Jin Mo Chung

Quantitative assessment of tactile allodynia in the rat paw

We applied and validated a quantitative allodynia assessment technique, using a recently developed rat surgical neuropathy model wherein nocifensive behaviors are evoked by light touch to the paw. Employing von Frey hairs from 0.41 to 15.1 g, we first characterized the percent response at each stimulus intensity. A smooth log-linear relationship was observed, with a median 50% threshold at 1.97 g (95% confidence limits, 1.12-3.57 g). Subsequently, we applied a paradigm using stimulus oscillation around the response threshold, which allowed more rapid, efficient measurements. Median 50% threshold by this up-down method was 2.4 g (1.81-2.76). Correlation coefficient between the two methods was 0.91. In neuropathic rats, good intra- and inter-observer reproducibility was found for the up-down paradigm; some variability was seen in normal rats, attributable to extensive testing. Thresholds in a sizable group of neuropathic rats showed insignificant variability over 20 days. After 50 days, 61% still met strict neuropathy criteria, using survival analysis. Threshold measurement using the up-down paradigm, in combination with the neuropathic pain model, represents a powerful tool for analyzing the effects of manipulations of the neuropathic pain state.

An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat

&NA; We attempted to develop an experimental animal model for peripheral neuropathic pain. Under sodium pentobarbital anesthesia, both the L5 and L6 spinal nerves (group 1) or the L5 spinal nerve alone (group 2) of one side of the rat were tightly ligated. For comparison, a parallel study was conducted with another group of rats (group 3) which received a partial tight sciatic nerve ligation, a paradigm developed previously as a neuropathy model. Withdrawal latencies to application of radiant heat to the foot were tested for the next 16 weeks in all 3 groups. Sensitivity of the hind paw to mechanical stimulation was tested with von Frey filaments. The general behavior of each rat was noted during the entire test period. Results suggested that the surgical procedure in all 3 groups produced a long‐lasting hyperalgesia to noxious heat (at least 5 weeks) and mechanical allodynia (at least 10 weeks) of the affected foot. In addition, there were behavioral signs of the presence of spontaneous pain in the affected foot. Therefore, we believe we have developed an experimental animal model for peripheral neuropathy using tight ligations of spinal nerves. The model manifests the symptoms of human patients with causalgia and is compatible with a previously developed neuropathy model. The present model has two unique features. First, the surgical procedure is stereotyped. Second, the levels of injured and intact spinal segments are completely separated, allowing independent experimental manipulations of the injured and intact spinal segments in future experiments to answer questions regarding mechanisms underlying causalgia.We attempted to develop an experimental animal model for peripheral neuropathic pain. Under sodium pentobarbital anesthesia, both the L5 and L6 spinal nerves (group 1) or the L5 spinal nerve alone (group 2) of one side of the rat were tightly ligated. For comparison, a parallel study was conducted with another group of rats (group 3) which received a partial tight sciatic nerve ligation, a paradigm developed previously as a neuropathy model. Withdrawal latencies to application of radiant heat to the foot were tested for the next 16 weeks in all 3 groups. Sensitivity of the hind paw to mechanical stimulation was tested with von Frey filaments. The general behavior of each rat was noted during the entire test period. Results suggested that the surgical procedure in all 3 groups produced a long-lasting hyperalgesia to noxious heat (at least 5 weeks) and mechanical allodynia (at least 10 weeks) of the affected foot. In addition, there were behavioral signs of the presence of spontaneous pain in the affected foot. Therefore, we believe we have developed an experimental animal model for peripheral neuropathy using tight ligations of spinal nerves. The model manifests the symptoms of human patients with causalgia and is compatible with a previously developed neuropathy model. The present model has two unique features. First, the surgical procedure is stereotyped. Second, the levels of injured and intact spinal segments are completely separated, allowing independent experimental manipulations of the injured and intact spinal segments in future experiments to answer questions regarding mechanisms underlying causalgia.

Comparison of three rodent neuropathic pain models

To characterize various animal models of neuropathic pain, we compared three previously developed rat models using the same behavioral testing methods. These models involve: (1) chronic constriction injury by loose ligation of the sciatic nerve (CCI); (2) tight ligation of the partial sciatic nerve (PSL); and (3) tight ligation of spinal nerves (SNL). Comparisons were made for the time course of behavioral signs representing various components of neuropathic pain as well as for the effects of surgical sympathectomy. In general, all three methods of peripheral nerve injury produced behavioral signs of both ongoing and evoked pain with similar time courses. However, there was a considerable difference in the magnitude of each pain component between models. Signs of mechanical allodynia were largest in the SNL injury and smallest in the CCI model. On the other hand, behavioral signs representing ongoing pain were much more prominent in the CCI model than in the other two. Although the behavioral signs of neuropathic pain tended to decrease after sympathectomy in all three models, the change was most evident in the SNL model. The results of the present study suggest that the three rat models tested have contrasting features, yet all are useful neuropathic pain models, possibly representing different populations of human neuropathic pain patients.

Reactive oxygen species (ROS) play an important role in a rat model of neuropathic pain

&NA; Reactive oxygen species (ROS) are free radicals produced in biological systems that are involved in various degenerative brain diseases. The present study tests the hypothesis that ROS also play an important role in neuropathic pain. In the rat spinal nerve ligation (SNL) model of neuropathic pain, mechanical allodynia develops fully 3 days after nerve ligation and persists for many weeks. Systemic injection of a ROS scavenger, phenyl‐N‐tert‐butylnitrone (PBN), relieves SNL‐induced mechanical allodynia in a dose‐dependent manner. Repeated injections cause no development of tolerance or no loss of potency. Preemptive treatment with PBN is also effective in preventing full development of neuropathic pain behavior. Systemic injection was mimicked by intrathecal injection with a little less efficacy, while intracerebroventricular administration produced a much smaller effect. These data suggest that PBN exerts its anti‐allodynic action mainly by spinal mechanisms. Systemic treatment with other spin‐trap reagents, 5,5‐dimethylpyrroline‐N‐oxide and nitrosobenzene, showed similar analgesic effects, suggesting that ROS are critically involved in the development and maintenance of neuropathic pain. Thus this study suggests that systemic administration of non‐toxic doses of free radical scavengers could be useful for treatment of neuropathic pain.

The anti-allodynic effects of amitriptyline, gabapentin, and lidocaine in a rat model of neuropathic pain.

The management of patients with neuropathic pain is challenging.There are only a few reports regarding the acute effects of the commonly used adjuvant drugs amitriptyline (AMI), gabapentin (GBP), and lidocaine (LDC) on neuropathic pain behaviors in animal models. Thus, the purpose of this study was to investigate the acute effects of AMI, GBP, and LDC on behavioral signs of mechanical allodynia and the site of action of these drugs using a rat model of neuropathic pain. Under general anesthesia with halothane, neuropathic injury was produced in rats by tightly ligating the left L5 and L6 spinal nerves. In Experiment 1, baseline mechanical allodynia data were recorded, and the animals were randomly divided into five groups: Group 1 received saline intraperitoneally (IP), Group 2 received AMI (1.5 mg/kg IP); Group 3 received GBP (50 mg/kg IP), Group 4 received an IV saline infusion for 10 min, and Group 5 received LDC (10-mg/kg IV infusion) for 10 min. Measurements of mechanical allodynia were repeated 0.5, 1, 2, and 4 h and 1, 3, and 7 days after treatment. In Experiment 2, rats were prepared similarly to the first experiment, and a single unit activity of continuous discharges of injured afferent fibers was recorded from the left L5 fascicles before and until 1 h after treatment. All animals developed neuropathic pain behavior within 7 days after surgery. All three tested drugs were effective in increasing the threshold for mechanical allodynia as early as 30 min after treatment, and the effect lasted for at least 1 h. Furthermore, AMI and LDC reduced the rate of continuing discharges of injured afferent fibers, whereas GBP did not influence these discharges. Our findings clearly demonstrate an attenuation of neuropathic pain behavior in rats treated with AMI, GBP, or LDC. Finally, the site of action of LDC seems to be primarily in the periphery, and that of GBP is exclusively central, whereas that of AMI seems to have both peripheral and central components. Implications: In the present study, we examined the effectiveness of three drugs commonly used for the treatment of neuropathic pain. Systemic injections of amitriptyline, gabapentin, or lidocaine produced pain-relieving effects in this established model for neuropathic pain in rats, which supports their clinical use in managing patients with neuropathic pain syndromes. (Anesth Analg 1998;87:1360-6)

Signs of neuropathic pain depend on signals from injured nerve fibers in a rat model

The present study was undertaken to determine the role of injured fibers in the development of neuropathic pain using our earlier established rat model. Our model was produced by placing tight ligatures to the L5 or both the L5 and L6 spinal nerves on one side in the rat. These rats showed long-lasting behavioral signs of mechanical allodynia and heat hyperalgesia. Using the uniqueness of our model, 3 specific questions are being asked concerning the initiation and maintenance of behavioral signs for neuropathic pain. The results of behavioral tests performed after various surgical manipulations suggest that: (1) peripheral nerve injury itself is the critical factor for the development of behavioral signs of neuropathic pain; (2) signs of neuropathic pain appear only when injury occurs at a part of the peripheral nerve distal to the dorsal root ganglion; and (3) signals (either electrical or chemical) entering the spinal cord from the injured fibers or the dorsal root ganglion cells play a critical role for both initiation and maintenance of the neuropathic pain state.

Contributions of injured and intact afferents to neuropathic pain in an experimental rat model

&NA; This study was conducted to determine the contribution of peripheral inputs from injured and intact afferent fibers to behavioral signs of neuropathic pain, using a previously developed neuropathic rat model. Neuropathic injury was produced by tightly ligating the left L5 and L6 spinal nerves; this procedure induced rats to display neuropathic pain behaviors in the ipsilateral hindlimb. The behaviors included signs of mechanical and cold allodynia, as well as ongoing pain. Five days after neuropathic injury, peripheral inputs from injured segments (L5 and L6) or intact segments (L3 and L4) were blocked by either transection of the dorsal roots or application of a local anesthetic (bupivacaine) to the roots. Dorsal rhizotomy of the injured segments reduced all components of neuropathic pain behaviors. In contrast, dorsal rhizotomy of the uninjured segments abolished behavioral signs of mechanical and cold allodynia, but signs of ongoing pain were preserved. Blocking afferent inputs by application of bupivacaine mimicked the results of dorsal rhizotomy, in a reversible manner. These results suggest that afferent signals from injured and intact fibers play distinctively different roles in neuropathic pain: inputs from injured afferents maintain all components of neuropathic pain, while those from intact afferents mediate evoked pain such as mechanical and cold allodynia. An hypothesis is proposed to explain the results of the present as well as other published studies.

Factors influencing peripheral nerve stimulation produced inhibition of primate spinothalamic tract cells

&NA; Several factors that influence the inhibition of primate spinothalamic tract (STT) cells produced by repetitive peripheral conditioning stimulation have been studied. Identified STT cells were recorded from the lumbosacral spinal cord in intact, anesthetized monkeys. In addition, presumed STT cells were recorded from unanesthetized, decerebrate or decerebrate, spinalized monkeys; these cells were identified by antidromic activation from the contralateral ventral lateral funiculus of the upper cervical spinal cord. Activity of the STT cells was evoked by electrically stimulating the sural nerve with pulses having an intensity strong enough to activate C fibers. The C fiber evoked STT cell activity was compared before, during and after repetitive conditioning stimuli applied to the tibial nerve for 5 min. By applying graded strengths of conditioning stimuli, it was found that the A&dgr; fiber group is the most important for producing inhibition of STT cells, although significant additional effects were also produced by the A&agr;&bgr; and C fiber groups. Conditioning stimuli with fixed intensity at different frequencies showed that the higher the frequency the more powerful the inhibition within the range we tested (0.5–20 Hz). The inhibition produced by peripheral nerve stimulation was segmentally organized, so the most effective nerve in producing inhibition amongst those tested was the ipsilateral tibial nerve. The contralateral sciatic nerve, the ipsilateral median nerve and the contralateral median nerve were less effective in that order. The results of the present experiments suggest that the most effective way to produce analgesia by peripheral nerve stimulation would be by high frequency stimulation of a nerve innervating the area from which pain originates with an intensity at least strong enough to activate A&dgr; fibers.

Sympathetic sprouting in the dorsal root ganglia of the injured peripheral nerve in a rat neuropathic pain model

The extent of the sprouting of sympathetic postganglionic fibers in the dorsal root ganglion (DRG) and the peripheral nerves was examined in neuropathic rats at different postoperative times. After the L5 and L6 spinal nerves were ligated on one side, three different pain behavior tests (representing mechanical allodynia, cold allodynia, ongoing pain exacerbated by cold stress) were performed at various time intervals. The sympathetic postganglionic fibers were visualized by immunostaining with antibodies to tyrosine hydroxylase (TH). In the neuropathic rats, all three pain behaviors were fully developed within 3 days after the surgery, maintained up to 2 weeks, and then started to decline gradually afterward. At 20 weeks after neuropathic surgery, pain behaviors were reduced significantly compared to the peak response, but were still higher than the presurgery levels. Sympathectomy, performed 4 days after neuropathic surgery, almost completely abolished the signs of mechanical allodynia and ongoing pain behaviors, and it reduced the behaviors of cold allodynia to approximately half. The numerical density of sympathetic fibers in the DRG of an injured segment was significantly higher at 1, 4, and 20 weeks after neuropathic surgery as compared to the normal, suggesting that there is sprouting of sympathetic fibers in the DRG after peripheral nerve injury. Sprouting of sympathetic fibers in the DRG was extensive as early as 2 days after the spinal nerve ligation, and the sprouted fibers were almost completely eliminated after sympathectomy. The data suggest that sympathetic innervation of the DRG may play an important role in the development and maintenance of sympathetically maintained neuropathic pain.

Characteristics of ectopic discharges in a rat neuropathic pain model

Injured afferent neurons produce spontaneous activity that is generated away from the normal impulse generation site. Since this activity, referred to as ectopic discharges, may play a significant role in neuropathic pain, it is important to systematically analyze the activity in various pain states. The present study used the segmental spinal nerve injury model of neuropathic pain to quantify the ectopic discharges from injured afferents in the neuropathic rat under various conditions. All aspects of measured ectopic discharges declined as postoperative time lengthened. Neuropathic pain behaviors declined in a similar fashion over the same time period. Surgical sympathectomy on neuropathic animals lowered the level of ectopic discharges along with neuropathic pain behaviors. The data indicate that the level of ectopic discharges is well correlated with that of pain behaviors in a rat neuropathic pain model, and this reinforces the supposition that ectopic discharges are important to the maintenance of neuropathic pain behaviors. The data suggest that there are two components of ectopic discharge generator mechanisms: sympathetically dependent and sympathetically independent components.Abstract Injured afferent neurons produce spontaneous activity that is generated away from the normal impulse generation site. Since this activity, referred to as ectopic discharges, may play a significant role in neuropathic pain, it is important to systematically analyze the activity in various pain states. The present study used the segmental spinal nerve injury model of neuropathic pain to quantify the ectopic discharges from injured afferents in the neuropathic rat under various conditions. All aspects of measured ectopic discharges declined as postoperative time lengthened. Neuropathic pain behaviors declined in a similar fashion over the same time period. Surgical sympathectomy on neuropathic animals lowered the level of ectopic discharges along with neuropathic pain behaviors. The data indicate that the level of ectopic discharges is well correlated with that of pain behaviors in a rat neuropathic pain model, and this reinforces the supposition that ectopic discharges are important to the maintenance of neuropathic pain behaviors. The data suggest that there are two components of ectopic discharge generator mechanisms: sympathetically dependent and sympathetically independent components.