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Mechanical and thermal hypersensitivity develops following kainate lesion of the ventral posterior lateral thalamus in rats
Neuroscience Letters 290 (2000) 79±83
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Mechanical and thermal hypersensitivity develops following kainate lesion of the ventral posterior lateral thalamus in rats Christopher J. LaBuda a, Todd D. Cutler a, Patrick M. Dougherty b, Perry N. Fuchs a,* a Department of Psychology, University of Texas at Arlington, Box 19528, Arlington, TX, USA Departments of Neurosurgery and Neuroscience, The Johns Hopkins School of Medicine, 600 N. Wolfe Street, Meyer 5±109, Baltimore, MD 21287, USA
b
Received 22 March 2000; received in revised form 26 June 2000; accepted 27 June 2000
Abstract Damage to the ventral-posterior lateral nucleus (VPL) of the thalamus or its afferent pathways can produce moderate to severe on-going pain and pain in response to normally innocuous stimuli (allodynia) and hypersensitivity to mildly noxious stimuli (hyperalgesia). The present study measured the responses to mechanical and thermal stimuli before and 2, 8, 24 and 48 h after a kainate-induced lesion of the VPL in male rats. Compared with control animals, hypersensitivity to mechanical stimulation of the hindpaw was apparent by 24 h post-lesion. At 48 h, the frequency of mechanical response increased from a baseline response frequency of 17 ^ 4.7 to 46 ^ 11.6%. Thermal withdrawal latencies 48 h after the lesion decreased from a baseline latency of 9.9 ^ 1.8 to 5.3 ^ 0.4 s. It is concluded that a neurotoxic lesion of the VPL results in a delayed onset of mechanical and thermal hyperalgesia. This study suggests a potential model for studying the basic mechanisms and potential therapies for central pain syndrome. q 2000 Published by Elsevier Science Ireland Ltd. Keywords: Allodynia; Hyperalgesia; Thalamus; Neurotoxic Lesion; Central pain syndrome
Chronic pain following thalamic lesion was ®rst described almost 100 years ago [5,6] and remains a substantial clinical problem [1,2,16]. The pain experienced by these patients varies in associative quality and intensity, but it is most often characterized by either reduced or elevated sensitivity to touch, temperature or pain in the affected body regions and described as a severe burning or lacerating sensation [8]. Additional symptoms may include hemiparesis, dysesthesias and hemiataxia contralateral or ipsilateral to the thalamic lesion [6]. The patients described by Dejerine and Roussy [6] had lesions involving the posterior and medial portions of the thalamus and adjacent structures. Although signi®cant advances have been made, the physiological mechanisms associated with the peculiar symptoms of this syndrome, including the delayed onset, remain poorly understood. As a result, few standards exist for the treatment of central pain syndrome (CPS). Exploration of the relationship between ventral-posterior lateral nucleus (VPL) lesion and CPS forms the basis for this study. Lesions in VPL, historically due to trauma or infarct * Corresponding author. Tel.: 11-817-272-3427; fax: 11-817272-2364. E-mail address: [email protected] (P.N. Fuchs).
[6], and more recently, following stereotactic electrolysis [11], often result in CPS. The question of interest is whether a similar pattern of behavioral changes can be seen following localized damage to the VPL in rats. As a means to explore this possibility, VPL thalamic lesions were induced in rats by unilateral microinjection of the excitatory amino acid kainate, which produces a dose related neuronal apoptosis with a sparing of axons of passage [13,15]. The present experiment examined mechanical and thermal responses to detect allodynia and hyperalgesia following thalamic damage and characterized the chronology of its onset. Male Sprague±Dawley rats (N 33; 300±400 g) were used in this study (University of Texas at Arlington vivarium). The animals were housed at a constant temperature of 218C on a 12-h alternating light/dark cycle with food and water available ad libitum. The experimental protocol was approved by the Institutional Animal Care and Use Committee at the University of Texas at Arlington and was conducted in accordance with the guidelines outlined by the International Association for the Study of Pain [19]. Animals were anesthetized with 0.1 ml/kg of ACE Promeline (s.c.) followed by 1 ml/kg of ketamine (50 mg/kg, i.m.) and ®tted into a stereotaxic device. With the skull level
0304-3940/00/$ - see front matter q 2000 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 0) 01 32 3- 9
80
C.J. LaBuda et al. / Neuroscience Letters 290 (2000) 79±83
between bregma and lambda, a guide cannula (23-gauge) was implanted such that the tip was 0.5 mm above the region of the VPL (AP: 23.1; L: 3.0; D: 5.5 mm). To prevent clogging of the guide cannula, a stylet (00 insect pin) was inserted and secured in place until the time of injection. Animals were allowed a 7-day recovery period prior to behavioral testing. Measurement of hind paw withdrawal response utilized a Plexiglas chamber (12.5 £ 25 £ 30 cm) positioned on an elevated 0.6 cm wire mesh screen. Following a 15-min habituation period, calibrated von Frey ®laments were pressed upward against the plantar surface of each hind paw for approximately 1 s. Four von Frey ®laments (4.11, 4.28, 5.67, 8.07 mN) were employed and administered in an ascending series to establish response threshold. The stimulus series consisted of alternately testing the left and right hind paws with each of the four ®laments beginning with the least (4.11 mN) and progressing in order to the greatest (8.07 mN) force von Frey. A withdrawal response was recorded when the animal actively lifted the stimulated paw during the stimulation period. The ascending stimulus series was repeated over ten trials and the frequency of withdrawal response was converted to percent response (% response (frequency of response/20) £ 100). Measurement of withdrawal latency to a thermal stimulus utilized an infrared heat source applied to the plantar surface of both hindpaws (Ugo Basile, Plantar Test #7370). Following a 15min habituation period, threshold testing was performed twice per paw with at least 2-min separating each measurement. The average value of the four measures was calculated as the withdrawal latency for each animal. Behavioral testing was performed prior to surgery, prior to injection and at 2, 8, 24 and 48 h post-injection. Animals randomly received either no injection (Surgery Control), an injection of physiological saline (Vehicle Control) or kainate. Under light anesthesia (2% Halothane), the stylet was removed and an injection cannula (AM Systems, Carlsburg, Wa., #8320), constructed so that it extended 0.5 mm below the guide cannula, was inserted. Kainate (10 mg/ml) or physiological saline was microinjected using a 1-ml Hamilton syringe attached to the injection cannula via PE 10 tubing. A total volume of 0.5 ml was injected over 90s and the injection cannula was left in place for an additional 60 s to allow for absorption into the surrounding brain region and to minimize spread of the injectate along the cannula track. There were transient signs (,24 h) of seizure-induced activity following kainate injection that was not evident in vehicle injected animals. However, behavioral testing was performed blind with respect to the location of microinjection (hit vs. miss) and the histological analysis of cannula tip location was performed blind with respect to the behavioral outcome. Following the experimental procedure, animals were sacri®ced and 80-mm coronal sections were mounted on slides and stained with thionin. Four groups were considered for data analysis: Surgery Control (n 8), Vehicle injection (n 13), Kainate Miss (n 6) and Kainate Hit (n 6).
Subjects that received kainate injections were designated as a miss or a hit based on histological examination of cannula tip placement and lesioning in the VPL (Fig. 1). Histological analysis revealed that the anterior/posterior extent of the kainate lesion was typically restricted too less than 1 mm (^0.5 mm from the cannula tip). The restricted extent of damage was expected since twice the volume of an injectate (1 ml) infused at a signi®cantly greater rate (1 ml/min) has dissociated drug effects in structures separated by as little as 0.5 mm [17]. The most anterior and the most posterior damage was very similar for both groups (Kainate Miss: anterior 21.3 mm, posterior 23.6 mm from bregma; Kainate Hit: anterior 21.8 mm, posterior 23.6 mm from bregma). Further analysis revealed a clear difference between the Kainate Miss and Kainate Hit groups. The primary damage produced within the Kainate Miss group never involved the VPL and was localized to either the ventroposterior medial thalamus (n 2), ventrolateral thalamus (n 1), reticular thalamic nucleus (n 1), globus pallidus (n 1) or internal capsule (n 1). On the other hand, the primary damage produced within the Kainate Hit group was always localized to the VPL, accompanied by slight glial proliferation in immediately adjacent structures such as the lateral portion of the ventroposterior medial thalamus (n 4), dorsal aspect of the ventrolateral thalamus (n 1), or the dorsal aspect of the reticular thalamic nucleus (n 1). In no case was there evidence of damage to the posterior thalamic nuclear group. The analysis of variance (ANOVA) comparing the percent of withdrawal response among the groups for each von Frey force across the six test periods (Fig. 2) revealed a signi®cant group £ force £ test period interaction (P 0:05). Additional exploration of the interaction was performed by ANOVA comparing the percent of withdrawal response across the six test periods for each of the separate groups. The analysis revealed that the Surgery Control, Vehicle and Kainate Miss groups maintained stable withdrawal responding across the test periods. However, the analysis of withdrawal responses for the Kainate Hit group revealed a signi®cant force £ test period interaction (P , 0:05). Post-hoc comparisons (protected t-test) revealed that, compared to pre-surgery baseline responding, there was a dramatic shift in the force/response function beginning at 24 h and was maximal at 48 h. An ANOVA comparing the withdrawal response at the 48 h test period to the highest force von Frey (8.07 mN) revealed a signi®cant difference among the groups (P , 0:001). The Kainate Hit group showing a signi®cant elevation of withdrawal responding compared to the other groups, which did not differ from each other (protected t-test). Three of the animals in the Kainate Miss group were not tested during the initial stage of the experimental procedure in thermal responding. Therefore, due to the small sample size, this group was excluded from the ANOVA of withdrawal latency to the thermal stimulus. The ANOVA of withdrawal latency to the thermal stimulus revealed a
C.J. LaBuda et al. / Neuroscience Letters 290 (2000) 79±83
signi®cant group X time interaction (P 0:05). Additional ANOVA revealed no difference in baseline withdrawal latency among the groups. However, at 48 h post-injection, the ANOVA indicated a signi®cant difference among the groups (P , 0:05). Additional post-hoc comparisons (protected t-test) indicated signi®cantly greater thermal paw withdrawal latencies for the vehicle and surgery control groups compared to the Kainate Hit group (Fig. 3). The present results demonstrate that kainate-induced lesion of the VPL results in a leftward shift in the force/ response function as revealed by a signi®cant increase in response to normally innocuous mechanical and thermal stimuli. The mean frequency of responses at 24 h after the kainate injection is more than double the baseline response frequency and at 48 h , the response percent is greater than three times the baseline response frequency (Fig. 2). In contrast, animals within the other groups exhibited no such increase in responsiveness over time. A similar pattern of thermal allodynia is also re¯ected as a decrease in response latency to thermal stimuli (Fig. 3). Our results con®rm and better de®ne the ®ndings of another recent
81
report that showed extensive electrolytic lesions of thalamus and cortex produce a long-lasting enhancement of reactivity to normally noxious mechanical and thermal stimuli (hyperalgesia) [14]. The selective and restricted nature of our lesions indicates that VPL damage is suf®cient to cause mechanical and thermal allodynia. These results are in general agreement with a proposed mechanism of CPS based on differential loss of lateral versus medial projections into the thalamus [9]. However, in con¯ict with the most recent hypothesis that accounts for hyperalgesia to thermal stimuli in CPS [3], we found no evidence that our lesions directly involved the posterior thalamic nuclear group, proposed as a key site for thermal and pain discrimination [4]. The present results cannot rule out the potential involvement of ®bers of passage. However, the lack of allodynia in the Kainate Miss group and the selectivity of kainate to damage cell bodies rather than axons of passage [13,15] makes it likely that loss of neurons in VPL proper is a key element in the generation of CPS. Future studies with restricted kainate injections con®ned to the anterior versus posterior thalamus or perhaps speci®c
Fig. 1. (A) Schematic representation of cannula tip locations for the Surgery (B), Vehicle injection (A), Kainate Miss (W) and Kainate Hit (X) groups based on plates from Paxinos and Watson [12]. (B) Photomicrograph of a subject that received vehicle injection within the region of the VPL. The arrow indicates the boundary of the cannula tip (dorsal aspect is right). (C) Photomicrograph of a subject that received kainate injection within the region of the VPL. The arrow indicates the boundary of the cannula tip and the star indicates the area of pronounced glial proliferation (dorsal aspect is right). All subjects included in the Kainate Hit group had cannula tip locations on target with the VPL and pronounced glial proliferation. Scale bar 250 mM.
82
C.J. LaBuda et al. / Neuroscience Letters 290 (2000) 79±83
Fig. 2. The symbol and line plots summarizes the effects of surgery alone (upper left panel), vehicle injection (upper right), injection of kainate con®ned to nuclei outside of the VPL (lower left) and injection of kainate localized to the VPL (lower right) on the mean percent of paw withdrawal response (^SEM) to punctate stimulation of the hindpaw. The responses to four intensities of punctate stimuli are shown at several time points including pre-surgery, pre-injection, 2, 8, 24 and 48 h post-injection. *P , 0:05, **P , 0:01, ***P , 0:001 compared to pre-surgery baseline.
lesion of the lamina I projection to Vmpo using the recently developed Substance P-saporin neurotoxin [18] should be useful in further dissecting this issue. Paradoxically, both thalamic lesions and thalamic stimulation have been used to relieve CPS [7]. Positron emission tomography (PET) studies in patients with post-traumatic neuropathic pain reveal a unilateral decrease in VP thalamic activity contralateral to the injury [10]. Moderate increases in the function of the VPL, such as that produced by electrical stimulation or by microinjection of physiological concentrations of excitatory neurotransmitters or reuptake inhibitors, may therefore be the most suitable means of treatment for CPS. The animal model we describe here mimics many of the peculiar symptoms of CPS including delayed onset of pain and hyperalgesia to mechanical and thermal stimuli, and may allow evaluation of this and other possible treatments for CPS. In addition, since the onset of
Fig. 3. The bar graph summarizes the mean withdrawal latency (^SEM) to radiant heat applied to the hind paw before (baseline, left group of bars) and then 48 h after injection for subjects that received no injection (surgery control), vehicle injection, or kainate injection within the VPL. **P , 0:01 compared to kainate hit group.
C.J. LaBuda et al. / Neuroscience Letters 290 (2000) 79±83
pain is most often delayed following the initiating event, the animal model we present provides an opportunity to investigate interventions that might prevent the onset of CPS following injury to the nervous system.
[11] [12]
[1] Bonica, J.J., Introduction: semantic, epidemiologic, and educational issues, In K.L. Casey (Ed.), Pain and central nervous system disease: the central pain syndromes, Raven Press, New York, 1991, pp. 13±29. [2] Casey, K.L., Pain and central nervous system disease: a summary and overview, In K.L. Casey (Ed.), Pain and central nervous system disease: the central pain syndromes, Raven Press, New York, 1991, pp. 1±11. [3] Craig, A.D., A new hypothesis of central pain, Pain Forum, 7 (1998) 1±14. [4] Craig, A.D., Bushnell, M.C., Zhang, E.-T. and Blomqvist, A., A thalamic nucleus speci®c for pain and temperature sensation, Nature, 372 (1995) 770±773. [5] Dejerine, J. and Egger, M., Le syndrome douloureaux thalamique, Rev. Neurol., 14 (1903) 521±532. [6] Dejerine, J. and Roussy, G., La syndrome thalamique, Rev. Neurol., 14 (1906) 521±532. [7] Duncan, G., Kupers, R., Marchand, S., Villemure, J., Gybels, J. and Bushnell, M., Stimulation of human thalamus for pain relief, J. Neurophysiol., 80 (1998) 3326±3330. [8] Fuchs, P.N., Lee, J.I. and Lenz, F.A., Central pain secondary to intracranial lesions, In K.J. Burichel (Ed.), Pain Surgery, Thieme, New York, 2000, in press. [9] Head, H. and Holmes, G., Sensory disturbances from cerebral lesions, Brain, 34 (1912) 102±254. [10] Iadarola, M.J., Max, M.B., Berman, K.F., Byas-Smith, M.G., Coghill, R.C., Gracely, R.H. and Bennett, G.J., Unilateral
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decrease in thalamic activity observed with positron emission tomography in patients with chronic neuropathic pain, Pain, 63 (1995) 55±64. Pagni, C.A., Central pain and painful anesthesia, Prog. Neurol. Surg., 8 (1976) 132±257. Paxinos, G. and Watson, C., The Rat Brain in Stereotaxic Coordinates, Academic Press, New York, 1986. Pisharodi, M. and Nauta, H., An animal model for neuronspeci®c spinal cord lesions by the microinjection of Nmethylaspartate, kainic acid and quisqualic acid, Appl. Neurophysiol., 48 (1985) 226±233. SaadeÂ, N.E., Kafrouni, A.I., Saab, C.Y., Atweh, S.F. and Jabbur, S.J., Chronic thalamotomy increases pain-related behavior in rats, Pain, 83 (1999) 401±409. Schwarcz, R., Zaczek, R. and Coyle, J., Microinjection of kainic acid into the rat hippocampus, Eur. J. Pharmacol., 50 (1978) 209±220. Tasker, R.R., DeCarvalho, G. and Dostrovsky, J.O., The history of central pain syndromes. with observations concerning pathophysiology and treatment, In K.L. Casey (Ed.), Pain and central nervous system disease: the central pain syndromes, Raven Press, New York, 1991, pp. 31±58. Waddington, J.L., Psychopharmacological studies in rodents: stereotaxic intracerebral injections and behavioural assessment, In M.H. Joseph, J.L. Waddington (Eds.), Working methods in neuropharmacology, University Press, Manchester, 1986, pp. 1±40. Wiley, R.G. and Lappi, D.A., Destruction of neurokinin-1 receptor expressing cells in vitro and in vivo using substance P-saporin in rats, Neurosci. Lett., 230 (1997) 97± 100. Zimmermann, M., Ethical guidelines for investigators of experimental pain unconscious animals, Pain, 16 (1983) 109±110.
www.elsevier.com/locate/neulet
Mechanical and thermal hypersensitivity develops following kainate lesion of the ventral posterior lateral thalamus in rats Christopher J. LaBuda a, Todd D. Cutler a, Patrick M. Dougherty b, Perry N. Fuchs a,* a Department of Psychology, University of Texas at Arlington, Box 19528, Arlington, TX, USA Departments of Neurosurgery and Neuroscience, The Johns Hopkins School of Medicine, 600 N. Wolfe Street, Meyer 5±109, Baltimore, MD 21287, USA
b
Received 22 March 2000; received in revised form 26 June 2000; accepted 27 June 2000
Abstract Damage to the ventral-posterior lateral nucleus (VPL) of the thalamus or its afferent pathways can produce moderate to severe on-going pain and pain in response to normally innocuous stimuli (allodynia) and hypersensitivity to mildly noxious stimuli (hyperalgesia). The present study measured the responses to mechanical and thermal stimuli before and 2, 8, 24 and 48 h after a kainate-induced lesion of the VPL in male rats. Compared with control animals, hypersensitivity to mechanical stimulation of the hindpaw was apparent by 24 h post-lesion. At 48 h, the frequency of mechanical response increased from a baseline response frequency of 17 ^ 4.7 to 46 ^ 11.6%. Thermal withdrawal latencies 48 h after the lesion decreased from a baseline latency of 9.9 ^ 1.8 to 5.3 ^ 0.4 s. It is concluded that a neurotoxic lesion of the VPL results in a delayed onset of mechanical and thermal hyperalgesia. This study suggests a potential model for studying the basic mechanisms and potential therapies for central pain syndrome. q 2000 Published by Elsevier Science Ireland Ltd. Keywords: Allodynia; Hyperalgesia; Thalamus; Neurotoxic Lesion; Central pain syndrome
Chronic pain following thalamic lesion was ®rst described almost 100 years ago [5,6] and remains a substantial clinical problem [1,2,16]. The pain experienced by these patients varies in associative quality and intensity, but it is most often characterized by either reduced or elevated sensitivity to touch, temperature or pain in the affected body regions and described as a severe burning or lacerating sensation [8]. Additional symptoms may include hemiparesis, dysesthesias and hemiataxia contralateral or ipsilateral to the thalamic lesion [6]. The patients described by Dejerine and Roussy [6] had lesions involving the posterior and medial portions of the thalamus and adjacent structures. Although signi®cant advances have been made, the physiological mechanisms associated with the peculiar symptoms of this syndrome, including the delayed onset, remain poorly understood. As a result, few standards exist for the treatment of central pain syndrome (CPS). Exploration of the relationship between ventral-posterior lateral nucleus (VPL) lesion and CPS forms the basis for this study. Lesions in VPL, historically due to trauma or infarct * Corresponding author. Tel.: 11-817-272-3427; fax: 11-817272-2364. E-mail address: [email protected] (P.N. Fuchs).
[6], and more recently, following stereotactic electrolysis [11], often result in CPS. The question of interest is whether a similar pattern of behavioral changes can be seen following localized damage to the VPL in rats. As a means to explore this possibility, VPL thalamic lesions were induced in rats by unilateral microinjection of the excitatory amino acid kainate, which produces a dose related neuronal apoptosis with a sparing of axons of passage [13,15]. The present experiment examined mechanical and thermal responses to detect allodynia and hyperalgesia following thalamic damage and characterized the chronology of its onset. Male Sprague±Dawley rats (N 33; 300±400 g) were used in this study (University of Texas at Arlington vivarium). The animals were housed at a constant temperature of 218C on a 12-h alternating light/dark cycle with food and water available ad libitum. The experimental protocol was approved by the Institutional Animal Care and Use Committee at the University of Texas at Arlington and was conducted in accordance with the guidelines outlined by the International Association for the Study of Pain [19]. Animals were anesthetized with 0.1 ml/kg of ACE Promeline (s.c.) followed by 1 ml/kg of ketamine (50 mg/kg, i.m.) and ®tted into a stereotaxic device. With the skull level
0304-3940/00/$ - see front matter q 2000 Published by Elsevier Science Ireland Ltd. PII: S03 04 - 394 0( 0 0) 01 32 3- 9
80
C.J. LaBuda et al. / Neuroscience Letters 290 (2000) 79±83
between bregma and lambda, a guide cannula (23-gauge) was implanted such that the tip was 0.5 mm above the region of the VPL (AP: 23.1; L: 3.0; D: 5.5 mm). To prevent clogging of the guide cannula, a stylet (00 insect pin) was inserted and secured in place until the time of injection. Animals were allowed a 7-day recovery period prior to behavioral testing. Measurement of hind paw withdrawal response utilized a Plexiglas chamber (12.5 £ 25 £ 30 cm) positioned on an elevated 0.6 cm wire mesh screen. Following a 15-min habituation period, calibrated von Frey ®laments were pressed upward against the plantar surface of each hind paw for approximately 1 s. Four von Frey ®laments (4.11, 4.28, 5.67, 8.07 mN) were employed and administered in an ascending series to establish response threshold. The stimulus series consisted of alternately testing the left and right hind paws with each of the four ®laments beginning with the least (4.11 mN) and progressing in order to the greatest (8.07 mN) force von Frey. A withdrawal response was recorded when the animal actively lifted the stimulated paw during the stimulation period. The ascending stimulus series was repeated over ten trials and the frequency of withdrawal response was converted to percent response (% response (frequency of response/20) £ 100). Measurement of withdrawal latency to a thermal stimulus utilized an infrared heat source applied to the plantar surface of both hindpaws (Ugo Basile, Plantar Test #7370). Following a 15min habituation period, threshold testing was performed twice per paw with at least 2-min separating each measurement. The average value of the four measures was calculated as the withdrawal latency for each animal. Behavioral testing was performed prior to surgery, prior to injection and at 2, 8, 24 and 48 h post-injection. Animals randomly received either no injection (Surgery Control), an injection of physiological saline (Vehicle Control) or kainate. Under light anesthesia (2% Halothane), the stylet was removed and an injection cannula (AM Systems, Carlsburg, Wa., #8320), constructed so that it extended 0.5 mm below the guide cannula, was inserted. Kainate (10 mg/ml) or physiological saline was microinjected using a 1-ml Hamilton syringe attached to the injection cannula via PE 10 tubing. A total volume of 0.5 ml was injected over 90s and the injection cannula was left in place for an additional 60 s to allow for absorption into the surrounding brain region and to minimize spread of the injectate along the cannula track. There were transient signs (,24 h) of seizure-induced activity following kainate injection that was not evident in vehicle injected animals. However, behavioral testing was performed blind with respect to the location of microinjection (hit vs. miss) and the histological analysis of cannula tip location was performed blind with respect to the behavioral outcome. Following the experimental procedure, animals were sacri®ced and 80-mm coronal sections were mounted on slides and stained with thionin. Four groups were considered for data analysis: Surgery Control (n 8), Vehicle injection (n 13), Kainate Miss (n 6) and Kainate Hit (n 6).
Subjects that received kainate injections were designated as a miss or a hit based on histological examination of cannula tip placement and lesioning in the VPL (Fig. 1). Histological analysis revealed that the anterior/posterior extent of the kainate lesion was typically restricted too less than 1 mm (^0.5 mm from the cannula tip). The restricted extent of damage was expected since twice the volume of an injectate (1 ml) infused at a signi®cantly greater rate (1 ml/min) has dissociated drug effects in structures separated by as little as 0.5 mm [17]. The most anterior and the most posterior damage was very similar for both groups (Kainate Miss: anterior 21.3 mm, posterior 23.6 mm from bregma; Kainate Hit: anterior 21.8 mm, posterior 23.6 mm from bregma). Further analysis revealed a clear difference between the Kainate Miss and Kainate Hit groups. The primary damage produced within the Kainate Miss group never involved the VPL and was localized to either the ventroposterior medial thalamus (n 2), ventrolateral thalamus (n 1), reticular thalamic nucleus (n 1), globus pallidus (n 1) or internal capsule (n 1). On the other hand, the primary damage produced within the Kainate Hit group was always localized to the VPL, accompanied by slight glial proliferation in immediately adjacent structures such as the lateral portion of the ventroposterior medial thalamus (n 4), dorsal aspect of the ventrolateral thalamus (n 1), or the dorsal aspect of the reticular thalamic nucleus (n 1). In no case was there evidence of damage to the posterior thalamic nuclear group. The analysis of variance (ANOVA) comparing the percent of withdrawal response among the groups for each von Frey force across the six test periods (Fig. 2) revealed a signi®cant group £ force £ test period interaction (P 0:05). Additional exploration of the interaction was performed by ANOVA comparing the percent of withdrawal response across the six test periods for each of the separate groups. The analysis revealed that the Surgery Control, Vehicle and Kainate Miss groups maintained stable withdrawal responding across the test periods. However, the analysis of withdrawal responses for the Kainate Hit group revealed a signi®cant force £ test period interaction (P , 0:05). Post-hoc comparisons (protected t-test) revealed that, compared to pre-surgery baseline responding, there was a dramatic shift in the force/response function beginning at 24 h and was maximal at 48 h. An ANOVA comparing the withdrawal response at the 48 h test period to the highest force von Frey (8.07 mN) revealed a signi®cant difference among the groups (P , 0:001). The Kainate Hit group showing a signi®cant elevation of withdrawal responding compared to the other groups, which did not differ from each other (protected t-test). Three of the animals in the Kainate Miss group were not tested during the initial stage of the experimental procedure in thermal responding. Therefore, due to the small sample size, this group was excluded from the ANOVA of withdrawal latency to the thermal stimulus. The ANOVA of withdrawal latency to the thermal stimulus revealed a
C.J. LaBuda et al. / Neuroscience Letters 290 (2000) 79±83
signi®cant group X time interaction (P 0:05). Additional ANOVA revealed no difference in baseline withdrawal latency among the groups. However, at 48 h post-injection, the ANOVA indicated a signi®cant difference among the groups (P , 0:05). Additional post-hoc comparisons (protected t-test) indicated signi®cantly greater thermal paw withdrawal latencies for the vehicle and surgery control groups compared to the Kainate Hit group (Fig. 3). The present results demonstrate that kainate-induced lesion of the VPL results in a leftward shift in the force/ response function as revealed by a signi®cant increase in response to normally innocuous mechanical and thermal stimuli. The mean frequency of responses at 24 h after the kainate injection is more than double the baseline response frequency and at 48 h , the response percent is greater than three times the baseline response frequency (Fig. 2). In contrast, animals within the other groups exhibited no such increase in responsiveness over time. A similar pattern of thermal allodynia is also re¯ected as a decrease in response latency to thermal stimuli (Fig. 3). Our results con®rm and better de®ne the ®ndings of another recent
81
report that showed extensive electrolytic lesions of thalamus and cortex produce a long-lasting enhancement of reactivity to normally noxious mechanical and thermal stimuli (hyperalgesia) [14]. The selective and restricted nature of our lesions indicates that VPL damage is suf®cient to cause mechanical and thermal allodynia. These results are in general agreement with a proposed mechanism of CPS based on differential loss of lateral versus medial projections into the thalamus [9]. However, in con¯ict with the most recent hypothesis that accounts for hyperalgesia to thermal stimuli in CPS [3], we found no evidence that our lesions directly involved the posterior thalamic nuclear group, proposed as a key site for thermal and pain discrimination [4]. The present results cannot rule out the potential involvement of ®bers of passage. However, the lack of allodynia in the Kainate Miss group and the selectivity of kainate to damage cell bodies rather than axons of passage [13,15] makes it likely that loss of neurons in VPL proper is a key element in the generation of CPS. Future studies with restricted kainate injections con®ned to the anterior versus posterior thalamus or perhaps speci®c
Fig. 1. (A) Schematic representation of cannula tip locations for the Surgery (B), Vehicle injection (A), Kainate Miss (W) and Kainate Hit (X) groups based on plates from Paxinos and Watson [12]. (B) Photomicrograph of a subject that received vehicle injection within the region of the VPL. The arrow indicates the boundary of the cannula tip (dorsal aspect is right). (C) Photomicrograph of a subject that received kainate injection within the region of the VPL. The arrow indicates the boundary of the cannula tip and the star indicates the area of pronounced glial proliferation (dorsal aspect is right). All subjects included in the Kainate Hit group had cannula tip locations on target with the VPL and pronounced glial proliferation. Scale bar 250 mM.
82
C.J. LaBuda et al. / Neuroscience Letters 290 (2000) 79±83
Fig. 2. The symbol and line plots summarizes the effects of surgery alone (upper left panel), vehicle injection (upper right), injection of kainate con®ned to nuclei outside of the VPL (lower left) and injection of kainate localized to the VPL (lower right) on the mean percent of paw withdrawal response (^SEM) to punctate stimulation of the hindpaw. The responses to four intensities of punctate stimuli are shown at several time points including pre-surgery, pre-injection, 2, 8, 24 and 48 h post-injection. *P , 0:05, **P , 0:01, ***P , 0:001 compared to pre-surgery baseline.
lesion of the lamina I projection to Vmpo using the recently developed Substance P-saporin neurotoxin [18] should be useful in further dissecting this issue. Paradoxically, both thalamic lesions and thalamic stimulation have been used to relieve CPS [7]. Positron emission tomography (PET) studies in patients with post-traumatic neuropathic pain reveal a unilateral decrease in VP thalamic activity contralateral to the injury [10]. Moderate increases in the function of the VPL, such as that produced by electrical stimulation or by microinjection of physiological concentrations of excitatory neurotransmitters or reuptake inhibitors, may therefore be the most suitable means of treatment for CPS. The animal model we describe here mimics many of the peculiar symptoms of CPS including delayed onset of pain and hyperalgesia to mechanical and thermal stimuli, and may allow evaluation of this and other possible treatments for CPS. In addition, since the onset of
Fig. 3. The bar graph summarizes the mean withdrawal latency (^SEM) to radiant heat applied to the hind paw before (baseline, left group of bars) and then 48 h after injection for subjects that received no injection (surgery control), vehicle injection, or kainate injection within the VPL. **P , 0:01 compared to kainate hit group.
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pain is most often delayed following the initiating event, the animal model we present provides an opportunity to investigate interventions that might prevent the onset of CPS following injury to the nervous system.
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