Sinomenine Alleviates Murine Experimental Autoimmune Encephalomyelitis Model of Multiple Sclerosis through Inhibiting NLRP3 Inflammasome.

Multiple sclerosis (MS) is known as a chronic neuroinflammatory disorder typified by an immune-mediated demyelination process with ensuing axonal damage and loss. Sinomenine is a natural alkaloid with different therapeutic benefits, including anti-inflammatory and immunosuppressive activities. In this study, possible beneficial effects of sinomenine in an MOG-induced model of MS were determined. Sinomenine was given to MOG35-55-immunized C57BL/6 mice at doses of 25 or 100 mg/kg/day after onset of MS clinical signs till day 30 post-immunization. Analyzed data showed that sinomenine reduces severity of the clinical signs and to some extent decreases tissue level of pro-inflammatory cytokines IL-1ß, IL-6, IL-18, TNFα, IL-17A, and increases level of anti-inflammatory IL-10. In addition, sinomenine successfully attenuated tissue levels of inflammasome NLRP3, ASC, and caspase 1 besides its reduction of intensity of neuroinflammation, demyelination, and axonal damage and loss in lumbar spinal cord specimens. Furthermore, immunoreactivity for MBP decreased and increased for GFAP and Iba1 after MOG-immunization, which was in part reversed upon sinomenine administration. Overall, sinomenine decreases EAE severity, which is attributed to its alleviation of microglial and astrocytic mobilization, demyelination, and axonal damage along with its suppression of neuroinflammation, and its beneficial effect is also associated with its inhibitory effects on inflammasome and pyroptotic pathways; this may be of potential benefit for the primary progressive phenotype of MS.

Comprehensive analysis of the differential expression profile of microRNAs in rats with spinal cord injury treated by electroacupuncture.

Abnormal microRNA (miRNA) expression has been implicated in spinal cord injury (SCI), but the underlying mechanisms are poorly understood. To observe the effect of electroacupuncture (EA) on miRNA expression profiles in SCI rats and investigate the potential mechanisms involved in this process, Sprague­Dawley rats were divided into sham, SCI and SCI+EA groups (n=6 each). Basso, Beattie and Bresnahan (BBB) scoring and hematoxylin­eosin staining of cortical tissues were used to evaluate spinal cord recovery with EA treatment 21 days post­surgery across the three groups. To investigate miRNA expression profiles, 6 Sprague­Dawley rats were randomly divided into SCI and SCI+EA groups (n=3 in each group) and examined using next­generation sequencing. Integrated miRNA­mRNA­pathway network analysis was performed to elucidate the interaction network of the candidate miRNAs, their target genes and the involved pathways. Behavioral scores suggested that hindlimb motor functions improved with EA treatments. Apoptotic indices were lower in the SCI+EA group compared with the SCI group. It was also observed that 168 miRNAs were differentially expressed between the SCI and SCI+EA groups, with 29 upregulated and 139 downregulated miRNAs in the SCI+EA group. Changes in miRNA expression are involved in SCI physiopathology, including inflammation and apoptosis. Reverse transcription­quantitative PCR measurement of the five candidate miRNAs, namely rno­miR­219a­5p, rno­miR­486, rno­miR­136­5p, rno­miR­128­3p, and rno­miR­7b, was consistent with RNA sequencing data. Integrated miRNA­mRNA­pathway analysis suggested that the MAPK, Wnt and NF­κB signaling pathways were involved in EA­mediated recovery from SCI. The present study evaluated the miRNA expression profiles involved in EA­treated SCI rats and demonstrated the potential mechanism and functional role of miRNAs in SCI in rats.

Alterations in lipid metabolism of spinal cord linked to amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is characterized by progressive loss of upper and lower motor neurons leading to muscle paralysis and death. While a link between dysregulated lipid metabolism and ALS has been proposed, lipidome alterations involved in disease progression are still understudied. Using a rodent model of ALS overexpressing mutant human Cu/Zn-superoxide dismutase gene (SOD1-G93A), we performed a comparative lipidomic analysis in motor cortex and spinal cord tissues of SOD1-G93A and WT rats at asymptomatic (~70 days) and symptomatic stages (~120 days). Interestingly, lipidome alterations in motor cortex were mostly related to age than ALS. In contrast, drastic changes were observed in spinal cord of SOD1-G93A 120d group, including decreased levels of cardiolipin and a 6-fold increase in several cholesteryl esters linked to polyunsaturated fatty acids. Consistent with previous studies, our findings suggest abnormal mitochondria in motor neurons and lipid droplets accumulation in aberrant astrocytes. Although the mechanism leading to cholesteryl esters accumulation remains to be established, we postulate a hypothetical model based on neuroprotection of polyunsaturated fatty acids into lipid droplets in response to increased oxidative stress. Implicated in the pathology of other neurodegenerative diseases, cholesteryl esters appear as attractive targets for further investigations.

Oxycodone concentrations in the central nervous system and cerebrospinal fluid after epidural administration to the pregnant ewe.

The main sites of the analgesic action of oxycodone are the brain and spinal cord. The present study describes the concentrations of oxycodone and its metabolites in the brain and spinal cord after epidural administration to the ewe. Twenty pregnant ewes undergoing laparotomy were randomized into two groups to receive epidural oxycodone: infusion group (n = 10, 0.1 mg·kg-1 bolus followed by continuous infusion of 0.05 mg·kg-1 ·h-1 for five days) or repeated boluses group (n = 10, 0.2 + 2x0.1 mg·kg-1 bolus followed by a 0.2 mg·kg-1 bolus every 12 hours for five days). After five days of oxycodone administration, arterial blood samples were collected, the sheep were killed, and a CSF sample and tissue samples from the cortex, thalamus, cerebellum and spinal cord were obtained for the quantification of oxycodone and its main metabolites. The median plasma and CSF concentrations of oxycodone were 9.0 and 14.2 ng·mL-1 after infusion and 0.4 and 1.1 ng·mL-1 after repeated boluses. In the infusion group, the cortex, thalamus and cerebellum oxycodone concentrations were 4-8 times higher and in the spinal cord 1310 times higher than in plasma. In the repeated boluses group, brain tissue concentrations were similar in the three areas, and in the spinal cord were 720 times higher than in plasma. Oxymorphone was the main metabolite detected, which accumulated in the brain and spinal cord tissue. In conclusion, first, accumulation of oxycodone and oxymorphone in the CNS was observed, and second, high spinal cord concentrations suggest that epidural oxycodone may provide segmental analgesia.

[Analgesic effect and central mechanisms of CQ prescription on cancer invasion induced mirror image pain in model mice].

This study aimed to analyze the analgesic effect and related central mechanisms of CQ prescription on cancer invasion induced mirror image pain (CIIMIP)in model mice.In the study, male BALB/c mice were randomly divided into normal group, operation control group (injected with 0.2 mL inactivated S180 sarcoma cell sap), model group (injected with 0.2 mL S180 sarcoma cell sap on the right leg near the greater trochanter of femur) and CQ prescription low dose group (intraperitoneally injected with CQ prescription 100 mg•kg⁻¹ on the basis of model mice), CQ prescription middle dose group (intraperitoneally injected with CQ prescription 150 mg•kg⁻¹ on the basis of model mice), and CQ prescription high dose group (intraperitoneally injected with CQ prescription 200 mg•kg⁻¹ on the basis of model mice). Mechanical withdraw threshold (MWT) of the mirror image lateral hind paws were evaluated by Von Frey hairs before modeling and after surgery. The levels of glutamate (Glu), gamma aminobutyric acid (GABA), glycine (Gly), and taurine (Tau) in the L3-L5 spinal cord were measured by the high performance liquid chromatography-fluorescence detector (HPLC-FLD); AimPlex detection technology with multiple factors was used to detect the levels of regulated on activation in normal T-cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP-3) in the L3-L5 spinal cord. Then we observed the influence of GABAa receptor antagonist (Bicuculline) on analgesic effect of CQ prescription.The results indicated that CQ prescription could remarkably increase MWT of model mice(P<0.01, P<0.05), decrease the level of Glu(P<0.01, P<0.05), improve the levels of GABA, Gly, Tau(P<0.01, P<0.05), lower the ratio of Glu/GABA(P<0.01, P<0.05), and reduce the levels of RANTES, MCP-3(P<0.05) in the L3-L5 spinal cord, and GABAa receptor antagonist significantly blocked the analgesic effect of CQ prescription at two time points(P<0.05).This study showed that CQ prescription had significant analgesic effect on CIIMIP model mice, and its mechanism was associated with regulating the balance between excitability amino acid(EAA) and inhibitory amino acid (IAA) transmitters in central nervous system, partially activating GABAa receptor, and reducing the release of RANTES and MCP-3 in the spinal cord.

Preemptive Analgesia with Acupuncture Monitored by c-Fos Expression in Rats.

Pain behavior and awareness are characterized by heightened alertness and anxiety, which begin to disappear as soon as the curative process starts. The present study aimed to quantify c-fos expression in rat spinal cords and brains after a surgical stimulus and with preoperative or postoperative acupuncture. Animals were randomly divided into preoperative and postoperative groups and were then further divided into control, manual acupuncture (MA), or electroacupuncture (EA) groups. Expression of c-fos was quantified using immunohistochemistry. The collected data were analyzed using the t test at a 5% probability level. Presurgery and postsurgery spinal cord c-fos expressions were similar in all of the treatment groups. In the control rats, c-fos expression was higher before surgery than after surgery, contradicting the expected outcome of acupuncture and preemptive analgesia. After treatment, the expression of c-fos in the brains of the rats in the MA and the EA groups was reduced compared with that of the rats in the control group. These findings suggest that acupuncture used as preemptive analgesia in rats is a useful model for studying its application in human treatment.

[Effect of Electroacupuncture Intervention on ß-endorphin Levels of Hypothalamus and Spinal Cord Tissues in Rats with Pelmatic Incisional Pain].

OBJECTIVE: To observe the effect of electroacupuncture (EA) intervention on pain thresholds (PT) and contents of ß-endorphin (EP) in the hypothalamus and spinal cord, and the expression of 5-HT in the dorsal raphe nucleus(DRN)in rats with pelmatic incisional pain, so as to investigate the underlying mechanisms of acupuncture in reducing post-operative pain. METHODS: Wistar rats were randomized into normal control, model, EA and non-acupoint groups (n=8/group). The pelmatic pain model was induced by making an incision (about 1 cm in length, to the fascia and muscle layers) from the heel towards the toes. EA (2 Hz, 1.5-2 V) was applied to "Zusanli" (ST 36) and "Kunlun" (BL 60) or non-acupoint (about 3 mm beside the ST 36 and BL 60) on the affected side for 20 min, once daily for three days. The thermal PT and mechanical PT were measured before and after operation and after EA. The contents of ß-EP in hypothalamus and L3-S4 spinal cord were detected using enzyme linked immunosorbent assay (ELISA) and the expressions of ß-EP in hypothalamus and 5-HT in DRN were measured with immunohistochemistry. RESULTS: After EA intervention, the markedly decreased mechanical and thermal pain thresholds on day 1 and 3 after paw incision were significantly increased in the EA group (P<0.05), but not in the non-acupoint group (P>0.05). The hypothalamic ß-EP content was significantly higher in the model group than in the normal group (P<0.05), and further up-regulated in the EA group (not the non-acupoint group) than in the model group (P<0.05). In addition, the hypothalamic ß-EP immunoreactive (IR)-positive cell number and 5-HT immunoactivity level in DRN were also considerably up-regulated in the EA group (P<0.05) but not in the non-acupoint group (P>0.05). No significant changes were found in the lumbar spinal ß-EP contents in the model, EA and non-acupoint groups (P>0.05). CONCLUSIONS: EA stimulation of "Zusanli"(ST 36) and "Kunlun" (BL 60) has an analgesic effect in pelmatic incision pain rats, which may be related to its effects in raising the level of hypothalamic ß-EP and the expression of 5-HT in DRN.

Nanowired Delivery of Growth Hormone Attenuates Pathophysiology of Spinal Cord Injury and Enhances Insulin-Like Growth Factor-1 Concentration in the Plasma and the Spinal Cord.

Previous studies from our laboratory showed that topical application of growth hormone (GH) induced neuroprotection 5 h after spinal cord injury (SCI) in a rat model. Since nanodelivery of drugs exerts superior neuroprotective effects, a possibility exists that nanodelivery of GH will induce long-term neuroprotection after a focal SCI. SCI induces GH deficiency that is coupled with insulin-like growth factor-1 (IGF-1) reduction in the plasma. Thus, an exogenous supplement of GH in SCI may enhance the IGF-1 levels in the cord and induce neuroprotection. In the present investigation, we delivered TiO2-nanowired growth hormone (NWGH) after a longitudinal incision of the right dorsal horn at the T10-11 segments in anesthetized rats and compared the results with normal GH therapy on IGF-1 and GH contents in the plasma and in the cord in relation to blood-spinal cord barrier (BSCB) disruption, edema formation, and neuronal injuries. Our results showed a progressive decline in IGF-1 and GH contents in the plasma and the T9 and T12 segments of the cord 12 and 24 h after SCI. Marked increase in the BSCB breakdown, as revealed by extravasation of Evans blue and radioiodine, was seen at these time points after SCI in association with edema and neuronal injuries. Administration of NWGH markedly enhanced the IGF-1 levels and GH contents in plasma and cord after SCI, whereas normal GH was unable to enhance IGF-1 or GH levels 12 or 24 h after SCI. Interestingly, NWGH was also able to reduce BSCB disruption, edema formation, and neuronal injuries after trauma. On the other hand, normal GH was ineffective on these parameters at all time points examined. Taken together, our results are the first to demonstrate that NWGH is quite effective in enhancing IGF-1 and GH levels in the cord and plasma that may be crucial in reducing pathophysiology of SCI.

[Colorectal nociceptive signal input facilitates impact of acupoint stimulation of "Zusanli" (ST 36) on electrical activities of wide dynamic range neurons in lumbar spinal cord in rats].

OBJECTIVE: To observe changes of electrical activities of wide dynamic neurons (WDR) in the lumbar spinal cord after electroacupuncture (EA) stimulation of "Zusanli" (ST 36) during colorectal distension in rats, so as to analyze alternations of acupoint's functions under pathological conditions. METHODS: Experiments were performed in male Sprague-Dawley rats. Visceral nociceptive information input was generated by colorectal distension (CRD). Extracellular discharges of WDR neu- rons in the dorsal horns of L(1-3) spinal cord were recorded using glass micropipettes and a microelectrode amplifier. EA stimulation (15 Hz, 1, 4, 7, 10 mA) was applied to ipsilateral "Zusanli" (ST 36) area. RESULTS: A total of 40 neurons were recorded in the present study. Under no CRD conditions, the firing rates of WDR neurons were significantly increased by (18.12 +/- 13.56)% (1 mA), (152.38 +/- 36.19)% (4 mA), (231.21 +/- 49.74)% (7 mA) and (331.54 +/- 61.89)% (10 mA) respectively after EA sti- mulation of ST 36. Under CRD conditions, the firing rates of these WDR neurons were increased by (226.78 +/- 39.59)% (1 mA), (282.80 +/- 47.54)% (4 mA), (343.06 +/- 58.35)% (7 mA), and (338.62 +/- 80.04)% (10 mA) respectively after EA stimulation. It showed a considerablely increased sensitivity of electrical activities of WDR neurons to EA stimulation during CRD nociceptive stimulation. CONCLUSION: colorectal distension (visceral nociceptive stimulation) can strengthen the sensitivity of acupoint-EA stimulation-induced increase of firing rates of WDR neurons in the dorsal horns of the lumbar spinal cord in rats, suggesting a potentiation of the acupoint action under visceral nociceptive signal inputs.

The acute antinociceptive effect of hyperbaric oxygen is not accompanied by an increase in markers of oxidative stress.

AIMS: Exposure to hyperbaric oxygen (HBO2) causes an antinociceptive response in mice. However, breathing oxygen (O2) at an elevated pressure can potentially cause oxygen toxicity. The aim of this study was to identify the determinants of HBO2 antinociception and the toxicity profile of HBO2. MAIN METHODS: Male NIH Swiss mice were assessed for acute antinociceptive responsiveness under room air or 100% O2 at 1.0 or 3.5 atmospheres absolute (ATA), using the acetic acid-induced abdominal constriction test. For the oxygen toxicity test, mice were exposed to 3.5 ATA oxygen for 11min, 60min, and 60min daily for 2days (120min) or 60min daily for 4days (240min), then assessed by analyzing the levels of two oxidative stress markers, MDA (malondialdehyde) and protein carbonyl in brain, spinal cord and lung. KEY FINDINGS: Only the combination of 100% O2 and 3.5 ATA caused significant antinociception. The antinociceptive effect of 100% O2 was pressure-dependent up to 3.5 ATA. In the oxygen toxicity test, mice exposed to HBO2 for different time intervals had levels of brain, spinal cord and lung MDA and protein carbonyl that were comparable to that of control animals exposed to room air. SIGNIFICANCE: Treatment with 100% O2 evokes a pressure-dependent antinociceptive effect. Since there was no significant increase in levels of the oxidative stress markers in the tested tissues, it is concluded that HBO2 at 3.5 ATA produces antinociception in the absence of oxidative stress in mice.

Analysis of interspike interval of dorsal horn neurons evoked by different needle manipulations at ST36.

OBJECTIVES: Previous research has suggested that different manual acupuncture (MA) manipulations may have different physiological effects. Recent studies have demonstrated that neural electrical signals are generated or changed when acupuncture is administered. In order to explore the effects of different MA manipulations on the neural system, an experiment was designed to record the discharges of wide dynamic range (WDR) neurons in the spinal dorsal horn evoked by MA at different frequencies (0.5, 1, 2 and 3 Hz) at ST36. METHODS: Microelectrode extracellular recordings were used to record the discharges of WDR neurons evoked by different MA manipulations. Approximate firing rate and coefficient of variation of interspike interval (ISI) were used to extract the characteristic parameters of the neural electrical signals after spike sorting, and the neural coding of the evoked discharges by different MA manipulations was obtained. RESULTS: Our results indicated that the neuronal firing rate and time sequences of ISI showed distinct clustering properties for different MA manipulations, which could distinguish them effectively. CONCLUSIONS: The combination of firing rate and ISI codes carries information about the acupuncture stimulus frequency. Different MA manipulations appear to change the neural coding of electrical signals in the spinal dorsal horn through WDR neurons.

Neuroanatomical basis for acupuncture point PC8 in the rat: neural tracing study with cholera toxin subunit B.

OBJECTIVES: This study was performed to investigate the innervations related to acupuncture point PC8 in rats using a neural tracing technique. METHODS: After 6 µL of 1% cholera toxin subunit B (CTB) was injected into the site between the second and third metacarpal bone in rats, a corresponding site to acupuncture point PC8 in the human body, CTB labelling was examined with immunofluorescence and immunohistochemistry in the dorsal root ganglia (DRG), spinal cord and brainstem. RESULTS: All CTB labelling appeared on the ipsilateral side of the injection. The labelled sensory neurons distributed from cervical (C)6 to thoracic (T)1 DRG, while the labelled motor neurons were located on the dorsolateral part of the spinal ventral horn ranging from the C6 to T1 segments. In addition, the transganglionically-labelled axonal terminals were found to be dense in the medial part of laminae 3-4 from C6 to the T1 spinal dorsal horn, as far as in the cuneate nucleus. CONCLUSIONS: These results indicate that sensory and motor neurons associated with PC8 distribute in a distinct segmental pattern. The sensory information from PC8 could be transganglionically transported to the spinal dorsal horn and cuneate nucleus.

Heat-shock protein 70 is involved in hyperbaric oxygen preconditioning on decompression sickness in rats.

Decompression sickness (DCS) is a major concern in diving and space walk. Hyperbaric oxygen (HBO) preconditioning has been proved to enhance tolerance to DCS via nitric oxide. Heat-shock protein (HSP) 70 was also found to have protective effects against DCS. We hypothesized that the beneficial effects of HBO preconditioning on DCS was related to levels of elevated HSP70. HSPs (70, 27 and 90) expressed in tissues of spinal cord and lung in rats was detected at different time points following HBO exposure by Western blot. HSP27 and HSP90 showed a slight but not significant increase after HBO. HSP70 increased and reached highest at 18 h following exposure before decreasing. Then rats were exposed to HBO and subjected to simulated air dive and rapid decompression to induce DCS 18 h after HBO. The severity of DCS, along with levels of HSP70 expression, as well as the extent of oxidative and apoptotic parameters in the lung and spinal cord were compared among different groups of rats pretreated with HBO, HBO plus NG-nitro-l-arginine-methyl ester (l-NAME), HBO plus quercetin or normobaric air. HBO preconditioning significantly reduced the morbidity of DCS (from 66.7% to 36.7%), reduced levels of oxidation (malondialdehyde, 8-hydroxyguanine and hydrogen peroxide) and apoptosis (caspase-3 and -9 activities and the number of apoptotic cells). l-NAME or quercetin eliminated most of the beneficial effects of HBO on DCS, and counteracted the stimulation of HSP70 by HBO. Bubbles in pulmonary artery were detected using ultrasound imaging to observe the possible effect of HBO preconditioning on DCS bubble formation. The amounts of bubbles in rats pretreated with HBO or air showed no difference. These results suggest that HSP70 was involved in the beneficial effects of HBO on DCS in rats, suspected be by the antioxidation and antiapoptosis effects.

AR-A014418 as a glycogen synthase kinase-3 inhibitor: anti-apoptotic and therapeutic potential in experimental spinal cord injury.

OBJECTIVES: We aimed to investigate the effects of AR-A014418, a strong inhibitor specific to GSK-3beta, on neuronal apoptosis and neuroprotection in the traumatic SCI model. MATERIALS AND METHODS: In this study, three groups were generated from 36 Wistar rats; (1) control, (2) spinal cord trauma group created by clip compression technique after laminectomy, and (3) AR-A014418 (4mg/kg, i.p., DMSO) treatment group after laminectomy and spinal cord trauma. The TUNEL assay for apoptosis detection, immunohistochemical staining for bax and TGF-beta were applied in spinal cord tissues. For light microscopic examination, necrotic, and apoptotic cells were counted, and PMNL counting was applied to detect inflammation. Functional recovery was tested by field locomotor test in the 3rd and 7th days following surgery. RESULTS: In the trauma group, diffuse hemorrhage, cavitation, necrosis and edematous regions, degeneration in motor neurons and leukocyte infiltration were observed in gray matter. In the AR-A014418-treated groups, healthy cells were observed in more places compared to the trauma groups, however, cavitation, hemorrhagic, and edematous areas were seen in gray matter. In the AR-A014418-treatment groups, the number of apoptotic cells in the 3rd and 7th days (respectively; p<0.05, p<0.01), were significantly decreased compared to the trauma groups, as were the levels of bax (p<0.01) and TGF-beta 1 immunoreactivity. Results of the locomotor test were significantly increased in the treatment group (p<0.001) as compared to the trauma group. CONCLUSIONS: In this experimental spinal cord trauma model study neural apoptosis was significantly triggered in secondary damage developed after trauma, however, neurological healing was expedited by preventing mitochondrial apoptosis and reducing the inflammation by the potent inhibitor AR-A014418, which is GSK-3beta selective.

Transgenic mice with high endogenous omega-3 fatty acids are protected from spinal cord injury.

Omega-3 polyunsaturated fatty acids have been shown to have therapeutic potential in a variety of neurological disorders, including acute traumatic injury of the spinal cord. We addressed the question whether the neuroprotective effect of these compounds after spinal cord injury could also be seen when their level is raised in tissues prophylactically, prior to injury. In this study we used transgenic fat-1 mice to examine whether enriching spinal cord tissue in endogenous omega-3 polyunsaturated fatty acids has an effect on the outcome after compression spinal cord injury. The results demonstrate that after thoracic compression spinal cord injury, fat-1 mice display better locomotor recovery compared with the wild-type mice on a high omega-6 diet (high omega-6 polyunsaturated fatty acids in tissues), and wild-type mice on a normal diet (controls). This is associated with a significant increase in neuronal and oligodendrocyte survival and a decrease in non-phosphorylated neurofilament loss. The protection from spinal cord injury in fat-1 mice was also correlated with a reduction in microglia/macrophage activation and in pro-inflammatory mediators. In vitro experiments in dorsal root ganglia primary sensory neurons further demonstrated that a fat-1 tissue background confers robust neuroprotection against a combined mechanical stretch and hypoxic injury. In conclusion, our studies support the hypothesis that a raised omega-3 polyunsaturated fatty acid level and an altered tissue omega-6/omega-3 ratio prior to injury leads to a much improved outcome after spinal cord injury.

Primo vascular system in the subarachnoid space of the spinal cord of a pig.

The primo vascular system was recently observed in the central nervous systems of rabbits and rats, but no investigations in large animals have been reported. In the present work we found a putative primo vascular system in the spinal cord of a pig. We obtained spines from four healthy pigs and fixed them with paraformaldehyde. The primo vessels were expected to lie in the subarachnoid space between the pia mater and the arachnoid mater. The composite of three membranes (the pia, the arachnoid, and the dura maters) wrapping the spinal cord was peeled off, isolated from the spine, and put on a slide glass. This composite was stained with 4',6'-Diamidino-2-phenylindole (DAPI) and phalloidin to show the nuclei and the f-actin, respectively, in the cells of the primo vessels. We observed eleven pieces of the putative primo vessels in the subarachnoid space of the spines at the thoracic spinal nerve area. They had the typical rod-shaped nuclei distributed in a broken line, and f-actin signals around nuclei. The lengths of the nuclei were 12-15 µm, and the thicknesses of the primo vessels were 8∼20 µm, which were consistent with other primo vessels that had been observed in the various organs of rabbits, rats, and mice. In addition, we observed branching of the primo vessels, which is again an expected result from previous works. In conclusion, a primo vessel was observed in the subarachnoid space of the spinal cord of a pig. This was the first observation of a primo vessel in a large animal, and the staining method used to observe the primo vessel in a fixed sample was newly developed in this work.

[Effects of small needle knife on the substance P in the dorsal root ganglion and spinal cord of rats].

OBJECTIVE: To study the mechanism of synthesis of substance P (SP) in the dorsal root ganglion (DRG) and the release of it in the dorsal horn of the spinal cord of rats after compression of skeletal muscle, and to observe the influence of small needle knife. METHODS: Sustained pressure of 70 kPa was applied to rats, muscular tissues for 2 hours. The rats were divided into three groups: normal, control and experiment group respectively. In all rats except the six normal ones, the lower legs were compressed once one day. The left leg was considered as the control group, the right left was experiment group, which were divided into the 1st day, the 2nd day and the 3rd day within the two groups. Experiment group was treated with small needle knife after the muscular tissue was compressed. After completing the stimulation, the DRG related to the muscle and part of spinal cord were removed for the qualification of SP-like immunoreactivity using immunohistochemistry. The dark brown stains on the DRG and on the REXed laminae I and II in the dorsal horn of the spinal cord were counted by Image-Pro Plus software. RESULTS: SP-like immunoreactivity in the side treated by the small needle knife was enhanced comparing with the counterpart in DRG in normal group (P < 0.01). The integrated optical density of SP like immunoreactivity of the DRG in the experiment group were significantly reduced compared with the control group (P < 0.05). However, the release of SP from spinal cord in experiment group was lower than that in the control group at the 1st day and the 3rd day (P < 0.01), with the opposite result of the 2nd day. CONCLUSION: Based on the fact that SP is a nociceptive neurotransmitter, the present study suggests that tension relaxation by small needle knife reduces expression of SP in the DRG, and shows no effects on the release of SP from the spinal cord in short-term (3 days).

What does the mechanism of spinal cord stimulation tell us about complex regional pain syndrome?

Spinal cord stimulation (SCS) can have dramatic effects on painful, vascular, and motor symptoms of complex regional pain syndrome (CRPS), but its precise mechanism of action is unclear. Better understanding of the physiologic effects of SCS may improve understanding not only of this treatment modality but also of CRPS pathophysiology. Effects of SCS on pain perception are likely to occur through activation of inhibitory GABA-ergic and cholinergic spinal interneurons. Increased release of both neurotransmitters has been demonstrated following SCS in animal models of neuropathic pain, with accompanying reductions in pain behaviors. Effects of SCS on vascular symptoms of CRPS are thought to occur through two main mechanisms: antidromic activation of spinal afferent neurons and inhibition of sympathetic efferents. Cutaneous vasodilation following SCS in animal models has been shown to involve antidromic release of calcitonin gene-related peptide and possibly nitric oxide, from small-diameter sensory neurons expressing the transient receptor potential V1 (TRPV1) receptor. The involvement of sympathetic efferents in the effects of SCS has not been studied in animal models of neuropathic pain, but has been demonstrated in models of angina pectoris. In conclusion, SCS is of clinical benefit in CRPS, and although its mechanism of action merits further elucidation, what little we do know is informative and can partially explain some of the pathophysiology of CRPS.

Primo-vessels and primo-nodes in rat brain, spine and sciatic nerve.

We report a method using Trypan blue staining to detect primo-vessels in the nervous system on internal organs or in the skin of rat. We applied this technique to visualize the primo-vessels and primo-nodes in the brain, spinal cord and sciatic nerve of a rat. Primo-vessels and primo-nodes were preferentially stained at nerves, blood vessels, or fascia-like membranes and turned blue after the spread and washing of Trypan blue. The physiological role of the primo-vessels within the nervous system is an important question warranting further investigation.

Identification of acupuncture-specific proteins in the process of electro-acupuncture after spinal cord injury.

Spinal cord injury (SCI) is a serious condition often affecting young and healthy individuals around the world. Electro-acupuncture (EA) has been proven to contribute towards neurologic and functional recoveries in SCI, but the underlying mechanism remains largely unknown especially regarding neural specific proteins involved in the development of EA. The protein expression profile of spinal cord in both SCI and EA treatment models was analyzed by using two-dimensional electrophoresis-based proteomics. Using a MALDI-TOF/TOF MS and subsequent protein database searching, we identified changes in 15 proteins in the spinal cord following Governor Vessel (GV) EA treatment on SCI. These proteins are involved in inflammation, cell adhesion and migration, signal transduction and apoptosis processes. We selected 2 proteins (ANXA5 and CRMP2) beneficial to neuronal survival and axonal regeneration, and further identified these protein changes using Western blot analysis. Subsequently, Nissl staining and immunofluorescence double labeling approaches were used to explore possible role of the two neural specific proteins in the process of GV-EA treatment on SCI. Our results suggest that ANXA5 and CRMP2 may be neural specific proteins in the process of GV-EA treatment on SCI. This work might contribute to the better understanding of the mechanism involved in EA treatment on SCI at protein levels and provide a new therapeutic strategy for SCI.