Exp Brain Res (1992) 91:229-235
Experimental BrainResearch 9 Springer-Verlag 1992
Systemic tocainide relieves mechanical hypersensitivity and normalizes the responses of hyperexcitable dorsal horn wide-dynamic-range neurons after transient spinal cord ischemia in rats J-X. Hao 1'2, Y-X. Yu 1*, A. Seiger 2, and Z. Wiesenfeld-Hallin i i Department of Clinical Physiology, Section of Clinical Neurophysiology, Huddinge University Hospital, S-141 86 Huddinge, Sweden 2 Department of Geriatric Medicine, Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden Received March 10, 1992 / Accepted May 29, 1992
Summary. In the present study we examined the effect of systemic tocainide on sensory hypersensitivity in rats after spinal cord ischemia induced by a photochemical technique. After induction of spinal cord ischemia the rats exhibited a sensory disturbance which was mainly expressed as vocalization to innocuous cutaneous mechanical stimuli (allodynia) in the flank area during the following several days. Tocainide at 75 mg/kg i.p., but not 50 mg/kg i.p., significantly increased the vocalization threshold to mechanical pressure for 2 h. The effect of intraarterial (i.a.) tocainide on the responses of dorsal horn wide-dynamic-range (WDR) neurons to suprathreshold electrical stimulation of their receptive fields was also examined in normal rats and after transient spinal cord ischemia, at a time when the animals exhibited typical behavioral allodynia in the dermatomes innervated by the ischemic spinal segments. In normal rats, tocainide (50 mg/kg i.a.) strongly suppressed the responses of WDR neurons to C fiber input with lesser effect on A fiber input. In allodynic rats, tocainide suppressed the augmented A and C fiber mediated responses of WDR neurons to the extent that their responses were similar to those seen in normal rats without tocainide. There was no difference in the overall depression of A and C fiber mediated input by tocainide between normal and allodynic rats. The present results demonstrated the analgesic effect of systemic tocainide in relieving allodynia in rats and indicated that systemic local anesthetics, at doses that do not block nerve conduction, can be effective in suppressing dorsal horn WDR neuronal activity. Although such drugs primarily suppress C fiber induced activity, the depression by local anesthetics of increased A fiber induced responses in allodynic conditions mediated by myelinated afferents may explain the analgesic effect of such drugs on behavior.
* Present address: Department of Physiology, Beijing Medical University, Beijing, China Correspondence to:
Z. Wiesenfeld-Hallin
Key words: Local anesthetics - Spinal cord - Ischemia Pain - Tocainide
Introduction Tocainide is a primary amine congener of lidocaine and is used as an antiarrhythmic agent. It has the advantages over lidocaine of longer duration of action and excellent bioavailability after oral administration (Young et al. 1980; Holmes et al. 1983). Clinically, tocainide has been demonstrated to have analgesic effects in patients with trigeminal neuralgia (Lindstr6m and Lindblom 1987). Thus, this drug, when administered systemically at doses that do not have an effect on nerve conduction, exerts effects on the central nervous system, presumably at central synapses. We have previously found that nontoxic doses of systemically applied tocainide caused longlasting thermal analgesia in rats (Wiesenfeld-Hallin and Lindblom 1985). In an electrophysiological study in rats, tocainide at 50 mg/kg injected systemically was found to block the polysynaptic flexor reflex, without blocking conduction in A[~, A~ or C afferents. Furthermore, at this dose tocainide did not depress the monosynaptic reflex. In its blocking of the flexor reflex, tocainide depressed the input from A[~ and Ag fibers to a lesser extent than from C afferents (Woolf and Wiesenfeld-Hallin 1985). Such a rather selective depressive effect on the C fiber input in the spinal cord by tocainide may therefore underlie the analgesic effect observed in behaving animals. We have recently developed an animal pain model of spinal origin in the rat, which has many of the features also seen in patients who have pain from spinal cord injuries. Spinal ischemia produced by the photochemical reaction of an organic dye combined with a laser beam evokes platelet aggregation (Watson et al. 1988) and induces a state of hypersensitivity in rats. Notably, the rats showed pain-like reactions to innocuous mechanical stimuli applied by brushing or pressure to the dermatomes innervated by the ischemic spinal segments
230 (mechanical allodynia). This aUodynia was transient after 1-min laser irradiation, lasting a b o u t 1 week, and was n o t a c c o m p a n i e d by severe m o t o r deficts or detectable m o r p h o l o g i c a l d a m a g e in the spinal cord, dorsal roots, and dorsal r o o t ganglia ( H a o et al. 1991a). W e have also s h o w n that transient spinal c o r d ischemia caused hypersensitivity o f dorsal h o r n w i d e - d y n a m i c - r a n g e ( W D R ) neurons that received input f r o m b o t h A a n d C afferents to c u t a n e o u s mechanical stimuli ( H a o et al. 1991b). The responses o f the W D R n e u r o n s were also increased in response to suprathreshold electrical stimuli, mainly expressed as an a u g m e n t e d A fiber response ( H a o et al. 1991b). The behavioral allodynia as well as the mechanical hypersensitivity o f dorsal h o r n W D R neurons, were reversed by a systemic low dose o f the G A B A B agonist baclofen ( H a o et al. 1991a a n d unpublished data). These results indicated that spinal c o r d ischemia t h a t resulted in transient hypersensitivity o f W D R n e u r o n s to cutaneous myelinated A fiber input could be due to dysfunction o f G A B A e r g i c n e u r o n s in the spinal cord. This was in agreement with o u r behavioral observations. The transient allodynia was n o t relieved by a n u m b e r o f drugs, including systemic morphine, guanethidine, and m u s c i m o l ( H a o et al. 1991a). Since previous d a t a suggested that systemic low doses o f local anesthetics m a y have an analgesic effect u p o n n e u r o p a t h i c pain, we initiated the present study to examine the effect o f systemic tocainide on the allodynia-like s y m p t o m s after transient spinal c o r d ischemia. W e observed t h a t tocainide effectively relieved allodynia at doses below those that p r o d u c e d side effects, This result was s o m e w h a t surprising, as previous evidence indicated that systemic local anesthetics interfere with the transfer o f afferent input primarily via slowly conducting, u n m y e l i n a t e d C fibers, rather t h a n rapidly conducting, myelinated A fibers ( W o o l f and Wiesenfeld-Hallin 1985; Sotgiu et al. 1991). This p r o m p t e d us to investigate the effect o f systemic tocainide on the W D R cells in the dorsal h o r n o f the spinal c o r d in n o r m a l rats and in rats after spinal cord ischemia when these n e u r o n s were hyperexcitable.
Materials and methods Experiments were performed on female Sprague-Dawley rats (Alab, Stockholm, Sweden) weighing 200-250 g. The experiments were approved by the local ethics committee.
Induction of transient spinal cord ischemia Transient spinal cord ischemia was produced by the photochemical technique developed by Watson et al. (1986). Detailed surgical procedure and technical parameters for laser irradiation have been described previously (Hao et al. 1991a). Briefly, rats were anesthetized with chloral hydrate (300 mg/kg i.p., Sigma) and one jugular vein was cannulated. Vertebra T10 or T13, which overly spinal segments T11-12 or L4-5, were exposed and an organic dye, erythrosin B (Red No. 3; Aldrich-Chemie, Steinheim, Germany) was injected intravenously at a dose of 32.5 mg/kg. Rats were then positioned under a tunable argon ion laser (Innova, Model 70, Coherent Laser Products Division, Palo Alto, Calif., USA) and irradiated for 1 min at vertebral segment T10 or T13 (spinal segments Tll-12 or L4-5) by a knife-edge beam (1 x 10 mm).
Behavioral measurement Allodynia after laser irradiation was evaluated by recording the vocalization threshold to mechanical pressure applied by a set of calibrated yon Frey hairs, with which graded pressures of 0.021 g410 g could be applied on the skin. Twenty-four hours after spinal cord ischemia at vertebra T10 (spinal segments T11-12), the rats were gently held by the experimenter and the von Frey hairs were pressed on the skin until the filaments bent. Vocalization in response to pressure in the skin area innervated by the irradiated segments was taken as threshold.
Preparation for electrophysiological experiments Experiments were performed in normal rats, as well as 1-4 days after irradiation of vertebra T13 (spinal segments L4-L5) while the rats were exhibiting typical behavioral allodynia. The rats were briefly anesthetized with methohexital (70 mg/kg i.p. ; Brietal, Lilly, Indianapolis, USA) and decerebrated by aspiration of the forebrain and midbrain and then artificially ventilated. One carotid artery was cannulated. A laminectomy was performed at midthoracic level, and the cord was transected at T7-8. A second laminectomy was performed to expose lumbar segments L3-6. The cord was held rigidly by clamping vertebral processes caudal and rostral to the exposed segments. The dura was opened and the cord was covered with paraffin oil. The rats were paralyzed with pancuronium bromide (Pavulon, Organon) 2.5 mg/kg i.p., and blood pressure, electrocardiogram, rectal temperature, and skin coloration were monitored. The activity of single dorsal horn cells was recorded extracellularly with glass-coated tungsten microelectrodes (tip diameter 1-2 gm) in lumbar segments L4-5, which were at the epicenter of the laser irradiation. The search stimuli for evoking responses from the cells were electric shocks (5 mA, 1 ms, 0.2 Hz) applied subcutaneously to the sciatic nerve innervation area of the left hindpaw. When a single unit was isolated, the response of the neuron was characterized with mechanical stimuli (light brushing, light pressure, and pinch) and its receptive field was mapped. Only WDR cells, which were characterized by graded responses to innocuous (brushing, light pressure) and noxious (pinch) stimuli, were studied. After the characterization of the neurons, two to three control trials were recorded at 2-min, intervals in response to suprathreshold electrical stimuli before drug administration. Each trial consisted of ten averaged responses to subcutaneously applied shocks (5 mA, 1 ms, 0.2 Hz) in the receptive field of the cell. The action potentials were amplified, filtered, and displayed on an oscilloscope and analyzed on line by a microcomputer with a poststimulus time histogram program. The data for quantitative analysis were compiled in 48-ms bins. Neuronal responses were monitored at 1, 5, and 10 min postinjection and at 10-rain intervals thereafter until the response was back to control level. The depth of the cell locations was between 100 and 800 gm below the cord surface as recorded from the microdrive. In most experiments, on completion of a recording track a glass electrode was inserted into the cord at the same site and to the same depth as the recording electrode and its tip was broken and left in the cord for histological localization.
Drugs Tocainide hydrochloride (Tonocard, Hfissle, Sweden) was diluted in 0.9% saline and injected in a volume of 0.5 ml at 25, 50, or 75 mg/kg.
Statistics The data were expressed as mean • SEM and were analyzed by the Zz test, Kruskal-Wallis one-way analysis of variance (ANOVA)
231
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Fig. 1. The effect of 50 mg/kg (squares) and 75 mg/kg (circles) tocainide i.p. on vocalization threshold to mechanical pressure in spinally injured rats 24 h postlesion, n= 5 in each group, and the drug was administered at time zero. Kruskal-Wallis one-way ANOVA indicated significant overall increases in vocalization threshold
after 75 mg/kg to cainide (P < 0.01), while no significant difference was found after 50 mg/kg tocainide (P>0.05). Individual comparisons were made with the Mann-Whitney U test (* P
Experimental BrainResearch 9 Springer-Verlag 1992
Systemic tocainide relieves mechanical hypersensitivity and normalizes the responses of hyperexcitable dorsal horn wide-dynamic-range neurons after transient spinal cord ischemia in rats J-X. Hao 1'2, Y-X. Yu 1*, A. Seiger 2, and Z. Wiesenfeld-Hallin i i Department of Clinical Physiology, Section of Clinical Neurophysiology, Huddinge University Hospital, S-141 86 Huddinge, Sweden 2 Department of Geriatric Medicine, Karolinska Institute, Huddinge University Hospital, S-141 86 Huddinge, Sweden Received March 10, 1992 / Accepted May 29, 1992
Summary. In the present study we examined the effect of systemic tocainide on sensory hypersensitivity in rats after spinal cord ischemia induced by a photochemical technique. After induction of spinal cord ischemia the rats exhibited a sensory disturbance which was mainly expressed as vocalization to innocuous cutaneous mechanical stimuli (allodynia) in the flank area during the following several days. Tocainide at 75 mg/kg i.p., but not 50 mg/kg i.p., significantly increased the vocalization threshold to mechanical pressure for 2 h. The effect of intraarterial (i.a.) tocainide on the responses of dorsal horn wide-dynamic-range (WDR) neurons to suprathreshold electrical stimulation of their receptive fields was also examined in normal rats and after transient spinal cord ischemia, at a time when the animals exhibited typical behavioral allodynia in the dermatomes innervated by the ischemic spinal segments. In normal rats, tocainide (50 mg/kg i.a.) strongly suppressed the responses of WDR neurons to C fiber input with lesser effect on A fiber input. In allodynic rats, tocainide suppressed the augmented A and C fiber mediated responses of WDR neurons to the extent that their responses were similar to those seen in normal rats without tocainide. There was no difference in the overall depression of A and C fiber mediated input by tocainide between normal and allodynic rats. The present results demonstrated the analgesic effect of systemic tocainide in relieving allodynia in rats and indicated that systemic local anesthetics, at doses that do not block nerve conduction, can be effective in suppressing dorsal horn WDR neuronal activity. Although such drugs primarily suppress C fiber induced activity, the depression by local anesthetics of increased A fiber induced responses in allodynic conditions mediated by myelinated afferents may explain the analgesic effect of such drugs on behavior.
* Present address: Department of Physiology, Beijing Medical University, Beijing, China Correspondence to:
Z. Wiesenfeld-Hallin
Key words: Local anesthetics - Spinal cord - Ischemia Pain - Tocainide
Introduction Tocainide is a primary amine congener of lidocaine and is used as an antiarrhythmic agent. It has the advantages over lidocaine of longer duration of action and excellent bioavailability after oral administration (Young et al. 1980; Holmes et al. 1983). Clinically, tocainide has been demonstrated to have analgesic effects in patients with trigeminal neuralgia (Lindstr6m and Lindblom 1987). Thus, this drug, when administered systemically at doses that do not have an effect on nerve conduction, exerts effects on the central nervous system, presumably at central synapses. We have previously found that nontoxic doses of systemically applied tocainide caused longlasting thermal analgesia in rats (Wiesenfeld-Hallin and Lindblom 1985). In an electrophysiological study in rats, tocainide at 50 mg/kg injected systemically was found to block the polysynaptic flexor reflex, without blocking conduction in A[~, A~ or C afferents. Furthermore, at this dose tocainide did not depress the monosynaptic reflex. In its blocking of the flexor reflex, tocainide depressed the input from A[~ and Ag fibers to a lesser extent than from C afferents (Woolf and Wiesenfeld-Hallin 1985). Such a rather selective depressive effect on the C fiber input in the spinal cord by tocainide may therefore underlie the analgesic effect observed in behaving animals. We have recently developed an animal pain model of spinal origin in the rat, which has many of the features also seen in patients who have pain from spinal cord injuries. Spinal ischemia produced by the photochemical reaction of an organic dye combined with a laser beam evokes platelet aggregation (Watson et al. 1988) and induces a state of hypersensitivity in rats. Notably, the rats showed pain-like reactions to innocuous mechanical stimuli applied by brushing or pressure to the dermatomes innervated by the ischemic spinal segments
230 (mechanical allodynia). This aUodynia was transient after 1-min laser irradiation, lasting a b o u t 1 week, and was n o t a c c o m p a n i e d by severe m o t o r deficts or detectable m o r p h o l o g i c a l d a m a g e in the spinal cord, dorsal roots, and dorsal r o o t ganglia ( H a o et al. 1991a). W e have also s h o w n that transient spinal c o r d ischemia caused hypersensitivity o f dorsal h o r n w i d e - d y n a m i c - r a n g e ( W D R ) neurons that received input f r o m b o t h A a n d C afferents to c u t a n e o u s mechanical stimuli ( H a o et al. 1991b). The responses o f the W D R n e u r o n s were also increased in response to suprathreshold electrical stimuli, mainly expressed as an a u g m e n t e d A fiber response ( H a o et al. 1991b). The behavioral allodynia as well as the mechanical hypersensitivity o f dorsal h o r n W D R neurons, were reversed by a systemic low dose o f the G A B A B agonist baclofen ( H a o et al. 1991a a n d unpublished data). These results indicated that spinal c o r d ischemia t h a t resulted in transient hypersensitivity o f W D R n e u r o n s to cutaneous myelinated A fiber input could be due to dysfunction o f G A B A e r g i c n e u r o n s in the spinal cord. This was in agreement with o u r behavioral observations. The transient allodynia was n o t relieved by a n u m b e r o f drugs, including systemic morphine, guanethidine, and m u s c i m o l ( H a o et al. 1991a). Since previous d a t a suggested that systemic low doses o f local anesthetics m a y have an analgesic effect u p o n n e u r o p a t h i c pain, we initiated the present study to examine the effect o f systemic tocainide on the allodynia-like s y m p t o m s after transient spinal c o r d ischemia. W e observed t h a t tocainide effectively relieved allodynia at doses below those that p r o d u c e d side effects, This result was s o m e w h a t surprising, as previous evidence indicated that systemic local anesthetics interfere with the transfer o f afferent input primarily via slowly conducting, u n m y e l i n a t e d C fibers, rather t h a n rapidly conducting, myelinated A fibers ( W o o l f and Wiesenfeld-Hallin 1985; Sotgiu et al. 1991). This p r o m p t e d us to investigate the effect o f systemic tocainide on the W D R cells in the dorsal h o r n o f the spinal c o r d in n o r m a l rats and in rats after spinal cord ischemia when these n e u r o n s were hyperexcitable.
Materials and methods Experiments were performed on female Sprague-Dawley rats (Alab, Stockholm, Sweden) weighing 200-250 g. The experiments were approved by the local ethics committee.
Induction of transient spinal cord ischemia Transient spinal cord ischemia was produced by the photochemical technique developed by Watson et al. (1986). Detailed surgical procedure and technical parameters for laser irradiation have been described previously (Hao et al. 1991a). Briefly, rats were anesthetized with chloral hydrate (300 mg/kg i.p., Sigma) and one jugular vein was cannulated. Vertebra T10 or T13, which overly spinal segments T11-12 or L4-5, were exposed and an organic dye, erythrosin B (Red No. 3; Aldrich-Chemie, Steinheim, Germany) was injected intravenously at a dose of 32.5 mg/kg. Rats were then positioned under a tunable argon ion laser (Innova, Model 70, Coherent Laser Products Division, Palo Alto, Calif., USA) and irradiated for 1 min at vertebral segment T10 or T13 (spinal segments Tll-12 or L4-5) by a knife-edge beam (1 x 10 mm).
Behavioral measurement Allodynia after laser irradiation was evaluated by recording the vocalization threshold to mechanical pressure applied by a set of calibrated yon Frey hairs, with which graded pressures of 0.021 g410 g could be applied on the skin. Twenty-four hours after spinal cord ischemia at vertebra T10 (spinal segments T11-12), the rats were gently held by the experimenter and the von Frey hairs were pressed on the skin until the filaments bent. Vocalization in response to pressure in the skin area innervated by the irradiated segments was taken as threshold.
Preparation for electrophysiological experiments Experiments were performed in normal rats, as well as 1-4 days after irradiation of vertebra T13 (spinal segments L4-L5) while the rats were exhibiting typical behavioral allodynia. The rats were briefly anesthetized with methohexital (70 mg/kg i.p. ; Brietal, Lilly, Indianapolis, USA) and decerebrated by aspiration of the forebrain and midbrain and then artificially ventilated. One carotid artery was cannulated. A laminectomy was performed at midthoracic level, and the cord was transected at T7-8. A second laminectomy was performed to expose lumbar segments L3-6. The cord was held rigidly by clamping vertebral processes caudal and rostral to the exposed segments. The dura was opened and the cord was covered with paraffin oil. The rats were paralyzed with pancuronium bromide (Pavulon, Organon) 2.5 mg/kg i.p., and blood pressure, electrocardiogram, rectal temperature, and skin coloration were monitored. The activity of single dorsal horn cells was recorded extracellularly with glass-coated tungsten microelectrodes (tip diameter 1-2 gm) in lumbar segments L4-5, which were at the epicenter of the laser irradiation. The search stimuli for evoking responses from the cells were electric shocks (5 mA, 1 ms, 0.2 Hz) applied subcutaneously to the sciatic nerve innervation area of the left hindpaw. When a single unit was isolated, the response of the neuron was characterized with mechanical stimuli (light brushing, light pressure, and pinch) and its receptive field was mapped. Only WDR cells, which were characterized by graded responses to innocuous (brushing, light pressure) and noxious (pinch) stimuli, were studied. After the characterization of the neurons, two to three control trials were recorded at 2-min, intervals in response to suprathreshold electrical stimuli before drug administration. Each trial consisted of ten averaged responses to subcutaneously applied shocks (5 mA, 1 ms, 0.2 Hz) in the receptive field of the cell. The action potentials were amplified, filtered, and displayed on an oscilloscope and analyzed on line by a microcomputer with a poststimulus time histogram program. The data for quantitative analysis were compiled in 48-ms bins. Neuronal responses were monitored at 1, 5, and 10 min postinjection and at 10-rain intervals thereafter until the response was back to control level. The depth of the cell locations was between 100 and 800 gm below the cord surface as recorded from the microdrive. In most experiments, on completion of a recording track a glass electrode was inserted into the cord at the same site and to the same depth as the recording electrode and its tip was broken and left in the cord for histological localization.
Drugs Tocainide hydrochloride (Tonocard, Hfissle, Sweden) was diluted in 0.9% saline and injected in a volume of 0.5 ml at 25, 50, or 75 mg/kg.
Statistics The data were expressed as mean • SEM and were analyzed by the Zz test, Kruskal-Wallis one-way analysis of variance (ANOVA)
231
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1~ O O 20~>
Y-r' r
T I
0
30
I
60
T
T
T
[
I
I
I
I
f
90
120
150
180
210
240
Time
(mini
Fig. 1. The effect of 50 mg/kg (squares) and 75 mg/kg (circles) tocainide i.p. on vocalization threshold to mechanical pressure in spinally injured rats 24 h postlesion, n= 5 in each group, and the drug was administered at time zero. Kruskal-Wallis one-way ANOVA indicated significant overall increases in vocalization threshold
after 75 mg/kg to cainide (P < 0.01), while no significant difference was found after 50 mg/kg tocainide (P>0.05). Individual comparisons were made with the Mann-Whitney U test (* P
