Método intratecal

JPM Vol. 32, No.4 December1994:197-200 ORIGINAL ARTICLES A Method to Perform Direct Transcutaneous Intrathecal Injection in Rats Christine Mestre, T6r6sa P61issier, J o s e p h Fialip, G e o r g e W i l c o x , and Alain Eschalier Department of Pharmacology, Faculty of Medicine (C.M., T.P., A.E.), Department of Pharmacology, Faculty of Pharmacy, NPPUA team (J.F.), 63001 Clermont-Ferrand, France; Department of Pharmacology and Graduate Program in Neuroscience, School of Medicine, University of Minnesota (G. W.), Minneapolis, USA; Department of Pharmacology, Faculty of Medicine, University of Chile (T.P.), Santiago, Chile This work describes a method for performing direct intrathecal (i.t.) injections in rats without introducing a spinal catheter. Its ease of use in awake animals yields rapid and reproducible results with no sign of motor impairment. The quality of each injection was ensured by the observation of an injection-induced tail-flick. A 10-p~L injection of methylene blue was well localized yielding a very limited diffusion along the spinal cord. The method was validated by demonstrating that morphine (i.t.) had a marked antinociceptive effect and that naloxone (i.t.) blocked the effect of systemic (s.c.) morphine in mononeuropathic rat. Keywords: Intrathecal injection; Rat; Analgesia Introduction Central administration of drugs is important for the study of their site or mechanism of action. Intrathecal administration is particularly useful for distinguishing spinal and supraspinal effects. Administrations at the spinal level in rats are usually performed via a catheter introduced along the spinal cord according the technique described by Yaksh and Rudy (1976). However, the necessity of anesthesia and surgery makes this technique complicated and this becomes more of an issue if only one injection is to be made. Furthermore, it is necessary to allow at least 1 week for subject animals to be tested to make certain that introduction of the catheter has been successful and has not resulted in motor lesions. Hylden and Wilcox (1980) have described a method for a direct intrathecal injection in the mouse. This technique allows a rapid, innocuous, reliable injection, which can be performed within a minute before testing. The aim of the present work was to adapt this method to Address reprint requests to Dr. A. Eschalier, Laboratoire de Pharmacologie M6dicale, Facult6 de MEdecine, 63001 Clermont-Ferrand Cedex, France. Received March l, 1994; revised and accepted May l, 1994. Journalof Pharmacologicaland ToxicologicalMethods32, 197-200(1994) © 1994ElsevierScienceInc. 655 Avenueof the Americas,New York,NY 10010 the rat. The procedure used and a pharmacological validation using morphine will be described. Methods Operative Procedure Male Sprague-Dawley rats weighing from 200 to 250 g were used. The injections were performed by holding the rat securely in one hand by the pelvic girdle and inserting a 25-Ga × 1" needle, connected to a 25-1xL Hamilton syringe, into the tissues between the dorsal aspects of L 5 and L6, perpendicular to the vertebral column (Fig. 1). This site was selected so that the injection was restricted to the region where the spinal cord ends and the cauda equina begins in order to reduce the possibility of spinal damage and increase the intervertebral accessibility. When the needle entered the subarachnoidal space, a sudden lateral movement of the tail was observed. This reflex was used as an indicator of successful puncture. No other specific behavior or sign of distress or pain was observed at this time. A constant 10-1xL volume was injected. Then, the syringe was held in position for a few seconds and progressively removed to avoid any outflow of the drug. 1056-8719/94/$7.00 t. a laminectomy was performed and the spinal cord examined. In a few instances.t.. Results Verification of the Site of Injection At the site of the needle penetration into the skin.t. were determined using a Ugo Basile analgesimeter (Apelex.198 JPM Vol.). . but no staining was found in the paravertebral muscles. the other receiving just the sham operation. 1991). i. to examine the degree of diffusion. Bennet.c. the brain was also examined to test for possible rostrai diffusion of dye. After obtaining control values of vocalization thresholds.c. between L 5 and L 6. Nociceptive thresholds. The site of injection was verified by the administration of methylene blue: 10 rats were injected intrathecally with 10-txL of methylene blue and were killed by cervical dislocation 10 min afterwards. After a longitudinal skin incision and muscle dissection. vocalization thresholds were measured immediately before the intrathecal injection of naloxone (10 ixg/rat) or saline (10 ixL/rat). Furthermore.. 1993). morphine has been shown to be more effective in this model than in normal rats (Attal et al. Diffuse staining was evident along 5 or 6 mm in the spinal cord and along the corresponding internal walls of the spinal canal. 12 (6 x 2) mononeuropathic rats were injected intrathecaUy with morphine (8 p~g/rat) or saline (10 /xL/rat). saline s.. At 14 days afterwards. a diffuse methylene blue staining was observed in paravertebral muscles. Mononeuropathic rats (n = 24) were subcutaneously injected with morphine (2 mg/kg) or saline (NaC1 0. four loose ligatures were placed around the common sciatic nerve on one side only (usually the right).p. and every 15 rain thereafter for a total duration of 120 rain. Diagram showing insertion of needle into the intervertebral space. Two separate experiments were performed as follows: 1. + naloxone i. a mechanical noxious stimulus was applied to the ligatured paw according to the method previously described by Randall and Selitto (1957). tip diameter of probe 1 mm. + saline i. a well-defined blue mark was observed. 4 December 1994:197-200 Spinous process \ Spinal cord ~'4~ I[ ~ (( equina Canda F i g u r e 1. weight 30 g) by applying increasing pressure to the right lesioned hind paw until vocalization was elicited.t. At 10 min after the injection. when the injection was not made correctly as indicated by absence of the tail flick upon penetration. 32.c. In addition. The animals were devised into the following four groups: saline s. morphine s.. Under sodium pentobarbital anesthesia (50 mg/kg. No staining was observed in the brain.9%) (2 mL/kg) after determination of control vocalization thresholds.c. A sample of 10 animals was observed for 1 week following injection and no evidence of motor impairment was observed. + saline i. morphine s. expressed in grams. 2. No. Vocalization thresholds were measured 10 min after the injection and every 10 min thereafter for a total duration of 90 min. 1988. Pharmacological Experiments Pharmacological validation of the method using morphine was performed in mononeuropathic rats. + naloxone i. This recently described model of persistent pain based on a chronic constriction injury of the sciatic nerve is one of the most often used to mimic human neuropathic pain (Bennet and Xie. might not be tolerated easily. easy to employ. this method permits the avoidance of anesthesia which in some animals with persistent pain (e. 2 (B)]. . .t.. morphine (+ saline) induced a marked antinociceptive effect (+ 64% ___ 25%) at the earliest time point tested after s.001 versus predrug values (time 0). ( • ) morphine + saline. (B) Subcutaneous administration of morphine (2 mg/kg) or saline (2 mL/kg) followed (10 min after) by i. and reproducible method can be used to perform intrathecal injection in rats without introducing a spinal catheter. (--A--) morphine + naloxone. reliable. zo . 4o /! 6o 4s T I M E (mln) B VOCALIZATION THRESHOLI~ 7OO-- Liptured p a w 60O 4OO .C. DIRECT TRANSCUTANEOUS INTRATHECAL INJECTIONS 199 While control groups (saline + saline or saline + naloxone) did not show any change in the vocalization thresholds.T. the results obtained with methylene blue. particularly in the field of pain. The intrathecal injection of naloxone. at this time. (---~--) saline + saline. or Saline Nsdoxone I. totally suppressed morphine-induced analgesia [Fig. similar to that used by Yaksh and Rudy (1976).t.~ Morphine S.C.05. maximum increase). (A) Intrathecal administration of morphine (8 txg/rat) ( • ) or saline (10 ixL/rat) ( ~ ) at time 0 (14 days after ligature of the sciatic nerve). administration and the antagonism of the effect of systemic administration of morphine by i. Guilbaud G (1991) Behavioural evidence that systemic morphine may modulate a phasic pain-related behaviour in a rat model of peripheral mononeuropathy. Time course of the antinociceptive effect of morphine in mononeuropathic rats submitted to a mechanical noxious stimulus. these data confirm the previously demonstrated antinociceptive effect of i. The injection volume corresponds to 4% of the total volume of the cerebrospinal fluid (CSF) in the rat (250 ~L) (Bass and Lundberg. or . innocuous. n = 6 in each group.01. A VOCALIZATION TflRESHOLI~$ (Ig) 600Liptured Imw 500- 40O- Discussion T 2OO- . References Attal N. The reliability of this method was demonstrated by both a specific behavior (a sudden lateral movement of the tail) only obtained when the needle was correctly introduced into the vertebral canal and a pharmacological validation. 2 (A)]. n = 6 in each group. Furthermore. Chen YL. This work demonstrates that a rapid.-injected morphine in the same model of peripheral mononeuropathy (Attal et al. reproducible. . and unaccompanied by motor impairment and can be useful for both physiological and pharmacological studies of spinal mechanisms. ~o~upm~ . Finally. pain tests can be performed without any motor dysfunction. (--A--) saline + naloxone.or stress-induced changes in pain scores (scores of control rats were not different. also demonstrate the limited diffusion.t. Kayser V. This effect had a long duration. Pharmacological Validation The intrathecal injection of saline failed to induce any change in the vocalization thresholds at any time after the injection. 1991). 1976). the volume of injection used (10 ~L).. . In conclusion. Additionally. suggesting an action limited to the spinal level. reversed the nerve ligation-induced hyperalgesia and induced a significant analgesia 10 min after the injection (164% _ 37%. naloxone. injection of either naloxone (10 Ixg/rat) or saline (10 p~L/rat). (1962) to avoid hemodynamic changes.~dine 60 90 120 T I M E (rain) Figure 2. Bars mean SEM.v.. . **p < 0. which agrees with the limit of 10% proposed by Rieselbach et al. beyond their interest in verifying the siting of the injection. Bars mean SEM. Morphine. It's also interesting to observe that. o . *p < 0. polyarthritic or diabetic rats). injection with a duration of 75 min. . ***p < 0. from predrug values). 1993. injected intrathecally. this method is rapid. Burns et al. P a i n 47:65-70. The well-known spinal effect of morphine was verified by both its marked effect after i. .c. Importantly. is also small enough to promote a wide diffusion. MESTRE ET AL. only 10 min after the injection. significant antinociception remaining at 90 min after the injection [Fig.g. N Engl J Med 267:12731278. Rudy TA (1976) Chronic catheterization of the spinal subarachnoid space. Mendels J (1976) A kinetic analysis of 5-hydroxyindoleacetic acid excretion from rat brain and CSF. Bennet GJ. Pain 33:87107. Muscle Nerve 16:1040-1048. 4 December 1994:197-200 Hylden JLK. No. DeChiro G. Yaksh TL. Xie YK (1988) A peripheral mononeuropathy in rat that produces abnormal pain sensation like those seen in man. JPM Vol. 32. Wilcox GL (1980) Intrathecal morphine in mice: A new technique. Burns D. Selitto JJ (1957) A method for measurement of analgesic activity on inflamed tissue. Physiol Behav 17:1031-1036.200 Bass NH. Brunswick DJ. Freireich EJ. Pring M. Eur J Pharmacol 67:313-316. Rieselbach RE. Bennet GJ (1993) An animal model of neuropathic pain: a review. Arch lnt Pharmacodyn 61:409-419. Randall LO. London J. Rail DP (1962) Subarachnoid distribution of drugs after lumbar injection. Rabinowitz JL. . Biol Psychiat 11:125-157. Brain Res 52:323-332. Garfinkel D. Lundborg P (1973) Postnatal development of bulk flow in the cerebrospinal fluid system of the albino rat: Clearance of carboxyl04C) inulin after intrathecal infusion.