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Decreasing sympathetic sprouting in pathologic sensory ganglia: a new mechanism for treating neuropathic pain using lidocaine
Pain 109 (2004) 143–149 www.elsevier.com/locate/pain
Decreasing sympathetic sprouting in pathologic sensory ganglia: a new mechanism for treating neuropathic pain using lidocaine Jun-Ming Zhanga,*, Huiqing Lia, Muhammad A. Munirb b
a Department of Anesthesiology, University of Arkansas for Medical Sciences, 4301 W. Markham St., #515, Little Rock, AR 72205, USA Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
Received 9 September 2003; received in revised form 7 January 2004; accepted 30 January 2004
Abstract Lidocaine brings relief to those suffering from certain neuropathic pain syndromes in humans and in animal models. Evidence suggests that some neuropathic pain behaviors are closely associated with extensive sprouting of noradrenergic sympathetic fibers in the dorsal root ganglia (DRG). Using immunohistochemistry, we examined lidocaine’s effects on abnormal sprouting of sympathetic fibers in two animal models: rats with unilateral spinal nerve ligation (SNL) and rats with complete sciatic nerve transection (CSNT). For the first time, we have demonstrated that systemic lidocaine beginning at the time of surgery via an implanted osmotic pump remarkably reduces sympathetic sprouting (2 – 3 fold) (e.g. the density of sympathetic fibers and the number of DRG neurons surrounded by sympathetic fibers) in axotomized DRGs in SNL rats. The effects of systemic lidocaine lasted more than 7 days after the termination of lidocaine administration. Similar results were obtained after topical application of lidocaine to the nerve trunk to block abnormal discharges originating in the neuroma in CSNT rats. Results strongly suggest that sympathetic sprouting in pathologic DRG may be associated with abnormal spontaneous activity originating in the DRG or the injured axons (e.g. neuroma). This finding provides new insight into the mechanisms underlying sympathetic sprouting and increases our current understanding of the prolonged therapeutic effects of lidocaine on neuropathic pain syndromes. q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Dorsal root ganglion; Neuropathic pain; Sympathetic sprouting
1. Introduction Systemic (e.g. oral, intravenous, or subcutaneous) and local (e.g. topical or epidural) administration of the local anesthetic lidocaine brings significant relief to those suffering from postherpetic neuralgia (Rowbotham et al., 1991) and some other neuropathic pain syndromes in humans and certain animal models (Abdi et al., 1998; Chabal et al., 1989; Glazer and Portenoy, 1991; Tanelian and MacIver, 1991; Wallace et al., 2000). It is generally believed that lidocaine alleviates pain by reducing or preventing pathophysiologically damaged primary sensory neurons from excessive discharges through the blockage of sodium channel activity (Chabal et al., 1989; Devor et al., 1992). Animal studies from various types of neuropathic pain animal models have demonstrated extensive sprouting of * Corresponding author. Tel.: þ 1-501-603-1936; fax: þ1-501-603-1216. E-mail addresses: [email protected], [email protected] (J.-M. Zhang).
sympathetic nerve fibers in lumbar dorsal root ganglia (DRG) and their adjacent peripheral nerves (Chung et al., 1996; Lee et al., 1998a; McLachlan et al., 1993; Ramer and Bisby, 1997; Ramer et al., 1999). Neuropathic pain behaviors such as allodynia and cold-stress-exacerbated ongoing pain are correlated with the density of sprouting and the number of basket formations in the DRG (Chung et al., 1996; McLachlan et al., 1993; Ramer et al., 1998). Surgical sympathectomy at spinal nerve level alleviates all sensory abnormalities such as cutaneous hypersensitivity to mechanical or thermal stimuli in rats with spinal nerve resection (Kinnman and Levine, 1995). Therefore, nerve injury-induced sympathetic sprouting is a major phenomenon implicated in neuropathic pain (for a review, see Devor, 1999). Neurotrophins (e.g. nerve growth factor and neurotrophin-3) (DiCicco-Bloom et al., 1993; Lee et al., 1998b; Zhou and Rush, 1996) and certain proinflammatory cytokines (e.g. interleukin-6 and leukemia inhibitory factor) (Ramer et al., 1998, 1999; Thompson and Majithia, 1998)
0304-3959/$20.00 q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2004.01.033
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are known factors that contribute to the sprouting of sympathetic fibers. However, neurotrophins or cytokines alone are not sufficient to explain the preference for largeand medium-sized neurons of sprouted fibers as demonstrated in the axotomized DRGs (McLachlan et al., 1993). These large- and medium-sized DRG neurons often exhibit high-frequency, spontaneous activity after injury of the peripheral axons. It has long been documented that the development of the vertebrate nervous system shows great dependency on neuronal activity (Fields and Nelson, 1992; Fields and Stevens-Graham, 2002). Thus, it is reasonable to assume that the triggering and, possibly, directing of sympathetic sprouting may be associated with abnormal spontaneous activity of injured sensory neurons. Here, we report for the first time that systemic lidocaine in the early stage of nerve injury remarkably reduced the sprouting of sympathetic nerve fibers in the axotomized lumbar ganglion. Similar results of reduced sympathetic sprouting were obtained with nerve blockade proximal to the nerve stump in the neuroma model. These results suggest that lidocaine may relieve neuropathic pain by reducing abnormal neuronal discharges and subsequently decreasing sympathetic sprouting.
and tightened just enough to close the side slit. An osmotic pump prefilled with 2% lidocaine or normal saline (volume 200 ml, flow rate 0.5 ml/h for 14 days) was connected to the cuff via silicon tubing. The osmotic pump and connecting tubing were immobilized using surgical ligatures through the surrounding tissue. The sciatic nerve was then tightly ligated (5-0 silk) distal to the cuff and transected 5 mm distally from the ligature as described previously (Dobretsov et al., 2001; Wall et al., 1974).
2. Methods
Tissue sections were incubated in antibodies to TH (raised from rabbit; obtained from Pel-Freeze, Rogers, AR) at a dilution of 1:1000 for 48 h at 4 8C, followed by the reaction with biotinylated secondary antibody and, finally, with Vector ABC reagent. Triton-X (0.3%) was used in all reaction solutions to enhance antibody penetration. Immunoreaction products were visualized by the diaminobenzidine method in the presence of H2O2 in 0.1 M phosphate buffer. Tissues were then mounted on gelatin-coated slides, air dried, dehydrated, and coverslipped for light-microscopic observation.
2.1. Surgery All the surgical procedures were reviewed and approved by the UAMS Institutional Animal Care and Use Committee (IACUC). 2.1.1. Surgical procedure for SNL rats and implantation of an osmotic pump for systemic administration of lidocaine The right L5 spinal nerve in adult Sprague– Dawley rats was isolated and tightly ligated with 6-0 silk thread under general anesthesia with isoflurane, as described previously (Kim and Chung, 1992). An osmotic mini-pump (volume 2 ml, flow rate 10 ml/h for 7 days) was then implanted in the peritoneal cavity of each rat. To avoid possible bias, the implanted pump was filled with either 20% lidocaine or normal saline by an investigator other than the one who would be performing the immunostaining procedure. The incision was closed in layers, and daily prophylactic amoxicillin-clavulanate potassium (7.52 g) was given via drinking water (500 ml) for three postoperative days (POD). 2.1.2. Surgical procedure for CSNT rats and implantation of an osmotic pump for persistent nerve block The right-side sciatic nerve was exposed at mid-thigh level and blocked with a piece of cotton soaked in 2% lidocaine. The nerve was then carefully placed into the cuff through the side slit (0.5 mm wide) of a ‘T-shaped’ catheter (4 –5 mm long) made of silastic laboratory tubing (Dow Corning, Auburn, MI). A ligature was placed around the cuff
2.2. Specimen preparation Rats were anesthetized with intraperitoneal pentobarbital sodium (40 mg/kg) and fixed by perfusing 200 –300 ml of Zamboni’s fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) through the left ventricle of the heart. The bilateral DRGs of L4 and L5 were removed, postfixed in the perfusion fixative for 1 h at 4 8C, and embedded in gelatin overnight. The ganglia were horizontally sectioned with a vibratome (Vibratome Company, St Louis, MO) at a thickness of 40 mm. 2.3. Tyrosine hydroxylase (TH) immunostaining of sympathetic fibers
2.4. Measurement of TH-immunoreactive (IR) fiber density in the DRG Slides from control and experimental groups were labeled with numbers in order to keep them blind from the person who would be performing the image analysis. Further, all images were captured and analyzed by an investigator other than the person who performed immunohistostaining to avoid possible bias. Each DRG was sectioned into 15– 20, 40-mm thick sections that were then mounted on a single slide. The numerical density of TH-IR fibers in the cellular regions was calculated by measuring TH-IR fibers within each section of the DRG. Using ImagePro Plus software (Media Cybernetics, Inc., Silver Spring, MD), images from all sections of each DRG were captured under a light microscope (20 £ ) equipped with a SPOT Insight colored digital camera (Diagnostic Instruments, Inc., Burlingame, CA), and stored in a Pentium IV computer for measurement. Using the ImagePro program, TH-IR fibers in each image
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were traced, and a new image containing all the traced fibers was generated from the original image (Fig. 3b). After applying a ‘thinning filter,’ all traced fibers were converted to single-pixel lines. The total number of pixels within each image was then counted and converted to fiber length (in mm). The numerical density of the TH-IR fiber within each image was obtained by dividing the total fiber length by the size of the measured area (area in mm2). The density of TH-IR fibers for each DRG was calculated after all images from all sections were measured and counted.
2.6. Statistical data analysis
2.5. Counting neurons with TH-IR basket-like structures or rings
3. Results
The number of neuronal somata surrounded by TH-IR basket-like structures or rings were counted from all sections of the TH-immunostained DRG. Only DRG neurons encircled by TH-IR fibers for at least two-thirds of the circumference of the somata, and which had a clearly visible nucleus, were counted. The average number of TH-IR baskets/rings within each DRG was obtained by dividing the total number of baskets/rings by the size of the total measured area (area in mm2).
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Data were expressed as the mean ^ the standard error of the mean. The numbers of cells with a TH-IR basket-like structure or TH-IR ring and the density of sympathetic fibers in the DRG of different groups were compared using Student’s t-test. The null hypothesis in each case was accepted or rejected using a significance level of P , 0:05:
This study utilized two of the most frequently employed animal models of neuropathic pain: rats with unilateral SNL or CSNT. We first examined the density of TH-IR fibers in lumbar DRGs from five normal rats that had not undergone surgery. In normal DRGs, scattered ‘dark cells’ with TH-IR ‘tails’ were found throughout the section. These dark cells represent a subpopulation of dopaminergic DRG neurons (Katz, 1983). TH-IR-positive sympathetic fibers formed varicose plexuses around vascular processes (Fig. 1a), as reported previously (McLachlan et al., 1993).
Fig. 1. Sympathetic sprouting in SNL rats with/without systemic lidocaine treatment. (a) Microscopic photographs of TH-IR axons in L5 DRGs from normal rats showing TH-IR vascular plexuses and ‘dark’ cells. (b) Sprouting TH-IR sympathetic fibers in the axotomized DRG form basket-like structures around large DRG neurons (arrows). (c) Comparison of TH-IR fiber density in axotomized and intact DRGs, with and without systemic lidocaine or saline-treatment, on POD 7. (d) Comparison of numbers of TH-IR baskets and rings in axotomized DRGs, with and without systemic lidocaine or saline-treatment. p p P , 0:01; Student’s t-test.
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3.1. Systemic lidocaine decreased sympathetic sprouting in DRGs ipsilateral and contralateral to the axotomy in SNL rats In subsequent experiments, we examined and compared the density of TH-IR fibers in DRGs from SNL rats with and without systemic lidocaine administration. Twenty-one rats received a unilateral SNL, followed by implantation of an osmotic pump prefilled with either 20% lidocaine HCl ðn ¼ 9Þ or normal saline ðn ¼ 7Þ in the peritoneal cavity. The remaining rats ðn ¼ 5Þ received SNL, but no pump was implanted. TH staining was performed on POD 7 when the pump became empty. In a select number of SNL rats, blood samples were collected to monitor plasma lidocaine levels. Immunostaining showed that, in saline-treated or untreated SNL rats, the density of TH-IR fibers was robustly increased compared to DRGs in normal rats (Fig. 1b and c). The majority of sprouting fibers originated from the spinal roots and, in most cases, filled the entire section by POD 7 (Fig. 1b). There was no statistical difference in the overall density of sprouted fibers between SNL rats with and without saline administration although fiber density in SNL rats with saline administration seemed to be higher than those without saline treatment (Fig. 1c). In some large- and medium-sized sensory neurons, sympathetic fibers wrapped around the somata and formed a basket-like structure; in other neurons, only a TH-IR ring could be found around the cell body (Fig. 1b). The numbers of TH-IR basket-structures or rings in the ipsilateral L5 DRG, with and without saline administration, were 13.8 ^ 2.9 and 19.2 ^ 2.1/mm2, respectively, with no statistical difference between the two groups. Increased density of TH-IR fibers was also found in the adjacent DRGs (e.g. ipsilateral L4) without axotomy, as well as in the contralateral DRGs (e.g. L5) (Fig. 1b). In lidocaine-treated SNL rats, sympathetic sprouting decreased significantly in both ipsilateral and contralateral DRGs as indicated by decreased fiber density (Fig. 1c). In certain sections, no TH-IR fibers could be observed; in other sections TH-IR fiber sprouts were limited to a small area. The number of TH-IR baskets or rings in the ipsilateral L5 DRG was reduced to 4.9 ^ 1.2/mm2 (Fig. 1d). We found that continuous lidocaine infusion via the implanted pump established a plasma lidocaine level of 0.97 ^ 0.46 mg/ml (ranging between 0.5 and 1.4 mg/ml, n ¼ 6) during the drug-delivery period (days 1 –6). 3.1.1. Effects of lidocaine on sympathetic sprouting persisted beyond the duration of drug administration In a total of 16 SNL rats, immunohistochemical staining was performed on POD 14; 7 days after the pump became empty. The osmotic pumps (10 ml/h, 7 days) implanted at the time of nerve injury were filled with lidocaine ðn ¼ 7Þ or normal saline ðn ¼ 9Þ as control. It was found that the overall density of TH-fibers in saline-treated SNL rats was similar on POD 7 and POD 14 (P . 0:05; t-test). The number of TH-IR basket-structures or rings, however, was
decreased on POD 14 as compared to that on POD 7 (P , 0:05; t-test). Although there was no statistical difference in the overall density of TH-fibers between saline- and lidocaine-treated SNL rats on POD 14 (Fig. 2a), the number of TH-IR basket-structures or rings in the ipsilateral L5 DRG on POD 14 was significantly lower in lidocaine-treated rats than in saline-treated rats (saline 4.7 ^ 0.8/mm 2 vs. lidocaine 2.7 ^ 0.2/mm 2, P , 0:05; t-test) (Fig. 2b). 3.1.2. Early nerve-block proximal to the injury site of sciatic nerve decreased sympathetic sprouting in CSNT rats To demonstrate that lidocaine-reduced sympathetic fiber sprouting may be associated with blocking sensory neuron discharges, and to rule out the possibility that systemic lidocaine reduces sprouting by directly acting on the sympathetic fibers or satellite glial cells rather than sensory neurons, we tested the effects of a peripheral nerve block on sympathetic sprouting in CSNT rats. Rats ðn ¼ 6Þ were anesthetized with isoflurane, and the right sciatic nerve was exposed and blocked by placing a piece of cotton soaked in 2% lidocaine around the nerve trunk. Nerve blockade was maintained by continuous infusion of the nerve with 2%
Fig. 2. Prolonged effects of systemic lidocaine on sympathetic sprouting in SNL rats. Immunohistochemical staining was performed on POD 14 in SNL rats with systemic lidocaine treatment during the first postoperative week. (a) Comparison of TH-IR fiber density in systemic lidocaine- and saline-treated axotomized L5 DRGs. (b) Comparison of the number of THIR baskets/rings in systemic lidocaine- and saline-treated axotomized L5 DRGs. p P , 0:05; Student’s t-test.
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lidocaine via a subcutaneously implanted osmotic pump. In control rats ðn ¼ 6Þ; the same procedure was used except the nerve was not blocked and the osmotic pumps were prefilled with normal saline. Immunohistochemical staining of the DRGs on POD 14 revealed apparent sprouting of sympathetic nerve fibers in the ipsilateral axotomized DRGs, as well as in the contralateral intact L4 and L5 DRGs. The density of sympathetic fibers in DRGs of CSNT rats was randomly distributed in the axotomized DRGs. Due to the relatively lower density of sympathetic sprouting in CSNT DRGs on POD 14, it was clear that the fine sprouting fibers were derived from the perivascular plexuses (Fig. 3a and b). Although basket formation could be observed in both L4 and L5 DRGs at an early stage of CSNT (see Fig. 3c), the number was very small (typically less than 10 per DRG) compared to the axotomized DRGs from SNL rats. Local nerve block via topical lidocaine application significantly decreased sympathetic fiber density within both L4 and L5 DRGs ipsilateral to the injury compared to saline-treated CSNT rats (Fig. 3d). Similar to the SNL rats receiving systemic lidocaine, regional nerve block also decreased sympathetic sprouting within the contralateral DRGs. The effect of lidocaine on basket formation was not assessed in CSNT rats because
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the small number of baskets or rings in each CSNT DRG at an early stage of injury were not sufficient for a reliable comparison.
4. Discussion In the present study, we first confirmed extensive sprouting of sympathetic fibers in axotomized DRG on POD 7 in SNL rats and day 14 in CSNT rats. Based on our results, sprouted sympathetic fibers in the axotomized DRGs have different origins in two different animal models. In SNL rats, the majority of fibers were from the spinal roots or fiber tracts, while in CSNT rats, sympathetic fibers normally accompany vascular processes sprouting into the DRG. The difference in the way sympathetic axons invade the DRG in different animal models may be attributed to the differential dependency on peripheral NGF (Ramer and Bisby, 1999). Previous animal studies have demonstrated that the functional coupling between sprouting sympathetic fibers and DRG cells is closely correlated with neuropathic pain behaviors (Chung et al., 1996; McLachlan et al., 1993;
Fig. 3. Effects of local nerve block on CSNT-induced sympathetic sprouting. (a) Color inverted image showing TH-IR fibers sprouting from perivascular plexuses in a DRG section of CSNT rats on POD 14. (b) Image is generated after tracing all the TH-IR fibers in (a). (c) Microscopic photographs of basket-like structures (arrows). (d) Nerve block decreased sympathetic fiber density measured on POD 14. p p P , 0:01; Student’s t-test.
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Ramer et al., 1998). Likely, the release of norepinephrine from sprouting sympathetic nerve endings excites myelinated or unmyelinated primary afferent neurons by activating a-adrenoceptors, contributing to central sensitization, and exaggerating ongoing pain and hyperalgesia (Gold et al., 1994; Liu et al., 1999; Michaelis et al., 1996; Sato and Perl, 1991; Xie et al., 1995). The most astonishing finding of our study is that lidocaine administration at an early stage of nerve injury remarkably reduces sympathetic sprouting. Lidocaineinduced anesthetic effects only last about 30 – 45 min, consistent with its suppressive effects on nerve pulse generation or neuronal spontaneous activity (Devor et al., 1992). However, the long-term effect after repetitive therapy with lidocaine in a subpopulation of neuropathic pain sufferers cannot be accounted for simply by its local anesthetic effects. Our results suggest that, in addition to delaying or preventing the development of central sensitization, decreasing sympathetic sprouting in pathologic DRGs may be a previously unfound mechanism underlying its prolonged therapeutic effect. Based on our findings it is reasonable to believe that lidocaine reduces sympathetic sprouting in the pathologic DRGs by depressing abnormal neuronal discharges. In support of this hypothesis, sprouting sympathetic fibers form basket structures preferentially in association with medium- and large-sized neurons (Chung et al., 1996; McLachlan et al., 1993), which are often present with highfrequency and/or bursting discharges after nerve injury (Zhang et al., 1997). Results from our preliminary study revealed that systemic administration of 4-aminopyridine, a Kþ channel blocker that enhances spontaneous activity, increased sympathetic sprouting, further, supporting our hypothesis. Because endogenous neurotrophins are proven factors that contribute to sympathetic sprouting (Ramer et al., 1999; Walsh et al., 1999; Zhou, 1996; Zhou et al., 1999), and there is sufficient evidence to suggest that neurotrophin expression/release is regulated by neuronal activity (Kim et al., 1994), it is possible that lidocaine may reduce sprouting by interfering with neurotrophin expression. However, additional studies are needed to demonstrate that neurotrophin expression can be reduced by systemic lidocaine or local nerve block, and that this effect is mediated by neuronal activity. The mechanisms underlying sympathetic sprouting in the adjacent intact DRGs or in DRGs contralateral to the nerve injury are not clear. However, as proposed by Ramer et al. (1999), sympathetic sprouting may involve the activation of satellite glia in the pathologic DRGs. If this is the case, then systemic lidocaine may interrupt the function of satellite glia through an unknown mechanism, which, in turn, leads to the reduction of sympathetic sprouting. The results of our studies have immediate clinical potential. Traumatic injury to soft-tissue, bone, and/or nerve often leads to a pathological pain state known as complex regional pain syndrome (Scadding, 1999).
Although there are conflicting laboratory reports with respect to the role of sympathetic sprouting in the development and/or persistence of neuropathic pain, clinically, there is an abundance of evidence that, in some patients, pain is maintained by efferent noradrenergic sympathetic activity and circulating catecholamines (sympathetically maintained pain, SMP) (Roberts, 1986). Pharmacologic sympathetic block or surgical sympathectomy may provide satisfactory pain relief in such patients. It is likely that the role of sympathetic sprouting in the development or persistence of neuropathic pain may very well depend on the type of nerve injury and/or the time after injury. Our results suggest that new drugs should be developed to disrupt the hyperexcitability cycle taking place in the DRG neurons and to reduce or prevent sympathetic sprouting. This will provide a new, non-opioid therapeutic approach for treating chronic pain resulting from peripheral nerve injury or injury to the lumbar ganglia.
Acknowledgements We would like to thank Drs Tony Yaksh and Steve Rossi of the Department of Anesthesiology, UCSD, for measuring the plasma level of lidocaine. This work was supported by National Institute of Neurological Disorders and Stroke (NINDS) Grant R01NS39568.
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Decreasing sympathetic sprouting in pathologic sensory ganglia: a new mechanism for treating neuropathic pain using lidocaine Jun-Ming Zhanga,*, Huiqing Lia, Muhammad A. Munirb b
a Department of Anesthesiology, University of Arkansas for Medical Sciences, 4301 W. Markham St., #515, Little Rock, AR 72205, USA Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA 02115, USA
Received 9 September 2003; received in revised form 7 January 2004; accepted 30 January 2004
Abstract Lidocaine brings relief to those suffering from certain neuropathic pain syndromes in humans and in animal models. Evidence suggests that some neuropathic pain behaviors are closely associated with extensive sprouting of noradrenergic sympathetic fibers in the dorsal root ganglia (DRG). Using immunohistochemistry, we examined lidocaine’s effects on abnormal sprouting of sympathetic fibers in two animal models: rats with unilateral spinal nerve ligation (SNL) and rats with complete sciatic nerve transection (CSNT). For the first time, we have demonstrated that systemic lidocaine beginning at the time of surgery via an implanted osmotic pump remarkably reduces sympathetic sprouting (2 – 3 fold) (e.g. the density of sympathetic fibers and the number of DRG neurons surrounded by sympathetic fibers) in axotomized DRGs in SNL rats. The effects of systemic lidocaine lasted more than 7 days after the termination of lidocaine administration. Similar results were obtained after topical application of lidocaine to the nerve trunk to block abnormal discharges originating in the neuroma in CSNT rats. Results strongly suggest that sympathetic sprouting in pathologic DRG may be associated with abnormal spontaneous activity originating in the DRG or the injured axons (e.g. neuroma). This finding provides new insight into the mechanisms underlying sympathetic sprouting and increases our current understanding of the prolonged therapeutic effects of lidocaine on neuropathic pain syndromes. q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. Keywords: Dorsal root ganglion; Neuropathic pain; Sympathetic sprouting
1. Introduction Systemic (e.g. oral, intravenous, or subcutaneous) and local (e.g. topical or epidural) administration of the local anesthetic lidocaine brings significant relief to those suffering from postherpetic neuralgia (Rowbotham et al., 1991) and some other neuropathic pain syndromes in humans and certain animal models (Abdi et al., 1998; Chabal et al., 1989; Glazer and Portenoy, 1991; Tanelian and MacIver, 1991; Wallace et al., 2000). It is generally believed that lidocaine alleviates pain by reducing or preventing pathophysiologically damaged primary sensory neurons from excessive discharges through the blockage of sodium channel activity (Chabal et al., 1989; Devor et al., 1992). Animal studies from various types of neuropathic pain animal models have demonstrated extensive sprouting of * Corresponding author. Tel.: þ 1-501-603-1936; fax: þ1-501-603-1216. E-mail addresses: [email protected], [email protected] (J.-M. Zhang).
sympathetic nerve fibers in lumbar dorsal root ganglia (DRG) and their adjacent peripheral nerves (Chung et al., 1996; Lee et al., 1998a; McLachlan et al., 1993; Ramer and Bisby, 1997; Ramer et al., 1999). Neuropathic pain behaviors such as allodynia and cold-stress-exacerbated ongoing pain are correlated with the density of sprouting and the number of basket formations in the DRG (Chung et al., 1996; McLachlan et al., 1993; Ramer et al., 1998). Surgical sympathectomy at spinal nerve level alleviates all sensory abnormalities such as cutaneous hypersensitivity to mechanical or thermal stimuli in rats with spinal nerve resection (Kinnman and Levine, 1995). Therefore, nerve injury-induced sympathetic sprouting is a major phenomenon implicated in neuropathic pain (for a review, see Devor, 1999). Neurotrophins (e.g. nerve growth factor and neurotrophin-3) (DiCicco-Bloom et al., 1993; Lee et al., 1998b; Zhou and Rush, 1996) and certain proinflammatory cytokines (e.g. interleukin-6 and leukemia inhibitory factor) (Ramer et al., 1998, 1999; Thompson and Majithia, 1998)
0304-3959/$20.00 q 2004 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.pain.2004.01.033
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are known factors that contribute to the sprouting of sympathetic fibers. However, neurotrophins or cytokines alone are not sufficient to explain the preference for largeand medium-sized neurons of sprouted fibers as demonstrated in the axotomized DRGs (McLachlan et al., 1993). These large- and medium-sized DRG neurons often exhibit high-frequency, spontaneous activity after injury of the peripheral axons. It has long been documented that the development of the vertebrate nervous system shows great dependency on neuronal activity (Fields and Nelson, 1992; Fields and Stevens-Graham, 2002). Thus, it is reasonable to assume that the triggering and, possibly, directing of sympathetic sprouting may be associated with abnormal spontaneous activity of injured sensory neurons. Here, we report for the first time that systemic lidocaine in the early stage of nerve injury remarkably reduced the sprouting of sympathetic nerve fibers in the axotomized lumbar ganglion. Similar results of reduced sympathetic sprouting were obtained with nerve blockade proximal to the nerve stump in the neuroma model. These results suggest that lidocaine may relieve neuropathic pain by reducing abnormal neuronal discharges and subsequently decreasing sympathetic sprouting.
and tightened just enough to close the side slit. An osmotic pump prefilled with 2% lidocaine or normal saline (volume 200 ml, flow rate 0.5 ml/h for 14 days) was connected to the cuff via silicon tubing. The osmotic pump and connecting tubing were immobilized using surgical ligatures through the surrounding tissue. The sciatic nerve was then tightly ligated (5-0 silk) distal to the cuff and transected 5 mm distally from the ligature as described previously (Dobretsov et al., 2001; Wall et al., 1974).
2. Methods
Tissue sections were incubated in antibodies to TH (raised from rabbit; obtained from Pel-Freeze, Rogers, AR) at a dilution of 1:1000 for 48 h at 4 8C, followed by the reaction with biotinylated secondary antibody and, finally, with Vector ABC reagent. Triton-X (0.3%) was used in all reaction solutions to enhance antibody penetration. Immunoreaction products were visualized by the diaminobenzidine method in the presence of H2O2 in 0.1 M phosphate buffer. Tissues were then mounted on gelatin-coated slides, air dried, dehydrated, and coverslipped for light-microscopic observation.
2.1. Surgery All the surgical procedures were reviewed and approved by the UAMS Institutional Animal Care and Use Committee (IACUC). 2.1.1. Surgical procedure for SNL rats and implantation of an osmotic pump for systemic administration of lidocaine The right L5 spinal nerve in adult Sprague– Dawley rats was isolated and tightly ligated with 6-0 silk thread under general anesthesia with isoflurane, as described previously (Kim and Chung, 1992). An osmotic mini-pump (volume 2 ml, flow rate 10 ml/h for 7 days) was then implanted in the peritoneal cavity of each rat. To avoid possible bias, the implanted pump was filled with either 20% lidocaine or normal saline by an investigator other than the one who would be performing the immunostaining procedure. The incision was closed in layers, and daily prophylactic amoxicillin-clavulanate potassium (7.52 g) was given via drinking water (500 ml) for three postoperative days (POD). 2.1.2. Surgical procedure for CSNT rats and implantation of an osmotic pump for persistent nerve block The right-side sciatic nerve was exposed at mid-thigh level and blocked with a piece of cotton soaked in 2% lidocaine. The nerve was then carefully placed into the cuff through the side slit (0.5 mm wide) of a ‘T-shaped’ catheter (4 –5 mm long) made of silastic laboratory tubing (Dow Corning, Auburn, MI). A ligature was placed around the cuff
2.2. Specimen preparation Rats were anesthetized with intraperitoneal pentobarbital sodium (40 mg/kg) and fixed by perfusing 200 –300 ml of Zamboni’s fixative (4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4) through the left ventricle of the heart. The bilateral DRGs of L4 and L5 were removed, postfixed in the perfusion fixative for 1 h at 4 8C, and embedded in gelatin overnight. The ganglia were horizontally sectioned with a vibratome (Vibratome Company, St Louis, MO) at a thickness of 40 mm. 2.3. Tyrosine hydroxylase (TH) immunostaining of sympathetic fibers
2.4. Measurement of TH-immunoreactive (IR) fiber density in the DRG Slides from control and experimental groups were labeled with numbers in order to keep them blind from the person who would be performing the image analysis. Further, all images were captured and analyzed by an investigator other than the person who performed immunohistostaining to avoid possible bias. Each DRG was sectioned into 15– 20, 40-mm thick sections that were then mounted on a single slide. The numerical density of TH-IR fibers in the cellular regions was calculated by measuring TH-IR fibers within each section of the DRG. Using ImagePro Plus software (Media Cybernetics, Inc., Silver Spring, MD), images from all sections of each DRG were captured under a light microscope (20 £ ) equipped with a SPOT Insight colored digital camera (Diagnostic Instruments, Inc., Burlingame, CA), and stored in a Pentium IV computer for measurement. Using the ImagePro program, TH-IR fibers in each image
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were traced, and a new image containing all the traced fibers was generated from the original image (Fig. 3b). After applying a ‘thinning filter,’ all traced fibers were converted to single-pixel lines. The total number of pixels within each image was then counted and converted to fiber length (in mm). The numerical density of the TH-IR fiber within each image was obtained by dividing the total fiber length by the size of the measured area (area in mm2). The density of TH-IR fibers for each DRG was calculated after all images from all sections were measured and counted.
2.6. Statistical data analysis
2.5. Counting neurons with TH-IR basket-like structures or rings
3. Results
The number of neuronal somata surrounded by TH-IR basket-like structures or rings were counted from all sections of the TH-immunostained DRG. Only DRG neurons encircled by TH-IR fibers for at least two-thirds of the circumference of the somata, and which had a clearly visible nucleus, were counted. The average number of TH-IR baskets/rings within each DRG was obtained by dividing the total number of baskets/rings by the size of the total measured area (area in mm2).
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Data were expressed as the mean ^ the standard error of the mean. The numbers of cells with a TH-IR basket-like structure or TH-IR ring and the density of sympathetic fibers in the DRG of different groups were compared using Student’s t-test. The null hypothesis in each case was accepted or rejected using a significance level of P , 0:05:
This study utilized two of the most frequently employed animal models of neuropathic pain: rats with unilateral SNL or CSNT. We first examined the density of TH-IR fibers in lumbar DRGs from five normal rats that had not undergone surgery. In normal DRGs, scattered ‘dark cells’ with TH-IR ‘tails’ were found throughout the section. These dark cells represent a subpopulation of dopaminergic DRG neurons (Katz, 1983). TH-IR-positive sympathetic fibers formed varicose plexuses around vascular processes (Fig. 1a), as reported previously (McLachlan et al., 1993).
Fig. 1. Sympathetic sprouting in SNL rats with/without systemic lidocaine treatment. (a) Microscopic photographs of TH-IR axons in L5 DRGs from normal rats showing TH-IR vascular plexuses and ‘dark’ cells. (b) Sprouting TH-IR sympathetic fibers in the axotomized DRG form basket-like structures around large DRG neurons (arrows). (c) Comparison of TH-IR fiber density in axotomized and intact DRGs, with and without systemic lidocaine or saline-treatment, on POD 7. (d) Comparison of numbers of TH-IR baskets and rings in axotomized DRGs, with and without systemic lidocaine or saline-treatment. p p P , 0:01; Student’s t-test.
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3.1. Systemic lidocaine decreased sympathetic sprouting in DRGs ipsilateral and contralateral to the axotomy in SNL rats In subsequent experiments, we examined and compared the density of TH-IR fibers in DRGs from SNL rats with and without systemic lidocaine administration. Twenty-one rats received a unilateral SNL, followed by implantation of an osmotic pump prefilled with either 20% lidocaine HCl ðn ¼ 9Þ or normal saline ðn ¼ 7Þ in the peritoneal cavity. The remaining rats ðn ¼ 5Þ received SNL, but no pump was implanted. TH staining was performed on POD 7 when the pump became empty. In a select number of SNL rats, blood samples were collected to monitor plasma lidocaine levels. Immunostaining showed that, in saline-treated or untreated SNL rats, the density of TH-IR fibers was robustly increased compared to DRGs in normal rats (Fig. 1b and c). The majority of sprouting fibers originated from the spinal roots and, in most cases, filled the entire section by POD 7 (Fig. 1b). There was no statistical difference in the overall density of sprouted fibers between SNL rats with and without saline administration although fiber density in SNL rats with saline administration seemed to be higher than those without saline treatment (Fig. 1c). In some large- and medium-sized sensory neurons, sympathetic fibers wrapped around the somata and formed a basket-like structure; in other neurons, only a TH-IR ring could be found around the cell body (Fig. 1b). The numbers of TH-IR basket-structures or rings in the ipsilateral L5 DRG, with and without saline administration, were 13.8 ^ 2.9 and 19.2 ^ 2.1/mm2, respectively, with no statistical difference between the two groups. Increased density of TH-IR fibers was also found in the adjacent DRGs (e.g. ipsilateral L4) without axotomy, as well as in the contralateral DRGs (e.g. L5) (Fig. 1b). In lidocaine-treated SNL rats, sympathetic sprouting decreased significantly in both ipsilateral and contralateral DRGs as indicated by decreased fiber density (Fig. 1c). In certain sections, no TH-IR fibers could be observed; in other sections TH-IR fiber sprouts were limited to a small area. The number of TH-IR baskets or rings in the ipsilateral L5 DRG was reduced to 4.9 ^ 1.2/mm2 (Fig. 1d). We found that continuous lidocaine infusion via the implanted pump established a plasma lidocaine level of 0.97 ^ 0.46 mg/ml (ranging between 0.5 and 1.4 mg/ml, n ¼ 6) during the drug-delivery period (days 1 –6). 3.1.1. Effects of lidocaine on sympathetic sprouting persisted beyond the duration of drug administration In a total of 16 SNL rats, immunohistochemical staining was performed on POD 14; 7 days after the pump became empty. The osmotic pumps (10 ml/h, 7 days) implanted at the time of nerve injury were filled with lidocaine ðn ¼ 7Þ or normal saline ðn ¼ 9Þ as control. It was found that the overall density of TH-fibers in saline-treated SNL rats was similar on POD 7 and POD 14 (P . 0:05; t-test). The number of TH-IR basket-structures or rings, however, was
decreased on POD 14 as compared to that on POD 7 (P , 0:05; t-test). Although there was no statistical difference in the overall density of TH-fibers between saline- and lidocaine-treated SNL rats on POD 14 (Fig. 2a), the number of TH-IR basket-structures or rings in the ipsilateral L5 DRG on POD 14 was significantly lower in lidocaine-treated rats than in saline-treated rats (saline 4.7 ^ 0.8/mm 2 vs. lidocaine 2.7 ^ 0.2/mm 2, P , 0:05; t-test) (Fig. 2b). 3.1.2. Early nerve-block proximal to the injury site of sciatic nerve decreased sympathetic sprouting in CSNT rats To demonstrate that lidocaine-reduced sympathetic fiber sprouting may be associated with blocking sensory neuron discharges, and to rule out the possibility that systemic lidocaine reduces sprouting by directly acting on the sympathetic fibers or satellite glial cells rather than sensory neurons, we tested the effects of a peripheral nerve block on sympathetic sprouting in CSNT rats. Rats ðn ¼ 6Þ were anesthetized with isoflurane, and the right sciatic nerve was exposed and blocked by placing a piece of cotton soaked in 2% lidocaine around the nerve trunk. Nerve blockade was maintained by continuous infusion of the nerve with 2%
Fig. 2. Prolonged effects of systemic lidocaine on sympathetic sprouting in SNL rats. Immunohistochemical staining was performed on POD 14 in SNL rats with systemic lidocaine treatment during the first postoperative week. (a) Comparison of TH-IR fiber density in systemic lidocaine- and saline-treated axotomized L5 DRGs. (b) Comparison of the number of THIR baskets/rings in systemic lidocaine- and saline-treated axotomized L5 DRGs. p P , 0:05; Student’s t-test.
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lidocaine via a subcutaneously implanted osmotic pump. In control rats ðn ¼ 6Þ; the same procedure was used except the nerve was not blocked and the osmotic pumps were prefilled with normal saline. Immunohistochemical staining of the DRGs on POD 14 revealed apparent sprouting of sympathetic nerve fibers in the ipsilateral axotomized DRGs, as well as in the contralateral intact L4 and L5 DRGs. The density of sympathetic fibers in DRGs of CSNT rats was randomly distributed in the axotomized DRGs. Due to the relatively lower density of sympathetic sprouting in CSNT DRGs on POD 14, it was clear that the fine sprouting fibers were derived from the perivascular plexuses (Fig. 3a and b). Although basket formation could be observed in both L4 and L5 DRGs at an early stage of CSNT (see Fig. 3c), the number was very small (typically less than 10 per DRG) compared to the axotomized DRGs from SNL rats. Local nerve block via topical lidocaine application significantly decreased sympathetic fiber density within both L4 and L5 DRGs ipsilateral to the injury compared to saline-treated CSNT rats (Fig. 3d). Similar to the SNL rats receiving systemic lidocaine, regional nerve block also decreased sympathetic sprouting within the contralateral DRGs. The effect of lidocaine on basket formation was not assessed in CSNT rats because
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the small number of baskets or rings in each CSNT DRG at an early stage of injury were not sufficient for a reliable comparison.
4. Discussion In the present study, we first confirmed extensive sprouting of sympathetic fibers in axotomized DRG on POD 7 in SNL rats and day 14 in CSNT rats. Based on our results, sprouted sympathetic fibers in the axotomized DRGs have different origins in two different animal models. In SNL rats, the majority of fibers were from the spinal roots or fiber tracts, while in CSNT rats, sympathetic fibers normally accompany vascular processes sprouting into the DRG. The difference in the way sympathetic axons invade the DRG in different animal models may be attributed to the differential dependency on peripheral NGF (Ramer and Bisby, 1999). Previous animal studies have demonstrated that the functional coupling between sprouting sympathetic fibers and DRG cells is closely correlated with neuropathic pain behaviors (Chung et al., 1996; McLachlan et al., 1993;
Fig. 3. Effects of local nerve block on CSNT-induced sympathetic sprouting. (a) Color inverted image showing TH-IR fibers sprouting from perivascular plexuses in a DRG section of CSNT rats on POD 14. (b) Image is generated after tracing all the TH-IR fibers in (a). (c) Microscopic photographs of basket-like structures (arrows). (d) Nerve block decreased sympathetic fiber density measured on POD 14. p p P , 0:01; Student’s t-test.
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Ramer et al., 1998). Likely, the release of norepinephrine from sprouting sympathetic nerve endings excites myelinated or unmyelinated primary afferent neurons by activating a-adrenoceptors, contributing to central sensitization, and exaggerating ongoing pain and hyperalgesia (Gold et al., 1994; Liu et al., 1999; Michaelis et al., 1996; Sato and Perl, 1991; Xie et al., 1995). The most astonishing finding of our study is that lidocaine administration at an early stage of nerve injury remarkably reduces sympathetic sprouting. Lidocaineinduced anesthetic effects only last about 30 – 45 min, consistent with its suppressive effects on nerve pulse generation or neuronal spontaneous activity (Devor et al., 1992). However, the long-term effect after repetitive therapy with lidocaine in a subpopulation of neuropathic pain sufferers cannot be accounted for simply by its local anesthetic effects. Our results suggest that, in addition to delaying or preventing the development of central sensitization, decreasing sympathetic sprouting in pathologic DRGs may be a previously unfound mechanism underlying its prolonged therapeutic effect. Based on our findings it is reasonable to believe that lidocaine reduces sympathetic sprouting in the pathologic DRGs by depressing abnormal neuronal discharges. In support of this hypothesis, sprouting sympathetic fibers form basket structures preferentially in association with medium- and large-sized neurons (Chung et al., 1996; McLachlan et al., 1993), which are often present with highfrequency and/or bursting discharges after nerve injury (Zhang et al., 1997). Results from our preliminary study revealed that systemic administration of 4-aminopyridine, a Kþ channel blocker that enhances spontaneous activity, increased sympathetic sprouting, further, supporting our hypothesis. Because endogenous neurotrophins are proven factors that contribute to sympathetic sprouting (Ramer et al., 1999; Walsh et al., 1999; Zhou, 1996; Zhou et al., 1999), and there is sufficient evidence to suggest that neurotrophin expression/release is regulated by neuronal activity (Kim et al., 1994), it is possible that lidocaine may reduce sprouting by interfering with neurotrophin expression. However, additional studies are needed to demonstrate that neurotrophin expression can be reduced by systemic lidocaine or local nerve block, and that this effect is mediated by neuronal activity. The mechanisms underlying sympathetic sprouting in the adjacent intact DRGs or in DRGs contralateral to the nerve injury are not clear. However, as proposed by Ramer et al. (1999), sympathetic sprouting may involve the activation of satellite glia in the pathologic DRGs. If this is the case, then systemic lidocaine may interrupt the function of satellite glia through an unknown mechanism, which, in turn, leads to the reduction of sympathetic sprouting. The results of our studies have immediate clinical potential. Traumatic injury to soft-tissue, bone, and/or nerve often leads to a pathological pain state known as complex regional pain syndrome (Scadding, 1999).
Although there are conflicting laboratory reports with respect to the role of sympathetic sprouting in the development and/or persistence of neuropathic pain, clinically, there is an abundance of evidence that, in some patients, pain is maintained by efferent noradrenergic sympathetic activity and circulating catecholamines (sympathetically maintained pain, SMP) (Roberts, 1986). Pharmacologic sympathetic block or surgical sympathectomy may provide satisfactory pain relief in such patients. It is likely that the role of sympathetic sprouting in the development or persistence of neuropathic pain may very well depend on the type of nerve injury and/or the time after injury. Our results suggest that new drugs should be developed to disrupt the hyperexcitability cycle taking place in the DRG neurons and to reduce or prevent sympathetic sprouting. This will provide a new, non-opioid therapeutic approach for treating chronic pain resulting from peripheral nerve injury or injury to the lumbar ganglia.
Acknowledgements We would like to thank Drs Tony Yaksh and Steve Rossi of the Department of Anesthesiology, UCSD, for measuring the plasma level of lidocaine. This work was supported by National Institute of Neurological Disorders and Stroke (NINDS) Grant R01NS39568.
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