Elevated microRNA-214-3p level ameliorates neuroinflammation after spinal cord ischemia-reperfusion injury by inhibiting Nmb/Cav3.2 pathway.

BACKGROUND: Neuromedin B (Nmb) plays a pivotal role in the transmission of neuroinflammation, particularly during spinal cord ischemia-reperfusion injury (SCII). However, the detailed molecular mechanisms underlying this process remain elusive. METHODS: The SCII model was established by clamping the abdominal aorta of male Sprague-Dawley (SD) rats for 60 min. The protein expression levels of Nmb, Cav3.2, and IL-1ß were detected by Western blotting, while miR-214-3p expression was quantified by qRT-PCR. The targeted regulation between miR-214-3p and Nmb was investigated using a dual-luciferase reporter gene assay. The cellular localization of Nmb and Cav3.2 with cell-specific markers was visualized by immunofluorescence staining. The specific roles of miR-214-3p on the Nmb/Cav3.2 interactions in SCII-injured rats were explored by intrathecal injection of Cav3.2-siRNA, PD168368 (a specific NmbR inhibitor) and synthetic miR-214-3p agomir and antagomir in separate experiments. Additionally, hind-limb motor function was evaluated using the modified Tarlov scores. RESULTS: Compared to the Sham group, the protein expression levels of Nmb, Cav3.2, and the proinflammatory factor Interleukin(IL)-1ß were significantly elevated at 24 h post-SCII. Intrathecal injection of PD168368 and Cav3.2-siRNA significantly suppressed the expression of Cav3.2 and IL-1ß compared to the SCII group. The miRDB database and dual-luciferase reporter gene assay identified Nmb as a direct target of miR-214-3p. As expected, in vivo overexpression of miR-214-3p by agomir-214-3p pretreatment significantly inhibited the increases in Nmb, Cav3.2 and IL-1ß expression and improved lower limb motor function in SCII-injured rats, while antagomiR-214-3p pretreatment reversed these effects. CONCLUSIONS: Nmb protein levels positively correlated with Cav3.2 expression in SCII rats. Upregulating miR-214-3p ameliorated hind-limb motor function and protected against neuroinflammation via inhibiting the aberrant Nmb/Cav3.2 interactions and downstream IL-1ß release. These findings provide novel therapeutic targets for clinical prevention and treatment of SCII.

Astaxanthin alleviates spinal cord ischemia-reperfusion injury via activation of PI3K/Akt/GSK-3ß pathway in rats.

BACKGROUND: Ischemia-reperfusion injury of the spinal cord (SCII) often leads to unalterable neurological deficits, which may be associated with apoptosis induced by oxidative stress and inflammation. Astaxanthin (AST) is a strong antioxidant and anti-inflammatory agent with multitarget neuroprotective effects. This study aimed to investigate the potential therapeutic effects of AST for SCII and the molecular mechanism. METHODS: Rat models of SCII with abdominal aortic occlusion for 40 min were carried out to investigate the effects of AST on the recovery of SCII. Tarlov's scores were used to assess the neuronal function; HE and TUNEL staining were used to observe the pathological morphology of lesions. Neuron oxidative stress and inflammation were measured using commercial detection kits. Flow cytometry was conducted to assess the mitochondrial swelling degree. Besides, Western blot assay was used to detect the expression of PI3K/Akt/GSK-3ß pathway-related proteins, as well as NOX2 and NLRP3 proteins. RESULTS: The results demonstrated that AST pretreatment promoted the hind limb motor function recovery and alleviated the pathological damage induced by SCII. Moreover, AST significantly enhanced the antioxidative stress response and attenuated mitochondrial swelling. However, AST pretreatment hardly inhibited the levels of proinflammatory cytokines after SCII. Most importantly, AST activated p-Akt and p-GSK-3ß expression levels. Meanwhile, cotreatment with LY294002 (a PI3K inhibitor) was found to abolish the above protective effects observed with the AST pretreatment. CONCLUSION: Overall, these results suggest that AST pretreatment not only mitigates pathological tissue damage but also effectively improves neural functional recovery following SCII, primarily by alleviating oxidative stress but not inhibiting inflammation. A possible underlying molecular mechanism of AST may be mainly attributed to the activation of PI3K/Akt/GSK-3ß pathway.

The transcription factor Foxd3 induces spinal cord ischemia-reperfusion injury by potentiating microRNA-214-dependent inhibition of Kcnk2.

Spinal cord injury after surgical repair of the thoracic or thoracoabdominal aorta is a devastating complication that is associated with pathological changes, including inflammation, edema, and nerve cell damage. Recently, microRNA (miRNA)-modulated control of spinal cord injury has been actively investigated. This study aims to clarify the regulatory effect of miR-214-mediated inhibition of Kcnk2 following spinal cord ischemia-reperfusion injury (SCII) and the possible underlying mechanisms. SCII was induced in rats by occluding the aortic arch followed by reperfusion. Gain-of-function and loss-of-function experiments were conducted to explore the modulatory effects of Foxd3, miR-214 and Kcnk2 on PC12 cells under hypoxia/reoxygenation (H/R) conditions. MiR-214 and Kcnk2 were poorly expressed, while Foxd3 was highly expressed in the rat spinal cord tissues and H/R-treated PC12 cells. Kcnk2 overexpression enhanced the viability and inhibited the apoptosis of the H/R-treated PC12 cells. Notably, Foxd3 activated miR-214, and miR-214 targeted Kcnk2. In addition, upregulation of Kcnk2 or knockdown of Foxd3 promoted the cell viability and reduced the apoptosis of the H/R-treated PC12 cells. Overall, our study identified a novel mechanism of Foxd3/miR-214/Kcnk2 involving SCII, suggesting that either Foxd3 or miR-214 may be a novel target for the treatment of SCII.

Altered expression of MiR-186-5p and its target genes after spinal cord ischemia-reperfusion injury in rats.

Spinal cord ischemia-reperfusion (I/R) injury remains an unresolved problem, and the mechanism is not fully elaborated. In a rat model of spinal cord I/R injury, we performed microarray analysis to examine the altered expression of microRNAs (miRs) at 24 h after the modelling. miR-186-5p was chosen for further study. An miR mimic or anti-miR oligonucleotide was intrathecally infused before the surgical procedure. The participation of miR-186-5p and its potential target genes based on bioinformatics analysis were analysed next. Pre-treatment with the miR-186-5p mimic improved neurological function and histological assessment scores; reduced Evans Blue extravasation; attenuated spinal cord oedema; and decreased interleukin 15 (IL-15), IL-6, IL-1ß, and tumour necrosis factor-α (TNF-α) expression at 24 h after the modelling. KEGG analysis showed that the group of potential target genes of miR-186-5p was notably enriched in several signalling cascades, such as the Wnt, Hippo, and PI3K-AKT pathways. Gene Ontology (GO) analysis revealed that the group of potential target genes of miR-186-5p was significantly enriched in several biological processes, such as 'Wnt signalling pathway', 'regulation of inflammatory response', and 'Toll-like receptor signalling pathway'. We further found that Wnt5a, TLR3, and chemokine (C-X-C motif) ligand 13 (CXCL13) were upregulated after the modelling and the miR-186-5p mimic reduced the induction of the aforementioned target genes. These data provide evidence that upregulation of miR-186-5p improves neurological outcomes induced by spinal cord I/R injury and may inhibit neuroinflammation through Wnt5a-, TLR3-, or CXCL13-mediated signal pathway in spinal cord I/R injury.

Abdominal Aortic Transplantation of Bone Marrow Mesenchymal Stem Cells Regulates the Expression of Ciliary Neurotrophic Factor and Inflammatory Cytokines in a Rat Model of Spinal Cord Ischemia-Reperfusion Injury.

BACKGROUND This study aimed to investigate the effects of abdominal aortic transplantation of bone marrow mesenchymal stem cells (BMMSCs) on the expression of inflammatory cytokines in a rat model of spinal cord ischemia-reperfusion injury. MATERIAL AND METHODS Adult female Sprague-Dawley rats (N=160) were divided into five groups: the sham operation group (N-32); the control group (N=32); the BMMSC transplanted group (N=32); the anti-ciliary neurotrophic factor (CNTF)-treated BMMSC transplanted group (N=32); and the CNTF small interfering RNA (siRNA)-treated BMMSC transplanted group (N=32). Motor behavior was assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. Motor evoked potentials (MEPs) and cortical somatosensory evoked potentials (CSEPs) were measured. Immunohistochemistry, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot analysis evaluated the expression of spinal inflammatory cytokines. RESULTS Following surgery, compared with the control group the findings in the BMMSC transplant groups included significantly increased BBB scores; the latency and the amplitude of MEP and CSEP were reduced and increased, respectively; spinal neuronal necrosis was reduced; the number of normal neurons increased; CNTF mRNA and protein expression levels increased; expression levels of interleukin-6 (IL-6) were reduced and IL-10 levels were significantly increased (P<0.05). The effects of abdominal aortic BMMSC transplantation were at least partially reversed by both anti-CNTF and CNTF siRNA treatment. CONCLUSIONS In a rat model of spinal cord ischemia-reperfusion injury, abdominal aortic transplantation of BMMSCs increased the expression of CNTF, which improved hindlimb locomotor recovery by regulating the expression of IL-6 and IL-10 to reduce inflammation of the spinal cord.

Downregulation of LncRNA TUG1 Inhibited TLR4 Signaling Pathway-Mediated Inflammatory Damage After Spinal Cord Ischemia Reperfusion in Rats via Suppressing TRIL Expression.

Toll-like receptor 4 (TLR4) and TLR4 interactor with leucine-rich repeats (TRIL) play a crucial role in the inflammatory response. This study investigated the role of long noncoding RNA taurine-upregulated gene 1 (lncRNA TUG1) in TRIL/TLR4 signaling in spinal cord ischemia reperfusion (IR) injury. IR injury was induced in experimental rats; knockdown of TUG1 and TRIL was induced by intrathecal injection of siRNAs and overexpression of TRIL was induced by pcDNA3.3-TRIL. The results showed that the mRNA levels of TUG1 were increased at 12 hours after IR; this was accompanied by increased expression of the TRIL- and TLR4-mediated NF-κB/IL-1ß signaling pathway. Activated microglia, detected with increased ionized calcium-binding adapter molecule 1 as a marker, exacerbated the hind-limb neurological impairment and blood-spinal cord barrier (BSCB) leakage after IR. TUG1 knockdown inhibited expression of TRIL and TLR4 signaling proinflammatory cytokines and microglial activation, and attenuated neurological deficit and BSCB leakage. TRIL knockdown inhibited the TLR4-mediated inflammatory response, while TRIL expression reversed the inhibited inflammatory effect caused by TUG1 knockdown. These data suggest that TUG1 knockdown inhibited inflammatory damage of the TLR4-mediated NF-κB/IL-1ß signaling pathway after IR via suppressing TRIL expression.

Breathing hydrogen sulfide prevents delayed paraplegia in mice.

Delayed paraplegia complicates the recovery from spinal cord ischemia or traumatic spinal cord injury. While delayed motor neuron apoptosis is implicated in the pathogenesis, no effective treatment or preventive measures is available for delayed paraplegia. Hydrogen sulfide exerts anti-apoptotic effects. Here, we examined effects of hydrogen sulfide breathing on the recovery from transient spinal cord ischemia. Breathing hydrogen sulfide starting 23 h after reperfusion for 5 h prevented delayed paraplegia after 5 min of spinal cord ischemia. Beneficial effects of hydrogen sulfide were mediated by upregulation of anti-apoptotic Bcl-XL and abolished by nitric oxide synthase 2 deficiency. S-nitrosylation of NFkB p65 subunit, which is induced by nitric oxide derived from nitric oxide synthase 2, facilitated subsequent sulfide-induced persulfidation of p65 and transcription of anti-apoptotic genes. These results uncover the molecular mechanism of the anti-apoptotic effects of sulfide based on the interaction between nitric oxide and sulfide. Exploitation of the anti-apoptotic effects of delayed hydrogen sulfide breathing may provide a new therapeutic approach for delayed paraplegia.

Effect of low temperatures on BAX and BCL2 proteins in rats with spinal cord ischemia reperfusion injury.

We evaluated changes in BAX and BCL2 expression levels after spinal cord ischemia/reperfusion injury (SCII) and hypothermia during operations in rats. Eighty rats were divided into four groups: Group A (N = 20, 18°C); Group B (N = 20, 28°C); Group C (N = 20, room temperature); and Group D (N = 20, sham operation control). Spinal cord ischemia was induced for 90 min. Hypothermia was induced 15 min before, and maintained during ischemia, followed by heating to normothermia for 30 min after reperfusion. Motor function of the lower limbs was evaluated according to the Tarlov score at 72 and 168 h. For each rat, spinal cord samples were taken at 6, 24, 72 h, and 1 week to evaluate the histopathological changes, neuronal apoptosis, and BAX and BCL2 expression levels. Compared with normothermia, hypothermia significantly improved hind limb function; Group B achieved a higher score than Group A. Group D showed no neurologic deficiency, while the other groups showed various degrees. Group C exhibited greater neuronal apoptosis, higher BAX expression, but lower BCL2 expression than the other groups. Compared with Group A, BAX was expressed less and BCL2 more in Group B, and there was less apoptosis in Group B. Hypothermia preserves hind limb motor function and reduces neuronal death, thereby protecting rats from SCII. The spinal cord may be protected from SCII by inhibition of BAX and activation of BCL2. However, deep hypothermia may inhibit the expression of BCL2, resulting in a worse outcome than mild hypothermia.

Inhibition of micro-ribonucleic acid-320 attenuates neurologic injuries after spinal cord ischemia.

OBJECTIVE: Micro ribonucleic acids (miRNAs) are involved in a wide range of biological functions, in multiple tissues, including the central nervous system. We investigated a novel neuroprotective strategy of down-regulation of miR-320 in the spinal cord under the condition of transient ischemia. METHODS: Spinal cord ischemia was induced in rats by cross-clamping the descending aorta for 14 minutes. Lentivirus vectors containing antisense oligonucleotides of rat miR-320 (antagomiR-320) were transfected into the experimental rats by means of intrathecal injection, 5 days before spinal cord ischemia. Control lentivirus vectors, or the vehicle, were given to the control animals. Hind-limb motor function was assessed during 48 hours after ischemia, using the Motor Deficit Index (MDI), and histologic examination was performed. In parallel experiments, expressions of miR-320, and the phosphorylation state of heat-shock protein 20 (phospho-Hsp20) in the spinal cord were evaluated by quantitative real-time polymerase chain reaction and western blot analysis. RESULTS: The time courses of expressions of miR-320 and phospho-Hsp20 in the spinal cord, after the transient ischemia, indicated that expression of phospho-Hsp20 was negatively correlated with expression of miR-320. Transfection of antagomiR-320 significantly reduced expression of miR-320 in the spinal cord and dramatically up-regulated expression of phospho-Hsp20. Compared with controls, inhibition of miR-320 markedly improved hind-limb motor function, as evidenced by lower MDI scores, at 6, 12, 24, and 48 hours after reperfusion, and increased the number of intact motor neurons in the lumbar spinal cord. CONCLUSIONS: Inhibition of miR-320 induces neuroprotection in the spinal cord, against ischemia-reperfusion injury, possibly via up-regulation of phospho-Hsp20.

Altered microRNA expression in the ischemic-reperfusion spinal cord with atorvastatin therapy.

We explored the neuroprotection by atorvastatin in the ischemia/reperfusion model of rat and its microRNA-related mechanisms. At first, we uncovered a previously unknown alteration in temporal expression of a large set of microRNAs following spinal cord ischemia-reperfusion injury (IRI). The target genes for the differentially expressed microRNAs include genes encoding components that are involved in the inflammation, apoptosis, and neural damage that are known to play important roles in IRI. Atorvastatin pretreatment restored part of the up or down regulations. These findings suggest that altered expression of microRNAs may contribute to the mechanism of neuroprotection of statins in spinal cord IRI.

Ischemic postconditioning protects the spinal cord from ischemia-reperfusion injury via modulation of redox signaling.

BACKGROUND: It is well known that ischemic postconditioning reduces ischemic-reperfusion injury, but the underlying mechanism is not fully understood. The current study investigated the role of reactive oxygen species-mediated upregulation of endogenous antioxidant enzymes in the generation of a protective effect induced by ischemic postconditioning against spinal cord reperfusion injury in the rabbit. METHODS: New Zealand White rabbits were randomly allocated to sham, ischemia-reperfusion, and postconditioning groups (3 cycles of 30 seconds of reperfusion and 30 seconds of occlusion during the onset of reperfusion). Spinal cord ischemia was induced by clamping the infrarenal abdominal aorta for 20 minutes in the ischemia-reperfusion and postconditioning groups. Forty-eight hours after reperfusion, the neurologic status of the lower limbs was assessed. Blood samples were collected for analysis of serum neuron-specific enolase levels, and the lumbar spinal cord segments (L5-7) were harvested for histopathologic and antioxidant enzyme activities and mRNA analysis with or without administration of N-2-mercaptopropionylglycine (an effective oxygen free radical scavenger) given at different reperfusion times. RESULTS: Continuous administration of N-2-mercaptopropionylglycine for 13 minutes, starting at 10 minutes before (but not 10 minutes after) the beginning of reperfusion, attenuated the neuroprotective effect of postconditioning against spinal cord ischemia and reversed the increase in activity of the antioxidant enzymes superoxide dismutase and catalase in spinal cord tissue subjected to ischemic postconditioning. CONCLUSIONS: The results indicate that reactive oxygen species-triggered upregulation of endogenous antioxidant enzyme activities may be involved in the mechanism of neuroprotection of ischemic postconditioning.

A first report on Hb Q-Iran in association with alpha-thalassemia in a case of spinal ischemia.

BACKGROUND: Hemoglobin Q-Iran is a rare variant which has not been described in association with alpha-thalassemia to date. We present the case of a Turkish patient who developed spinal ischemia. METHODS: Spinal ischemia was diagnosed clinically, via magnetic resonance imaging and angiographically. Blood samples were analyzed by high performance liquid chromatography, electrophoresis, gene sequencing, hematological and biochemical analysis. RESULTS: We detected hemoglobin Q-Iran in association with alpha-thalassemia. The same hemoglobinopathy was detected in two members of the patient's family. CONCLUSIONS: As various differential diagnosis approaches failed to reveal the cause of spinal ischemia, the combined hemoglobinopathy was eventually postulated.

Acute paraplegia due to spinal arteriovenous fistula in two patients with hereditary hemorrhagic telangiectasia.

Hereditary hemorrhagic telangiectasia (HHT) is an autosomal dominant disorder characterized by recurrent epistaxis, cutaneous telangiectasia, and visceral arteriovenous malformations (AVM). Of these, spinal AVM is a rare manifestation that concerns mainly children. In this report, we describe two cases of spinal AVM revealed by acute paraparesis due to subarachnoid hemorrhage in children with HHT and reviewed the literature on spinal arteriovenous malformations in HHT. In most of the cases reported, the clinical presentation was acute in the pediatric population and insidious during adulthood. The prognosis of spinal AVM mainly depends on the presence or not of medullar signs and symptoms and on the delay before treatment. In conclusion, any child with a family history of HHT should be considered at risk for spinal AVM in order to improve management of such complications and to decrease the risk of neurological sequellae.

Contrasting genetic effects of major histocompatibility complex on ischemic peripheral nerve and spinal cord injury in female rats.

We have recently shown that the major histocompatibility complex (MHC) exerts a regulatory influence on the development of neuropathic pain-like behaviors after partial sciatic nerve injury in male rats. In the present study, we assessed the role of the MHC in peripheral nerve injury-induced pain as well as central pain following spinal cord injury in female rats using the following inbred strains: Dark Agouti (DA; RT1(av1)), Piebald Virol Glaxo (PVG; RT1(c)) and in the MHC-congenic strain PVG-RT1(av1). In line with our previous observation in male rats, PVG-RT1(c) displayed more severe allodynia compared to the strains carrying the RT1(av1) haplotype (PVG-RT1(av1) and DA-RT1(av1)) following sciatic nerve injury in female rats. However, the MHC did not seem to impact the development of allodynia following spinal cord injury since the two congenic strains PVG-RT1(c) and PVG-RT1(av) did not differ after spinal cord injury. Interestingly, the DA-RT1(av1) strain displayed significantly more severe allodynia than both PVG strains and this difference was not explained by the extent of spinal cord injury. These results suggest that there are differences in the genetic mechanisms for neuropathic pain development following peripheral or central nervous system injury, both in regarding to the role of the MHC complex as well as non-MHC genes.

Over-expression of Hsp27 does not influence disease in the mutant SOD1(G93A) mouse model of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a chronic, adult-onset neurodegenerative disorder characterized by the selective loss of upper and lower motor neurons, resulting in severe atrophy of muscles and death. Although the exact pathogenic mechanism of mutant superoxide dismutase 1 (SOD1) causing familial ALS is still elusive, toxic protein aggregation leading to insufficiency of chaperones is one of the main hypotheses. In this study, we investigated the effect of over-expressing one of these chaperones, heat shock protein 27 (Hsp27), in ALS. Mice over-expressing the human, mutant SOD1(G93A) were crossed with mice that ubiquitously over-expressed human Hsp27. Even though the single transgenic hHsp27 mice showed protection against spinal cord ischemia, the double transgenic SOD1(G93A)/hHsp27 mice did not live longer, and did not show a significant delay in the onset of disease compared to their SOD1(G93A) littermates. There was no protective effect of hHsp27 over-expression on the motor neurons and on the mutant SOD1 aggregates in the double transgenic SOD1(G93A)/hHsp27 mice. In conclusion, despite the protective action against acute motor neuron injury, Hsp27 alone is not sufficient to protect against the chronic motor neuron injury due to the presence of mutant SOD1.

Spinal astrocyte glutamate receptor 1 overexpression after ischemic insult facilitates behavioral signs of spasticity and rigidity.

Using a rat model of ischemic paraplegia, we examined the expression of spinal AMPA receptors and their role in mediating spasticity and rigidity. Spinal ischemia was induced by transient occlusion of the descending aorta combined with systemic hypotension. Spasticity/rigidity were identified by simultaneous measurements of peripheral muscle resistance (PMR) and electromyography (EMG) before and during ankle flexion. In addition, Hoffman reflex (H-reflex) and motor evoked potentials (MEPs) were recorded from the gastrocnemius muscle. Animals were implanted with intrathecal catheters for drug delivery and injected with the AMPA receptor antagonist NGX424 (tezampanel), glutamate receptor 1 (GluR1) antisense, or vehicle. Where intrathecal vehicle had no effect, intrathecal NGX424 produced a dose-dependent suppression of PMR [ED50 of 0.44 microg (0.33-0.58)], as well as tonic and ankle flexion-evoked EMG activity. Similar suppression of MEP and H-reflex were also seen. Western blot analyses of lumbar spinal cord tissue from spastic animals showed a significant increase in GluR1 but decreased GluR2 and GluR4 proteins. Confocal and electron microscopic analyses of spinal cord sections from spastic animals revealed increased GluR1 immunoreactivity in reactive astrocytes. Selective GluR1 knockdown by intrathecal antisense treatment resulted in a potent reduction of spasticiy and rigidity and concurrent downregulation of neuronal/astrocytic GluR1 in the lumbar spinal cord. Treatment of rat astrocyte cultures with AMPA led to dose-dependent glutamate release, an effect blocked by NGX424. These data suggest that an AMPA/kainate receptor antagonist can represent a novel therapy in modulating spasticity/rigidity of spinal origin and that astrocytes may be a potential target for such treatment.

A hypoxia-inducible gene expression system using erythropoietin 3' untranslated region for the gene therapy of rat spinal cord injury.

Many neurologic disorders are accompanied by ischemic injury during the pathologic process. To develop a controllable and injury-specific gene therapy system for the neurologic disorders, we constructed a hypoxia inducible plasmid with the erythropoietin (Epo) 3' untranslated region (UTR), which can enhance the stability of target mRNAs in response to hypoxia. The Epo 3' UTR was inserted at the 3' flanking region of luciferase gene in pSV-Luc, resulting in the construction of pSV-Luc-EpoUTR. In pEpo-SV-Luc-EpoUTR, the Epo enhancer was inserted into the upstream of the SV40 promoter to increase the hypoxia inducibility. The plasmids were evaluated in N2a mouse neuroblastoma cells under hypoxic conditions and in a rat spinal cord injury (SCI) model. The results showed that the Epo 3' UTR alone showed a three-fold increase in luciferase activity in hypoxic N2a cells as well as in the rat SCI model when compared to the sham control. In contrast, the Epo 3' UTR showed no effect on the luciferase activity in the presence of the Epo enhancer, probably because the Epo enhancer was more sensitive to hypoxia and showed a dominant effect. However, the Epo enhancer itself showed high level of luciferase activity even in normoxia (about five to eight-folds increase), while the Epo 3' UTR did not show enhanced background activity. Immunohistochemical staining showed expression of luciferase from pSV-Luc-EpoUTR both in neurons and astrocytes around the injured spinal cord of rat. These results suggest that the Epo 3' UTR could provide a specific and safe system for the hypoxia-inducible gene therapy of the neurologic disorders including SCI.

Effects of buyang huanwu decoction on apoptosis of nervous cells and expressions of Bcl-2 and bax in the spinal cord of ischemia-reperfusion injury in rabbits.

OBJECTIVE: To study the effect of buyang huanwu decoction ([Chinese characters: see text] BYHWD) on spinal ischemia-reperfusion injury and explore the possible mechanism involved. METHOD: 27 model rabbits with ischemia-reperfusion injury were randomly divided into a sham operation group, a model group and a BYHWD-pre-treated group. 24 and 48 hours after the ischemia-reperfusion injury, the motor functions of hind limbs were observed, the expressions of apoptosis-related proteins bcl-2 and Bax in the spinal tissue were investigated with the immunohistochemical methods and apoptosis of nervous cells with TUNEL straining; and the malondialdehyde (MDA) level and superoxide dismutase (SOD) activity in the spinal cord were then determined. RESULTS: BYHWD can improve significantly the motor function of hind limbs, increase SOD activity and decrease the level of MDA, up-regulate expression of Bcl-2 and down-regulated expression of Bax, and depress apoptosis of nervous cells in the spinal cord caused by the ischemia reperfusion in the rabbits. CONCLUSION: BYHWD has a protective action on the spinal ischemia-reperfusion injury, and the mechanisms may be related to the decrease of lipid peroxidation and inhibition of apoptosis of nervous cells.

Ischemic injury-specific gene expression in the rat spinal cord injury model using hypoxia-inducible system.

STUDY DESIGN: A spinal cord injury and in vitro neural hypoxia models were used to evaluate the hypoxia responsive gene expression. OBJECTIVES: To limit the risk of unwanted overexpression of therapeutic genes, we developed a hypoxia-inducible gene therapy system using the erythropoietin (Epo) enhancer and the RTP801 promoter. SUMMARY OF BACKGROUND DATA: Gene therapy is an emerging therapeutic technique to treat spinal cord injury. However, uncontrolled overexpression of therapeutic genes in nondisease tissues during gene therapy raises a doubt about its safety. Post-traumatic ischemia is an important factor worsening the spinal cord damage, and hypoxia could regulate the gene expressions using a hypoxia-inducible promoter. METHODS: The plasmids, pEpo-SV-Luc and pRTP801-Luc, were constructed. Mouse neuroblastoma cells (N2A) were used to evaluate the hypoxia-inducible gene expression in vitro.- Gene transfection and expression were allowed for 24 hours under normoxia (pO2, 152 mm Hg) or hypoxia (pO2, 7.6 mm Hg). Spinal cord injury was made using clip compression. Plasmids were injected directly into the injured spinal cord immediately following injury. The gene expression was assessed by luciferase assay. RESULTS: pEpo-SV-Luc and pRTP801-Luc showed more than three times higher gene expression in N2A cells under hypoxia than normoxia. The expression level of luciferase in the injured spinal cord was higher than in the normal spinal cord. Immunostaining demonstrated that neurons, astrocytes, and capillary endothelial cells expressed luciferase in the cytoplasm. CONCLUSIONS: The pEpo-SV-Luc and pRTP801-Luc systems are effective in that they induce gene expression specifically in neurons under the hypoxic condition and spinal cord injury.