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Anesth Analg 2005;101:1422-1426 © 2005 International Anesthesia Research Society doi: 10.1213/01.ANE.0000180197.32577.9B
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Abstract |
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Gabapentin acts primarily on the central nervous system. Therefore, we hypothesized that the direct epidural administration of gabapentin could have various advantages over its oral administration with respect to required dose, side effects, and efficacy. However, before administering gabapentin into the epidural space in a clinical setting, its neurotoxicity must be examined in animals. Thus, we evaluated neurotoxicity of epidural gabapentin by observing behavioral and sensory-motor changes, and by histopathological examinations of spinal cords and dorsal root ganglia in the rat. Twenty-seven rats were randomly divided into 3 groups, which were administered 0.3 mL (30 mg) of epidural gabapentin (group G, n = 9) and the same volume of epidural alcohol (group A, n = 9) or normal saline (group N, n = 9). No rats in groups G and N showed sensory-motor dysfunction, behavioral change, or histopathological abnormalities over a 3-wk observation period, whereas all rats in group A showed abnormalities. We conclude that the direct epidural injection of gabapentin in rats did not show any neurotoxic evidence in terms of sensory-motor functions and behavior, or by a microscopic histopathological evaluation. This study represents a first promising step toward the trial of epidural gabapentin in a clinical setting.
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Introduction |
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The effects of spinoaxial or oral gabapentin on neuropathic pain have been well established by animal (1–4) or clinical studies (5–7). The mechanism of gabapentin is unclear, but may involve binding to the spinal
2
calcium channel subunit (8). Previous studies had shown that the mRNAs for the 2 subunits are expressed at high levels in sensory neurons of dorsal root ganglion and in the spinal cord dorsal horn (9,10). In addition, binding studies have shown that the 2-1 and 2-2 subunits bind gabapentin with high affinities (8). In a clinical setting, the main purpose of spinoaxial administration of drugs is to reduce the dosage, maximize efficacy, and minimize side effects when the action site of the drug involved is the central nervous system. In addition, pain clinicians prefer the epidural administration of analgesics to intrathecal administration because it is less invasive and is likely to reduce the risk of neurotoxicity, especially when the administration is conducted over an extended period (11). These observations led us to hypothesize that the spinoaxial administration of gabapentin could have many advantages over its oral administration in a clinical setting.
After the introduction of spinoaxial catheterization at the end of 19th century, various drugs were injected clinically or experimentally via the spinoaxial route. Moreover, some of these drugs were either neurotoxic or potentially neurotoxic. In addition, preservatives added to drugs may be neurotoxic when injected spinoaxially (12). Therefore, if a drug is to be administered intrathecally or epidurally in a clinical setting, its safety must be determined initially by preliminary animal studies, because any toxic effect could have serious consequences (13). However, no animal studies have been performed on the possible neurotoxicity of spinoaxially injected gabapentin despite its excellent analgesic effect, and thus we undertook the present study to evaluate the neurotoxicity of epidurally injected gabapentin in a rat model.
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Methods |
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The experimental protocol used was reviewed and approved by our institutional Animal Care and Use Committee. All rats had free access to food and water and were individually housed under a 12-h light/dark cycle for 1 wk.
Anesthesia was induced by placing a rat in a closed box containing 4% enflurane in oxygen (3 L/min) with spontaneous ventilation. After loss of consciousness, anesthesia was maintained with 2%–3% enflurane via a loose-fitting mask. After applying a sterile dressing, epidural catheterization was performed, as we described previously (14,15) with some modification. Briefly, a 3-cm midline skin incision was made at the T13–L1 intervertebral space. Using a fine microscissors, a small hole was made at the center of the ligament flavum and a PE-10 catheter (Natsume, Japan) was inserted and gently advanced about 3 cm caudally. The catheter tip was placed in the space between L4 and L5. Cases were excluded if blood or cerebrospinal fluid was aspirated. A drop of cyanoacrylate (Aron-Alpha, Toagosei, Japan) was applied at the epidural catheter entry site. To confirm correct catheter positioning, we injected 0.15 mL of 2% lidocaine through the catheter after complete recovery from anesthesia, and we defined a correct epidural catheter placement as one that showed paralysis of the hindlimbs while the forelimbs retained normal motor power. If the test solution was accidentally injected intrathecally or IV, sudden respiratory arrest with or without cardiac arrest was observed; such cases were excluded from the study. The fascia and skin were sutured and antibiotic ointment was applied. After confirming correct epidural catheter placement, we examined gait, spinal deformity, and behavioral abnormalities for 3 days. If the rats showed abnormal findings during the 3-day observation period, they were excluded from this study.
Twenty-seven male Sprague-Dawley rats, weighing 250–350 g, were successfully prepared for this study, and these rats were divided equally into 3 groups. Under general anesthesia, 30 mg (0.3 mL, 100 mg of gabapentin dissolved in 1 mL of distilled water) of preservative-free gabapentin (Sigma-Aldrich, St. Louis, MO) was injected via an epidural catheter in group G (n = 9). In groups A (n = 9) and N (n = 9), the same volume of 40% alcohol and 0.9% normal saline were injected via an epidural catheter, respectively. After recovery from anesthesia, rats were individually housed under a 12-h light/dark cycle.
In the pilot study, we evaluated the extent of spreading of contrast medium by fluoroscopy, and found that the total spreading spinal segments of 0.3 mL of contrast medium was 10 or 11, which was enough to affect the entire spinal cord segment cropped for histopathological examination.
The epidural gabapentin dosage was determined in this study by observing the development of behavioral changes, e.g., motor deficit, crying, or aggressive behavior. Epidural gabapentin was started from 0.3 mg (0.3 mL, 1 mg of gabapentin was dissolved in 1 mL of distilled water), which was the maximal amount injected intrathecally in previous studies (2–4). We did not find any motor deficit of grade 2 or more or any behavioral changes at the dose of 30 mg (0.3 mL, 100 mg of gabapentin dissolved in 1 mL of distilled water), which was 100 times the previously reported maximal intrathecal dose (2–4).
Acute toxicity was evaluated 2 days after injection and chronic toxicity 7 and 21 days after injection. One examiner unaware of the study details observed motor and sensory deficits. Pinch-toe testing and motor-function evaluation were started 2 days after drug injection to exclude the possibility of the systemic effect of gabapentin. Pinch-toe testing was used to evaluate motor and sensory deficits (16). Motor function was assessed using a previously devised scoring system with some modification (17). Grades were defined as follows: grade 1 = normal gait with no evidence of motor paresis; grade 2 = normal gait with slight hindpaw deformity, such as plantar flexion of toes; grade 3 = slight gait disturbance with motor weakness and/or an inverted hindpaw; and grade 4 = a prominent limping gait with a dropped hindpaw. The rats with a motor disturbance of grade 2 or above were considered to a have motor deficit.
The spinal cord was cropped in 3 rats of each group 2 days after injection to evaluate acute toxicity. To evaluate chronic toxicity, the spinal cords of the remaining 6 rats/group were cropped on the 7th (3 rats/group) and on the 21st days (3 rats/group) after injection. Rats were killed under general anesthesia (as described above), by transcardial perfusion with 4% paraformaldehyde in 0.1 M phosphate buffer. Approximately 1-cm lengths of spinal cord, caudal and rostral to the catheter tip, were obtained. For light microscopy, spinal cords were fixed with 10% neutral formalin solution. Using standard tissue slide preparation methods, three blocks were made per sample. The slides were prepared from 4- to 5-µm sections stained with hematoxylin and eosin. To evaluate myelin loss, Luxol fast blue (a specific myelin stain) was added. Microscopic tissue analysis results (15,18,19) were classified in 7 ways, namely as, dural hypertrophy, adhesion of meninges to surrounding tissues, local neuritis, meningeal inflammation, local myelopathy, myelin loss, and peripheral neuropathy. One pathologist blinded to this study analyzed histological change.
Intergroup comparisons of body weights were analyzed using Kruskal-Wallis one-way ANOVA on ranks and this was followed by Dunn's method. Motor and sensory deficit and histopathological spinal cord data were analyzed using Fisher's exact test with a Bonferroni correction. A P value < 0.05 was considered significant.
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Results |
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All rats in groups N and G showed normal behavior throughout the study period, whereas all rats in group A showed both reduced activity and appetite. Rats in group A showed significantly low body weights 7 and 21 days after injection (P < 0.05, Table 1). All rats in groups N and G responded normally to pinch-toe testing and had a normal gait at each observation point. However, all rats in group A, except one rat on day 2 after injection, showed an abnormal response to pinch-toe testing. These animals also showed hindpaw deformity, and a gait disturbance of grade 2 or more (P < 0.05, Table 2).
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No histological lesions on hematoxylin and eosin and Luxol fast blue stains were observed in groups N or G at any time. However, in group A, 2 of the 3 spinal cords obtained 2 days after injection showed myelin loss and peripheral neuropathy and all of those obtained 7 and 21 days after injection showed various neuropathies (P < 0.05, Table 3 and Figs. 1 and 2).
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Discussion |
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We administered 30 mg of preservative-free gabapentin into the epidural space. In humans, this 30 mg (approximately 100 mg/kg) is equivalent to an epidural dose of 6000 mg. Moreover, the required dose for the epidural route is about 1/30 of that required for oral administration (20). Thus, the 30 mg of epidural gabapentin administered may be equivalent to an oral administration of about 180,000 mg for a human adult. Because the maximal recommended oral dose for gabapentin is 3600 mg in human adults, and because the dura mater of small animals has greater diffusibility than that of the human (21), we decided that a 30-mg dose of epidural gabapentin was sufficient for neurotoxicity evaluation purposes in the rat.
In the present study, all rats in group A showed reduced activity and appetite, poor weight gain, and acute and chronic neurotoxicity findings on histopathological examination, all of which might be considered as a sequela of alcohol-induced neurotoxicity (22–24). However, all rats in group G, similar to group N, showed no motor or sensory deficits and no abnormal histopathological findings at any time.
The symptoms of neuronal damage might be present even in the absence of histological change (25). Thus, neurotoxicity should also be assessed by examining sensory, motor, and behavioral changes. In the present study, no findings of neuronal damage, such as persistent motor function changes or sensory losses, were observed in rats administered gabapentin epidurally.
In this study, spinal cord neurotoxicity was determined by light microscopy. The use of electron microscope with morphometric methods for analyzing cell loss might have provided more information about the safety of epidural gabapentin. We are considering a further study including electron microscopy.
In conclusion, gabapentin administered epidurally to rats did not cause sensory, motor, behavioral, or histopathological changes that might suggest neurotoxicity, which implies that gabapentin could be directly administered epidurally in a clinical setting. However, similar studies on different animals are required to obtain reliable safety data on the epidural application of gabapentin before use in a clinical setting.
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Footnotes |
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Accepted for publication April 25, 2005.
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