Anesth Analg 2004;98:148-152
© 2004 International Anesthesia Research Society
PAIN MEDICINE
Antinociceptive and Neurotoxicologic Screening of Chronic Intrathecal Administration of Ketorolac Tromethamine in the Rat
H. Ayben Korkmaz, MD*,
Fikret Maltepe, MD*,
Serhat Erbayraktar, MD
,
Osman Yilmaz
,
Merih Güray, MD
,
M. 
erefettin Canda, MD,
Ali Günerli, MD*, and
Necati Gökmen, MD*
Departments of *Anesthesiology and Reanimation,
Neurosurgery,the
Animal Research Center, and the
Department of Pathology, Dokuz Eylül University, School of Medicine, 
zmir, Turkey
Address correspondence and reprint requests to Fikret Maltepe, MD, Dokuz Eylül Üniversitesi T
p Fakültesi, Anesteziyoloji ve Reanimasyon AD, 35340 INCIRALTI, IZMIR, TÜRKYE. Address email to fikret.maltepe{at}deu.edu.tr
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Abstract
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Many drugs are tested intrathecally to investigate alternatives to opioids. We aimed to explore the analgesic and possible neurotoxic effects of chronic intrathecally-administered ketorolac tromethamine in rats. Catheters were placed via atlantoaxial interval in 28 Wistar rats under anesthesia of intraperitoneally-injected thiopental 30 mg/kg. Rats were randomized into 4 groups and administered 4 repeated intrathecal doses of therapy with 5-day intervals. The control group received 10 µL of saline, and the other groups received 50, 150, and 400 µg of ketorolac tromethamine respectively. The formalin test, behavioral test, and histopathological examination of four different spinal cord levels were performed. Neither behavioral testing nor histopathological examination revealed abnormalities that would suggest neurotoxicity. Formalin tests showed that both phase I and phase II responses of ketorolac tromethamine groups were significantly less than those of the control group. Although phase I responses did not differ during comparisons among ketorolac tromethamine-administered groups, phase II responses decreased significantly in groups that received 150 and 400 µg of ketorolac tromethamine. Intrathecally administered ketorolac tromethamine reduced nociceptive responses and exhibited no untoward neurological effect even at large doses. However, its intrathecal use as a safe alternative drug for chronic pain remains to be investigated in other species.
IMPLICATIONS: The present study is unique because it has demonstrated that chronic intrathecal administration of ketorolac tromethamine in rats, even at considerably large doses, showed a potent analgesic effect during the formalin test without exhibiting any neurotoxic side effect.
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Introduction
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Ketorolac tromethamine (KT), a pyrolacetic acid derivative, is a nonsteroidal antiinflammatory drug and is frequently used for the management of postoperative pain, renal colic, sickle cell crisis, arthritis, lumbago, headache, and cancer pain either intrathecally or IM (1). KT has also been reported to provide successful analgesia when used via intracerebroventricular (2), intrathecal (3–5,7), and epidural (8) routes in experimental models. Analgesic effects of intrathecal administration of KT have been investigated in mouse (2), rat (4–7), and dog (8) models before its recent use in humans by Eisenach et al. (9). In those studies, various techniques, such as subcutaneous formalin injection (3), strychnine administration into the subarachnoid space (7), spinal nerve ligation (5), hot plate and tail-flick test (6), were used to induce pain. It has commonly been thought that single intrathecal KT doses produce a dose-dependent antinociceptive effect and have no side effects. However, direct proof of possible antinociceptive and neurotoxic effects of chronic intrathecal KT administration is still lacking. There were two goals of the present study. First, to determine whether intrathecal KT, administered chronically at different doses and concentrations, would provide analgesia in the rat formalin test. Second, to explore whether chronic administration would produce behavioral and/or histological evidence of neurologic damage in rats.
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Methods
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The study protocol was approved by the Animal Research Committee of Dokuz Eylül University. Wistar rats (male) weighing 240–260 g were used in this study. Animals were maintained in a 12-h light/dark cycle at a room temperature of 20°C to 22°C with water and food freely available.
Under general anesthesia with 30 mg/kg of intraperitoneal thiopental, catheters were placed intrathecally with modification of the method described by Yaksh and Rudy (10), by using the interval between the C1-C2 vertebrae. Polyethylene catheters (Perifix® Paed, B. Braun Melsungen AG, Melsungen, Germany) were shortened to a length of 11 cm and introduced 8 cm caudally to the level of lumbal enlargement. The remaining 3 cm of the catheter was fixed in a screw connector. To confirm correct placement of the catheters, a bilateral motor block of the hindlimbs was induced by injecting 10 µL of lidocaine 2% followed by 10 µL of saline flush. Animals with correctly placed catheters were allowed to recover for 5 days after surgical procedure and lidocaine test. The rats that developed functional deficits in either their fore- or hindlimbs were excluded.
KT was supplied by DEVA Laboratories in sterile packages without preservatives (MW 376.41 Dalton, Ketrodol; DEVA, stanbul, Turkey) and 5 mg/mL, 15 mg/mL, and 40 mg/mL of KT solutions were prepared with 0.9% NaCl with a pH between 6.5–7. The intrathecal KT was injected 4 times with 5 days between injections. Catheters were flushed with saline after administration of 10 µL of either the saline or the drug. According to the type of analgesic regimen, rats were randomized into 4 groups as follows (n = 7 rats in each group).
- Group S: 10 µL of saline.
- Group K50: 50 µg of KT.
- Group K150: 150 µg of KT.
- Group K400: 400 µg of KT.
Assessment of behavioral neurotoxicity was performed as described by Yamamoto and Yaksh (11). Motor function of rats was assessed before and after treatment using the placing/stepping and righting reflexes. The observed responses were graded according to the system used by Chiari et al. (12).
For the evaluation of pain, the formalin test modified by Abram et al. (13) was performed 15 min after the last intrathecal injection. Rats were anesthestized with ether, and 50 µL of 5% formalin was injected subcutaneously into the dorsal surface of right hindpaw with a 26-gauge needle. After formalin injection, flinches were counted for 1-min periods at 1 to 2, 5 to 6, and 5-min intervals during the interval from 10 to 60 min.
Two phases of spontaneous flinching behavior were observed as previously described (11). Phase I beginning immediately after formalin injection and lasting to the second observation interval (5–6 min) and phase II beginning at the 10th min and lasting through the 60th min. Thus, the mean of the first two measurements was the phase I value whereas that of the remaining 9 measurements was phase II value.
After the formalin test at completion of the study (day 20), the rats were killed by large doses of ether, followed by left ventricle cannulation and perfusion with phosphate buffer, then 4% paraformaldehyde. Bilateral laminectomy was performed and spinal cords with the accompanying catheter tips located at the lumbar enlargements were removed from the vertebral canal and fixed in formalin. Frozen transverse sections at four different levels of spinal cords (C3–4, C7–8, T8, L1-2) were embedded in paraffin and sectioned. The sections (6–7 µm thick) were stained with hematoxylin and eosin and examined under light microscopy. Histopathological changes were evaluated in a blinded fashion by the senior pathologist (MC) according to a scoring system described by Malinovsky et al. (14).
Statistical analysis was performed using SPSS (Version 10.0) for Windows. The results are given as mean ± SD. The results of neurobehavioral tests and histopathological examinations were assessed with using 
2 analysis, and the Fisher’s exact test was used when appropriate. A P value <0.05 was considered significant. The data analysis for comparisons between the formalin test results was performed with using the Kruskal-Wallis followed by Mann-Whitney U-test. For multiple comparisons, Bonferroni correction was performed and a P value <0.008 was considered significant.
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Results
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One of the 28 rats was excluded from the study as paraplegia was observed on the fourth postoperative day. The catheter tip in two rats from the saline group was found to be located at the level of the thoracic subarachnoid space during laminectomy and their specimens were not studied histopathologically. No excitation or pain behavior was observed during intrathecal injections.
No abnormality of placing, stepping, or righting reflexes were exhibited at any time among the control or KT treated animals. All animals, after even the largest dose of intrathecal KT, displayed normal symmetrical ambulation and no detectable changes in motor strength (Table 1).
The effects of intrathecal administration on formalin test responses are presented in Figure 1. The responses to formalin test were assessed as phase I and phase II. When group S was compared with groups K50, K150, and K400, significant decreases related to phase I and II responses were found in the groups treated with KT (P = 0.001). When phase I responses of groups K50, K150, and K400 were compared, no significant difference was observed. Comparisons of phase II responses belonging to the same groups revealed that phase II responses of groups K150 and K400 were significantly less than those of group K50 (P = 0.001). However, no significant difference was found during comparisons between phase II responses of the groups K150 and K400 (Fig. 2).
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Figure 1. Time-effect curve for the number of flinches per minute observed after the subcutaneous injection of formalin. Each group represents the group mean ± SD. *P < 0.008 for comparisons with group S, #P < 0.008 for comparisons with group K50.
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Figure 2. Dose-response curves for intrathecal ketorolac tromethamine (KT), presenting the number of formalin-evoked flinches during the first and second phases of the formalin test. *P < 0.008 for comparisons between KT and saline; #P < 0.008 for comparisons with 50 µg of KT.
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Macroscopic examinations of the removed spinal cords showed no abnormality such as hemorrhage or necrosis. Except for two rats, spinal sections of all rats demonstrated normal histological appearance (Table 2). No sign of gliosis, demyelination, fibrosis, or inflammation was found during microscopic examinations (Fig. 3). In a section removed from the upper cervical cord of a rat in group S, significant hemorrhagic foci was observed in all of the zones of gray and white matter. Limited hemorrhage of the white matter was also seen in another section obtained from the lower cervical spinal cord of the same rat. In another rat from group K50 hemorrhagic foci in all zones of the gray and white matter of the thoracic cord were observed. Lumbar sections of the same rat also demonstrated hemorrhage limited to the white matter.
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Figure 3. Photomicrographs showing the panoramic histopathological appearances of spinal cord sections obtained from L1–2 levels of 4 different groups. a: group S, b: group K 50, c: group K 150, and d: group K 400. H&E, x20. All demonstrated similar histological features of the intact white and gray matter substances.
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Discussion
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Several authors have shown acute analgesic effects of intrathecal KT in the management of pain (2,6,7) and Malmberg and Yaksh (4) have demonstrated that a single intrathecal dose of KT is capable of producing analgesia in the model used in the present study. Although repeated doses of some drugs, such as a variety of opiates (15), neostigmine (16), or A1 receptor agonist (R-PIA) (17), have shown no untoward reaction, even a single injection of some other drugs such as somatostatin (18) or dynorphin (19) induces significant irreversible motor dysfunction and histopathological changes. Recently, a phase I safety assessment of a single intrathecal dose of KT in humans revealed no effect on reported pain to acute noxious stimuli and no long-term side effects (9). However, the authors could not conclude whether repeated doses of intrathecal KT for chronic use were either effective or safe. The present study is the first to investigate 4 doses over 20 days and specifically to examine neurotoxicity of intrathecal KT in the rat.
It would probably have been better to have obtained information on possible changes in efficacy with repeated administration by performing the formalin test after each intrathecal KT administration. Nevertheless, we found that repeated intrathecal administration of KT to rats exhibited analgesic effects at doses of 50, 150, and 400 µg and its maximum effective dose was observed to be 150 µg. Especially, intrathecal KT prevented nociceptive pain and showed limited effects on phase I responses in the formalin test contrary to its strong dose-dependent suppressor effect on phase II responses. Fifty micrograms of ketorolac decreased phase II responses by about 65%, whereas 150 µg of ketorolac decreased responses by approximately 90%. No difference was detected in the phase II inhibitory responses during comparisons between the doses of 150 and 400 µg of KT, but 20 days might not be enough time for evaluating the neurotoxic effects of chronic intrathecal drug administration, and it would have been much better if pathological specimens of the animals after 3 or 6 months of drug administration were available. However, significantly, even 400 µg of intrathecal KT did not cause signs of behavioral or histopathological neurotoxicity. Hemorrhagic foci observed in sections of the two rats were attributed to the long-term catheterization rather than to the administration of KT because similar changes were also reported after intrathecal catheterization in several previous studies (20,21). However, one of our rats developed a bilateral lower limb motor deficit on the fourth day of intrathecal catheterization. This might have resulted from either neurotoxicity of the administered drug or spinal cord ischemia (because of hypotension or anterior spinal artery trauma and/or spasm) as mentioned by Aldrete et al. (22). The occurrence of paraplegia during the recovery period without any drug and saline administration suggested that the most likely cause of this complication might have been an acute injury probably induced by a change in the position of the catheter in the spinal subarachnoid space. Detection of a hematoma in the thoracic cord at the time of laminectomy of the rat with paraplegia may also confirm this suggestion.
We observed that chronic intrathecal injections of KT did not lead to any histopathological sign of injury of the spinal cord and reduced nociceptive responses. Thus, KT might become an alternative drug in the treatment of chronic pain with intrathecal administrations. However, extrapolating from rat to humans must be made with caution, and neurotoxicologic testing with different doses and therapy regimens in some other animal species remains to be completed and followed by some other safety trials in humans before clinical use.
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Accepted for publication August 13, 2003.
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Abstract
Introduction
