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Effects of intrathecal lidocaine on hyperalgesia and allodynia following chronic constriction injury in rats Jie Tian a,1, Yiwen Gu a,1, Diansan Su a, Yichao Wu b, Xiangrui Wang a,* a b

Department of Anesthesiology, Renji Hospital, Medical School of Shanghai Jiaotong University, Shanghai, China CBR Institute for Biomedical Research, Harvard Medical School, Boston, MA, USA

a r t i c l e

i n f o

Article history: Received 9 September 2007 Received in revised form 6 March 2008 Accepted 29 March 2008 Available online xxxx Keywords: Intrathecal lidocaine Neuropathic pain Thermal hyperalgesia Tactile allodynia Chronic constriction injury

a b s t r a c t The present study investigated the effects of different doses of intrathecal lidocaine on established thermal hyperalgesia and tactile allodynia in the chronic constriction injury model of neuropathic pain, defined the effective drug dose range, the duration of pain-relief effects, and the influence of this treatment on the body and tissues. Male Sprague–Dawley rats were divided into five groups and received intrathecal saline or lidocaine (2, 6.5, 15, and 35 mg/kg) 7 days after loose sciatic ligation. Respiratory depression and hemodynamic instability were found to become more severe as doses of lidocaine increased during intrathecal therapy. Two animals in the group receiving 35 mg/kg lidocaine developed pulmonary oedema and died. Behavioral tests indicated that 6.5, 15, and 35 mg/kg intrathecal lidocaine showed different degrees of reversal of thermal hyperalgesia, and lasted for 2–8 days, while 2 mg/kg lidocaine did not. The inhibition of tactile allodynia was only observed in rats receiving 15 and 35 mg/kg lidocaine, and the anti-allodynic effects were identical in these two groups. Histopathologic examinations on the spinal cords revealed mild changes in rats receiving 2–15 mg/kg lidocaine. However, lesions were severe after administration of 35 mg/kg lidocaine. These findings indicate that intrathecal lidocaine has prolonged therapeutic effects on established neuropathic pain. The balance between sympathetic and parasympathetic nervous activities could be well preserved in most cases, except for 35 mg/kg. Considering the ratio between useful effects and side effects, doses of 15 mg/kg are suitable for intrathecal injection for relief of neuropathic pain. Ó 2008 European Federation of Chapters of the International Association for the Study of Pain. Published by Elsevier Ltd. All rights reserved.

1. Introduction Neuropathic pain is one of the most significant health problems in the world. Although numerous methods have been applied in the clinics to treat neuropathic pain, many patients report resistance to the available treatments. And issues such as tolerance to and physical dependence on analgesics, efficacy of treatment alternatives, and high treatment costs plague both the clinicians and patients. Studies in the implementation of improved therapies for neuropathic pain, which consider not only analgesic effects, but also other benefits (e.g., long-term pain-relief effect, improved quality of life, low cost-effectiveness) have always generated considerable interest. The use of systemic lidocaine has been extensively studied for the treatment of chronic pain in the past years. Now there is accumulating evidence for the efficacy of systemic lidocaine to treat neuropathic pain in both animal models and in clinical patients (Mao and Chen, 2000; Smith et al., 2002). However, the effective* Corresponding author. Tel.: +86 2158752345x3198; fax: +86 2150903239. E-mail address: [email protected] (X. Wang). 1 These authors contributed equally to this work.

ness seems to be dose-related (Kalso, 2005). The doses of systemic lidocaine that are sufficient to relieve neuropathic pain are often associated with intolerable side effects, such as dizziness, nausea, seizures, and significant cardiac system injury, thus precluding a routine use of this therapy (Martin and Eisenach, 2001). And only certain types of neuropathic pain behaviors are responsive to systemic lidocaine administration (Sinnott et al., 1999; Mao and Chen, 2000). Recently, evidence that lidocaine exerts some of its analgesic effects via actions in the spinal cord (Olschewski et al., 1998; Ness, 2000) has led to an interest in intrathecal therapy with this agent for chronic pain treatment. Several studies have reported that large doses of intrathecal lidocaine, which will generally lead to total spinal anesthesia (TSA) in the clinics, produced reduced pain scores for up to 7 days in patients (Yokoyama et al., 2002). The unexpected long-term pain-relief effect of intrathecal lidocaine has added a new modality to the treatment regimen for intractable neuropathic pain. However, a number of issues regarding this treatment, including the effective, meaningful drug dose range, the durability of pain-relief effects, and long-term outcomes of intrathecal anesthetic, remain unclear, because in this kind of clinical study, further investigation is obviously too dangerous and

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unethical. Therefore, there is a need to use animal models of intrathecal lidocaine treatment in the neuropathic states, to broaden our knowledge of this therapy, and to help to have a rational application, rather than an empirical clinical practice of this treatment. In the present study, we examined the dose–response and detailed time course of pain-relieving effect of single intrathecally administered lidocaine in a rat model of neuropathic pain induced by chronic constriction injury (CCI) of the sciatic nerve, which has been reported to develop reliable, long-lasting hyperalgesia and allodynia comparable in character to the clinical syndrome of neuropathic pain (Bennett and Xie, 1988). Histopathologic changes that intrathecal lidocaine might induce on the spinal cord of these animals were further evaluated to explore the safety of this treatment. 2. Methods Male Sprague–Dawley rats weighing between 250 and 300 g were used. The rats were fed rat chow with free access to tap water and housed in temperature- and humidity-controlled animal quarters with 12 h light/dark cycle. All rats were housed for a minimum of 1 week prior to use. The procedures were approved by the Institutional Animal Care Committee. 2.1. Chronic constriction injury (CCI) surgery CCI surgery was carried out as described previously (Bennett and Xie, 1988). Briefly, rats were anesthetized with 10% chloral hydrate (3 ml/kg, i.p.), and a 7-mm segment of the left common sciatic nerve was exposed at the mid-thigh level, proximal to the sciatic trifurcation. Four 4–0 chromic gut sutures were tired around the nerve at intervals of 1 mm, and ligatures were tied loosely enough so that, on visual inspection, blood flow was not obstructed. The right side of the body was exposed but not ligated. The surgical incision was completed by closing the muscles and skin in layers. The CCI surgery day was regarded as post-operative day (POD) 0. 2.2. Intrathecal catheter implantation On the fourth day after CCI surgery (POD 4), the rats were anesthetized by intraperitoneal injection of 10% chloral hydrate, and intrathecal catheters composed of polyethylene (PE-10) tubing were introduced into the subarachnoid space using a previously described modification (Størkson et al., 1996) of the method of Yaksh and Rudy (1976). Catheters were passed through a slit in the L5-6 interspace, and advanced 2–3 cm into the intrathecal space so that the tip terminated near the lumbar enlargement. The right position of the catheters was verified by cautious aspiration of cerebrospinal fluid (CSF). The catheters were then implanted subcutaneously on the back of the rats, and externalized between the ears for injection. Rats exhibiting any evidence of motor or additional sensory dysfunction were excluded in the further study 3 days after catheters implantation. 2.3. Intrathecal lidocaine injection On POD 7, the rats equipped with catheters were anesthetized under inhalational anesthesia with ether delivered through a facesnout mask. The right jugular vein and caudal artery of the rats were cannulated with 22-G intravenous catheters (BD AngiocahTM; Brazil), for administration of vasoconstrictors, and for measurements of mean arterial blood pressure (MAP), respectively. Refitted 16-G intravenous catheters (BD AngiocahTM; Brazil) were advanced endotracheally for preparation of mechanical ventilation.

The rats were then randomly divided into five groups (n = 6–10 in each group), and received intrathecal solutions as follows: (1) control group: 0.5 ml 0.9% saline; (2–5) lidocaine groups: 0.5 ml of 2, 6.5, 15, or 35 mg/kg lidocaine (pH ranged from 4.48 to 5.38), in group L2, L6.5, L15, or L35, respectively. Lidocaine was supplied as a 2% solution, and diluted in normal saline to 0.5 ml equally in all the groups, to avoid the influence of different intracranial pressures on the development of pain. Such a volume was selected because previous studies have shown that a saline injection of 0.5 ml did not induce any brain stem responses (Yamada et al., 1994, 1997). All the solutions were injected through the intrathecal catheters with an injection pump (PerfusorÒSpace, B. Braun, Germany) at a constant rate of 0.10 ml/min. Ether inhalation was stopped immediately after onset of intrathecal injection. At the end of each injection, the intrathecal catheter was flushed with 15 ll of 0.9% saline solution for the dead space of the catheter. Rectal temperature was monitored and maintained within 0.5 °C throughout the procedure by means of a heat pad. Respiration of the rats was observed carefully once intrathecal injection had begun. The rats were mechanically ventilated (Rat Ventilator, RSP1002, Kent Scientific Inc., USA) with an oxygen/air mixture immediately at the sight of irregular respiration. Onset time of mechanical ventilation was recorded. MAP was continuously monitored during intrathecal lidocaine therapy. A hypotension episode was defined as a drop in pressure to less than 30% from the baseline value, and was treated by continuous intravenous administration of dopamine at an initial rate of 100 lg kg 1 min 1. The rate of administration was decreased by 20 lg kg 1 min 1 from its current level (80, 60, 40, 20, and 0 lg kg 1 min 1), when blood pressure returned to normal range and remained stable for at least 5 min. In the case that the administration rate was kept at 100 lg kg 1 min 1 for more than 5 min, Voluven (HAES-steril 130/0.4, 6%, Fresenius Kabi, Germany), which is a clinically commonly used colloidal fluid, would be infused continuously using a B. Braun injection pump at a rate of 0.2 ml/min, and was stopped when the rate of dopamine decreased. The total doses of dopamine and Voluven administration were recorded. The total duration time was comprised between the beginning of injection and complete recovery from the block, which was defined as comeback of spontaneous ventilation and free movement of hind limbs without any limitation. The wounds of the animals were closed with suture after recovery. They were then housed in groups of 2 or 3 in clear plastic cages with solid floors covered with 3–6 cm of soft bedding, and fed with free access to food and water. 2.4. Behavioral tests Yiwen Gu, who was blinded to the animal groups, performed the behavioral tests. The thermal hind paw withdrawal latency and Von Frey withdrawal threshold of all animals was obtained 3 days prior to CCI surgery (pre-lesion baseline, PRE), on POD 1, POD 3, POD 6, and each day after intrathecal lidocaine therapy (post-therapeutic day [PTD] 1, PTD 2, PTD 3, . . .) until PTD 10, as the pilot study showed that the values returned to pre-therapeutic levels in all the groups on PTD 10. Those responding abnormal at baseline values were excluded from the beginning. 2.4.1. Thermal paw withdrawal latency Thermal hyperalgesia was assessed with a test apparatus consisting of a movable radiant heat source and a controller (BME410A, Institute of Biomedical Engineering, CAMS) as previously described (Villetti et al., 2003). The rat was placed on a smooth glass surface in a box measuring 17  22  14 cm. The temperature of the glass surface was maintained at 25 ± 1 °C. The radiant heat source under the glass floor was positioned directly under the

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desired hind paw, and the controller detected the paw withdrawal latency (PWL) in 0.1-s steps, and switched off the heat source when the animal withdrew its hind paw. The intensity of the heat stimulus was maintained constant throughout all experiments, and was pre-calibrated to give a baseline PWL of approximately 10– 15 s in control rats. Cut-off latency was set at 30 s to prevent tissue damage of the plantar zone. Thermal stimulus was delivered five times to each hind paw at 10-min intervals. The five responses per side were averaged and a difference score was computed by subtracting the average latency of the control side from the average latency of the ligated side. 2.4.2. Von Frey withdrawal threshold The pressure algometer was used to test mechanical threshold as performed by Tabo et al. (1999). Rats were placed on a metal mesh cage, which allowed access to the paws. A set of Von Frey filaments was applied, in ascending order, to the mid-plantar left hind paws. Each filament was delivered five times at approximately 5-s intervals; if the rat did not withdraw the paw, a filament with the next higher bending force was similarly delivered. The procedure was continued until application of the filament elicited a paw withdrawal on each of the five applications. The procedure was repeated two times, and the average force was taken as threshold. 2.5. Histopathologic study In another set of experiments, animals underwent the same treatments. The set of rats was killed immediately after recovery from intrathecal lidocaine therapy (POD 7 or PTD 0), for histopathologic examination of the spinal cord (lumbar enlargement). Rats were killed with a chloral hydrate overdose. Hearts were perfused in situ with 0.9% saline (37 °C) followed by 4% paraformaldehyde, first at 37 °C, then at 4 °C. The lumbar spinal cord, including both the anterior and posterior roots, was sampled by laminectomy. All specimens were stored in 4% paraformaldehyde (4 °C) until histopathologic examination. Spinal cords were embedded in paraffin, and then cut with a microtome in 6-lm section slices. Examinations were performed on 6 hematoxylin- and eosin-stained slices in each segment under an Olympus B40 microscope. A neuropathologist, who was blinded to intrathecal administration, examined the morphologic pathology. 2.6. Statistics All data were expressed as mean ± SEM. The mean values after nerve injury or after intrathecal therapy vs. pre-lesion baseline values (within group), and a control mean vs. each group mean at each time point (between group) comparisons were done by repeated measures analysis of variance, and post hoc Student–Newman–Keuls test. P < 0.05 was considered significant.

3. Results 3.1. General observations After CCI surgery, the rats developed neuropathic pain syndrome as previously described by Bennett and Xie (1988). Unusual gait and posture of the rats could be seen as early as on the first day after surgery (POD 1). In general, the affected toes, which are normally spread and apart while walking or standing, became together and slightly ventroflexed, with the corresponding hind paws everted, and placed clumsily while walking. The rats were often seen to raise the affected hind paws from the floor and hold them in a protected position. Licks of the affected paws were frequently seen. 18.6% (8 of 43) rats were excluded from the study after intrathecal catheter implantation due to traumatic hind limb palsy caused by catheterization (n = 6), or insufficient fixation (n = 2). After recovery from intrathecal solutions injections, the unusual appearance of the affected hind paws was retained in all the rats. However, raising and licks of the paws were rarely seen in groups L15 and L35, which were still frequent in control group and the groups receiving lower doses of lidocaine. Besides the same posture and different action above, all the animals were in good health, and the behavior was generally normal. No palsy or additional sensory dysfunction was encountered. The fur of all rats was sleek and well groomed. There was no significant difference among the weights of the five groups (data not shown). 3.2. Systemic effects of intrathecal lidocaine Two rats in group L35 died during recovery from intrathecal lidocaine therapy. Frothy and pink sputum was seen in the dying rats. Post-mortem dissection revealed extensive pulmonary oedema upon gross examination. A total of 33 rats were analyzed by behavioral tests. The number of rats in each group was as follows: control group, n = 6; group L2, n = 6; group L6.5, n = 6; group L15, n = 8; group L35, n = 7. In all groups, a total volume of 0.5 ml liquid was injected through the catheter over 5 min. The saline injection failed to induce any significant changes in respiration, or in hemodynamics (Table 1, Fig. 1). With the lowest dose (2 mg/kg) of lidocaine, no rats presented respiratory inhibition, while mechanical ventilation was needed in five of six rats in group L6.5, and all the animals in groups L15 and L35 (Table 1). Significant hypotension was observed soon after lidocaine injection (Fig. 1). Dopamine administration was required in all the rats with 6.5 mg/kg or higher doses of lidocaine, while only in two-thirds of rats receiving 2 mg/kg lidocaine. The dose of dopamine ranged from 0.31 to 1.94 mg, with the volume of Voluven infusion ranging from 0 to 2.06 ml (Table 1). Complete recovery of intrathecal anesthesia was observed in all rats in control group, group L2, L6.5, and L15, but only in 77.8% rats in group

Table 1 Systemic effects of intrathecal saline or 2–35 mg/kg lidocaine in rats (mean ± SEM) Group

Onset time (min)

Dopamine (mg)

Voluven (ml)

Total time (min)

Control (n = 6) L2 (n = 6) L6.5 (n = 6) L15 (n = 8) L35 (n = 7)

na na 3.60 ± 0.62 (n = 5) 1.59 ± 0.21 1.01 ± 0.17

0 0.31 ± 0.10 0.61 ± 0.13 0.93 ± 0.06 1.94 ± 0.14

0 0 0.50 ± 0.22 1.13 ± 0.08 2.06 ± 0.12

13.83 ± 0.70 32.67 ± 0.84 40.50 ± 4.98 76.25 ± 4.40 94.29 ± 11.79

na = not applicable. Onset time = Onset time of mechanical ventilation; dopamine was used to treat drop in mean arterial pressure; Voluven treatment was used if blood pressure remained low after dopamine administration at highest level for more than 5 min; total time = from beginning of saline/lidocaine injection until complete recovery from the block.

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Fig. 1. Variations of mean arterial blood pressure in rats receiving saline or 2–35 mg/kg lidocaine during intrathecal therapy. Significant hypotension occurred immediately with intrathecal lidocaine, and required the use of dopamine in some groups (see text). *Indicates significantly different from control saline group at the same time at P < 0.05. Mean values are shown, n = 6–8 in each group.

Fig. 2. Effects of intrathecal lidocaine on established thermal hyperalgesia caused by chronic constriction injury (CCI). Scores are expressed as the mean of difference between ipsilateral and contralateral paw withdrawal latency (PWL). The PWL was markedly reduced on the ligated side since post-operative day (POD) 1 after CCI surgery, yielding negative mean difference scores. The difference enlarged on POD 6. Single intrathecal injection of 6.5–35 mg/kg lidocaine significantly reversed the thermal hyperalgesia from post-therapeutic day (PTD) 1. The difference scores were turned to positive values in rats receiving 15 and 35 mg/kg lidocaine. The anti-hyperalgesic effects remained for 2, 8, and 8 days by 6.5, 15, and 35 mg/kg lidocaine, respectively. *Indicates significantly different from control saline group on the same day at P < 0.05. Mean values are shown, n = 6–8 in each group.

L35, taking from 13.83 to 94.29 min (Table 1). As the intrathecal drug dose increased, the doses of dopamine and Voluven administration also increased, and the total duration time became prolonged. At the end of intrathecal infusion in each group, the extent of anesthesia level was determined by a skin clamp applied progressively cephalad until a response was elicited. No response was encountered throughout the body in animals given 15 and 35 mg/kg lidocaine. The rats in the two groups were found unconscious, and dilation of pupils was observed. The level of sensory block extended to the fore limb in groups L6.5, to the hind limb in groups L2, and no block was revealed in control group.

3.3. Behavioral tests 3.3.1. Thermal paw withdrawal latency The difference latency scores between the left side and the right side of normal animals clustered around zero, as shown in Fig. 2. The PWL was markedly reduced on the ligated side starting on POD 1 after CCI surgery, yielding negative mean difference scores. The difference enlarged on POD 6, and the scores remained stable around 7 s during the following days in control group. No significant difference was found between group L2 and control group on all the days. In contrast, infusion of 6.5, 15 and 35 mg/kg lidocaine significantly reversed the thermal hyperalgesia beginning the first

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day after intrathecal therapy (PTD 1). The elevation of PWL on the ligated side in group L6.5 was modest, and the difference between the left side and the right side returned to the pre-therapeutic value 3 days after injection. Higher doses of 15 and 35 mg/kg lidocaine were able to turn the difference scores to positive values. In other words, the PWL on the ligated side was even longer than the normal side after treatment in these two groups. Lidocaine (15 mg/kg) continued to elevate the PWL of the ligated side above that of the normal side for 4 days, and the reversal of hyperalgesia remained for 8 days. The therapeutic effect was even greater in rats receiving 35 mg/kg lidocaine, with the PWL of the ligated side above that of the normal side for 7 days. But the persistent effect was no greater in group L35 than group L15. 3.3.2. Von Frey withdrawal threshold There was no significant difference in Von Frey withdrawal threshold among any of the tested groups at pre-lesion baseline. CCI surgery was found to significantly lower the threshold on the ligated side (Fig. 3), indicating that tactile allodynia had developed. The mechanical withdrawal threshold was acutely elevated from 6.70 ± 1.17 and 5.44 ± 0.47 g before therapy to 17.79 ± 3.46 and 16.86 ± 2.28 g on PTD1 for groups L15 and L35, respectively, significantly above the pre-therapeutic levels. Infusion of saline or 2 and 6.5 mg/kg lidocaine, however, did not change the mechanical threshold. The anti-allodynic effects in groups L15 and L35 peaked on PTD 2 and 3, respectively, and both remained for 8 days. Statistical examination revealed no significant difference between these two groups on all the days. The mechanical withdrawal threshold values in the rats receiving 2 and 6.5 mg/kg intrathecal lidocaine were not different from saline-infused animals throughout the same period. 3.4. Histopathologic study No specific histopathologic changes were seen in the spinal cords in rats receiving saline, as shown in Fig. 4A. In contrast, intrathecal lidocaine caused different degrees of lesions in the treated rats.

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In rats receiving 2 mg/kg lidocaine, mild infiltration of inflammatory cells and endoneuronal oedema were found in spinal cord. The lesions were limited to the posterior root and posterior horn. Other areas, such as the anterior horn, were intact. In groups L6.5 and L15, histopathologic changes were similar to those observed in L2 group (data not shown for groups L2 and L6.5, representative picture of group L15 was shown in Fig. 4B). However, rats receiving 35 mg/kg lidocaine presented more prominent lesions in the spinal cords. In addition to inflammation and oedema, severe neurotoxicity, such as destruction of myelin sheaths and axonal structures was frequently seen (Fig. 4C). The lesions were no longer restricted, extending to the anterior horn. In some cases in group L35, red blood cell infiltration was also found in the posterior root. Table 2 shows the incidence of the lesions with each drug dose. 4. Discussion The present study investigated effects of different doses of single intrathecal lidocaine on established thermal hyperalgesia and tactile allodynia caused by chronic constriction injury. In addition to 2, 6.5, 15, and 35 mg/kg, we also studied several doses in between, including 4, 10, and 25 mg/kg. The results showed that the anti-hyperalgesic effect of 4 mg/kg lidocaine was between that of 2 and 6.5 mg/kg, 10 mg/kg between that of 6.5 and 15 mg/kg, and 25 mg/kg between that of 15 and 35 mg/kg, whereas the anti-allodynic effect of 4, 10, and 25 mg/kg was identical to that of 2, 6.5, and 15 mg/kg, respectively (data not shown). These results, together with the present findings, indicate that certain doses of single intrathecal lidocaine can produce long-term alleviation of established thermal hyperalgesia and tactile allodynia. But the effects of lidocaine on the two kinds of pain do not appear to share a common pattern. The degree and persistence of the antihyperalgesic effect is related to the dose of intrathecally administered lidocaine, and the doses of approximately 6.5 mg/kg and higher are effective for reversal of hyperalgesia. Meanwhile, the anti-allodynic effect of the drug demonstrates a ‘‘threshold” value of about 15 mg/kg, and a ‘‘ceiling effect”, as 25 and 35 mg/kg lidocaine exerts no greater magnitude and durability of anti-allodynic

Fig. 3. Effects of intrathecal lidocaine on established tactile allodynia caused by chronic constriction injury (CCI). The Von Frey withdrawal threshold on the ligated side was significantly decreased after CCI surgery. While the threshold was not changed by intrathecal saline or 2 and 6.5 mg/kg lidocaine, it was significantly elevated after injection of 15 and 35 mg/kg lidocaine. The value returned to the level identical to control group both on post-therapeutic day (PTD) 8 in the two groups. Statistical examination revealed no significant difference between 15 and 35 mg/kg groups on all the days. *Indicates significantly different from control saline group on the same day at P < 0.05. Mean values are shown, n = 6–8 in each group. POD = post-operative day.

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Fig. 4. Representative pictures of hematoxylin- and eosin-stained slides in the spinal cords of rats receiving intrathecal saline or lidocaine (original magnification  100). (A) No specific lesion was observed in the spinal cord after 0.5 ml intrathecal saline. (B) Mild infiltration of inflammatory cells and endoneuronal oedema could be seen in the posterior root and posterior horn of the spinal cord after 15 mg/kg intrathecal lidocaine. (C) The lesions in the spinal cord after 35 mg/kg intrathecal lidocaine were extensive. In addition to massive polynuclear infiltration and oedema, diffuse destruction of myelin sheaths and axon could be seen.

Table 2 Injury scores for spinal cords obtained from rats receiving saline or 2–35 mg/kg lidocaine during intrathecal therapy (mean ± SEM) Group

Spinal cord

Control L2 L6.5 L15 L35

0.22 ± 0.11 0.89 ± 0.06 0.89 ± 0.15 1.11 ± 0.06 2.61 ± 0.15

Injury scores for each animal were based on all slices presented in a cross-section. Each slice was assigned an injury score of 0–3, where 0 = normal (no lesions), 1 = mild (lesions were rare, seen in occasional fields), 2 = moderate (lesions could be seen in less than 50% of all fields), and 3 = severe (lesions could be seen in more than 50% of all fields). The injury score for each animal was then calculated as the average score of all slices in the segment. n = 3 in each group.

effect than 15 mg/kg lidocaine. Our study also shows that increasing doses of intrathecal drug are associated with increasing severity of respiratory depression and hemodynamic instability. Especially, the highest dose of intrathecal lidocaine, 35 mg/kg, induces increased mortality, as well as significant spinal lesions, as revealed by histopathologic examinations. It is a common practice to develop spinal anesthesia in rats by intrathecal injection of 2 mg/kg lidocaine (Chaplan et al., 1995). Yamada et al. demonstrated that the dose of lidocaine required to cause TSA in rats ranged from 15.0 to 38.1 mg/kg (Yamada et al., 1997). We therefore tested a dose-range paradigm from 2 to 35 mg/kg in our study, to explore the dose–response of painrelieving effect of intrathecal lidocaine. Subarachnoid anesthetics are known to block the sympathetic system, but the results from the current study indicated that the balance between sympathetic and parasympathetic nervous activities could be well preserved

with the use of vasoconstrictors and breathing machine in most cases. Only with the highest dose of lidocaine (35 mg/kg), hypotension and respiratory depression could not be corrected in some rats, and severe lesions in the spinal cords occurred. The more prominent histopathologic changes observed in group L35 than other groups might have been the consequence of severe arterial hypotension-induced decreased spinal cord blood flow, or might be related to the effects of large amount of vasoconstrictors on the tissue circulation. The possible neurotoxicity of intrathecal lidocaine, as demonstrated by histologic evidence in the present study, raises the possibility that lidocaine induced changes in neurologic function towards stimulation rather than it reversed hyperalgesia and allodynia. In order to discriminate between the effects of intrathecal lidocaine per se and neurological deficits induced by its toxicity, we tested the sensory threshold by means of thermal hind paw withdrawal latency in normal rats before and each day after intrathecal injection of 2–35 mg/kg lidocaine, until the 10th day. Another set of normal rats were sacrificed after recovery from lidocaine injection, and the spinal cords were prepared for histopathologic examination. The results showed that the morphologic changes induced by different doses of intrathecal lidocaine in normal rats were similar to the same dose in the neuropathic rats, and no significant difference in the hind paw withdrawal latency was observed in all the rats at any time points after receiving lidocaine injection as compared with their pre-injection baseline (data not shown). This suggests that intrathecal lidocaine, even at the highest dose given, was not interfering with normal sensorimotor integration. Our results of the pilot study were consistent with the findings by Takenami et al., who reported normal sensory threshold in the rats that exhibited comparable severe histopathologic lesions in the spinal cord after receiving high doses of intrathecal

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lidocaine (Takenami et al., 2004). Therefore, the elevation of thermal withdrawal latency and mechanical withdrawal threshold after intrathecal drug injection in the neuropathic rats in the present study should have been caused by the pain-relieving effect of lidocaine. To our knowledge, this is the first study to demonstrate the detailed dose–response as well as the magnitude and duration of pain-relieving effects of intrathecal lidocaine. In a previous study, Chaplan et al. reported that intrathecal lidocaine failed to attenuate tactile allodynia following spinal nerve ligation (Chaplan et al., 1995). Their result was not contrary to our findings, as they used only 500 lg lidocaine, which equaled approximately 2 mg/kg in 250 g rats. Since no response was elicited by a skin clamp through out the body in animals receiving 15 and 35 mg/kg lidocaine, suggesting that these doses of drugs also caused TSA in rats in the present study, our behavioral result is in agreement with the clinical study by Yokoyama et al., who reported sustained analgesic effects of TSA therapy with lidocaine for the relief of neuropathic pain (Yokoyama et al., 2002). In addition, our results extend this previous study by showing that lower doses of intrathecal lidocaine, which are not sufficient to cause TSA, also induce long-term suppression of hyperalgesia, and that the role of intrathecal lidocaine in providing relief of allodynia has a ‘‘ceiling effect”, which means higher doses of intrathecal lidocaine do not result in any greater anti-allodynic effects once the dose reaches the TSA level. Ma et al. (2003) demonstrated in their report, however, that ongoing allodynia was significantly reversed at 2 h as well as 3 days after intrathecal injection of only 100–300 lg lidocaine in partial spinal nerve ligation rats. The inconsistency between their study and ours may be caused by the differences in injury models. At the same time, it is necessary to point out that examination of the effects of intrathecal lidocaine was started 24 h after infusion in the current study. An acute anti-nociceptive effect of lower doses of intrathecal lidocaine thus can not be excluded based on the present findings. The mechanisms by which intrathecal lidocaine reduces thermal hyperalgesia and tactile allodynia are yet to be understood. While the suppression of spontaneous ectopic discharges on the peripheral injured nerve is not likely involved, it is more possible that intrathecal lidocaine exerts its effect through a central suppression effect. Actually, several previous studies have demonstrated a central action of lidocaine. Jaffe et al. found that with electrophysiological examinations, the electrical responses in spinal cord were suppressed during perfusion with lidocaine (Jaffe and Rowe, 1995). Olschewski et al. (1998) showed that lidocaine inhibited spinal neuron activity, likely by blocking sodium and potassium currents evoked in dorsal horn neurons. These evidence support the assumption that the central action plays an important role in the pain-relieving effects of intrathecal lidocaine. Meanwhile, our findings that the effects of lidocaine on hyperalgesia and allodynia are different also lead to speculation that the mechanisms of lidocaine on reversal of these two behavioral manifestations are distinct. Actually, the development of thermal hyperalgesia and tactile allodynia is known to involve separate pathways (Ossipov et al., 2000). While noxious thermal stimuli is thought to be mediated through high-threshold, thin unmyelinated primary afferent C-fibers, non-noxious tactile stimulation is believed to be mediated through large diameter, low threshold Ab afferent fibers, and processed at supraspinal sites receiving input through the dorsal columns (Yeomans et al., 1996; Ossipov et al., 1999; Willis et al., 1999). We are currently using this intrathecal lidocaine therapy in neuropathic pain rat model to investigate the actual effective site (spinal cord or supraspinal site, or both) and means of action of intrathecal lidocaine. In summary, we have shown that doses of 6.5 mg/kg or 15 mg/ kg and higher intrathecal lidocaine could produce prolonged rever-

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sal of established hyperalgesia or allodynia, respectively, but severe lesions in the spinal cord will develop after administration of 35 mg/kg lidocaine. Considering the ratio between useful effects and side effects, doses of 15 mg/kg are suitable for intrathecal injection for the relief of neuropathic pain. The mechanisms of action remain to be elucidated. Although extrapolation to clinical practice must be made with caution, the present finding carries important fundamental and methodological consideration for the basic and clinical study of treatment for neuropathic pain. For the patients who have failed more conservative treatments and with no surgical treatable pathology, intrathecal lidocaine therapy can provide an alternative approach to the treatment of their intractable neuropathic pain. Declaration of interests The work was supported by Doctoral Innovation Program Foundation from Medical School of Shanghai Jiaotong University (BXJ0720), Shanghai, China. Acknowledgement We thank Dr. Xu Zhang and Dr. Lan Bao for their contribution to the design and for showing us the technique of behavioral tests. References Bennett GJ, Xie YK. A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 1988;33:87–107. Chaplan SR, Bach FW, Shafer SL, Yaksh TL. Prolonged alleviation of tactile allodynia by intravenous lidocaine in neuropathic rats. Anesthesiology 1995;83:775–85. Jaffe RA, Rowe MA. Subanesthetic concentrations of lidocaine selectively inhibit a nociceptive response in the isolated rat spinal cord. Pain 1995;60:167–74. Kalso E. Sodium channel blockers in neuropathic pain. Curr Pharm Des 2005;11:3005–11. Ma W, Du W, Eisenach JC. Intrathecal lidocaine reverses tactile allodynia caused by nerve injuries and potentiates the antiallodynic effect of the COX inhibitor ketorolac. Anesthesiology 2003;98:203–8. Mao J, Chen LL. Systemic lidocaine for neuropathic pain relief. Pain 2000;87:7–17. Martin TJ, Eisenach JC. Pharmacology of opioid and nonopioid analgesics in chronic pain states. J Pharmacol Exp Ther 2001;299:811–7. Ness TJ. Evidence for ascending visceral nociceptive information in the dorsal midline and lateral spinal cord. Pain 2000;87:83–8. Olschewski A, Hempelmann G, Vogel W, Safronov BV. Blockade of Na+ and K+ currents by local anesthetics in the dorsal horn neurons of the spinal cord. Anesthesiology 1998;88:172–9. Ossipov MH, Bian D, Malan Jr TP, Lai J, Porreca F. Lack of involvement of capsaicinsensitive primary afferents in nerve-ligation injury induced tactile allodynia in rats. Pain 1999;79:127–33. Ossipov MH, Lai J, Malan Jr TP, Porreca F. Spinal and supraspinal mechanisms of neuropathic pain. Ann NY Acad Sci 2000;909:12–24. Sinnott CJ, Garfield JM, Strichartz GR. Differential efficacy of intravenous lidocaine in alleviating ipsilateral versus contralateral neuropathic pain in the rat. Pain 1999;80:521–31. Smith LJ, Shih A, Miletic G, Miletic V. Continual systemic infusion of lidocaine provides analgesia in an animal model of neuropathic pain. Pain 2002;97:267–73. Størkson RV, Kjørsvik A, Tjølsen A, Hole K. Lumbar catheterization of the spinal subarachnoid space in the rat. J Neurosci Methods 1996;65:167–72. Tabo E, Jinks SL, Eisele Jr JH, Carstens E. Behavioral manifestations of neuropathic pain and mechanical allodynia, and changes in spinal dorsal horn neurons, following L4–L6 dorsal root constriction in rats. Pain 1999;80:503–20. Takenami T, Yagishita S, Nara Y, Hoka S. Intrathecal mepivacaine and prilocaine are less neurotoxic than lidocaine in a rat intrathecal model. Reg Anesth Pain Med 2004;29:446–53. Villetti G, Bergamaschi M, Bassani F, Bolzoni PT, Maiorino M, Pietra C, et al. Antinociceptive activity of the N-methyl-D-aspartate receptor antagonist N-(2Indanyl)-glycinamide hydrochloride (CHF3381) in experimental models of inflammatory and neuropathic pain. J Pharmacol Exp Ther 2003;306:804–14. Willis WD, Al-Chaer ED, Quast MJ, Westlund KN. A visceral pain pathway in the dorsal column of the spinal cord. Proc Natl Acad Sci USA 1999;96:7675–9. Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17:1031–6. Yamada K, Kaga K, Sakata H, Uno A, Tsuzuku T. Auditory evoked responses under total spinal anesthesia in rats. Ann Otol Rhinol Laryngol 1997;106:1087–92. Yamada K, Kaga K, Tsuzuku T, Uno A. Analysis of auditory brain stem response with lidocaine injection into the cerebrospinal fluid in rats. Ann Otol Rhinol Laryngol 1994;103:796–800.

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Please cite this article in press as: Tian J et al., Effects of intrathecal lidocaine on hyperalgesia and allodynia following chronic ..., Eur J Pain (2008), doi:10.1016/j.ejpain.2008.03.013