The Antiallodynic Effects of Intrathecal Cholinesterase Inhibitors in a Rat Model of Neuropathic Pain

Background: This study determined the effect of intrathecally administered cholinesterase inhibitors, edrophonium and neostigmine, on nerve injury-induced, touch-evoked allodynia and identified the pharmacologic characteristics of this action.

Methods: Rats were prepared with tight ligation of the left L5 and L6 spinal nerves and with lumbar intrathecal catheters fitted for long-term monitoring. Edrophonium (3, 10, 30, or 100 [micro sign]g) or neostigmine (0.3, 1, 3, or 10 [micro sign]g) was administered intrathecally. Tactile allodynia and motor weakness were assessed. To evaluate the pharmacologic characteristics of the activity, a muscarinic receptor antagonist or a nicotinic receptor antagonist was administered intrathecally before edrophonium or neostigmine was injected. To compare the action of subtype antagonists, the M1 muscarinic receptor antagonist pirenzepine, the M (2) antagonist methoctramine, the M3 antagonist 4-DAMP (diphenylacetoxy-N-methypiperidine), and the M4 antagonist tropicamide were administered intrathecally before cholinesterase inhibitors were injected.

Results: Intrathecal edrophonium or neostigmine produced a dose-dependent antagonism of the touch-evoked allodynia. Neostigmine resulted in a moderate effect on motor weakness at doses of 3 and 10 [micro sign]g. Pretreated with intrathecal atropine but not mecamylamine yielded a complete antagonism of the effects of the cholinesterase inhibitors. In addition, antiallodynia produced by edrophonium (100 [micro sign]g) was reversed by pretreatment with methoctramine, 4-DAMP, tropicamide, and pirenzepine. In the neostigmine (10 [micro sign]g) group, only M1 antagonist pirenzepine had a moderate effect on reversal of increased allodynic threshold.     

Development of Neuropathic Pain in the Rat Spared Nerve Injury Model Is Not Prevented by a Peripheral Nerve Block

Background: The mechanisms responsible for initiation of persistent neuropathic pain after peripheral nerve injury are unclear. One hypothesis is that injury discharge and early ectopic discharges in injured nerves produce activity-dependent irreversible changes in the central nervous system. The aim of this study was to determine whether blockade of peripheral discharge by blocking nerve conduction before and 1 week after nerve injury could prevent the development and persistence of neuropathic pain-like behavior in the spared nerve injury model.

Methods: Bupivacaine-loaded biodegradable microspheres embedded in fibrin glue were placed in a silicone tube around the sciatic nerve to produce a conduction block. After sensory-motor testing of block efficacy, a spared nerve injury procedure was performed. Development of neuropathic pain behavior was assessed for 4 weeks by withdrawal responses to stimulation (i.e., von Frey filaments, acetone, pinprick, radiant heat) in bupivacaine microspheres-treated animals (n = 12) and in controls (n = 11).

Results: Bupivacaine microspheres treatment produced conduction blockade with a complete lack of sensory responsiveness in the sural territory for 6 to 10 days. Once the block wore off, the degree of hypersensitivity to stimuli was similar in both groups.     

Role of Spinal NO in Antiallodynic Effect of Intrathecal Clonidine in Neuropathic Rats

Background: The role of spinal nitric oxide (NO) in neuropathic pain remains uncertain. Although intrathecal clonidine causes NO release in the spinal cord, the functional role of spinal No in clonidine-produced analgesia has not been examined. The objectives of this study were to assess the role of spinal NO in maintenance of allodynia and to determine the role of spinal NO in the antiallodynic effect of intrathecal clonidine.

Methods: Allodynia was produced in rats by tight ligation of the left L5-L6 spinal nerves. Intrathecal catheters were inserted with tips in the lumbar intrathecal space. Mechanical allodynia was determined by application of von Frey filaments to the left hindpaw. In the first series of experiments, allodynia was assessed before and after intrathecal injection of saline, L-arginine, an NO donor (SNAP), two NO synthase inhibitors (TRIM and NMMA), or an NO scavenger (PTIO). In the second series of experiments, 20 [micro sign]g of clonidine was injected intrathecally 15 min after intrathecal injection of saline, TRIM, NMMA, or PTIO.

Results: Allodynia was not affected significantly by intrathecal injection of L-arginine, SNAP, TRIM, NMMA, or PTIO. The antiallodynic effect produced by intrathecal injection of clonidine was attenuated significantly by pretreatment with TRIM, NMMA, or PTIO.     

Effect of Systemic and Intrathecal Morphine in a Rat Model of Postoperative Pain

Background: To learn more about persistent pain after an incision, a rat model for postoperative pain has been developed. To further evaluate this model, the authors examined the effect of intrathecal (IT) and subcutaneous (SC) morphine, effective for postoperative pain relief in patients, on pain behaviors immediately after surgery and 1 day after surgery.

Methods: Rats were anesthetized with halothane, and a 1-cm incision was made in the plantar aspect of the foot and closed. After recovery, the rats were placed on an elevated plastic mesh floor, and withdrawal threshold was determined using calibrated von Frey filaments (15-522 mN) applied from beneath the test cage to an area adjacent to the wound at the heel. Pain behaviors also were assessed using the response frequency to a nonpunctate mechanical stimulus and a cumulative pain score.

Results: Mechanical hyperalgesia and nonevoked pain behaviors were present on the day of surgery and 1 day after surgery. Administration of either SC (0.3-3.0 mg/kg) or IT (0.16-5.0 micro gram) morphine reversibly increased the withdrawal threshold. The response frequency to the nonpunctate stimulus and the nonevoked pain scores also were decreased by 3 mg/kg of SC or 5 micro gram of IT morphine. Naloxone (1 mg/kg) reversed morphine-produced hypoalgesia.     

Antiallodynic Effect of Intrathecal Neostigmine Is Mediated by Spinal Nitric Oxide in a Rat Model of Diabetic Neuropathic Pain

Background: Intrathecal administration of acetylcholinesterase inhibitors produces antinociception in both animals and humans, but their effect on diabetic neuropathic pain has not been studied. In the current study, we determined the antiallodynic effect of intrathecal injection of an acetylcholinesterase inhibitor, neostigmine, in a rat model of diabetic neuropathic pain. In addition, since acetylcholine can increase release of nitric oxide in the spinal cord, we studied the role of spinal endogenous nitric oxide in the action of intrathecal neostigmine in diabetic neuropathic pain.

Methods: Rats were rendered diabetic with an intraperitoneal 50-mg/kg injection of streptozotocin. Intrathecal catheters were inserted, with tips in the lumbar intrathecal space. Mechanical allodynia was determined by application of von Frey filaments to the hind paw. We first determined the dose-dependent effect of intrathecal neostigmine on allodynia. The role of spinal nitric oxide in the action of intrathecal neostigmine was then examined through intrathecal treatments with a neuronal nitric oxide synthase inhibitor (TRIM), a nitric oxide scavenger (PTIO), l-arginine, or d-arginine.

Results: The diabetic rats developed a sustained tactile allodynia within 4 weeks after streptozotocin injection. Intrathecal injection of 0.1-0.5 [mu]g neostigmine dose-dependently increased the withdrawal threshold in response to application of von Frey filaments. Intrathecal pretreatment with 30 [mu]g TRIM or 30 [mu]g PTIO abolished the antiallodynic effect of intrathecal neostigmine. Furthermore, the inhibitory effect of TRIM on the action of intrathecal neostigmine was reversed by intrathecal injection of 100 [mu]g l-arginine but not d-arginine.     

Detection of Neuropathic Pain in a Rat Model of Peripheral Nerve Injury

Background: Behavioral criteria that confirm neuropathic pain in animal injury models are undefined. Therefore, the authors sought clinically relevant measures that distinguish pain behavior of rats with peripheral nerve injury from those with sham injury.

Methods: The authors examined mechanical and thermal sensory sensitivity, comparing responses at baseline to responses after spinal nerve ligation (SNL group), sham nerve injury (sham group), or skin incision alone (control group).

Results: Substantial variance was evident in all sensory tests at baseline. After surgery, tests using brush, cold, or heat stimulation showed minimal distinctions between surgical groups. Postsurgical thresholds for flexion withdrawal from mechanical stimulation with von Frey fibers were decreased bilaterally in SNL and sham groups. In contrast, the probability of a complex hyperalgesia-type response with prolonged elevation, shaking, or licking of the paw was selectively increased on the ipsilateral side in the SNL group. Nonetheless, the effect of SNL on behavior was inconsistent, regardless of the sensory test. The behavioral measure that best distinguishes between SNL and sham groups and thereby best identifies animals with successful SNL-induced neuropathic pain is increased ipsilateral postsurgical probability of a hyperalgesia-type response to noxious mechanical stimulation. Using receiver operating characteristics analysis, mechanical hyperalgesia identifies a local SNL effect in approximately 60% of animals when specificity is required to be 90% or higher.     

Consumption of Soy Diet before Nerve Injury Preempts the Development of Neuropathic Pain in Rats

Background : A previous report using a partial sciatic nerve ligation (PSL) model for neuropathic pain in rats demonstrated that consumption of soy-containing diets preoperatively and postoperatively suppressed development of mechanical and heat allodynia, as well as hyperalgesia. The current study examined whether dietary soy suppresses these neuropathic sensory disorders when consumed either before or after PSL injury.

Methods : Male Wistar rats were grouped into seven different feeding regimens. These rats were fed SOY (RMH-1000, PMI Feeds, St. Louis, MO), a diet containing 85% soy protein since weaning, and were then switched to noSOY (Bio-Serv Co., Frenchtown, NJ), a diet devoid of soy at certain time points before PSL injury (14, 7, 1 days, or 15 and 0 h). Postoperatively, these rats were fed SOY or noSOY diets. Levels of mechanical and heat allodynia and hyperalgesia were determined preoperatively and 3, 8, and 14 days after PSL injury.

Results : Compared with groups fed preoperative noSOY, consumption of SOY before PSL injury significantly blunted postoperative levels of allodynia and hyperalgesia. Administering the SOY diet both before and after PSL injury provided no additional suppression of neuropathic pain. No pain suppression was noted in rats fed a noSOY diet preoperatively and SOY diet after PSL injury. Switching from SOY to noSOY feeding within 15 h of PSL injury was sufficient to allow for the full development of allodynia and hyperalgesia.     

Antihyperalgesic and Side Effects of Intrathecal Clonidine and Tizanidine in a Rat Model of Neuropathic Pain

Background: Although intrathecal clonidine produces pronounced analgesia, antinociceptive doses of intrathecal clonidine produce several side effects, including hypotension, bradycardia, and sedation. Intrathecal tizanidine, another [alpha]2-adrenergic agonist, has provided antinociception without producing pronounced hemodynamic changes in animal studies. However, it has been unclear whether antihyperalgesic doses of intrathecal clonidine and tizanidine produce hypotension and bradycardia in a neuropathic pain state. This study was designed to evaluate the antihyperalgesic effects and side effects of intrathecal clonidine and tizanidine in a rat model of neuropathic pain.

Methods: Male Sprague-Dawley rats were chronically implanted with lumbar intrathecal catheters, and the sciatic nerve was loosely ligated. After 21-28 days after surgery, the rats received intrathecal clonidine (0.3, 1.0, and 3.0 [mu]g) and tizanidine (1.0, 2.0, and 5.0 [mu]g), and the antihyperalgesic effects of thermal and mechanical stimuli were examined. In addition, the changes in blood pressure and heart rate, sedation level, and other side effects after intrathecal administration of drugs were recorded.

Results: The administration of 3.0 [mu]g intrathecal clonidine or 5.0 [mu]g tizanidine significantly reversed both thermal and mechanical hyperalgesia. The administration of 3.0 [mu]g intrathecal clonidine, but not 5.0 [mu]g tizanidine, significantly decreased mean blood pressure and heart rate and produced urinary voiding. A greater sedative effect was produced by 3.0 [mu]g intrathecal clonidine than by 5.0 [mu]g tizanidine.     

Antiallodynic Effect of Intrathecal Gabapentin and Its Interaction with Clonidine in a Rat Model of Postoperative Pain

Background: Systemic administration of gabapentin was shown previously to attenuate mechanical allodynia in a rat model of postoperative pain. Because intrathecal administration of gabapentin is effective in other hypersensitivity states, the authors tested its effect in the postoperative model, its interaction with another antiallodynic agent (clonidine), and a possible mechanism of gabapentin action (entry into sites of action via an L-amino acid transporter).

Methods: Male Sprague-Dawley rats were anesthetized with halothane, and an incision of the plantaris muscle of right hind paw induced punctate mechanical allodynia. Withdrawal threshold to von Frey filament application near the incision site was determined before and 2 h after surgery. Then, an intrathecal injection was performed and thresholds were determined every 30 min for 3 h thereafter.

Results: Paw incision induced a mechanical hypersensitivity (mechanical threshold > 25 g before incision and < 5 g after). Intrathecal gabapentin dose-dependently (10-100 [mu]g) reduced mechanical allodynia. Intrathecal injection of an inhibitor of L-amino acid transporters or a competitor for this transporter, L-leucine, did not reverse the intrathecal effect of gabapentin. The ED50 of intrathecal gabapentin, clonidine, and their combination were 51, 31, and 9 [mu]g, respectively, and isobolographic analysis showed synergy between gabapentin and clonidine.     

Magnesium Chloride and Ruthenium Red Attenuate the Antiallodynic Effect of Intrathecal Gabapentin in a Rat Model of Postoperative Pain

Background: Gabapentin, a [gamma]-aminobutyric acid analog anticonvulsant, has been shown to possess antinociceptive effects in animal models and clinical trials. An endogenous binding site of [3H]gabapentin has been revealed to be the [alpha]2[delta] subunit of voltage-dependent Ca2+ channels. Magnesium chloride, ruthenium red, and spermine have been shown to modulate [3H]gabapentin binding to this binding site in vitro. In this study, the authors examined whether intrathecal magnesium chloride, ruthenium red, or spermine could affect the antiallodynic effect of intrathecal gabapentin in a rat model of postoperative pain.

Methods: Under isoflurane anesthesia, male Sprague-Dawley rats received an incision over the plantar surface of the right hind paw to produce punctate mechanical allodynia. Withdrawal thresholds to von Frey filament stimulation near the incision site were measured before incision, 2 h after incision, and every 30 min after intrathecal coadministration of gabapentin with normal saline or different doses of magnesium chloride, ruthenium red, or spermine for 2 h.

Results: Intrathecal gabapentin (30, 100, 200 [mu]g) dose-dependently reduced incision-induced allodynia. Hexahydrated magnesium chloride (5, 10, 20 [mu]g) and ruthenium red (0.2, 2, 20 ng) noncompetitively inhibited the antiallodynic effect of gabapentin. Spermine at doses not inducing motor weakness (30, 60 [mu]g) did not affect the antiallodynic effect of gabapentin. The antiallodynic effect of intrathecal morphine (1.5 [mu]g) was not affected by hexahydrated magnesium chloride (20 [mu]g), ruthenium red (20 ng), or spermine (60 [mu]g).     

Comparison of Pre- versus Post-incision Administration of Intrathecal Bupivacaine and Intrathecal Morphine in a Rat Model of Postoperative Pain

Background: Preclinical studies in experimental animals suggest that preemptive analgesia may improve postoperative pain management. The beneficial effects of preemptive analgesia appear less remarkable clinically. The purpose of this study is to examine the effect of pre- and post-incision administration of intrathecal bupivacaine and intrathecal morphine in a rat model for postoperative pain.

Methods: Rats with intrathecal catheters were anesthetized with halothane, and the surgical field was prepared. A saline vehicle or the test drug was administered 15 min before an incision was made in the plantar aspect of the hindpaw or after the incision was completed. After recovery, mechanical hyperalgesia to punctate and nonpunctate stimuli was measured. Rats were tested on the day of surgery for the first 5 h and each day for 6 days.

Results: In saline vehicle-treated rats, the median withdrawal threshold decreased from 522 mN to 54 mN or less, and the response frequency to pressure from application of the plastic disc increased from 0 +/- 0% to 96 +/- 12% or greater after incision. Hyperalgesia was persistent through 2 days after surgery and then gradually returned toward preincision values over the next 4 days. Pre- or postincision administration of either intrathecal morphine or intrathecal bupivacaine reduced hyperalgesia on the day of surgery; at all subsequent times, there were no differences between the saline vehicle groups and the drug treatment groups. There were never any significant differences between pre- and postincision treatments.     

miR-195通过抑制小胶质细胞自噬促进神经术后持续性疼痛的研究

目的探讨大鼠神经结扎术后miR-195在脊髓小胶质细胞自噬及在持续性疼痛中的作用。方法随机选取成年健康雄性SD大鼠36只,按随机数字表法分为假手术组(Sham组)与脊髓神经结扎模型组(SNL组)。SNL组结扎大鼠L5段神经,Sham组术中不予结扎。术后指定时间点以Percoll密度梯度离心法分离脊髓背角小胶质细胞,Real-time PCR检测miR-195表达水平,Western Blot检测LC3及p62表达量。另以上法选取36只SD大鼠分组手术并在脊神经周围给鞘内注射miR-195模拟物或抑制物,行为学检测大鼠50%机械性痛阈和热痛反应,Western Blot检测脊髓小胶质细胞炎症因子白细胞介素-1β,肿瘤坏死因子-α和诱导性一氧化氮合酶表达水平及自噬水平。结果SNL组术后脊髓小胶质细胞内miR-195的表达水平显著高于Sham组,同时自噬水平低于Sham组;与对照组相比,鞘内给予miR-195模拟物后大鼠机械刺激缩足阈值降低并对热刺激敏感,小胶质细胞炎症因子表达水平增高且自噬水平明显降低。结论miR-195通过抑制小胶质细胞自噬从而提高炎症反应,参与调节大鼠术后持续性疼痛的形成与维持。     

Analysis of Drug Interaction between Intrathecal Clonidine and MK-801 in Peripheral Neuropathic Pain Rat Model

Background: Spinally delivered alpha2 -adrenoceptor agonists and N-methyl-D-aspartate antagonists each have been shown to have actions attenuating the hyperesthesia in rat models of nerve injury pain. Using a fixed-dose analysis and an isobolographic paradigm, the spinal interaction between the alpha2 -adrenoceptor agonist clonidine and the N-methyl-D-aspartate antagonist MK-801 is characterized in a rat model of nerve injury-induced tactile hyperesthesia.

Methods: Male Sprague Dawley rats were anesthetized with halothane, and the left L5 and L6 spinal nerves were ligated (Chung model). After 7-10 days' recovery, a Polyethylene tubing catheter was implanted into the lumbar intrathecal space. After recovery from catheter implantations (5-7 days), intrathecal dose-response curves were established for the antihyperesthesia effects of clonidine (3, 6, 10, and 20 micro gram) and MK-801 (1, 3, 10, and 20 micro gram) alone to obtain the ED50 for each agent. In separate studies, three doses of clonidine (l, 3, and 10 micro gram) were injected mixed with one dose of MK-801 (1 micro gram) for fixed-dose analysis, and three doses of the two agents (2, 6, and 20 micro gram) were injected jointly in a fraction of the dose ratio 1:1 for isobolographic analysis. Thresholds for left hind paw withdrawal to von Frey hair application were assessed.

Results: Rats with nerve ligation showed a reliable tactile hyperesthesia (mechanical threshold 1-3 g vs. normal > 15 g). Intrathecal clonidine and MK-801 alone produced dose-dependent reductions of tactile hyperesthesia: ED50 9 micro gram and 10 micro gram, respectively. With the fixed-dose analysis, the log dose-response curves showed a left shift that considerably exceeds the theoretical curve made by a simple sum of the effects of clonidine alone and with MK-801 (1 micro gram). With the isobolographic analysis, the combination ED50 was found to be statistically less than the theoretical additive dose combination. lntrathecal atipamezole, an alpha2 antagonist, reversed the effects of clonidine and the clonidine/MK-801 mixture but not MK-801 alone. The side effect of clonidine was sedation and urination and that of MK-801 was motor weakness at doses above 10 micro gram. These effects were considerably less severe in rats after equiactive doses in the combination group.     

An Obligatory Role for Spinal Cholinergic Neurons in the Antiallodynic Effects of Clonidine after Peripheral Nerve Injury

Background: Indirect evidence supports a role of spinal cholinergic neurons in tonically reducing response to noxious mechanical stimulation and in effecting analgesia from [alpha]2-adrenergic agonists. This study directly assessed the role of cholinergic neurons in regulating the level of mechanical allodynia and in participating in the antiallodynic effect of the clinically used [alpha]2-adrenergic agonist, clonidine, in an animal model of neuropathic pain.

Methods: Allodynia was produced in rats by ligation of the left L5 and L6 spinal nerves. Rats received a single intrathecal injection of saline or one of three different doses of the cholinergic neurotoxin, ethylcholine mustard aziridinium ion (AF64-A; 2, 5, and 15 nmol). Seven days later, allodynia was assessed before and after intrathecal injection of 15 [mu]g clonidine. The spinal cord was removed, and spinal cord acetylcholine content, cholinergic neuron number and distribution, and [alpha]2-adrenergic receptor expression were determined.

Results: AF64-A administration reduced both the number of cholinergic cells and the acetylcholine content of the lumbar dorsal spinal cord by 20-50% but did not affect level of mechanical allodynia. AF64-A did, however, completely block the antiallodynic effect of clonidine. AF64-A did not reduce [alpha]2-adrenergic ligand binding in dorsal lumbar cord.     

Intrathecal Morphine Reduces Allodynia after Peripheral Nerve Injury in Rats via Activation of a Spinal A1 Adenosine Receptor

Background: The degree to which intrathecally administered morphine can alleviate hypersensitivity in animals after peripheral nerve injury is controversial, and the mechanisms by which morphine works in these circumstances are uncertain. In normal animals, morphine induces adenosine release, and in vitro data suggest that this link is disrupted after peripheral nerve injury. Therefore, using a controlled, blinded study design, the authors tested intrathecal morphine efficacy in rats with peripheral nerve injury and the role of spinal A1 adenosine receptors in the action of morphine.

Methods: Male rats underwent intrathecal catheter implantation and lumbar spinal nerve ligation, resulting in hypersensitivity to tactile stimulation of the paw. Intrathecal morphine alone or with naloxone or the specific A1 adenosine receptor antagonist, 1,3-dipropyl-8-cyclopentyxanthine (DPCPX), was administered, and withdrawal threshold to von Frey filament application to the hind paw was determined.

Results: Intrathecal morphine (0.25-30 [mu]g) dose-dependently reversed mechanical hypersensitivity after spinal nerve ligation, with an ED50 of 0.79 [mu]g. The effect of morphine was blocked by intrathecal naloxone. Intrathecal DPCPX alone had no effect on withdrawal threshold after spinal nerve ligation but completely reversed the effect of morphine, with an ID50 of 5.6 [mu]g.     

Stereospecific Effect of Pregabalin on Ectopic Afferent Discharges and Neuropathic Pain Induced by Sciatic Nerve Ligation in Rats

Background : The new anticonvulsants, gabapentin and pregabalin, are effective in the treatment of neuropathic pain. The sites and mechanisms of their analgesic action are not fully known. The authors have previously demonstrated that systemic gabapentin suppresses ectopic afferent discharges recorded from injured sciatic nerves in rats. In the current study, they further examined the stereospecific effect of pregabalin on neuropathic pain and afferent ectopic discharges in a rodent model of neuropathic pain.

Methods : Tactile allodynia and thermal hyperalgesia were induced by partial ligation of the left sciatic nerve in rats. Single-unit activity of afferent ectopic discharges was recorded from the sciatic nerve proximal to the site of ligation.

Results : Intravenous injection of 10-30 mg/kg pregabalin dose-dependently attenuated tactile allodynia (n = 10) and thermal hyperalgesia (n = 8). The stereoisomer of pregabalin, R-3-isobutylgaba, had no analgesic effect in this dose range. Furthermore, intravenous injection of pregabalin, but not R-3-isobutylgaba, significantly inhibited the ectopic discharges from injured afferents in a dose-dependent manner (from 20.8 +/- 2.4 impulses/s during control to 2.3 +/- 0.7 impulses/s after treatment with 30 mg/kg pregabalin, n = 15). Pregabalin did not affect the conduction velocity of afferent fibers and the response of normal afferent nerves to mechanical stimulation.