitive NMDA receptor antagonist, on mitochondrial ultramicrostructure of neurons in the ipsilateral cingulate cortex and hippocampus after TBI in neonatal rats, and to analyze the optimal time interval of MK-801 administration （1 mg/kg）. DESIGN： Completely randomized controlled study. SETTING： Shanghai Jiao Tong University. MATERIALS： Eight 7-day-old neonatal SD rats, irrespective of gender, were provided by Experimental Animal Center, Medical College of Fudan University. The experiment was approved by a local ethics committee. MK-801 was provided by Sigma. A CM-120 transmission electron microscope （Philips, Holland） was used for tissue analysis. METHODS： This study was performed at the Departments of Anatomy, Neuromorphology, and Biophysics, Medical College of Shanghai, Jiaotong University, between October 2006 and January 2007. Focal models of contusion and laceration of brain were established by the free-falling impact method. Eight rats were randomly divided into a normal control group （n = 2 ） and a MK-801 group （n = 6）. Rats in the normal control group did not receive model establishment and administration, and they were only analyzed by an electron microscope. In the MK-801 group, the cingulate cortex was damaged using a contusion device. MK-801 （1 mg/kg） was intraperitoneally injected 30 minutes before lesion, immediately after lesion, and 30 minutes after lesion （n = 2 for each time point）.MAIN OUTCOME MEASURES： The cingulate cortex and hippocampal tissues from the injured side were removed 24 hours after lesion and routinely processed for analysis of neuronal ultramicrostructure using transmission electron microscopy. RESULTS： Differential therapeutic effects of MK-801 （1 mg/kg） at distinct administration time points： thirty minutes before lesion, the shape of cortical and hippocampal neurons was similar to that observed during excitotoxicity-induced cell death. Organelles were enlarged, the nuclear membrane of cortical neurons was complete with gear wheel-like changes, and the nuclear chromatin was irregularly aggregated around the edge. When MK-801 was applied 30 minutes after lesion, the cingulate cortex contained apoptotic neurons in early and late stages. The nuclear membrane of hippocampal neurons displayed incisures. The chromatin shape was not similar to necrosis in an early stage. Immediate administration of MK-801 after lesion slightly altered the neuronal architecture, such that mitochondria were enlarged. The neuronal shape in the control group was normal. Effects of immediate administration of MK-801 on mitochondrial injury following TBI were that the mitochondria in cortical and hippocampal neurons were damaged to a certain degree in the MK-801 group. Mitochondrial injury was reversible, when MK-801 was applied 30 minutes before lesion and immediately after lesion. Application 30 minutes after lesion produced irreversible changes. In addition, mitochondrial injury occurred earlier than other organelle and nuclear changes. CONCLUSION： Mitochondrial injury occurs earlier than other organelle and nuclear changes. Early administration of MK-801 （1 mg/kg） can prevent or reduce necrosis following TBI, decrease the degree of neuronal injury, and protect nerve cells.

Foxg1 mRNA overexpression in neonatal rats following hypoxic brain injury

Forkhead box G1 （Foxgl） is expressed during the embryonic stage and in postnatal brain regions sensitive to hypoxia/ischemia injury, such as the hippocampus and cerebral cortex. To date, very little is known about Foxgl expression changes in the brain following hypoxia injury （HI）. The present study measured Foxgl mRNA expression using reverse-transcription polymerase chain reaction on days 3, 7, 14, 28, and 56 following HI to determine self-restorative features in the injured brain. In addition, mRNA expression of other related layer markers, such as Reelin, RORB, Foxpl, Foxp2, ER81, and Otx-1, was detected following HI. Results revealed significantly decreased Foxgl mRNA expression at 3 days after HI, which significantly increased by 56 days. Reelin and Foxp2 mRNA expression were upregulated until 56 days after HI, but Foxpl and ER81 mRNA expression decreased from day 14 to 56 following HI. In addition, Otx-1 and RORI3 mRNA expression decreased from day 3 to 28 after HI. These findings revealed Fxogl mRNA overexpression and varying degrees of restoration in the neonatal rat brain following HI.

Assessment of the Effect of Cardiomyocyte Transplantation on Left Ventricular Remodeling and Function in Post-Infarction Wister Rats by Using High-frequency Ultrasound

The effects of cardiomyocyte grafting on left ventricular （LV） remodeling and function in rats with chronic myocardial infarction were evaluated using high-frequency ultrasound. Chronic myocardial infarction was induced in 50 Wister rats by ligating the left anterior descending artery. They were randomized into two groups： a trial group that received neonatal rat cardiomyocyte trans- plantation （n=25） and a control group which were given intramyocardial injection of culture medium （n=25）. The left ventricular （LV） geometry and function were evaluated by high-frequency ultrasound before and 4 weeks after the cell transplantation. After the final evaluation, all rats were sacrificed for histological study. The results showed that 4 weeks after the cell transplantation, as compared with the control group, the LV end-systolic dimension, end-diastolic dimension, end-systolic volume and end-diastolic volume were significantly decreased and the LV anterior wall end-diastolic thickness, LV ejection fraction and fractional shortening were significantly increased in the trial group （P〈0.01）. Histological study showed that transplanted neonatal rat cardiomyocytes were found in all host hearts and identified by Brdu staining. It was suggested that transplantation of neonatal rat cardiomyocytes can reverse cardiac remodeling and improve heart function in chronic myocardial infarction rats. High-frequency ultrasound can be used as a reliable technique for the non-invasive evaluation of the effect of cardiomyocyte transplantation.

EFFECT OF MILD HYPOTHERMIA ON ACTIVITY NITRIC OXIDE SYNTHASE IN CORTICAL NEURONS AND GLYCEMIA LEVELS OF NEONATAL RATS WITH HYPOXIC ISCHEMIC BRAIN DAMAGE

Through investigating the effect of mild hypothermia on activity of nitric oxide snythase (NOS) in cortical neurons and glycemia levels of neonatal rats with hypoxic ischemic brain damage (HIBD). We studied the mechanism of protecting hypoxic ischemic neurons of mild hypothermia. We established neonatal rat HIBD models, used NOS immunohistochemistry and glycemia determination by micromethod. The number of cortical NOS positive neurons after hypoxic ischemia was significantly decreased as compared with controls. The glycemia levels was significantly increased than that controls. No significant difference was found in number of cortical NOS positive neurons and glycemia levels between 31℃ and 34℃ mild hypothemia. The results imply that hypothermia can decrease overproduction of NO through inhibiting the increase of the activity of NOS, and increase the glycemia levels, thus protect the hypoxic ischemic neurons.

Alterations in Caspase-3 in Juvenile Rats Treated Neonatally with Domoic Acid

The clinical presentation of schizophrenia involves a variety of symptoms, which in many cases include hallucinations and delusions. Experimentally revealed alterations in both pre-pulse inhibition (PPI) and latent inhibition (LI) are also apparent in individuals afflicted with this disorder. Many have speculated that altered synaptic connections are, in part, responsible for this subset of behavioral abnormalities. We have previously reported that neonatal chronic low-dose injections of domoic acid (DOM) produce adult rats with deficits in PPI and LI. The current study was conducted to determine whether this toxin-treatment would alter the degree of apoptosis occurring in the developing brain. Results revealed significant decreases in caspase-3 within the right prelimbic cortex (PrL) in both male and female DOM-treated rats suggesting that even modest alterations in glutamate (Glu) signaling during critical periods of central nervous system (CNS) maturation will modify ontogenetic processes in the prefrontal cortex (PFC) of the juvenile rat.

Effect of polygonatum polysaccharide on the hypoxia-induced apoptosis and necrosis in in vitro cultured cerebral cortical neurons from neonatal rats

BACKGROUND： Cardiocerebrovascular diseases induced cerebral circulation insufficiency and senile vascular dementia can result in ischemic/hypoxic apoptosis of central neurons, which we should pay more attention to and prevent and treat as early as possible. Traditional Chinese medicine possesses the unique advantage in this field. Polygonatum, a Chinese herb for invigorating qi, may play a role against the hypoxic apoptosis of brain neurons. OBJECTIVE ： To observe the protective effect of polygonatum polysaccharide on hypoxia-induced apoptosis and necrosis in cerebral cortical neurons cultured in vitro. DESIGN： A comparative experiment.SETTING： Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine. MATERIALS： The experiment was carried out in the Laboratory of Cell Biology, Institute of Basic Medical Sciences, Jiangxi Provincial Academy of Traditional Chinese Medicine from November 2003 to April 2005. Totally 218 Wistar rats （male or female） of clean degree within 24 hours after birth were purchased from the animal center of Jiangxi Medical College （certification number was 021-97-03）. METHODS：① Preparation of cerebral cortical neurons of rats： The cerebral cortical tissues were isolated from the Wistar rats within 24 hours after birth, and prepared to single cell suspension, and the cerebral cortical neurons of neonatal rats were in vitro cultured in serum free medium with Neurobasal plus B27 Supplement. ② Observation on the non-toxic dosage of polygonatum polysaccharide on neurons： After the neurons were cultured for 4 days, polygonatum polysaccharide of different dosages （1-20 g/L） was added for continuous culture for 48 hours, the toxicity and non-toxic dosage of polygonatum polysaccharide on neurons were observed and detected with trypan blue staining. ③Grouping： After hypoxia/reoxygenation, the cultured neurons were divided into normal control group, positive apoptotic group and polygonatum polysaccharide group. In the normal control group, the neurons were cultured at 37℃ in CO2 with the volume fraction of 0.05 under saturated humidity for 6 days. In the apoptotic positive group, the neurons were cultured with hypoxia for 12 hours after 4-day culture, and followed by reoxygenation for 48 hours. In the polygonatum polysaccharide group, polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to some neurons at 10 hours before the hypoxia culture, and then the neurons were cultured with hypoxia for 12 hours, followed by reoxygenation for 48 hours; polygonatum polysaccharide with the terminal concentration of 0.5, 1 and 1.5 g/L was added to the other neurons at 12 hours after hypoxia followed by reoxygenation for 48 hours.④ The Hoechst33342 fluorescence staining, Annexin V/PI flow cytometer, appearance of DNA agarose gel electrophoresis gradient strap and immunohistochemical staining were used to observe the expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax, and observe the effect of polygonatum polysaccharide against the hypoxic apoptosis of cerebral cortical neurons of neonatal rats. MAIN OUTCOME MEASURES： ① Toxicity and non-toxic dosage of polygonatum polysaccharide on neurons;② Apoptotic rate of neurons detected with Hoechst33342 fluorescence staining;③ Early apoptotic rate and necrotic rate of neurons detected with Annexin V/PI flow cytometer; ④DNA agarose gel electrophoresis ladder-like strap appeared or not;⑤ Expressions of Bcl-2, Bax and Caspase-3 apoptotic and anti-apoptotic proteins and the ratio of Bcl-2/Bax. RESULTS：① Polygonatum polysaccharide within 6 g/L had no cytotoxicity on the normal cultured cerebral cortical neurons （P 〉 0.05）. ②The apoptotic rates of neurons detected with Hoechst33342 fluorescence staining had significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 10 hours before the hypoxia culture [（13.00±4.52）%,（12.72±2.15）%, （11.80±1.18）%,（38.03±1.05）%, P 〈 0.01], and had no significant differences between the polygonatum polysaccharide groups and positive apoptosis group added to neurons at 12 hours after the hypoxia culture （36.77±1.45）%, （36.60±1.61）%, （36.37±2.02）%, （38.03±1.05）%, P 〉 0.05].③ Annexin V/PI flow cytometer detected that the anti-necrotic effect was enhanced with the increased concentration of polygonatum polysaccharide within 0.5-1.5 g/L （P 〈 0.01）. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia could significantly decrease the apoptotic rate of neurons induced by hypoxia/reoxygenation （P 〈 0.01）. ④ No DNA agarose gel electrophoresis ladder-like strap appeared in the groups with polygonatum polysaccharide of 0.5-1.5 g/L added at 10 hours before hypoxia;⑤ After Polygonatum polysaccharide of 0.5-1.5 g/L was added before hypoxia, the expression of Bcl-2 protein of hypoxic neurons was increased （P 〈 0.01）, and those of Bax protein and Caspase-3 protein were reduced （P 〈 0.01）, and the ratio of Bcl-2/Bax was increased （P 〈 0.01）. CONCLUSION： Polygonatum polysaccharide within 6 g/L has no cytotoxicity on the normal cultured cerebral cortical neurons. Polygonatum polysaccharide of 0.5-1.5 g/L added before hypoxia plays a role agains necrosis of neurons induced by hypoxia. Polygonatum polysaccharide of 0.5-1.5 g/L can significantly reduce the apoptosis of neurons induced by hypoxia through up-regulating the expression of Bcl-2 protein, down-regulating the expressions of Bax protein and Caspase-3 protein, and increasing the ratio of Bcl-2/Bax.

Embryonic skeleton development and neonatal learning and memory ability of rats anesthetized with pentobarbital sodium: Differences of administration occasion and time

BACKGROUND: Generally speaking, anesthesia is often used in gravid body and it has been already proved that many kind of medicine can result in malformation. OBJECTIVE: To explore embryonic skeleton development and neonatal learning and memory of rats anesthetized with pentobarbital sodium in gravid rats. DESIGN: A randomized control trial. SETTING: Laboratory Animal Center of Xuzhou Medical College. MATERIALS: A total of 80 adult female SD rats, of clean grade and weighing 220-240 g, were selected in this study. The main reagents were detailed as follows: pentobarbital sodium (Shanghai Xingzhi Chemical Plant, batch number: 921019); MG-2 maze test apparatus (Zhangjiagang Biomedical Instrument Factory); somatotype microscope (Beijing Taike Instrument Co., Ltd.). METHODS: ① A total of 160 SD rats of half males and females were selected in this study. All rats were copulated. The day that the plug was checked out in the vagina next day was looked as the first day of pregnancy. Gravid rats were divided randomly into four groups, including early anesthesia group, second anesthesia group, late anesthesia group and control group with 20 in each group. Rats in the early anesthesia group were injected with 25 mg/kg soluble pentobarbitone on the 7th day of pregnancy for once; rats in the second anesthesia group were anesthetized with 25 mg/kg soluble pentobarbitone on the 7th and the 14th days of pregnancy for once; rats in the late anesthesia group were anesthetized with 25 mg/kg soluble pentobarbitone on the 14th day of pregnancy for once; rats in the control group did not treat with anything. The time of anesthetizing was controlled in 3 to 4 hours and ether was absorbed while the time was not enough. ② Half of each group was sacrificed on day 20th of pregnancy and the fetus was taken out to be stained with alizarin red S. After stained, the fetal skeleton was examined. The learning and memorizing of one-month rats that were given birth by the rest gravid rats were tested through electric mare method. Determine their study ability according to their correct rate of 90% or above of arrival at the safe area in 20 s. After they finally learned to arrive at the safe area correctly, test them once more in 24 hours and record the correct rate of 15 times. MAIN OUTCOME MEASURES: The rate of malformation in fetus and ability of learning and memory in one-month rats. RESULTS: A total of 80 female rats were anesthetized in this experiment. Totally 490 immature rats were tested with maze testing machine and 196 fetuses were stained with alizarin red S to observe the development of their skeleton. However, one of the 80 female rats was led to death because of overdose. ① Malformation experiment: Learning ability of second anesthesia group was evidently different from the control group while the other two groups were not in the electric mare method. The fetal skeleton malformation rate of three experimental groups was 87.0%, 60.9% and 17.9%, respectively, while it was 5.6% in the control group. ② Electric mare method: Times of rats which arrived at the safe regions were respectively 49.0±31.0, 68.0±35.0, 47.0±31.0 and 44.0±21.0 in early anesthesia group, second anesthesia group, late anesthesia group and control group; and then, there was significant difference between the second anesthesia group and the control group (P < 0.05). Exact rates of memory of rats were respectively (64.36±14.35)%, (62.15±18.33)%, (54.19±12.28)% and (68.24±15.91)% in early anesthesia group, second anesthesia group, late anesthesia group and control group; and then, there were no significant differences as compared with the control group (P > 0.05). CONCLUSION: The influence of anesthesia with pentobarbital sodium is obvious in fetal skeleton development and learning and memory ability.

Culture and identification of neonatal rat brain-derived neural stem cells

BACKGROUND Timing of passaging,passage number,passaging approaches and methods for cell identification are critical factors influencing the quality of neural stem cells(NSCs)culture.How to effectively culture and identify NSCs is a continuous interest in NSCs study while these factors are comprehensively considered.AIM To establish a simplified and efficient method for culture and identification of neonatal rat brain-derived NSCs.METHODS First,curved tip operating scissors were used to dissect brain tissues from new born rats(2 to 3 d)and the brain tissues were cut into approximately 1 mm^(3)sections.Filter the single cell suspension through a nylon mesh(200-mesh)and culture the sections in suspensions.Passaging was conducted with TrypLTM Express combined with mechanical tapping and pipetting techniques.Second,identify the 5th generation of passaged NSCs as well as the revived NSCs from cryopreservation.BrdU incorporation method was used to detect self-renew and proliferation capabilities of cells.Different NSCs specific antibodies(anti-nestin,NF200,NSE and GFAP antibodies)were used to identify NSCs specific surface markers and muti-differentiation capabilities by immunofluorescence staining.RESULTS Brain derived cells from newborn rats(2 to 3 d)proliferate and aggregate into spherical-shaped clusters with sustained continuous and stable passaging.When BrdU was incorporated into the 5th generation of passaged cells,positive BrdU cells and nestin cells were observed by immunofluorescence staining.After induction of dissociation using 5%fetal bovine serum,positive NF200,NSE and GFAP cells were observed by immunofluorescence staining.CONCLUSION This is a simplified and efficient method for neonatal rat brain-derived neural stem cell culture and identification.

Puerarin exhibits greater distribution and longer retention time in neurons than astrocytes in a co-cultured system

The phytoestrogen puerarin has been shown to protect neurons and astrocytes in the brain, and is therefore an attractive drug in the treatment of Alzheimer’s disease, Parkinson’s disease and cerebral ischemia. Whether puerarin exhibits the same biological processes in neurons and astro-cytesin vitro has rarely been reported. In this study, cortical neurons and astrocytes of newborn Sprague-Dawley rats were separated, identiifed and co-cultured in a system based on Transwell membranes. The retention time and distribution of puerarin in each cell type was detected by lfuorescence spectrophotometry and lfuorescence microscope. The concentration of puerarin in both co-cultured and separately cultured neurons was greater than that of astrocytes. Puerarin concentration reached a maximum 20 minutes after it was added. At 60 minutes after its addi-tion, a scant amount of drug was detected in astrocytes; however in both separately cultured and co-cultured neurons, the concentration of puerarin achieved a stable level of about 12.8 ng/mL. The results indicate that puerarin had a higher concentration and longer retention time in neu-rons than that observed in astrocytes.

Baicalin protects neonatal rat brains against hypoxicischemic injury by upregulating glutamate transporter 1 via the phosphoinositide 3-kinase/protein kinase B signaling pathway

Baicalin is a flavonoid compound extracted from Scutellaria baicalensis root.Recent evidence indicates that baicalin is neuroprotective in models of ischemic stroke.Here,we investigate the neuroprotective effect of baicalin in a neonatal rat model of hypoxic-ischemic encephalopathy.Seven-day-old pups underwent left common carotid artery ligation followed by hypoxia（8% oxygen at 37°C） for 2 hours,before being injected with baicalin（120 mg/kg intraperitoneally） and examined 24 hours later.Baicalin effectively reduced cerebral infarct volume and neuronal loss,inhibited apoptosis,and upregulated the expression of p-Akt and glutamate transporter 1.Intracerebroventricular injection of the phosphoinositide 3-kinase/protein kinase B（PI3 K/Akt） inhibitor LY294002 30 minutes before injury blocked the effect of baicalin on p-Akt and glutamate transporter 1,and weakened the associated neuroprotective effect.Our findings provide the first evidence,to our knowledge that baicalin can protect neonatal rat brains against hypoxic-ischemic injury by upregulating glutamate transporter 1 via the PI3 K/Akt signaling pathway.

Experimental nerve transfer model in the neonatal rat

Clinically,peripheral nerve reconstructions in neonates are most frequently applied in brachial plexus birth injuries.Most surgical concepts,however,have investigated nerve reconstructions in adult animal models.The immature neuromuscular system reacts differently to the effects of nerve lesion and surgery and is poorly investigated due to the lack of reliable experimental models.Here,we describe an experimental forelimb model in the neonatal rat,to study these effects on both the peripheral and central nervous systems.Within 24 hours after birth,three groups were prepared:In the nerve transfer group,a lesion of the musculocutaneous nerve was reconstructed by selectively transferring the ulnar nerve.In the negative control group,the musculocutaneous nerve was divided and not reconstructed and in the positive control group,a sham surgery was performed.The animal's ability to adapt to nerve lesions and progressive improvement over time were depict by the Bertelli test,which observes the development of grooming.Twelve weeks postoperatively,animals were fully matured and the nerve transfer successfully reinnervated their target muscles,which was indicated by muscle force,muscle weight,and cross sectional area evaluation.On the contrary,no spontaneous regeneration was found in the negative control group.In the positive control group,reference values were established.Retrograde labeling indicated that the motoneuron pool of the ulnar nerve was reduced following nerve transfer.Due to this post-axotomy motoneuron death,a diminished amount of motoneurons reinnervated the biceps muscle in the nerve transfer group,when compared to the native motoneuron pool of the musculocutaneous nerve.These findings indicate that the immature neuromuscular system behaves profoundly different than similar lesions in adult rats and explains reduced muscle force.Ultimately,pathophysiologic adaptations are inevitable.The maturing neuromuscular system,however,utilizes neonatal capacity of regeneration and seizes a variety of compensation mechanism to restore a functional extremity.The above described neonatal rat model demonstrates a constant anatomy,suitable for nerve transfers and allows all standard neuromuscular analyses.Hence,detailed investigations on the pathophysiological changes and subsequent effects of trauma on the various levels within the neuromuscular system as well as neural reorganization of the neonatal rat may be elucidated.This study was approved by the Ethics Committee of the Medical University of Vienna and the Austrian Ministry for Research and Science(BMWF-66.009/0187-WF/V/3 b/2015)on March 20,2015.

Recombinant human erythropoietin for repair of white matter damage

Erythropoietin has been shown to exhibit neuroprotective effects in animal models. A neonatal rat model of hypoxic-ischemic white matter damage was established via bilateral carotid artery ligation in 4-day-old Sprague-Dawley rats. The rats were subsequently treated with recombinant human erythropoietin to observe pathological changes in the brain and long-term neurobehavioral functions before and after intervention. Results showed that the number of myelin basic protein-positive cells, which reflected myelin/oligodendrocyte damage, significantly increased, although the number of amyloid precursor protein-positive cells, which reflected axonaf injury, significantly decreased in periventricular white matter at 72 hours and 7 days following erythropoietin intervention. The number of glial fibrillary acidic protein-positive cells, indicating astrocytic damage, significantly decreased in periventricular white matter of erythropoietin-treated rats at 48 hours, 72 hours, 7 days and 26 days. Following erythropoietin intervention in the 30-day-old rats, head-turning time in the slope test was shortened and open-field test scores increased. These results suggested that erythropoietJn promoted repair of white matter damage, as well as improved neurobehavioral functions in a rat model of hypoxic-ischemic injury.

Detection of Circulating Autoantibodiestoβ_2-Adrenergic Receptors in Patients with Asthma

To investigate the roles of autoantibodies to β 2 adrenergic receptors in the pathogenesis of asthma, the positive chronotropic action of the β 2selective adrenergic agonist, clenbuterol, was investigated on cultured neonatal rat cardiomyocytes, firstly. Then we detected the autoantibodies to β 2adrenergic receptors through the sera of patients with asthma could inhibit the positive chronotropic action of clenbuterol. The results showed that all the sera of the patients with asthma (16 cases) had the autoantibodies to β 2 adrenergic receptors; in contrast, none of the control subjects (20 cases) had the autoantibodies to β 2 adrenergic receptors, and that the inhibitory autoantibodies were IgG type. This study suggests that the autoantibodies to β 2 adrenergic receptors may play an important role in the pathogenesis of asthma.

Transplantation of placenta-derived mesenchymal stem cells reduces hypoxic-ischemic brain damage in rats by ameliorating the inflammatory response

Hypoxic-ischemic brain damage(HIBD)is a common cause of infant death.The purpose of our research was to explore the immunoregulatory mechanism of placenta-derived mesenchymal stem cells(PD-MSCs)in HIBD treatment.Seven-day-old rat pups were randomly divided into HIBD,PD-MSC,fibroblast,and control groups.Forty-eight hours after HIBD induction,cells at a density of 5×104 cells/10μl were injected into the cerebral tissue in the PD-MSC and fibroblast groups.The TNF-α,interleukin-17(IL-17),interferon-γ(IFN-γ),and IL-10 levels were detected through quantitative real-time polymerase chain reaction(RT-PCR)and enzyme-linked immunosorbent assay(ELISA).Regulatory T cell(Tregs)populations were detected through flow cytometry,and forkhead box P3(Foxp3)was measured through western blot analysis.Behavioral tests and gross and pathological examinations showed that PD-MSC treatment exerted significantly stronger neuroprotective effects than the other treatments.The expression levels of pro-inflammatory cytokines were substantially upregulated after HI injury.Compared with fibroblast treatment,PD-MSC treatment inhibited the production of pro-inflammatory cytokines and increased the production of IL-10 in the ischemic hemispheres and peripheral blood serum(all P<0.01).Flow cytometry results showed a notable increase in the number of Tregs within the spleen of the HIBD group.Moreover,the number of Tregs and the Foxp3 expression levels were higher in the PD-MSC treatment group than in the HIBD and fibroblast groups(all P<0.01).Our research suggests that the mechanism of PD-MSC treatment for HIBD partially involves inflammatory response suppression.