Modulation of C-nociceptive Activities by Inputs from Myelinated Fibers.

To understand the mechanisms of neuropathic pain caused by demyelination, a rapid-onset, completed but reversible demyelination of peripheral A-fibers and neuropathic pain behaviors in adult rats by single injection of cobra venom into the sciatic nerve, was created. Microfilament recording revealed that cobra venom selectively blocked A-fibers, but not C-fibers. Selective blockade of A-fibers may result from A-fiber demyelination at the site of venom injection as demonstrated by microscope examination. Neuropathic pain behaviors including inflammatory response appeared almost immediately after venom injection and lasted about 3 weeks. Electrophysiological studies indicated that venom injection induced loss of conduction in A-fibers, increased sensitivity of C-polymodal nociceptors to innocuous stimuli, and triggered spontaneous activity from peripheral and central terminals of C-fiber nociceptors. Neurogenic inflammatory responses were also observed in the affected skin via Evans blue extravasation experiments. Both antidromic C-fiber spontaneous activity and neurogenic inflammation were substantially decreased by continuous A-fiber threshold electric stimuli applied proximally to the venom injection site. The data suggest that normal activity of peripheral A-fibers may produce inhibitory modulation of C-polymodal nociceptors. Removal of inhibition to C-fiber polymodal nociceptors following demyelination of A-fibers may result in pain and neurogenic inflammation in the affected receptive field.

Mechanisms of topical analgesics in relieving pain in an animal model of muscular inflammation.

OBJECTIVE: To investigate the possible mechanisms of topical analgesics in relieving pain in an animal model of muscular inflammation. METHODS: Adult Sprague-Dawley rats of both sexes were injected with complete Freund's adjuvant to induce inflammation in the anterior tibialis muscle of left hindlimb. One of two types of topical analgesics: Xiaotong Tiegao (XTT), a Tibetan herb compound, or Capzasin (CAP), a cream containing 0.1% capsaicin, was applied to the skin over the inflamed anterior tibialis muscle. The following experiments were performed: pain behavioral tests, evaluation of plasma extravasation in the affected limb, and electrophysiological recordings of afferent nerve fibers. RESULTS: The behavioral experiments demonstrated that applications of either type of topical analgesic to the skin over the inflamed muscle significantly reduced muscular inflammatory pain, as indicated by the increased weight bearing capacity on the affected hindlimb (with latencies of 10 minutes for XTT and 1-2 hours for CAP). Meanwhile, both analgesics caused plasma extravasation in the affected skin. Electrophysiological recordings from the afferent fibers in the related cutaneous nerve indicated that topical analgesics selectively activated C-fibers, but not A-fibers innervating the same region of receptive field. The latency and duration of C-fiber activation was similar to those of the reduction of muscular inflammatory pain. On the contrary, topical analgesics substantially decreased C-fiber afferent spontaneous firing in the nerve innervating the inflamed muscle. Moreover, denervation of the affected skin blocked the analgesic effects of both topical analgesics in muscular inflammatory pain. CONCLUSION: This study suggests that topical analgesics may reduce the nociceptive input from inflamed muscles via a reflex mechanism by activating the cutaneous nociceptive afferents.

A new animal model of trigeminal neuralgia produced by administration of cobra venom to the infraorbital nerve in the rat.

BACKGROUND: Understanding the mechanism of trigeminal neuralgia may be elucidated by developing laboratory animal models that closely mimic the features of this specific type of neuropathic pain. We have developed an experimental animal model for trigeminal neuralgia using a technique of injecting cobra venom into the infraorbital nerve (ION) trunk. METHODS: Male Sprague-Dawley rats were subjected to the administration of cobra venom or saline into the ION trunk. Mechanical stimuli were applied to the ION territory in consecutive days after surgery. Mechanical thresholds were measured over a 90-day period on the bilateral facial region. Vascular permeability in the ION territory was measured using Evans blue dye. RESULTS: The cobra venom-treated rats developed mechanical allodynia 3 days after surgery that lasted for 60 days postoperatively at the ipsilateral side. The mechanical thresholds of the contralateral ION territory also showed a profound decrease but were sustained for only approximately 30 days. There was no change of mechanical thresholds in the control groups. The extravasation of Evans blue increased significantly in the skin after administration of cobra venom to the ION compared with control rats (P < 0.05). CONCLUSION: The cobra venom model may provide a reasonable model for investigating the mechanism of trigeminal neuropathic pain.

Glycosylation-induced depolarization facilitates subthreshold membrane oscillation in injured primary sensory neurons.

Subthreshold membrane potential oscillations (SMPO) in the injured dorsal root ganglion (DRG) neurons are involved in the generation of spontaneous activity, which can directly evoke neuropathic pain. Nerve injury usually triggers the synthesis of large quantities of membrane protein in nerve injured DRG neurons. Membrane proteins are glycosylated by addition of sugars, especially negatively charged sialic acid residues, which may depolarize the resting membrane potential (Vm), open voltage-gated channels in injured neurons, and cause spontaneous activity. In the present study, we aimed to determine if increased negative charge on the cell surface, carried by the sialic acid residues, could contribute to the generation of SMPO in injured DRG neurons. Intracellular recording was performed in DRG neurons following chronic constrictive injury (CCI) of the sciatic nerve. Results indicated that both A- and C-type injured DRG neurons exhibited a higher incidence of SMPO and more depolarized Vm than those of the control neurons. Ca(2+), Mg(2+), Mn(2+), or poly-lysine, a positively charged organic compound, when topically applied to the DRG, not only reduced SMPO but also caused a rapid hyperpolarizing shift in Vm. Topical application of neuraminidase to selectively remove sialic acid residues on the extracellular membrane normalized the depolarized Vm and inhibited both spontaneous and evoked SMPO. However, application of Ca(2+), Mg(2+), Mn(2+) or neuraminidase had no effect on excitability and Vm in normal neurons. The results demonstrated that the increase in negatively charged sialic acid residues on the extracellular membrane of neuronal somata is a critical factor in the generation of SMPO and hyperexcitability in injured sensory neurons.

Human acupuncture points mapped in rats are associated with excitable muscle/skin-nerve complexes with enriched nerve endings.

As part of our ongoing investigation into the neurological mechanisms of acupuncture, we have tried to correlate the distribution of afferent nerve endings with acupuncture points (AP) in the rat hind limbs. In vivo extracellular microfilament recordings of Aalpha/Abeta/Adelta fibers were taken from peripheral nerves to search for units with nerve endings or receptive fields (RF) in the skin or the muscles. The location of the RFs for each identified unit was marked on scaled diagrams of the hind limb. Noxious antidromic stimulation-induced Evans blue extravasation was used to map the RFs of C-fibers in the skin or muscles. Results indicate that, for both A- and C-fibers, the distribution of RFs was closely associated with the APs. In the skin, the RFs concentrate either at the sites of APs or along the orbit of meridian channels. Similarly, the majority of sarcous sensory receptors are located at the APs in the muscle. Results from our studies strongly suggest that APs in humans may be excitable muscle/skin-nerve complexes with high density of nerve endings.

Sialic acid contributes to hyperexcitability of dorsal root ganglion neurons in rats with peripheral nerve injury.

Axonal injury of the dorsal root ganglion (DRG) neurons may alter the synthesis of certain membrane proteins that are responsible for the development of abnormal hyperexcitability. The external domains of most of these membrane proteins are sialylated. Because sialic acid carries heavy negative charges, the increase of sialylated proteins may increase neurons' negative surface charges, which will have predictable effects on the voltage-gated channels, and affect the excitability of injured neurons. Using intracellular electrophysiological recording, we demonstrated that following chronic constriction injury (CCI) of the sciatic nerve, Aalpha/beta DRG neurons become hyperexcitable, as indicated by a more depolarized resting membrane potential (Vm) and a lowered threshold current (TIC). More interestingly, the excitability of injured DRG neurons was reduced substantially when the extracellular sialic acid was removed by pretreatment with neuraminidase. The Vm was less depolarized and the TIC increased robustly as compared to the CCI neurons without neuraminidase treatment. However, desialylation of normal, intact neurons had no significant effect on the Vm and less effect on the TIC. Our results suggest that the hyperexcitability of injured sensory neurons may be associated with increased negatively charged sialic acid residues on the surface of the neuronal somata.

Sialic acid contributes to generation of ectopic spontaneous discharges in rats with neuropathic pain.

Ectopic spontaneous discharges (ESD) of teased myelinated fibers were recorded from the sciatic nerve proximal to the site of 'chronic constriction nerve injury' in the rat. Ca(2+), Mg(2+), Mn(2+), Ni(2+), La(3+) and some positively charged organic compounds (hexamethonium and poly-lysine) when applied topically to the injured site abolished or significantly reduced the rate of ESD. After enzymatic removal of sialic acid by neuraminidase (2 units/ml), the ESD was silenced in 11, reduced in four and unchanged in four of 19 fibers. However, divalent cations failed to depress the reappeared ESD evoked by 4-aminopyridine in the desialylated silenced fibers. Moreover, the mean incidence of ESD was significantly reduced after neuraminidase treatment. These results indicate that an increase in negative charges on the external membrane surface of injured neuron caused by sialylation is a key factor in ESD generation.

[Dendritic projections from meridian-related motoneurons to sympathic preganglion neurons in the spinal cord of rats].

OBJECTIVE: Previous studies indicated a close involvement of reflex activities of motoneurons in the spinal cord in the mechanism of meridian phenomena. The present study was designed to investigate the dendrite projections of meridian-related motoneurons among the motoneurons and sympathetic preganglionic neurons in the spinal cord. METHODS: A total of 41 Sprague-Dawley rats were used in the present study. Cholera toxin B-subunit conjugated horseradish peroxidase (CB-HRP) containing 1.0% HRP was respectively injected to acupoint "Chengman" (ST 20), "Liangmen" (ST 21), "Guanmen" (ST 22), "Taiyi" (ST 23), "Huaroumen" (ST 24), "Tianshu" (ST 25) and "Wailing" (ST 26) of the Stomach Meridian, and "Ganshu" (BL 18), "Danshu" (BL 19), "Pishu" (BL 20), "Weishu" (BL 21) and "Sanjiaoshu" (BL 22) of the Bladder Meridian, and "Daimai" (GB 26), "Wushu" (GB 27), "Weidao" (GB 28), "Juliao" (GB 29), "Huantiao" (GB 30), "Fengshi" (GB 31), "Zhongdu" (GB 32), "Xiyangguan" (GB 33) and "Yanglingquan" (GB 34) of the Gallbladder Meridian (for labeling preganglionic neurons), and the celiac ganglion and superior mesenteric ganglion for labeling sympathetic preganglionic neurons. Three days after injection, the animals anesthetized were transcardia-cally perfused with 1.5% paraformaldehyde, the spinal cord was removed to be fixed routinely and then cut into sections for observing the labeled cells under microscope. RESULTS: In the ipsilateral ventral horn of the spinal cord, the motoneurons retrogradely labeled by CB-HRP formed dendritic projections oriented only to those motoneurons innervating the same meridian. In the longitudinal sections of spinal cord, the labeled motoneurons formed a bead-like column with a prominent network of longitudinal dendrites connecting the motoneurons innervating acupoints from the same meridian. In the transverse sections of spinal cord, two groups of dendrites from the labeled motoneurons projected to the identified sympathetic preganglionic regions: one group extended dorsolateraly to the intermediolateral gray, another group extended intermediolateraly toward the central canal. In rats with injection of CB-HRP into both acupoint regions and ipsilateral celiac ganglion, the dendrites originated from the labeled motoneurons projected directly to the labeled sympathetic preganglionic neurons. CONCLUSION: Each of the ST, BL and GB meridians is innervated by a specific group of motoneurons in the spinal cord. The motoneurons form a column with distinct border in the ventral horn of spinal cord, and the dendritic projections from the motoneurons oriented only to those innervating the same meridian. The dendrites from the meridian-related motoneurons can specifically project to the sympathetic preganglionic neurons at the thoracolumbar level.

[Dense innervation of acupoints and its easier reflex excitatory character in rats].

OBJECTIVE: To study the innervating character of tissues around the acupoints and along the meridian course and to analyze the reflex activity correlation between acupuncture points in a given meridian in the rat. METHODS: Forty Wistar and 15 SD rats were used in this study. Electrical activities of microfilaments of the afferent nerves (deep tibial nerve, peroneal nerve and the lateral cutaneous nerve of the leg) were observed for identifying their receptive field and the type of nerve fibers. Nerve stem-antidromic stimulation induced Evan's blue extravasation method was employed to compare the difference of the nerve ending distribution in the acupoint area and non-acupoint area. The reflex activities evoked by electric stimulation of the acupoint were used to analyze the interrelation between acupoint and meridian. RESULTS: Findings showed that a great majority of the afferent nerve endings supplying the tibialis anterior/rectus femoris muscle and the foot skin distributed in an uneven pattern in the sites being in accord with acupoints or with the orbit of meridians. Antidromic stimulation of C-fibers in the deep tibial nerve evoked extravasation of Evan's blue from the plasma into the interstitial fluid, blue foci appeared at the acupoints of the Stomach Meridian and along the orbit of the Stomach Meridian. The special distribution of the afferent nerve endings in the acupoint was also associated with the special reflex activity originating from the acupoints of the muscle group of a given meridian. CONCLUSION: The acupoint is an excitable muscle/skin-nerve complex with greatest concentration of nerve endings. The meridian consists of acupoints that possess a close interaction in physiological reflex activities.