105
Neuroscience Letters, 128 (1991) 105-108 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100339X NSL 07857
Hypersensitivity of dorsal horn wide dynamic range neurons to cutaneous mechanical stimuli after transient spinal cord ischemia in the rat J.-X. Hao 1'2, X.-J. Xu 1, Y.-X. Yu ~, ,~. Seiger2 and Z. Wiesenfeld-Hallin1 Karolinska Institute, 1Department of Clinical Physiology, Section of Clinical Neurophysiology and 2Department of Geriatric Medicine, Huddinge University Hospital, Huddinge (Sweden) (Received 22 February 1991; Revised version received 8 April 1991; Accepted 8 April 1991)
Key words: Wide dynamic range neurons; Ischemia; Spinal cord; Central Pain; Allodynia; Mechanoreceptors The responsiveness of dorsal horn wide dynamic range (WDR) neurons to cutaneous mechanical stimuli was studied in decerebrate, spinalized, unanesthetized rats before and after transient photochemically induced spinal cord ischemia. In normal rats, the discharges of dorsal horn WDR neurons to the graded mechanical stimuli applied with calibrated von Frey hairs increase linearly. One to four days after spinal ischemia, when the rats exhibit a strong allodynia-like behavioral reaction to cutaneous stimuli, the sensitivity of dorsal horn WDR neurons to mechanical pressure is greatly increased. There is a significant decrease in the threshold pressure to evoke neuronal discharges and the exponential stimulus-response curve is shifted to the left. Thus, transient ischemia of the spinal cord results in hyperexcitability of dorsal horn WDR neurons, which may underly the allodynia-like sensory abnormalities observed in behaving animals. The present results may contribute to understanding the mechanism of the development of chronic central pain in patients after central nervous system injury involving ischemia.
Injury to the central nervous system (CNS) can lead to neuronal hyperexcitability and chronic pain [see refs 7 and 9 for review]. The mechanisms underlying the development of central pain are unclear and no effective treatments are available [7]. We have recently shown that a photochemically induced spinal cord ischemic injury evokes pain-related sensations in rats [2]. The syndrome is mainly expressed as allodynia, which is defined as nociceptive r~actions to non-noxious stimuli. The allodynia is observed as abnormal reactions, including vocalization and agitation, to graded, weak mechanical stimuli applied to the dermatomes innervated by the influenced spinal segments. The allodynic behavior persists for several days after initiation of spinal cord ischemia. It is present even after transient spinal ischemia, which does not cause severe motor deficits or detectable morphological damage in the dorsal root ganglia, dorsal roots and spinal cord in the majority of rats [2]. The allodynia is reversed by the GABAB agonist baclofen, but not the GABAA agonist muscimol or the opioid agonist morphine [2]. Since this sensory abnormality is a consequence of spinal cord ischemia, it may be viewed as an animal model for central pain. The present study was conducted to explore the possible neuronal mechanisms Correspondence: Z. Wiesenfeld-Hallin, Department of Clinical Neurophysiology, Huddinge University Hospital, S-141 86 Huddinge, Sweden.
responsible for this allodynic phenomenon by examining the responsiveness of dorsal horn wide dynamic range neurons [3] to cutaneous mechanical stimuli in normal rats and those exhibiting allodynia after spinal cord ischemia. Experiments were performed on 34 female SpragueDawley rats weighing 200 g (Alab, Stockholm, Sweden). The spinal cord ischemia was produced with a recently developed photochemical technique [2,5,8]. This method utilizes an intravascular photochemical reaction with a chemical dye, which is activated by an argon ion laser, to produce single oxygen molecules at the endothelial surface of spinal cord vessels, resulting in an intense platelet response leading to vessel occlusion and subsequent parenchymal tissue infarction. The rats were anesthetized with chloral hydrate (Sigma, 300 mg/kg, i.p.) and one jugular vein was cannulated. After a midline incision of the skin on the back, the underlying vertebrae were exposed (T12-L1). The animals were then positioned under the laser. Erythrosin B (Red No. 3, Aldrich-Chemic, Steinheim, F.R.G.), dissolved in 0.9% saline, was injected i.v. at a dose of 32.5 mg/kg. Immediately following the injection, the rats were irradiated for 1 min under the laser beam at vertebral segment T13 (spinal segments L4-5). A tunable argon ion laser (Innova, model 70, Coherent Laser Products Division, Palo Alto, CA, U.S.A.) operating at 514.5 nm, which is near the absorption maximum of erythrosin
106 TABLE I D I S T R I B U T I O N OF N E U R O N A L TYPES IN T H E D O R S A L H O R N OF N O R M A L A N D A L L O D Y N I C RATS The neurons were characterized by their responses to mechanical stimuli (brushing, light pressure and pinch). The wide dynamic range neurons gave a graded response to innocuous and noxious mechanical stimuli. High-threshold neurons responded exclusively to noxious stimuli, whereas low-threshold neurons responded maximally to innocuous stimuli. Spinal ischemia and the presence of allodynia did not influence the distribution of neuronal types (2'2 = 0.33, P > 0.05).
Wide dynamic range High threshold Low threshold
Normal rats
Allodynic rats
31 4 2
29 3 1
B, was used for irradiation. The vertebra and the spinal cord were irradiated with an average power of 0.16 W delivered via a beam chopper operating at 500 Hz (2.4 W peak power, 6.7% duty circle). A knife edge beam was used to cover the single T13 vertebra. In order to reduce the amount of heat introduced into the tissue during the irradiation, two piezoelectric fans (Piezo System Inc., Cambridge, MA, U.S.A.) operating at peak air velocity of 6 m/s were placed as close to the spinal column as possible (approximately 5 mm distance). During the period of irradiation, the body temperature of the rat was maintained between 37°C and 38°C. In acute electrophysiological experiments, normal rats and rats that had been exposed to laser irradiation 1-4 days earlier and were exhibiting typical hypersensitivity to innocuous mechanical stimuli were briefly anesthetized with methohexital (Brietal, Lilly, 70 mg/kg, i.p.), decerebrated by aspiration of the forebrain and midbrain and then artificially ventilated. A small laminectomy was performed at midthoracic level and the spinal cord was transected at Th7-8. A second laminectomy was performed to expose lumbar segments L3-6. The
T A B L E II RESPONSE C H A R A C T E R I S T I C S O F D O R S A L H O R N W I D E D Y N A M I C R A N G E N E U R O N S TO S U P R A T H R E S H O L D PERIP H E R A L E L E C T R I C A L S T I M U L A T I O N IN N O R M A L A N D A L L O D Y N I C RATS Data are presented as mean + S.E.M.
Separation of A and C response Response duration (ms) Background activity (imp/s)
Normal
Allodynia
27/31 (87%) 356.1 _+34.2 9.0+ 3.7
9/29 (31%p 704.4 _ 194.7 b 8 . 8 _ 2.9 c
a Z2= 32.51, P < 0.001; b t =2.82, P < 0.01; c not significant.
dura was opened and the cord was covered with mineral oil. The rats were paralyzed with pancuronium bromide (Pavalon, Organon, 2.5 mg/kg, i.p.) and the electrocardiogram, body temperature and skin coloration were monitored. Extracellular recordings of the activity of single cells in the dorsal horn at lumbar segments L4-5, the level where the cord was irradiated, were made with glasscoated tungsten microelectrodes. The action potentials were amplified, filtered and displayed on an oscilloscope and analyzed on-line by a microcomputer with a poststimulus time histogram program. Search stimuli for evoking cell responses were electric shocks applied subcutaneously to the hind paw or to the sciatic nerve, which was dissected at the popliteal fossa and suspended on a pair of silver hook electrodes. The shocks activated both A and C fibers (5 mA, 1 ms, 0.2 Hz). Once a single unit was isolated and its response to electrical stimulation was recorded, the response of the neuron was characterized with mechanical stimuli (light brushing, light pressure and pinch) and its receptive field was mapped. The quantitative response of the neuron to mechanical stimulation was then examined by pressing calibrated yon Frey hairs against the skin at the center of the receptor field for 8 l0 s. The data collection was initiated after the dynamic response of the cell to the application of the von Frey hair had terminated. Thus, only the maintained response to mechanical pressure was analyzed. A total of 70 neurons were recorded from 34 rats, 37 from normal rats, 33 from rats after spinal ischemia, through the entire depth of the dorsal horn. The depth of the recorded cells was between 100 and 800 pm below the cord surface. 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 electrodes and its tip was broken off and left in the cord. The recording site was then localized histologically in Rexed's laminae I-V. There was no difference in the distribution of the depth of the recording sites in the normal and allodynic rats. The majority of the neurons encountered in normal rats were wide dynamic range (WDR) cells, as they could be activated by both innocuous (brushing, light pressure) and noxious (pinch) stimuli (Table I). A few cells could be maximally activated by innocuous stimuli and were characterized as low threshold (LT) neurons. Some others responded only to noxious pressure and were thus characterized as high threshold (HT) neurons. Spinal ischemia did not change the distribution of the neuronal classification (Table I). In normal rats, the response of dorsal horn W D R neurons to suprathreshold electrical stimulation of their re-
107 B
A .,+iii.i.
I
iJ,,,/t ....................
L
I
__2
:.,,,
~ir .+ ~ - i +=++ k ~ l l+~.+ ~ " *! ,+. q --~,+-1 . - , - , - 1 .+r . ,
+
I
.+
.•JlOOpV
t
lOOms
Fig. 1. Photographic illustration of typical responses of two dorsal horn WDR neurons to subcutaneous electrical stimuli (5 mA, 1 ms) applied to their cutaneous receptive fields in a normal rat (A) and at two days after spinal ischemia (B). Note the increased A-fiberresponse and prolongation of the overall response duration of the neuron in the allodynic rat. The stimulus is indicated by the arrows in A and B. ceptive field or of the sciatic nerve consists of short latency A-fiber ( < 1-40 ms) and long latency C-fiber responses (90-400 ms) (Table II, Fig. 1A). In a few cases, afterdischarges (400-900 ms) were observed. Regression analysis revealed that there was a highly significant positive linear correlation between the neuronal discharges and the cutaneous pressure applied via the von Frey hairs (Fig. 2). The threshold in these neurons to evoke a clear neuronal response, defined as 20% over background activity, was 13.8 _ 2.2 g (range 4.7-73 g, n = 31). One to four days after induction of spinal ischemia, at which time the rats exhibited behavioral allodynia, the neuronal responsiveness of dorsal horn W D R neurons to cutaneous stimuli was dramatically increased. The pattern of the neuronal responses to suprathreshold electrical stimulation was altered. The A-fiber response became prolonged and, in most cases, the interval between A- and C-fiber responses became indistinguishable (Table II, Fig. 1B). The duration of the neuronal response of the cells to electrical stimulation also increased in most cases (Table II, Fig. 1B). The rate of background activity was similar in normal and allodynic animals (Table II), but the sensitivity of these neurons to mechanical pressure increased significantly. The neurons began to respond at much lower pressure and the rate of increase of discharges during pressure became much more rapid (Fig. 2). Analysis of the discharge pattern indicated that there was a highly significant positive correlation between neuronal discharges and mechanical pressure and the regression analysis revealed that the response curve of these cells was best fitted by a quadratic equation (Fig. 2). The threshold of pressure that evoked a neuronal response 20% over background activity in ischemic and allodynic rats was 2.1 _ 0.4 g (range 0.097.1 g, n = 29), which is significantly different from the value in normal animals (t = 4.557, P < 0.0001). We have previously shown that transient spinal ischemia induces a state of mechanical hyperpathia in rats for
several days [2]. The present results indicate that such transient spinal ischemia results in hypersensitivity of dorsal horn W D R neurons to electrical and mechanical stimuli, thus providing a neuronal basis for the behavioral observations. The remarkable similarities between the behavioral and electrophysiological results suggest that the increased sensitivity of dorsal horn W D R neurons may underly the development of abnormal pain-related sensations in rats after spinal cord ischemia. What is the mechanism for this neuronal hypersensitivity after transient spinal ischemia? It is well known that small GABAergic interneurons located in the region of the substantia gelatinosa are involved in spinal inhibition [1,4]. The behavioral allodynia seen after spinal ischemia is relieved by low doses of systemic baclofen [2], suggesting that abnormal hypersensitivity may be related to the loss of inhibitory control normally exerted by GABAergic neurons; through baclofen-sensitive GABAB receptors presynaptically on afferent terminals and/or postsynaptically on W D R neurons, following transient spinal ischemia. In support of this hypothesis, it has been shown that GABAergic inhibitory neurons in the brain are highly susceptible to ischemic damage [6].
350
300-
1
250 -
i2 200"
0
|
.~
100 '
50
0 0
10
20
30
40
50
60
70
80
go
1~10
Pressure (g)
Fig. 2. Responses of dorsal horn WDR neurons to graded mechanical stimuli applied by von Frey hairs in normal rats (circle) and 1~, days after spinal ischemia(square), at which time the animals exhibited typical allodynia-like behavioral responses to mechanical stimuli. The neuronal responses were calculated as percent increase in the number of action potentials over background activity during mechanicalstimulation. The center of the receptivefield of each cell was stimulated with calibrated von Frey hairs for 8-10 s. Each data point represents responses of 20-30 cells and is presented as mean+S.E.M. The regression line for normal rats is y = 3.313x-2.972 (r = 0.623); for allodynic rats it is y=-0.089x2+ 12.522x+3.990 (r=0.657). Analysis of variance indicated that both regressions are highly significant (Fl.176= 110.982 and F2,296 = 111.366, respectively,P< 0.0001).
108
This work was supported by the Swedish MRC (Project 07913, 06555), the Bank of Sweden Tercentenary Foundation, the Miami Project Foundation, the Wenner-Gren Center Foundation, the Daniel Henmann Fund for Spinal Cord Research, the King Gustav V and Queen Victoria Foundation, the Petrus and Augusta Hedlunds Foundation, the Axelsson-Johnson Foundation and research funds of the Karolinska Institute. We wish to thank Dr V. Pieribone for providing us the software used for data analysis and Dr B.D. Watson for designing and installing our laser set-up. 1 Game, C.J.A. and Lodge, D., The pharmacology of the inhibition of dorsal horn neurones by impulses in myelinated cutaneous afterens in the cat, Exp. Brain Res., 23 (1975) 75-84. 2 Hao, J.-X., Xu, X.-J., Aldskogius, H., Seiger, .~. and WiesenfeldHallin, Z., Allodynia-like effect in rat after ischemic spinal cord injury photochemically induced by laser irradiation, Pain, in press. 3 Mendell, L.M., Physiological properties of unmyelinated fiber projection to the spinal cord, Exp. Neurol., 16 (1966) 316-332.
40tsuka, M. and Konishi, S., GABA in the spinal cord. In E. Roberts, T.N. Chase and D.B. Tower (Eds.), GABA in Nervous System Function, Raven, New York, 1976, pp. 197-202. 5 Prado, R., Dietrich, W.D., Watson, B.D., Ginsberg, M.D. and Green, B.A., Photoehemically induced graded spinal cord infarction: behavioral, electrophysiological, and morphological correlates, J. Neurosurg., 67 (1987) 743-753. 6 Sloper, J.J., Johnson, P. and Powell, T.P.S., Selective degeneration of interneurons in the motor cortex of infant monkeys following controlled hypoxia: a possible cause of epilepsy, Brain Res., 198 (1980) 204-209. 7 Tasker, R.R., Pain resulting from central nervous system pathology (central pain). In J.J. Bonica (Ed.), The Management of Pain, Vol. 1, 2nd edn., Lea and Febiger, Philadelphia, 1990, pp. 264-283. 8 Watson, B.D., Prado, R. and Dietrich, W.D., PhotochemicaUy induced spinal cord injury in the rat, Brain Res., 367 (1986) 296300. 9 Zimmermann, M., Central nervous mechanisms modulating painrelated information. In K.L. Casey (Ed.), Pain and Central Nervous System Disease: The Central Pain Syndromes, Raven, New York, in press.
Neuroscience Letters, 128 (1991) 105-108 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100339X NSL 07857
Hypersensitivity of dorsal horn wide dynamic range neurons to cutaneous mechanical stimuli after transient spinal cord ischemia in the rat J.-X. Hao 1'2, X.-J. Xu 1, Y.-X. Yu ~, ,~. Seiger2 and Z. Wiesenfeld-Hallin1 Karolinska Institute, 1Department of Clinical Physiology, Section of Clinical Neurophysiology and 2Department of Geriatric Medicine, Huddinge University Hospital, Huddinge (Sweden) (Received 22 February 1991; Revised version received 8 April 1991; Accepted 8 April 1991)
Key words: Wide dynamic range neurons; Ischemia; Spinal cord; Central Pain; Allodynia; Mechanoreceptors The responsiveness of dorsal horn wide dynamic range (WDR) neurons to cutaneous mechanical stimuli was studied in decerebrate, spinalized, unanesthetized rats before and after transient photochemically induced spinal cord ischemia. In normal rats, the discharges of dorsal horn WDR neurons to the graded mechanical stimuli applied with calibrated von Frey hairs increase linearly. One to four days after spinal ischemia, when the rats exhibit a strong allodynia-like behavioral reaction to cutaneous stimuli, the sensitivity of dorsal horn WDR neurons to mechanical pressure is greatly increased. There is a significant decrease in the threshold pressure to evoke neuronal discharges and the exponential stimulus-response curve is shifted to the left. Thus, transient ischemia of the spinal cord results in hyperexcitability of dorsal horn WDR neurons, which may underly the allodynia-like sensory abnormalities observed in behaving animals. The present results may contribute to understanding the mechanism of the development of chronic central pain in patients after central nervous system injury involving ischemia.
Injury to the central nervous system (CNS) can lead to neuronal hyperexcitability and chronic pain [see refs 7 and 9 for review]. The mechanisms underlying the development of central pain are unclear and no effective treatments are available [7]. We have recently shown that a photochemically induced spinal cord ischemic injury evokes pain-related sensations in rats [2]. The syndrome is mainly expressed as allodynia, which is defined as nociceptive r~actions to non-noxious stimuli. The allodynia is observed as abnormal reactions, including vocalization and agitation, to graded, weak mechanical stimuli applied to the dermatomes innervated by the influenced spinal segments. The allodynic behavior persists for several days after initiation of spinal cord ischemia. It is present even after transient spinal ischemia, which does not cause severe motor deficits or detectable morphological damage in the dorsal root ganglia, dorsal roots and spinal cord in the majority of rats [2]. The allodynia is reversed by the GABAB agonist baclofen, but not the GABAA agonist muscimol or the opioid agonist morphine [2]. Since this sensory abnormality is a consequence of spinal cord ischemia, it may be viewed as an animal model for central pain. The present study was conducted to explore the possible neuronal mechanisms Correspondence: Z. Wiesenfeld-Hallin, Department of Clinical Neurophysiology, Huddinge University Hospital, S-141 86 Huddinge, Sweden.
responsible for this allodynic phenomenon by examining the responsiveness of dorsal horn wide dynamic range neurons [3] to cutaneous mechanical stimuli in normal rats and those exhibiting allodynia after spinal cord ischemia. Experiments were performed on 34 female SpragueDawley rats weighing 200 g (Alab, Stockholm, Sweden). The spinal cord ischemia was produced with a recently developed photochemical technique [2,5,8]. This method utilizes an intravascular photochemical reaction with a chemical dye, which is activated by an argon ion laser, to produce single oxygen molecules at the endothelial surface of spinal cord vessels, resulting in an intense platelet response leading to vessel occlusion and subsequent parenchymal tissue infarction. The rats were anesthetized with chloral hydrate (Sigma, 300 mg/kg, i.p.) and one jugular vein was cannulated. After a midline incision of the skin on the back, the underlying vertebrae were exposed (T12-L1). The animals were then positioned under the laser. Erythrosin B (Red No. 3, Aldrich-Chemic, Steinheim, F.R.G.), dissolved in 0.9% saline, was injected i.v. at a dose of 32.5 mg/kg. Immediately following the injection, the rats were irradiated for 1 min under the laser beam at vertebral segment T13 (spinal segments L4-5). A tunable argon ion laser (Innova, model 70, Coherent Laser Products Division, Palo Alto, CA, U.S.A.) operating at 514.5 nm, which is near the absorption maximum of erythrosin
106 TABLE I D I S T R I B U T I O N OF N E U R O N A L TYPES IN T H E D O R S A L H O R N OF N O R M A L A N D A L L O D Y N I C RATS The neurons were characterized by their responses to mechanical stimuli (brushing, light pressure and pinch). The wide dynamic range neurons gave a graded response to innocuous and noxious mechanical stimuli. High-threshold neurons responded exclusively to noxious stimuli, whereas low-threshold neurons responded maximally to innocuous stimuli. Spinal ischemia and the presence of allodynia did not influence the distribution of neuronal types (2'2 = 0.33, P > 0.05).
Wide dynamic range High threshold Low threshold
Normal rats
Allodynic rats
31 4 2
29 3 1
B, was used for irradiation. The vertebra and the spinal cord were irradiated with an average power of 0.16 W delivered via a beam chopper operating at 500 Hz (2.4 W peak power, 6.7% duty circle). A knife edge beam was used to cover the single T13 vertebra. In order to reduce the amount of heat introduced into the tissue during the irradiation, two piezoelectric fans (Piezo System Inc., Cambridge, MA, U.S.A.) operating at peak air velocity of 6 m/s were placed as close to the spinal column as possible (approximately 5 mm distance). During the period of irradiation, the body temperature of the rat was maintained between 37°C and 38°C. In acute electrophysiological experiments, normal rats and rats that had been exposed to laser irradiation 1-4 days earlier and were exhibiting typical hypersensitivity to innocuous mechanical stimuli were briefly anesthetized with methohexital (Brietal, Lilly, 70 mg/kg, i.p.), decerebrated by aspiration of the forebrain and midbrain and then artificially ventilated. A small laminectomy was performed at midthoracic level and the spinal cord was transected at Th7-8. A second laminectomy was performed to expose lumbar segments L3-6. The
T A B L E II RESPONSE C H A R A C T E R I S T I C S O F D O R S A L H O R N W I D E D Y N A M I C R A N G E N E U R O N S TO S U P R A T H R E S H O L D PERIP H E R A L E L E C T R I C A L S T I M U L A T I O N IN N O R M A L A N D A L L O D Y N I C RATS Data are presented as mean + S.E.M.
Separation of A and C response Response duration (ms) Background activity (imp/s)
Normal
Allodynia
27/31 (87%) 356.1 _+34.2 9.0+ 3.7
9/29 (31%p 704.4 _ 194.7 b 8 . 8 _ 2.9 c
a Z2= 32.51, P < 0.001; b t =2.82, P < 0.01; c not significant.
dura was opened and the cord was covered with mineral oil. The rats were paralyzed with pancuronium bromide (Pavalon, Organon, 2.5 mg/kg, i.p.) and the electrocardiogram, body temperature and skin coloration were monitored. Extracellular recordings of the activity of single cells in the dorsal horn at lumbar segments L4-5, the level where the cord was irradiated, were made with glasscoated tungsten microelectrodes. The action potentials were amplified, filtered and displayed on an oscilloscope and analyzed on-line by a microcomputer with a poststimulus time histogram program. Search stimuli for evoking cell responses were electric shocks applied subcutaneously to the hind paw or to the sciatic nerve, which was dissected at the popliteal fossa and suspended on a pair of silver hook electrodes. The shocks activated both A and C fibers (5 mA, 1 ms, 0.2 Hz). Once a single unit was isolated and its response to electrical stimulation was recorded, the response of the neuron was characterized with mechanical stimuli (light brushing, light pressure and pinch) and its receptive field was mapped. The quantitative response of the neuron to mechanical stimulation was then examined by pressing calibrated yon Frey hairs against the skin at the center of the receptor field for 8 l0 s. The data collection was initiated after the dynamic response of the cell to the application of the von Frey hair had terminated. Thus, only the maintained response to mechanical pressure was analyzed. A total of 70 neurons were recorded from 34 rats, 37 from normal rats, 33 from rats after spinal ischemia, through the entire depth of the dorsal horn. The depth of the recorded cells was between 100 and 800 pm below the cord surface. 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 electrodes and its tip was broken off and left in the cord. The recording site was then localized histologically in Rexed's laminae I-V. There was no difference in the distribution of the depth of the recording sites in the normal and allodynic rats. The majority of the neurons encountered in normal rats were wide dynamic range (WDR) cells, as they could be activated by both innocuous (brushing, light pressure) and noxious (pinch) stimuli (Table I). A few cells could be maximally activated by innocuous stimuli and were characterized as low threshold (LT) neurons. Some others responded only to noxious pressure and were thus characterized as high threshold (HT) neurons. Spinal ischemia did not change the distribution of the neuronal classification (Table I). In normal rats, the response of dorsal horn W D R neurons to suprathreshold electrical stimulation of their re-
107 B
A .,+iii.i.
I
iJ,,,/t ....................
L
I
__2
:.,,,
~ir .+ ~ - i +=++ k ~ l l+~.+ ~ " *! ,+. q --~,+-1 . - , - , - 1 .+r . ,
+
I
.+
.•JlOOpV
t
lOOms
Fig. 1. Photographic illustration of typical responses of two dorsal horn WDR neurons to subcutaneous electrical stimuli (5 mA, 1 ms) applied to their cutaneous receptive fields in a normal rat (A) and at two days after spinal ischemia (B). Note the increased A-fiberresponse and prolongation of the overall response duration of the neuron in the allodynic rat. The stimulus is indicated by the arrows in A and B. ceptive field or of the sciatic nerve consists of short latency A-fiber ( < 1-40 ms) and long latency C-fiber responses (90-400 ms) (Table II, Fig. 1A). In a few cases, afterdischarges (400-900 ms) were observed. Regression analysis revealed that there was a highly significant positive linear correlation between the neuronal discharges and the cutaneous pressure applied via the von Frey hairs (Fig. 2). The threshold in these neurons to evoke a clear neuronal response, defined as 20% over background activity, was 13.8 _ 2.2 g (range 4.7-73 g, n = 31). One to four days after induction of spinal ischemia, at which time the rats exhibited behavioral allodynia, the neuronal responsiveness of dorsal horn W D R neurons to cutaneous stimuli was dramatically increased. The pattern of the neuronal responses to suprathreshold electrical stimulation was altered. The A-fiber response became prolonged and, in most cases, the interval between A- and C-fiber responses became indistinguishable (Table II, Fig. 1B). The duration of the neuronal response of the cells to electrical stimulation also increased in most cases (Table II, Fig. 1B). The rate of background activity was similar in normal and allodynic animals (Table II), but the sensitivity of these neurons to mechanical pressure increased significantly. The neurons began to respond at much lower pressure and the rate of increase of discharges during pressure became much more rapid (Fig. 2). Analysis of the discharge pattern indicated that there was a highly significant positive correlation between neuronal discharges and mechanical pressure and the regression analysis revealed that the response curve of these cells was best fitted by a quadratic equation (Fig. 2). The threshold of pressure that evoked a neuronal response 20% over background activity in ischemic and allodynic rats was 2.1 _ 0.4 g (range 0.097.1 g, n = 29), which is significantly different from the value in normal animals (t = 4.557, P < 0.0001). We have previously shown that transient spinal ischemia induces a state of mechanical hyperpathia in rats for
several days [2]. The present results indicate that such transient spinal ischemia results in hypersensitivity of dorsal horn W D R neurons to electrical and mechanical stimuli, thus providing a neuronal basis for the behavioral observations. The remarkable similarities between the behavioral and electrophysiological results suggest that the increased sensitivity of dorsal horn W D R neurons may underly the development of abnormal pain-related sensations in rats after spinal cord ischemia. What is the mechanism for this neuronal hypersensitivity after transient spinal ischemia? It is well known that small GABAergic interneurons located in the region of the substantia gelatinosa are involved in spinal inhibition [1,4]. The behavioral allodynia seen after spinal ischemia is relieved by low doses of systemic baclofen [2], suggesting that abnormal hypersensitivity may be related to the loss of inhibitory control normally exerted by GABAergic neurons; through baclofen-sensitive GABAB receptors presynaptically on afferent terminals and/or postsynaptically on W D R neurons, following transient spinal ischemia. In support of this hypothesis, it has been shown that GABAergic inhibitory neurons in the brain are highly susceptible to ischemic damage [6].
350
300-
1
250 -
i2 200"
0
|
.~
100 '
50
0 0
10
20
30
40
50
60
70
80
go
1~10
Pressure (g)
Fig. 2. Responses of dorsal horn WDR neurons to graded mechanical stimuli applied by von Frey hairs in normal rats (circle) and 1~, days after spinal ischemia(square), at which time the animals exhibited typical allodynia-like behavioral responses to mechanical stimuli. The neuronal responses were calculated as percent increase in the number of action potentials over background activity during mechanicalstimulation. The center of the receptivefield of each cell was stimulated with calibrated von Frey hairs for 8-10 s. Each data point represents responses of 20-30 cells and is presented as mean+S.E.M. The regression line for normal rats is y = 3.313x-2.972 (r = 0.623); for allodynic rats it is y=-0.089x2+ 12.522x+3.990 (r=0.657). Analysis of variance indicated that both regressions are highly significant (Fl.176= 110.982 and F2,296 = 111.366, respectively,P< 0.0001).
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This work was supported by the Swedish MRC (Project 07913, 06555), the Bank of Sweden Tercentenary Foundation, the Miami Project Foundation, the Wenner-Gren Center Foundation, the Daniel Henmann Fund for Spinal Cord Research, the King Gustav V and Queen Victoria Foundation, the Petrus and Augusta Hedlunds Foundation, the Axelsson-Johnson Foundation and research funds of the Karolinska Institute. We wish to thank Dr V. Pieribone for providing us the software used for data analysis and Dr B.D. Watson for designing and installing our laser set-up. 1 Game, C.J.A. and Lodge, D., The pharmacology of the inhibition of dorsal horn neurones by impulses in myelinated cutaneous afterens in the cat, Exp. Brain Res., 23 (1975) 75-84. 2 Hao, J.-X., Xu, X.-J., Aldskogius, H., Seiger, .~. and WiesenfeldHallin, Z., Allodynia-like effect in rat after ischemic spinal cord injury photochemically induced by laser irradiation, Pain, in press. 3 Mendell, L.M., Physiological properties of unmyelinated fiber projection to the spinal cord, Exp. Neurol., 16 (1966) 316-332.
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