Cathepsin B mRNA and protein expression following contusion spinal cord injury in rats

We provide the first data that cathepsin B (Cath B), a lysosomal cysteine protease, is up-regulated following contusion-spinal cord injury (SCI). Following T12 laminectomy and moderate contusion, Cath B mRNA and protein expression profiles were examined from 2 to 168 h post-injury in rats using real-time PCR and immunoblots, respectively. Contusion injury significantly increased [mRNA]Cath B in the injury site and adjacent segments over sham injury levels. While the largest [mRNA]Cath B induction (20-fold over naive) was seen in the injury site, the caudal segment routinely yielded [mRNA]Cath B levels greater than 10-fold over naive. Interestingly, sham injury animals also experienced mRNA induction at several time points at the injury site and in segments rostral and caudal to the injury site. Contusion injury also significantly elevated levels of Cath B proenzyme protein (37 kDa) over sham injury in the injury site (48, 72 and 168 h post-injury). Furthermore, significant protein increases of single and double chain Cath B (both active forms) occurred at the injury site at 72 and 168 h post-injury. Similar significant increases in Cath B protein levels were seen in areas adjacent to the injury site. The induction of Cath B mRNA and protein expression following contusion injury is previously undescribed and suggests that Cath B may potentially be involved in the secondary injury cascade, perhaps for as long as 1 week post-injury.     

Increased dynorphin immunoreactivity in spinal cord after traumatic injury

Opiate antagonists, at high doses, have been shown to improve physiological variables and outcome after experimental spinal injury. Dynorphin appears to be unique amongst opioids in producing hindlimb paralysis after intrathecal injection. Taken together, these findings suggest a possible pathophysiological role for endogenous opioids, particularly dynorphin, in spinal injury. In the present studies we examined the relationship between changes in dynorphin immunoreactivity (Dyn-ir) in rat spinal cord after traumatic injury and the subsequent motor dysfunction. Trauma was associated with significantly increased Dyn-ir at the injury site, but not distant from the lesion. Dyn-ir was found elevated as early as 2 h and as late as 2 weeks after trauma, and was significantly correlated with the degree of injury. These data are consistent with the hypothesis that dynorphin systems may be involved in the secondary injury that follows spinal trauma.     

Increased expression of spinal cord Fos protein induced by bladder stimulation after spinal cord injury

These studies examined Fos protein expression in spinal cord neurons synaptically activated by stimulation of bladder afferent pathways after spinal cord injury (SCI). In urethan-anesthetized Wistar rats after SCI for 6 wk, intravesical saline distension significantly (P 相似文献   

Effects of nitric oxide synthase inhibitors on systemic hypotension, cytokines and inducible nitric oxide synthase expression and lung injury following endotoxin administration in rats

Endotoxin shock is characterized by systemic hypotension, hyporeactiveness to vasoconstrictors and acute lung edema. A nitric oxide synthase (NOS) inhibitor, N^G-monomethyl-L-arginine (L-NMMA) has been shown to be effective in reversing acute lung injury. In the present study, we evaluated the effects of NOS blockade by different mechanisms on the endotoxin-induced changes. In anesthetized rats, lipopolysaccharide (LPS,Klebsiella pneumoniae) was administered intravenously in a dose of 10 mg/kg. LPS caused sustained systemic hypotension accompanied by an eightfold increase of exhaled NO during an observation period of 4 h. After the experiment, the lung weight was obtained and lung tissues were taken for the determination of mRNA expressions of inducible NOS (iNOS), interleukin-1 (IL-1) and tumor necrosis factor--(TNF-). Histological examination of the lungs was also performed. In the control group injected with saline solution, mRNA expressions of iNOS, IL-1 and TNF- were absent. Four hours after LPS, the mRNA expressions of iNOS and IL-1 were still significantly enhanced, but TNF- was not discernibly expressed. LPS also caused a twofold increase in lung weight. Pathological examination revealed endothelial damage and interstitial edema. Various NOS inhibitors were given 1 h after LPS administration. These agents included N-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg), a constitutive NOS and iNOS inhibitor; S,S-1,4-phenylene-bis-(1,2-ethanedinyl) bis-isothiourea dihydrobromide (1,4-PBIT, 10 mg/kg), a relatively specific iNOS inhibitor, and dexamethasone (3 mg/kg), an inhibitor of iNOS expression. These NOS inhibitors all effectively reversed the systemic hypotension, reduced the exhaled NO concentration and prevented acute lung injury. The LPS-induced mRNA expressions of iNOS and IL-1 were also significantly depressed by these NOS inhibitors. Our results suggest that NO production through the iNOS pathway is responsible for endotoxin-induced lung injury. Certain cytokines such as IL-1 are possibly involved. These changes are minimized by NOS inhibitors through different mechanisms.     

Neurochemical plasticity of nitric oxide synthase isoforms in neurogenic detrusor overactivity after spinal cord injury

Nitric oxide (NO) participates in the neural pathways controlling the lower urinary tract (LUT). Expression of NO synthase (NOS) can be upregulated after spinal cord injury (SCI), and altered NOS activity may participate in resulting LUT dysfunction. To investigate distribution of NOS-immunoreactivity (NOS-IR) in neurons of rats following SCI and the possible effects of NOS inhibitors. Expression of neuronal and inducible NOS-IR in lumbosacral spinal cord was assessed in rats. Cystometry was performed to examine effects of intrathecal injection of NOS inhibitor. There was increased expression of neuronal NOS-IR after trauma. Maximum bladder capacity was increased by neuronal NOS (nNOS) inhibitors. Upregulation of nNOS may facilitate emergence of the spinal micturition reflex following SCI; nNOS inhibitor suppressed SCI-induced urinary incontinence by increasing bladder capacity. Our results indicate manipulation of NO production could help treat LUT dysfunction after SCI.     

Attenuated nitric oxide synthase activity and protein expression accompany intestinal ischemia/reperfusion injury in rats

Intestinal ischemia/reperfusion (I/R) leads to bowel impairment via the release of reactive oxygen species (ROS) and neutrophil infiltration. In addition to modulating intestinal integrity, nitric oxide (NO(*)) inhibits neutrophil activation and scavenges ROS. Attenuated endogenous NO(*) formation may result in the accrual of these deleterious stimuli. Therefore, we determined nitric oxide synthase (NOS) activity in anesthetized rats subjected to 1 h of superior mesenteric ischemia or ischemia followed by reflow. NOS activity was measured in intestinal tissue homogenates as the conversion rate of (3)H-L-arginine to (3)H-L-citrulline. Our results demonstrate that intestinal ischemia leads to a decrease in NOS activity indicating lower NO(*) formation in the animal model. The attenuation in NOS activity was not reversed following 4 h of reperfusion. Western blot analysis revealed that the decline in enzyme activity was accompanied by reduced intestinal NOS III (endothelial constitutive NOS) expression. These findings provide biochemical evidence for impaired NO(*) formation machinery in intestinal I/R injury.     

NOSIP, a novel modulator of endothelial nitric oxide synthase activity.

Production of nitric oxide (NO) in endothelial cells is regulated by direct interactions of endothelial nitric oxide synthase (eNOS) with effector proteins such as Ca2+-calmodulin, by posttranslational modifications such as phosphorylation via protein kinase B, and by translocation of the enzyme from the plasma membrane caveolae to intracellular compartments. Reversible acylation of eNOS is thought to contribute to the intracellular trafficking of the enzyme; however, protein factor(s) that govern the translocation of the enzyme are still unknown. Here we have used the yeast two-hybrid system and identified a novel 34 kDa protein, termed NOSIP (eNOS interacting protein), which avidly binds to the carboxyl-terminal region of the eNOS oxygenase domain. Coimmunoprecipitation studies demonstrated the specific interaction of eNOS and NOSIP in vitro and in vivo, and complex formation was inhibited by a synthetic peptide of the caveolin-1 scaffolding domain. NO production was significantly reduced in eNOS-expressing CHO cells (CHO-eNOS) that transiently overexpressed NOSIP. Stimulation with the calcium ionophore A23187 induced the reversible translocation of eNOS from the detergent-insoluble to the detergent-soluble fractions of CHO-eNOS, and this translocation was completely prevented by transient coexpression of NOSIP in CHO-eNOS. Immunofluorescence studies revealed a prominent plasma membrane staining for eNOS in CHO-eNOS that was abolished in the presence of NOSIP. Subcellular fractionation studies identified eNOS in the caveolin-rich membrane fractions of CHO-eNOS, and coexpression of NOSIP caused a shift of eNOS to intracellular compartments. We conclude that NOSIP is a novel type of modulator that promotes translocation of eNOS from the plasma membrane to intracellular sites, thereby uncoupling eNOS from plasma membrane caveolae and inhibiting NO synthesis.     

Increased expression of CDK11p58 and cyclin D3 following spinal cord injury in rats

Protein kinases are critical signalling molecules for normal cell growth and development. CDK11^p58 is a p34^cdc2-related protein kinase, and plays an important role in normal cell cycle progression. However its distribution and function in the central nervous system (CNS) lesion remain unclear. In this study, we mainly investigated the protein expression and cellular localization of CDK11 during spinal cord injury (SCI). Western blot analysis revealed that CDK11^p58 was not detected in normal spinal cord. It gradually increased, reached a peak at 3 day after SCI, and then decreased. The protein expression of CDK11^p58 was further analyzed by immunohistochemistry. The variable immunostaining patterns of CDK11^p58 were visualized at different periods of injury. Double immunofluorescence staining showed that CDK11 was co-expressed with NeuN, CNPase and GFAP. Co-localization of CDK11/active caspase-3 and CDK11/proliferating cell nuclear antigen (PCNA) were detected in some cells. Cyclin D3, which was associated with CDK11^p58 and could enhance kinase activity, was detected in the normal and injured spinal cord. The cyclin D3 protein underwent a similar pattern with CDK11^p58 during SCI. Double immunofluorescence staining indicated that CDK11 co-expressed with cyclin D3 in neurons and glial cells. Coimmunoprecipitation further showed that CDK11^p58 and cyclin D3 interacted with each other in the damaged spinal cord. Thus, it is likely CDK11^p58 and cyclin D3 could interact with each other after acute SCI. Another partner of CDK11^p58 was β-1,4-galactosyltransferase 1 (β-1,4-GT 1). The co-localization of CDK11/β-1,4-GT 1 in the damaged spinal cord was revealed by immunofluorescence analysis. The cyclin D3-CDK4 complexes were also present by coimmunoprecipitation analysis. Taken together, these data suggested that both CDK11 and cyclin D3 may play important roles in spinal cord pathophysiology. The authors Yuhong Ji and Feng Xiao contributed equally to this work.     

脊髓水平iNOS在吗啡依赖大鼠戒断反应中的作用

目的:探讨脊髓水平诱导型一氧化氮合酶在吗啡依赖大鼠戒断反应中的作用。方法:健康雄性SD大鼠72只,体重200~250 g,吗啡剂量每次10 mg/kg,每日2次,隔日每次增加10 mg/kg,至第6天末次注射50 mg/kg,大鼠腹腔注射纳洛酮4 mg/kg建立吗啡依赖及戒断模型,在纳洛酮激发戒断前30 min鞘内注射iNOS特异性抑制剂氨基胍(AG)150μg。分为正常对照组、吗啡依赖组、吗啡戒断组、AG组。采用行为学(n=8)、免疫组织化学(n=6)和Western blot(n=4)方法观察鞘内应用iNOS特异性抑制剂氨基胍对吗啡依赖大鼠纳洛酮催促戒断反应和脊髓神经元iNOS表达的影响。结果:AG组戒断症状评分和戒断组促诱发痛评分均低于戒断组(P<0.05)。免疫组织化学和Western blot显示戒断组大鼠脊髓iNOS阳性神经元的数目和蛋白的表达增高,而AG组大鼠脊髓iNOS阳性神经元的数目和iNOS蛋白的表达低于戒断组(P<0.05)。结论:脊髓水平iNOS表达上调可能参与介导吗啡戒断反应。     

Regulation of clusterin expression following spinal cord injury

We have investigated the localization and regulation of a putative extracellular chaperone, clusterin, in the rat spinal cord after lesion. In control animals, clusterin is expressed in motoneurons, in meningeal and ependymal cells, and in astrocytes mainly located beneath the pial surface. Beginning at day 2 after hemisection at segmental level C6, clusterin levels increase in GFAP-positive astrocytes within the lesioned segment. Three weeks after trauma, clusterin mRNA and protein are elevated in neurons close to the lesion site and in glial elements within scar tissue and within degenerating fiber tracts rostral and caudal to the lesion. This study provides evidence for a role of clusterin in the subacute and late phase of spinal cord injury.     

Alterations in growth-associated protein (GAP-43) expression in lower urinary tract pathways following chronic spinal cord injury

Alterations in the expression of growth-associated protein 43 (GAP-43) were examined in lower urinary tract micturition reflex pathways 6 or 8 weeks following complete spinal cord transection (approximately T9). In control animals, expression of GAP-43 was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the corticospinal tract; (3) the dorsal horn; and (4) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (5) in the lateral collateral pathway of Lissauer in L6-S1 spinal segments. Densitometry analysis has demonstrated significant increases (p < or =0.001; 1.3-6.4-fold increase) in GAP-43-immunoreactivity (IR) in these regions of the rostral lumbar (L1-L2) and caudal lumbosacral (L6-S1) spinal cord 6 weeks following spinal cord injury. Changes in GAP-43-IR were restricted to the L1-L2 and L6-S1 segments that are involved in lower urinary tract reflexes. Changes in GAP-43-IR were not observed at the L5 segmental level except for an increase in GAP-43-IR in the superficial, dorsal horn at 6 weeks post-injury. In all segments examined, GAP-43-IR was decreased (2-5-fold) in the corticospinal tract (dorsal division) 6 and 8 weeks following spinal cord injury. Eight weeks following spinal cord injury, changes in GAP-43-IR had returned to control levels except for the persistence of increased GAP-43-IR in the region of the sacral parasympathetic nucleus and the lateral collateral pathway in the S1 spinal segment. Alterations in GAP-43-IR following chronic spinal cord injury may suggest a reorganization of bladder afferent projections and spinal elements involved in urinary bladder reflexes consistent with alterations in urinary bladder function (hyperreflexia) observed in animals following spinal cord injury above the lumbosacral spinal cord.     

Alterations in growth-associated protein (GAP-43) expression in lower urinary tract pathways following chronic spinal cord injury

Alterations in the expression of growth-associated protein 43 (GAP-43) were examined in lower urinary tract micturition reflex pathways 6 or 8 weeks following complete spinal cord transection (~ T9). In control animals, expression of GAP-43 was present in specific regions of the gray matter in the rostral lumbar and caudal lumbosacral spinal cord, including: (1) the dorsal commissure; (2) the corticospinal tract; (3) the dorsal horn; and (4) the regions of the intermediolateral cell column (L1-L2) and the sacral parasympathetic nucleus (L6-S1); and (5) in the lateral collateral pathway of Lissauer in L6-S1 spinal segments. Densitometry analysis has demonstrated significant increases (p 0.001; 1.3-6.4-fold increase) in GAP-43-immunoreactivity (IR) in these regions of the rostral lumbar (L1-L2) and caudal lumbosacral (L6-S1) spinal cord 6 weeks following spinal cord injury. Changes in GAP-43-IR were restricted to the L1-L2 and L6-S1 segments that are involved in lower urinary tract reflexes. Changes in GAP-43-IR were not observed at the L5 segmental level except for an increase in GAP-43-IR in the superficial, dorsal horn at 6 weeks post-injury. In all segments examined, GAP-43-IR was decreased (2-5-fold) in the corticospinal tract (dorsal division) 6 and 8 weeks following spinal cord injury. Eight weeks following spinal cord injury, changes in GAP-43-IR had returned to control levels except for the persistence of increased GAP-43-IR in the region of the sacral parasympathetic nucleus and the lateral collateral pathway in the S1 spinal segment. Alterations in GAP-43-IR following chronic spinal cord injury may suggest a reorganization of bladder afferent projections and spinal elements involved in urinary bladder reflexes consistent with alterations in urinary bladder function (hyperreflexia) observed in animals following spinal cord injury above the lumbosacral spinal cord.     

Effects of nerve graft on nitric oxide synthase, NAD(P)H oxidase, and antioxidant enzymes in chronic spinal cord injury

Oxidative stress and nitrosative stress play important roles in the pathogenesis of secondary spinal cord injury. Recently, we demonstrated that peripheral nerve grafts (PNG) with acidic fibroblast growth factor (aFGF) partially restore hind limb locomotion in adult rats with completely transected spinal cords. This study investigated the protein abundances of the superoxide (O2*)-generating enzyme nicotinamide adenine dinucleotide (phosphate) oxidase (NAD(P)H oxidase; gp91phox subunit), nitric oxide synthases (NOS), antioxidant enzymes, superoxide dismutases (Cu Zn SOD, Mn SOD), catalase, and glutathione peroxidase (GPX) as well as nitrotyrosine in the spinal cord tissue 4 months after spinal cord transection in rats with and without PNG and aFGF. The protein abundances of the gp91phox subunit of NAD(P)H oxidase, Mn SOD, catalase, GPX, eNOS, and nitrotyrosine were significantly upregulated, whereas Cu Zn SOD and nNOS were unchanged in the injury group compared to the sham controls. The nerve graft with aFGF treated group showed significantly better hind limb locomotion recovery than the injury group. Although the protein abundances of gp91phox, nitrotyrosine, and Cu Zn SOD were similar in the treated group (nerve graft with aFGF) compared to the injury group, Mn SOD, GPX, catalase, and eNOS protein abundances were significantly higher, whereas nNOS was markedly lower in the treated group. We conclude that the combination of nerve graft and aFGF enhances the local antioxidant defense system after spinal cord transection in rats.     

Induction of NADPH diaphorase/nitric oxide synthase in the spinal cord motor neurons of rats following a single and multiple non-penetrative blasts

The present study has demonstrated the induction of nicotinamide adenine dinucleotide phosphate-diaphorase (NADPH-d) reactivity and nitric oxide synthase-like immunoreactivity (NOS-LI) in the ventral horn motoneurons of the spinal cord in rats subjected to a single or multiple underground, or a single surface blast. Both enzyme activities were first detected in some motoneurons in laminae VIII and IX of Rexed, 3 hours after the blast. Some NADPH-d and NOS-LI positive neurons were also distributed in laminae VI and VII. The number and intensity of the labelled cells appeared to increase progressively, peaking at 2-3 days after the blast but were drastically reduced thereafter, so that at 7 days after the blast only a few positive neurons were observed. In rats killed at 2 weeks and in longer surviving intervals, i.e. up to 1 month, NADPH-d/NOS reactivity in the ventral horn motor neurons had diminished. The functional significance of the transient expression of neuronal NADPH-d/NOS after the blasts remains uncertain, although from a speculative point of view, the induction of these enzymes probably would reflect an increased production of nitric oxide (NO). In view of the lack of atrophic changes in most, if not all, of motor neurons, it is suggested that the increased levels of NO production after the blast injury may be involved in a neuroprotective function.     

Endothelium-derived nitric oxide synthase protein expression in ovine placental arteries

During the third trimester, fetoplacental and uterine blood flows increase dramatically to meet the high metabolic demands of the growing fetus. We hypothesized that the expression of endothelial nitric oxide synthase (eNOS) in fetoplacental artery endothelium and the concentrations of nitric oxide (NO) and cyclic GMP (cGMP) in amniotic fluid (AF) are increased during the third trimester of ovine gestation. Placental arteries and AF were collected from ewes at 110, 120, 130, and 142 days of gestation (n = 24; mean +/- SEM term = 145 +/- 3 days). Expression of eNOS protein was measured in intact and denuded placental arteries and in endothelium-derived protein by Western analysis and confirmed by immunohistochemistry. Concentrations of NO (nitrates plus nitrites) and cGMP were determined in AF. Placental artery eNOS protein expression was localized to the endothelium, where it was markedly greater than in vascular smooth muscle. Placental artery endothelium-derived eNOS expression and AF cGMP concentrations were similar at 110 and 120 days of gestation; however, both peaked at 130 days at levels two- to threefold above baseline (P < 0.05) before returning to baseline at 142 days of pregnancy. The AF NO (nitrates plus nitrites) levels, however, increased progressively between 120 days of gestation and term (P < 0.05). We concluded that endothelium-derived placental artery eNOS levels, AF NO (nitrates plus nitrites), and AF cGMP were markedly increased during the third trimester, thus supporting a role for NO-mediated elevations in cGMP in the control of fetoplacental blood flow.     

Altered levels of PGF in cat spinal cord tissue following traumatic injury

Previous studies by others indicated that PGs were present in brain, spinal cord, and c.s.f. of several mammalian species. In the present study we compared levels of PGE and PGF by R.I.A. in spinal cord tissue from traumatized cats and cats pretreated with indomethacin prior to trauma to those of baseline and sham operated controls in order to assess for the first time, to our knowledge, whether meaningful changes in levels of PGE and PGF could be detected which might shed new light on the etiology of spinal cord trauma.Levels of PGF (nanograms/gram wet wt) in the cord segment immediately adjacent to the point of trauma were 8.05 ± 1.50, and 13.13 ± 1.38 for baseline and sham operated cats respectively. Spinal trauma led to more than a 100% increase in PGF levels to 29.26 ± 3.58. Although pretreatment with indomethacin 30 min prior to trauma gave the expected blockade of the PGF response to trauma, a measurable level of PGF (2.55 ± 0.17) was found in the cord after indomethacin. Cord levels of PGF declined after 3 hr in both sham operated and traumatized animals. PGF was maximally stimulated by trauma during the first 3 hr with little effect at 72 hr. Although carefully examined, PGE levels in cat spinal cord appeared to be virtually unaffected by trauma.These findings clearly demonstrate for the first time that traumatic injury to the spinal cord is accompanied by marked increases in PG levels at the site of trauma, and that the observed elevation in PGF in response to trauma can be blocked by indomethacin $\text{in vivo}$. Whether PGF changes are causally related to the etiology of spinal cord trauma, or merely represent a manifestation of PG release as a result of non-specific tissue injury, remains to be seen.     

Effects of magnesium sulphate following spinal cord injury in rats

Neutrophil infiltration has been implicated in the secondary destructive pathomechanisms after initial mechanical injury to the spinal cord. Tissue myeloperoxidase (MPO) activity has been shown to be an exclusive indicator of the extent of post-traumatic neutrophil infiltration. We have studied the effect of magnesium sulphate on MPO activity after spinal cord injury in rats. Rats were randomly allocated into 5 groups. Group 1 was control and normal spinal cord samples were obtained after clinical examination. Forty g-cm contusion injury was introduced to Group 2. Group 3 was vehicle, 1 ml of physiological saline was injected post-trauma. Group 4 was given 30 mg/kg methylprednisolone sodium succinate (MPSS) immediately after trauma. Group 5 was given 600 mg/kg magnesium sulphate immediately after trauma. Animals were examined by inclined plane technique of Rivlin and Tator 24 h after trauma. Spinal cord samples obtained following clinical evaluations. Magnesium sulphate treatment improved early functional scores and decreased MPO activity. These findings revealed that magnesium sulphate treatment possesses neuroprotection on early clinical results and on neutrophil infiltration after acute contusion injury to the rat spinal cord.