Induction of GADD45α protects M17 neuroblastoma cells against MPP*.

Growth arrest and DNA-damage-inducible protein 45α (GADD45α) is an important member of the family of growth arrest and DNA damage-inducible (GADD) proteins. The expression patterns and possible roles of GADD45α in Parkinson's disease (PD) are so far less understood. In this study, we found that 1-methyl-4-phenylpyridinium (MPP+) treatment up-regulates the expression of GADD45α in both a time-dependent manner and a dose-dependent manner in human dopamine neuroblastoma M17 cells. The up-regulation of GADD45α was abolished by pretreatment with the c-Jun N-terminal kinases (JNK) inhibitor SP600125 but not the p38 specific inhibitor SB203580. Further study revealed that c-Jun silencing abolished the effects of MPP+ on the expression of GADD45α. Important, ChIP studies verified the ability of c-Jun to bind to the GADD45 promoter. In addition, we found that inhibition of GADD45α by small RNA interference exacerbates the impaired cell viability, LDH release, and apoptosis induced by MPP+. Correspondingly, silence of GADD45 exacerbated Caspase-3 activation induced by MPP+. These data suggested a neuroprotective effect of GADD45α against MPP+ neurotoxicity.

[A rat model of focal lymph encephalopathy established by partial ligation of the cerebral superficial artery].

OBJECTIVE: To establish a rat model of focal lymphatic encephalopathy by partial ligation of the cerebral superficial artery for observation of the changes of Virchow-Robin spaces (VRS). METHODS: Thirty male SD rats were randomized into 3 groups (n=10), including two model groups and a sham-operated group. The rats in the model groups were subjected to partial ligation of the cerebral superficial arteries under EEG monitoring to induce focal lymphatic encephalopathy, and those in the sham-operated group underwent only dissociation of the cerebral superficial artery without ligation. The rats in the two model groups were executed at 24 and 48 h, and those in the sham-operated group at 48 h following the operation, respectively. Frozen sections of the brain tissues were prepared for microscopic morphological observation and quantitative analysis of the VRS using HE staining and an image analysis system, respectively. RESULTS: EEG remained normal during the operations suggesting intact brain function. Partial ligation of the cerebral superficial arteries resulted in obvious dilation of the VRS in the cerebral cortex and subcortical medulla, and the tissues around the dilated VRSs appeared pale and structurally loosened. The two model groups showed significantly enlarged VRS areas as compared to the sham-operated group (P<0.01), but no significant differences were found in the mean VRS areas between the two model groups. CONCLUSION: Partial dilation of the cerebral superficial artery is effective and convenient to induce focal lymphatic encephalopathy in rats, and this model can be ideal for studying focal cerebral lymph circulation.

[Correlation of ABO groups to hypertensive intracerebral hemorrhage].

OBJECTIVE: To study the relationship between ABO blood groups and hypertensive intracerebral hemorrhage (HICH). METHODS: The clinical data of 425 patients with HICH admitted to Nanfang Hospital were collected to analyze the relationship between the ABO blood groups and the occurrence of HICH, with normal Han Chinese subjects serving as the controls. RESULTS: Compared to the officially documented distribution of ABO groups in Chinese population (O 34.11%, B 28.98%, A 28.29%, AB 8.69%) and in Guangzhou residents (O 46.00%, B 25.00%, A 23.00%, AB 6.00%), a significant difference was noted in the blood group distribution in this cohort (O 45.10%, A 26.00%, B 24.00%, AB 4.90%). O blood type individuals with HICH showed a higher morbidity than others. Th ABO blood type distribution in this cohort showed no significant difference from that in the control group (P>0.05), but differed significantly from the Chinese norm (P<0.05). CONCLUSION: The ABO blood group is a factor contributing to the occurrence of HICH. O blood type is related to cerebral hemorrhage, and may serve as a risk factor for HICH.

[Association of cytochrome P4502D6 gene polymorphism with the susceptibility of heroin spongiform leucoencephalopathy].

OBJECTIVE: To elucidate the relation between cytochrome P4502D6 (CYP2D6) gene polymorphism and the susceptibility of heroin spongiform leucoencephalopathy (HSLE). METHODS: With polymerase chain reaction-restriction fragment length polymorphism technique, the cytochrome P4502D6 gene polymorphisms were analyzed in HSLE cases and control subjects. RESULTS: The frequencies of CYP2D6 (CYP2D6/C188, CYP2D6/L2938, CYP2D6/G4268) gene mutations were higher in HSLE patients than in the controls. CONCLUSION: The CYP2D6 gene mutation is associated with a high risk of HSLE.

[Clinical and neuroimaging features of heroin spongiform leukoencephalopathy].

OBJECTIVE: To summarize the clinical and radiographic characteristics of heroin spongiform leukoencephalopathy (HSLE). METHODS: A clinical analysis of 42 cases of HSLE was conducted. RESULTS: Clinically, the patients with HSLE all had a positive history of inhalation of heated heroin vapor with acute or subacute onset in most cases, presenting initially cerebellar signs. Pyramidal tract lesion was frequently involved, but the sensory system usually remained normal. The consciousness disturbances may occur in the serious cases. Brain CT and magnetic resonance imaging (MRI) revealed extensive symmetric white matter lesions in the cerebrum and cerebellum, and in serious cases, the midbrain and pons could be damaged. Spongiform vacuoles degeneration of white matter characterized the predominant pathological changes. CONCLUSION: Spongiform leukoencephalopathy should be considered in a patient who shows acute cerebellar signs and reports a history of inhaling heated heroin vapor, and a definite diagnosis of HSLE can be made in such a case upon the identification of typical CT or MRI findings.

[Correlation between diffusion anisotropy of the white matter fibers and cognitive function in patients with leukoaraiosis].

OBJECTIVE: To investigate the correlation between the diffusion anisotropy of the white matter fibers and the cognitive function in patients with leukoaraiosis (LA). METHODS: Thirty-one LA patients were enrolled in this study, including 13 with grade LA-1 (mild), 12 with grade LA-2 (moderate) and 6 with grade LA-3 (severe) condition. The control group consisted of 18 subjects who were free of obvious clinical symptoms or had only mild dizziness and headache but with negative history for neural system diseases and in the absence of cognitive dysfunction, brain trauma, positive signs in neurological examinations, or abnormities in MRI examination. The Mini-mental State Examination (MMSE) was applied to evaluate the patients' cognitive function. The LA patients underwent examination with diffusion tensor MR imaging (DTI), and the FA and MD values in the normal-appearing white matter (NAWM) were measured. RESULTS: The cognitive function of the LA patients tended to decline with the decrease of the MMSE scores, and their scores for time orientation, place orientation and calculation were significantly lower than those of the control group (P<0.05). No significant difference was found in memory, language and comprehensive abilities between the LA and control groups. In LA-1, LA-2 and total LA cases, the FA value in the NAWM was positively, and the MD value inversely, correlated to the cognitive function with correlation coefficients ranging from 0.5 to 0.8 (P<0.05). CONCLUSION: The DTI parameters of NAWM region are correlated to the cognitive function of LA patients. DTI is far more sensitive than MRI in evaluating cognitive dysfunction in LA patients.

[Correlation between white matter lesion and memory impairment in patients with obstructive sleep apnea syndrome].

OBJECTIVE: To investigate the nature of white matter lesion and correlation to memory impairment in patients with obstructive sleep apnea syndrome (OSAS) by diffusion tensor imaging ( DTI). METHOD: We conducted a cross-sectional study of 20 middle-aged male people (10 with OSAS and 10 healthy controls) group-matched by age, educational level, and socioeconomic status. DTI was performed on those people with OSAS and on matched controls. Fractional anisotropy(FA) and average diffusion coefficient(ADC) values were measured respectively in bilateral white matter of the frontal lobe, anterior cingulate gyrus, anterior cingulum, etc. At the same time, WMS values were detected respectively. RESULTS: Relative to controls, patients with OSAS had a lower FA in the white matter of right frontal lobe (0.444-/+0.025), splenium of corpus callosum (0.776-/+0.028 ), left anterior cingulate gyrus (0.154-/+0.021), right anterior cingulate gyrus (0.152-/+0.017), left anterior cingulum (0.372-/+0.022), right anterior cingulum (0.351-/+0.029), left centrum semiovale (0.501-/+0.029), peripheric white matter of left anterior angle (0.242-/+0.031), peripheric white matter of left posterior angle (0.338-/+0.029), peripheric white matter of right posterior angle (0.360-/+0.022 ), left parahippocampal gyrus (0.167-/+0.027), right parahippocampal gyrus (0.177-/+0.023). Besides, FA values of OSAS patients in the white matter of right frontal lobe and left centrum semiovale were strongly positively correlated with WMS values. While in comparison with that of healthy controls, ADC of OSAS patients was significantly higher in the white matter of right frontal lobe (8.589-/+0.264 x e(-10) mm(2)/s), trunk of corpus callosum (8.197-/+0.253 x e(-10) mm(2)/s), splenium of corpus callosum (8.218-/+0.194 x e(-10) mm(2)/s), left anterior cingulate gyrus (12.151-/+0.454 x e(-10) mm(2)/s), right anterior cingulate gyrus (12.113-/+0.524 x e(-10) mm(2)/s), right anterior cingulum (8.954-/+0.177 x e(-10) mm(2)/s),left centrum semiovale (7.333-/+0.220 x e(-10) mm(2)/s) and peripheric white matter of left anterior angle (9.186-/+0.465 x e(-10) mm(2)/s). CONCLUSIONS: This study indicated that white matter and gray matter was both remarkably damaged in OSAS patients, which could contribute to memory impairment.

[Modified neurological tests for evaluating the severity of cerebral infarction and estimating the infarct area in rats].

OBJECTIVE: To test the reliability of quantitative neurological tests in evaluating the neurological deficits in rat models of cerebral ischemia. METHODS: Forty male SD rats (7 to 9 weeks old) were subjected to transient (1 h) middle cerebral artery occlusion (MCAO) to induce cerebral infarction and 9 received sham operation only. The motor and sensory functions of the rats were measured before and 1, 3, 7 and 14 days after MCAO by accelerating rotarod test and modified adhesive removal test. Fourteen days later, the rats were sacrificed for histological examination, and the lesion size was determined by Winroof image analysis. The neurological deficits of the rats with different lesion sizes and at different time points were analyzed. RESULTS: According to the infarct size, the rats after MCAO were divided into two subgroups with large lesions [(68.56+/-12.54)%] affecting both the cortical area and basal ganglia and small lesions [(12.45+/-9.06)%] affecting the basal ganglia. Significant differences in the results of modified adhesive removal test and rotarod test were found between the two groups, and a liner relationship was noted between the lesion size and the results of the neurological tests. CONCLUSION: The modified quantitative neurological tests can reliably evaluate the severity of the infarction and estimate the infarct size in rats with cerebral infarction.

[Roles of cyclooxygenase 2/2', 3'-cyclic nucleotide3' phosphohydrolase in the oligodendrocyte apoptosis in heroin-induced spongiform leucoencephalopathy].

OBJECTIVE: To investigate the regulation of cyclooxygenase (Cox)-2/2', 3'-cyclic nucleotide3' phosphohydrolase (CNPase) on the oligodendrocyte apoptosis in the pathogenesis of the heroin-induced spongiform leucoencephalopathy (HSLE). METHODS: Samples of frontal lobe, cerebellum, and corpus callosum were obtained from the brains during autopsy of 4 HSLE patients and 5 patients who died of diseases other than cerebral diseases (controls) and underwent light microscopy and electron microscopy. Immunocytochemistry was carried out to detect the expression of myelin basic protein (MBP), caspase-3, COX-2, and CNPase protein. Apoptosis was examined by TUNEL staining. RESULTS: Widespread demyelination was seen in the white matter of the frontal lobe, cerebellum, and corpus callosum of the HSLE cases, most severely in cerebellum. In he HSLE group, the levels of caspase-3 and COX-2 expression were significantly higher, and the level of CNPase was significantly lower than those of the control group (all P < 0.05). CONCLUSION: Widespread demyelination in the white matter is a prevailing pathological change of HSLE. Oligodendrocyte apoptosis is one of the causes of HSLE. The upregulation of COX-2 and downregulation of CNPase may contribute to the pathogenesis.

[Roles of bcl-2/bax expression and oligodendrocyte apoptosis in the pathogenesis of heroin-induced spongiform leucoencephalopathy].

OBJECTIVE: To investigate the role of oligodendrocyte apoptosis under the regulation of the bcl-2/bax protein expression in brain white matter in the pathogenesis of heroin-induced spongiform leucoencephalopathy (HSLE). METHODS: Samples of frontal lobe, cerebellum, and corpus callosum were obtained from the brains during autopsy of 4 HSLE cases and 5 normal controls and underwent light microscopy and electron microscopy. Immunocytochemistry was used to detect the expression of myelin basic protein (MBP), caspase-3, bcl-2 protein, and bax protein. RESULTS: Widespread demyelination was seen in the white matter of the frontal lobe, cerebellum and corpus callosum of the HSLE cases, most severely in the cerebellum. The levels of caspase-3 and bax expression of the HSLE group were significantly higher than those of the control group (both P <0.05) , however, the bcl-2 level of the HSLE group was no significantly different from that of the control group (P > 0.05). CONCLUSION: Widespread demyelination in the white matter is a prevailed pathological change of HSLE. Oligodendrocyte apoptosis under induced by the decrease of bcl-2/bax ratio may contribute to the pathogenesis.

[Effect of Tongxinluo on the proliferation and differentiation of rat embryonic neural stem cells].

OBJECTIVE: To investigate the effect of Tongxinluo in on the proliferation and differentiation of rat embryonic neural stem cells (NSCs). METHODS: NSCs were isolated from 12- to 14-day SD rat embryo and treated with Tongxinluo at different doses, and the proliferation and differentiation of the cells were observed by immunofluorescence staining at different time points. RESULTS: The ratio of embryonic NSCs labeled with nestin decreased soon after Tongxinluo treatment, but increased afterwards. Significant difference was noted in the number of cells labeled with beta-tubulin between Tongxinluo group and the control group 3 and 7 days after the treatment, and also between high-dose and low-dose Tongxinluo groups at 7 days. CONCLUSION: Tongxinluo can induce the proliferation and neuronal differentiation of rat embryonic NSCs, and the effect is related to the dose of Tongxinluo administered.

[Event-related potentials of relational memory in target and delay phases of working memory].

OBJECTIVE: To compare the spatiotemporal pattern of event-related potentials (ERPs) of relational memory retention between color-location binding and semantics-location binding. METHODS: The ERPs of 19 channels were recorded from 14 healthy subjects while performing the two binding delayed-match-to-sample tasks. A paired-sample t test was used for analysis of both the behavioral performance and ERPs, and statistical parametric mapping (SPM) of the t values was applied to ERPs. RESULTS: Behavioral performances (reaction time and accuracy) in color-location binding were significant better than those of semantics-location binding. The SPM(t) of the ERPs showed significant effects in the middle frontal region and most areas of the parietal lobe (about 200 ms), the bilateral prefrontal and frontal regions (260 approximately 320 ms), and the left occipital region (500 approximately 580 ms). CONCLUSION: Retention of semantics-location relation involves more brain regions than that of color-location relation, and the largest effect occurs in the frontal region.

[A diffusion tensor magnetic resonance imaging study of age-related cerebral white matter diffusion anisotropy in normal human adult].

OBJECTIVE: To investigate the correlation between cerebral white matter fraction anisotropy (FA) in normal human adults using the diffusion tensor magnetic resonance (MR) imaging (DTI). METHODS: Forty-five adults with normal cerebral white matter MRI findings in 3 age groups (n=15), namely 25 approximately 35 years (young), 45 approximately 55 years (middle-aged) and 65 years or above (elderly), underwent conventional MRI and diffusion tensor MR imaging. FA was measured in different regions of interest (ROIs) including the genu and splenium of the corpus callosum, posterior limb and anterior limb of the internal capsule, centrum semiovale, frontal white matter, thalamus and head of the caudate nucleus. RESULTS: The FA values of the corresponding regions were similar between the left and right hemispheres. The FA value in the genu of the corpus callosum, centrum semiovale and the frontal white matter decreased with age, showing significant differences between the 3 age groups (P<0.05). The FA value in the splenium of the corpus callosum decreased significantly with age, with significant differences between the elderly and young groups and between the elderly and middle-aged groups (P<0.05). The values in the posterior limb and anterior limb of the internal capsule also decreased significantly with age as shown by comparison between the elderly and young groups (P<0.05). No significant difference was found in the FA value of the thalamus and head of the caudate nucleus between the three groups (P>0.05). CONCLUSION: The FA values decrease with age, especially in the genu of corpus callosum, centrum semiovale and frontal white matter. The patient's age and age-related white matter degradation must be considered in DTI-based diagnosis of cerebral diseases.

[Pathological analysis of heroin spongiform leukoencephalopathy].

OBJECTIVE: To investigate the pathological characteristics of heroin spongiform leukoencephalopathy (HSLE). METHODS: Cerebral tissue specimens were obtained from 15 patients with HSLE and the histological observations under optical and electron microscopes were carried out by HE, Bielschowsky's, and chromotrope 2R-brilliant green staining. RESULTS: HSLE was characterized primarily by spongiform vacuolar degeneration of the cerebral white matter. Neurons in the gray matter, Purkinje and granular cells in the cerebella remain intact in all the cases. Numerous vacuoles, which merged to form larger cavities, appeared in the damaged white matter, and the axons survived in the deep white matter. The myelin sheath in the cerebellar white matter sustained more severe damages than those in the cerebral white matter. No vacuoles or lymphocyte infiltration occurred in the small peripheral vessels. CONCLUSION: HSLE is pathologically characterized by vacuolar degeneration due to primary damage of the myelin, and the spongiform vacuolar degeneration is closely associated with the severity of demyelination in the white matter.

[Relationship between polymorphisms of interleukin 10 promoter and serum levels of lipoprotein in the Chinese Han population].

OBJECTIVE: To study the relationship between polymorphisms of interleukin 10 (IL10QX) promoter and serum levels of lipoprotein in the healthy Chinese Han population. METHODS: PCR restriction fragment length polymorphism assay was used to detect the distribution of genotypes of IL10 -592,-819,-1082 in 200 healthy Chinese Han subjects. Serum levels of total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), low density lipoprotein-cholesterol (LDL-C) and very low-density lipoprotein (VLDL) in all subjects were measured to analyze the relationship with the polymorphisms of IL10 promoter. RESULTS: Comparing with AA genotype, the group with GA genotype at IL10 promoter -1082 position had a significant elevation of serum HDL-C level [(1.514+/-0.501) mmol/L vs. (1.261+/-0.346) mmol/L, t=-2.225, P=0.028] and a lower serum TG level[(1.701+/-1.836) mmol/L vs. (0.981+/-0.314) mmol/L,Z=-2.096,P=0.036]. The TG, TC, HDL-C, LDL-C and VLDL levels did not show any statistically significant differences among different genotypes (CC, AA, CA) of the IL10 -592, as well as the genotypes (TT, TA, AA) ofIL10 -819 (P>0.05). CONCLUSION: The results suggest that in the Chinese Han population, the polymorphism at position -1082 in the promoter region of IL10 gene may be associated with the serum HDL-C level and TG level.

[Therapeutic effect of glucose-6-phosphate polyclonal antibody on vasogenic brain edema in rats].

OBJECTIVE: To observe the therapeutic effect of glucose-6-phosphate polyclonal antibody (G-6-P pAb) on vasogenic brain edema (VBE) in rats. METHODS: Sixty Wistar rats were randomly divided into normal control group, VBE group, mannitol-treated edema group, and G-6-P pAb-treated edema group. After establishment of rat models of VBE by intraperitoneal injection of phenylephrine in the latter 3 groups, mannitol was injected through the femoral vein in mannitol group and G-6-P pAb injected intraperitoneally in G-6-P pAb group. The permeability of the blood-brain barrier (BBB) was determined by Evans blue (EB) extravasation method, and the brain water content in the gray and white matter measured with a moisture analyzer. RESULTS: G-6-P pAb administration significantly reduced the permeability of BBB as well as the water content in the white matter in comparison with mannitol treatment (P<0.01), but the two treatments showed no obvious difference in reducing the water content in the gray matter (P>0.05). CONCLUSION: Changes in G-6-P activity results in BBB permeability alteration in the condition of VBE, and G-6-P pAb has a selective therapeutic effect against VBE, especially white matter edema.

[Cloning and sequencing of the junction fragment of dystrophin gene with exons 3 to 5 deletion].

OBJECTIVE: To study the mechanisms of dystrophin gene deletion by cloning and sequencing the junction fragment of dystrophin gene with exons 3 to 5 deletion. METHODS: PCR was performed to verify dystrophin gene exons 3 to 5 deletion in a patient with Duchenne muscular dystrophy. A PCR-based genome-walking method was used to localize the breakpoint in introns 2 and 5, and the deletion-junction fragment was directly amplified by PCR approach with forward and reverse primers annealing to a DNA sequence as close as possible to the breakpoint in the introns 2 and 5. The sequencing result of the deletion-junction fragment was compared with the normal intron sequences. RESULTS: A sequence of 2113 bp containing the junction fragment was obtained. The 5' breakpoint was located in SINE/Alu element of intron 2, and the 3' breakpoint was located in the unique sequence near the sequence TTTAAA. The breakpoints were associated with a strong topoisomerase II cleavage site. A 26-bp fragment was inserted into the breakpoint and formed 3 duplications (GGCTTATATTTAA) of 13 bp around the deletion-junction fragment. CONCLUSION: Repeat sequence and strong topoisomerase II cleavage site around the breakpoint may predispose double-strand DNA breaks and recombination, which, in addition to the nonhomologous end-joining mechanism, may contribute as important factors to the gene deletion.

[Nestin activation after rat cerebral ischemia-reperfusion injury and its changes in response to Tongxinluo treatment].

OBJECTIVE: To investigate nestin activation in rat brain subjected to ischemia-reperfusion injury and its changes in response to Tongxinluo treatment. METHODS: Cerebral ischemia was induced by temporary middle cerebral artery occlusion (MCAO) in rats. At 3, 7, 14 and 21 days after MCAO, nestin expression in the ependyma, subventricular zone (SVZ), hippocampal subdentate gyrus zone (HDG) of the rats treated with Tongxinluo were guantified by immunohistochemistry. RESULTS: Compared with the sham operation group, nestin was significantly increased 7, 14 and 21 days after MCAO (P<0.05), and immunofluorescence of BrdU+nestin-positive neurons significantly increased in the SVZ. After treatment with Tongxinluo, the number of BrdU-positive neurons and BrdU+nestin-positive neurons significantly increased as compared with MCAO group (P<0.05). CONCLUSION: Focal cerebral ischemia in the rat results in rapid response and proliferation of neural stem cells in the SVZ and HDG in the ischemic hemisphere, and Tongxinluo may enhance the differentiation and proliferation capacity of the neural stem cells after MCAO.

Cloning and sequencing of junction fragment with exons 45-54 deletion of dystrophin gene.

OBJECTIVE: To study the mechanisms of dystrophin gene deletion, the junction fragment with exons 45-54 deletion were cloned and sequenced. METHODS: A Duchenne muscular dystrophy (DMD) patient with exons 45-54 deletion has been substantiated by PCR amplification of the exons. Then we used a PCR-based genome-walking method for localizing the breakpoints in introns 44 and 54. At last, the deletion-junction fragment was directly amplified by PCR approach with forward and reverse primers annealing to a DNA sequence as close as possible to the breakpoints in introns 45 and 54. The sequencing result of the deletion-junction fragment was compared with the normal intronic sequences. RESULTS: A total of 2716 bp sequence containing the junction fragment was obtained. The 5' breakpoint was located in LINE/L1 element of intron 44 and close to a matrix attachment region (MAR). The 3' breakpoint was located in the minor potential MAR with topoisomerase II cleavage sites around. Beside the 3' breakpoint there was a 6 bp palindromic sequence. A 4 bp microhomologous sequence (AGAG) was in the joint of the deletion-junction fragment. CONCLUSION: The nonhomologous recombination caused by L1 repeated element, topoisomerase II cleavage sites, MARs and the nonhomologous end joining of microhomologous sequence may be the important factors in this huge gene fragment deletion.