Effect of Propolis on Neurological Recovery After Experimental Spinal Cord Injury.

AIM: To examine the effect of propolis on the healing process in terms of both electrophysiological and ultrastructural parameters in a rat model of experimental spinal cord injury. MATERIAL AND METHODS: Thirty rats were divided into control, spinal cord trauma, and treated trauma groups with 10 rats per group. The rats were sacrificed after 10 days. Before sacrifice, all rats were neurologically assessed by electrophysiological monitoring, and immediately after sacrifice, the spinal cord was examined ultrastructurally by transmission electron microscopy (TEM). RESULTS: According to the electrophysiological examination, the treatment group was statistically significantly different from the trauma group. However, no statistically significant difference was found between the control and treatment groups. In terms of the TEM examination, the treatment group was significantly different from the trauma group. CONCLUSION: In this study, propolis was administered just before the induction of trauma, and the findings suggest that the use of propolis has a positive effect on the healing process. This implies that in order to prevent postoperative deficits, this treatment may be preferably applied before spinal cord surgery for trauma.

Improved spinal cord gray matter morphology induced by Spirulina platensis following spinal cord injury in rat models.

Despite intense preclinical research focusing on developing potential strategies of mitigating spinal cord injury (SCI), SCI still results in permanent, debilitating symptoms for which there are currently no effective pharmacological interventions to improve the recovery of the fine ultrastructure of the spinal cord. Spirulina platensis is thought to have potential neuroprotective effects. We have previously demonstrated its protective potential on the lesioned corticospinal tracts and behavioral recovery. In this study, spirulina, known for its neuroprotective properties was used to further explore its protective effects on spinal cord gray matter ultrastructural. Twenty-four Sprague-Dawley rats were used and divided into sham group (laminectomy without SCI), control group (SCI without S. platensis), and S. platensis group (SCI + 180 mg/kg S. platensis). All animals were anesthetized via intramuscular injection. A partial crush injury was induced at the level of T12. The rats were humanely sacrificed for 28 days postinjury for ultrastructural study. There were significant mean differences with respect to pairwise comparisons between the ultrastructural grading score of neuronal perikarya of control and the S. platensis following injury at day 28, which correlates with the functional locomotor recovery at this timepoint in our previous study. The group supplemented with spirulina, thus, revealed a better improvement in the fine ultrastructure of the spinal cord gray matter when compared to the control group thereby suggesting neuroprotective potentials of spirulina in mitigating the effects of spinal cord injury and inducing functional recovery.

Enhanced Pain Sensitivity with Systemic Ultrastructural Changes of the Nervous Systems after Cobra Venom Injection is Reversed by Electroacupuncture Treatment.

BACKGROUND: Electroacupuncture (EA) has been proved to be effective in treating certain neuropathic pain conditions. The mechanisms of pain relief by EA are not fully understood. There have been sporadic reports of damage in the peripheral nervous system (PNS) and regions of the central nervous system (CNS) at the ultrastructural level following peripheral nerve injury. However, information about possible systemic changes in the PNS and CNS after nerve injury is scarce. OBJECTIVES: The goal of this study was to examine the ultrastructural changes of the nervous system induced by a local injection of cobra venom into the sciatic nerve and to compare the ultrastructural changes in rats with or without treatment with EA or pregabalin. STUDY DESIGN: An experimental study. SETTING: Department of Anesthesiology, Pain Medicine, and Critical Care Medicine, Aviation General Hospital of China Medical University. METHODS: In this study, using an established model of sciatic neuralgia induced by local injection of cobra venom into the sciatic nerve, we examined ultrastructural changes of the PNS and CNS and how they respond to EA and pregabalin treatment. EA and pregabalin were given daily from postoperative day (POD) 14 to 36. Based on previous works, the frequency of EA stimulation of the ST36 and GB34 acupoints was held to 2/100 Hz variable. Pain sensitivity in the sciatic neuralgia rats with and without treatments was assessed using the von Frey test. Ultrastructural alterations were examined bilaterally in the prefrontal cortex, hippocampus, medulla oblongata; and the cervical, thoracic, and lumbar spinal cords on PODs 14, 40, and 60. Ultrastructural examinations were also carried out on the bilateral sciatic nerves and dorsal root ganglion (DRG) at the cervical, thoracic and lumbar levels. In rats treated with EA or pregabalin, the ultrastructure was examined on PODs 40 and 60. RESULTS: Behavioral signs of pain and systemic ultrastructural changes including demyelination were observed at all levels of the PNS and CNS in rats with sciatic neuralgia. After intervention, the mechanical withdrawal thresholds of the EA group and pregabalin group were significantly higher than that of the cobra venom group (P < 0.05). Both EA and pregabalin treatments partially reversed increased cutaneous sensitivity to mechanical stimulation. However, only the EA treatment was able to repair the ultrastructural damages caused by cobra venom. LIMITATIONS: The results confirm that peripheral nerve injury led to the ultrastructural damage at different levels of the CNS as demonstrated with electron microscopy; however, we need to further verify this at both the molecular level and in light microscope level. Sciatic neuralgia induced by cobra venom is a chemical injury, and whether this exactly mimics a peripheral nerve mechanical injury is still unclear. CONCLUSIONS: Local cobra venom injection leads to systemic neurotoxicity. EA and pregabalin alleviate pain via different mechanisms. KEY WORDS: Sciatic neuralgia, cobra venom, demyelination, electroacupuncture, pregabalin, rat model.

Transplantation of human bone marrow stem cells into symptomatic ALS mice enhances structural and functional blood-spinal cord barrier repair.

Accumulating evidence shows alterations in the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) in ALS patients and in animal models of disease, mainly by endothelial cell (EC) damage. Repair of the altered barrier in the CNS by replacement of ECs via cell transplantation may be a new therapeutic approach for ALS. Recently, we demonstrated positive effects towards BSCB repair by intravenous administration of unmodified human bone marrow CD34+ (hBM34+) cells at different doses into symptomatic ALS mice. However, particular benefits of these transplanted cells on microvascular integrity in symptomatic ALS mice are still unclear. The aim of the present study was to determine the structural and functional spinal cord capillary integrity in symptomatic ALS mice after intravenous administration of hBM34+ cells. The G93A mice at 13 weeks of age intravenously received one of three different cell doses (5 × 104, 5 × 105, or 1 × 106) and were euthanized at 17 weeks of age (4 weeks post-transplant). Control groups were media-treated and non-carrier mutant SOD1 gene mice. Capillary ultrastructural (electron microscopy), immunohistochemical (laminin and HuNu), and histological (myelin and capillary density) analyses were performed in the cervical and lumbar spinal cords. Capillary permeability in the spinal cords was determined by Evans Blue (EB) injection. Results showed significant restoration of ultrastructural capillary morphology, improvement of basement membrane integrity, enhancement of axonal myelin coherence, and stabilization of capillary density in the spinal cords primarily of ALS mice receiving the high dose of 1 × 106 cells. Moreover, substantial reduction of parenchymal EB levels was determined in these mice, confirming our previous results on capillary permeability. Additionally, transplanted cells were detected in blood smears of sacrificed late symptomatic mice by HuNu marker. Altogether, these results provide novel evidence that unmodified bone marrow hematopoietic stem cell treatment at optimal dose might be beneficial for structural and functional repair of the damaged BSCB in advanced stage of ALS, potentially resulting in delayed disease progression by increased motor neuron survival.

[Icariin alleviates lipid peroxidation after spinal cord injury in rats].

OBJECTIVE: To assess the effects of intragastric administration of icariin on lipid peroxidation after spinal cord injury in rats. METHODS: Seventy-two healthy adult male SD rats were randomized equally into icariin group, control group and sham-operated group. In the control and icariin groups, spinal cord injury was induced using modified Allen's method, and the rats in the sham-operated group underwent laminotomy without damaging the spinal cord. Immediately after the surgery, the rats in icariin group were subjected to intragastric administration of icariin (100 mg/kg), and those in the control and sham-operated groups received an equal volume of saline in the same manner once a day. At 24 h after the operation, malondialdehyde (MDA) content was detected using thiobarbituric acid method, superoxide dismutase (SOD) activity was measured with xanthine oxidase method, and the water content in the spinal cord was measured using dry-wet weight method. At 48 h after the operation, the ultrastructure of the spinal cord was observed with transmission electron microscopy and scored using Kaptanoglu scoring method. The motor function of the rats was assessed using BBB scoring at 7, 14, 21 and 28 days after the operation. RESULTS: At 24 h after the operation, MDA content was significantly higher in the control group and icariin group than in the sham-operated group, and was significantly lower in icariin group than in the control group (P<0.05); SOD activity was significantly higher in icariin group than in the control group, and was both significantly lower than that in the sham-operated group (P<0.05). At 48 h after operation, the water content and ultrastructure score of the spinal cord were the highest in sham-operated group (P<0.05), and were significantly lower in icariin group than in the control group (P<0.05). At all the time points of measurement, the BBB scores were significantly lower in the control and icariin groups than in the sham-operated group (P<0.05), and were significantly higher in icariin group than in the control group (P<0.05). CONCLUSION: Icariin can significantly reduce MDA content, increase SOD activity, and ameliorate lipid peroxidation, spinal cord edema, and histopathological damage of the spinal cord to improve motor function of rats with spinal cord injury.

Bu Shen Yi Sui capsule promotes remyelination correlating with Sema3A/NRP-1, LIF/LIFR and Nkx6.2 in mice with experimental autoimmune encephalomyelitis.

ETHNOPHARMACOLOGICAL RELEVANCE: Bu Shen Yi Sui capsule (BSYSC), based on traditional Chinese formula Liu Wei Di Huang pill, is effective for the treatment of multiple sclerosis (MS) in clinical experience and trials. Our previous studies confirmed that BSYSC had the neuroprotective effect in MS and its animal model, experimental autoimmune encephalomyelitis (EAE); however, its mechanism of action was not clear. Thus, the effect of BSYSC on remyelination and the underlying mechanisms were investigated in the EAE mice. MATERIALS AND METHODS: The EAE model was established by injecting subcutaneously myelin oligodendrocyte protein (MOG) 35-55 in mice. Mice were treated with BSYSC (3.02 g/kg) or vehicle daily by oral gavage for 40 days. The body weight and clinical score of mice were evaluated. Brain was observed by magnetic resonance imaging. The inflammation infiltrate of brain and spinal cord was determined by hematoxylin-eosin staining, while the structure of myelin sheath was visualized by transmission electron microscopy on days 23 and 40 post immunization (dpi), respectively. The protein and mRNA levels of platelets-derived growth factor receptor (PDGFR) α and 2', 3'-cyclic nucleotide-3'-phosphodiesterase (CNPase) were measured by immunohistochemistry, western blot and quantitative real-time polymerase chain reaction. The protein expressions of semaphorins (Sema) 3A, Neuropilin (NRP) - 1, leukemia inhibitory factor (LIF), LIF receptor (LIFR) and Nkx6.2 were further investigated by western blot. RESULTS: BSYSC treatment improved the body weight and clinical score of EAE mice, alleviated inflammatory infiltration and nerve fiber injuries. It also protected the ultrastructural integrity of myelin sheath. BSYSC significantly increased expressions of PDGFRα and CNPase in mice with EAE on 40 dpi. Furthermore, BSYSC treatment increased the expressions of LIF, LIFR and Nkx6.2 and reduced Sema3A and NRP-1 in EAE mice on 40 dpi. CONCLUSIONS: The data demonstrated that BSYSC exhibited the neuroprotective effect against EAE by promoting oligodendrocyte progenitor cells (OPCs) proliferation and differentiation, thus facilitating remyelination. Sema3A/NRP-1, LIF/LIFR and Nkx6.2 are likely contributed to the effects of BSYSC on OPCs.

Assessment of Neuroprotective Properties of Melissa officinalis in Combination With Human Umbilical Cord Blood Stem Cells After Spinal Cord Injury.

INTRODUCTION: The pathophysiology of spinal cord injury (SCI) has a classically bad prognosis. It has been demonstrated that human umbilical cord blood stem cells (hUCBSCs) and Melissa officinalis (MO) are useful for the prevention of neurological disease. METHODS: Thirty-six adult male rats were randomly divided into intact, sham, control (SCI), MO, hUCBSC, and MO-hUCBSC groups. Intraperitoneal injection of MO (150 mg/kg) was commenced 24 hr post-SCI and continued once a day for 14 days. Intraspinal grafting of hUCBSCs was commenced immediately in the next day. The motor and sensory functions of all animals were evaluated once a week after the commencement of SCI. Electromyography (EMG) was performed in the last day in order to measure the recruitment index. Immunohistochemistry, reverse transcription-polymerase chain reaction, and transmission electron microscopy evaluations were performed to determine the level of astrogliosis and myelination. RESULTS: The results revealed that motor function (MO-hUCBSC: 15 ± 0.3, SCI: 8.2 ± 0.37, p < .001), sensory function (MO-hUCBSC: 3.57 ± 0.19, SCI: 6.38 ± 0.23, p < .001), and EMG recruitment index (MO-hUCBSC: 3.71 ± 0.18, SCI: 1.6 ± 0.1, p < .001) were significantly improved in the MO-hUCBSC group compared with SCI group. Mean cavity area (MO-hUCBSC: 0.03 ± 0.03, SCI: 0.07 ± 0.004, p < .001) was reduced and loss of lower motor neurons (MO-hUCBSC: 7.6 ± 0.43, SCI: 3 ± 0.12, p < .001) and astrogliosis density (MO-hUCBSC: 3.1 ± 0.15, SCI: 6.25 ± 1.42, p < 0.001) in the ventral horn of spinal cord were prevented in MO-hUCBSC group compared with SCI group. CONCLUSION: The results revealed that the combination of MO and hUCBSCs in comparison with the control group has neuroprotective effects in SCI.

You-Gui pills promote nerve regeneration by regulating netrin1, DCC and Rho family GTPases RhoA, Racl, Cdc42 in C57BL/6 mice with experimental autoimmune encephalomyelitis.

ETHNOPHARMACOLOGICAL RELEVANCE: You-Gui pills (YGPs) are an effective traditional Chinese formula being used clinically for the treatment of multiple sclerosis (MS). Previous studies demonstrated that YGPs exerted the potent neuroprotective effects in murine models of experimental autoimmune encephalomyelitis (EAE), which is an equivalent animal model for multiple sclerosis (MS). However, the mechanism of YGPs functions remained unclear. AIM OF THIS STUDY: The aim of this study was to evaluate the therapeutic effect of YGPs in MOG35-55-induced EAE mice and to further elucidate the underlying molecular mechanism. METHODS: Female C57BL/6 mice were divided into six groups, including the non-treated EAE model, prednisone acetate- and 1.2, 2.4 or 4.8g/kg YGPs-treated EAE groups, and a normal control group. The EAE model was established by injecting the mice subcutaneously with MOG35-55 antigen. The body weights were measured and the neurological functions were scored in each group. The pathology and morphology of the brain and spinal cord was examined. The expression of MAP-2 was detected by immunofluorescent staining. The levels of netrin1, DCC, RhoA, Rac1, and Cdc42 were assayed by immunohistochemistry, qRT-PCR and Western blot on day 40 post-immunization (PI). RESULTS: YGPs treatments significantly reduced neurological function scores in EAE mice, where the inflammatory infiltration was reduced and the axon and myelin damage in both brain and spinal cord was alleviated. In the brain and spinal cord tissues, YGPs increased the expression of neuronal factors MAP-2, netrin1 and DCC. The expression of Rac1 and Cdc42 were increased, while RhoA was reduced following YGPs treatments. CONCLUSION: Our results demonstrated that YGPs exhibited a neuroprotective effect on promoting nerve regeneration at the brain and spinal cord in EAE mice induced by MOG35-55. Netrin1, DCC and the Rho family GTPases of RhoA, Racl, Cdc42 were involved in mediating the effects of YGPs on nerve regeneration.

Analgesic Effects and Neuropathology Changes of Electroacupuncture on Curing a Rat Model of Brachial Plexus Neuralgia Induced by Cobra Venom.

BACKGROUND: Electroacupuncture (EA) is widely applied to treat neuropathic pain. Brachial plexus neuralgia (BPN) is a common form of chronic persistent pain. Few studies have evaluated the analgesic effects and mechanism of EA using the novel animal model of BPN. OBJECTIVE: To observe the curative effects of repeated EA on curing BPN induced by administration of cobra venom to the lower trunk of the right brachial plexus. STUDY DESIGN: Controlled animal study. SETTING: Department of Anesthesiology, Pain Medicine & Critical Care Medicine, Aviation General Hospital of China Medical University. METHODS: Sixty-six adult male Sprague-Dawley rats were equally and randomly divided into the following groups: normal control (NC), brachial plexus neuralgia (BPN), BPN with sham EA stimulation, BPN with EA stimulation starting on postoperative day 1 (EA1), and BPN with EA stimulation starting on postoperative day 12 (EA12). The BPN model was established by administration of cobra venom to the lower trunk of the right brachial plexus. On postoperative day 1 or day 12, EA (constant aquare wave, 2 Hz and 100 Hz alternating frequencies, intensities ranging from 1 - 1.5 - 2 mA) was applied to the right "Shousanli" (LI10) and "Quchi" (LI11) acupoints for 30 minutes, once every other day for 12 times in both groups. Mechanical withdrawal thresholds (MWT) were tested with von Frey filaments. Video recordings were conducted to analyze the spontaneous exploratory behaviors. Moreover, the organizational and structural alterations of the right brachial plexus and cervical cord (C8-T1) were examined via light and electron microscopy. RESULTS: Following the production of the BPN model, the MWT of both ipsilateral and contralateral paws demonstrated a profound decrease (P < 0.05). But after EA interventions, the MWT showed a significant increase (P < 0.05). In comparison to the EA12 group, the analgesic effects of the EA1 group were more significant, and similar results were observed in exploratory behaviors. However, grooming behaviors did not demonstrate significant differences. Meanwhile, on day 12 after surgery it was observed under light microscopy that the inflammatory response in the right brachial plexus and cervical cord (C8-T1) were significantly attenuated after EA stimulation. Furthermore, the demyelination of the brachial plexus and cervical cord (C8-T1) were also reversed. LIMITATIONS: Limitations include the fact that there was demyelination of the cervical cord (C8-T1) in the control group because of inappropriate manipulation. CONCLUSION: Repeated EA contributes significant analgesic effects in the treatment of BPN.

Enhanced Neuroprotective Effects of Combination Therapy with Bone Marrow-Derived Mesenchymal Stem Cells and Ginkgo biloba Extract (EGb761) in a Rat Model of Experimental Autoimmune Encephalomyelitis.

OBJECTIVES: We investigated whether Ginkgo biloba extract (EGb761) can provide neuroprotective effects and enhance the efficacy of bone marrow-derived mesenchymal stem cells (BMSCs) in a rat model of experimental autoimmune encephalomyelitis (EAE). METHODS: We examined the synergistic action of BMSCs combined with EGb761 treatment in EAE rats. The immunized rats received an intravenous injection of BMSCs or intraperitoneal administration of EGb761 or both on the day of the onset of clinical symptoms and for the following 21 days. Clinical severity scores were recorded daily and histopathological examination of the spinal cord and cytokine concentrations in the serum were studied on days 14 and 31 postimmunization. RESULTS: Our results showed that combined treatment with BMSCs and EGb761 further decreased the disease severity, maximal clinical score and number of infiltrated mononuclear cells, especially CD3-positive T cells. We observed that the demyelination score and the density of axonal loss in the spinal cord were significantly reduced in mice receiving the combination therapy. The serum concentrations of the phosphorylated neurofilament heavy chain, tumor necrosis factor-α and interferon-γ were reduced in the combination-treatment group. CONCLUSION: Our results suggest that combined treatment with BMSCs and EGb761 have a synergistic effect in rats with EAE by inhibiting the secretion of proinflammatory cytokines, demyelination and protecting axons and neurons.

Expression of iron homeostasis proteins in the spinal cord in experimental autoimmune encephalomyelitis and their implications for iron accumulation.

Iron accumulation occurs in the CNS in multiple sclerosis (MS) and in experimental autoimmune encephalomyelitis (EAE). However, the mechanisms underlying such iron accumulation are not fully understood. We studied the expression and cellular localization of molecules involved in cellular iron influx, storage, and efflux. This was assessed in two mouse models of EAE: relapsing-remitting (RR-EAE) and chronic (CH-EAE). The expression of molecules involved in iron homeostasis was assessed at the onset, peak, remission/progressive and late stages of the disease. We provide several lines of evidence for iron accumulation in the EAE spinal cord which increases with disease progression and duration, is worse in CH-EAE, and is localized in macrophages and microglia. We also provide evidence that there is a disruption of the iron efflux mechanism in macrophages/microglia that underlie the iron accumulation seen in these cells. Macrophages/microglia also lack expression of the ferroxidases (ceruloplasmin and hephaestin) which have antioxidant effects. In contrast, astrocytes which do not accumulate iron, show robust expression of several iron influx and efflux proteins and the ferroxidase ceruloplasmin which detoxifies ferrous iron. Astrocytes therefore are capable of efficiently recycling iron from sites of EAE lesions likely into the circulation. We also provide evidence of marked dysregulation of mitochondrial function and energy metabolism genes, as well as of NADPH oxidase genes in the EAE spinal cord. This data provides the basis for the selective iron accumulation in macrophage/microglia and further evidence of severe mitochondrial dysfunction in EAE. It may provide insights into processes underling iron accumulation in MS and other neurodegenerative diseases in which iron accumulation occurs.

Hyperbaric oxygen treatment at various stages following chronic constriction injury produces different antinociceptive effects via regulation of P2X4R expression and apoptosis.

PURPOSE: The aims of this study were to investigate the effect of hyperbaric oxygen (HBO) treatment at various stages following chronic constriction injury (CCI) and to explore the underlying mechanisms of HBO treatment. METHODS: Forty adult male Sprague-Dawley rats were randomly assigned to five groups (n = 8 for each group): the sham group, CCI group, HBO1 group, HBO2 group, and HBO3 group. Neuropathic pain was induced by CCI of the sciatic nerve. HBO treatment began on postoperative days 1, 6, and 11 and continued for 5 days. The mechanical withdrawal threshold and thermal withdrawal latency were tested on preoperative day 3 and postoperative days 1, 3, 5, 7, 10, 14, and 21. The expression of P2X4R was determined by immunohistochemistry and western blot analysis. Cell apoptosis was measured using TUNEL staining. The expression of caspase 3 was measured using reverse transcription polymerase chain reaction (RT-PCR). Electron microscopy was used to determine the ultrastructural changes. RESULTS: Early HBO treatment beginning on postoperative day 1 produced a persistent antinociceptive effect and inhibited the CCI-induced increase in the expression of P2X4R without changing CCI-induced apoptosis. In contrast, late HBO treatment beginning on postoperative day 11 produced a persistent antinociceptive effect and inhibited CCI-induced apoptosis and upregulation of caspase-3 without changing the expression of P2X4R. In addition, late HBO treatment reduced CCI-induced ultrastructural damage. However, HBO treatment beginning on postoperative day 6 produced a transient antinociceptive effect without changing the expression of P2X4R or CCI-induced apoptosis. CONCLUSION: HBO treatment at various stages following CCI can produce antinociceptive effects via different mechanisms. Early HBO treatment is associated with inhibition of P2X4R expression, and late HBO treatment is associated with inhibition of cell apoptosis.

N-acetylcysteine amide preserves mitochondrial bioenergetics and improves functional recovery following spinal trauma.

Mitochondrial dysfunction is becoming a pivotal target for neuroprotective strategies following contusion spinal cord injury (SCI) and the pharmacological compounds that maintain mitochondrial function confer neuroprotection and improve long-term hindlimb function after injury. In the current study we evaluated the efficacy of cell-permeating thiol, N-acetylcysteine amide (NACA), a precursor of endogenous antioxidant glutathione (GSH), on mitochondrial function acutely, and long-term tissue sparing and hindlimb locomotor recovery following upper lumbar contusion SCI. Some designated injured adult female Sprague-Dawley rats (n=120) received either vehicle or NACA (75, 150, 300 or 600mg/kg) at 15min and 6h post-injury. After 24h the total, synaptic, and non-synaptic mitochondrial populations were isolated from a single 1.5cm spinal cord segment (centered at injury site) and assessed for mitochondrial bioenergetics. Results showed compromised total mitochondrial bioenergetics following acute SCI that was significantly improved with NACA treatment in a dose-dependent manner, with maximum effects at 300mg/kg (n=4/group). For synaptic and non-synaptic mitochondria, only 300mg/kg NACA dosage showed efficacy. Similar dosage (300mg/kg) also maintained mitochondrial GSH near normal levels. Other designated injured rats (n=21) received continuous NACA (150 or 300mg/kg/day) treatment starting at 15min post-injury for one week to assess long-term functional recovery over 6weeks post-injury. Locomotor testing and novel gait analyses showed significantly improved hindlimb function with NACA that were associated with increased tissue sparing at the injury site. Overall, NACA treatment significantly maintained acute mitochondrial bioenergetics and normalized GSH levels following SCI, and prolonged delivery resulted in significant tissue sparing and improved recovery of hindlimb function.

Reversible nerve conduction block using kilohertz frequency alternating current.

OBJECTIVES: The features and clinical applications of balanced-charge kilohertz frequency alternating currents (KHFAC) are reviewed. Preclinical studies of KHFAC block have demonstrated that it can produce an extremely rapid and reversible block of nerve conduction. Recent systematic analysis and experimentation utilizing KHFAC block have resulted in a significant increase in interest in KHFAC block, both scientifically and clinically. MATERIALS AND METHODS: We review the history and characteristics of KHFAC block, the methods used to investigate this type of block, the experimental evaluation of block, and the electrical parameters and electrode designs needed to achieve successful block. We then analyze the existing clinical applications of high-frequency currents, comparing the early results with the known features of KHFAC block. RESULTS: Although many features of KHFAC block have been characterized, there is still much that is unknown regarding the response of neural structures to rapidly fluctuating electrical fields. The clinical reports to date do not provide sufficient information to properly evaluate the mechanisms that result in successful or unsuccessful treatment. CONCLUSIONS: KHFAC nerve block has significant potential as a means of controlling nerve activity for the purpose of treating disease. However, early clinical studies in the use of high-frequency currents for the treatment of pain have not been designed to elucidate mechanisms or allow direct comparisons to preclinical data. We strongly encourage the careful reporting of the parameters utilized in these clinical studies, as well as the development of outcome measures that could illuminate the mechanisms of this modality.

Neuroprotective effects of gabapentin on spinal cord ischemia-reperfusion injury in rabbits.

OBJECT: Extensive research has been focused on neuroprotection after spinal cord trauma to alleviate the effects of secondary injury. This study aims to investigate the neuroprotective effects of gabapentin in an experimental spinal cord ischemia reperfusion injury. METHODS: Thirty-two adult male New Zealand white rabbits received spinal cord ischemic injury using the aortic occlusion model. Animals were divided into 4 groups (sham, control, low-dose, and high-dose treatment groups; 8 rabbits in each group). High (200 mg/kg) and low (30 mg/kg) doses of gabapentin were administered to the animals in the treatment groups after spinal cord ischemic injury. Neurological status of the animals, ultrastructural findings in injured tissue samples, and levels of tissue injury markers in these 2 groups were compared with findings in the animals that did not receive the ischemic procedure (sham-operated group) and those that received normal saline after administration of ischemia. RESULTS: Regarding levels of tissue injury marker levels after ischemic injury, animals in the gabapentin-treated groups demonstrated better results than animals in the other groups. The ultrastructural findings and caspase-3 activity were similar. The treatment groups demonstrated better results than the other groups. CONCLUSIONS: Gabapentin demonstrated significant neuroprotection after early phases of ischemic injury. Further studies with different experimental settings including neurological outcome are required to achieve conclusive results.

Ligand-directed trafficking of the δ-opioid receptor in vivo: two paths toward analgesic tolerance.

δ-Opioid receptors are G-protein-coupled receptors that regulate nociceptive and emotional responses. It has been well established that distinct agonists acting at the same G-protein-coupled receptor can engage different signaling or regulatory responses. This concept, known as biased agonism, has important biological and therapeutic implications. Ligand-biased responses are well described in cellular models, however, demonstrating the physiological relevance of biased agonism in vivo remains a major challenge. The aim of this study was to investigate the long-term consequences of ligand-biased trafficking of the δ-opioid receptor, at both the cellular and behavioral level. We used δ agonists with similar binding and analgesic properties, but high [SNC80 ((+)-4-[(αR)-α-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide)]- or low [ARM390 (N,N-diethyl-4-(phenyl-piperidin-4-ylidenemethyl)-benzamide)]-internalization potencies. As we found previously, a single SNC80-but not ARM390-administration triggered acute desensitization of the analgesic response in mice. However, daily injections of either compound over 5 d produced full analgesic tolerance. SNC80-tolerant animals showed widespread receptor downregulation, and tolerance to analgesic, locomotor and anxiolytic effects of the agonist. Hence, internalization-dependent tolerance developed, as a result of generalized receptor degradation. In contrast, ARM390-tolerant mice showed intact receptor expression, but δ-opioid receptor coupling to Ca²+ channels was abolished in dorsal root ganglia. Concomitantly, tolerance developed for agonist-induced analgesia, but not locomotor or anxiolytic responses. Therefore, internalization-independent tolerance was produced by anatomically restricted adaptations leading to pain-specific tolerance. Hence, ligand-directed receptor trafficking of the δ-opioid receptor engages distinct adaptive responses, and this study reveals a novel aspect of biased agonism in vivo.

Effect of cyclosporin A on functional recovery in the spinal cord following contusion injury.

Considerable evidence has shown that the immunosuppressant drug cyclosporin A (CsA) may have neuroprotective properties which can be exploited in the treatment of spinal cord injury. The aim of this study was to investigate the cellular environment within the spinal cord following injury and determine whether CsA has an effect on altering cellular interactions to promote a growth-permissive environment. CsA was administered to a group of rats 4 days after they endured a moderate contusion injury. Functional recovery was assessed using the Basso Beattie Bresnahan (BBB) locomotor rating scale at 3, 5 and 7 weeks post-injury. The rats were sacrificed 3 and 7 weeks post-injury and the spinal cords were sectioned, stained using histological and immunohistochemical methods and analysed. Using stereology, the lesion size and cellular environment in the CsA-treated and control groups was examined. Little difference in lesion volume was observed between the two groups. An improvement in functional recovery was observed within CsA-treated animals at 3 weeks. Although we did not see significant reduction in tissue damage, there were some notable differences in the proportion of individual cells contributing to the lesion. CsA administration may be used as a technique to control the cell population of the lesion, making it more permissive to neuronal regeneration once the ideal environment for regeneration and the effects of CsA administration at different time points post-injury have been identified.

The effects of an herbal medicine Bu-Wang-San on learning and memory of ovariectomized female rat.

ETHNOPHARMACOLOGICAL SIGNIFICANCE: Bu-Wang-San (BWS) is a traditional Chinese herbal medicine for the treatment of learning and memory impairment. The effect of BWS on neuroprotection and how BWS increases CA1 dendritic spine synapse density in menopaused women was investigated in the model of ovariectomized (OVX) rats. MATERIALS AND METHODS: Sixteen OVX rats were divided into two groups, the OVX group and OVX+BWS group. After 3 months, Morris water maze was used to assess spatial acquisition and spatial retention. Swim time, swim distance, swim speed, quadrant time and platform crossing were recorded. The ultrastructure of the pyramidal cell and spine synapse density were examined by transmission electron microscopy (TEM). RESULTS: In the spatial acquisition and spatial retention phase of testing, BWS group functioned significantly better than control group. Ultrastructural observation of the hippocampal CA1 region of OVX group showed swelling of mitochondria, the broken and reduced cristas and even crista dissolution; however, the mitochondria were protected well in BWS group. In addition, BWS significantly increased spine synapse density. CONCLUSIONS: These results suggested that BWS could improve cognitive ability of menopause-induced learning and memory impairment. The positive effect of BWS on rat learning and memory was associated with increase of spinal synapse density and protection of mitochondrial function of the pyramidal cell in hippocampal CA1 region from menopause-induced injury.

Vesicular glutamate transporters define two sets of glutamatergic afferents to the somatosensory thalamus and two thalamocortical projections in the mouse.

The ventral posterior nucleus of the thalamus (VP) receives two major sets of excitatory inputs, one from the ascending somatosensory pathways originating in the dorsal horn, dorsal column nuclei, and trigeminal nuclei, and the other originating from the cerebral cortex. Both systems use glutamate as neurotransmitter, as do the thalamocortical axons relaying somatosensory information from the VP to the primary somatosensory cortex (SI). The synapses formed by these projection systems differ anatomically, physiologically, and in their capacity for short-term synaptic plasticity. Glutamate uptake into synaptic vesicles and its release at central synapses depend on two isoforms of vesicular glutamate transporters, VGluT1 and VGluT2. Despite ample evidence of their complementary distribution, some instances exist of co-localization in the same brain areas or at the same synapses. In the thalamus, the two transcripts coexist in cells of the VP and other nuclei but not in the posterior or intralaminar nuclei. We show that the two isoforms are completely segregated at VP synapses, despite their widespread expression throughout the dorsal and ventral thalamus. We present immunocytochemical, ultrastructural, gene expression, and connectional evidence that VGluT1 in the VP is only found at corticothalamic synapses, whereas VGluT2 is only found at terminals made by axons originating in the spinal cord and brainstem. By contrast, the two VGluT isoforms are co-localized in thalamocortical axon terminals targeting layer IV, but not in those targeting layer I, suggesting the presence of two distinct projection systems related to the core/matrix pattern of organization of thalamocortical connectivity described in other mammals.

Overexpression of CCS in G93A-SOD1 mice leads to accelerated neurological deficits with severe mitochondrial pathology.

Cu, Zn superoxide dismutase (SOD1) has been detected within spinal cord mitochondria of mutant SOD1 transgenic mice, a model of familial ALS. The copper chaperone for SOD1 (CCS) provides SOD1 with copper, facilitates the conversion of immature apo-SOD1 to a mature holoform, and influences in yeast the cytosolic/mitochondrial partitioning of SOD1. To determine how CCS affects G93A-SOD1-induced disease, we generated transgenic mice overexpressing CCS and crossed them to G93A-SOD1 or wild-type SOD1 transgenic mice. Both CCS transgenic mice and CCS/wild-type-SOD1 dual transgenic mice are neurologically normal. In contrast, CCS/G93A-SOD1 dual transgenic mice develop accelerated neurological deficits, with a mean survival of 36 days, compared with 242 days for G93A-SOD1 mice. Immuno-EM and subcellular fractionation studies on the spinal cord show that G93A-SOD1 is enriched within mitochondria in the presence of CCS overexpression. Our results indicate that CCS overexpression in G93A-SOD1 mice produces severe mitochondrial pathology and accelerates disease course.