Pten regulates neuronal arborization and social interaction in mice

CNS deletion of Pten in the mouse has revealed its roles in controlling cell size and number, thus providing compelling etiology for macrocephaly and Lhermitte-Duclos disease. PTEN mutations in individuals with autism spectrum disorders (ASD) have also been reported, although a causal link between PTEN and ASD remains unclear. In the present study, we deleted Pten in limited differentiated neuronal populations in the cerebral cortex and hippocampus of mice. Resulting mutant mice showed abnormal social interaction and exaggerated responses to sensory stimuli. We observed macrocephaly and neuronal hypertrophy, including hypertrophic and ectopic dendrites and axonal tracts with increased synapses. This abnormal morphology was associated with activation of the Akt/mTor/S6k pathway and inactivation of Gsk3beta. Thus, our data suggest that abnormal activation of the PI3K/AKT pathway in specific neuronal populations can underlie macrocephaly and behavioral abnormalities reminiscent of certain features of human ASD.     

Assessing the developmental trajectory of mouse models of neurodevelopmental disorders: Social and communication deficits in mice with Neurexin 1α deletion

Neurexin 1α mutations are strongly associated with neurodevelopmental disorders such as autism spectrum disorders and schizophrenia in humans. Studies using the Neurexin 1α knock‐out mouse have showed behavioral abnormalities of relevance to these disorders and baseline deficits in excitatory synaptic function have been described. However, little is known about the effect of Neurexin 1α deletion on behavior during development. This study examined the effects of Neurexin 1α deletion on behavior across a range of developmental time points to determine whether potential abnormalities follow a developmental trajectory. Pups lacking Neurexin 1α emitted a reduced number of ultrasonic vocalizations early in development combined with a restricted repertoire of calls indicative of a loss in complexity in vocal production and showed delays in reaching certain developmental milestones. Behavioral testing showed that juvenile and adult male Neurexin 1α knock‐out mice exhibited social deficits and increased levels of aggression, confirming previous findings. No increases in repetitive behaviors or deficits in motor learning or olfaction were seen. In conclusion, this research showed that Neurexin 1α deletion does result in social and communication deficits that follow a developmental trajectory. These are the first experimental data that associate a deletion of Neurexin 1α with alterations in behaviors relevant to autism spectrum disorder across development and highlight the importance of assessing the developmental trajectory in mouse models of neurodevelopmental disorders.     

Microinjection of Valproic Acid into the Ventrolateral Orbital Cortex Enhances Stress-Related Memory Formation

There is collecting evidence suggesting that the process of chromatin remodeling such as changes in histone acetylation contribute to the formation of stress-related memory. Recently, the ventrolateral orbital cortex (VLO), a major subdivision of orbitofrontal cortex (OFC), was shown to be involved in antidepressant-like actions through epigenetic mechanisms. Here, we further investigated the effects of the histone deacetylase inhibitor (HDACi) valproic acid (VPA) on stress-related memory formation and the underlying molecular mechanisms by using the traditional two-day forced swimming test (FST). The results showed that VPA significantly increased the immobility time on day 2 when infused into the VLO before the initial forced swim stress on day 1. The learned immobility response to the stress was associated with increased phosphorylation of extracellular signal-regulated kinase (ERK) in VLO and hippocampus on the first day. The levels of phosphorylated ERK (phospho-ERK) in VLO and hippocampus were significantly decreased when retested 24 h later. The pretreatment with intra-VLO VPA infusion further reduced the activation of ERK on day 2 and day 7 compared with the saline controls. Moreover, the VPA infusion pretreatment also induced a significantly decreased BDNF level in the VLO on day 2, whereas no change was detected in the hippocampus. These findings suggest that VPA enhance the memories of emotionally stressful events and the ERK activity is implicated in stimulating adaptive and mnemonic processes in case the event would recur.     

mGluR5-antagonist mediated reversal of elevated stereotyped, repetitive behaviors in the VPA model of autism

Autism spectrum disorders (ASD) are highly disabling developmental disorders with a population prevalence of 1-3%. Despite a strong genetic etiology, there are no current therapeutic options that target the core symptoms of ASD. Emerging evidence suggests that dysfunction of glutamatergic signaling, in particular through metabotropic glutamate receptor 5 (mGluR5) receptors, may contribute to phenotypic deficits and may be appropriate targets for pharmacologic intervention. This study assessed the therapeutic potential of 2-methyl-6-phenylethyl-pyrididine (MPEP), an mGluR5-receptor antagonist, on repetitive and anxiety-like behaviors in the valproic acid (VPA) mouse model of autism. Mice were exposed prenatally on day E13 to VPA and assessed for repetitive self-grooming and marble burying behaviors as adults. Anxiety-like behavior and locomotor activity were measured in an open-field. VPA-exposed mice displayed increased repetitive and anxiety-like behaviors, consistent with previously published results. Across both marble burying and self-grooming assays, MPEP significantly reduced repetitive behaviors in VPA-treated mice, but had no effect on locomotor activity. These results are consistent with emerging preclinical literature that mGluR5-antagonists may have therapeutic efficacy for core symptoms of autism.     

Longitudinal in vivo developmental changes of metabolites in the hippocampus of Fmr1 knockout mice

Fragile X syndrome (FXS) is the most common form of inherited mental retardation and is studied in the Fmr1 knockout (KO) mouse, which models both the anatomical and behavioral changes observed in FXS patients. In vitro studies have shown many alterations in synaptic plasticity and increased density of immature dendritic spines in the hippocampus, a region involved in learning and memory. In this study, magnetic resonance imaging (MRI) and ¹H magnetic resonance spectroscopy (MRS) were used to determine in vivo longitudinal changes in volume and metabolites in the hippocampus during the critical period of early myelination and synaptogenesis at post‐natal days (PND) 18, 21, and 30 in Fmr1 KO mice compared with wild‐type (WT) controls. MRI demonstrated an increase in volume of the hippocampus in the Fmr1 KO mouse compared with controls. MRS revealed significant developmental changes in the ratios of hippocampal metabolites N‐acetylaspartate (NAA), myo‐inositol (Ins), and taurine to total creatine (tCr) in Fmr1 KO mice compared with WT controls. Ins was decreased at PND 30, and taurine was increased at all ages studied in Fmr1 KO mice compared with controls. An imbalance of brain metabolites in the hippocampus of Fmr1 KO mice during the critical developmental period of synaptogenesis and early myelination could have long‐lasting effects that adversely affect brain development and contribute to ongoing alterations in brain function.     

Valproic acid stimulates proliferation of glial precursors during cortical gliogenesis in developing rat

Valproic acid (VPA) is a neurotherapeutic drug prescribed for seizures, bipolar disorder, and migraine, including women of reproductive age. VPA is a well‐known teratogen that produces congenital malformations in many organs including the nervous system, as well as later neurodevelopmental disorders, including mental retardation and autism. In developing brain, few studies have examined VPA effects on glial cells, particularly astrocytes. To investigate effects on primary glial precursors, we developed new cell culture and in vivo models using frontal cerebral cortex of postnatal day (P2) rat. In vitro, VPA exposure elicited dose‐dependent, biphasic effects on DNA synthesis and proliferation. In vivo VPA (300 mg/kg) exposure from P2 to P4 increased both DNA synthesis and cell proliferation, affecting primarily astrocyte precursors, as >75% of mitotic cells expressed brain lipid‐binding protein. Significantly, the consequence of early VPA exposure was increased astrocytes, as both S100‐β+ cells and glial fibrillary acidic protein were increased in adolescent brain. Molecularly, VPA served as an HDAC inhibitor in vitro and in vivo as enhanced proliferation was accompanied by increased histone acetylation, whereas it elicited changes in culture in cell‐cycle regulators, including cyclin D1 and E, and cyclin‐dependent kinase (CDK) inhibitors, p21 and p27. Collectively, these data suggest clinically relevant VPA exposures stimulate glial precursor proliferation, though at higher doses can elicit inhibition through differential regulation of CDK inhibitors. Because changes in glial cell functions are proposed as mechanisms contributing to neuropsychiatric disorders, these observations suggest that VPA teratogenic actions may be mediated through changes in astrocyte generation during development. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 780–798, 2016     

Aberrant induction of p19Arf-mediated cellular senescence contributes to neurodevelopmental defects

Valproic acid (VPA) is a widely prescribed drug to treat epilepsy, bipolar disorder, and migraine. If taken during pregnancy, however, exposure to the developing embryo can cause birth defects, cognitive impairment, and autism spectrum disorder. How VPA causes these developmental defects remains unknown. We used embryonic mice and human organoids to model key features of VPA drug exposure, including exencephaly, microcephaly, and spinal defects. In the malformed tissues, in which neurogenesis is defective, we find pronounced induction of cellular senescence in the neuroepithelial (NE) cells. Critically, through genetic and functional studies, we identified p19^Arf as the instrumental mediator of senescence and microcephaly, but, surprisingly, not exencephaly and spinal defects. Together, these findings demonstrate that misregulated senescence in NE cells can contribute to developmental defects.

Does cellular senescence contribute to developmental birth defects? This study shows that valproic acid induces cellular senescence in neuroepithelial cells, the precursors of all adult brain cells, and blocks their differentiation, suggesting that aberrant senescence in the embryo may contribute to postnatal cognitive defects.     

Subchronic Treatment of Donepezil Rescues Impaired Social,Hyperactive, and Stereotypic Behavior in Valproic Acid-Induced Animal Model of Autism

Autism spectrum disorder (ASD) is a group of pervasive developmental disorders with core symptoms such as sociability deficit, language impairment, and repetitive/restricted behaviors. Although worldwide prevalence of ASD has been increased continuously, therapeutic agents to ameliorate the core symptoms especially social deficits, are very limited. In this study, we investigated therapeutic potential of donepezil for ASD using valproic acid-induced autistic animal model (VPA animal model). We found that prenatal exposure of valproic acid (VPA) induced dysregulation of cholinergic neuronal development, most notably the up-regulation of acetylcholinesterase (AChE) in the prefrontal cortex of affected rat and mouse offspring. Similarly, differentiating cortical neural progenitor cell in culture treated with VPA showed increased expression of AChE in vitro. Chromatin precipitation experiments revealed that acetylation of histone H3 bound to AChE promoter region was increased by VPA. In addition, other histone deacetyalse inhibitors (HDACIs) such as trichostatin A and sodium butyrate also increased the expression of AChE in differentiating neural progenitor cells suggesting the essential role of HDACIs in the regulation of AChE expression. For behavioral analysis, we injected PBS or donepezil (0.3 mg/kg) intraperitoneally to control and VPA mice once daily from postnatal day 14 all throughout the experiment. Subchronic treatment of donepezil improved sociability and prevented repetitive behavior and hyperactivity of VPA-treated mice offspring. Taken together, these results provide evidence that dysregulation of ACh system represented by the up-regulation of AChE may serve as an effective pharmacological therapeutic target against autistic behaviors in VPA animal model of ASD, which should be subjected for further investigation to verify the clinical relevance.     

Prenatal exposure to valproate induces sex-, age-, and tissue-dependent alterations of cholesterol metabolism: Potential implications on autism

Here, we investigated the protein network regulating cholesterol metabolism in the liver and brain of adolescent and adult male and female rats prenatally exposed to valproate (VPA), a well validated experimental model of autism spectrum disorders (ASD). We were aimed at studying whether prenatal VPA exposure affected the proteins involved in cholesterol homeostasis in a sex-dependent manner. To this aim the protein network of cholesterol metabolism, in term of synthesis and plasma membrane trafficking, was analyzed by western blot in the liver and different brain areas (amygdala, cerebellum, cortex, hippocampus, nucleus accumbens, and dorsal striatum) of adolescent and adult male and female rats prenatally exposed to VPA. Our results show that physiological sex-dependent differences are present both in the liver and in brain of rats. Interestingly, VPA affects specifically the brain in an age- and region-specific manner; indeed, cerebellum, cortex, hippocampus and nucleus accumbens are affected in a sex-dependent way, while this does not occur in amygdala and dorsal striatum. Overall, we demonstrate that each brain area responds differently to the same external stimulus and males and females respond in a different way, suggesting that this could be related to the diverse incidences, between the sexes, of some neurodevelopmental pathologies such as autism, which displays a 3:1 male to female ratio.     

孤独症谱系障碍小鼠肠道屏障功能研究

研究孤独症谱系障碍（ASD）模型小鼠肠道屏障功能及肠道动力的改变情况,为微生物−肠−脑轴在ASD发病中的作用提供理论基础。

C57bl/6J雌鼠和雄鼠交配,丙戊酸钠（VPA）诱导的ASD模型及对照组小鼠分别于孕12.5天皮下注射VPA或生理盐水。BTBR模型组为BTBR T⁺Itpr3^tf/J小鼠交配所产幼雄鼠。各组小鼠出生3周后,每窝随机选择2只雄鼠,各组5窝,共计10只,纳入相应组别。各组小鼠6周开始按如下顺序进行行为学检测：旷场实验、埋珠实验、三室社交实验、发声检测和自我梳理实验,各检测间隔5 d。评估小鼠肠道动力（粪便含水量及小肠推进率）和小鼠肠道屏障功能：FITC-葡聚糖灌胃后光谱仪检测小鼠血清中FITC-葡聚糖水平;Western blot检测各组小鼠结肠TREK1,CLDN1,CLDN3,OCLN及ZO1蛋白的表达;qPCR检测各组小鼠结肠Il6,Tnf-α和Ifn-γ mRNA的表达情况。

行为学检测结果显示：与对照组相比,BTBR组和VPA组小鼠穿越旷场中间区次数和停留时间显著降低（P＜0.05）;于目标小鼠侧室的停留时间率显著降低（P＜0.05）;埋珠率和自我梳理时间均显著升高（P＜0.05）;在（20～50）kHz和（50～100）kHz频率范围的发声次数及持续时间均显著降低（P＜0.05）。与对照组相比,BTBR组小鼠粪便含水量显著降低（P＜0.05）,而VPA组小鼠的差异无统计学意义（P＞0.05）。BTBR组和VPA组小鼠小肠推进率与对照组相比无差异（P＞0.05）。此外,与对照组相比,BTBR组和VPA组小鼠血清FITC−葡聚糖水平均升高,结肠TREK1,CLDN1,CLDN3,OCLD蛋白的表达水平降低,结肠Il6,Tnf-α和Ifn-γ mRNA的表达显著升高,差异均具有统计学意义（P＜0.05）,而ZO1的表达差异无统计学意义（P＞0.05）。

鉴于BTBR及VPA模型小鼠均表现出与ASD患者类似的广泛行为障碍,上述模型可作为ASD研究的可靠参考。与此同时,两种ASD小鼠模型均出现肠道紧密连接蛋白表达下调,肠道屏障通透性和促炎细胞因子水平的增高,表明微生物−肠−脑轴在ASD发病中扮演重要角色,且对治疗ASD症状具有潜在临床价值。

     

Hippocampal and visuospatial learning defects in mice with a deletion of frizzled 9, a gene in the Williams syndrome deletion interval

Wnt signaling regulates hippocampal development but little is known about the functions of specific Wnt receptors in this structure. Frizzled 9 is selectively expressed in the hippocampus and is one of about 20 genes typically deleted in Williams syndrome. Since Williams syndrome is associated with severe visuospatial processing defects, we generated a targeted null allele for frizzled 9 to examine its role in hippocampal development. Frizzled 9-null mice had generally normal gross anatomical hippocampal organization but showed large increases in apoptotic cell death in the developing dentate gyrus. This increase in programmed cell death commenced with the onset of dentate gyrus development and persisted into the first postnatal week of life. There was also a perhaps compensatory increase in the number of dividing precursors in the dentate gyrus, which may have been a compensatory response to the increased cell death. These changes in the mutants resulted in a moderate decrease in the number of adult dentate granule cells in null mice and an increase in the number of hilar mossy cells. Heterozygous mice (the same frizzled 9 genotype as Williams syndrome patients) were intermediate between wild type and null mice for all developmental neuronanatomic defects. All mice with a mutant allele had diminished seizure thresholds, and frizzled 9 null mice had severe deficits on tests of visuospatial learning/memory. We conclude that frizzled 9 is a critical determinant of hippocampal development and is very likely to be a contributing factor to the neurodevelopmental and behavioral phenotype of patients with Williams syndrome.     

Expression of LKB1 and PTEN tumor suppressor genes during mouse embryonic development.

Germ-line mutations of LKB1 and PTEN tumor suppressor genes underlie the phenotypically related Peutz-Jeghers syndrome (PJS) and Cowden disease (CD), respectively. To analyze possible developmental roles of PTEN and LKB1, we have studied their mRNA expression during mouse embryonic development (E7-17.5) by in situ hybridization. Ubiquitous expression of both genes during early stages (E7-11) became more restricted in later embryonic development (E15-19) where LKB1 and PTEN showed prominent overlapping expression in e.g. gastrointestinal tract and lung. In contrast, LKB1 was selectively expressed at high levels in testis and PTEN was prominently expressed in skin epithelium and underlying mesenchyme. These results indicate that LKB1 and PTEN display largely overlapping expression patterns during embryonic development. Moreover, a high expression of these genes was observed in the tissues and organs affected in PJS and CD patients and in PTEN+/- mice.     

Chronic Powder Diet After Weaning Induces Sleep,Behavioral, Neuroanatomical,and Neurophysiological Changes in Mice

The purpose of this study is to clarify the effects of chronic powder diet feeding on sleep patterns and other physiological/anatomical changes in mice. C57BL/6 male mice were divided into two groups from weaning: a group fed with solid food (SD) and a group fed with powder food (PD), and sleep and physiological and anatomical changes were compared between the groups. PD exhibited less cranial bone structure development and a significant weight gain. Furthermore, these PD mice showed reduced number of neurogenesis in the hippocampus. Sleep analysis showed that PD induced attenuated diurnal sleep/wake rhythm, characterized by increased sleep during active period and decreased sleep during rest period. With food deprivation (FD), PD showed less enhancement of wake/locomotor activity compared to SD, indicating reduced food-seeking behavior during FD. These results suggest that powder feeding in mice results in a cluster of detrimental symptoms caused by abnormal energy metabolism and anatomical/neurological changes.     

Phosphorylation of PTEN and Akt in astrocytes of the rat hippocampus following transient forebrain ischemia

To ascertain whether the PTEN (phosphatase and tensin homolog deleted on chromosome 10)/Akt signaling pathway is activated during ischemic brain injury, we investigated the expression and phosphorylation of PTEN and Akt by immunohistochemistry in the rat hippocampus after transient forebrain ischemia. Weak immunoreactivity for PTEN and its phosphorylated form (p-PTEN) was constitutively expressed in hippocampal neurons and astrocytes of the control rats, but their upregulation was detected mainly in reactive astrocytes in the ischemic hippocampus. Increased immunoreactivity for PTEN and p-PTEN occurred specifically in astrocytes by day 1 and was sustained for more than 2 weeks. The spatiotemporal activation of Akt in the ischemic hippocampus mirrored that of p-PTEN expression. Post-ischemic activation of Akt, revealed by phosphorylated Akt (p-Akt) immunoreactivity, was first detected at day 1 and was maintained for at least 2 weeks. Double-labeling experiments revealed that the cells expressing PTEN, p-PTEN, or p-Akt were reactive astrocytes expressing glial fibrillary acidic protein. These results demonstrate the increased phosphorylation of PTEN and Akt in reactive astrocytes of the post-ischemic hippocampus, suggesting that the PTEN/Akt pathway is involved in the astroglial reaction in the rat hippocampus after transient forebrain ischemia.This research was supported by Korea Science and Engineering Foundation (R01-2002-000-00334-0(2002)).     

MicroRNA-21 protects from mesangial cell proliferation induced by diabetic nephropathy in db/db mice

Diabetic nephropathy (DN) is a major diabetic complication. But the initiating molecular events triggering DN are unknown. Recent researches have addressed the role of microRNAs in diabetes and its complications. In this study, we looked for microRNAs expression during early DN, and showed microRNA-21 (miR-21) expression was downregulated in response to early DN in vitro and in vivo. Over-expression of miR-21 inhibited proliferation of mesangial cells and decreased the 24-h urine albumin excretion rate in diabetic db/db mice. Moreover, we identified PTEN as a target of miR-21. We also found PI3 K and p-Akt increased in miR-21 treated mesangial cells and db/db mice. Overall, these studies for the first time provide evidence for the potential role of miR-21 in early DN.     

Effects of an H3R Antagonist on the Animal Model of Autism Induced by Prenatal Exposure to Valproic Acid

Autism spectrum disorders (ASD) are a group of neurodevelopmental disorders primarily characterized by impaired social interaction and communication, and by restricted repetitive behaviors and interests. Ligands of histamine receptor 3 (H3R) are considered potential therapeutic agents for the treatment of different brain disorders and cognitive impairments. Considering this, the aim of the present study is to evaluate the actions of ciproxifan (CPX), an H3R antagonist, on the animal model of autism induced by prenatal exposure to valproic acid (VPA). Swiss mice were prenatally exposed to VPA on embryonic day 11 and assessed for social behavior, nociceptive threshold and repetitive behavior at 50 days of life. The treatment with CPX (3 mg/kg) or saline was administered 30 minutes before each behavioral test. The VPA group presented lower sociability index compared to VPA animals that were treated with CPX. Compared to the Control group, VPA animals presented a significantly higher nociceptive threshold, and treatment with CPX was not able to modify this parameter. In the marble burying test, the number of marbles buried by VPA animals was consistent with markedly repetitive behavior. VPA animals that received CPX buried a reduced amount of marbles. In summary, we report that an acute dose of CPX is able to attenuate sociability deficits and stereotypies present in the VPA model of autism. Our findings have the potential to help the investigations of both the molecular underpinnings of ASD and of possible treatments to ameliorate the ASD symptomatology, although more research is still necessary to corroborate and expand this initial data.     

Delays in GABAergic interneuron development and behavioral inhibition after prenatal stress

Prenatal stress is associated with altered behavioral, cognitive, and psychiatric outcomes in offspring. Due to the importance of GABAergic systems in normal development and in psychiatric disorders, prenatal stress effects on these neurons have been investigated in animal models. Prenatal stress delays GABAergic progenitor migration, but the significance of these early developmental disruptions for the continued development of GABAergic cells in the juvenile brain is unclear. Here, we examined effects of prenatal stress on populations of GABAergic neurons in juvenile and adult medial frontal cortex (mFC) and hippocampus through stereological counting, gene expression, and relevant anxiety‐like and social behaviors. Postnatally, the total GABAergic cell number that peaks in adolescence showed altered trajectories in mFC and hippocampus. Parvalbumin neuron proportion in juvenile brain was altered by prenatal stress, but parvalbumin gene expression showed no differences. In adult brain, parvalbumin neuron proportions were altered by prenatal stress with opposite gene expression changes. Adult prenatally stressed offspring showed a lack of social preference on a three‐chambered task, increased anxiety‐like behavior on the elevated plus maze, and reduced center time in an open field. Despite a lack of significant group differences in adult total GABAergic cell populations, performance of these tasks was correlated with GABAergic populations in mFC and hippocampus. In conclusion, prenatal stress resulted in a delay in GABAergic cell number and maturation of the parvalbumin subtype. Influences of prenatal stress on GABAergic populations during developmentally dynamic periods and during adulthood may be relevant to the anxiety‐like behaviors that occur after prenatal stress. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 1078–1091, 2016     

Increases in tumor necrosis factor-alpha following transient global cerebral ischemia do not contribute to neuron death in mouse hippocampus

The actions of tumor necrosis factor-alpha (TNF-alpha) produced by resident brain cells and bone marrow-derived cells in brain following a transient global ischemia were evaluated. In wild-type mice (C57Bl/6J) following 20 min ischemia with bilateral common carotid artery occlusion (BCCAo), TNF-alpha mRNA expression levels in the hippocampus were significantly increased at 3 h and 36 h and exhibited a biphasic expression pattern. There were no hippocampal TNF-alpha mRNA expression levels at early time points in either wild-type mice bone marrow transplanted (BMT)-chimeric-TNF-alpha gene-deficient (T/W) or TNF-alpha gene-deficient mice BMT-TNF-alpha gene-deficient mice (T/T), although TNF-alpha mRNA levels were detectable in T/W BMT mice at 36 h. Histopathological findings showed no intergroup differences between wild-type and TNF-alpha gene-deficient mice at 4 and 7 days after transient ischemia. In addition, nuclear factor-kappaB (NF-kappaB) was activated within 12 h after global cerebral ischemia, but electrophoretic mobility shift assays (EMSA) showed no intergroup differences between wild type and TNF-alpha gene-deficient mice. In summary, early hippocampal TNF-alpha mRNA expression may not be related to bone marrow-derived cells, and secondary TNF-alpha expression as early as 36 h after ischemia probably resulted mainly from endogenous brain cells and possibly a few bone marrow-derived cells. Although we cannot exclude the possibility of the TNF-alpha contribution to the physiologic changes of hippocampus after transient global ischemia, these results indicate that TNF-alpha does not influence the morphological changes of the hippocampal neurons under our study condition.