Cyclooxygenase-2 contributes to N-methyl-D-aspartate-mediated neuronal cell death in primary cortical cell culture

Cyclooxygenase isozymes (COX-1 and COX-2) are found to be constitutively expressed in brain, with neuronal expression of COX-2 being rapidly induced after numerous insults, including cerebral ischemia. Because overactivation of N-methyl-D-aspartate (NMDA) receptors has been implicated in the cell loss associated with ischemia, we characterized the expression of the COX isozymes in murine mixed cortical cell cultures and used isozyme-selective inhibitors to determine their relative contribution to NMDA receptor-stimulated prostaglandin (PG) production and excitotoxic neuronal cell death. Immunocytochemical analysis of mixed cortical cell cultures revealed that COX-2 expression was restricted to neurons, whereas COX-1 was expressed in both neurons and astrocytes. Brief exposure to NMDA (5 min; 100 microM) elicited a time-dependent accumulation of PGs in the culture medium that preceded neuronal cell death and correlated with the induction of COX-2 mRNA. COX-1 expression remained unchanged. Flurbiprofen, a nonselective COX-1/COX-2 inhibitor, blocked NMDA-stimulated PG production and attenuated neuronal death in a concentration-dependent manner. Similar results were obtained with the specific COX-2 inhibitor NS-398 (10-30 microM) but not with the selective COX-1 inhibitor valeryl salicylate (10-300 microM). Inhibition of total constitutive COX activity with aspirin (100 microM, 1.5 h) before NMDA exposure did not prevent subsequent NMDA-mediated neuronal cell death. However, neuronal injury in aspirin-pretreated cultures was attenuated by flurbiprofen administration after NMDA exposure. Finally, the protection afforded by COX-2 inhibition was specific for NMDA because neither flurbiprofen nor NS-398 protected neurons against kainate-mediated neurotoxicity. Together, these results support the conclusion that newly synthesized COX-2 protein contributes to NMDA-induced neuronal injury.     

Stereospecific analysis of omeprazole supports artemisinin as a potent inducer of CYP2C19

The purpose of the study was to determine the enantiomer pharmacokinetics of omeprazole and 5-hydroxy-omeprazole before and after administration of the antimalarial artemisinin to confirm artemisinin's ability to induce CYP2C19. Nine healthy male Vietnamese subjects were given a single 20 mg dose of omeprazole orally 1 week before (day - 7) artemisinin administration. Artemisinin was then given orally (500 mg) for 7 days (days 1-7). On days 1 and 7, a single 20 mg dose of omeprazole was coadministered with artemisinin. After a washout period of 6 days, a single 20 mg dose of omeprazole was again administered together with a single 500 mg of artemisinin (day 14). Stereoselective pharmacokinetics of omeprazole and 5-hydroxyomeprazole was determined on days of omeprazole administration. Seven days of artemisinin administration significantly decreased the AUC of both omeprazole enantiomers (day 7), compared with day 1 (P < 0.001). All values were normalized after the washout period. Artemisinin increased the AUC ratio of R-5-hydroxyomeprazole/R-omeprazole significantly (P < 0.01) on day 7. The AUC ratio of omeprazole sulphone/S-omeprazole did not differ between study days. Artemisinin decreased the AUC of S-omeprazole to the same extent as that of R-omeprazole in extensive CYP2C19 metabolizers. suggesting that artemisinin induces a different enzyme in addition to CYP2C19. These results support and strengthen earlier findings that artemisinin induces CYP2C19 as well as at least one enzyme other than CYP3A4.     

Optogenetic control of iPS cell‐derived neurons in 2D and 3D culture systems using channelrhodopsin‐2 expression driven by the synapsin‐1 and calcium‐calmodulin kinase II promoters

Development of an optogenetically controllable human neural network model in three‐dimensional (3D) cultures can provide an investigative system that is more physiologically relevant and better able to mimic aspects of human brain function. Light‐sensitive neurons were generated by transducing channelrhodopsin‐2 (ChR2) into human induced pluripotent stem cell (hiPSC) derived neural progenitor cells (Axol) using lentiviruses and cell‐type specific promoters. A mixed population of human iPSC‐derived cortical neurons, astrocytes and progenitor cells were obtained (Axol‐ChR2) upon neural differentiation. Pan‐neuronal promoter synapsin‐1 (SYN1) and excitatory neuron‐specific promoter calcium‐calmodulin kinase II (CaMKII) were used to drive reporter gene expression in order to assess the differentiation status of the targeted cells. Expression of ChR2 and characterisation of subpopulations in differentiated Axol‐ChR2 cells were evaluated using flow cytometry and immunofluorescent staining. These cells were transferred from 2D culture to 3D alginate hydrogel functionalised with arginine‐glycine‐aspartate (RGD) and small molecules (Y‐27632). Improved RGD‐alginate hydrogel was physically characterised and assessed for cell viability to serve as a generic 3D culture system for human pluripotent stem cells (hPSCs) and neuronal cells. Prior to cell encapsulation, neural network activities of Axol‐ChR2 cells and primary neurons were investigated using calcium imaging. Results demonstrate that functional activities were successfully achieved through expression of ChR2‐ by both the CaMKII and SYN1 promoters. The RGD‐alginate hydrogel system supports the growth of differentiated Axol‐ChR2 cells whilst allowing detection of ChR2 expression upon light stimulation. This allows precise and non‐invasive control of human neural networks in 3D.     

Protective effect of mesenchymal stem cell-derived exosomal treatment of hippocampal neurons against oxygen-glucose deprivation/reperfusion-induced injury

BACKGROUND:Individuals who survive a cardiac arrest often sustain cognitive impairments due to ischemia-reperfusion injury.Mesenchymal stem cell(MSC)transplantation is used to reduce tissue damage,but exosomes are more stable and highly conserved than MSCs.This study was conducted to investigate the therapeutic effects of MSC-derived exosomes(MSC-Exo)on cerebral ischemia-reperfusion injury in an in vitro model of oxygen-glucose deprivation/reperfusion(OGD/R),and to explore the underlying mechanisms.METHODS:Primary hippocampal neurons obtained from 18-day Sprague-Dawley rat embryos were subjected to OGD/R treatment,with or without MSC-Exo treatment.Exosomal integration,cell viability,mitochondrial membrane potential,and generation of reactive oxygen species(ROS)were examined.Terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nickend labeling(TUNEL)staining was performed to detect neuronal apoptosis.Moreover,mitochondrial function-associated gene expression,Nrf2 translocation,and expression of downstream antioxidant proteins were determined.RESULTS:MSC-Exo attenuated OGD/R-induced neuronal apoptosis and decreased ROS generation(P<0.05).The exosomes reduced OGD/R-induced Nrf2 translocation into the nucleus(2.14±0.65 vs.5.48±1.09,P<0.01)and increased the intracellular expression of antioxidative proteins,including superoxide dismutase and glutathione peroxidase(17.18±0.97 vs.14.40±0.62,and 20.65±2.23 vs.16.44±2.05,respectively;P<0.05 for both).OGD/R significantly impaired the mitochondrial membrane potential and modulated the expression of mitochondrial functionassociated genes,such as PINK,DJ1,LRRK2,Mfn-1,Mfn-2,and OPA1.The abovementioned changes were partially reversed by exosomal treatment of the hippocampal neurons.CONCLUSIONS:MSC-Exo treatment can alleviate OGD/R-induced oxidative stress and dysregulation of mitochondrial function-associated genes in hippocampal neurons.Therefore,MSCExo might be a potential therapeutic strategy to prevent OGD/R-induced neuronal injury.     

Mitochondrial complex I deficiency leads to increased production of superoxide radicals and induction of superoxide dismutase.

Mitochondria were isolated from skin fibroblast cultures derived from healthy individuals (controls) and from a group patients with complex I (NADH-CoQ reductase) deficiency of the mitochondrial respiratory chain. The complex I deficient patients included those with fatal infantile lactic acidosis (FILA), cardiomyopathy with cataracts (CC), hepatopathy with tubulopathy (HT), Leigh's disease (LD), cataracts and developmental delay (CD), and lactic acidemia in the neonatal period followed by mild symptoms (MS). Production of superoxide radicals, on addition of NADH, were measured using the luminometric probe lucigenin with isolated fibroblast mitochondrial membranes. Superoxide production rates were highest with CD and decreased in the order CD >> MS > LD > control > HT > FILA = CC. The quantity of Mn-superoxide dismutase (MnSOD), as measured by ELISA techniques, however, was highest in CC and FILA and lowest in CD. Plots of MnSOD quantity versus superoxide production showed an inverse relationship for most conditions with complex I deficiency. We hypothesize that oxygen radical production is increased when complex I activity is compromised. However, the observed superoxide production rates are modulated by the variant induction of MnSOD which decreases the rates, sometimes below those seen in control fibroblast mitochondria. In turn, we show that the variant induction of MnSOD is most likely a function of the change in the redox state of the cell experienced rather than a result of the complex I defect per se.     

Mechanism of the tumor suppressive effect of MnSOD overexpression.

The mitochondrial antioxidant protein manganese-containing superoxide dismutase (MnSOD) has been shown to be a new type of tumor suppressor protein. Overexpression of MnSOD protein inhibits growth in a wide variety of cancer types. This review examines the molecular mechanism of the tumor suppressive effect of MnSOD. Three species have been proposed to cause the tumor suppressive effect: superoxide radical, hydrogen peroxide and nitric oxide. At the present time, the evidence appears strongest that hydrogen peroxide is the effector molecule since both catalase and glutathione peroxidase has been shown to modulate the effect. Surprisingly, in different cancer cell lines, overexpression of GPx has been found to both decrease and increase the growth inhibitory effect of MnSOD overexpression. Knowledge of which molecule causes the tumor suppressive effect of MnSOD and the mechanism of action will likely lead to new therapies for the treatment of cancer.     

Astrocytes‐derived exosomes induce neuronal recovery after traumatic brain injury via delivering gap junction alpha 1‐20 k

Astrocytes are more resistant to ischemia and hypoxia in the acute phase of brain injury after traumatic brain injury (TBI). Previous study showed that gap junction alpha 1 (GJA1) phosphorylation can increase the survival of damaged astrocytes. The GJA1‐20 k expression in neurons co‐culture with astrocytes was positively correlated with exosomes uptake. This study aims to explore the effect of exogenous GJA1‐20 k carried by astrocyte‐derived exosomes on neurons apoptosis and mitochondrial function after TBI. Astrocytes were co‐cultured with the neuron with/without damage from air pressure. Exosomes were isolated, extracted from the culture medium by differential ultra‐centrifugation, and verified by electron microscopy. Immunofluorescence staining, tunnel, western blot were employed to detect exosomes marker CD60, apoptosis, and mitochondrial function related protein expression and GJA1‐20 k in cell culture. A rat model of hydraulic injury TBI was built, and exosomes was transferred. 2,3,5‐Triphenyltetrazolium chloride (TTC) staining and immunohistochemistry staining of Nissl and microtubule associated protein 2 were used to detect the brain damage. A transwell stereo culture model of astrocytes and TBI‐like injured neuron was constructed. The exosomes derived from astrocytes promoted the recovery of damaged neuron by in vitro exosome treatment. Compared with GJA1‐20 k knockout exosome control group, GJA1‐20 k exosomes were uptaken by neuron and downregulated the apoptosis rate and upregulated mitochondrial function to promote neuronal recovery. Finally, the results were validated by TTC staining and damaged tissue sections of rat TBI model. This study contributes to a better understanding of the astrocyte‐neuron protection mechanism in TBI and provides a potential new target for the treatment of TBI.     

Neurotoxicity of artemisinin analogs in vitro.

The sesquiterpene endoperoxide antimalarial agents arteether and artemether have been reported to cause neurotoxicity with a discrete distribution in the brain stems of rats and dogs after multiple doses. The nature and distribution of the brain lesions suggest a specific neuronal target, the identity of which is unknown. In order to further investigate artemisinin analog-induced neurotoxicity, we evaluated several in vitro models: fetal rat primary neuronal cultures, fetal rat secondary astrocyte cultures, and transformed neuronal cultures (rat-derived neuroblastoma NG108-15 and mouse-derived neuroblastoma Neuro-2a). Results indicate that toxicity was specific for neuronal cell types but not glial cells. Neurotoxicity, as indexed by liberation of lactate dehydrogenase and/or inhibition of radiolabelled-leucine uptake, was seen in all three neuronal culture types, implicating a common target. In vitro neurotoxicity was dose and time dependent. Acute exposure to drug results in delayed, but not immediate, manifestations of cell toxicity. Structure-activity comparisons indicate that substitutions at positions 9 and 10 and stereoisomerism at position 10 of the artemisinin backbone influence the degree of toxicity. The endoperoxide is necessary but not sufficient for toxicity. Sodium artesunate and dihydroartemisinin, a metabolite common to all artemisinin analogs currently being developed for clinical use, are the most potent of all analogs tested. These results are consistent with a specific neuronal target, but the identity of the target(s) remains unknown.     

Mechanisms of N-methyl-D-aspartate-induced apoptosis in phencyclidine-treated cultured forebrain neurons

Chronic administration of phencyclidine (PCP) to rats has been demonstrated to produce a sensitized locomotor response to PCP challenge that is associated with apoptotic cell death and an up-regulation of the N-methyl-D-aspartate (NMDA) receptor. To determine the underlying mechanisms, dissociated forebrain cultures were treated for 2 days with 3 microM PCP. After washout of PCP, NMDA was added (in the presence of Mg(2+)) for 20 h. The uptake of a vital dye and the release of lactate dehydrogenase measured cell viability. Apoptosis was assessed by an enzyme-linked immunosorbent assay that was specific for fragmented (histone-associated) DNA and an in situ assay for nicked DNA, terminal dUTP nick-end labeling. These assays showed that the effect of a nontoxic concentration of NMDA (30 microM) became lethal to approximately one-third of the neurons after chronic (48-h) PCP treatment. This treatment also resulted in a 47% increase in NR1 subunit mRNA, suggesting that NMDA-induced neuronal cell death after chronic PCP is due to NMDA receptor up-regulation. Furthermore, exposure of PCP-treated cultures to NMDA led to increased expression of Bax and decreased expression of Bcl-X(L). The Bcl-X(L)/Bax ratio was markedly decreased by 30 microM NMDA in the PCP-treated, but not control, cultures. Addition of superoxide dismutase and catalase prevented the decrease in Bcl-X(L)/Bax. This study suggests that NMDA-induced changes in Bax and/or Bcl-X(L) involve the formation of reactive oxygen species. By extrapolation, these data suggest that PCP-induced apoptosis in vivo may involve similar mechanisms and that cultured neurons may be a suitable model for the mechanistic study PCP toxicity in vivo.     

Modulation of skin tumorigenesis by SOD.

Generation of reactive oxygen species (ROS) has been implicated in the development of cancer. Groundwork establishing mitochondria as a critical source of ROS generation and the role of manganese superoxide dismutase (MnSOD) in preventing mitochondria-mediated cell death have been well established. In a seemingly contradictory role, it also is well documented that increased MnSOD expression suppresses the carcinogenesis effect of ROS. Our recent studies demonstrated that overexpression of MnSOD reduced tumor incidence in the two-stage 7,12-dimethylbenz(a)-anthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate (TPA) skin carcinogenesis model. However, reduction of MnSOD by heterozygous knockout of the MnSOD gene (Sod 2+/-) did not lead to an increase in tumor incidence. Thus, how modulation of mitochondrial ROS levels alter the outcome of developing cancer is unclear. This review will provide background information on the sequence of ROS-mediated events in the mitochondria and evidence that suggests that the antioxidant and tumor suppressor functions of MnSOD are indeed inter-related. It also will offer insights into the mechanisms by which MnSOD modulates the outcome of early stage skin carcinogenesis.     

丹红注射液对体外神经元N-甲基-D-天冬氨酸损伤后的保护作用

目的：胎鼠原代皮层神经元培养,建立N-甲基-D-天冬氨酸（NMDA）诱导的兴奋性毒性损伤模型。研究丹红注射液对于神经元NMDA损伤后的保护作用。方法：胎鼠原代神经元培养并鉴定。培养至10d时,给予不同浓度NMDA,采用MTT法检测细胞生存率并测定LDH释放率,建立NMDA损伤模型。给予两种剂量的丹红注射液干预,测定细胞生存率及LDH释放率。采用Hoechst染色及TUNEL检测小剂量丹红干预组细胞凋亡情况。结果：随着NMDA浓度的增高,神经元生存率逐步下降,LDH释放率依次增加,以NMDA300μmol·L^-1组损伤最明显。丹红干预组细胞生存率提高,LDH释放率下降。小剂量丹红干预组凋亡细胞百分率减低。结论：丹红注射液可减轻NMDA诱导的体外神经元损伤,抑制神经元凋亡。     

Stem cell factor stimulates neurogenesis in vitro and in vivo

Cerebral ischemia stimulates neurogenesis in proliferative zones of the rodent forebrain. To identify the signaling factors involved, cerebral cortical cultures prepared from embryonic mouse brains were deprived of oxygen. Hypoxia increased bromodeoxyuridine (BrdU) incorporation into cells that expressed proliferation markers and immature neuronal markers and that lacked evidence of DNA damage or caspase-3 activation. Hypoxia-conditioned medium and stem cell factor (SCF), which was present in hypoxia-conditioned medium at increased levels, also stimulated BrdU incorporation into normoxic cultures. The SCF receptor, c-kit, was expressed in neuronal cultures and in neuroproliferative zones of the adult rat brain, and in vivo administration of SCF increased BrdU labeling of immature neurons in these regions. Cerebral hypoxia and ischemia may stimulate neurogenesis through trophic factors, including SCF.     

制备神经细胞实验性缺氧缺糖模型的简易方法

背景:在神经细胞培养中实验性缺氧缺糖在一定程度上模拟缺血性卒中,对于研究缺血性神经元损伤的进程和病理生理学机制有非常重要的用处.目的:在神经元培养时制作实验性缺氧缺糖模型.设计、时间及地点:分组对照观察,实验于2007-01/2008-03在北京大学第三医院中心实验室完成. 材料:17-19 d胎龄的Wistar大鼠.方法:细胞培养取17～19 d胎龄的Wistar大鼠的皮质神经元做原代细胞培养,并且去掉污染的非神经原细胞.缺氧缺糖的诱导分为3组:实验组将第7天的皮质神经元置于无糖甲衡盐溶液和2%去氧酶中,在37℃的潮湿保温箱中培育.空白对照组培养基为含20 mmol/L葡萄糖的无去氧酶平衡盐溶液.假性实验组培养基为含20 mmol/L葡萄糖和失活的去氧酶平衡盐溶液.主要观察指标:以血气分析进行氧浓度的测定;以相差显微镜观察实验组培养细胞神经元死亡状况;以用乳酸脱氢酶检测盒检测乳酸脱氢酶活性;以锥虫蓝染色观察缺氧缺糖对神经元存活力的影响.结果:氧浓度测定显示在加入去氧酶后培养基迅速产生缺氧状态;乳酸脱氢酶检测显示在用去氧酶和无糖平衡盐处理后,培养基中乳酸脱氢酶释放显著增加;锥虫蓝染色和相差显微镜检查显示经去氧酶和无糖平衡盐处理后实验组的细胞活力明显下降,大部分神经元在6 h死亡.结论:实验结果显示去氧酶与无精平衡盐液可联合用于神经元培养时产生缺氧缺糖状态,其在体外模拟脑缺血的相关研究中有重要作用.     

Treatment with recombinant human tumor necrosis factor-alpha protects rats against the lethality, hypotension, and hypothermia of gram-negative sepsis.

Tumor necrosis factor (TNF) is a peptide secreted by macrophages in response to endotoxin that can produce many of the changes seen in septic shock. After cecal ligation and puncture (CLP) rats gradually develop tachycardia, hypotension, tachypnea, and hypothermia. At 5 h post-CLP, rats have a peak in serum levels of endotoxin and 60% of rats have blood cultures that grow Gram-negative rods (Escherichia coli and Klebsiella pneumonia). At 20 h post-CLP all rats develop positive blood cultures. Serum levels of TNF are not reproducibly measurable in rats following CLP. Rats undergoing CLP have a 50-80% mortality with deaths usually occurring 24-72 h postinjury. Repetitive (twice daily x 6 d) i.p. injection of sublethal doses of recombinant human TNF-alpha (100 micrograms/kg) to rats undergoing CLP 1 d after the treatment period resulted in a significant reduction in mortality compared to control rats previously unexposed to rTNF (P less than 0.03). Animals treated with rTNF had no hypotension or hypothermia after CLP and regained normal food intake faster than control rats. 12 h after CLP the gene expression for manganous superoxide dismutase (MnSOD), an inducible mitochondrial metalloenzyme responsible for cellular resistance to injury from toxic reactive oxygen species, was higher in livers of rats treated with rTNF suggesting that the TNF treatment augmented expression of this protective enzyme. Unlike MnSOD, expression of the gene for copper-zinc SOD was not affected by CLP or rTNF treatment. The results suggest that prior treatment with recombinant TNF can ameliorate the lethality, hypotension, hypothermia, and anorexia of Gram-negative sepsis in rats and that the mechanism may be related to enhanced hepatic expression of the gene for MnSOD. Repeated administration of recombinant TNF may be a strategy to minimize mortality and morbidity of Gram-negative sepsis.     

Enhanced peroxynitrite formation is associated with vascular aging

Vascular aging is mainly characterized by endothelial dysfunction. We found decreased free nitric oxide (NO) levels in aged rat aortas, in conjunction with a sevenfold higher expression and activity of endothelial NO synthase (eNOS). This is shown to be a consequence of age-associated enhanced superoxide (.O(2)(-)) production with concomitant quenching of NO by the formation of peroxynitrite leading to nitrotyrosilation of mitochondrial manganese superoxide dismutase (MnSOD), a molecular footprint of increased peroxynitrite levels, which also increased with age. Thus, vascular aging appears to be initiated by augmented.O(2)(-) release, trapping of vasorelaxant NO, and subsequent peroxynitrite formation, followed by the nitration and inhibition of MnSOD. Increased eNOS expression and activity is a compensatory, but eventually futile, mechanism to counter regulate the loss of NO. The ultrastructural distribution of 3-nitrotyrosyl suggests that mitochondrial dysfunction plays a major role in the vascular aging process.     

Manganese superoxide dismutase promotes interaction of actin,S100A4 and Talin,and enhances rat gastric tumor cell invasion

It has been demonstrated that cancer cells are under high levels of oxidative stress and express high levels of Manganese superoxide dismutase (MnSOD) to protect themselves and support the anabolic metabolism needed for growth and cell motility. The aim of this study was to identify proteins that may have a correlation with invasion and redox regulation by mitochondrial reactive oxygen species (ROS). MnSOD scavenges superoxide anions generated from mitochondria and is an important regulator of cellular redox status. Oxidative posttranslational modification of cysteine residues is a key mechanism that regulates protein structure and function. We hypothesized that MnSOD regulates intracellular reduced thiol status and promotes cancer invasion. A proteomic thiol-labeling approach with 5-iodoacetamidofluorescein was used to identify changes in intracellular reduced thiol-containing proteins. Our results demonstrate that overexpression of MnSOD maintained the major structural protein, actin, in a reduced state, and enhanced the invasion ability in gastric mucosal cancer cells, RGK1. We also found that the expression of Talin and S100A4 were increased in MnSOD-overexpressed RGK1 cells. Moreover, Talin bound not only with actin but also with S100A4, suggesting that the interaction of these proteins may, in part, contribute to the invasive ability of rat gastric cancer.     

Normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental parkinsonism with intravenous nonviral gene therapy and a brain-specific promoter

The goal of this work was to normalize striatal tyrosine hydroxylase (TH) activity with intravenous nonviral TH gene therapy and at the same time eliminate ectopic TH gene expression in peripheral organs such as liver in the rat. TH-expression plasmids, containing either the SV40 promoter or the glial fibrillary acidic protein (GFAP) gene promoter, were globally delivered to the brain across the blood-brain barrier (BBB) after intravenous administration of pegylated immunoliposomes (PILs). The GFAP-TH- or SV40-TH-expression plasmids were encapsulated in the interior of 85-nm PILs, which were targeted across both the BBB and the neuronal cell membrane with a monoclonal antibody (mAb) to the transferrin receptor (TfR). Striatal TH activity was 98% depleted with the unilateral intracerebral injection of 6-hydroxydopamine. TH in the striatum ipsilateral to the lesion was normalized 3 days after the intravenous injection of 10 microg per rat of either the SV40-TH or the GFAP-TH plasmid DNA. Whereas the SV40-TH gene caused a 10-fold increase in hepatic TH activity, there was no increase in liver TH with the GFAP-TH gene. The GFAP-TH gene therapy caused an 82% reduction in apomorphine-induced rotation in the lesioned rats. Confocal microscopy using antibodies to TH, GFAP, and neuronal nuclei (NeuN) showed the GFAP-TH gene was selectively expressed in nigra-striatal neurons, with no expression in either cortical neurons, or astrocytes. These studies demonstrate that global delivery of exogenous genes to the brain is possible with intravenous nonviral gene transfer, and that ectopic gene expression is eliminated with the use of brain-specific gene promoters.     

高压氧对帕金森病大鼠多巴胺神经元保护作用的研究

目的探讨高压氧对帕金森病大鼠多巴胺神经元的保护作用。方法将68只雌性Sprague—Dawley大鼠随机分成5组：注射生理盐水高压氧处理组（A组，n=7）、全程高压氧处理模型组（B组，n=18）、未经高压氧处理模型组（C组，n=7）、造模后高压氧处理组（D组，n=18）、造模前高压氧处理组（E组，n=18）。在实验第1天至第7天给予A组、B组和E组大鼠高压氧治疗；而在实验第8天时，分别向B组、C组、D组及E组大鼠单侧脑黑质内定位注射6-羟基多巴胺以制作偏侧帕金森病大鼠模型，给予A组等量生理盐水定位注射。从实验第8天至结束，分别给予A组、B组及D组大鼠高压氧处理；并于造模后第9天，16天及21天每组各处死6只大鼠，取其纹状体用分光光度计测定超氧化物歧化酶（SOD）、丙二醛（MDA）和谷胱甘肽过氧化物酶（GSH—Px）含量，选用免疫组织化学方法测定黑质区域内酪氨酸羟化酶（TH）阳性细胞数量及胶质纤维酸性蛋白（GFAP）的表达。结果与C组比较，B、D、E组大鼠病变侧纹状体内SOD及GSH—Px活性显著增高，MDA含量及GFAP表达明显降低，6-羟基多巴胺毁损黑质区残存的TH阳性细胞数目明显增加。结论高压氧治疗可以显著提高机体抗自由基损伤功能、减弱胶质细胞效应发挥，从而有效保护脑黑质区多巴胺（DA）能神经元功能。     

Hematopoietic Stem Cell Regeneration Enhanced by Ectopic Expression of ROS-detoxifying Enzymes in Transplant Mice

High levels of reactive oxygen species (ROS) can exhaust hematopoietic stem cells (HSCs). Thus, maintaining a low state of redox in HSCs by modulating ROS-detoxifying enzymes may augment the regeneration potential of HSCs. Our results show that basal expression of manganese superoxide dismutase (MnSOD) and catalase were at low levels in long-term and short-term repopulating HSCs, and administration of a MnSOD plasmid and lipofectin complex (MnSOD-PL) conferred radiation protection on irradiated recipient mice. To assess the intrinsic role of elevated MnSOD or catalase in HSCs and hematopoietic progenitor cells, the MnSOD or catalase gene was overexpressed in mouse hematopoietic cells via retroviral transduction. The impact of MnSOD and catalase on hematopoietic progenitor cells was mild, as measured by colony-forming units (CFUs). However, overexpressed catalase had a significant beneficial effect on long-term engraftment of transplanted HSCs, and this effect was further enhanced after an insult of low-dose γ-irradiation in the transplant mice. In contrast, overexpressed MnSOD exhibited an insignificant effect on long-term engraftment of transplanted HSCs, but had a significant beneficial effect after an insult of sublethal irradiation. Taken together, these results demonstrate that HSC function can be enhanced by ectopic expression of ROS-detoxifying enzymes, especially after radiation exposure in vivo.