Neuropathic pain is common after surgery1 and traumatic injury.2 Once established, chronic pain in general, and chronic neuropathic pain specifically, are difficult to treat, and the treatments that are available for neuropathic pain have limited efficacy. The antiepileptic drugs gabapentin and pregabalin are often included in the treatment of neuropathic pain and have been shown in human studies to provide analgesia.3–5 Furthermore, a recent report suggests that these drugs can reduce or prevent chronic postsurgical pain if administered perioperatively.6 While effective, both gabapentin and pregabalin use are often accompanied by serious side effects, sometimes sufficient to contraindicate continued treatment.7 Although the mechanism of action of these 2 drugs is not completely understood, both drugs appear to interact with the voltage gated calcium channel α(2)δ-1 (VaCα2δ-1) subunits in the central nervous system.5,8–20
Chronic neuropathic pain seems to be maintained, if not both caused and maintained, by peripheral generators.21 Therefore, if the peripheral generator can be silenced or reduced early in the pain event, chronic neuropathic pain may be reduced or never initiated. Only a few previous studies have investigated peripheral effects of gabapentin or pregabalin.22–24 Two of these22,23 investigated the effects on neuropathic pain models, but both applied the pregabalin to the peripheral site to which the pain was referred. In this study, we investigated the analgesic effect of the immediate postinjury application of perineural pregabalin directly to the injured nerve, sustained for 7 days in a rat sciatic nerve neuropathic pain model, and compared it with systemic pregabalin. Our hypothesis was that perineural pregabalin would provide greater analgesic effect than systemic pregabalin.
This study was approved by and performed in accordance with our local institutional animal care and use committee. Forty genetically identical male Sprague Dawley rats (Harlan, Indianapolis) 8 to 9 weeks old were used in these experiments. Rats were acclimated for at least 24 hours on a 12-hour light/dark cycle with food and water available ad libitum. Rats were randomized into 4 study groups: sciatic nerve crush injury with perineural pregabalin infusion (treatment, n = 10), sciatic crush injury with perineural saline infusion (saline control, n = 10), sciatic nerve crush injury with subcutaneous pregabalin infusion (systemic drug control, n = 10), and a sham surgery group (sham surgery, n = 10). No animals received any additional analgesic medication other than the study drug.
Sciatic Nerve Crush Injury
Nerve crush injury was performed on the perineural pregabalin treatment group, the systemic drug control group, and the saline control group. The sham surgery group and the pregabalin toxicity testing group (see later) did not have nerve crush.
Sciatic nerve crush injury was performed as previously described.25 Briefly, general anesthesia was induced and maintained using 2% to 3% isoflurane. The left leg was shaved and aseptically prepped with 2% chlorhexidine gluconate and 70% isopropyl alcohol (ChloraPrep, Carefusion). A 1-cm incision was made over the lateral aspect of the rat’s left hindlimb at the mid-femur. The sciatic nerve was exposed via blunt dissection between the vastus lateralis and biceps femoris muscles. For animals in the crush injury groups, a small locking jeweler’s forceps was applied to the sciatic nerve 0.5 cm proximal to the trifurcation of the sciatic nerve. The locking forceps was engaged on the nerve for 15 seconds and then removed.
Osmotic Pump Implantation and Loading
A 2-cm horizontal incision was made at the scruff of the back at the level of the scapula. A 2 × 2 cm subcutaneous space was created using blunt dissection. A 2-mL osmotic micropump (Alzet, Durect Corp. Cupertino, CA) filled with either 0.9% saline (sham surgery and saline control groups) or 1% pregabalin in normal saline (treatment group and systemic drug control group) was implanted into the subcutaneous space. Pumps delivered approximately 10 mg/kg of pregabalin per day. This dose, when given systemically, causes analgesia in a rat neuropathic pain model without signs of lethargy or other side effects of pregabalin.26 In the treatment and saline control rats, the pump’s micro-bore catheter was tunneled through the subcutaneous tissue of the back and the left gluteal muscle. The terminal end of the catheter was positioned alongside the sciatic nerve proximal to the sciatic nerve injury site. The catheter was secured in place using 4-0 silk suture attached to subcutaneous tissue and fascia at the gluteal muscle. The muscle layer overlying the sciatic nerve was reapproximated using 4-0 silk sutures, and the pump was secured to subcutaneous tissue using the same sutures. In the systemic drug control group, the catheter was placed as described above but was then severed at 2 separate locations, one near its attachment to the pump and the second just proximal to the entry to the gluteal muscle to ensure subcutaneous delivery of the pregabalin distant from the sciatic nerve. Thus, accumulation of systemic pregabalin should not be different between these 2 groups of rats, but the concentration of pregabalin should be much higher near the injured nerve in the treatment group. In the sham surgery group, the sciatic nerve was exposed, and the catheter and pump were placed as discussed above; however, no crush injury was applied to the sciatic nerve. To establish a drug level immediately after nerve injury in the treatment group, the sciatic nerve was bathed in 0.3 mL (volume approximately equivalent to 1 day of pump solution delivery) of 1% pregabalin solution. The saline control group was similarly bathed with 0.3 mL saline solution before closure of the muscle layer overlying the sciatic nerve. Rats in the systemic drug control group received a subcutaneous injection of 0.3 mL of 1% pregabalin distant to the sciatic nerve before completion of surgery. Hemostasis was confirmed, and skin incisions were closed using 4-0 silk sutures and dermabond. After surgery, the animals were allowed to recover and housed 1 rat per cage with food and water available ad libitum. Postoperatively, no signs of motor impairment were observed.
Pure pregabalin in powder form was obtained through Pfizer as a gift. The purified pregabalin dissolved readily in 0.9% saline to yield a concentration of 1% pregabalin solution. The osmotic micropump was filled with 2 mL solution (saline or pregabalin) and allowed to prime the micro-bore catheter for 24 hours before surgery by placing the pump in 0.9% saline bath at room temperature. The pumps were capable of delivering a continuous steady infusion rate of 10 μL/h over 7 days. Before the initiation of the study, pump function and perineural delivery were confirmed in vivo by infusing methylene blue via the implanted osmotic pump to the sciatic nerve in 3 animals. After 7 days, the rats were euthanized, and sciatic nerves examined showing circumferential staining with methylene blue to the sciatic nerve and surrounding tissue in all animals. Pump function was evaluated at the conclusion of the study by aspirating the volume of solution remaining in the pump after removal of the pumps from euthanized animals.
The investigator performing behavioral testing was blinded to the rats’ assigned groups. Before surgery, and 1, 2, 4, and 7 days after surgery, behavioral responses were measured using Hargreaves’ radiant heat withdrawal method,27 guarding behavior,28 and incapacitance meter.29 Animals were acclimated to each of the testing environments once daily for a week before beginning testing. Animals were allowed to acclimate to each testing environment for approximately 30 minutes before initiation of testing on each testing day.
The radiant heat withdrawal latency testing (Hargreaves) was performed using a device specifically designed for this purpose (IITC Life Science, Woodland Hills, CA). The rats were placed on the glass surface and allowed to acclimate. The heat lamp was then positioned under the plantar aspect of the hindpaw and the withdrawal latency measured in seconds, with a maximum time of 15 seconds. The animal was allowed to rest for 5 minutes and the test repeated for a total of 3 measurements.
The guarding behavioral assay consisted of a wire mesh stand (IITC Life Science, Woodland Hills, CA) with holes measuring approximately 6 x 6 mm. The rats were placed atop the wire mesh stand and covered with a clear plastic cage. The guarding score testing was done for 60 minutes per session. At 5-minute intervals, the amount of pressure placed on each hindpaw was observed, and a numerical value of 0, 1, or 2 was assigned. A score of zero indicated full pressure on the hindpaw causing blanching of the skin on the plantar surface of the foot. A score of 1 indicated light pressure on the hindpaw but not enough to cause blanching of the plantar surface of the foot. A score of 2 denoted no contact between the plantar surface of the foot and the wire mesh. Twelve data points were collected on both the ipsilateral and contralateral hindpaws on each day of testing. The animals received a score based on the sum of all scores over the 60-minute period with a maximum score of 24.
Incapacitance testing consisted of placing the animal in an incapacitance meter (IITC Life Science, IITC Life Science, Woodland Hills, CA) with an incline shelf that forced the animal to stand on its hindpaws. Underneath each hindpaw was an independent pressure plate. Care was taken to ensure that the animal’s ipsilateral and contralateral hindpaws were placed completely on their respective pressure plates. Also, care was taken to ensure that the animal was not leaning on the sides of the cage and was completely upright on the pressure plates. The incapacitance meter measured the amount of weight (g) on each plate averaged over 5 seconds. Five separate weight readings were taken from both the ipsilateral and contralateral plates with at least 1-minute time gap between each reading. The readings of each foot were then averaged, and the incapacitance score was reported as a percentage of total weight applied to the injured (left) hindpaw based on the following formula: incapacitance score = left/(left + right) × 100.
Histology and Immunohistochemistry
On postoperative day 7, after completion of behavioral testing, the animals were again anesthetized using 2% to 3% isoflurane. The ipsilateral and contralateral sciatic nerves of each rat were harvested from distal to the trifurcation to as far proximal as possible close to the sciatic notch. Samples were either immediately fixed in formalin for histology or frozen in medium using a snap-freeze protocol for immunohistochemistry. All samples undergoing histopathology were sectioned and stained using standard hematoxylin and eosin staining protocol and were read by a board-certified pathologist. Immunohistochemistry samples were prepared using mouse anti-CACNA2D1 (Abcam, catalog #ab2864) in a 1:50 concentration and Rabbit anti-Mouse IgG (Abcam, catalog #ab98784) in a 1:500 concentration. Preabsorption controls were performed omitting the primary antibody. These controls had some background labeling in the perineural connective tissues in all samples but only faint background labeling within the nerve. Samples were interpreted by a board-certified pathologist.
Ten animals were randomized into 2 groups of 5 animals each. One group had an osmotic pump filled with saline and perineural catheter implanted as above without injury to the nerve. The other group had an osmotic pump filled with 1% pregabalin solution and perineural catheter implanted as described above also without injury to the nerve. After 7 days, the sciatic nerves were harvested and fixed in formalin and then sectioned and stained with hematoxylin and eosin. Samples were evaluated by a pathologist for potential neurotoxicity from pregabalin.
The power analysis to determine sample size was computed before the initiation of the study. The power analysis determined that for a power of 0.80, the sample size would have to be slightly >8 for each group for the main effect of group for an effect size of 0.46 (mean difference) and standard deviations (SDs) of 0.9. For the main effect of time, a similar effect and SD size would be achieved with this same sample size. We increased the sample size to 10 for each group to account for any subject loss during the experimental procedures. Post hoc analysis of our data showed the effect sizes for 1-way analysis of variance (ANOVA) on each day were ≥0.69, and the observed power for incapacitance meter on each of the days closely approximated 1.0, although this is likely overestimated due to very small P values. Confidence intervals (CIs) are included for results that did not reach statistical significance. Based on this information, it appears that the study was adequately powered.
Analysis of the data was performed using SPSS 17 (IBM, Armonk, NY). Overall group, time, and group × time interactions were assessed using 4 (group) × 5 (time) repeated-measures ANOVA. Because the data are not normally distributed and variance is not equal among groups, significance was set at a more stringent α of 0.01. When significant group effects or group × time interactions were noted, further analysis of group differences across time as well as group differences at each time point was done using ANOVA. Post hoc Student t tests with unequal variances were used to determine significant differences among groups. Bonferroni adjustment for multiple comparisons (n = 6) resulted in an adjusted α of 0.002 (0.01/6).
There were no differences in the weight of the animals at the beginning or at the end of the study. Average remaining volume in the pumps was 0.17 ± 0.07 mL at the conclusion of the study, and there were no significant differences in residual pump volume among groups (P = 0.42). The average daily dose of pregabalin was 10.13 mg/kg/d. All perineural catheter tips remained in close proximity to the sciatic nerve as observed during nerve harvesting at the conclusion of the study.
All rats underwent baseline nociceptive behavioral testing before surgery (day 0). There were no significant differences among any of the test groups for any of the behavior tests on day 0. Subsequently, all rats again underwent behavioral testing using the tests described on postoperative days 1, 2, 4, and 7. For guarding and incapacitance, 4 × 5 RM ANOVA indicated significant time, group, and group × time interaction effects (P < 0.0001), and further analyses were performed and results described below. For Hargreaves results, the overall 4 × 5 RM ANOVA showed a significant time effect (P = 0.005), but no significant effects for group (P = 0.15) or group × time interaction (P = 0.07).
Guarding Behavior Caused by Nerve Crush Was Greatly Reduced at All Times by Perineural Pregabalin Treatment but Was Only Marginally Improved by Systemic Pregabalin Treatment
Analyses of the overall group effect across all postoperative time points indicated that the sham surgery group (nerve exposure, but not nerve crush, saline osmotic pump) showed significantly lower guarding scores than all other groups (P < 0.0001) (Fig. 1 open squares). The perineural pregabalin treatment group (Fig. 1, filled triangles) had significantly lower guarding scores than the systemic drug control group (Fig. 1, filled squares) (P < 0.0001). The perineural pregabalin treatment group also had lower guarding scores than the saline control group (Fig. 1, filled diamonds) (P < 0.0001). The saline control group had marginally significantly higher guarding scores compared with that of the systemic control group (P = 0.018, 99% CI, 0.3–4.1) (Fig. 1, compare triangles with filled diamonds). When comparing between-group effects by day, ANOVAs indicated significant group differences at each postoperative time point (P < 0.0001). Post hoc analyses (required adjusted α = P≤ 0.002) determined that the perineural pregabalin treatment group scores were significantly lower than both the systemic drug control (P < 0.0001) and the saline control (P < 0.0001) groups on all days. The systemic drug control group had marginally significantly lower scores than the Saline Control group on day 2 (P = 0.007) and significantly lower scores on day 4 (P = 0.001), but not on day 1 (P = 0.74, 99% CI, −4.4 to 1.2) or day 7 (P = 0.32, 99% CI, −6.4 to 1.0).
Perineural Pregabalin Treatment Prevented Pain Behavior at All Times After Nerve Injury While Systemic Pregabalin Only Reduced Pain Behavior Caused by Nerve Crush as Measured by the Incapacitance Meter
Analyses of the overall group effect across all postoperative time points indicated that the sham surgery group (no nerve injury) (Fig. 2, open squares) had significantly higher incapacitance scores compared with that of systemic drug control (P < 0.0001) (Fig. 2, filled squares) and the saline control groups (P <0.0001) (Fig. 2, filled diamonds). There was no significant difference between the sham surgery group and the treatment group (Fig. 2, filled triangles) (P = 0.42, 99% CI, −0.8 to 3.7). The treatment group had significantly higher incapacitance scores compared with that of systemic drug control (P = 0.001) and saline control (P < 0.0001) groups. The systemic drug control group had significantly higher incapacitance scores compared with that of the saline control group (P < 0.0001). When comparing between-group effects by day, ANOVAs indicated significant group differences at each postoperative time point (P < 0.0001). Post hoc analyses determined that treatment group scores were not significantly different than the sham surgery group on any day (P ≥ 0.24). There were marginally significant differences between the treatment and systemic control groups on days 1, 2, and 4 (P = 0.007, 99% CI, 1.2–8.2; P = 0.018, 99% CI, 0.6–8.2; and P = 0.004, 99% CI, 1.4–8.2, respectively), but no difference on day 7 (P = 0.87, 99% CI, −4.7 to 5.3). Treatment group and systemic drug control scores were significantly different than the saline control group on all postoperative days (P < 0.0001 and P ≤ 0.002, respectively).
Hargreaves Heat Lamp Test Indicated Hypoalgesia in All Nerve Crush Injured Rats on the Plantar Surface of the Foot Regardless of Treatment
A 3 (group) × 5 (time) RM ANOVA showed no difference between the saline control, systemic drug control, or treatment groups in Hargreaves test (P = 0.80), with injured animals showing hypoalgesia on postoperative days 1 to 4 and then returned close to normal on postoperative day 7.
All sciatic nerve specimens that underwent left sciatic nerve crush injury showed significant axonal degeneration and necrosis with proliferation of Schwann cells and an influx of macrophages (Figs. 4 and 5, B–D in both). Nerve damage was typically moderate (mean injury severity score 2.8 ± 0.1, Table 1) and located near the sciatic trifurcation end along a 3- to 5-mm zone. The damaged nerve was characterized by eosinophilic debris within axons across most of the width of the nerve (Fig. 4, B–D with details in inset in Fig. 3C). Histiocytes were observed phagocytizing this debris, and there were proliferating Schwann cells often arranged in bands of Bungner (details in inset of Fig. 4C). There were likely proliferating fibroblasts in the damaged area as well. All left-sided (ipsilateral) specimens showed similar levels of perineural inflammation (injured and noninjured nerves). The sciatic nerve sections of injured groups appeared relatively normal a short distance away from the injury site (Fig. 5, B–D). Specimens from the contralateral sides of all rats were normal and did not show signs of axonal degeneration or necrosis.
Nerve specimens that received a 7-day infusion of pregabalin without nerve injury showed perineural inflammation with no abnormalities within the nerve. The perineural inflammation was similar to that seen in the sham surgery group (data not shown).
In the sham surgery group, there was minimal to mild neutrophilic and histiocytic inflammation in the perineural connective tissue and fat. Nerve damage was absent in all but 1 sample where it was graded as minimal. VaCα2δ-1 expression was very low (minimal-grade 1) in these nerve samples (Fig. 4A).
There was similar VaCα2δ-1 expression (Figs. 4 and 5, brown stain) between the groups that received the nerve crush (treatment, systemic drug control, or saline control groups; Fig. 4, B–D). VaCα2δ-1 expression was moderate to marked (mean score 3.9 ± 0.1) (Table 2, Fig. 3) in the injured region of all nerve samples where nerve crush was done. This increased expression was observed only at the injury site, not at other locations without apparent histologic nerve damage proximal to the injury site (Fig. 5, B–D). The increased expression was observed in numerous cell types within the nerve proper, including potential Schwann cells, fibroblasts, macrophages, smooth muscle cells, and endothelial cells of capillaries and small blood vessels (details in Fig. 4C). Axonal expression of VaCα2δ-1 was not an obvious feature in the sections. Proximal to the region of nerve damage the rest of the nerve appeared normal histologically with no increase in VaCα2δ-1 expression (Fig. 5, compare A with B, C, D).
In this study, we demonstrated that pregabalin applied to the site of an injured sciatic nerve soon after injury can inhibit the development and severity of neuropathic pain. Continued administration reduced pain for at least 7 days (the last day sampled here). We also demonstrated that the pain relief caused by perineural pregabalin was more profound than equal doses of pregabalin administered systemically. These findings suggest that peripherally targeted pregabalin may be an effective means of reducing acute pain caused by nerve injury and could be useful for preventing long-term pain if administered early in acute neuropathic pain caused by traumatic injuries. These findings also suggest that the observed effects were mediated locally, at the injury site, and not through incorporation of the injected pregabalin into the central nervous system. This indicates that administration of pregabalin at the site of a peripheral nerve may be an effective means for treating both acute and long-term neuropathic pain. These results are similar to findings in previous studies22,23 that peripheral application of pregabalin can block neuropathic pain in rat models and that analgesic effects can be produced by application directly to a nerve.
Hyposensitivity to heat developed on the plantar surface of the affected paws, the same surface that was tested with the Hargreaves test. Previous studies have demonstrated similar hyposensitivity to heat on the plantar surface of affected paws after nerve crush injury presumably caused by the extensive denervation of this surface of the foot.30,31 Thus, the interpretation could be that at least some peripheral input is necessary for evoked neuropathic pain, so that completely anesthetic regions, when stimulated, may not evoke any responses, and the only responses that are observed are due to small amounts of mechanical inputs from sites remote from the previous innervation regions of the damaged nerve. As the nerve grows back, neuropathic pain may appear as was shown by Kingery et al.30 In our present study, pregabalin did not appear to modulate the hyposensitivity to heat experienced by the animals in which the nerve was crushed. Whether the lack of pregabalin’s effect on the Hargreaves test resulted from the profound hypoalgesia caused by the severity of nerve injury or from the lack of effectiveness of pregabalin on heat pain cannot be determined from our study. Pain behavior demonstrated in the guarding and incapacitance meter testing, however, was profound and significantly reduced by treatment with pregabalin.
Future studies should focus on several questions that arise from our study as follows:
1. Dose–response of peripherally applied pregabalin to the injury site on blocking the development of neuropathic pain.
2. Window of opportunity for analgesic action of peripherally applied pregabalin; that is, does the pregabalin have to be applied immediately after injury? Can it alleviate chronic neuropathic pain?
3. Whether a single application of pregabalin can prevent development of neuropathic pain. If not, for how long does it need to be applied?
4. Whether pain will recur if pregabalin administration is discontinued.
5. At what dose (compared to the effective dose) will peripherally applied pregabalin become toxic?
The present study also suggests a possible mechanism for the peripheral actions of pregabalin analgesia when applied peripherally. Previous studies have demonstrated an increase in the expression of the VaCα2δ-1 receptor in the dorsal columns of the spinal cord and along the presynaptic ends of the spinal nerve root that is thought to be causally related to the development of neuropathic pain states.32–39 Pregabalin and gabapentin have shown efficacy in treating neuropathic pain in animal neuropathic pain models presumably by effects on the VaCα2δ-1 receptor. Some studies have suggested that these effects are mediated by interference with the transport of VaCα2δ-1 to the receptor regions of spinal neurons.12,32,33,40 Our data suggest that VaCα2δ-1 receptor expression is also increased in nonneural cells in the injured peripheral nerve. Thus, injury may induce VaCα2δ-1 receptor expression in immune and supportive cells within the nerve, which may lead to ectopic discharge in the surrounding nerve fibers. However, it appears that pregabalin does not reduce this expression. Whether this peripheral expression of VaCα2δ-1 receptor is related to the development of neuropathic pain needs further study.
We have demonstrated a novel finding that locally targeted delivery of pregabalin to the injury site of the sciatic nerve provided superior analgesia to equal doses of systemic pregabalin. Given the dose limitations of systemic pregabalin due to systemic side effects, it is possible that targeted perineural delivery of pregabalin could provide superior analgesia with fewer systemic side effects than current oral pregabalin routes of administration. Further work needs to be done to examine the potential for targeted use of pregabalin around a peripheral nerve for neuropathic pain relief.
Name: Michael J. Buys, MD.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Attestation: Michael J. Buys has seen the original study data, reviewed the analysis of the data, approved the final manuscript, and is the author responsible for archiving the study files.
Name: Carlo Alphonso, MD.
Contribution: This author helped design and conduct the study, analyze the data, and write the manuscript.
Attestation: Carlo Alphonso has seen the original study data, reviewed the analysis of the data, and approved the final manuscript.
This manuscript was handled by: Jianren Mao, MD, PhD.
The authors would like to thank Alan Light, PhD, and Andrea White, PhD, for their help editing the manuscript.
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