An animal model of nociceptive peripheral neuropathy following repeated cisplatin injections

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Experimental Neurology 182 (2003) 12–20

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An animal model of nociceptive peripheral neuropathy following repeated cisplatin injections Nicolas Authier,a Jean-Pierre Gillet,d Joseph Fialip,b Alain Eschalier,c and Franc¸ois Coudorea a

Laboratoire de Toxicologie, Faculte´s de Me´decine et Pharmacie, EMI INSERM/UdA 9904, 28 place Henri Dunant, BP 38, 63001 Clermont-Ferrand, France, b Laboratoire de Pharmacologie, Faculte´s de Me´decine et Pharmacie, EMI INSERM/UdA 9904, 28 place Henri Dunant, BP 38, 63001 Clermont-Ferrand, France, c Laboratoire de Pharmacologie Me´dicale, Faculte´s de Me´decine et Pharmacie, EMI INSERM/UdA 9904, 28 place Henri Dunant, BP 38, 63001 Clermont-Ferrand, France d Centre de Recherche, 63200 Riom, France Received 11 February 2002; accepted 21 August 2002

Abstract We report the assessment of motor and sensory behaviors using an electrophysiologic and an histologic approach, in a rat model of cisplatin peripheral neuropathy. Cisplatin was injected intraperitoneally one (3 mg/ kg), two (2 mg/kg), or three (1 mg/kg) times a week up to a cumulative dose of 15 or 20 mg/kg. With regard to nociceptive signs, we observed mechanical and thermal (cold stimuli) hyperalgesia and allodynia associated with minor motor disorders for the 3 mg/kg dose. Peripheral nerve conduction velocities were decreased in the cisplatin-(3 mg/kg) treated group. In addition, the histologic approach revealed that large axons were more frequently affected than the small ones, and nonmyelinated axons were unaffected. However, even in the most severe cases, myelin sheaths remained within normal limits. This animal model of nociceptive neuropathy would be suitable to study the pathophysiologic mechanisms of neuropathic pain and to test potential neuroprotective agents. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Nociception; Hyperalgesia; Allodynia; Neurotoxicity; Rat; Chemotherapy

Introduction Neuropathic pain is usually difficult to treat because the etiology is heterogeneous and the underlying pathophysiology is complex. The clinical features of neuropathic pain include the paradoxical combination of sensory loss in the painful neuropathic area and hypersensitivity phenomena such as allodynia and hyperalgesia (Bennett, 1994). Because no universally efficacious therapy exists for these nociceptive signs, a better understanding of the mechanisms responsible for these nociceptive signs is required to develop more specific therapies. Among the toxic neuropathies, those that are a consequence of certain chemotherapies (paclitaxel, vincristine, and cisplatin) frequently involve the development of painful sensory disorders and sometimes require the cessation of

anticancer therapy (Airiau, 1999; Hilkens and ven den Bent, 1997; Kaplan and Wiernick, 1982; Windebank, 1999). Cisplatin is used extensively in the treatment of cancers (testicular, ovarian, breath, bladder, lung), alone or in combination with another neurotoxic agent, paclitaxel, which is associated with serious adverse effects including nephrotoxicity and peripheral neurotoxicity. Renal toxicity is limited by chloride diuresis before, during, and after treatment. Sensory neuropathy, with complaints of paresthesiae and dysesthesiae in the distal extremities, is the major side effect (Krarup-Hansen et al., 1993). This neurotoxicity is dosedependent and appears in patients who receive more than 400 mg/m2 cisplatin (Cavaletti et al., 1992a; Siegal and Haim, 1990; Thompson et al., 1984). Cisplatin neuropathy involves predominantly large myelinated sensory nerve fibers and may continue to deteriorate for up to 3 to 4 months

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N. Authier et al. / Experimental Neurology 182 (2003) 12–20

after cessation of therapy with a gradual but often incomplete recovery (Hovestadt et al., 1992). These observations led to the hypothesis that cisplatin induces a primary neuronopathy followed by an axonopathy involving large myelinated fibers (Verdu et al., 1999). Electrophysiologic evaluations show that sensory nerve conduction is slowed and there is a reduction in the amplitude of the sensory action potentials (Cavaletti et al., 1992b; Lo Monaco et al., 1992; Pirovano et al., 1992; Thompson et al., 1984). In animal studies, behavioral assessments have shown sensory (thermal hypoalgesia) and motor (coordination and motor force decrease) disorders after repeated injections of cisplatin in rats or mice (Authier et al., 2000a; Barajon et al., 1996; Boyle et al., 1999; Tredici et al., 1998; Verdu et al., 1999). Numerous neurophysiologic studies have shown that cisplatin decreases sensory nerve conduction velocities and reduces the amplitude of nerve action potentials, with minimal or no motor involvement (Boyle et al., 1999; De Koning et al., 1987). Morphologic ultrastructural observations indicate that the nucleolus in the nuclei of primary sensory neurons is the structure most compromised in experimentally induced cisplatin neuronopathy (Cavaletti et al., 1992b; Mu¨ ller et al., 1990; Tomiwa et al., 1986). The pathologic changes found in peripheral nerves included a decrease in the number of large- and medium-size neurons indicating neuronal atrophy and advanced Wallerian degeneration of some large myelinated fibers (Barajon et al., 1996; Cavaletti et al., 1992b). Cisplatin may be expected to accumulate in dorsal root ganglia, leading to nucleolar damage and an alteration in the peptide content, and would also exert its neurotoxic effects through Schawnn cells (Hol et al., 1994; Yamamoto et al., 1997). We have previously described an initial study of the sensitive neurotoxicity of repeated cisplatin injections in rats using behavioral tests that showed a mechanical hyperalgesia and allodynia in treated rats (Authier et al., 2000a). However, the schedule of cisplatin administration and the clinical status of rats were not optimal, and improvements were possible. The objective of this study was to fully describe the behavioral, histologic, and electrophysiologic disorders characterizing an animal model of painful neuropathy following cisplatin administration, which is associated with a good clinical status (i.e., absence of abnormal clinical signs compared to controls). Further studies of the effects of cisplatin therapy are needed to ensure the prevention and treatment of the painful symptoms associated with the peripheral neuropathy that can occur clinically.

Materials and methods Animals Seventy-two male Sprague-Dawley rats (Charles River, St-Aubin-Le`s-Elbeuf, France), weighing 180 to 200 g at the beginning of the experiment, were used and housed in

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plastic cages on a 12-h light/12-h dark cycle with access to water and food ad libitum. Room temperature was maintained at 22°C and relative humidity was usually between 50 and 60%. Animals were allowed a 1-week acclimatization period before use in experiments. Drugs Cisplatin-treated groups Cisplatin (10 mg/20 ml, Laboratoires Qualimed Levallois-Perret, France) was diluted in normal saline (0.9% NaCl) just before administration to a final concentration between 0.08 and 0.15 mg/ml, depending on the animal weight, while ensuring that volumes of less than 5 ml would be injected into the peritoneal cavity. Control groups Injected volumes of saline (0.9% NaCl) were calculated according to the weight of the rat. Experimental procedures Cisplatin was administered intraperitoneally once a week (Monday, day 0) at a dose of 3 mg/kg (cumulative dose, 15 mg/kg), twice a week (Monday and Friday) at a dose of 2 mg/kg (cumulative dose, 20 mg/kg), and three time a week (Monday, Wednesday, and Friday) at a dose of 1 mg/kg (cumulative dose, 15 mg/kg) for 5 weeks. Before each injection, 2 ml of sterile saline solution was given subcutaneously to prevent renal damage via hyperhydration. Treatments were randomized within each cage of four rats (three dose and saline). To avoid acute effects, the injections were therefore given after the tests were performed. Rats were divided in two groups of 36 (9 rats/dose) as follows: group 1 for testing mechanical stimuli, motor activity, and histologic studies and group 2 for testing thermal stimuli, grip strength, and electrophysiologic studies. Assessment of general toxicity Body weights (g) were measured before each injection and every 5 days after the last injection. Motor activity was monitored in a dark room once a week (Saturday) using an actimeter Actisystem (Apelex, Passy, France) over a 10-min interval. The scoring system quantified rat’s movements for 10 min in a cage using an electromagnetic field. Superficial temperature (°C) was assessed with an electronic thermometer (Thermoscan IRT2020, Braun, Germany) in the right ear, twice a week (Monday and Thursday) after performing the tests. In addition, all rats were examined every day to detect abnormal clinical signs such as piloerection, hindlimb weakness, or gastrointestinal disorders.

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Behavioral assessment All behavioral tests were conducted before each injection of cisplatin. The guidelines of the IASP Committee for Research and Ethical Issues were followed for all experiments (Zimmermann, 1983). Grip strength test (Meyer et al., 1979) After the strain gauge was zeroed, the rat was positioned with both forepaws inside the front grip grid. When the rat gripped the grid, it was steadily pulled backward by the tail until its grip was broken. The reading on the strain gauge was recorded (N), the strain gauge was zeroed, and the rat retested until three successive readings were obtained. Paw pressure test The paw pressure test has been previously described by (Randall and Selitto, 1957). Nociceptive thresholds, expressed in grams, were measured by applying increasing pressure to the right hind paw using an Ugo Basile analgesimeter (Apelex). The parameter used to quantify the nociceptive threshold was the vocalization of the animal. Rats were habituated to the testing procedures and handling by the investigator during the week before the experiment. Experiments were performed until two similar consecutive pressure values were obtained. The cutoff pressure was 450 g. Von Frey hair test (Chaplan et al., 1994) To assess changes in mechanical nociceptive thresholds, rats were placed on a plastic mesh floor covered by transparent Plexiglas cages. On the week before the first administration of cisplatin or saline, rats were placed in the test environment each day for 15 min. For testing, rats were allowed to acclimatize for 10 min. Successively greater diameter von Frey nylon monofilaments (Stoelting, Wood Dale, IL) (pressures 1.202, 1.479, 2.041, 3.630, 5.495, 8.511, 11.749, 15.136, and 28.840 g) were applied to the medial plantar surface of both hindpaws. For each filament, a series of five stimuli (1-s stimulus) were applied within a 5-s interstimulus period per paw. Paw withdrawal threshold was defined as the minimum pressure required to elicit a withdrawal reflex of the paw. Plantar test (Hargreaves et al., 1988) The rats were placed on a glass plate under a Plexiglas cage for 15 min before each experiment. On the week before the first administration of cisplatin or saline, rats were placed in the test environment each day for 15 min. A radiant heat stimulus was applied to the plantar surface of each hindpaw by aiming a beam of light produced by a 50-W projection lamp (Apelex). When the rat lifted the paw, the light beam was turned off automatically allowing measurement of the time between the start of the light stimulus and the foot lift. This time was defined as the withdrawal latency. Heat stimuli were stopped at 20.8 s to

avoid skin injury. Three trials were conducted on each hindpaw, with 5 min between each trial. Tail immersion test (Necker and Hellon 1978) The tail of the rat was immersed in cold or hot water maintained at noxious (4 and 46°C) or nonnoxious (10 and 42°C) temperatures, until the tail was withdrawn. The duration of immersion was recorded and a cutoff time of 15 s was used. Rats were habituated to the testing procedures and to handling by the investigator during the week before the experiment. Electrophysiologic study Six rats in the saline and 3 mg/kg cisplatin-treated group were anesthetized by intraperitoneal injection of ethyl carbamate (2.5 mg/kg) 5 days after the last injection. Samples of sciatic nerves were quickly taken, preserved in a RingerLocke solution and then placed between two electrodes separated by 2.5 cm. After estimating the depolarization threshold of the nerve, the nerve was stimulated 50 times with a supramaximal stimulus. Nerve conduction velocity was directly calculated using a computer and locally made software. All measurements were performed under constant conditions in a Faraday cage. Microscopic examination Fixation of tissues At the time of euthanization, based on satisfactory results of behavioral tests (i.e., significant decrease of sensory thresholds), selected rats were deeply anesthetized by an intraperitoneal injection of a 6% aqueous pentobarbital solution (0.5 ml/100 g body wt, i.e., 300 mg/kg). After opening the thoracic cavity, the vascular system was intracardiacally perfused using a phosphate buffer solution (pH 7.2– 7.4, 200 ml), followed by a 4% paraformaldehyde ⫹ 1% glutaraldehyde (200 ml) solution (in the same phosphate buffer solution). Samples of the lumbar spinal cord, sciatic nerve, and skin with the subcutaneous tissue of the paws were stored in the fixative solution as above, until trimmed for histology and examination of the ultrastructure. Histology Samples of lumbar spinal cord, sciatic nerve, and skin with subcutaneous tissue of the paws were paraffin embedded, cut 4 ␮m thick, and stained by the hematoxylin and eosin method before examination using light microscopy. Electron microscopy Blocks of lumbar spinal cord, sciatic nerve and skin with subcutaneous tissue of the paws of approximately 2 ⫻ 2 ⫻ 1 mm were postfixed in a 1% osmium tetroxide solution and embbeded in epon after graded dehydration in alcohol. Semithin and ultrathin sections were obtained using a Reichert Ultracut E ultramicrotome. Semithin sections were

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stained with toluidine blue before light microscopy. Ultrathin sections were contrasted by the uranyle acetate-lead citrate method before examination with a Jeol 1200EX.

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(P ⬍ 0.001; ⫺1.7°C) and after 4 weeks for both the 1 (P ⬍ 0.001; ⫺1.3°C) and the 3 mg/kg (P ⬍ 0.01; ⫺1.2°C) groups with recovery days after the last injection for the 3 mg/kg group.

Statistical analysis Behavioral examinations Behavioral data were evaluated using analysis of variance (Fisher test) followed by Student’s t test to detect either differences within treatment groups between baseline and postinjection values or differences between each treatment group on each day tested. Investigators performing the behavioral and histologic studies were blinded with respect to the treatment received by each group.

Results Assessment of general toxicity All animals received the injections as scheduled in the protocol. Three rats died in the 2 mg/kg group (30%), 3, 10, and 15 days, respectively, after the last injection. In the 3 mg/kg group, one rat died (10%) 10 days after the last injection. No rat in the saline control group died throughout the course of the experiment, and no alterations in temperature, clinical signs, or motor activity were observed. In the 1 mg/kg cisplatin group, body weight gain was significantly decreased compared to saline from the third injection with a maximal decrease (P ⬍ 0.001; ⫺50%; 218 ⫾ 27 vs 436 ⫾ 21 g) at the end of the experiment. Rats receiving 2 mg/kg showed a significantly lower body weight gain compared to saline from the first injection (fourth day) to the end of the study with a maximal decrease after the last injection which then remained stable (P ⬍ 0.001; ⫺55%; 170 ⫾ 20 vs 377 ⫾ 11 g). The highest dose, 3 mg/kg, induced a significant decrease in body weight gain compared to saline 1 week after the first injection until the end of the experiment, with a maximal decrease 1 week after the last injection (P ⬍ 0.001; ⫺30%; 263 ⫾ 17 vs 377 ⫾ 11 g). No rapid body weight recovery was observed except for the 3 mg/kg group who regained weight after the last injection. Motor activity was decreased in all groups for the 1 mg/kg group from the sixth injection to the end with a maximal decrease after the last injection (P ⬍ 0.001; ⫺80%; 68 ⫾ 5 vs 336 ⫾ 40), for the 2 mg/kg group from the fourth injection to the end with a maximal decrease after the last injection (P ⬍ 0.001; ⫺90%; 32 ⫾ 6 vs 336 ⫾ 40), and finally for the 3 mg/kg group from the third injection to the end of injection corresponding to the maximal decrease (P ⬍ 0.01; ⫺55%; 151 ⫾ 15 vs 336 ⫾ 40). Most cisplatintreated rats showed a definite change in motor activity that was characterized by a neuropathic gait consisting of toe walking with an arched back. Significant variation was observed in the temperature assessment. Compared to saline, a slight hypothermia was observed after 2 weeks of treatment for the 2 mg/kg group

Before the first injection, mean thresholds were not significantly different between the saline- and cisplatin-treated groups. Grip strength test Grip strength was similar irrespective of treatment until the 27th day. In the 2 mg/kg group a significant decrease, compared to saline, was then observed to the end of the experiment with a maximal decrease after the last injection (P ⬍ 0.01, ⫺30%; 10.1 ⫾ 0.7 vs 14.4 ⫾ 0.7 N). In the 1 mg/kg group, a significant decrease compared to saline was observed from the last injection to the end (maximal decrease, P ⬍ 0.01, ⫺26%; 11.9 ⫾ 0.7 vs 16.2 ⫾ 0.5 N). Finally, the 3 mg/kg group showed a significant decrease only after the last injection (P ⬍ 0.05, ⫺15%; 12.2 ⫾ 0.5 vs 14.4 ⫾ 0.7 N) followed by a complete recovery over the last 2 weeks of the experiment. Paw pressure test (Fig. 1) One-hundred percent of rats treated with the 1 mg/kg dose showed a significant decrease in vocalization threshold compared to saline, from the fifth injection to the end of the experiment, with a maximum decrease 3 days after the last injection (P ⬍ 0.001; ⫺30%). A significant reduction in the vocalization threshold compared to the saline group was observed in the 2 mg/kg group from the second injection to the end of the study (100% of treated rats). The maximum decrease appeared 3 days after the eighth injection (P ⬍ 0.001; ⫺39%). In 100% of rats in the 3 mg/kg group, vocalization thresholds were significantly decreased compared to the saline group, from the first injection to the end of the experiment. The maximum decrease appeared 1 week after the last injection (P ⬍ 0.001; ⫺35%). With regard to the saline group, no decrease in vocalization threshold was observed during the course of the study. Von Frey hairs test (Fig. 2) In the 1 mg/kg group, a significant decrease in withdrawal thresholds compared to the saline group was observed in 88% of rats, from the eighth injection to the end of the study, with a maximum decrease 3 days after the last injection (P ⬍ 0.01; ⫺61%). A significant reduction in the withdrawal thresholds compared to the saline group was noted in 75% of rats in the 2 mg/kg group from the fourth injection to the end of the experiment, with a maximum decrease 5 days after eighth injection (P ⬍ 0.01; ⫺74%). In 100% of rats in the 3 mg/kg group, withdrawal thresholds were significantly decreased compared to saline group, from the second injection to the end of the experiment. The

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Fig. 1. Mechanical hyperalgesia. Time course of paw withdrawal thresholds of treated (‚, 2 mg/kg cisplatin, n ⫽ 8; E, 3 mg/kg cisplatin, n ⫽ 8), and control (}, saline, n ⫽ 8) rats submitted to an increasing pressure on a hindpaw. Each group received two, one, and two intraperitoneal injections, respectively, per week for 5 weeks. Scores were determined twice a week for 6 weeks. The bars represent SEM. No significant variation was observed for control rats. A significant difference (*P ⬍ 0.05. **P ⬍ 0.01, ***P ⬍ 0.001, analysis of variance followed by Student t test in unpaired series) compared to the saline group was observed from day 7 to the end of the study for both the 2 and the 3 mg/kg cisplatin groups.

was observed at any time in either the control rats or the cisplatin-treated rats, irrespective of the dose used. On the other hand, application of a hot noxious thermal stimulus (46°C) induced a significant increase in withdrawal latency in the 1 mg/kg group (100% of rats) compared to saline from the 12th injection to the end of the study (maximal increase, P ⬍ 0.01, ⫹30%). The withdrawal latency of the 2 mg/kg group was also significantly increased (100% of rats) compared to saline from the 6th injection to the end of experiment (maximal increase ⫹35%, P ⬍ 0.01). Finally, we observed a significant increase compared to saline in the 3 mg/kg group (100% of rats), from the fourth injection to the end of the study (maximal increase ⫹25%, P ⬍ 0.01). When a cold noxious (4°C) stimulus was used (Fig. 3), in 63% of the 1 mg/kg cisplatin-treated group we observed a significant decrease from the 14th injection with a maximal decrease after the last injection (P ⬍ 0.001, ⫺45%) and a complete recovery 1 week later. A significant decrease in the withdrawal latency compared to saline occurred in 88% of rats in the 2 mg/kg group from the 5th injection to 8 days after the last injection, with a maximal decrease just before the last injection (P ⬍ 0.001, ⫺60%). Sixty-three percent of the 3 mg/kg group also showed a significant decrease compared to saline in withdrawal latencies, from the 4th injection to 17 days

maximum decrease was observed just before the fifth injection (P ⬍ 0.001; ⫺79%). No modification of the withdrawal threshold was observed at any time in the saline-treated group. Plantar test In the 1 mg/kg group, 75% of rats showed a significant increase in withdrawal latencies, compared to saline, from the 12th injection to the end of the experiment, with a maximal increase 6 days after the last injection (P ⬍ 0.01, ⫹26%; 10.7 ⫾ 0.2 vs 8.5 ⫾ 0.3 s). With regard to the 2 mg/kg group, we observed a significant increase compared to saline in 88% of rats from the 6th injection to the end of the study, with a maximal increase 6 days after the last injection (P ⬍ 0.01, ⫹35%; 11.4 ⫾ 0.5 vs 8.5 ⫾ 0.3 s). A significant increase in the withdrawal latencies compared to saline was observed in 88% of rats in the 3 mg/kg group from the fourth injection to the end of the experiment and with a maximal increase 6 days after the last injection (P ⬍ 0.01, ⫹23%; 10.4 ⫾ 0.4 vs 8.5 ⫾ 0.3 s). Tail immersion test Whatever the temperature applied, the control group did not show modification of the withdrawal latency at any time. With regard to nonnoxious thermal stimulus (42°C), no significant variation in tail withdrawal times

Fig. 2. Mechanical allodynia. Paw withdrawal thresholds of treated (‚, 2 mg/kg cisplatin, n ⫽ 8; E, 3 mg/kg cisplatin, n ⫽ 8) and control (}, saline, n ⫽ 8) rats to von Frey hairs applied to the plantar surface of the hind paw are shown. Each group received two, one, and two intraperitoneal injections, respectively, per week for 5 weeks. Scores were determined twice a week for 6 weeks. The bars represent SEM. No significant variation was observed for control rats. A significant decrease in mechanical threshold (*P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001, analysis of variance followed by Student t test in unpaired series) compared to the saline group was observed from day 14 to the end of the study for both the 2 and the 3 mg/kg cisplatin groups.

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Fig. 3. Cold thermal hyperalgesia. Tail withdrawal latencies of treated (‚, 2 mg/kg cisplatin, n ⫽ 8; E, 3 mg/kg cisplatin, n ⫽ 8) and control (}, saline, n ⫽ 8) rats after immersion in a cold (4°C) water bath are shown. Each group received two, one and two intraperitoneal injections, respectively, per week for 5 weeks. Scores were determined twice a week for 7 weeks. The bars represent SEM. No significant variation was observed for control rats. A significant difference (*P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001, analysis of variance followed by Student t test in unpaired series) compared to the saline group was observed from day 17 to day 42 for the 2 mg/kg cisplatin group and from day 24 to day 45 for the 3 mg/kg cisplatin group.

after the last injection, with a maximal decrease 3 days after the 5th injection (P ⬍ 0.01, ⫺42%). After a cold nonnoxious (10°C) stimulus was applied (Fig. 4), the tail withdrawal latency of the 1 mg/kg group showed a significant decrease just after the last injection (P ⬍ 0.05, ⫺29%). In the 2 mg/kg group, we observed a significant decrease in the tail withdrawal latency from the 8th injection to the end of the study, with a maximal decrease 13 days after the last injection (P ⬍ 0.001, ⫺43%). Finally, 75% of rats treated with the 3 mg/kg dose showed a significant decrease in the tail withdrawal latency from the 4th injection to the end of the experiment with a maximal decrease 17 days after the last injection (P ⬍ 0.01, ⫺30%).

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Fig. 4. Cold thermal allodynia. Tail withdrawal latencies of treated (‚, 2 mg/kg cisplatin, n ⫽ 8; E, 3 mg/kg cisplatin, n ⫽ 8) and control (}, saline, n ⫽ 8) rats after immersion in a cold (10°C) water bath are shown. Each group received two, one and two intraperitoneal injections, respectively, per week for 5 weeks. Scores were determined twice a week for 7 weeks. The bars represent SEM. No significant variation was observed for control rats. A significant difference (*P ⬍ 0.05, **P ⬍ 0.01, ***P ⬍ 0.001, analysis of variance followed by Student t test in unpaired series) compared to the saline group was observed from day 28 to the end of the study for both the 2 and the 3 mg/kg cisplatin groups.

cally and ultrastructurally within normal limits, and the axon was the sole affected component of the nerve. Among the range of the dosage levels, 3- and 2-mg groups showed similar degenerative lesions, in terms of incidence and severity, while at 1 mg, axonal degenerations were rare and of low severity. Electrophysiologic study In the 3 mg/kg cisplatin-treated group, we observed a significant decrease (P ⬍ 0.05, ⫺28%) in the peripheral nerve conduction velocity (34 m/s), compared to the saline

Histologic study Microscopic and ultrastructural evaluation of the three examined organs (lumbar spinal cord, sciatic nerve, paw subcutaneous tissue) showed various degrees of axonal degeneration (Figs. 5–9). The most striking changes were found in the fine nerve fibers from the subcutis. Large axons were more frequently affected than the small ones, and nonmyelinated axons were unaffected. However, even in the most severe cases, myelin sheaths remained histologi-

Fig. 5. Normal sciatic nerve. Transversal section of the sciatic nerve in a control rat. Semithin section. Toluidine blue. ⫻1700.

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Fig. 6. Degenerated sciatic nerve. Transversal section of the sciatic nerve in a cisplatin-treated rat (3 mg/kg) showing several degenerative changes (arrows). Semithin section. Toluidine blue. ⫻1700.

Fig. 8. Normal distal nervous fiber. Transversal section of a normal subcutaneous nervous fiber from a control rats. Ultrathin section. Lead citrate and uranyl acetate. ⫻11,500.

group (43.5 m/s), after the fifth and last injections (cumulative dose of 15 mg/kg).

This study describes a new model of peripheral nociceptive neuropathy produced by the neurotoxicity of cisplatin, an antineoplastic agent. This model displays nociceptive signs such as a mechanical hyperalgesia and allodynia, a cold thermal hyperalgesia and allodynia, and a heat thermal hypoalgesia associated with significant electrophysiologic and histologic disorders. The five weekly intraperitoneal injections of 3 mg/kg are a good compromise between the preservation of good general clinical status of the rats and the induction of nociceptive neuropathy. Using the same cumulative dose of cisplatin compared to the model previously published (Authier et al., 2000a), we have simplified the administration schedule, while retaining or enhancing the nociceptive signs and confirming that peripheral neuropathy reveals a decrease in nerve conduction velocity associated with peripheral histologic lesions. Contrary to the vincristine- or the paclitaxel-induced models of peripheral neuropathy, a decrease in motor activity and motor strength could not be completely pre-

vented; however, the decrease in motor activity could be related to spontaneous nociception that could also be responsible for the neuropathic gait observed (Authier et al., 1999, 2000b). Heat thermal hypoalgesia demonstrated using two different behavioral tests could be partially explained by an association with significant superficial hypothermia. Biochemical studies have measured the levels of peptides such as substance P, calcitonin gene-related peptide (CGRP), galanin, and somatostatin revealing alterations in the peptide content of dorsal root ganglia and in the sciatic nerve following cisplatin (Barajon et al., 1996). A reduction in the content of neuropeptides such as CGRP and VIP in the peripheral nerve fibers innervating cutaneous structures in foot pads as described by (Verdu et al., 1999) could be linked to this thermal hypoalgesia. This increase in heat thresholds was not significant in our previous study (Authier et al., 2000a) and was not associated with a significant decrease in cutaneous temperature. Moreover, this lost of sensibility has already been observed in numerous animal studies (Barajon et al., 1996; Boyle et al., 1999; Rebert et al., 1984; Tredici et al., 1998; Verdu et al., 1999). Although this hypothermia is probably implicated in the heat thermal hypoalgesia observed, it probably underestimates the decrease in response to cold thermal stimuli.

Fig. 7. Degenerated distal nerve fiber. Subcutaneous tissue (paw) from a cisplatin-treated rat (3 mg/kg) with a fine nerve showing numerous degenerative changes. Semithin section. Toluidine blue. ⫻1100.

Fig. 9. Axonal lesions. Subcutaneous tissue (paw) from a cisplatin-treated rat (3 mg/kg). Transversal section of a fine nerve fiber showing several axonal lesions (star) while myelin sheath remains normal. Ultrathin section. Lead citrate and uranyl acetate. ⫻9000.

Discussion

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Alteration of axonal transport also induces an accumulation of neuropeptides in the soma of affected neurons and in the dorsal root ganglia. This is in accordance with a possible central sensitisation which is reflected in the allodynia noted. The increase in peptide concentration in the dorsal root ganglia is probably due to cisplatin-related damage to the axonal transport system rather than to an increase neuropeptide synthesis (Barajon et al., 1996). According to Lajer and Daugaard (1999), hypomagnesemia is a frequent complication during chemotherapy with cisplatin, affecting 90% of patients. Magnesium is known to bind to N-methyl-D-aspartate (NMDA) receptor channels. A decrease in magnesium levels could enhance nociceptive signs by increasing binding between glutamate and NMDA receptors (Begon et al., 2000). This is probably not the main cause but could explain in part the hyperalgesia and allodynia observed. The previous cisplatin model published (Authier et al., 2000a) assessed sensory thresholds after the same cumulative dose (15 mg/kg) of cisplatin administered using a different dosage schedule (10 injections). Mechanical thresholds were similar; however, thermal thresholds displayed significant differences in this new model. Therefore, the reduction in the number of injections is associated with a better clinical status of the rats due to the longer period of recovery between each dose of cisplatin. The ex vivo electrophysiologic study demonstrated for the 3 mg/kg cisplatin group a significant decrease in nerve conduction velocity compared to the saline group. This observation agrees with cisplatin results published previously describing a decrease in sensory nerve conduction velocity and the amplitude of nerve action potentials, with minimal or no motor involvement (Barajon et al., 1996; Lajer and Daugaard, 1999; Gerritsen van der Hoop et al., 1994; Hamers et al., 1991; Hargreaves et al., 1988; Hilkens and ven den Bent, 1997; Rebert et al., 1984; Tredici et al., 1994, 1998; Verdu et al., 1999). The present histologic and ultrastructural findings are consistent with the changes described in the literature, except for the degeneration of the myelin sheaths. This change, absent under the conditions of the present study, was sometimes cited among the lesions seen after cisplatin administration. Thus, loss of large myelinated fiber, especially in the distal part of the nerve and abnormalities in the myelin sheath of medium fibers, have also been observed following cisplatin (Luo et al., 1999). Furthermore, cisplatin slows the rate of microtubule disassembly and alters the axonal transport system. The dorsal root ganglia (DRG) has been suggested to be a primary site of damage as cisplatin concentrations in the DRG are 5 to 20 times higher than in the central nervous system and spinal cord and similar to those found in tumors (Thompson et al., 1984). This model of cisplatin-induced peripheral nociceptive neuropathy in conjunction with previously published models using paclitaxel and vincristine (Authier et al., 1999, 2000a,b) will enable study of the pathophysiologic mecha-

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nisms of neuropathic pain as induced by neurotoxic drugs. In spite of the similar nociceptive signs, these mechanisms may differ for different etiologies of neuropathy such as trauma, diabetes, and chemical toxicity (Bennett and Xie, 1988; De Koning et al., 1987; Courteix et al., 1993).

Acknowledgments This work was supported by the Association pour la Recherche sur le Cancer (ARC). We thank Dr. P. Duprat and Dr. S. Molon-Noblot for advice and support, Dr. A. Gross for comments on the manuscript, and C. Graulie`re and M. Levasseur for technical assistance.

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