Induction of heme oxygenase-1 (HO-1) in the contused spinal cord of the rat

The induction of heme oxygenase-1 (HO-1) was studied in intact spinal cords and injured spinal cords after a moderate, thoracic contusion injury. HO-1 was immunolocalized in the normal cord and along the axis of the cord at 1, 2, 3 and 4 days after contusion. Induction of this enzyme in astrocytes and microglia/macrophages was evaluated using immunofluorescent double labeling with monoclonal antibodies to HO-1 and either glial fibrillary acidic protein or the complement C3bi receptor. HO-1 was expressed in neurons in the normal spinal cord. After contusion, HO-1 was induced in both gray and white matter at the impact site. In segments of cord that were 1 cm proximal or distal to the injury, HO-1 was primarily induced in the dorsal columns and occasionally in the lateral white matter. This pattern of induction was noted at all time points. The HO-1 was induced primarily in microglia/macrophages. The distribution of the HO-1 positive cells closely correlated with the pattern of intraparenchymal hemorrhage. These findings demonstrate acute induction of HO-1 in non-neuronal cells in the injured spinal cord. Induction of HO-1 in glia may be a consequence of multiple factors including exposure to heme proteins, hypoxia and oxidative stress.     

Co-induction of HSP70 and heme oxygenase-1 in macrophages and glia after spinal cord contusion in the rat

HSP70 and heme oxygenase-1 (HO-1) are thought to be markers of cell injury and oxidative stress, respectively. We have immunolocalized these proteins in the spinal cord at 1-14 days after contusion. HSP70 and HO-1 were co-induced in glia and macrophages within the injured segment at all time points. This co-induction may reflect complementary functions that serve to protect these cells as they respond to the postcontusional environment.     

血红素氧合酶-2基因缺失对氧化应激性脑损伤的保护作用

目的 探讨血红素氧合酶-2基因缺失对血红素诱导氧化应激性脑损伤的保护作用.方法 分别将6 μl (8 μmol/L)灭菌氯高铁血红素定向注入野生型小鼠和基因(HO-2)敲除小鼠的纹状体内,72 h后分别检测纹状体细胞生存率,蛋白和脂类的氧化作用.用蛋白质印迹法检测血红素氧合酶-1,2(HO-1)的表达.结果 与野生型相比,基因(HO-2)敲除小鼠纹状体内蛋白和脂类的氧化作用显著降低,而纹状体细胞的存活率显著增加;HO-1的表达在两种小鼠注射前后没有明显差异.结论 结果提示,血红素氧合酶-2基因缺失对血红素诱导的氧化应激性脑损伤具有保护作用;选择性抑制神经元血红素氧合酶-2基因的表达可减轻氧化应激性脑损伤.     

Endothelin-mediated induction of heme oxygenase-1 in the spinal cord is attenuated in transgenic mice overexpressing superoxide dismutase

Spinal cord blood flow and the induction of heme oxygenase-1 (HO-1), an indicator of oxidative stress, were studied in the spinal cords of adult wild-type and transgenic mice overexpressing the antioxidant copper, zinc superoxide dismutase (CuZn SOD) after intrathecal administration of the potent vasoactive peptide endothelin-1 (ET-1). Gelfoam, saturated with ET-1 (40, 80, or 400 micromol/L), was positioned in the intrathecal space at the midthoracic level in anesthetized animals. Blood flow was continuously monitored by laser Doppler for 10 min after the intrathecal application of ET-1. There was a significant reduction in spinal cord blood flow to approximately 40% of control values by 10 min after the intrathecal application of the peptide in both wild-type and transgenic mice. Moreover, SB209670, a nonselective endothelin receptor antagonist, blocked this reduction in flow. Each animal was euthanized 24 h after the intrathecal administration of ET-1, and the spinal cord was prepared for quantitative immunocytochemistry. HO-1 was primarily induced in astrocytes near the dorsal surface of the spinal cord in wild-type mice. This induction was attenuated in both wild-type, treated with SB209670, and untreated transgenic mice. Together, these findings suggest that ET-1 mediates oxidative stress in the spinal cord through the modulation of spinal cord blood flow.     

Sustained induction of heme oxygenase-1 in the traumatized spinal cord

Oxidative stress contributes to secondary injury after spinal cord trauma. Among the consequences of oxidative stress is the induction of heme oxygenase-1 (HO-1), an inducible isozyme that metabolizes heme to iron, biliverdin, and carbon monoxide. Here we examine the induction of HO-1 in the hemisected spinal cord, a model that results in reproducible degeneration in the ipsilateral white matter. HO-1 was induced in microglia and macrophages from 24 h to at least 42 days after injury. Within the first week after injury, HO-1 was induced in both the gray and the white matter. Thereafter, HO-1 expression was limited to degenerating fiber tracts. HSP70, a heat shock protein induced mainly by the presence of denatured proteins, was consistently colocalized with HO-1 in the microglia and macrophages. This study to demonstrates long-term induction of HO-1 and HSP70 in microglia and macrophages after traumatic injury and an association between induction of HO-1 and Wallerian degeneration. White matter degeneration is characterized by phagocytosis of cellular debris and remodeling of surviving tissue. This results in the metabolism, synthesis, and turnover of heme and heme proteins. Thus, sustained induction of HO-1 and HSP70 in microglia and macrophages suggests that tissue degeneration is an ongoing process, lasting 6 weeks and perhaps even longer.     

Distinct roles of oxidative stress and antioxidants in the nucleus dorsalis and red nucleus following spinal cord hemisection

Oxidative stress plays an important role in the pathogenesis of neurodegeneration after the acute central nervous system injury. We reported previously that increased nitric oxide (NO) production following spinal cord hemisection tends to lead to neurodegeneration in neurons of the nucleus dorsalis (ND) that normally lacks expression of neuronal NO synthase (nNOS) in opposition to those in the red nucleus (RN) that constitutively expresses nNOS. We wondered whether oxidative stress could be a mechanism underlying this NO involved neurodegeneration. In the present study, we examined oxidative damage evaluated by the presence of 4-hydroxynonenal (HNE) and iron accumulation and expression of putative antioxidant enzymes heme oxygenase-1 (HO-1) and superoxide dismutase (SOD) in neurons of the ND and RN after spinal cord hemisection. We found that HNE expression was induced in neurons of the ipsilateral ND from 1 to 14 days following spinal cord hemisection. Concomitantly, iron staining was seen from 7 to 14 days after lesion. HO-1, however, was only transiently induced in ipsilateral ND neurons between 3 and 7 days after lesion. In contrast to the ND neurons, HNE was undetectable and iron level was unaltered in the RN neurons after spinal cord hemisection. HO-1, SOD-Cu/Zn and SOD-Mn were constitutively expressed in RN neurons, and lesion to the spinal cord did not change their expression. These results suggest that oxidative stress is involved in the degeneration of the lesioned ND neurons; whereas constitutive antioxidant enzymes may protect the RN neurons from oxidative damage.     

Lithium promotes recovery after spinal cord injury

Lithium is associated with oxidative stress and apoptosis,but the mechanism by which lithium protects against spinal cord injury remains poorly understood.In this study,we found that intraperitoneal administration of lithium chloride(LiCl)in a rat model of spinal cord injury alleviated pathological spinal cord injury and inhibited expression of tumor necrosis factorα,interleukin-6,and interleukin 1β.Lithium inhibited pyroptosis and reduced inflammation by inhibiting Caspase-1 expression,reducing the oxidative stress response,and inhibiting activation of the Nod-like receptor protein 3 inflammasome.We also investigated the neuroprotective effects of lithium intervention on oxygen/glucose-deprived PC12 cells.We found that lithium reduced inflammation,oxidative damage,apoptosis,and necrosis and up-regulated nuclear factor E2-related factor 2(Nrf2)and heme oxygenase-1 in PC12 cells.All-trans retinoic acid,an Nrf2 inhibitor,reversed the effects of lithium.These results suggest that lithium exerts anti-inflammatory,anti-oxidant,and anti-pyroptotic effects through the Nrf2/heme oxygenase-1 pathway to promote recovery after spinal cord injury.This study was approved by the Animal Ethics Committee of Xi’an Jiaotong University(approval No.2018-2053)on October 23,2018.     

Beta-amyloid 42 accumulation in the lumbar spinal cord motor neurons of amyotrophic lateral sclerosis patients

Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of large motor neurons in the brain and spinal cord. Amyloid precursor protein (APP), the transmembrane precursor of beta-amyloid (A beta), accumulates in the anterior horn motor neurons of ALS patients with mild lesions. APP undergoes an alternative proteolysis mediated by caspase-3, which is activated in motor neurons in a mouse model of ALS. The ALS spinal cord motor neurons also show evidence of increased oxidative damage, which is thought to alter APP processing. We sought to determine whether A beta42, the more pathogenic A beta species, accumulates in the postmortem lumbar spinal cord of ALS patients. While there was little or no A beta42 labeling in control spinal cord tissues, elevated A beta42 immunoreactivity occurred in ALS motor neuronal perikarya and axonal swellings in the anterior horn. A few A beta42-positive neurons exhibited thioflavine S staining. No extracellular A beta42 deposits were found. A beta42 coexisted with the oxidative damage markers malondialdehyde, 8-hydroxydeoxyguanosine, heme oxygenase-1, and nitrotyrosine in abnormal neurons. The neurons with intracellular A beta42 accumulation also displayed robust cleaved caspase-3 immunoreactivity. Very little A beta40 immunoreactivity occurred in motor neurons of both control and ALS. These results suggest that aberrant accumulation of A beta42 in ALS spinal cord motor neurons is associated with oxidative stress, and may play a role in the pathogenesis of neurodegeneration in ALS.     

Heme oxygenase-1 stabilizes the blood-spinal cord barrier and limits oxidative stress and white matter damage in the acutely injured murine spinal cord.

We hypothesized that heme oxygenase (HO)-1, the inducible form of HO, represents an important defense against early oxidative injury in the traumatized spinal cord by stabilizing the blood-spinal cord barrier and limiting the infiltration of leukocytes. To test this hypothesis, we first examined the immunoexpression of HO-1 and compared barrier permeability and leukocyte infiltration in spinal cord-injured HO-1-deficient (+/-) and wild-type (WT, +/+) mice. Heme oxygenase was expressed in both endothelial cells and glia of the injured cord. Barrier disruption to luciferase and infiltration of neutrophils were significantly greater in the HO-1+/- than WT mice at 24 h postinjury (P相似文献   

Heme oxygenase-1 in vascular smooth muscle cells counteracts cardiovascular damage induced by angiotensin II

Heme oxygenase (HO) is a microsomal enzyme that catalyzes the degradation of heme into biliverdin, which is subsequently reduced to bilirubin, free iron and carbon monoxide (CO), and induction of heme oxygenase-1 (HO-1) is potentially associated with cellular protection, especially against oxidative insults. Using transgenic mice that overexpress HO-1 (HO-1 Tg) specifically in vascular smooth muscle cells, we investigated the organ-protective effects of HO-1 against angiotensin II (Ang II). Following administration of Ang II and a high- salt diet for 14 days, marked intimal hyperplasia as well as inflammatory changes were observed in coronary arteries of Ang II/salt-treated wild type (Wt) mice. In Wt mice, Ang II/salt loading increased urinary excretion of 8- hydroxydeoxyguanosine (8-OHdG) and 8-lso-Prostaglandin F2 alpha. Cardiac levels of MDA and 4-HAE, markers of lipid peroxidation, and GSSG/GSH were also increased in Wt. mice after Ang II/salt loading, but not in HO-1 Tg mice. Consistently, immunostaining for both 8-0HdG, a marker of oxidative DNA damage, and 3-nitrotyrosine, the metabolites of reactive oxygen species, were apparently increased in the Ang II/salt-treated heart of Wt. mice; however, no significant changes in these responses were detected in HO-1 Tg mice after Ang II/salt loading. These data suggest that increased oxidative stress might be involved in the coronary artery changes induced by Ang II/salt loading. The evidence presented in the current study indicates that vascular HO-1 exerts its protective effect against cardiovascular damage, possibly through the inhibition of oxidative stress.     

Differential upregulation of heme oxygenase-1 (HSP32) in glial cells after oxidative stress and in demyelinating disorders

Oxidative stress is implicated in the pathogenesis of demyelinating disorders and inflammatory responses. Heme oxygenase-1 (HO-1; HSP32) is a small heat shock protein (HSP) with enzymatic activity, which is inducible by oxidative stress. In this study we analyzed autopsy and biopsy brain samples of patients with multiple sclerosis (MS) and ADEM (acute disseminated leucoencephalomyelits) and spinal cord lesions of mouse EAE (experimental autoimmune encephalomyelitis), which was actively induced by immunization with myelin oligodendrocyte glycoprotein (MOG_35–55) peptide, for the presence of HO-1. HO-1 was observed in glial cells during different stages: (1) during acute phases of mainly inflammatory diseases (EAE and ADEM) expression of HO-1 was prominent in microglia/macrophages and astrocytes, and upregulation correlated with inflammation, and (2) in early MS lesions HO-1 was expressed in oligodendrocytes. Furthermore, in glial cell cultures, we can show that upregulation of HO-1 in oligodendrocytes was paralleled by severe morphological damage. Oligodendrocytes underwent apoptotic cell death at a concentration of hydrogen peroxide (50–200 μM) which did not affect astrocytes or microglia. Using oligodendroglial OLN-93 cells, we demonstrate that oxidative stress led to mitochondrial impairment and the disorganization of the microtubule network. Zinc protoporphyrin, an inhibitor of HO-1, augmented the cytotoxic consequences of hydrogen peroxide in OLN-93 cells. Hence, the presence of HO-1 in EAE, ADEM, and MS points to the involvement of oxidative stress and a role of HO-1 in the pathogenesis of the diseases. The data suggest that stress-induced HO-1 initially plays a protective role, while its chronic upregulation, might contribute to oligodendroglial cell death rather than providing protection.     

NMDA受体激活诱导甲醛炎性痛大鼠脊髓HO-1蛋白表达增加

目的本实验通过在甲醛炎性痛大鼠鞘内注射N-甲基-D-d门冬氨酸(NMDA)受体抑制剂地卓西平马来酸盐(MK-801)以及在正常大鼠鞘内注射NMDA受体激动剂NMDA,观察二者对甲醛炎性痛大鼠以及正常大鼠脊髓血红素氧合酶-1(HO-1)表达的影响,探讨甲醛炎性痛诱导的大鼠脊髓HO-1蛋白表达改变是否受NMDA受体激活的影响。方法采用右后掌足底注射甲醛复制炎性痛模型,采用免疫组织化学方法观察脊髓HO-1蛋白表达。结果甲醛炎性痛大鼠L5脊髓后角Ⅰ-Ⅱ板层HO-1蛋白免疫反应阳性细胞数目、平均光密度值均明显大于正常对照组,预先鞘内注射MK-801可明显抑制甲醛炎性痛诱导的大鼠双侧脊髓后角Ⅰ-Ⅱ板层HO-1蛋白的表达,正常大鼠鞘内注射NMDA可诱导脊髓后角HO-1蛋白表达增加。结论 NMDA受体激活可促进脊髓神经元HO-1蛋白的表达。     

Induction of heme oxygenase-1 after hyperosmotic opening of the blood-brain barrier

The induction of the stress protein heme oxygenase-1 (HO-1) was studied in the rat brain after intracarotid administration of hyperosmolar mannitol. HO-1 was immunolocalized in fixed sections of brain 24 h to 7 days after injection. Immunoglobulin G (IgG) was immunolocalized in adjacent sections to demonstrate areas of breakdown of the blood–brain barrier. Induction of HO-1 was also evaluated by Western immunoblots, performed at 24 h after the insult. Immunofluorescent double labelling with monoclonal antibodies to HO-1 and either glial fibrillary acidic protein or the complement C3bi receptor was used to determine if glia/macrophages expressed HO-1. There was pronounced, widespread induction of HO-1 in the ipsilateral hemisphere and cerebellum by 24 h both by immunocytochemistry and by Western blots. This induction was markedly attenuated at later times. HO-1 was induced in astrocytes and microglia/macrophages in the ipsilateral hemisphere. In addition, the protein was induced in Bergmann glia and scattered microglia/macrophages in the cerebellum. The mechanism of induction of HO-1 in glia after opening of the blood–brain barrier could include exposure to heme proteins, denatured proteins and other plasma constituents known to induce HO-1. This glial induction may reflect a protective response of these cells.     

Long-term expression of heme oxygenase-1 (HO-1, HSP-32) following focal cerebral infarctions and traumatic brain injury in humans

Extracellular heme derived from hemoglobin following hemorrhage or released from dying cells induces the expression of heme oxygenase-1 (HO-1, HSP-32) which metabolizes heme to the gaseous mediator carbon monoxide (CO), iron (Fe) and biliverdin. Biliverdin and its product bilirubin are powerful antioxidants. Thus, expression of HO-1 is considered to be a protective mechanism against oxidative stress and has been described in microglia, astrocytes and neurons following distinct experimental models of pathological alterations to the brain such as subarachnoidal hemorrhage, ischemia and traumatic brain injury (TBI) and in human neurodegenerative diseases. We have now analyzed the expression of HO-1 in human brains following TBI (n = 28; survival times: few minutes up to 6 months) and focal cerebral infarctions (FCI; n = 17; survival time: < 1 day up to months) by immunohistochemistry. Follwing TBI, accumulation of HO-1+ microglia/macrophages at the hemorrhagic lesion was detected as early as 6 h post trauma and was still pronounced after 6 months. In contrast, after FCI HO-1+ microglia/macrophages accumulated within focal hemorrhages only and were absent in non-hemorrhagic regions. Further, HO-1 was weakly expressed in astrocytes in the perifocal penumbra. In contrast to experimental data derived from rat focal ischemia, these results indicate a prolonged HO-1 expression in humans after brain injury.     

Protective effects of heme oxygenase-1 against MPP+-induced cytotoxicity in PC-12 cells

Heme oxygenase-1 (HO-1) catalyses the rate-limiting step of heme degradation to biliverdin, which is in turn reduced to bilirubin, CO and free iron. HO-1 can be induced by several harmful stimuli including oxidative stress, and it has a protective role against the cytotoxicity in different cells. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridinium (MPP⁺) is a neurotoxic substance that induces the degeneration of dopaminergic neurons. This study examined whether HO-1 can be induced by MPP⁺ and whether HO-1 has a protective role against the MPP⁺-induced cytotoxicity in PC-12 cells. MPP⁺ triggered a relatively rapid induction of HO-1. The MPP⁺-induced cytotoxicity and reactive oxygen species (ROS) production markedly increased by HO-1 inhibitor, zinc protoporphyrin-IX (ZnPP-IX). The increase of ROS production by ZnPP-IX was completely abrogated by either two products of HO (biliverdin or bilirubin) while the increase of cytotoxicity by ZnPP-IX was attenuated partially. These suggest that HO-1 expression might have some cytoprotective effect against MPP⁺-induced cytotoxicity.     

A dopamine receptor antagonist L-745,870 suppresses microglia activation in spinal cord and mitigates the progression in ALS model mice

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motor neurons in the motor cortex, brainstem, and spinal cord. It has been shown that oxidative stress plays a pivotal role in the progression of this motor neuron loss. We have previously reported that L-745,870, a dopamine D4 receptor antagonist, selectively inhibits oxidative stress-induced cell death in vitro and exerts a potent neuroprotective effect against ischemia-induced neural cell damage in gerbil. To investigate the efficacy of L-745,870 in the treatment of ALS, we here conducted a chronic administration of L-745,870 to transgenic mice expressing a mutated form of human superoxide dismutase gene (SOD1^H46R); a mouse model of familial ALS, and assessed whether the mice benefit from this treatment. The pre-onset administration of L-745,870 significantly delayed the onset of motor deficits, slowed the disease progression, and extended a life span in transgenic mice. These animals showed a delayed loss of anterior horn cells in the spinal cord concomitant with a reduced level of microglial activation at a late symptomatic stage. Further, the post-onset administration of L-745,870 to the SOD1^H46R transgenic mice remarkably slowed the disease progression and extended their life spans. Taken together, our findings in a rodent model of ALS may have implication that L-745,870 is a possible novel therapeutic means to the treatment of ALS.     

Monitoring systemic oxidative stress in an animal model of amyotrophic lateral sclerosis

A mutant form of the ubiquitous copper/zinc superoxide dismutase (SOD1) protein has been found in some patients with amyotrophic lateral sclerosis (ALS). We monitored oxidative stress in an animal model of ALS, the SOD^G93A mouse, which develops a disease similar to ALS with an accelerated course. The aim of this work was to show that ALS damages several organs and tissues, from an oxidative stress point of view. We measured lipid and protein oxidative damage in different tissue homogenates of SOD^G93A mice. The biomarkers that we analyzed were malondialdehyde + 4-hydroxyalkenal (MDA + 4-HDA) and carbonyls, respectively. The spinal cord and brain of SOD^G93A mice showed increased lipid peroxidation after 100 or 130 days compared to age-matched littermate controls. The CNS was most affected, but lipid peroxidation was also detected in the skeletal muscle and liver on day 130. No changes were observed in protein carbonylation in the homogenates. Our results are consistent with a multisystem etiology of ALS and suggest that oxidative stress may play a primary role in ALS pathogenesis. Thus, oxidative stress represents a potential biomarker that might be useful in developing new therapeutic strategies for ALS.     

Immunohistochemical study on the distribution of glycogen synthase kinase 3α in the central nervous system of SOD1G93A transgenic mice

Abstract

Objective: To identify glycogen synthase kinase (GSK) 3α expression in a mouse model of familial amyotrophic lateral sclerosis (ALS), we investigated the changes of GSK3α in the central nervous system of SOD1^G93A transgenic mice by immunohistochemistry.

Methods: We used 12 SOD1^G93A transgenic and ten wild-type (wt) SOD1 transgenic mice bred by 'The Jackson Laboratory' under the strain designations B6SJL-TgN (SOD1^G93A) 1 Gur/J and B6SJL-TgN (SOD1) 2 Gur/J, respectively. Immunohistochemistry was performed in accordance with the free-floating method described earlier.

Results: In symptomatic transgenic mice, GSK3α-immunoreactive astrocytes were detected in the spinal cord, brainstem and cerebellum of symptomatic SOD1^G93A transgenic mice. In contrast to symptomatic mice, no GSK3α-immunoreactive astrocytes were observed in any brain region of wtSOD1 and pre-symptomatic mice, and the number and intensity of stained cells were not different at the age of 8 and 13 weeks.

Discussion: These results provide the first evidence that GSK3α-immunoreactive astrocytes were found in the CNS of SOD1^G93A transgenic mice after clinical symptoms, suggesting a possible role in the pathologic process of ALS. However, the mechanisms underlying the increased immunoreactivity for GSK3α and the functional implications require elucidation.