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Chronic lumbar catheterization of the spinal subarachnoid space in mice
Journal of Neuroscience Methods 133 (2004) 65–69
Chronic lumbar catheterization of the spinal subarachnoid space in mice Wei-Ping Wu, Xiao-Jun Xu, Jing-Xia Hao∗ Department of Laboratory Medicine, Division of Clinical Neurophysiology, Huddinge University Hospital, S-141 86 Stockholm, Sweden Received 18 July 2003; received in revised form 12 September 2003; accepted 15 September 2003
Abstract A simple method for chronic intrathecal (i.t.) catheterization of the lumbar subarachnoid space in mice is described. The procedure does not require major surgery and does not produce neurological deficits. The intrathecal catheter stayed in place and was functional for at least 10 days. Morphological studies revealed no histological damage in the spinal cord after catheter implantation. The effects of acute and chronic intrathecal morphine were studied and compared between the current method and that of acute lumbar puncture. Morphine produced similar antinociceptive effect when administered acutely. All mice with catheters responded reliably to daily morphine injection up to 5 days whereas it was difficult to inject drugs repeatedly with lumbar puncture. It is concluded that this novel method of chronic lumbar catheterization in mice has advantages over the existing lumbar puncture technique for intrathecal delivery of drugs upon repeated administration. This method may be particularly useful in studies of genetically modified mice where the number of mice available is often limited. © 2003 Elsevier B.V. All rights reserved. Keywords: Intrathecal; Lumbar puncture; Mice; Subarachnoid space; Spinal cord
1. Introduction
2. Materials and methods
Several methods of implanting chronic indwelling catheters for intrathecal (i.t.) delivery of drugs in rats are available (Yaksh and Rudy, 1976; Storkson et al., 1996). These methods have been invaluable for research on spinal cord function and pharmacology. There are however no methods of chronic i.t. catheterization for mice. i.t. drug delivery in mice is most commonly through acute lumbar puncture (Hylden and Wilcox, 1980). This method, although extremely valuable, is not particularly suitable for repeated drug administration. Moreover, since the mice cannot be repeatedly used, large numbers of subjects are often required, as for testing dose–response relationships. This is not optimal for experiments using genetically modified mice since the supply of animals is often limited. We now describe a method of implanting chronic i.t. catheters in mice which is based on a modification of the technique developed for rats (Storkson et al., 1996). We demonstrate that this new method offers some advantages over lumber puncture for i.t. drug delivery in mice.
2.1. Animals
∗
Corresponding author. Tel.: +46-8-58582213; fax: +46-8-7748856. E-mail address: [email protected] (J.-X. Hao).
0165-0270/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2003.09.015
Male C57 black mice weighting 20–30 g (B&K Universal, Sweden) were used. The animals were housed at 21–24 ◦ C with free access to food and water and a 12-h light/dark cycle. The mice were kept in individual cages after catheterization. The study was carried out according to the Ethical Guidelines of the International Association for the Study of Pain (Zimmermann, 1983) and was approved by the local animal research ethics committee. 2.2. Preparation of the catheter Polyethylene tube, PE 10 (Clay Adams, Becton Dickinson Co., MD, USA) with outer diameter 0.6 mm was used. A thin tungsten wire (diameter 0.12 mm, Luma Metall, Kalmar, Sweden) was inserted into the PE 10 tube as a guide wire. One end of the catheter was then slowly stretched to about 0.3 mm in outer diameter (Fig. 1). Two beads of about 1.5 mm diameter were made on the catheter, one was made at just beside the stretched part and second one was made on the unstretched part (Fig. 1). The bead was made by briefly heating the catheter with a flame and compressing
66
W.-P. Wu et al. / Journal of Neuroscience Methods 133 (2004) 65–69
11 cm 2 cm
2.5 cm
OD 0.6mm
OD 0.3mm first bead streched PE 10 catheter
second bead
guide wire
unstreched PE 10 catheter
Fig. 1. Schematic illustration of construction of the i.t. catheter.
the catheter slightly (Fig. 1). The wire inside prevented the catheter from occlusion and saline was injected through the catheter to check for leakage. The distance between the tip of the catheter and the first bead was 1.8–1.9 cm for mice weighing 20 g and 2.0–2.1 cm for 30 g mice. These distances were determined for placement of the tip of the catheter at L4 when the penetration was made between L5 and L6 spinal processes (see below). The distance between the end of the catheter and the second bead was 2.0–2.5 cm. The full length of the catheter was 11 cm with a dead space of about 5 l. 2.3. Implantation of the catheter The mice were anesthetized by chloral hydrate (300 mg/kg, i.p.). A 2 cm longitudinal skin incision was made above vertebrae L5 and L6. The mouse was held firmly by the pelvic girdle in one hand and vertebra was accessed through a hole in the muscle made by a 23 gauge needle. The stretched catheter with the metal wire inside was pushed into one side of the L5–6 process at an angle of about 20–30◦ above the vertebral column (Fig. 2B) and 20–30◦ to the midline of the vertebra (Fig. 2A). The catheter was then pushed through the intervertebral space and dura. When the sign of dura penetration (sudden movement of tail or the hindlimb) was observed, the guide wire was withdrawn for about 2 cm in order to avoid spinal damage by the metal wire. The catheter was then pushed gently upward to reach L4 at the lumbar enlargement. The first bead was then fixed by suturing the bead into the superficial muscle (Fig. 2C). Another skin incision was then made at the neck area and the catheter was tunneled under the skin and pulled out of the cut at the neck where the second bead was fixed into the muscle. All incisions were then carefully sutured and the outer end of the catheter was sealed by melting (Fig. 2C). Heat melting was also used during chronic experiment after each injection.
Fig. 2. (A) The overview of a mouse in prone position. The catheter with the guide wire was inserted at the space between lumbar vertebrae L5 and L6 with an angle of about 20–30◦ left to the vertebral column. (B) The side view of the mouse. The catheter with guide wire was inserted at an angle of 20–30◦ above the vertebral column. (C) Fixation of the catheter. The black circles are beads which are fixed in the muscle. The solid and dashed line represents the external and internal portion of the catheter, respectively.
10–20 min was taken as an indication for a correct intrathecal catheter location.
2.4. Verification of catheter localization
2.5. Motor function
One or 2 days after catheter insertion the free end was cut to allow intrathecal injection of lidocaine (2.5 l, 20 mg/ml, Astra, Södertälje, Sweden) followed by 5 l saline. Motor paralysis of the hindlimbs within 15 s and lasting for
The motor function of the mice were examined 1 day after implantation by observing if there was any walking dysfunction, weakened extension withdrawal reflex of the hindlimb and reduced toe spread.
W.-P. Wu et al. / Journal of Neuroscience Methods 133 (2004) 65–69
2.6. Morphological studies Nine mice were used for morphological examination. Three were normal controls. Three mice were sacrificed 4 and 12 days after catheterization, respectively. The animals were deeply anesthetized with chloral hydrate and spinal cord segments L2–L5 were removed after laminectomy and placed in fixative of 2% glutaraldehyde in 0.1 M cacodylate buffer (pH = 7.2) at room temperature for 2 h. The segments were then stored in the fixative at 4 ◦ C until further process. The segments were rinsed and were osmicated in 2% OsO4 for 2 h at 0 ◦ C, dehydrated serially with alcohol and acetone and embedded in LX-112 plastic (Ladd Res. Industries Inc., VT, USA). Transverse sections (1 m thick) were cut from the slices and stained with toluidine blue.
67
and the Wilcoxon signed rank test was used for the chronic morphine experiment.
3. Results 3.1. General conditions and response to lidocaine after implantation Around 20% of mice implanted with i.t. catheters exhibited some degree of motor impairment 1 day after implantation and were sacrificed. In the remaining mice, the position of catheter was checked by i.t. lidocaine 1–2 days after implantation. It was found that 25 of 27 (93%) mice had total bilateral paralysis of the hindlimbs within 15 s lasting for 10–20 min.
2.7. Lumbar puncture
3.2. Morphologic analysis
A 2 cm long skin incision was made over the lumber region. The mice were rested for 20–30 min before drug injection. The injection was made at the space between L5 and L6 where the cord ends and cauda equina begins (Sidman et al., 1971). A 27 gauge needle was used to puncture the muscle, spinal process and the dura until a characterized brief motor response of tail or hindlimbs was observed indicating dura penetration. The needle was connected to a microsyringe through a PE 50 polyester catheter (12 cm).
After laminectomy, the tip of catheter was located in the subarachnoid space at the level of the lumbar enlargement. No signs of morphological abnormalities could be seen under light microscopic level in the lumbar spinal cord removed 4 or 12 days after catheter implantation (data not shown).
2.8. Drugs Morphine hydrochloride (10 mg/ml, Pharmacia & Upjohn, Sweden) was diluted with physiological saline to desired concentrations. Morphine was injected in a 5 l volume followed by 5 l saline to flush the catheter. 2.9. Tail flick test The effect of i.t. morphine was evaluated with the tail flick test. The mice were held gently and a radiant heat source was focused 1–2 cm from the tip of the tail and the latency to tail flick was recorded automatically (Ugo Basile, Italy). The intensity of the stimulation was adjusted so that the baseline latency was 4–6 s and the cut-off value was set at 15 s. 2.10. Statistics The data are expressed as mean ± S.E.M. The percentage maximal possible effect (%MPE) was calculated based on the formula: post-morphine response–baseline % MPE = × 100 cut-off(15 s)–baseline The significance level was set at P < 0.05. Unpaired student’s t-test was used for the acute morphine experiment
3.3. The effects of acute intrathecal morphine The experiments were conducted 3 days after catheter implantation. Intrathecal saline had no effect on the tail flick latency whereas intrathecal morphine induced a dosedependent prolongation of the tail flick latency when administered either via the chronic catheter or through lumbar puncture and the effect of morphine was not significantly different between the two groups (Fig. 3). 3.4. Comparison of the effect of repeated morphine administration Five mice with a chronic catheter were administered 100 ng morphine for 5 days starting 3 days after catheter implantation and all mice responded up to day 5 (Fig. 4A). In the mice with lumbar puncture, we only observed significant antinociception on day 1 (Fig. 4B) and it became gradually difficult to perform lumbar puncture repeatedly and reliably mainly due to the difficulty in recognizing muscle structure overlying the vertebra resulting from previous cut and scar formation. Only two mice could be successfully punctured on day 5 (Fig. 4B).
4. Discussion The method of chronically implanting an intrathecal catheter in mice is simple to perform with a fairly high success rate and offers some advantages over the existing method of lumbar puncture (Hylden and Wilcox, 1980).
68
W.-P. Wu et al. / Journal of Neuroscience Methods 133 (2004) 65–69
100 (10) catheter puncture
% MPE (Tail Flick Latency)
80
(12)
60
40 (5)
20
(5) (5) (5)
(3)
Mor.100ng
-20
Mor.10ng
saline
Mor.1ng
0
Tail Flick Latency (s)
Fig. 3. The effect of acute i.t. morphine on tail flick latency in mice injected through the catheter (open bar) or lumbar puncture (filled bar). The doses are expressed on the X-axis and %MPE at 30 min after morphine administration are shown on the Y-axis. N is indicated in the brackets. Unpaired Student’s t-test indicated that there are no significant differences between the two groups at any doses.
18 16 14 12 10 8 6 4 2 0
*
(5)
(5)
1 2 3 4 Times of injection (days)
5
*
(5)
(5)
0
Tail Flick Latency (s)
*
(5)
(A)
20 18 16 14 12 10 8 6 4 2 0
before after
*
(6) (6)
(4)
(3) (2)
0 (B)
*
*
1 2 3 4 Times of injection (days)
5
Fig. 4. The effect of daily injection of morphine on the tail flick latency in mice through the chronic catheter (A) or lumbar puncture (B). The data are shown as baseline value for each day (circles) and 30 min after morphine administration. The number of mice in which the injection was successful is indicated in the brackets. Wilcoxon signed-ranks test was used to examine the difference between tail flick latency before and after morphine administration (∗ P < 0.05).
The majority of mice had proper location of the catheter, no neurological deficits and no damage to the spinal cord. Successfully implantated mice showed reliable response to intrathecal morphine and since the catheter was
implanted prior to drug administration, the proper position of the catheter tip could be evaluated with lidocaine. The mice may also be less stressed when the catheter was pre-implanted than with acute injection. Furthermore, although both methods require practice, the implantation is done under anesthesia. Another important practical advantage is that with the implantation method the person who performs the implantation does not need to be present at the subsequent pharmacological experiments. The implanted catheters are clearly functional as lidocaine and morphine produced expected effects. Repeated daily injection of morphine through the chronically implanted catheter produced consistent antinociceptive effect in mice tested for up to 5 days. The mice behaved normally and there was no blockade of the catheters. However, repeated injection through lumbar puncture proved to be a difficult task even for a highly skilled experimenter. This is primarily due to the difficulty in recognizing muscle structure overlying the vertebra resulting from scar formation. In conclusion, we showed that intrathecal implantation of a chronic intrathecal catheter is feasible in mice. The method offers several advantages over existing lumbar puncture technique particularly for chronic repeated administration. This is preferable in experiments involving studies of drug tolerance and in situation where the number of mice may be limited, such as in studies of genetically modified mice.
Acknowledgements The present study is supported by Swedish Science Council (12168), Clinical Research Center of the Huddinge University Hospital and the research funds of the Karolinska Institutet.
W.-P. Wu et al. / Journal of Neuroscience Methods 133 (2004) 65–69
References Hylden JLK, Wilcox GL. Intrathecal morphine in mice: a new technique. Eur J Pharmacol 1980;67:313–6. Sidman RL, Angevine JB, Pierce ET. Atlas of the mouse brain and spinal cord. Cambridge, MA, USA: Harvard University Press; 1971.
69
Storkson RV, Kjorsvik A, Tjolsen A, Hole K. Lumbar catheterization of the spinal subarachnoid space in the rat. J Neurosci Meth 1996;65:167–72. Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17:1036–7. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals (editorial). Pain 1983;16:109–10.
Chronic lumbar catheterization of the spinal subarachnoid space in mice Wei-Ping Wu, Xiao-Jun Xu, Jing-Xia Hao∗ Department of Laboratory Medicine, Division of Clinical Neurophysiology, Huddinge University Hospital, S-141 86 Stockholm, Sweden Received 18 July 2003; received in revised form 12 September 2003; accepted 15 September 2003
Abstract A simple method for chronic intrathecal (i.t.) catheterization of the lumbar subarachnoid space in mice is described. The procedure does not require major surgery and does not produce neurological deficits. The intrathecal catheter stayed in place and was functional for at least 10 days. Morphological studies revealed no histological damage in the spinal cord after catheter implantation. The effects of acute and chronic intrathecal morphine were studied and compared between the current method and that of acute lumbar puncture. Morphine produced similar antinociceptive effect when administered acutely. All mice with catheters responded reliably to daily morphine injection up to 5 days whereas it was difficult to inject drugs repeatedly with lumbar puncture. It is concluded that this novel method of chronic lumbar catheterization in mice has advantages over the existing lumbar puncture technique for intrathecal delivery of drugs upon repeated administration. This method may be particularly useful in studies of genetically modified mice where the number of mice available is often limited. © 2003 Elsevier B.V. All rights reserved. Keywords: Intrathecal; Lumbar puncture; Mice; Subarachnoid space; Spinal cord
1. Introduction
2. Materials and methods
Several methods of implanting chronic indwelling catheters for intrathecal (i.t.) delivery of drugs in rats are available (Yaksh and Rudy, 1976; Storkson et al., 1996). These methods have been invaluable for research on spinal cord function and pharmacology. There are however no methods of chronic i.t. catheterization for mice. i.t. drug delivery in mice is most commonly through acute lumbar puncture (Hylden and Wilcox, 1980). This method, although extremely valuable, is not particularly suitable for repeated drug administration. Moreover, since the mice cannot be repeatedly used, large numbers of subjects are often required, as for testing dose–response relationships. This is not optimal for experiments using genetically modified mice since the supply of animals is often limited. We now describe a method of implanting chronic i.t. catheters in mice which is based on a modification of the technique developed for rats (Storkson et al., 1996). We demonstrate that this new method offers some advantages over lumber puncture for i.t. drug delivery in mice.
2.1. Animals
∗
Corresponding author. Tel.: +46-8-58582213; fax: +46-8-7748856. E-mail address: [email protected] (J.-X. Hao).
0165-0270/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.jneumeth.2003.09.015
Male C57 black mice weighting 20–30 g (B&K Universal, Sweden) were used. The animals were housed at 21–24 ◦ C with free access to food and water and a 12-h light/dark cycle. The mice were kept in individual cages after catheterization. The study was carried out according to the Ethical Guidelines of the International Association for the Study of Pain (Zimmermann, 1983) and was approved by the local animal research ethics committee. 2.2. Preparation of the catheter Polyethylene tube, PE 10 (Clay Adams, Becton Dickinson Co., MD, USA) with outer diameter 0.6 mm was used. A thin tungsten wire (diameter 0.12 mm, Luma Metall, Kalmar, Sweden) was inserted into the PE 10 tube as a guide wire. One end of the catheter was then slowly stretched to about 0.3 mm in outer diameter (Fig. 1). Two beads of about 1.5 mm diameter were made on the catheter, one was made at just beside the stretched part and second one was made on the unstretched part (Fig. 1). The bead was made by briefly heating the catheter with a flame and compressing
66
W.-P. Wu et al. / Journal of Neuroscience Methods 133 (2004) 65–69
11 cm 2 cm
2.5 cm
OD 0.6mm
OD 0.3mm first bead streched PE 10 catheter
second bead
guide wire
unstreched PE 10 catheter
Fig. 1. Schematic illustration of construction of the i.t. catheter.
the catheter slightly (Fig. 1). The wire inside prevented the catheter from occlusion and saline was injected through the catheter to check for leakage. The distance between the tip of the catheter and the first bead was 1.8–1.9 cm for mice weighing 20 g and 2.0–2.1 cm for 30 g mice. These distances were determined for placement of the tip of the catheter at L4 when the penetration was made between L5 and L6 spinal processes (see below). The distance between the end of the catheter and the second bead was 2.0–2.5 cm. The full length of the catheter was 11 cm with a dead space of about 5 l. 2.3. Implantation of the catheter The mice were anesthetized by chloral hydrate (300 mg/kg, i.p.). A 2 cm longitudinal skin incision was made above vertebrae L5 and L6. The mouse was held firmly by the pelvic girdle in one hand and vertebra was accessed through a hole in the muscle made by a 23 gauge needle. The stretched catheter with the metal wire inside was pushed into one side of the L5–6 process at an angle of about 20–30◦ above the vertebral column (Fig. 2B) and 20–30◦ to the midline of the vertebra (Fig. 2A). The catheter was then pushed through the intervertebral space and dura. When the sign of dura penetration (sudden movement of tail or the hindlimb) was observed, the guide wire was withdrawn for about 2 cm in order to avoid spinal damage by the metal wire. The catheter was then pushed gently upward to reach L4 at the lumbar enlargement. The first bead was then fixed by suturing the bead into the superficial muscle (Fig. 2C). Another skin incision was then made at the neck area and the catheter was tunneled under the skin and pulled out of the cut at the neck where the second bead was fixed into the muscle. All incisions were then carefully sutured and the outer end of the catheter was sealed by melting (Fig. 2C). Heat melting was also used during chronic experiment after each injection.
Fig. 2. (A) The overview of a mouse in prone position. The catheter with the guide wire was inserted at the space between lumbar vertebrae L5 and L6 with an angle of about 20–30◦ left to the vertebral column. (B) The side view of the mouse. The catheter with guide wire was inserted at an angle of 20–30◦ above the vertebral column. (C) Fixation of the catheter. The black circles are beads which are fixed in the muscle. The solid and dashed line represents the external and internal portion of the catheter, respectively.
10–20 min was taken as an indication for a correct intrathecal catheter location.
2.4. Verification of catheter localization
2.5. Motor function
One or 2 days after catheter insertion the free end was cut to allow intrathecal injection of lidocaine (2.5 l, 20 mg/ml, Astra, Södertälje, Sweden) followed by 5 l saline. Motor paralysis of the hindlimbs within 15 s and lasting for
The motor function of the mice were examined 1 day after implantation by observing if there was any walking dysfunction, weakened extension withdrawal reflex of the hindlimb and reduced toe spread.
W.-P. Wu et al. / Journal of Neuroscience Methods 133 (2004) 65–69
2.6. Morphological studies Nine mice were used for morphological examination. Three were normal controls. Three mice were sacrificed 4 and 12 days after catheterization, respectively. The animals were deeply anesthetized with chloral hydrate and spinal cord segments L2–L5 were removed after laminectomy and placed in fixative of 2% glutaraldehyde in 0.1 M cacodylate buffer (pH = 7.2) at room temperature for 2 h. The segments were then stored in the fixative at 4 ◦ C until further process. The segments were rinsed and were osmicated in 2% OsO4 for 2 h at 0 ◦ C, dehydrated serially with alcohol and acetone and embedded in LX-112 plastic (Ladd Res. Industries Inc., VT, USA). Transverse sections (1 m thick) were cut from the slices and stained with toluidine blue.
67
and the Wilcoxon signed rank test was used for the chronic morphine experiment.
3. Results 3.1. General conditions and response to lidocaine after implantation Around 20% of mice implanted with i.t. catheters exhibited some degree of motor impairment 1 day after implantation and were sacrificed. In the remaining mice, the position of catheter was checked by i.t. lidocaine 1–2 days after implantation. It was found that 25 of 27 (93%) mice had total bilateral paralysis of the hindlimbs within 15 s lasting for 10–20 min.
2.7. Lumbar puncture
3.2. Morphologic analysis
A 2 cm long skin incision was made over the lumber region. The mice were rested for 20–30 min before drug injection. The injection was made at the space between L5 and L6 where the cord ends and cauda equina begins (Sidman et al., 1971). A 27 gauge needle was used to puncture the muscle, spinal process and the dura until a characterized brief motor response of tail or hindlimbs was observed indicating dura penetration. The needle was connected to a microsyringe through a PE 50 polyester catheter (12 cm).
After laminectomy, the tip of catheter was located in the subarachnoid space at the level of the lumbar enlargement. No signs of morphological abnormalities could be seen under light microscopic level in the lumbar spinal cord removed 4 or 12 days after catheter implantation (data not shown).
2.8. Drugs Morphine hydrochloride (10 mg/ml, Pharmacia & Upjohn, Sweden) was diluted with physiological saline to desired concentrations. Morphine was injected in a 5 l volume followed by 5 l saline to flush the catheter. 2.9. Tail flick test The effect of i.t. morphine was evaluated with the tail flick test. The mice were held gently and a radiant heat source was focused 1–2 cm from the tip of the tail and the latency to tail flick was recorded automatically (Ugo Basile, Italy). The intensity of the stimulation was adjusted so that the baseline latency was 4–6 s and the cut-off value was set at 15 s. 2.10. Statistics The data are expressed as mean ± S.E.M. The percentage maximal possible effect (%MPE) was calculated based on the formula: post-morphine response–baseline % MPE = × 100 cut-off(15 s)–baseline The significance level was set at P < 0.05. Unpaired student’s t-test was used for the acute morphine experiment
3.3. The effects of acute intrathecal morphine The experiments were conducted 3 days after catheter implantation. Intrathecal saline had no effect on the tail flick latency whereas intrathecal morphine induced a dosedependent prolongation of the tail flick latency when administered either via the chronic catheter or through lumbar puncture and the effect of morphine was not significantly different between the two groups (Fig. 3). 3.4. Comparison of the effect of repeated morphine administration Five mice with a chronic catheter were administered 100 ng morphine for 5 days starting 3 days after catheter implantation and all mice responded up to day 5 (Fig. 4A). In the mice with lumbar puncture, we only observed significant antinociception on day 1 (Fig. 4B) and it became gradually difficult to perform lumbar puncture repeatedly and reliably mainly due to the difficulty in recognizing muscle structure overlying the vertebra resulting from previous cut and scar formation. Only two mice could be successfully punctured on day 5 (Fig. 4B).
4. Discussion The method of chronically implanting an intrathecal catheter in mice is simple to perform with a fairly high success rate and offers some advantages over the existing method of lumbar puncture (Hylden and Wilcox, 1980).
68
W.-P. Wu et al. / Journal of Neuroscience Methods 133 (2004) 65–69
100 (10) catheter puncture
% MPE (Tail Flick Latency)
80
(12)
60
40 (5)
20
(5) (5) (5)
(3)
Mor.100ng
-20
Mor.10ng
saline
Mor.1ng
0
Tail Flick Latency (s)
Fig. 3. The effect of acute i.t. morphine on tail flick latency in mice injected through the catheter (open bar) or lumbar puncture (filled bar). The doses are expressed on the X-axis and %MPE at 30 min after morphine administration are shown on the Y-axis. N is indicated in the brackets. Unpaired Student’s t-test indicated that there are no significant differences between the two groups at any doses.
18 16 14 12 10 8 6 4 2 0
*
(5)
(5)
1 2 3 4 Times of injection (days)
5
*
(5)
(5)
0
Tail Flick Latency (s)
*
(5)
(A)
20 18 16 14 12 10 8 6 4 2 0
before after
*
(6) (6)
(4)
(3) (2)
0 (B)
*
*
1 2 3 4 Times of injection (days)
5
Fig. 4. The effect of daily injection of morphine on the tail flick latency in mice through the chronic catheter (A) or lumbar puncture (B). The data are shown as baseline value for each day (circles) and 30 min after morphine administration. The number of mice in which the injection was successful is indicated in the brackets. Wilcoxon signed-ranks test was used to examine the difference between tail flick latency before and after morphine administration (∗ P < 0.05).
The majority of mice had proper location of the catheter, no neurological deficits and no damage to the spinal cord. Successfully implantated mice showed reliable response to intrathecal morphine and since the catheter was
implanted prior to drug administration, the proper position of the catheter tip could be evaluated with lidocaine. The mice may also be less stressed when the catheter was pre-implanted than with acute injection. Furthermore, although both methods require practice, the implantation is done under anesthesia. Another important practical advantage is that with the implantation method the person who performs the implantation does not need to be present at the subsequent pharmacological experiments. The implanted catheters are clearly functional as lidocaine and morphine produced expected effects. Repeated daily injection of morphine through the chronically implanted catheter produced consistent antinociceptive effect in mice tested for up to 5 days. The mice behaved normally and there was no blockade of the catheters. However, repeated injection through lumbar puncture proved to be a difficult task even for a highly skilled experimenter. This is primarily due to the difficulty in recognizing muscle structure overlying the vertebra resulting from scar formation. In conclusion, we showed that intrathecal implantation of a chronic intrathecal catheter is feasible in mice. The method offers several advantages over existing lumbar puncture technique particularly for chronic repeated administration. This is preferable in experiments involving studies of drug tolerance and in situation where the number of mice may be limited, such as in studies of genetically modified mice.
Acknowledgements The present study is supported by Swedish Science Council (12168), Clinical Research Center of the Huddinge University Hospital and the research funds of the Karolinska Institutet.
W.-P. Wu et al. / Journal of Neuroscience Methods 133 (2004) 65–69
References Hylden JLK, Wilcox GL. Intrathecal morphine in mice: a new technique. Eur J Pharmacol 1980;67:313–6. Sidman RL, Angevine JB, Pierce ET. Atlas of the mouse brain and spinal cord. Cambridge, MA, USA: Harvard University Press; 1971.
69
Storkson RV, Kjorsvik A, Tjolsen A, Hole K. Lumbar catheterization of the spinal subarachnoid space in the rat. J Neurosci Meth 1996;65:167–72. Yaksh TL, Rudy TA. Chronic catheterization of the spinal subarachnoid space. Physiol Behav 1976;17:1036–7. Zimmermann M. Ethical guidelines for investigations of experimental pain in conscious animals (editorial). Pain 1983;16:109–10.