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.     

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).     

Antiallodynic Effects of Systemic and Intrathecal Morphine in the Spared Nerve Injury Model of Neuropathic Pain in Rats

Background: The efficacy of opioids for neuropathic pain remains controversial. The effects of morphine on pain behavior were investigated in two animal models of neuropathic pain: the spared nerve injury (SNI) model and the spinal nerve ligation (SNL) model.

Methods: Nerve injuries were created in rats either by tight ligation and section of the left tibial and common peroneal nerves (SNI) or by unilateral ligation of L5 and L6 spinal nerves (SNL). Paw withdrawal threshold to mechanical stimuli was measured using the up-down method in the hairy and glabrous skin territories of the sural nerve for SNI rats or in the mid-plantar paw of SNL rats.

Results: Before SNI, the median paw withdrawal thresholds in hairy and glabrous skin were similar (26 g [25%, 75% quartiles: 26, 26 g]). The paw withdrawal threshold decreased after SNI in both hairy and glabrous skin (P < 0.001). Thirty days after the SNI, the threshold in hairy skin (0.3 g) was significantly lower than in glabrous skin (1.9 g; P < 0.001). In blinded experiments, both subcutaneous and intrathecal morphine (0.1-10 [mu]g) dose-dependently attenuated mechanical allodynia induced by SNI measured in the hairy skin, an effect that was naloxone reversible. The ED50 for the intrathecal morphine was 0.52 [mu]g (95% confidence interval, 0.31-0.90 [mu]g). Morphine (1 [mu]g intrathecal) attenuated SNI-induced mechanical allodynia in glabrous skin with potency similar to that in hairy skin. In SNL rats, morphine (30 [mu]g intrathecal) almost completely reversed the SNL-induced mechanical allodynia.     

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.     

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.     

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.     

Epidural and Intrathecal n-Butyl-p-Aminobenzoate Solution in the Rat: Comparison with Bupivacaine

Background: Epidural administration of an aqueous suspension of n-butyl-p-aminobenzoate (BAB) to humans results in long-lasting sensory blockade without motor block. The dose-response of BAB administered epidurally and intrathecally as a solution was studied in rats to define the local anesthetic properties in an established animal model.

Methods: The time course of changes in tail withdrawal latency and motor function were determined in rats after epidural or intrathecal administration of solutions of BAB or bupivacaine. The dose-response relation was determined and median effective dose values were calculated.

Results: After epidural and intrathecal administration of BAB solutions, the onset and duration of the antinociceptive action were comparable to bupivacaine. Median effective dose values for tail-withdrawal latency of 6 s or more were significantly greater for BAB. After both routes of administration, BAB clearly affected motor function.     

Intrathecal Lidocaine Reverses Tactile Allodynia Caused by Nerve Injuries and Potentiates the Antiallodynic Effect of the COX Inhibitor Ketorolac

Background: Systemic lidocaine and other local anesthetics reduce hypersensitivity states induced by both acute inflammation and peripheral nerve injury in animals and produce analgesia in some patients with chronic pain. The mechanisms underlying the antiallodynic effect of systemic lidocaine are unclear, although most focus is on peripheral mechanisms. Central mechanisms, particularly at the spinal dorsal horn level, are less known. In this study, the authors aimed to determine whether intrathecal lidocaine has an antiallodynic effect on established mechanical allodynia in two well-characterized neuropathic pain rat models: partial sciatic nerve ligation (PSNL) and spinal nerve ligation (SNL).

Methods: Lidocaine (100-300 [mu]g) was intrathecally injected in PSNL and SNL rats. The withdrawal threshold of both hind paws in response to mechanical stimulation was measured using a series of calibrated von Frey filaments.

Results: This single injection reduced ongoing tactile allodynia in PSNL and SNL rats. The antiallodynic effect of intrathecal lidocaine lasted longer in PSNL (> 3 days) than in SNL rats (< 3 days). Intraperitoneal lidocaine (300 [mu]g) had no effect on tactile allodynia in PSNL rats. In SNL rats, prior intrathecal lidocaine (200 and 300 [mu]g) potentiated the antiallodynic effect of intrathecal ketorolac, a nonselective cyclooxygenase inhibitor. Intrathecal ketorolac alone had no antiallodynic effect on SNL rats. However, prior intrathecal lidocaine (100 [mu]g) failed to potentiate the antiallodynic effect of intrathecal ketorolac.     

Additive Effects of Hypothermia and Phenobarbitol upon Cerebral Oxygen Consumption in the Rat

The quantitative effects of a combination of hypothermia and phenobarbital on cerebral oxygen uptake (CMRo2) was studied in rats, curarized and artificially ventilated with 70% nitrous oxide in oxygen. Cerebral blood flow (CBF) was measured with a modification of the KETY & SCHMIDT (1948) technique, using 133xenon as a tracer. Arteriovenous difference in oxygen content over the brain was measured and CMRo2 was calculated. Four groups were studied. Group 1 was a control group. The three experimental groups were injected with phenobarbital intraperitoneally: Group 2 with 50 mg/kg body weight; Group 3 with 150 mg/kg; and Group 4 with 50 mg/kg of phenobarbital, and, in addition, body temperature was lowered to 32 degrees C in this group. CMRo2 in groups 2, 3 and 4 was reduced by 22, 37 and 43%, respectively, compared to Group 1. The changes in CBF were of the same magnitude. In a previous study we have found that CMRo2 decreases by 5% per 1 degree C decrease in body temperature. The value for CMRo2 in Group 4 is close to the value obtained if the effect of 50 mg/kg body weight of phenobarbital on CMRo2 is added to the effect of a temperature reduction of 5 degrees C. It is concluded that the effects of barbiturates and hypothermia on CMRo2 are additive.     

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.     

Intrathecal Gabapentin Enhances the Analgesic Effects of Subtherapeutic Dose Morphine in a Rat Experimental Pancreatitis Model

Background: Morphine sulfate has long been used for analgesia, but clinical applications can be limited by side effects, tolerance, and potential for addiction at therapeutic doses. An agent that produces therapeutic analgesia when coadministered with low-dose morphine could have important clinical uses. The anticonvulsant agent gabapentin has been identified as having antihyperalgesic properties acting on the [alpha]2[delta]1 subunit of N-type voltage-activated calcium channels on dorsal root ganglia neurons. In this study, intrathecal gabapentin, which by itself is ineffective when administered spinally, was combined with low-dose morphine and tested in an acute bradykinin-induced pancreatitis model in rats.

Methods: An intrathecal catheter was surgically inserted into the subarachnoid space of male Sprague-Dawley rats. A laparotomy was performed for ligation and cannulation of the bile-pancreatic duct. Rats were pretreated intrathecally with artificial cerebrospinal fluid, gabapentin, morphine, or combined gabapentin and morphine 30 min before bradykinin injection into the bile-pancreatic duct. Spontaneous behavioral activity (cage crossing, rearing, and hind limb extension) was monitored before drug injection (baseline) and after bradykinin injection into the bile-pancreatic duct to assess visceral pain.

Results: Spinal pretreatment with up to 300 [mu]g gabapentin alone was not effective in reducing hind limb extension in this model, but did restore some cage crossing and rearing behaviors. Spinal treatment with low-dose morphine reduced hind limb extension only. Spinal pretreatment with combined gabapentin and subtherapeutic doses of morphine sulfate resulted in restoration of all spontaneous behaviors to surgical baseline levels including elimination of hind limb extension.     

Intrathecal Catheterization in the Rat: Improved Technique for Morphologic Analysis of Drug-induced Injury

Background: The authors previously described an in vivo model suitable for investigation of functional impairment induced by intrathecally injected local anesthetic. However, meaningful histologic analysis could not be performed because catheterization, per se, induced morphologic changes in control animals. In the current experiments, the authors sought to identify an alternative, less reactive, catheterization technique for intrathecal drug administration.

Methods: Twenty-five rats received an intrathecal infusion of normal saline through a catheter composed of either 28-gauge polyurethane, 32-gauge polyimide, 32-gauge polyurethane, PE-10 polyethylene, or PE-10 polyethylene that had been stretched to twice its original length. Seven days after infusion, sensory function was assessed using the tail-flick test, and the spinal cord and nerve roots were prepared for neuropathologic evaluation.

Results: There was no significant difference in sensory function among groups. Animals in which 28-gauge polyurethane, 32-gauge polyimide, PE-10, and double-stretched PE-10 had been implanted had moderate to severe nerve injury in 11%, 14%, 23%, and 8% of fascicles, respectively, whereas none of the animals in which 32-gauge polyurethane was implanted had any evidence of moderate or severe damage.     

Continuous Intrathecal Administration of Short-lasting micro Opioids Remifentanil and Alfentanil in the Rat

Background: Lipid soluble micro opioids given intrathecally produce a potent, dose-dependent analgesic response, which because of rapid clearance, is of short duration. Such agents delivered by continuous infusion can result in systemic accumulation and significant extraspinally mediated side effects. The effects of intrathecal infusions of two lipid-soluble micro opioids were investigated: remifentanil, an esterase metabolized agent with an inactive metabolite, and alfentanil.

Methods: Rats with chronic lumbar intrathecal catheters received intrathecal infusions (in flow rates of 1.0 micro liter/min and 0.1 micro liter/min) of remifentanil or alfentanil and were tested for hind paw thermal withdrawal latency, supraspinal side effects (sedation, block of pinna, and corneal responses) and motor impairment. Remifentanil was delivered either in a glycine formulation (Rg) or in a saline vehicle (Rs). Separate studies with the glycine vehicle also were undertaken.

Results: At an infusion rate of 0.1 micro liter/min, remifentanil and alfentanil produced naloxone-reversible, dose-dependent analgesia and supraspinal side effects with the intrathecal ED50 (micro gram/min; 95% confidence interval) for analgesia: Rs = 1.5 (1.2-1.8), Rg = 1.2 (0.7-2.3); alfentanil = 1.5 (1.4-1.6) and for supraspinal side effects: Rs = 1.7 (1.4-1.9); Rg = 1.9 (1.6-2.4); alfentanil = 1.5 (1.4-1.7). There was no difference in potency or time until onset for analgesia at either delivery rate (12-20 min), whereas for supraspinal side effects, 1.0 micro liter/min resulted in a faster onset for Rg. Recovery of normal thresholds after equianalgesic doses was faster in R sub s than alfentanil and for the supraspinal index faster in Rs and Rg groups. Rg, but not Rs or alfentanil, produced a dose-dependent motor impairment after 90 min of intrathecal infusion at a flow rate of 0.1 micro liter/min. Both glycine in Rg and glycine (matching glycine dose) alone showed parallel time courses for motor impairment and similar intrathecal ED50 (6.6 vs. 6.4 micro gram/min over 90 min) for this nonnaloxone reversible effect. Intrathecal bolus administration of the same total dose of glycine showed no significant motor effects.     

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.