OBJECTIVETo 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.

METHODSSustained 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.

RESULTSSP-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.

CONCLUSIONBased 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).

Animals ; Female ; Ganglia, Spinal ; chemistry ; Immunohistochemistry ; Male ; Medicine, Chinese Traditional ; Needles ; Rats ; Rats, Sprague-Dawley ; Spinal Cord ; chemistry ; Substance P ; analysis ; secretion

BACKGROUND: The primary mode of conduction bldegrees Ckade by ldegrees Cal anesthetics degrees Ccurs through the inhibition of voltage-dependent sodium current and, inhibitory potency of ldegrees Cal anesthetics are correlated with their hydrophobicity, expressed as degrees Ctanol/buffer partition coefficients(PC). The homologous structural bidegrees Chemistry and analogous physiology of voltage-dependent sodium and calcium channels prompted us to examine the relationship between potency of various ldegrees Cal anesthetics for the inhibition of voltage-dependent calcium channels(VDCC) and their PC values. METHODS: Whole cell patch clamp recordings were made from acutely dissdegrees Ciated rat dorsal root ganglion neurons, and voltage dependent calcium current(ICa) was evoked by depolarizing pulse. The concentrations of various ldegrees Cal anesthetics(bupivacaine, liddegrees Caine, prildegrees Caine, prdegrees Caine, tetracaine) that bldegrees Ck 50% of the control ICa(IC50) were calculated from dose-response curves. The relationship between IC50 and PC values of various ldegrees Cal anesthetics were investigated. RESULTS: Ldegrees Cal anesthetics inhibited ICa with neglegible effect on the current- voltage relatonship. IC50 values of tetracaine, bupivacaine, liddegrees Caine, prildegrees Caine and prdegrees Caine were 98, 142, 2710, 10400, 16900 uM respectively, and linear regression of the plot of log(IC50) against log(PC) was statistically significant (p<0.001). CONCLUSIONS: It is speculated that inhibitory effects of ldegrees Cal anesthetics on the VDCC when used in epidural and spinal anesthesia may contribute to their analgesic and anesthetic actions. Inhibitory potency of ldegrees Cal anesthetics on the VDCC, as for voltage-dependent sodium channels, was correlated with their hydrophobicity.
Anesthesia, Spinal ; Anesthetics* ; Animals ; Bupivacaine ; Calcium Channels ; Calcium* ; Chemistry ; Ganglia, Spinal ; Hydrophobic and Hydrophilic Interactions* ; Inhibitory Concentration 50 ; Linear Models ; Neurons ; Physiology ; Rats ; Sodium ; Sodium Channels ; Tetracaine

OBJECTIVETo investigate the effect of high-fat diet on the expression of transient receptor potential vanilloid 1 (TRPV1) in the respiratory system and the dorsal root ganglion (DRG) of mice, as well as its effect on the excitability of sensory neurons.

METHODSA total of 20 C57BL/6 mice were randomly divided into normal-diet (ND) group and high-fat diet (HFD) group, with 10 mice in each group. The mice were given corresponding diets and body weights were monitored. After 7 weeks of feeding, lung tissue, bronchial tissue, and DRG at thoracic segments 3-4 were collected and immunohistochemical staining was performed. A patch clamp was used to measure the number of action potentials and TRPV1 current intensity in the DRG.

RESULTSAfter 7 weeks of feeding, the HFD group had significantly greater mean weight gain than the ND group (6.4±2.6 g vs 2.3±0.5 g; P<0.001). The HFD group had significantly higher expression of TRPV1 in the bronchus, pulmonary alveoli, and DRG than the ND group (P<0.05). Compared with the ND group, the HFD group had significant increases in the TRPV1 current intensity and number of action potentials in the DRG (P<0.05).

CONCLUSIONSHigh-fat diet induces a significant increase in body weight and leads to high expression of TRPV1 and high excitability in the respiratory system and the peripheral sensory neurons. This suggests that TRPV1 may be an important factor in the physiopathological mechanisms of bronchial hyperresponsiveness.

Action Potentials ; Animals ; Body Weight ; Diet, High-Fat ; Ganglia, Spinal ; chemistry ; Male ; Mice ; Mice, Inbred C57BL ; Respiratory System ; chemistry ; TRPV Cation Channels ; analysis ; physiology

OBJECTIVETo detect the effects of lumbar vertebrae Gucuofeng on the substance P content of hypothalamus and dorsal root ganglia in rat models.

METHODSA hundred and twenty SPF level SD male rats with the weight of 350 to 450 g were randomly divided into rotary fixation group (RF group), simple fixation group (SF group) and sham-operation group (Sham group). The external link fixation system was implanted into the L4-L6 of rats in RF group and SF group; and in RF group, that the L5 spinous process was rotated to the right resulted in L4, L5, L6 spinous process not collinear; in SF group, the external link fixation system was simply implanted and not rotated. The rats of Sham group were not implanted the external link fixation system and only open and suture. The substance P content of hypothalamus and dorsal root ganglia were detected at 1, 4, 8, 12 weeks after operation.

RESULTSSubstance P content of hypothalamus in RF group and SF group was lower than Sham group at 1, 4, 8 weeks after operation (P<0.05). Substance P content of dorsal root ganglia was higher than Sham group at 1, 4, 8, 12 weeks after operation (P<0.05). There was no significant differences in the substance P content of hypothalamus among three groups at 12 weeks after operation (P>0.05).

CONCLUSIONLumbar vertebrae Gucuofeng can inhibit the analgesic activity of substance P in hypothalamus and promote the synthesis and transmission of substance P in dorsal root ganglia, so as to cause or aggravate the pain.

Animals ; Disease Models, Animal ; Ganglia, Spinal ; chemistry ; Hypothalamus ; chemistry ; Joint Dislocations ; metabolism ; Lumbar Vertebrae ; injuries ; Male ; Rats ; Rats, Sprague-Dawley ; Substance P ; analysis ; physiology

Animal models for human chronic pain syndromes have been developed and widely used for pain research. One of these neuropathic pain models by Kim and Chung (1992) has many advantages for operation and pain elicitation. In this neuropathic model we have examined the c-fos protein, substance P, CGRP immunoreactivity in dorsal root ganglia and dorsal horn. 50 Sprague-Dawley rats were used for this study. L5 and L6 spinal nerves were ligated tightly to produce the neuropathic pain model. After 2, 4, 8, 16, and 24 hours and 1 week of surgery, rats were anesthetized and sacrificed by perfusion. After confirmation of the roots transected by the surgery, the L5 and L6 dorsal root ganglions and spinal cord were removed and processed for immunohistochemistry. All tissue sections were immunohistochemically stained for substance P, CGRP and c-fos using the peroxidase-antiperoxidase (PAP) method. The number of immunostained substance P and CGRP dorsal root ganglion cells and c-fos immunoreactive dorsal horn cells were counted and analyzed statistically with Mann-Whitney U test. The results are as follows. The number of c-fos protein immunoreactive neurons in the superficial layer of dorsal horn were increased markedly 2 hours after operation, and gradually decreased to normal level 1 week after operation. The number of c-fos protein immunoreactive neurons in the deep layer of the dorsal horn gradually increased to a peak 24 hours after operation, then decreased to the normal level 1 week after operation. The number of substance P and CGRP immunoreactive L5 and L6 dorsal root ganglion neurons were decreased markedly 1 week after the pain model operation. In conclusion, after neuropathic pain model operation, c-fos proteins were immediately expressed in the superficial layer of spinal dorsal horn, thereafter c-fos proteins in the deep layer of spinal dorsal horn were expressed. CGRP and substance P immunoreactive neurons in DRG were decreased markedly 1 week after neuropathic pain model operation. These decrements do not coincide with the other chronic pain models, which show great increases in these pain transmitting substances. Therefore, the relationship between pain and c-fos, SP and CGRP should be investigated further.
Animal ; Calcitonin Gene-Related Peptide/analysis* ; Ganglia, Spinal/chemistry* ; Immunohistochemistry ; Neurotransmitters/analysis* ; Pain/metabolism* ; Peripheral Nervous System Diseases/metabolism* ; Proto-Oncogene Proteins c-fos/analysis* ; Rats ; Rats, Sprague-Dawley ; Spinal Cord/chemistry* ; Substance P/analysis*

OBJECTIVETo study the relationship between substance P (SP) and/or calcitonin gene-related peptide (CGRP) immunoreactive neurons in dorsal root ganglia (DRG) and the transmission of nociception in the penile frenulum of rats.

METHODSThe fluoro-gold (FG) retrograde tracing method was used to trace the origin of nerve terminals in the penile frenulum of rats. And SP and/or CGRP immunofluorescence labeling was employed to detect the distribution of SP and/or CGRP immunoreactive neurons in DRG.

RESULTSFG retrograde tracing showed that the FG retrolabeled neurons were localized in L6-DRG and S1-DRG. SP and/or CGRP immunofluorescence labeling indicated that a large number of DRG neurons were SP- and CGRP-immunoreactive, different in size, bright red and bright green respectively in color, and arranged in rows or spots among nerve bundles. All the FG/SP and FG/CGRP double-labeled neurons were medium or small-sized. One third of the FG-labeled neurons were SP-immunoreactive, and a half of them CGRP-immunoreactive in L6-DRG and S1-DRG respectively. The FG/SP/CGRP-labeled neurons accounted for one fifth of the FG retro labeled neurons.

CONCLUSIONSP- and CGRP-immunoreactive neurons in L6-DRG and SI-DRG of rats may be involved in the transmission of nociception in rat penile frenulum.

Animals ; Calcitonin Gene-Related Peptide ; analysis ; Ganglia, Spinal ; chemistry ; cytology ; Male ; Microscopy, Fluorescence ; Neurons ; chemistry ; physiology ; Neurons, Afferent ; chemistry ; physiology ; Penis ; innervation ; Rats ; Rats, Sprague-Dawley ; Substance P ; analysis

BACKGROUNDPeripheral nerve regeneration across large gaps is clinically challenging. Scaffold design plays a pivotal role in nerve tissue engineering. Recently, nanofibrous scaffolds have proven a suitable environment for cell attachment and proliferation due to similarities of their physical properties to natural extracellular matrix. Poly(propylene carbonate) (PPC) nanofibrous scaffolds have been investigated for vascular tissue engineering. However, no reports exist of PPC nanofibrous scaffolds for nerve tissue engineering. This study aimed to evaluate the potential role of aligned and random PPC nanofibrous scaffolds as substrates for peripheral nerve tissue and cells in nerve tissue engineering.

METHODSAligned and random PPC nanofibrous scaffolds were fabricated by electrospinning and their chemical characterization were carried out using scanning electron microscopy (SEM). Dorsal root ganglia (DRG) from Sprague-Dawley rats were cultured on the nanofibrous substrates for 7 days. Neurite outgrowth and Schwann-cell migration from DRG were observed and quantified using immunocytochemistry and SEM. Schwann cells derived from rat sciatic nerves were cultured in electrospun PPC scaffold-extract fluid for 24, 48, 72 hours and 7 days. The viability of Schwann cells was evaluated by 3-[4,5-dimethyl(thiazol-2-yl)-2,5-diphenyl] tetrazolium bromide (MTT) assay.

RESULTSThe diameter of aligned and random fibers ranged between 800 nm and 1200 nm, and the thickness of the films was approximately 10 - 20 µm. Quantification of aligned fiber films revealed approximately 90% alignment of all fibers along the longitudinal axis. However, with random fiber films, the alignment of fibers was random through all angle bins. Rat DRG explants were grown on PPC nanofiber films for up to 1 week. On the aligned fiber films, the majority of neurite outgrowth and Schwann cell migration from the DRG extended unidirectionally, parallel to the aligned fibers. However, on the random fiber films, neurite outgrowth and Schwann cell migration were randomly distributed. A comparison of cumulative neurite lengths from cultured DRGs indicated that neurites grew faster on aligned PPC films ((2537.6 ± 987.3) µm) than randomly-distributed fibers ((493.5 ± 50.6) µm). The average distance of Schwann cell migration on aligned PPC nanofibrous films ((2803.5 ± 943.6) µm) were significantly greater than those on random fibers ((625.3 ± 47.8) µm). The viability of Schwann cells cultured in aligned PPC scaffold extract fluid was not significantly different from that in the plain DMEM/F12 medium at all time points after seeding.

CONCLUSIONSThe aligned PPC nanofibrous film, but not the randomly-oriented fibers, significantly enhanced peripheral nerve regeneration in vitro, indicating the substantial role of topographical cues in stimulating endogenous nerve repair mechanisms. Aligned PPC nanofibrous scaffolds may be a promising biomaterial for nerve regeneration.

Animals ; Biocompatible Materials ; chemistry ; Cells, Cultured ; Ganglia, Spinal ; cytology ; metabolism ; ultrastructure ; Immunohistochemistry ; Microscopy, Electron, Scanning ; Nanofibers ; chemistry ; Nerve Regeneration ; physiology ; Nerve Tissue ; cytology ; metabolism ; ultrastructure ; Polymers ; chemistry ; Propane ; analogs & derivatives ; chemistry ; Rats ; Rats, Sprague-Dawley ; Schwann Cells ; cytology ; metabolism ; ultrastructure ; Tissue Engineering ; methods ; Tissue Scaffolds ; chemistry

Using patch clamp technique the effects of dragon's blood and its component loureirin B on tetrodotoxin-sensitive sodium channel currents in dorsal root ganglion cells were observed. The experimental data were simulated with Hodgkin-Huxley model and the corresponding parameters were estimated. In addition, computer-simulated neuron action potentials in the absence and presence of drugs were produced using Hodgkin-Huxley model. The results show that the conductance of tetrodotoxin-sensitive sodium channel was fitted with m3h model well, the half-activated potentials of the sodium channel in the presence of drugs were shifted to the depolarizing direction and the threshold intensity of the cells in the presence of drugs was increased. These results demonstrate that dragon's blood and loureirin B did not resemble the tetrodotoxin which inhibited tetrodotoxin-sensitive sodium channel currents completely. Perhaps the analgesic effects of dragon's blood were partly caused by loureirin B affecting the activation, blocking the action potential generation and interfering with the transmission of painful signals into the central nervous system.
Action Potentials ; drug effects ; Analgesics, Non-Narcotic ; pharmacology ; Animals ; Computer Simulation ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Ganglia, Spinal ; cytology ; Models, Biological ; Neurons ; drug effects ; Rats ; Resins, Plant ; pharmacology ; Sodium Channels ; drug effects ; Tetrodotoxin ; pharmacology

To further uncover the scientific significance and molecular mechanism of the Chinese herbs with pungent hot or warm natures, endogenous and exogenous expression systems were established by isolation of dorsal root ganglion (DRG) neurons and transfection of HEK293 cells with TRPV1 channel gene separately. On this basis, the regulation action of capsaicin, one main ingredient from chili pepper, on TRPV1 channel was further explored by using confocal microscope. Besides, the three-sites one-unit technique and method were constructed based on the brown adipose tissue (BAT), anal and tail skin temperatures. Then the effect of capsaicin on mouse energy metabolism was evaluated. Both endogenous and exogenous TRPV1 channel could be activated and this action could be specifically blocked by the TRPV1 channel inhibitor capsazepine. Simultaneously, the mice's core body temperature and BAT temperature fall down and then go up, accompanied by the increase of temperature of the mice's tail skin. Promotion of the energy metabolism by activation of TRPV1 channel might be the common way for the pungent-hot (warm) herbs to demonstrate their natures.
Adipose Tissue, Brown ; drug effects ; physiology ; Animals ; Capsaicin ; analogs & derivatives ; pharmacology ; Energy Metabolism ; Ganglia, Spinal ; cytology ; HEK293 Cells ; Humans ; Mice ; Neurons ; drug effects ; physiology ; Plants, Medicinal ; chemistry ; TRPV Cation Channels ; physiology ; Temperature ; Thermogenesis

In this study, we prepared PLLA/bpV(pic) microspheres, a bpV(pic) controlled release system and examined their ability to protect nerve cells and promote axonal growth. PLLA microspheres were prepared by employing the o/w single emulsification-evaporation technique. Neural stem cells and dorsal root ganglia were divided into 3 groups in terms of the treatment they received: a routine medium group (cultured in DMEM), a PLLA microsphere group (DMEM containing PLLA microspheres alone) and a PLLA/bpV(pic) group [DMEM containing PLLA/bpV(pic) microspheres]. The effects of PLLA/bpV(pic) microspheres were evaluated by the live-dead test and measurement of axonal length. Our results showed that PLLA/bpV(pic) granulation rate was (88.2±5.6)%; particle size was (16.8±3.1)%, drug loading was (4.05±0.3)%; encapsulation efficiency was (48.5±1.8)%. The release time lasted for 30 days. In PLLA/bpV(pic) microsphere group, the cell survival rate was (95.2 ±4.77)%, and the length of dorsal root ganglion (DRG) was 718±95 μm, which were all significantly greater than those in ordinary routine medium group and PLLA microsphere group. This preliminary test results showed the PLLA/bpV(pic) microspheres were successfully prepared and they could promote the survival and growth of neural cells in DRG.
Animals ; Axons ; drug effects ; physiology ; Cells, Cultured ; Delayed-Action Preparations ; chemistry ; pharmacokinetics ; pharmacology ; Drug Compounding ; Female ; Ganglia, Spinal ; drug effects ; metabolism ; physiology ; Immunohistochemistry ; Lactic Acid ; chemistry ; pharmacokinetics ; pharmacology ; Microscopy, Electron ; Microspheres ; Neural Stem Cells ; drug effects ; physiology ; Neurofilament Proteins ; metabolism ; Neurons ; drug effects ; metabolism ; Organometallic Compounds ; chemistry ; pharmacokinetics ; pharmacology ; Polyesters ; Polymers ; chemistry ; pharmacokinetics ; pharmacology ; Pregnancy ; Rats