周围神经病损引起慢性痛的形态学和生理功能改变

周围神经受损后可导致痛觉过敏、感觉倒错、烧灼性痛等感觉障碍。本文主要对目前国内外普遍采用的、用于研究慢性神经病损性疼痛机制的两种动物模型进行综述,着重介绍神经开矿学和功能的改变。     

Ethanol metabolism by the rat heart and alcohol dehydrogenase activity.

Rat hearts perfused with oxygenated buffer containing [1-14C]ethanol metabolized small amounts of the ethanol to carbon dioxide. Very sensitive techniques are required to separate the resulting 14CO2 from the ethanol. This metabolism is not inhibited by levels of pyrazole which markedly inhibit NAD dependent liver alcohol dehydrogenase (EC 1.1.1.1). In vitro studies suggest that NADP functions as a cofactor for the rat heart alcohol dehydrogenase activity of crude heart homogenates. The kinetics parameters, the specific activity, and the pH dependence of the enzyme activity measured in these experiments suggest that it may have a minor role in ethanol metabolism by the rat.     

Early painful diabetic neuropathy is associated with differential changes in tetrodotoxin-sensitive and -resistant sodium channels in dorsal root ganglion neurons in the rat

Diabetic neuropathy is a common form of peripheral neuropathy, yet the mechanisms responsible for pain in this disease are poorly understood. Alterations in the expression and function of voltage-gated tetrodotoxin-resistant (TTX-R) sodium channels have been implicated in animal models of neuropathic pain, including models of diabetic neuropathy. We investigated the expression and function of TTX-sensitive (TTX-S) and TTX-R sodium channels in dorsal root ganglion (DRG) neurons and the responses to thermal hyperalgesia and mechanical allodynia in streptozotocin-treated rats between 4-8 weeks after onset of diabetes. Diabetic rats demonstrated a significant reduction in the threshold for escape from innocuous mechanical pressure (allodynia) and a reduction in the latency to withdrawal from a noxious thermal stimulus (hyperalgesia). Both TTX-S and TTX-R sodium currents increased significantly in small DRG neurons isolated from diabetic rats. The voltage-dependent activation and steady-state inactivation curves for these currents were shifted negatively. TTX-S currents induced by fast or slow voltage ramps increased markedly in neurons from diabetic rats. Immunoblots and immunofluorescence staining demonstrated significant increases in the expression of Na(v)1.3 (TTX-S) and Na(v) 1.7 (TTX-S) and decreases in the expression of Na(v) 1.6 (TTX-S) and Na(v)1.8 (TTX-R) in diabetic rats. The level of serine/threonine phosphorylation of Na(v) 1.6 and In Na(v)1.8 increased in response to diabetes. addition, increased tyrosine phosphorylation of Na(v)1.6 and Na(v)1.7 was observed in DRGs from diabetic rats. These results suggest that both TTX-S and TTX-R sodium channels play important roles and that differential phosphorylation of sodium channels involving both serine/threonine and tyrosine sites contributes to painful diabetic neuropathy.     

Zonisamide: Antihyperalgesic efficacy,the role of serotonergic receptors on efficacy in a rat model for painful diabetic neuropathy

Aims

The purpose of this study was to compare the changes of antihyperalgesic effectiveness of zonisamide (25 and 50 mg/kg), an antiepileptic drug, on the early and late phases of neuropathy and to investigate the role of serotonergic descending inhibitory pain pathways in antihyperalgesic effectiveness of zonisamide in the streptozotocin-induced rat model for painful diabetic neuropathy.

Main methods

The hot-plate and tail-immersion, to determine thermal thresholds, and paw pressure withdrawal tests, to determine mechanical thresholds, were performed as hyperalgesia tests. To investigate the role of serotonergic pathway, 1 mg/kg ketanserin (5-HT_2A/2C antagonist) and ondansetron (serotonin 5-HT₃ receptor antagonist) were used.

Key findings

Zonisamide enhanced pain thresholds significantly in the 3rd, 6th and 8th weeks as the reference drugs morphine (5 mg/kg) and carbamazepine (32 mg/kg, tested only in the 3rd week). There were no observed differences on the potency of antihyperalgesic effect between weeks and between doses. Each antagonist reversed the effect of zonisamide in the hot-plate and tail-immersion tests significantly, but, relatively in the paw pressure withdrawal tests.

Significance

These results support the role for zonisamide in the management of diabetic neuropathic pain in all phases. Serotonin 5-HT_2A/2C and 5-HT₃ receptors are involved in the antihyperalgesic effect of zonisamide by enhancement of thermal threshold, and partially by mechanical threshold, so they may not mediate mechanical hyperalgesia in diabetic neuropathy.     

Pulmonary C-fiber receptor activation abolishes uncoupled facial nerve activity from phrenic bursting during positive end-expired pressure in the rat.

Phasic respiratory bursting in the facial nerve (FN) can be uncoupled from phrenic bursting by application of 9 cmH(2)O positive end-expired pressure (PEEP). This response reflects excitation of expiratory-inspiratory (EI) and preinspiratory (Pre-I) facial neurons during the Pre-I period and inhibition of EI neurons during inspiration (I). Because activation of pulmonary C-fiber (PCF) receptors can inhibit the discharge of EI and Pre-I neurons, we hypothesized that PCF receptor activation via capsaicin would attenuate or abolish uncoupled FN bursting with an increase from 3 cmH(2)O (baseline) to 9 cmH(2)O PEEP. Neurograms were recorded in the FN and phrenic nerve in anesthetized, ventilated, vagally intact adult Wistar rats. Increasing PEEP to 9 cmH(2)O resulted in a persistent rhythmic discharge in the FN during phrenic quiescence (i.e., uncoupled bursting). Combination of PEEP with intrajugular capsaicin injection severely attenuated or eliminated uncoupled bursting in the FN (P < 0.05). Additional experiments examined the pattern of facial motoneuron (vs. neurogram) bursting during PEEP application and capsaicin treatment. These single-fiber recordings confirmed that Pre-I and EI (but not I) neurons continued to burst during PEEP-induced phrenic apnea. Capsaicin treatment during PEEP substantially inhibited Pre-I and EI neuron discharge. Finally, analyses of FN and motoneuron bursting across the respiratory cycle indicated that the inhibitory effects of capsaicin were more pronounced during the Pre-I period. We conclude that activation of PCF receptors can inhibit FN bursting during PEEP-induced phrenic apnea by inhibiting EI and I facial motoneuron discharge.     

Superoxide dismutase activity in rat brain during acute and chronic alcohol intoxication

The effect of acute and chronic ethanol administration on rat brain superoxide dismutase (SOD) activity was studied. Intraperitoneal injections of ethanol led to an inhibition of SOD activity. When ethanol was fed as the sole fluid, the SOD activity decreased progressively, reaching a plateau after 6 weeks of treatment. Withdrawal of ethanol produced a recovery of control values within 48 hr. SOD activity was also decreased in rats born from ethanol-drinking mothers. Inhibition of SOD activity by ethanol may allow an accumulation of cytotoxic O₂ ^– radicals; this may account for some nervous system disorders during alcohol intoxication.     

Dynorphin increases in the dorsal spinal cord in rats with a painful peripheral neuropathy

It is known that painful tissue injury evokes an increase in dynorphin in spinal neurons. It is not known, however, whether dynorphinergic systems respond similarly to the pain that accompanies peripheral neuropathy. Radioimmunoassays and immunocytochemistry were used to evaluate changes in dynorphin A(1-8) in the spinal cord of rats with a painful peripheral neuropathy. The neuropathy is the result of a constriction injury that is created by tying loose ligatures around the common sciatic nerve. Signs of abnormal pain sensations, hyperalgesia, allodynia (pain after normally innocuous stimuli), and spontaneous pain (or dysesthesia), are first detected 2-5 days after injury, reach peak severity in about 10 days, and persist for 2-3 months (Bennett, G. J.; Xie, Y.-K. Pain 33:87-107; 1988). Dynorphin increased by 5 days in cells in laminae I-II and V-VII in the lumbar spinal cord ipsilateral to the injury. This increase, maximal at 10 days (262%), was still present 20 days after the injury but was now seen only in neurons in the deep laminae (V-VII). Thus, the spinal dynorphinergic system appears to respond to neuropathic pain. Furthermore, our results suggest that dynorphinergic cells in the superficial and deep laminae may have different roles in nociception.     

Liver alcohol dehydrogenase activity in the female rat: Effects of ovariectomy and estradiol administration

The effects of ovariectomy and administration of estradiol on the activity of liver alcohol dehydrogenase and on the rate of ethanol elimination were determined in female Sprague-Dawley rats. The activity of the enzyme and the rates of ethanol elimination in the female sham-operated animals were higher than obtained previously in male rats of the same age. Ovariectomy had no effect on liver alcohol dehydrogenase and on rates of ethanol elimination. Estradiol administration resulted in an increase in liver weight and in total liver alcohol dehydrogenase activity per animal in sham-operated but not in ovariectomized animals. The increase in enzyme activity after estradiol administration in sham-operated animals was not associated with a significant increase in the rate of ethanol elimination, suggesting that the enzyme activity in female rats is not rate-limiting in in vivo ethanol oxidation.     

Suppressing PKC-dependent membrane P2X3 receptor upregulation in dorsal root ganglia mediated electroacupuncture analgesia in rat painful diabetic neuropathy

Painful diabetic neuropathy (PDN) is a common and troublesome diabetes complication. Protein kinase C (PKC)-mediated dorsal root ganglia (DRG) P2X3 receptor upregulation is one important mechanism underlying PDN. Accumulating evidence demonstrated that electroacupuncture (EA) at low frequency could effectively attenuate neuropathic pain. Our previous study showed that 2-Hz EA could relieve pain well in PDN. The study aimed to investigate whether 2-Hz EA relieves pain in PDN through suppressing PKC-mediated DRG P2X3 receptor upregulation. A 7-week feeding of high-fat and high-sugar diet plus a single injection of streptozotocin (STZ) in a dose of 35 mg/kg after a 5-week feeding of the diet successfully induced type 2 PDN in rats as revealed by the elevated body weight, fasting blood glucose, fasting insulin and insulin resistance, and the reduced paw withdrawal threshold (PWT), as well as the destructive ultrastructural change of sciatic nerve. DRG plasma membrane P2X3 receptor level and DRG PKC expression were elevated. Two-hertz EA failed to improve peripheral neuropathy; however, it reduced PWT, DRG plasma membrane P2X3 receptor level, and DRG PKC expression in PDN rats. Intraperitoneal administration of P2X3 receptor agonist αβ-meATP or PKC activator phorbol 12-myristate 13-acetate (PMA) blocked 2-Hz EA analgesia. Furthermore, PMA administration increased DRG plasma membrane P2X3 receptor level in PDN rats subject to 2-Hz EA treatment. These findings together indicated that the analgesic effect of EA in PDN is mediated by suppressing PKC-dependent membrane P2X3 upregulation in DRG. EA at low frequency is a valuable approach for PDN control.     

Calcium-activated thiol-proteinase activity in the fusion of rat erythrocytes induced by benzyl alcohol.

1. Rat erythrocytes were fused by incubation with benzyl alcohol and Ca2+. 2. Cell fusion was inhibited by EGTA, N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine, Tos-Lys-CH2Cl, and to a lesser extent by Tos-Phe-CH2Cl. Phenylmethanesulphonyl fluoride, Tos-Arg-OMe and histamine did not inhibit cell fusion. 3. Gel electrophoresis of membrane proteins from "ghosts" of the erythrocytes treated with benzyl alcohol showed that a high-molecular-weight polymer was present: this was consistent with the entry into the cells of Ca2+ and the activation of a transglutaminase enzyme. 4. In the treated cells the proteins corresponding to bands 2 and 3 in human erythrocytes were decreased, and a polypeptide with a slightly greater mobility than band 3 was produced. 5. These changes were inhibited by EGTA, N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine, and Tos-Lys-CH2Cl, but not by phenylmethanesulphonyl fluoride, Tos-Arg-OMe, or histamine. 6. The intramembraneous particles of the P-fracture face of cells treated with benzyl alcohol to induce fusion were decreased in number and were susceptible to cold-induced aggregation; both of these phenomena were markedly inhibited to EGTA, and partially inhibited by Tos-Lys-CH2Cl and N-ethylmaleimide. 7. These several observations indicate that a Ca2+-activated thiol-proteinase, which acts to degrade membrane proteins and to give freedom of lateral movement to intramembranous particles, may be essential feature of membrane fusion in this system. 8. It is suggested that this proteinase may act to degrade spectrin-binding proteins that attach band-3 protein to the erythrocyte cytoskeleton.     

Effects of human eosinophil granule-derived cationic proteins on C-fiber afferents in the rat lung.

Experiments were performed to test the hypothesis that human eosinophil granule-derived cationic proteins stimulate vagal C-fiber afferents in the lungs and elicit pulmonary chemoreflex responses in anesthetized Sprague-Dawley rats. Intratracheal instillation of eosinophil cationic protein (ECP; 1-2 mg/ml, 0.1 ml) consistently induced an irregular breathing pattern, characterized by tachypnea (change in breathing frequency of 44.7%) and small unstable tidal volume (VT). The tachypnea, accompanied by decreased heart rate and arterial blood pressure, started within 30 s after the delivery of ECP and lasted for >30 min. These ECP-induced cardiorespiratory responses were completely prevented by perineural capsaicin treatment of both cervical vagi, which selectively blocked C-fiber conduction, suggesting the involvement of these afferents. Indeed, direct recording of single-unit activities of pulmonary C-fibers further demonstrated that the same dose of ECP evoked a pronounced and sustained (>30-min) stimulatory effect on pulmonary C-fibers. Furthermore, the sensitivity of these afferents to lung inflation was also markedly elevated after the ECP instillation, whereas the vehicle of ECP administered in the same manner had no effect. Other types of eosinophil granule cationic proteins, such as major basic protein and eosinophil peroxidase, induced very similar respiratory and cardiovascular reflex responses. In conclusion, these results show that eosinophil granule-derived cationic proteins induce a distinct stimulatory effect on vagal pulmonary C-fiber endings, which may play an important role in the airway hyperresponsiveness associated with eosinophil infiltration in the airways.     

Long-term alcohol consumption increases matrix metalloproteinase-2 activity in rat aorta.

Altered degradation of extracellular matrix (ECM) underlies vascular remodeling, a hallmark in the pathogenesis of cardiovascular diseases including hypertension and aneurysmal dilatation. Although alcohol is recognized as a risk factor for certain cardiovascular disease states, its role in vascular remodeling has not been completely explored. We studied the effect of chronic alcohol consumption on upregulation of the enzymatic activity of matrix metalloproteinase-2 (MMP-2) as a possible pathway for large vessel remodeling. For this purpose, female rats were placed on one of three diets: a modified Lieber-DeCarli liquid diet containing 35% ethanol-derived calories, a pair-fed liquid diet with ethanol replaced by isocaloric maltose-dextrin, or a standard rat pellet. Weekly blood alcohol concentration averaged 117+/-7.9 mg/dl for the alcohol-fed rats. At 2, 4, and 72 weeks, aortas were removed and processed for measuring MMPs activity by gelatin zymography. Aortic extracts from rats on long-term (72 weeks), but not the short-term (2 and 4 weeks), alcohol diets showed increased MMP-2 activity. Furthermore, histochemical analysis of the aortas showed distinct disruption of the elastic fibers only in the 72 weeks alcohol-fed rats, compared to the control animals. These observations demonstrate that long-term alcohol consumption up-regulates MMP-2 activity, which is coincident with the alteration of aortic ECM composition through the degradation of vascular elastin components.     

Response of respiratory-related hypoglossal nerve activity to capsaicin-induced pulmonary C-fiber activation in rats

This study was designed to examine respiratory-related hypoglossal nerve activity in response to activation of pulmonary C-fibers by capsaicin. Rats were anesthetized with urethane (1.2 g/kg, i.p.). Tracheostomy was performed. Catheters were introduced into the femoral vein and artery. Another catheter was placed near the entrance of the right atrium via the right jugular vein. Rats were paralyzed with gallamine triethiodide (5 mg/kg, i.v.), and ventilated artificially. Activities of the phrenic nerve (PNA) and the hypoglossal nerve (HNA) were recorded simultaneously. Varied doses of capsaicin (0.625, 1.25, and 5 µg/kg) were delivered into the right atrium to activate pulmonary C-fibers. Before bilateral vagotomy, apnea, decreases in PNA and HNA were observed in response to pulmonary C-fiber activation by the low and moderate doses of capsaicin. The high dose of capsaicin evoked an increase in PNA, an immediate tonic discharge of the hypoglossal nerve, and a decrease in phasic HNA. The onset time of HNA preceding PNA was abolished and replaced by a time lagged pattern as pulmonary C-fibers were activated. Raising CO₂ concentration did not attenuate the inhibitory effect of pulmonary C-fiber activation upon PNA and HNA. After bilateral sectioning of the vagi, administration of the moderate dose of capsaicin to activate non-vagal C-fibers produced increases in PNA and HNA. These results suggest that pulmonary vagal C-fiber activation may narrow the diameter at the oropharyngeal level by a decrease in phasic HNA, which may be disadvantageous for the maintenance of a patent upper airway.     

CRMP-2 peptide mediated decrease of high and low voltage-activated calcium channels, attenuation of nociceptor excitability, and anti-nociception in a model of AIDS therapy-induced painful peripheral neuropathy

ABSTRACT: BACKGROUND: The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al..., Nature Medicine 17:822[EN DASH]829 (2011)]. RESULTS AND DISCUSSION: Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K) that bound with greater affinity to Ca2+ channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion) observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca2+ channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP) release by compared to vehicle control. CONCLUSIONS: Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against particular subset of voltage-gated calcium channels resulting in a multipharmacology of action on the target.     

Changes in the activity of alcohol dehydrogenase isoenzymes during the estrus cycle in the vagina of the rat

In rodents, the vaginal epithelium undergoes cyclical changes with an alternating pattern of keratinization and mucification. It has been known for decades that vitamin A and its active form retinoic acid are responsible for normal epithelial homeostasis. However, it has not so far been certain which enzymes catalyze the first and rate-limiting step in retinoic acid synthesis. By means of microdissection and ultrathin-layer gel electrophoresis, alcohol dehydrogenase isoenzyme activity was determined quantitatively in the various layers of the vaginal mucous membrane. It was found that, in the rat, only alcohol dehydrogenase 3 and 4 are expressed. Marked cyclical changes of alcohol dehydrogenase 4 activity in the stratum germinativum of the vaginal epithelium strongly support the assumption that this isoenzyme is responsible for retinoic acid synthesis, and that it is essential for the changes accompanying keratinization and mucification.     

Early painful diabetic neuropathy is associated with differential changes in the expression and function of vanilloid receptor 1

Diabetes mellitus is associated with one or more kinds of stimulus-evoked pain including hyperalgesia and allodynia. The mechanisms underlying painful diabetic neuropathy remain poorly understood. Previous studies demonstrate an important role of vanilloid receptor 1 (VR1) in inflammation and injury-induced pain. Here we investigated the function and expression of VR1 in dorsal root ganglion (DRG) neurons isolated from streptozotocin-induced diabetic rats between 4 and 8 weeks after onset of diabetes. DRG neurons from diabetic rats showed significant increases in capsaicin- and proton-activated inward currents. These evoked currents were completely blocked by the capsaicin antagonist capsazepine. Capsaicin-induced desensitization of VR1 was down-regulated, whereas VR1 re-sensitization was up-regulated in DRG neurons from diabetic rats. The protein kinase C (PKC) activator phorbol 12-myristate 13-acetate blunted VR1 desensitization, and this effect was reversible in the presence of the PKC inhibitor bisindolylmaleimide I. Compared with the controls, VR1 protein was decreased in DRG whole-cell homogenates from diabetic rats, but increased levels of VR1 protein were observed on plasma membranes. Of interest, the tetrameric form of VR1 increased significantly in DRGs from diabetic rats. Increased phosphorylation levels of VR1 were also observed in DRG neurons from diabetic rats. Colocalization studies demonstrated that VR1 expression was increased in large myelinated A-fiber DRG neurons, whereas it was decreased in small unmyelinated C-fiber neurons as a result of diabetes. These results suggest that painful diabetic neuropathy is associated with altered cell-specific expression of the VR1 receptor that is coupled to increased function through PKC-mediated phosphorylation, oligomerization, and targeted expression on the cell surface membrane.