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.

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.

Effect of emulsified isoflurane on apoptosis of anoxia-reoxygenation neonatal rat cardiomyocytes

Objective:To explore the effect of emulsified isoflurane(EI)on apoptosis of anoxia-reoxygenation neonatal rat cardiomyocytea and relevant protein expression.Methods:Cardiac muscle anoxiareoxygenation damage model was established with culture in vitro neonatal rat cardiomyocytes.The cardiomyocytes were divided into control group,model group,fat emulsion group and EI group.The cardiomyocytes apoptosis rates and lactic dehydrogenase(LDH),superoxide dismutase(SOD)and malondialdehyde(MDA)index standardization were detected after relevant treatment The expression of apoptosis-related proteins Bel-2,Bax and Caspase-3 were detected with Western blot approach.Results:After hypoxia/reoxygenation(H/R)model was treated by EI,the cells apoptosis rate decreased and was dramatically below the fat emulsion group(P<0.05),Cardiomyocytes biochemical index detection presented that,compared with the control group that the LDH activity and MDA content dramatically increased(P<0.05),while the SOD activity notably decreased(P<0.05);compared with the H/R group,the SOD activity of the fat emulsion group and EI group increased(P<0.05);while the LDH activity and MDA content decreased(P<0.05).And the change of the EI group was more remarkable than the fat emulsion group(P<0.05).The Western blot analysis presented that,compared with the control group,the Bcl-2 protein expression of the other groups significantly decreased(P<0.05),the expressions of Bax protein and Caspase-3protein increased significantly(P<0.05);compared with H/R group,cardiomyocytes Bc1-2protein expression of EI group increased significantly(P<0.05),the expressions of Bax protein and Caspase-3 protein decreased significantly(P<0.05),and the change of EI group was more remarkable than the fat emulsion group(P<0.05).Conclusions:EI can inhabit the apoptosis of anoxia-reoxygenation damage model cardiomyocytes,and may he related to the up-regulation of expression of Bcl-2 and down-regulation of expression of Caspase-3 protein.

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.

Effects of the viability of Lactobacillus rhamnosus GG on rotavirus infection in neonatal rats

AIM:To study the effects of live and dead Lactobacillus rhamnosus GG(GG) on rotavirus infection in a neonatal rat model.METHODS:At the age of 2 d,suckling Lewis rat pups were supplemented with either live or dead GG and the treatment was continued daily throughout the experi-ment.At the age of 5 and 6 d the pups received oral rotavirus(RV) SA-11 strain.The pups were sacrificed at the age of 7 or 8 d by decapitation.The gastrointestinal tract was removed and macroscopic observations were done.The consistency of feces in the colon was classified using a four-tier system.RV was detected from the plasma,small intestine,colon and feces by real-time quantitative polymerase chain reaction(PCR).RESULTS:In this neonatal rat model,RV induced a mild-to-moderate diarrhea in all except one pup of the RV-inoculated rats.RV moderately reduced body weight development from day 6 onwards.On day 7,after 2 d of RV infection,live and dead GG groups gained significantly more weight than the RV group without probiotics [36%(P = 0.001) and 28%(P = 0.031),respectively].In addition,when compared with the RV control group,both live and dead GG reduced the weight ratio of colon/animal body weight to the same level as in the healthy control group,with reductions of 22%(P = 0.002) and 28%(P < 0.001),respectively.Diarrhea increased moderately in both GG groups.However,the diarrhea incidence and severity in the GG groups were not statistically significantly different as compared with the RV control group.Moreover,observed diarrhea did not provoke weight loss or death.The RV control group had the largest amount of RV PCR-positive samples among the RV-infected groups,and the live GG group had the smallest amount.Rats receiving live GG had significantly less RV in the colon(P = 0.027) when compared with the RV control group.Live GG was also more effective over dead GG in reducing the quantity of RV from plasma(P = 0.047).CONCLUSION:Both live and dead GG have beneficial effects in RV infection.GG may increase RV clearance from the body and reduce colon swelling.

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.

Glucocorticoid receptor expression in neonatal rat cortex following recurrent seizures The role in developing brain injury

BACKGROUND： Studies have explored changes in neonatal rat glucocorticoid receptor （GR） expression changes following mature brain injury. OBJECTIVE： To investigate the temporal and special changes of GR during brain development in rats with recurrent seizures. DESIGN, TIME AND SE＇n＇ING： A randomized, controlled animal experiment was performed at the Department of Pediatrics, Second Xiangya Hospital of Central South University, from February 2008 to March 2009. MATERIALS： Rabbit anti-rat GR monoclonal antibody was purchased from Santa Cruz Biotechnology, USA; goat anti-rabbit IgG was purchased from Zhongshan Goldenbridge Biotechnology, China. METHODS： A total of 48 Sprague-Dawley rats, 7 days old, were randomly assigned to control and seizure groups, with 24 animals in each group. Seizures were induced by inhalant flurothyl. MAIN OUTCOME MEASURES： Changes in GR protein expression in the rat cerebral cortex were detected by Western blotting analysis and immunohistochemistry. RESULTS： GR expression in the cerebral cortex of control rats significantly increased with aging （P 〈 0.05）, and varied in the frontal lobe, temporal lobe, and parietal lobe. GR was predominantly expressed in the cytoplasm early and rapidly increased in the nuclei. GR protein expression in the cerebral cortex after seizure was lower in the cytoplasm at 15 days and in nuclear protein at 19 days. CONCLUSION： GR expression displayed temporal and spatial changes in brain development. Recurrent seizures in neonatal rats cause abnormal GR expression and might play an important role in developing brain injury.

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.

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.

Effect of G-CSF and TPO on HIBD in neonatal rats

Objective:To observe effect of granulocyte colony-stimulating factor(G-CSF) and restructure human thrombopoietin on hypoxic-ischemic brain damage(HIBD) in new born rats.Methods:A total of 60 neonatal SD rats were selected and divided into 4 groups,with 15 in each group.Group A served as control group.Rats of Groups B-D were prepared for HIBD model by ligation of left common carotid artery combined with hypoxia method.Rats of Group A were only completed with free left common carotid artery without ligation and hypoxia operation.After HIBD model preparation,Group B was administrated with subcutaneous injection of normal saline for placebo treatment;Group C was administrated with cervical subcutaneous injection of 0.5 μg/10 g granulocyte colony stimulating factor(G-CSF) for 5 d(Once a day);Group D was administrated with intraperitoneal injection of 15 U/10 g recombinant human thromobopoietin(rhTPO) for treatment.After modeling for 7,14 and 21 d,5 rate were sacrificed in each group,respectively.Brain quality damage(%) conditions of experimental animals in each group were compared in different time points,and cerebral histopathological changes of each group were observed.Expression of nestin in rats of each group was detected by immunohistochemical method.Results:After modeling for 7,14 and 21 d,brain quality damages(%) of Groups B,C and D were significant higher than that of in Group A(P<0.05),while brain quality damage(%) degree of Group B was the highest in different time points,followed by Groups D and C,respectively.It was significant different compared among groups(P<0.05).The histopathological observation showed that degrees of brain damages in Groups C and D were significant lower than that of in Group B.After modeling for 7,14 and 21 d,nestin positive cell populations in Groups B,C,and D were significant higher than Group A(P<0.05).Nestin cell populations of Group C in different time points were significant higher than Groups B and D(P<0.05).There was no significant difference in nestin positive cell papulations,in the above time points between Groups B and D(P>0.05).Conclusions:Both G-CSF and TPO can protect the nervous system of HIBD neonatal rats.G-CSF can promote the proliferation and differentiation of neural precursor cells to decrease the degeneration and necrosis of nerve cell.TPO can obviously ameliorate morphology index of HIBD rats.Through regulating ratio of TIMP-1 and MMP-9,TPO can maintain the integrity of blood brain barrier to relieve the occurrence of brain damage.

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.

Effects of Neonatal Undernutrition on Development of the Dorsolateral Prefrontal Cortex Pyramidal Cells in the Rat

The dorsolateral prefrontal cortex (dlPFC) of the rat plays a role in the encoding of neuronal signals involved in conflict-induced behavioral adjustment, working memory, planning and executive abilities, attentional control and other cognitive responses. In altricial species, early perinatal undernutrition interferes with the morphofunctional organization of a number of central nervous system (CNS) structures including the prefrontal cortex. The effects of neonatal undernutrition on dendritic arbor density, perikaryon measurements, and the number of spines (detected by rapid-Golgi) of basilar dendritic segments in layer III pyramidal neurons of the dlPFC were examined in male Wistar rats on postnatal (PDs) 12, 20, and 30. In the underfed (U) subjects the distal portions of the dendritic arbors had a consistent hipoplasia, mainly on PD 30, with reduced cross sectional area, perimeter, and spine densities on the basilar dendrites on all days studied. Thus, the alterations of the dlPFC pyramidal neurons may interfere with the plastic synaptic activity and cognitive performance of rats subjected to the stress of early underfeeding. Characterizing these anatomical alterations may help to understand the disrupted cognitive processes associated with neonatal undernutrition.

Juvenile Play Behavior in Neonatally Underfed and Sensory Stimulated Wistar Rats

Play development in juvenile rats depends on specific sensory signals integrated at cortical, limbic and brain stem levels to modulate motoric, metabolic, motivational and social responses. Neonatal undernourishment disrupts the morphological and functional organization of the brain for adaptive responses including play performance. These alterations may be restored by preweaning exposure to sensory-enriched environments. This study was designed to determine in four experimental groups, Control (LC), Underfed (LU), Control Ligated/Stimulated (LCS), and Underfed Ligated/Stimulated (LUS), whether changes in juvenile play of neonatally underfed male rats by the nipple-ligated procedure of F0 dams and/or the handling of F1 rats may restore the deficiencies in juvenile play performance. The pinning frequency values in LC, LCS and LUS groups consistently increased until reaching a significant peak between postnatal days (PDs) 25 and 50 and then gradually declining until PD 60, when the play in pairs was significantly higher compared with the play in groups that follows the same sequence but with lower values in the stimulated groups. The results may reflect poor maternal care and lower somatosensory stimulation;and the sensory massage of LU F1 pups compared with the LC, LUS, and LCS rats. Fewer dorsal body contacts occurred in LU and LUS rats when playing in pairs than in groups. Results suggest that although handling has salutary effects on neuronal play structures, the reduced levels of total pinning and dorsal contacts, mainly in the play of rat pairs in LCS vs. LUS groups, were not fully recovered.

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.

Wound Closure on the Neonatal Rat Skin I. The Modulation of the Thickness of Epidermis at the Closing Incisional Wounds

Full-thickness incisional wounds were made on the dorsal skin of 1-day-old rats to elucidate the mechanism of the fluctuation of the epidermal thickness after the wound closure. The thickness of the epidermis covering the wound reached a peak around 96 h post-wounding (PW), and became thinner thereafter. The analyses of the cell proliferation and apoptosis at the epidermal wound regions revealed that the rate of TUNEL-positive cells that displays the cells undergoing apoptosis increased as the epidermis became thinner around 120 h PW. Next, immunohistochemical analyses using antibodies against keratinocyte differentiation marker proteins indicated that the delay or interruption of the spinous to granular transition from 96 to 120 h PW might result in the epidermal thickening in the wound region. Third, the region undyed with anti-caspase-14 antibody extended downward in the thickened epidermis by 96 h PW, and in turn, it became intensely and widely stained with this antibody in the thinning epidermis by 120 h PW. Taken together, it is likely that the delay and acceleration of the terminal differentiation, including cornification of the epidermal keratinocytes may coordinately cause the fluctuation of the thickness of the epidermis at the wound site in rat neonates.