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Spinal neurons involved in the generation of at-level pain following spinal injury in the rat
Neuroscience Letters 361 (2004) 232–236 www.elsevier.com/locate/neulet
Spinal neurons involved in the generation of at-level pain following spinal injury in the rat Robert P. Yezierskia,b,*, Chen-Guang Yua,b, Patrick W. Mantyhc,d,e, Charles J. Viercka,b, Douglas A. Lappif a
Department of Orthodontics, Comprehensive Center for Pain Research, McKnight Brain Institute, University of Florida, 1600 S.W. Archer Road, Rm D10– 19, P.O. Box 100444, Gainesville, FL 32610, USA b Department of Neuroscience, University of Florida, 1600 S.W. Archer Road, Gainesville, FL 32610, USA c Department of Preventive Sciences, University of Minnesota, Minneapolis, MN, USA d Department of Psychiatry, University of Minnesota, Minneapolis, MN, USA e Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA f Advanced Targeting Systems, San Diego, CA, USA
Abstract Using a conjugate of substance P and the ribosome-inactivating protein saporin, neurons expressing the neurokinin-1 receptor in lamina I of the spinal cord were targeted to determine their role in the expression of a spontaneous pain behavior following intraspinal injections of quisqualic acid in the rat. Treatment was carried out at the time of injury in order to prevent the onset of the behavior, and following onset in order to evaluate the potential clinical utility of this intervention. Treatment at the time of injury resulted in significant decreases in onsettime and severity of pain behavior, while treatment at the time of onset led to a significant reduction of the spontaneous self-directed behavior. The results suggest that the substrate for at-level pain following spinal cord injury includes a population of spinal neurons expressing the neurokinin-1 receptor in the superficial laminae of the spinal cord. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Central pain; Quisqualic acid; Excitotoxicity; Lamina I; Substance P; Saporin
Chronic pain affects nearly 70% of patients with pathology resulting from traumatic or ischemic injury of the spinal cord [12]. Although loss of sensory and motor function represent the most commonly described deficits associated with spinal injury, abnormal sensations including pain are among the most difficult complications to deal with following injury [15]. For many patients with spinal injury, elimination of pain represents the possibility of returning to work, improving social and family relationships as well as reducing pain medications. For a significant percentage of patients elimination of pain is even more important than recovery of motor function, further emphasizing the impact of injury induced pain [12]. Pain associated with spinal cord injury (SCI) can be divided into nociceptive (including musculoskeletal and visceral) and neuropathic (including above-, at- and below-level) [12]. The primary characteristic of at-level pain is a hypersensitive band of skin in dermatomes associated with spinal segments adjacent to the site of injury. Injury-induced loss of intrinsic * Corresponding author. Tel.: þ1-352-392-4081; fax: þ 1-352-392-3031. E-mail address: [email protected] (R.P. Yezierski).
inhibitory control and changes in the functional state of neurons adjacent to the site of injury represent the current view of a mechanism responsible for this condition [17]. Unfortunately, the precise neural substrate responsible for this type of pain remains undefined. Consequently, effective treatments are largely inadequate, ranging from implanted stimulators and ablative surgery [2] to a wide-range of pharmacological interventions [1]. Intraspinal injection of the AMPA/metabotropic receptor agonist quisqualic acid (QUIS) has been used to examine the underlying pathophysiological changes responsible for altered sensibilities, including pain, associated with SCI [17]. By creating an excitotoxic challenge to surrounding tissue, thus simulating an important chemical change occurring at the time of injury, pathological characteristics similar to those associated with ischemic and traumatic injury are produced [17]. The transient elevation of excitatory amino acids initiates a secondary injury cascade which has a profound impact on the anatomical organization and functional state of neuronal circuits within the spinal gray matter [11]. The pathological characteristics, including neuronal loss, glial response, and syrinx formation,
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.12.035
R.P. Yezierski et al. / Neuroscience Letters 361 (2004) 232–236
associated with this injury model parallel those observed in humans with SCI, especially those associated with posttraumatic syringomyelia [5]. One of the clinical complications associated with this condition is the onset of chronic pain that develops at and below the level of injury [5]. The behavioral sequelae of the excitotoxic injury model includes the onset of evoked and spontaneous pain-like behaviors commonly associated with animal models of neuropathic and inflammatory pain [17]. One of the hallmarks of this model is an injury-induced biting behavior, referred to as excessive grooming, which has been proposed as a model of SCI-induced at-level pain. The delayed onset and association with dermatomes adjacent to the site of injury are characteristics making this behavior similar to those defining the human condition. Although the exact quality of the eliciting stimulus is unknown, e.g. paresthesia, dysesthesia, pain, the sensation evokes a spontaneous self-directed behavior similar to that reported in humans with neuropathic pain [6]. Consistent with the clinical profile of at-level pain, hypersensitivity to thermal and mechanical stimuli are characteristics of the area targeted for excessive grooming behavior. Efforts to elucidate the underlying mechanism of this behavior have included studies focusing on anatomical [3], physiological [17], and molecular [10] changes coinciding with the onset and progression of excessive grooming behavior. Available evidence suggests the involvement of neurons in the superficial laminae of the spinal cord as a critical component of a spinal pain generator responsible for the onset of this behavior [17]. Additionally, a recent report described the importance of a spatial threshold of tissue injury along the rostrocaudal axis of the cord that must be exceeded in order to achieve expression of excessive grooming behavior [18]. Recently, a population of neurons expressing the substance P receptor (SPR) in the superficial dorsal horn was shown to have a unique role in the processing of nociceptive information and the development of chronic pain and hyperalgesia. Using internalization of the SPR as a vehicle to transport a substance P-saporin conjugate into the cell, lamina I SPR-expressing neurons have been targeted and eliminated by the ribosome inactivating protein saporin. This strategy reduces nocifensive behaviors enhanced by capsaicin [7], inflammation, and nerve injury [9]. The involvement of this population of neurons in the activation of supraspinal pain centers was demonstrated by a reduction in operant escape behavior after intrathecal application of SP-saporin to the lumbar spinal cord [14]. These studies suggest the functional importance of SPR-expressing neurons in hyperalgesia. The importance of these neurons in the development of central sensitization, a critical step in the cascade of central events responsible for the conversion of acute to chronic pain, has also been described [4]. Furthermore, evidence suggesting that neuronal excitability in the dorsal horn is influenced by lamina I neurons activating descending facilitatory pathways was recently described [13]. The conclusion from these studies, in combination with
233
previous reports emphasizing the importance of the superficial dorsal horn as part of the substrate responsible for atlevel pain, makes the population of SPR expressing neurons in this region of the cord an obvious target for therapeutic strategies. In the present study, the effects of [Sar9Met(O2)11] substance P-saporin (SSP-SAP) neurotoxin [16] were evaluated against the onset and progression of injury-induced excessive grooming behavior. This reagent has been shown to specifically eliminate neurokinin-1 receptor (NK-1R) expressing neurons in the brain and spinal cord [7 –9,16]. Two strategies were used for delivery of SSP-SAP: (a) treatment at the time of injury for the purpose of delaying the onset of excessive grooming behavior; and (b) post-hoc treatment following injury for the purpose of slowing the progression and/or eliminating the pain behavior. Excessive grooming behavior was assessed on a regular schedule following injury and evaluated according to: (a) time of onset; (b) area of skin targeted for excessive grooming behavior; and (c) severity (class I– IV) [18]. The first group of 35 animals received SSP-SAP, SAP, or vehicle treatment at the time of QUIS injections. A solution containing SSP-SAP or SAP (Advanced Targeting Systems, San Diego, CA) was placed in a paraffin well made at the injection site 30 min after the QUIS injection (total volume 100 ml). SSP-SAP (25 mg) was dissolved in 833 ml of PBS to give 300 ng/10 ml. SAP (100 mg) was dissolved in 30 ml of PBS to give 33.3 ng/10 ml. Fluid was left on the cord for 10 min and then removed. Animals in this group received either 150 ng (n ¼ 13) or 300 ng (n ¼ 13) of SSP-SAP, vehicle (PBS), or SAP-alone (300 ng) (n ¼ 9). The second group of 26 animals included those receiving SSP-SAP treatment within 3– 5 days following onset of excessive grooming behavior. Since the time to grooming onset ranged from 12 to 17 days post-injury it was necessary to reopen the surgery site and remove scar tissue at the site of injection. The same surgical procedure as described above was used in preparing the cord for treatment with different agents. The following groups received solutions containing SSP-SAP or SAP were placed on the cord as described above: (1) grooming behavior þ SSP-SAP 150 ng (n ¼ 7); (2) grooming behavior þ SSP-SAP 300 ng (n ¼ 9); and (3) grooming behavior þ vehicle or SAP (300 ng) (n ¼ 10). Beginning 7 days post-injection, all animals were inspected daily for signs of excessive grooming. When grooming behavior was observed the area of skin targeted was reconstructed on a standardized drawing [18]. Assessment of grooming behavior continued for 30 days or until tissue damage progressed beyond the superficial layers of skin, at which time animals were sacrificed. The severity of grooming was divided into four classes (I–IV) as described previously [3]. Analysis of grooming behavior consisted of plotting the mean area of skin damage along with evaluations of onsettime post-injection, and severity of grooming (class I–IV). Data were compared between the pre-treated, post-treated and vehicle or SAP-treated groups using one-way analysis of
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Table 1 Effects of treatment delivered at the time of injury (mean ^ SEM) Group
Onset
Area
Class (median ^ IQR)
Survival days
Incidence (%)
I II III
14.8 ^ 2.2 22.8 ^ 1.9** 27.2 ^ 1.5***
4.5 ^ 1.0 1.2 ^ 0.4*** 0.5 ^ 0.3****
4.0 ^ 0.0 1.5 ^ 3.0** 0.0 ^ 1.3***
24.3 ^ 2.2 29.4 ^ 0.7* 29.2 ^ 0.9*
78 46 31
I, QUIS þ SAP (n ¼ 9); II, QUIS þ SSP-SAP 150 ng (n ¼ 13); and III, QUIS þ SSP-SAP 300 ng (n ¼ 13). Mean ^ SEM: t-test, *P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001. Median ^ IQR: Kruskal– Wallis test and Mann–Whitney test, **P , 0.01, ***P , 0.001.
variance and Fisher’s protected least significant difference test. Grooming class data (I–IV) are expressed as median ^ interquartile range (IQR). For the analysis of these data the non-parametric Kruskal–Wallis test was used for multiple comparisons, followed by the Mann–Whitney U-test to compare individual groups. All behavioral and histological analyzes were carried out by individuals blinded to the drug treatment of each animal. P-values of P , 0:05 were used for statistical significance. Elimination of NK-1R expressing neurons in the superficial dorsal horn was confirmed by immunohistochemistry targeting the NK-1R. Immunohistochemistry was carried out as described previously [7,9]. Results related to the delivery of SSP-SAP at the time of QUIS injections showed that animals receiving treatment with SAP or vehicle (PBS) developed excessive grooming with an average onset time of 14.8 ^ 2.2 days (Table 1). SAP or vehicle treated animals displayed a significant increase in grooming area over time, reaching a maximum of 4.5 ^ 1.0 cm2 (Table 1). The severity of grooming in SAP/vehicle-treated animals progressed to Class 4.0 ^ 0.0 and targeted peripheral dermatomes associated with spinal segments at or caudal to the site of injury (Table 1). Following treatment with SSP-SAP the same three outcome measures were evaluated and compared against results from SAP or vehicle-treated animals. Excessive grooming behavior developed in 7/9 (78%) SAP/vehicle-treated animals. SSP-SAP (150 or 300 ng) decreased the incidence of spontaneous grooming behavior to 46% (6/13) in animals treated with 150 ng, while only 4/13 rats (31%) treated with 300 ng developed the behavior. Treatment with 150 or 300 ng SSP-SAP delayed the onset of grooming behavior by 54 and 84%, respectively, compared with SAP/vehicle-treated animals (SSP-SAP 150 ng, 22.8 ^ 1.9 days vs 14.8 ^ 2.2, P , 0:01; and SSP-SAP 300 ng, 27.2 ^ 1.5 days vs 14.8 ^ 2.2, P , 0:001) (Table 1). The area of skin targeted for excessive grooming behavior following 150 or 300 ng SSP-SAP was reduced by 73 and 89%, respectively, compared to SAP/vehicle-treated animals (SSP-SAP 150 ng, 1.2 ^ 0.4 cm2 vs 4.5 ^ 1.0, P , 0:001; and SSP-SAP 300 ng, 0.5 ^ 0.3 cm2 vs 4.5 ^ 1.1, P , 0:0001) (Table 1). The final class of grooming was also significantly reduced relative to SAP/vehicle-treated control animals (SSP-SAP 150 ng, 1.5 ^ 3.0 (P , 0:01) vs SAP/ vehicle-treated animals 4.0 ^ 0.0, and SSP-SAP 300 ng, 0.0 ^ 1.3 vs 4.0 ^ 0.0, P , 0:001 (Table 1 and Fig. 1). The second part of this study was designed to evaluate the
potential clinical utility of SSP-SAP treatment and consisted of a post-hoc evaluation of animals expressing injury-induced excessive grooming behavior. Animals were randomly divided into three post-injury treatment groups: (a) SSPSAP; (b) SAP; and (c) vehicle. No significant differences in area, onset-time, or class of grooming behavior were observed among the groups prior to the start of treatment. The effects of SSP-SAP, SAP and vehicle treatment on grooming area and severity are shown in Fig. 2. After treatment SAP/vehicletreated animals exhibited a significant increase in grooming area over time (6.3 ^ 1.1 cm2), whereas SSP-SAP (150 ng) and SSP-SAP (300 ng) treatment groups showed a 67 and 71% reduction, respectively, in the area of skin damage targeted for
Fig. 1. Effects of SSP-SAP on the area of skin targeted for excessive grooming behavior, days post injury to grooming onset, and severity of grooming behavior (data are represented as mean ^ SEM). Individual data points for the final class of grooming are represented by circles. SSP-SAP (300 or 150 ng), PBS or SAP (300 ng) were delivered directly to the dorsal surface of the cord for 10 min immediately after QUIS injection. **P , 0:01; ***P , 0:001; ****P , 0:0001:
R.P. Yezierski et al. / Neuroscience Letters 361 (2004) 232–236
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Table 2 Effects of treatment delivered following onset of excessive grooming behavior (Mean ^ SEM) Group
Grooming area
Grooming class (median ^ IQR)
Survival days
n
I II III
6.3 ^ 1.1 2.1 ^ 0.6** 1.8 ^ 0.5***
4.0 ^ 0.0 2.5 ^ 0.5** 2.5 ^ 1.0***
23.5 ^ 1.4 28.7 ^ 1.2* 29.9 ^ 2.1*
10 7 9
I, Q-grooming þ vehicle (PBS or SAP); II, Q-grooming þ SSP-SAP 150 ng; and III, Q-grooming þ SSP-SAP 300 ng. Mean ^ SEM: t-test, *P , 0.05, **P , 0.01, ***P , 0.001. Median ^ IQR: Kruskal– Wallis test and Mann–Whitney test, **P , 0.01, ***P , 0.001.
excessive grooming behavior: SSP-SAP (150 ng) 2.1 ^ 0.6 cm, P , 0:01; SSP-SAP (300 ng) 1.8 ^ 0.5 vs 6.3 ^ 1.1, P , 0:001; Table 2). SSP-SAP (150 or 300 ng) delivered to the dorsal surface of the cord also significantly reduced the final class of grooming compared to vehicle-treated control animals: SSP-SAP (150 ng): 2.5 ^ 0.5 vs 4.0 ^ 0.0, P , 0:01 and SSP-SAP (300 ng): 2.5 ^ 1.0, P , 0:001; (Table 2). Staining for the substance P receptor (SPR) provided evidence supporting the specificity of SSP-SAP in eliminating NK-1R expressing neurons in the spinal cord (Fig. 3). The results are consistent with those of previous studies documenting the specificity of the substance P-saporin conjugate in the spinal cord and selected brain regions [4, 7 –9,14]. Figs. 3A –C shows staining of tissue taken from an animal injected with QUIS followed 14 days later by the development of excessive grooming behavior. The sections in Fig. 3 were taken 2 mm from the epicenter of the QUIS injection site. NeuN staining failed to show a substantial loss
Fig. 2. Effects of SSP-SAP on the area of skin targeted for excessive grooming behavior and the severity of grooming behavior (data are represented as mean ^ SEM). Individual data points for the final class of grooming are represented by circles. SSP-SAP (300 or 150 ng), PBS or SAP (300 ng) were delivered directly to the dorsal surface of the cord for 10 min following the onset of excessive grooming behavior. ***P , 0:001; **P , 0:01:
of neurons in the superficial laminae of the dorsal horn following QUIS injections (Fig. 3B). A robust astroglial reaction to the QUIS injection is indicated by the intense staining for GFAP (Fig. 3C). By contrast to animals treated with SSP-SAP (Fig. 3A) those treated with SAP (Fig. 3D) showed no decrease in NK-1R staining following treatment. The dense staining for GFAP (Fig. 3F) and NeuN staining (Fig. 3E) are consistent with the pathological characteristics of the QUIS injury. Consistent with previous reports dense
Fig. 3. Immunostaining for the SPR (A, D); cell stain (B, E); and GFAP (C, F). Sections in A–C were taken from an animal that was injected with QUIS and 14 days after injection developed excessive grooming behavior. Following treatment with SSP-SAP (150 ng) the animal survived a total of 30 days. Sections in D –F were taken from an animal that was injected with QUIS and 12 days after injection developed excessive grooming behavior. Following treatment with SAP (300 ng) the animal survived a total of 29 days. Note the reduced staining for the substance P receptor in the superficial lamina of the dorsal horn (A) compared to the SAP treated animal (D). The cell stain (NeuN) in B and E shows that there was a significant number of neurons remaining in the dorsal horn of animals treated with SSP-SAP and SAP. The intense staining for GFAP immunoreactivity reflects the glial response to the excitotoxic injury (C, F).
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staining in lamina I for SPR-immunoreactivity (Fig. 3D) supports the conclusion that neurons in this region were not affected by SAP treatment [7 – 9]. Neuronal loss in the neck of the dorsal horn with partial or complete sparing of the superficial laminae is a distinguishing pathological characteristic associated with excessive grooming behavior in the QUIS model of SCI [3]. After-discharges to mechanical stimuli, lowered response thresholds and increases in background discharge of neurons adjacent to the site of injury are believed to contribute to the central mechanism underlying excessive grooming behavior as well as the clinical condition of injury-induced at-level neuropathic pain [17]. These physiological characteristics, reflecting an increased excitability of spinal neurons, are consistent with those described in patients with SCI induced pain [17]. The present results together with current understanding of secondary events following SCI support a spinal mechanism for at-level pain that includes: (a) a secondary pathological process consisting of a cascade of cellular events leading to changes in the functional state of NK-1R expressing neurons in the superficial dorsal horn; and (b) an increase in spontaneous activity along with a lower activation threshold that contribute to the clinical conditions of allodynia, hyperalgesia and spontaneous pain. Although it is acknowledged that the functional properties of neurons other than those in lamina I are affected by spinal injury [17], previous evidence supporting the involvement of lamina I in the expression of injury-induced at-level pain provided ample justification for targeting NK-1R expressing lamina I neurons with SSP-SAP. The fact that this treatment had a significant affect on the onset and progression of excessive grooming behavior suggests that this population of neurons plays a critical role in the expression of this injury-induced behavior. Given the compromising influence on quality of life experienced by individuals with pain associated with SCI combined with the significant clinical challenge of managing this type of pain, the present study targeting a population of chemically identified neurons offers a novel treatment strategy to those presently being used to manage this condition. Finally, the present results extend to the condition of SCI induced pain descriptions of NK-1R expressing neurons in the superficial dorsal horn or an important component of the spinal mechanism responsible for central sensitization, hyperalgesia, and chronic pain [4,7,9,13].
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Acknowledgements This work was supported by NS40096 (RPY) and MH56368 (DAL). The authors thank Victoria Gority and Brandi Baker for their expert help in preparation of the manuscript. References [1] N.P. Finnerup, I.L. Johannesen, S.H. Sindrup, F.W. Bach, T.S. Jensen,
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Spinal neurons involved in the generation of at-level pain following spinal injury in the rat Robert P. Yezierskia,b,*, Chen-Guang Yua,b, Patrick W. Mantyhc,d,e, Charles J. Viercka,b, Douglas A. Lappif a
Department of Orthodontics, Comprehensive Center for Pain Research, McKnight Brain Institute, University of Florida, 1600 S.W. Archer Road, Rm D10– 19, P.O. Box 100444, Gainesville, FL 32610, USA b Department of Neuroscience, University of Florida, 1600 S.W. Archer Road, Gainesville, FL 32610, USA c Department of Preventive Sciences, University of Minnesota, Minneapolis, MN, USA d Department of Psychiatry, University of Minnesota, Minneapolis, MN, USA e Department of Neuroscience, University of Minnesota, Minneapolis, MN, USA f Advanced Targeting Systems, San Diego, CA, USA
Abstract Using a conjugate of substance P and the ribosome-inactivating protein saporin, neurons expressing the neurokinin-1 receptor in lamina I of the spinal cord were targeted to determine their role in the expression of a spontaneous pain behavior following intraspinal injections of quisqualic acid in the rat. Treatment was carried out at the time of injury in order to prevent the onset of the behavior, and following onset in order to evaluate the potential clinical utility of this intervention. Treatment at the time of injury resulted in significant decreases in onsettime and severity of pain behavior, while treatment at the time of onset led to a significant reduction of the spontaneous self-directed behavior. The results suggest that the substrate for at-level pain following spinal cord injury includes a population of spinal neurons expressing the neurokinin-1 receptor in the superficial laminae of the spinal cord. q 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Central pain; Quisqualic acid; Excitotoxicity; Lamina I; Substance P; Saporin
Chronic pain affects nearly 70% of patients with pathology resulting from traumatic or ischemic injury of the spinal cord [12]. Although loss of sensory and motor function represent the most commonly described deficits associated with spinal injury, abnormal sensations including pain are among the most difficult complications to deal with following injury [15]. For many patients with spinal injury, elimination of pain represents the possibility of returning to work, improving social and family relationships as well as reducing pain medications. For a significant percentage of patients elimination of pain is even more important than recovery of motor function, further emphasizing the impact of injury induced pain [12]. Pain associated with spinal cord injury (SCI) can be divided into nociceptive (including musculoskeletal and visceral) and neuropathic (including above-, at- and below-level) [12]. The primary characteristic of at-level pain is a hypersensitive band of skin in dermatomes associated with spinal segments adjacent to the site of injury. Injury-induced loss of intrinsic * Corresponding author. Tel.: þ1-352-392-4081; fax: þ 1-352-392-3031. E-mail address: [email protected] (R.P. Yezierski).
inhibitory control and changes in the functional state of neurons adjacent to the site of injury represent the current view of a mechanism responsible for this condition [17]. Unfortunately, the precise neural substrate responsible for this type of pain remains undefined. Consequently, effective treatments are largely inadequate, ranging from implanted stimulators and ablative surgery [2] to a wide-range of pharmacological interventions [1]. Intraspinal injection of the AMPA/metabotropic receptor agonist quisqualic acid (QUIS) has been used to examine the underlying pathophysiological changes responsible for altered sensibilities, including pain, associated with SCI [17]. By creating an excitotoxic challenge to surrounding tissue, thus simulating an important chemical change occurring at the time of injury, pathological characteristics similar to those associated with ischemic and traumatic injury are produced [17]. The transient elevation of excitatory amino acids initiates a secondary injury cascade which has a profound impact on the anatomical organization and functional state of neuronal circuits within the spinal gray matter [11]. The pathological characteristics, including neuronal loss, glial response, and syrinx formation,
0304-3940/03/$ - see front matter q 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2003.12.035
R.P. Yezierski et al. / Neuroscience Letters 361 (2004) 232–236
associated with this injury model parallel those observed in humans with SCI, especially those associated with posttraumatic syringomyelia [5]. One of the clinical complications associated with this condition is the onset of chronic pain that develops at and below the level of injury [5]. The behavioral sequelae of the excitotoxic injury model includes the onset of evoked and spontaneous pain-like behaviors commonly associated with animal models of neuropathic and inflammatory pain [17]. One of the hallmarks of this model is an injury-induced biting behavior, referred to as excessive grooming, which has been proposed as a model of SCI-induced at-level pain. The delayed onset and association with dermatomes adjacent to the site of injury are characteristics making this behavior similar to those defining the human condition. Although the exact quality of the eliciting stimulus is unknown, e.g. paresthesia, dysesthesia, pain, the sensation evokes a spontaneous self-directed behavior similar to that reported in humans with neuropathic pain [6]. Consistent with the clinical profile of at-level pain, hypersensitivity to thermal and mechanical stimuli are characteristics of the area targeted for excessive grooming behavior. Efforts to elucidate the underlying mechanism of this behavior have included studies focusing on anatomical [3], physiological [17], and molecular [10] changes coinciding with the onset and progression of excessive grooming behavior. Available evidence suggests the involvement of neurons in the superficial laminae of the spinal cord as a critical component of a spinal pain generator responsible for the onset of this behavior [17]. Additionally, a recent report described the importance of a spatial threshold of tissue injury along the rostrocaudal axis of the cord that must be exceeded in order to achieve expression of excessive grooming behavior [18]. Recently, a population of neurons expressing the substance P receptor (SPR) in the superficial dorsal horn was shown to have a unique role in the processing of nociceptive information and the development of chronic pain and hyperalgesia. Using internalization of the SPR as a vehicle to transport a substance P-saporin conjugate into the cell, lamina I SPR-expressing neurons have been targeted and eliminated by the ribosome inactivating protein saporin. This strategy reduces nocifensive behaviors enhanced by capsaicin [7], inflammation, and nerve injury [9]. The involvement of this population of neurons in the activation of supraspinal pain centers was demonstrated by a reduction in operant escape behavior after intrathecal application of SP-saporin to the lumbar spinal cord [14]. These studies suggest the functional importance of SPR-expressing neurons in hyperalgesia. The importance of these neurons in the development of central sensitization, a critical step in the cascade of central events responsible for the conversion of acute to chronic pain, has also been described [4]. Furthermore, evidence suggesting that neuronal excitability in the dorsal horn is influenced by lamina I neurons activating descending facilitatory pathways was recently described [13]. The conclusion from these studies, in combination with
233
previous reports emphasizing the importance of the superficial dorsal horn as part of the substrate responsible for atlevel pain, makes the population of SPR expressing neurons in this region of the cord an obvious target for therapeutic strategies. In the present study, the effects of [Sar9Met(O2)11] substance P-saporin (SSP-SAP) neurotoxin [16] were evaluated against the onset and progression of injury-induced excessive grooming behavior. This reagent has been shown to specifically eliminate neurokinin-1 receptor (NK-1R) expressing neurons in the brain and spinal cord [7 –9,16]. Two strategies were used for delivery of SSP-SAP: (a) treatment at the time of injury for the purpose of delaying the onset of excessive grooming behavior; and (b) post-hoc treatment following injury for the purpose of slowing the progression and/or eliminating the pain behavior. Excessive grooming behavior was assessed on a regular schedule following injury and evaluated according to: (a) time of onset; (b) area of skin targeted for excessive grooming behavior; and (c) severity (class I– IV) [18]. The first group of 35 animals received SSP-SAP, SAP, or vehicle treatment at the time of QUIS injections. A solution containing SSP-SAP or SAP (Advanced Targeting Systems, San Diego, CA) was placed in a paraffin well made at the injection site 30 min after the QUIS injection (total volume 100 ml). SSP-SAP (25 mg) was dissolved in 833 ml of PBS to give 300 ng/10 ml. SAP (100 mg) was dissolved in 30 ml of PBS to give 33.3 ng/10 ml. Fluid was left on the cord for 10 min and then removed. Animals in this group received either 150 ng (n ¼ 13) or 300 ng (n ¼ 13) of SSP-SAP, vehicle (PBS), or SAP-alone (300 ng) (n ¼ 9). The second group of 26 animals included those receiving SSP-SAP treatment within 3– 5 days following onset of excessive grooming behavior. Since the time to grooming onset ranged from 12 to 17 days post-injury it was necessary to reopen the surgery site and remove scar tissue at the site of injection. The same surgical procedure as described above was used in preparing the cord for treatment with different agents. The following groups received solutions containing SSP-SAP or SAP were placed on the cord as described above: (1) grooming behavior þ SSP-SAP 150 ng (n ¼ 7); (2) grooming behavior þ SSP-SAP 300 ng (n ¼ 9); and (3) grooming behavior þ vehicle or SAP (300 ng) (n ¼ 10). Beginning 7 days post-injection, all animals were inspected daily for signs of excessive grooming. When grooming behavior was observed the area of skin targeted was reconstructed on a standardized drawing [18]. Assessment of grooming behavior continued for 30 days or until tissue damage progressed beyond the superficial layers of skin, at which time animals were sacrificed. The severity of grooming was divided into four classes (I–IV) as described previously [3]. Analysis of grooming behavior consisted of plotting the mean area of skin damage along with evaluations of onsettime post-injection, and severity of grooming (class I–IV). Data were compared between the pre-treated, post-treated and vehicle or SAP-treated groups using one-way analysis of
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Table 1 Effects of treatment delivered at the time of injury (mean ^ SEM) Group
Onset
Area
Class (median ^ IQR)
Survival days
Incidence (%)
I II III
14.8 ^ 2.2 22.8 ^ 1.9** 27.2 ^ 1.5***
4.5 ^ 1.0 1.2 ^ 0.4*** 0.5 ^ 0.3****
4.0 ^ 0.0 1.5 ^ 3.0** 0.0 ^ 1.3***
24.3 ^ 2.2 29.4 ^ 0.7* 29.2 ^ 0.9*
78 46 31
I, QUIS þ SAP (n ¼ 9); II, QUIS þ SSP-SAP 150 ng (n ¼ 13); and III, QUIS þ SSP-SAP 300 ng (n ¼ 13). Mean ^ SEM: t-test, *P , 0.05, **P , 0.01, ***P , 0.001, ****P , 0.0001. Median ^ IQR: Kruskal– Wallis test and Mann–Whitney test, **P , 0.01, ***P , 0.001.
variance and Fisher’s protected least significant difference test. Grooming class data (I–IV) are expressed as median ^ interquartile range (IQR). For the analysis of these data the non-parametric Kruskal–Wallis test was used for multiple comparisons, followed by the Mann–Whitney U-test to compare individual groups. All behavioral and histological analyzes were carried out by individuals blinded to the drug treatment of each animal. P-values of P , 0:05 were used for statistical significance. Elimination of NK-1R expressing neurons in the superficial dorsal horn was confirmed by immunohistochemistry targeting the NK-1R. Immunohistochemistry was carried out as described previously [7,9]. Results related to the delivery of SSP-SAP at the time of QUIS injections showed that animals receiving treatment with SAP or vehicle (PBS) developed excessive grooming with an average onset time of 14.8 ^ 2.2 days (Table 1). SAP or vehicle treated animals displayed a significant increase in grooming area over time, reaching a maximum of 4.5 ^ 1.0 cm2 (Table 1). The severity of grooming in SAP/vehicle-treated animals progressed to Class 4.0 ^ 0.0 and targeted peripheral dermatomes associated with spinal segments at or caudal to the site of injury (Table 1). Following treatment with SSP-SAP the same three outcome measures were evaluated and compared against results from SAP or vehicle-treated animals. Excessive grooming behavior developed in 7/9 (78%) SAP/vehicle-treated animals. SSP-SAP (150 or 300 ng) decreased the incidence of spontaneous grooming behavior to 46% (6/13) in animals treated with 150 ng, while only 4/13 rats (31%) treated with 300 ng developed the behavior. Treatment with 150 or 300 ng SSP-SAP delayed the onset of grooming behavior by 54 and 84%, respectively, compared with SAP/vehicle-treated animals (SSP-SAP 150 ng, 22.8 ^ 1.9 days vs 14.8 ^ 2.2, P , 0:01; and SSP-SAP 300 ng, 27.2 ^ 1.5 days vs 14.8 ^ 2.2, P , 0:001) (Table 1). The area of skin targeted for excessive grooming behavior following 150 or 300 ng SSP-SAP was reduced by 73 and 89%, respectively, compared to SAP/vehicle-treated animals (SSP-SAP 150 ng, 1.2 ^ 0.4 cm2 vs 4.5 ^ 1.0, P , 0:001; and SSP-SAP 300 ng, 0.5 ^ 0.3 cm2 vs 4.5 ^ 1.1, P , 0:0001) (Table 1). The final class of grooming was also significantly reduced relative to SAP/vehicle-treated control animals (SSP-SAP 150 ng, 1.5 ^ 3.0 (P , 0:01) vs SAP/ vehicle-treated animals 4.0 ^ 0.0, and SSP-SAP 300 ng, 0.0 ^ 1.3 vs 4.0 ^ 0.0, P , 0:001 (Table 1 and Fig. 1). The second part of this study was designed to evaluate the
potential clinical utility of SSP-SAP treatment and consisted of a post-hoc evaluation of animals expressing injury-induced excessive grooming behavior. Animals were randomly divided into three post-injury treatment groups: (a) SSPSAP; (b) SAP; and (c) vehicle. No significant differences in area, onset-time, or class of grooming behavior were observed among the groups prior to the start of treatment. The effects of SSP-SAP, SAP and vehicle treatment on grooming area and severity are shown in Fig. 2. After treatment SAP/vehicletreated animals exhibited a significant increase in grooming area over time (6.3 ^ 1.1 cm2), whereas SSP-SAP (150 ng) and SSP-SAP (300 ng) treatment groups showed a 67 and 71% reduction, respectively, in the area of skin damage targeted for
Fig. 1. Effects of SSP-SAP on the area of skin targeted for excessive grooming behavior, days post injury to grooming onset, and severity of grooming behavior (data are represented as mean ^ SEM). Individual data points for the final class of grooming are represented by circles. SSP-SAP (300 or 150 ng), PBS or SAP (300 ng) were delivered directly to the dorsal surface of the cord for 10 min immediately after QUIS injection. **P , 0:01; ***P , 0:001; ****P , 0:0001:
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Table 2 Effects of treatment delivered following onset of excessive grooming behavior (Mean ^ SEM) Group
Grooming area
Grooming class (median ^ IQR)
Survival days
n
I II III
6.3 ^ 1.1 2.1 ^ 0.6** 1.8 ^ 0.5***
4.0 ^ 0.0 2.5 ^ 0.5** 2.5 ^ 1.0***
23.5 ^ 1.4 28.7 ^ 1.2* 29.9 ^ 2.1*
10 7 9
I, Q-grooming þ vehicle (PBS or SAP); II, Q-grooming þ SSP-SAP 150 ng; and III, Q-grooming þ SSP-SAP 300 ng. Mean ^ SEM: t-test, *P , 0.05, **P , 0.01, ***P , 0.001. Median ^ IQR: Kruskal– Wallis test and Mann–Whitney test, **P , 0.01, ***P , 0.001.
excessive grooming behavior: SSP-SAP (150 ng) 2.1 ^ 0.6 cm, P , 0:01; SSP-SAP (300 ng) 1.8 ^ 0.5 vs 6.3 ^ 1.1, P , 0:001; Table 2). SSP-SAP (150 or 300 ng) delivered to the dorsal surface of the cord also significantly reduced the final class of grooming compared to vehicle-treated control animals: SSP-SAP (150 ng): 2.5 ^ 0.5 vs 4.0 ^ 0.0, P , 0:01 and SSP-SAP (300 ng): 2.5 ^ 1.0, P , 0:001; (Table 2). Staining for the substance P receptor (SPR) provided evidence supporting the specificity of SSP-SAP in eliminating NK-1R expressing neurons in the spinal cord (Fig. 3). The results are consistent with those of previous studies documenting the specificity of the substance P-saporin conjugate in the spinal cord and selected brain regions [4, 7 –9,14]. Figs. 3A –C shows staining of tissue taken from an animal injected with QUIS followed 14 days later by the development of excessive grooming behavior. The sections in Fig. 3 were taken 2 mm from the epicenter of the QUIS injection site. NeuN staining failed to show a substantial loss
Fig. 2. Effects of SSP-SAP on the area of skin targeted for excessive grooming behavior and the severity of grooming behavior (data are represented as mean ^ SEM). Individual data points for the final class of grooming are represented by circles. SSP-SAP (300 or 150 ng), PBS or SAP (300 ng) were delivered directly to the dorsal surface of the cord for 10 min following the onset of excessive grooming behavior. ***P , 0:001; **P , 0:01:
of neurons in the superficial laminae of the dorsal horn following QUIS injections (Fig. 3B). A robust astroglial reaction to the QUIS injection is indicated by the intense staining for GFAP (Fig. 3C). By contrast to animals treated with SSP-SAP (Fig. 3A) those treated with SAP (Fig. 3D) showed no decrease in NK-1R staining following treatment. The dense staining for GFAP (Fig. 3F) and NeuN staining (Fig. 3E) are consistent with the pathological characteristics of the QUIS injury. Consistent with previous reports dense
Fig. 3. Immunostaining for the SPR (A, D); cell stain (B, E); and GFAP (C, F). Sections in A–C were taken from an animal that was injected with QUIS and 14 days after injection developed excessive grooming behavior. Following treatment with SSP-SAP (150 ng) the animal survived a total of 30 days. Sections in D –F were taken from an animal that was injected with QUIS and 12 days after injection developed excessive grooming behavior. Following treatment with SAP (300 ng) the animal survived a total of 29 days. Note the reduced staining for the substance P receptor in the superficial lamina of the dorsal horn (A) compared to the SAP treated animal (D). The cell stain (NeuN) in B and E shows that there was a significant number of neurons remaining in the dorsal horn of animals treated with SSP-SAP and SAP. The intense staining for GFAP immunoreactivity reflects the glial response to the excitotoxic injury (C, F).
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staining in lamina I for SPR-immunoreactivity (Fig. 3D) supports the conclusion that neurons in this region were not affected by SAP treatment [7 – 9]. Neuronal loss in the neck of the dorsal horn with partial or complete sparing of the superficial laminae is a distinguishing pathological characteristic associated with excessive grooming behavior in the QUIS model of SCI [3]. After-discharges to mechanical stimuli, lowered response thresholds and increases in background discharge of neurons adjacent to the site of injury are believed to contribute to the central mechanism underlying excessive grooming behavior as well as the clinical condition of injury-induced at-level neuropathic pain [17]. These physiological characteristics, reflecting an increased excitability of spinal neurons, are consistent with those described in patients with SCI induced pain [17]. The present results together with current understanding of secondary events following SCI support a spinal mechanism for at-level pain that includes: (a) a secondary pathological process consisting of a cascade of cellular events leading to changes in the functional state of NK-1R expressing neurons in the superficial dorsal horn; and (b) an increase in spontaneous activity along with a lower activation threshold that contribute to the clinical conditions of allodynia, hyperalgesia and spontaneous pain. Although it is acknowledged that the functional properties of neurons other than those in lamina I are affected by spinal injury [17], previous evidence supporting the involvement of lamina I in the expression of injury-induced at-level pain provided ample justification for targeting NK-1R expressing lamina I neurons with SSP-SAP. The fact that this treatment had a significant affect on the onset and progression of excessive grooming behavior suggests that this population of neurons plays a critical role in the expression of this injury-induced behavior. Given the compromising influence on quality of life experienced by individuals with pain associated with SCI combined with the significant clinical challenge of managing this type of pain, the present study targeting a population of chemically identified neurons offers a novel treatment strategy to those presently being used to manage this condition. Finally, the present results extend to the condition of SCI induced pain descriptions of NK-1R expressing neurons in the superficial dorsal horn or an important component of the spinal mechanism responsible for central sensitization, hyperalgesia, and chronic pain [4,7,9,13].
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