Xin-Min Li

Excessive autophagy contributes to neuron death in cerebral ischemia.

Aims: To determine the extent to which autophagy contributes to neuronal death in cerebral hypoxia and ischemia. Methods: We performed immunocytochemistry, western blot, cell viability assay, and electron microscopy to analyze autophagy activities in vitro and in vivo.Results: In both primary cortical neurons and SH‐SY5Y cells exposed to oxygen and glucose deprivation (OGD)for 6 h and reperfusion (RP) for 24, 48, and 72 h, respectively, an increase of autophagy was observed as determined by the increased ratio of LC3‐II to LC3‐I and Beclin‐1 (BECN1) expression. Using Fluoro‐Jade C and monodansylcadaverine double‐staining, and electron microscopy we found the increment in autophagy after OGD/RP was accompanied by increased autophagic cell death, and this increased cell death was inhibited by the specific autophagy inhibitor, 3‐methyladenine. The presence of large autolysosomes and numerous autophagosomes in cortical neurons were confirmed by electron microscopy. Autophagy activities were increased dramatically in the ischemic brains 3–7 days postinjury from a rat model of neonatal cerebral hypoxia/ischemia as shown by increased punctate LC3 staining and BECN1 expression. Conclusion: Excessive activation of autophagy contributes to neuronal death in cerebral ischemia.

Increased hippocampal neurogenesis in the progressive stage of Alzheimer's disease phenotype in an APP/PS1 double transgenic mouse model

Alzheimers disease (AD) is a progressive neurodegenerative disease associated with senile β‐amyloid (Aβ) plaques and cognitive decline. Neurogenesis in the adult hippocampus is implicated in regulating learning and memory, and is increased in human postmortem brain of AD patients. However, little is currently known about the changes of hippocampal neurogenesis in the progression of AD. As brain tissues from patients during the progression of AD are generally not available, an amyloid precursor protein (APP)/presenilin1 (PS1) double transgenic mouse model of AD was studied. Bromodeoxyuridine (BrdU) labeling supported by doublecortin staining was used to detect proliferating hippocampal cells in the mice. Compared with age‐matched wild‐type controls, 9‐month‐old transgenic mice with memory impairment and numerous brain Aβ deposits showed increased numbers of proliferating hippocampal cells. However, 3‐month‐old transgenic mice with normal memory and subtle brain Aβ deposits showed normal hippocampal proliferation. Double immunofluorescent labeling with BrdU and either NeuN or glial fibrillary acidic protein was conducted in mice at 10 months (28 days after the last BrdU injection) to determine the differentiation of proliferating cells. The number of hippocampal BrdU‐positive cells and BrdU‐positive cells differentiating into neurons (neurogenesis) in 10‐month‐old mice was greater in transgenic mice compared with age‐matched controls, but the ratio of hippocampal BrdU‐positive cells differentiating into neurons and astroglia was comparable. These results suggest hippocampal neurogenesis may increase during the progression of AD. Targeting this change in neurogenesis and understanding the underlying mechanism could lead to the development of a new treatment to control the progression of AD.

Quetiapine alleviates the cuprizone-induced white matter pathology in the brain of C57BL/6 mouse.

Recent human studies employing new magnetic resonance imaging techniques and micro-array analyses feature schizophrenia as a brain disease with alterations in white matter (WM), which is mainly composed of oligodendrocytes (OLs) and their processes wrapping around neuronal axons. To examine the putative role of OLs in the pathophysiology and treatment of schizophrenia, animal studies are essential. In the present study, C57BL/6 mice were given 0.2% cuprizone (CPZ) in their diet for five weeks during which they drank distilled water without or with quetiapine (QTP, 10 mg/kg). The mice fed with normal chow were used as controls. CPZ is a copper chelator and has been reported to induce consistent demyelination in the brain of C57BL/6 mouse by specifically damaging OLs. QTP is an atypical antipsychotic widely used in the treatment of schizophrenia and other psychotic disorders. In accordance with previous studies, CPZ-exposed mice showed pervasive myelin breakdown and demyelination. The amount of myelin basic protein (MBP) in the cerebral cortex was decreased by CPZ-exposure as shown in Western-blot analysis. In addition, the demyelinated sites were teemed with activated microglia and astrocytes but a few myelin forming OLs. Moreover, the activity of copper-zinc superoxide dismutase decreased in the cerebral cortex of CPZ-exposed mice. However, all of these pathological changes in WM were either prevented or alleviated in CPZ-exposed mice co-administered with QTP. These results suggest that the CPZ-exposed C57BL/6 mouse is a potential animal model to study possible roles of OLs in the pathogenesis and treatment of schizophrenia.

Behavioral and neurobiological changes in C57BL/6 mice exposed to cuprizone.

C57BL/6 mice were given 0.2% cuprizone (CPZ) for 2 to 6 weeks while controls ate the same diet without CPZ. At various time points the animals were subjected to behavioral tests and their brains were analyzed. Mice exposed to CPZ for 2 and 3 weeks displayed more climbing behavior and lower prepulse inhibition, suggesting an increase in central nervous system activity and impaired sensorimotor gating. In addition, they showed lower activities of monoamine oxidase and dopamine beta hydroxylase in the hippocampus and prefrontal cortex, and had higher dopamine but lower norepinephrine levels in the prefrontal cortex. Mice exposed to CPZ for 4 to 6 weeks had less social interaction, which is an animal correlate of social withdrawal of patients with schizophrenia. Also, these CPZ-exposed mice showed evident brain demyelination, myelin break down, and loss of oligodendrocytes. At all time points the CPZ-exposed mice spent more time in the open arms of an elevated plus maze and exhibited spatial working memory impairment. These data are in line with evidence from human studies suggesting a putative role of white matter abnormality in the pathophysiology of schizophrenia.

Quantitative MRI and ultrastructural examination of the cuprizone mouse model of demyelination

The cuprizone mouse model of demyelination was used to investigate the influence that white matter changes have on different magnetic resonance imaging results. In vivo T2‐weighted and magnetization transfer images (MTIs) were acquired weekly in control (n = 5) and cuprizone‐fed (n = 5) mice, with significant increases in signal intensity in T2‐weighted images (p < 0.001) and lower magnetization transfer ratio (p < 0.001) in the corpus callosum of the cuprizone‐fed mice starting at 3 weeks and peaking at 4 and 5 weeks, respectively. Diffusion tensor imaging (DTI), quantitative MTI (qMTI), and T1/T2 measurements were used to analyze freshly excised tissue after 6 weeks of cuprizone administration. In multicomponent T2 analysis with 10 ms echo spacing, there was no visible myelin water component associated with the short T2 value. Quantitative MTI metrics showed significant differences in the corpus callosum and external capsule of the cuprizone‐fed mice, similar to previous studies of multiple sclerosis in humans and animal models of demyelination. Fractional anisotropy was significantly lower and mean, axial, and radial diffusivity were significantly higher in the cuprizone‐fed mice. Cellular distributions measured in electron micrographs of the corpus callosum correlated strongly to several different quantitative MRI metrics. The largest Spearman correlation coefficient varied depending on cellular type: T1 versus the myelinated axon fraction (ρ = −0.90), the bound pool fraction (ƒ) versus the myelin sheath fraction (ρ = 0.93), and axial diffusivity versus the non‐myelinated cell fraction (ρ = 0.92). Using Pearsons correlation coefficient, ƒ was strongly correlated to the myelin sheath fraction (r = 0.98) with a linear equation predicting myelin content (5.37ƒ − 0.25). Of the calculated MRI metrics, ƒ was the strongest indicator of myelin content, while longitudinal relaxation rates and diffusivity measurements were the strongest indicators of changes in tissue structure. Copyright

Behavioral and neurobiological changes in C57BL/6 mouse exposed to cuprizone: effects of antipsychotics.

Recent human studies suggest a role for altered oligodendrocytes in the pathophysiology of schizophrenia. Our recent animal study has reported some schizophrenia-like behaviors in mice exposed to cuprizone (Xu et al., 2009), a copper chelator that has been shown to selectively damage the white matter. This study was to explore mechanisms underlying the behavioral changes in cuprizone-exposed mice and to examine effects of the antipsychotics haloperidol, clozapine and quetiapine on the changes in the mice. Mice given cuprizone for 14 days showed a deficit in the prepulse inhibition of acoustic startle response and higher dopamine in the prefrontal cortex (PFC), which changes were not seen in mice given cuprizone plus antipsychotics. Mice given cuprizone for 21 days showed lower spontaneous alternations in Y-maze, which was not seen in mice treated with cuprizone plus the antipsychotics. Mice given cuprizone for 28 days displayed less social interactions, which was not seen in mice given cuprizone plus clozapine/quetiapine, but was seen in mice given cuprizone plus haloperidol. Mice given cuprizone for 42 days showed myelin sheath loss and lower myelin basic protein in PFC, caudate putamen, and hippocampus. The white matter damage in PFC was attenuated in mice given cuprizone plus clozapine/haloperidol. But the white matter damage in caudate putamen and hippocampus was only attenuated by clozapine and quetiapine, not by haloperidol. These results help us to understand the behavioral changes and provide experimental evidence for the protective effects of antipsychotics on white matter damage in cuprizone-exposed mice.

Quetiapine enhances oligodendrocyte regeneration and myelin repair after cuprizone-induced demyelination

Myelin and oligodendrocyte dysfunctions have been consistently found in patients with schizophrenia. The effect of antipsychotics on myelin disturbances is unknown. The present study examined the effects of quetiapine on oligodendrocyte regeneration and myelin repair in a demyelination animal model. C57BL/6 mice were fed with cuprizone (0.2% w/w) for 12 weeks to induce chronic demyelination and oligodendrocyte degeneration, after which cuprizone was withdrawn to allow recovery. Quetiapine (10mg/kg/day) or vehicle (water) was administrated orally to mice for 0, 2, 3, or 4 weeks after cuprizone withdrawal. Locomotor activity and Y-maze tests were used to evaluate behavioral changes in the mice. Immunohistochemical staining was used to detect morphological and biological changes in the brains. Cuprizone administration for 12 weeks resulted in severe demyelination, locomotor hyperactivity, and working memory impairment in mice. Remyelination occurred when cuprizone was withdrawn. Quetiapine treatment during the recovery period significantly improved the spatial working memory and increased myelin restoration. Quetiapine treatment also enhanced the repopulation of mature oligodendrocytes in the demyelinated lesions, which was associated with down-regulation of transcription factor olig2 in the process of cell maturation. The results of this study demonstrated that quetiapine treatment during the recovery period improves spatial working memory and promotes oligodendrocyte development and remyelination. This study supports the role of oligodendrocyte dysfunction in memory deficits in a schizophrenia mouse model and suggests that quetiapine may target oligodendrocytes and improve cognitive function.

The response of synaptophysin and microtubule‐associated protein 1 to restraint stress in rat hippocampus and its modulation by venlafaxine

As part of our continuing study of neural plasticity in rat hippocampus, we examined two structural proteins involved in neuronal plasticity, synaptophysin (SYP) and microtubule‐associated protein 1 (MAP1) for their response to repeated restraint stress and modulation of such response by the antidepressant drug venlafaxine. This drug has the pharmacological action of inhibiting the reuptake of serotonin and norepinephrine in nerve terminals. We subjected the rats to restraint stress for 4 h per day for three days, and then injected the animals intraperitoneally (i.p.) with vehicle or 5 mg/kg/day of venlafaxine for various time periods. In all, eight groups of 10 rats each were used. The expression of these two proteins in hippocampal tissue of the rats was examined by means of western blot and immunohistochemical staining techniques. We found that restraint stress decreased the expression of SYP in the rat hippocampus by 50% (p < 0.01), and increased the expression of MAP1 by 60% (p < 0.01). SYP returned to the pre‐stress levels in three weeks and MAP1 in two weeks. In animals treated with venlafaxine post‐stress, SYP returned to pre‐stress levels after 2 weeks and MAP1 after 1 week. These findings enhance our understanding of the compromise of the hippocampus by stressful assaults, and may be relevant to the action of venlafaxine in the treatment of patients with major depression, a mental disease thought to be related to the mal‐adaptation of subjects to environmental stressors.

Region-specific susceptibilities to cuprizone-induced lesions in the mouse forebrain: Implications for the pathophysiology of schizophrenia.

Cuprizone (CPZ) is a neurotoxic agent acting as a copper chelator. In our recent study, C57BL/6 mice given dietary CPZ (0.2%) showed impairments in spatial working memory, social interaction, and prepulse inhibition. These abnormalities are reminiscent of certain schizophrenia symptoms and are not likely due to damage in the whole brain or in any single white matter tract/brain region. We hypothesized that white matter damage resulting from CPZ-treatment may be site-specific rather than universal. We examined the forebrains of C57BL/6 mice given the CPZ-containing diet and compared them with those of controls. We assessed CPZ-induced demyelination in main white matter tracts of the forebrain, evaluated myelin break down in the neuropil of the main olfactory bulb (MOB), cerebral cortex (CTX), caudate putamen (CP), hippocampus (HP), thalamus (TH), and hypothalamus (HY), and counted the number of myelin sheath forming oligodendrocytes (OLs) in CTX, CP, TH, and HY. Obvious demyelination was observed in the corpus callosum, external capsule, CP, and dorsal hippocampal commissure whereas other tracts seemed to be unaffected. The neuropil of CTX, HP and MOB showed myelin break down, which was mild in TH and HY. The number of OLs was decreased in all the above regions of CPZ-treated mice although the degree of OL loss was not consistent across regions. The data provide further support for white matter abnormalities contributing to schizophrenia-like behaviors in mice.

Quetiapine attenuates the depressive and anxiolytic-like behavioural changes induced by global cerebral ischemia in mice

Recently, we have reported that quetiapine, an atypical antipsychotic drug, prevents memory impairment and hippocampus neurodegeneration induced by global cerebral ischemia (GCI). In the present study, we examined the possible effects of quetiapine on other behavioural deficits, including the depressive and anxiolytic-like behavioural consequences of GCI. Mice were treated with quetiapine (5 or 10mg/kg/day; intraperitoneal (i.p.)) for 14 days. On Day 15, the animals were subjected to GCI. GCI resulted in a decrease of striatal tyrosine hydroxylase (TH) immunostaining and induced depressive and anxiolytic-like behavioural changes. The behavioural changes were indicated by a significant increase in the immobility duration in a tail-suspension test, and an increase in the time spent in the light box in a light/dark box test. Pre-administration of quetiapine significantly alleviated the decreased TH immunostaining and attenuated the depressive and anxiolytic-like behavioural changes induced by GCI. These results enhance our understanding about the mechanisms of quetiapine and suggest a wider perspective for the clinical use of quetiapine.