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PAIN MECHANISMS

Refinement of Symptoms of Neuropathic Pain Measurements After Various Transections of the Nerve Endings of the Sciatic and Femoral Nerve in Rats: An Exploratory Behavioral Analysis

Marie P. Van Remoortere, MD^*, Theo F. Meert, PhD, PhD, Kris C. Vissers, MD, PhD, FIPP, Hans Coppenolle, PhD, and Hugo Adriaensen, MD, PhD^*

From the *Department of Anesthesiology, University hospital of Antwerp, Edegem, Belgium; CNS, Pain and Alzheimer Discovery, Johnson & Johnson Pharmaceutical Research & Development, Beerse, Belgium; Department of Anesthesiology and Palliative Care, UMC St. Radboud, Nijmegen, The Netherlands; and Department of Biometrics & Clinical Informatics, Johnson & Johnson Pharmaceuticals Research & Development, Beerse, Belgium.

Address correspondence and reprint requests to Prof. Dr. K. Vissers, Hoogleraar Palliatieve Zorg, UMC St Radboud, Huispost 550 Anesthesiologie/Palliatieve Zorg, Postbus 9101, 6500 HB Nijmegen Route 548, Netherland. Address e-mail to k.vissers{at}anes.umcn.nl.

Abstract

BACKGROUND: Many animal models can be used to study the underlying pathophysiological mechanisms of neuropathic pain. Most of these models rely on a partial denervation of the limb of the animal by ligating a selected nerve. In this study, we performed nerve lesions on three peripheral nerves supplying the plantar side of the rat hindpaw by differentially transecting the saphenous, the tibial, and the sural nerves alone or in paired combinations.

METHODS: The development of neuropathic pain symptoms at three different anatomical areas (medial, central, and lateral) of the glabrous skin of the hindpaw was evaluated by sensory testing over a 12-wk period. Mechanical hyperalgesia (pinprick), cold allodynia (acetone), and abnormalities of hindpaw posture were continuously present in animals with tibial and tibial and saphenous nerve transection.

RESULTS: Transection of the tibial and sural nerves induced cold allodynia and moderate mechanical hyperalgesia. Transection of the sural, the saphenous, or both nerves simultaneously induced no signs of specific neuropathic pain behavior and no abnormalities in posture of the affected hindpaw were noted after adequate stimulation.

CONCLUSIONS: The overlapping innervation of nerve distribution can complicate the interpretation of nerve ligation studies of peripheral neuropathies.

Peripheral nerve injuries initiate and maintain neuropathic pain (1–4), which is characterized by spontaneous pain, allodynia and hyperalgesia for mechanical, thermal, and chemical stimuli (5–10). The underlying mechanisms of neuropathic pain are not fully understood and the prevention or treatment with drugs remains disappointing (11–14). Several animal models have been developed to study specific pathophysiological processes associated with neuropathic pain. Many of these models involve denervation of the rat hind limb (1–3,15). Mechanical and thermal hyperalgesia together with spontaneous pain have been observed in the chronic constriction injury model of Bennett and Xie (1), the partial sciatic nerve ligation of Seltzer et al., (3) the spinal nerve ligation of Kim and Chung (2), and the transection of different combinations of the sciatic nerve of Lee et al., (1–3,15,16).

In most studies of animal neuropathic pain, the exact location of paw stimulation is not characterized with sufficient precision to permit attribution to specific nerves (15). The saphenous nerve is a branch of the femoral nerve that innervates the medial part of the hind paw. The tibial nerve is a branch of the sciatic nerve that innervates the plantar surface of the hindpaw. The sural nerve is a branch of the sciatic nerve that innervates the lateral aspect of the hind paw (17). Despite these standard definitions, there is uncertainty about the precise boundaries between the areas innervated by the sural, saphenous, and tibial nerves. Swett et al. (18) and Decosterd and Woolf (19) defined them as closely corresponding with the hairline margin of the glabrous skin on the foot, implying that the sural and saphenous nerves do not innervate the plantar side of the paw. However, Decosterd and Woolf (19) concluded the opposite, and demonstrated that the sural and saphenous nerves innervate the lateral and medial aspects respectively of the plantar side of the paw. Other authors have reported that the saphenous nerve innervates the medial area of the plantar side of the paw (20,21).

We explored different combinations of peripheral nerve transection of the saphenous, tibial, and sural nerves. Transection of each nerve individually or a combination of two of the three nerves was performed to explore (1), which model of specific nerve transection will induce a clear neuropathic pain behavior after stimulation of the plantar side of the affected hind paw, and (2) which areas (lateral, medial, and central) of the glabrous skin are sensitized in each of the transection models.

METHODS

Animals
After IRB approval, we studied 84 adult male Sprague–Dawley rats (HSD, Harlan Winkelman GmbH, Borchen, Germany) weighing 250–270 g. For identification, animals were equipped with a subcutaneous chip. The animals were individually housed in ventilated cages under artificial lighting with a fixed 12-h light–dark cycle and with unrestricted access to soya-free food and water.

The rats were randomly divided into the different test conditions. The general physical condition of each animal was monitored regularly and body weight for each animal was followed and reported on day 0 and at 12 weeks after surgery. Guidelines for animal research by the International Association for the Study of Pain were followed (22).

Surgical Procedure for Selective Nerve Transection
Rats were anesthetized using a combination of fentanyl 50 µg/kg/rat subcutaneously (J&J, Beerse, Belgium) and Na-pentobarbital, (Abbott Laboratories, Montreal, Canada) 40 mg/100 mg intraperitoneally. The same person performed all surgical procedures under sterile conditions and did all manipulations. The type of surgery was noted in order to allow correlation with the animal, identified by the subcutaneously implanted chip. The list was sealed and inaccessible for the person performing the behavioral testing. The seal was broken only after all results of the behavioral testing were encoded.

Animals were divided into 7 study groups; each group consisted of 12 animals:

1. Control animals: these rats did not undergo any anesthesia or surgery (controls).
   
2. Transection of the sural nerve (sural).
   
3. Transection of the tibial nerve (tibial).
   
4. Transection of the saphenous nerve (saphenous).
   
5. Transection of the tibial and sural nerves (tibial and sural).
   
6. Transection of the tibial and saphenous nerves (tibial and saphenous).
   
7. Transection of the sural and saphenous nerves (sural and saphenous).
   

Transection of the Specific Branches of the Sciatic Nerve
A skin incision was made from the hip to the knee, 5 mm caudal to the femur in the lateral side of the left hind paw. After blunt dissection of the muscles, the sciatic nerve was exposed. Transverse transection with scissors of the targeted distal branch (sural or tibial nerve) was made 5 mm distal to the trifurcation. No distal excision or translocation of the nerve was performed.

Transection of the Saphenous Nerve
A skin incision was made from the knee to the ankle, dorsal to the course of the saphenous vein and artery, on the medial part of the left. The nerve was found ventral of the vessels and was sharply cut 5 mm proximal of the bifurcation with scissors. The transections were controlled for completeness by microscopic view with a 40x magnification.

After all the different types of transection, the muscles were sutured with Vicryl 6/0 (Ethicon®, Somerville, NJ) and the skin was closed with Vicryl 4/0 (Ethicon®, Somerville, NJ).

Behavioral Evaluation of Neuropathic Pain Symptoms
For behavioral testing, animals were placed in rectangular Plexiglas cages with a wire-mesh grid floor, which allows access to the plantar surface of the paws from below. The rats were allowed to habituate to the test environment for 30 min. Preoperatively, 4 days and 1, 2, 3, 4, 5, 7, 9, and 12 weeks postoperatively, the animals were tested for mechanical hyperalgesia, cold allodynia, and paw posture.

Mechanical Hyperalgesia
To evaluate mechanical hyperalgesia, a pinprick test with a safety pin, as described by Tal and Bennett (23), was performed. The plantar surface of the paw was briefly stimulated at three specific anatomical sites of the with a 5-min interval between stimuli. The animals were stimulated at the medial area (proximal to the toes, medial from the first tarsometatarsal joint), the lateral area (proximal to the toes, lateral from the fifth tarsometatarsal joint) and the central area (under the third tarsometatarsal joint, avoiding the less sensitive pads) (Fig. 1).

View larger version (11K): [in this window] [in a new window]   Figure 1. Drawing of the plantar surface of the hindpaw of the rat. The three areas where a pinprick was performed are marked with numbers: 1 (lateral), 2 (central), and 3 (medial). (Adapted from Devor et al. J Comp. Neurol 1979) (20). Reproduced with the written permission of the authors.

First, the sensory functioning was evaluated for each specific anatomical location by classifying the response to a pinprick as "present" or "absent." The ratio between the number of animals showing reaction and the total number of animals in each group was defined as the response rate and is represented as a percentage. Second, the degree of mechanical hyperalgesia was evaluated by recording the duration (in seconds) of the paw withdrawal with a stopwatch. An arbitrary minimal value of 0.5 s was used when the animals had only a very limited reflex reaction and a cut-off of 20 s was used for extended responding. When no reaction was observed, 0 s was used (23).

Cold Allodynia
To evaluate cold allodynia a droplet of acetone was gently applied to the most proximal side of the plantar surface of the operated paw using a syringe connected to a thin polyethylene tube while the rats were standing on a metal mesh floor (24). The duration of the withdrawal response was recorded with a stopwatch. An arbitrary minimal value of 0.5 s was used when the animals had only a very limited reflex reaction, and a cut-off of 20 s was used for extended responding (19). Hindpaw withdrawal responses associated with continuing pain behavior, with locomotion or body repositioning was not recorded in order to clearly differentiate between continuing and acetone-evoked behavioral responses.

Acetone was applied three times, with an interval of 5 min. For each animal, the total duration of withdrawal of the operated hindpaw after the three acetone applications was cumulated with a maximum of 60 s.

Paw Posture by a Four-Grade Scoring Method
The normal hindpaw posture of intact rats is characterized by a full extension of the toes and a maximal mediolateral spread of the toes away from each other. Animals with a peripheral nerve transection might alter their paw positioning. This was evaluated by placing the animals on a metal runway covered by household paper. The animals were stimulated to walk. While walking, the animals were observed laterally for the posture of the operated paw during at least three consecutive steps. Their paw positioning was evaluated using the following scoring system [adapted from Attal et al. (25): 0 (normal position), 1 (slight ventroflexion of the toes), 2 (hindpaw placement on the medial side), 3 (dorsoflexed hindpaw leaning on the heel)]. For each group, the percentage per score was plotted over time.

Order of Behavioral Testing
The behavioral testing of each animal was standardized: 1) pinprick test, 2) acetone test, 3) paw posture scoring, and 4) body weight measurement when applicable. A person unaware of the specific surgical procedures performed testing.

Statistics
The statistical analysis was performed using SAS software (26).

A generalized linear mixed modeling technique was used to analyze the duration of lifting for the lateral, medial, and central pinprick simultaneously (27,28). Simultaneous analysis of the different pinprick locations permitted modeling the nerve distribution to each location. In model building, a logarithmic link function for the response and a Poisson distribution for the error term were specified. In this way, the duration of lifting was modeled as a function of the fixed factors group, time, place of the pinprick, together with their pairwise two-factor and the three-factor interaction terms.

Dependencies of the response originate from the longitudinal nature of the animal-specific measurements over time, as well as from possible correlations between the duration of lifting for the different places of the pinprick. The dependencies were accounted for by defining a Kronecker product of an autoregressive covariance matrix for the different time points and an unstructured covariance matrix for the place of the pinprick. The use of the autoregressive structure was prompted by convergence issues. Next to common statistical contrasts, the integral of the duration of lifting response as a function of time was used as a measure to test differences among treatment groups for the different locations of the pinprick. The integrals were estimated as specific linear combinations of the model parameter estimates. The resulting area under the curve was considered to be an informative criterion for the total amount of pain. The acetone data were analyzed in a similar manner. Here, the response was modeled as a function of the fixed factors group and time, and their interaction. An autoregressive covariance matrix was specified to define the repeated structure of the data (28,29). A P value of <0.05 was accepted as significant.

To study differences among study groups for body weight, a model was fit with group, time, and its interaction as explanatory variables. The repeated time structure was accounted for by defining an unstructured covariance matrix. Differences among study groups were evaluated based on a test for the fixed effect of group and a test for the interaction between group and time.

RESULTS

Preoperatively, the body weight of the animals in the different treatment groups varied from 258.25 ± 0.74 g (tibial) to 259.92 ± 0.71 g (tibial and saphenous). Twelve weeks later, they ranged from 438.33 ± 3.38 g (tibial and saphenous) to 465.08 ± 3.55 g (sural and saphenous). No statistical differences were found among the experimental groups in relation to increases in bodyweight. Automutilation was not observed in any of the animals over the entire testing period. One animal from the tibial group died immediately after the operation, only preoperative data were noted for this animal.

Mechanical Hyperalgesia (Fig. 2 and Fig. 3)

View larger version (20K): [in this window] [in a new window]   Figure 2. Response rates in % (A) and average duration of lifting in seconds (B–D) after pinprick on different areas of the plantar side of the paw. Each treatment group is compared to the control group per time point in relation to previous measurements. Significant differences are noted with * (*: P < 0.05, **: P < 0.01, ***: P < 0.001).

View larger version (23K): [in this window] [in a new window]   Figure 3. Response rates in % (A) and average duration of lifting in seconds (B–D) after pinprick on different areas of the plantar side of the paw. Each treatment group is compared to the control group per time point in relation to previous measurements. Significant differences are noted with * (*: P < 0.05, **: P < 0.01, ***: P < 0.001).

Sensory Functioning
In control animals (n = 12), the response rate to the pinprick was 100% over the entire surface of the foot sole for the entire evaluation period as tested by stimulation of the medial, lateral and central area, except on the fourth day for the medial part, and during the third week for the lateral part where a response rate of 91.7% was found (data not shown).

There was no sensory deficit after transection of the sural (n = 12), saphenous (n = 12) and sural and saphenous (n = 12) nerves (Fig. 2A). Transection of the tibial nerve caused no sensory deficit on the lateral area, only a moderate deficit on the medial area during the first 4 wk postoperatively, but induced a clear hypoalgesia in the central area until the third week postoperatively (Fig. 3A). Transection of the tibial and sural nerves caused a sensory deficit in the medial and lateral area during the first three weeks and in the central area for a period of five weeks (Fig. 3A). After transection of the tibial and saphenous nerves, the sensory deficit was most pronounced in the medial and central areas during the first five weeks postoperatively and absent in the lateral area (Fig. 3A).

Presence of Hyperalgesia
The mean duration of lifting after pinprick for each group for the lateral, medial and the central area of the foot sole was compared to the control group. For the sural and sural & saphenous groups, no significant differences were found compared to the control group (Figs. 2B–D). For the saphenous group a significantly longer duration of lifting was found on the second week as compared to controls, after a central pinprick. For all other timepoints in the lateral and medial part of the foot sole, no significant differences were present (Fig. 2B–D). For the tibial group a significantly longer duration of lifting was measured after a central pinprick from the third until the seventh week (P < 0.02), after a lateral pinprick from the fourth postoperative day until the third week (P < 0.02) and on the seventh and the 12th week (P < 0.03) and after a medial pinprick on the first, the third and the ninth week (P < 0.04) (Fig. 4B–D).

View larger version (18K): [in this window] [in a new window]   Figure 4. Average duration of lifting after 3 acetone applications on the affected hindpaw per treatment group. Each group is compared to the control group per time point in relation to previous measurements. Significant differences are noted with * (*: P < 0.05, **: P < 0.01, ***: P < 0.001).

For the tibial and sural group a significantly higher lifting behavior was found on the third (P = 0.04) and the seventh week (P < 0.008) after a central pinprick, on the ninth (P < 0.004) and the 12th week (P < 0.02) after a lateral pinprick and on the fifth (P < 0.01) and the ninth week (P < 0.03) after a medial pinprick (Fig. 4B–D).

For the tibial and saphenous group a significantly longer duration of lifting after a central pinprick was found from the third until the seventh week (P < 0.05), after a lateral pinprick between the second and the seventh week (P < 0.05) and after a medial pinprick between the second and the fifth week (P < 0.05) (Fig. 3B–D).

The proportion of the measured durations that exceeded the cut-off was 4.5%. The proportion of censored observations by study group was highest for the tibial and saphenous (11%) and for the tibial (10%) study group. The occurrence of censoring makes the analysis rather conservative for the detection of pain sensitivity, but its low proportion justified the use of the proposed statistical techniques.

To define the most sensitive area of the sole of the foot for each test condition, the area under the curve of the duration of lifting was calculated for the lateral, medial, and central area and the groups were statistically compared with each other. In the control, sural, tibial, and tibial and saphenous groups, all areas showed the same sensitivity to a pinprick in the lateral, medial, and central area of the sole of the foot. In the saphenous group, the central and the medial part were significantly more sensitive to pinprick compared to the lateral part (P < 0.001). In the sural and saphenous group, the central part of the foot was more sensitive to pinprick compared to the lateral part (P = 0.03), but not to the medial part. In the tibial and sural group, the medial part was more sensitive to pinprick compared to the central (P = 0.005) and the lateral part (P < 0.001).

Cold Allodynia (Fig. 4)
In the control group, a slight but significant sensitization over time was measured. (Preoperative data compared to data of the 12th week, P < 0.001) In the sural and the sural and saphenous groups, a significant difference from the controls was observed on the seventh postoperative week in the duration of lifting compared to the control group (P < 0.007 and P < 0.05 respectively) (Figs. 4A and C). In the saphenous group no differences were seen from the control group (Fig. 4B).

In the tibial and the tibial and saphenous groups, an increase in cold allodynia was measured from the fourth postoperative day up to the ninth week (P < 0.002 for both groups), except for the fifth week. Peak durations of lifting were situated in the third week (19.1 ± 4.9 s for the tibial and 26.8 ± 6.5 s for the tibial and saphenous group) (Fig. 4C and E). In the tibial and sural group, a difference from the control group was found from the fourth postoperative day until the 12th week (P < 0.03), with peak duration of lifting during week 3 (35.9 ± 5.5 s) (Fig. 4D).

Paw Posture
As compared to the controls, animals in the sural, the sural and saphenous, and the saphenous group did not show abnormalities in positioning of their affected paw (Fig. 5). The hindpaws of the other three test conditions showed clear abnormalities: the paw was averted and the toes were held together and ventroflexed. In the tibial group, the period of peak deformity was observed on the second postoperative week when 25% of the animals had score 3 and 75% score 2 and on the third week postoperatively with 50% of the rats with a score of 3, 41.7% with a score of 2, and 8.3% with a score 1 (Fig. 5). The animals of the tibial and saphenous group had the highest deviating scores on the third week postoperatively, with 50% of the animals with a score 3, 41.7% having a score 2, and 8.3% with a score 1 (Fig. 5). In the tibial and sural group, the highest score was seen on the third postoperative week with 25% of the animals with score 3, 66.7% with score 2, and 8.3% with score 1 (Fig. 5).

View larger version (15K): [in this window] [in a new window]   Figure 5. Posture of the denervated foot: percentage of animals per score per treatment group over time. With 0 (normal position), 1 (slight ventroflexion of the toes), 2 (hindpaw placement on the medial side), 3 (dorsoflexed hindpaw leaning on the heel).

DISCUSSIONS

This study demonstrates that the transection of a combination of the tibial and the tibial and saphenous nerves resulted in a clear and stable tactile and thermal hyperalgesia to pinprick and to an acetone droplet test respectively. Furthermore, the combined transection of the tibial and the sural nerve results in a less pronounced development of mechanical hyperalgesia. These data are in contrast with the results reported by Lee et al. (15) and Dowdall et al. (16) who showed a clear and stable development of mechanical hyperalgesia for this group. There are some differences between both studies: in the Lee et al. study (1) the different nerves were ligated and transected, which prevented regeneration (2); a transection of the saphenous nerve was not included, and (3) mechanical allodynia was evaluated using von Frey hairs, which is in contrast with the pinprick test in our study. In a study by De Koning et al., (30) the tibial and saphenous nerves were transected, resulting in an absence of sensory function in the central area with a locally applied electrical current even after 20 days. Such a current is also used in other experiments (31–33) to evaluate the return of sensory function, but this type of stimulus completely bypasses peripheral receptors, stimulating nerve fibers directly with electrical thresholds related to their diameters (Aß < A < C), (34) whereas a pinprick causes a stimulation of superficial cutaneous nociceptors of A-fibers (35).

In this study, the transection of the saphenous nerve induced no characteristic presentation of neuropathic pain behavior. In the study by Walczak et al., (36) partial ligation of the saphenous nerve showed a clear cold and mechanical allodynia, thermal hyperalgesia and, to a lesser extent, mechanical hyperalgesia. These remarkable differences may have been caused by a specific inflammation due to the ligature.

Our conclusions regarding the exact location of sensory testing on the affected hindpaw are as follows: 1) the sensory deficit in the central area on the fourth postoperative day was much more pronounced in the tibial and sural group compared with the tibial and the tibial and saphenous groups and was absent in the sural and the sural and saphenous group and 2) the sensory deficit in the lateral area was present in the tibial and sural group but absent in the tibial group and in the sural group. These data suggest overlapping innervation between the tibial and sural nerves in the lateral and even the central area. Because collateral sprouting from uninjured nerves into a denervated area starts from the fourth postoperative day, the sensory deficit on the fourth postoperative day probably underestimates the initial effect of the partial denervation (20).

The sensory deficit in the medial area of the hindpaw sole in the tibial and saphenous group was much more pronounced compared with the tibial and the tibial and sural group and was absent in the saphenous group, confirming overlapping innervation between the tibial and the saphenous nerves (21,29). Consequently, after transection of the tibial nerve, the plantar side remains partially innervated by the sural and the saphenous nerves on the lateral and central area and on the medial area respectively. Mechanical hyperalgesia was present in both the injured as well as in the uninjured areas, which is in accordance with other studies (23,37).

The measurement of the tibial group can be compared to that in the Decosterd and Woolf study (19) in which the tibial and peroneus nerves were ligated and transected and animals were also tested behaviorally to detect neuropathic pain symptoms. As the peroneal nerve innervates the central dorsum of the paw and the hyperalgesia to pinprick and allodynia for acetone were tested on the lateral area of the plantar side of the paw, similar results from the tibial group in our study were observed (38). Indeed, in the Decosterd and Woolf study, hyperalgesia to pinprick and allodynia to acetone on the lateral plantar area developed early after operation, and lasted for 9 weeks comparable to our results in the tibial group, but after the third week a decrease in mechanical hyperalgesia was observed where after only borderline significant differences compared to the control group were found until the 12th week (19). Additionally, Decosterd and Woolf (19) found that the medial area was less sensitive to mechanical allodynia (as tested with von Frey hairs) compared to the lateral area. We made a comparable observation regarding the development of mechanical hyperalgesia. Although the area under the curve for the duration of lifting after pinprick in the tibial group was not significantly different for the three anatomical areas of the plantar paw, the intensity of mechanical hyperalgesia on the medial part was less pronounced than on the lateral part. Therefore, this study confirms the hypothesis of Decosterd and Woolf (19) that in the saphenous nerve (innervating the medial part), there is minimal co-mingling of injured and uninjured cell bodies in the same dorsal root ganglion (DRG), whereas in the sural nerve (innervating the lateral part) a considerable co-mingling is present. The tibial, peroneus, and sural nerves, all branches of the sciatic nerve, enter the spinal cord via the fourth, fifth, and sixth DRG whereas the saphenous nerve, a branch of the femoral nerve, enters the spinal cord via the second and the third DRG (38).

Except for the lateral area in the tibial group, where mechanical hyperalgesia was present on the first postoperative day, hyperalgesia to pinprick in the different groups developed more gradually over time compared to other pain models (3,19). This can possibly be explained by the fact that no epineurial inflammatory reaction due to foreign material was present after simply transecting the nerves (39). But even in the absence of this foreign material, Watkins and Maier (4) demonstrated that manipulation of nerves always induces neurogenic inflammation.

Behavioral tests were started only on the fourth postoperative day, so we do not know if the hyperalgesia in the tibial group was present immediately after operation. In the transection models, cold allodynia developed sooner and lasted longer than mechanical hyperalgesia. From the fourth postoperative day, cold allodynia was present for at least 9 weeks, after which a decrease towards control levels was seen on the 12th week. These data are in accordance with the findings of others (40).

A hypersensitivity to pinprick was only present for a maximum four or five consecutive weeks after which a decrease towards control values occurred. This was probably due to spontaneous regeneration that can occur in transected nerves as in this study (32,41–44). It was also mentioned that the persistence of behavioral changes requires that the injured neurons do not regenerate their peripheral targets (19).

Nine weeks after ligation, mechanical hyperalgesia was present on the lateral aspect in the tibial and sural group, whereas mechanical hyperalgesia on the medial part in the tibial and saphenous group was already present after the second postoperative week. This difference can be explained by the more pronounced collateral sprouting of the sural nerve into the injured area than of the saphenous nerve. Since the sural nerve showed a sensory overlap with the tibial nerve on the lateral and the central part, it is more likely that sprouts of the sural nerve reached the medial part in the tibial and saphenous group sooner, already leading to the development of mechanical hyperalgesia after the second postoperative week.

The abnormalities in the hindpaw posture seen in this study were similar to those seen after a chronic constriction injury (1,25). Although we observed parallel evolution in the severity of paw posture abnormalities and the symptoms of cold allodynia, changes in hindpaw posture after a peripheral nerve lesion are due to both sensory and motor abnormalities (45). Paw posture did not return to normal over the 3 month observation period in this study.

We conclude that the transection of the tibial nerve alone, or in combination with the sural or the saphenous nerve, induces the development of neuropathic pain behavior characterized by the presence of mechanical hyperalgesia and cold allodynia. Using the tibial nerve transection as a model to study neuropathic pain, mechanical hyperalgesia should be tested laterally or centrally. The advantage of this model is that only one incision is needed and only one nerve has to be cut, allowing better standardization. Moreover, mechanical hyperalgesia develops immediately after the operation on the lateral part of the paw. Another study showed the development of pronounced mechanical allodynia and, to a lesser extent, hyperalgesia with the hotplate test (46). The moderate sensory deficit in this treatment group was probably due to overlapping of the innervation between the tibial, the sural, and the saphenous nerves.

After transection of the tibial and saphenous nerves, behavioral testing for mechanical hyperalgesia can be done over the whole hindpaw sole. A disadvantage of this model is that two incisions have to be made. After transection of the tibial and sural nerves, the development of allodynia for a cold and chemical stimulus is more pronounced than hyperalgesia to a mechanical stimulus.

Future research using immunochemistry and electrophysiological techniques can further define the roles of regeneration, overlapping innervation, and collateral sprouting in the induction and maintenance of neuropathic pain.

ACKNOWLEDGMENTS

The authors thank H. Borghys and E. Van Eynde for the technical assistance during surgery, H. Coppenolle and L. Bijnens for the statistical support, L. Leijssen for the assistance with the figures. This work is supported by a grant of COSAT (J&J).

Footnotes

Accepted for publication January 4, 2007.

Supported by COSAT (J&J).

This work is completely attributed to Johnson & Johnson, Pharmaceutical Research & Development, CNS, Pain and Alzheimer. Turnhoutsebaan, 30, B-2340 Beerse.

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