Role of Microglia and Astrocytes in Spinal Cord Injury Induced Neuropathic Pain.

Background: Spinal cord injuries incite varying degrees of symptoms in patients, ranging from weakness and incoordination to paralysis. Common amongst spinal cord injury (SCI) patients, neuropathic pain (NP) is a debilitating medical condition. Unfortunately, there remain many clinical impediments in treating NP because there is a lack of understanding regarding the mechanisms behind SCI-induced NP (SCINP). Given that more than 450,000 people in the United States alone suffer from SCI, it is unsatisfactory that current treatments yield poor results in alleviating and treating NP. Summary: In this review, we briefly discussed the models of SCINP along with the mechanisms of NP progression. Further, current treatment modalities are herein explored for SCINP involving pharmacological interventions targeting glia cells and astrocytes. Key message: The studies presented in this review provide insight for new directions regarding SCINP alleviation. Given the severity and incapacitating effects of SCINP, it is imperative to study the pathways involved and find new therapeutic targets in coordination with stem cell research, and to develop a new gold-standard in SCINP treatment.

Altered Expression of Heat Shock Protein-27 and Monocyte Chemoattractant Protein-1 after Acute Spinal Cord Injury: A Pilot Study.

Background Spinal cord injury (SCI) leads to serious complications involving primary trauma and progressive loss due to inflammation, local ischemia, or infection. Despite a worldwide annual incidence of 15 to 40 cases per million, methylprednisolone is the only treatment available to alleviate neurologic dysfunction; therefore, research is currently focused on identifying novel targets by biochemical and molecular studies. Purpose Here, we investigated the expression of various molecular markers at the messenger ribonucleic acid (mRNA) and protein level at day 0 and day 30 post-SCI. Methods Enzyme-linked immunosorbent assay (ELISA) was performed to determine the expression of CASPASE-3 and heat shock protein-27 (HSP-27) in serum samples. Real-time polymerase chain reaction (RT-PCR) was performed to determine the level of mRNA expression of vascular endothelial growth factor receptor-1 (VEGFR-1), VEGFR-2, HSP-27, monocyte chemoattractant protein-1 (MCP-1), and CASPASE-3. Results HSP-27 expression at day 30, as compared with day 0, showed significant downregulation. In contrast, there was elevated expression of MCP-1. ELISA analysis showed no significant change in the expression of CASPASE-3 or HSP-27. Conclusion There may be possible opposing role of HSP-27 and MCP-1 governing SCI. Their association can be studied by designing in vitro studies.

Folic Acid Modulates Matrix Metalloproteinase-9 Expression Following Spinal Cord Injury.

BACKGROUND: Treatment of spinal cord injury (SCI) induced neuropathic pain (NP) proves to be extremely clinically challenging as the mechanism behind SCINP is poorly understood. Matrix metalloproteinase (MMP) is largely responsible for the early disruption of the blood spinal cord barrier. This system initiates macrophage infiltration and degradation of myelin, which plays a pivotal role in how NP occurs. In a recent study, we demonstrated that folic acid (FA) treatment to cSCI rats reduced NP and improved functional recovery by repressing MMP-2 expression. We hypothesize that MMP-2 expression is suppressed because FA actively methylates the DNA sequence that encodes for the MMP-2 protein. However, modulation of MMP-2 expression for alleviation of NP is only pertinent to the mid- to late-phase of injury. Therefore, we need to explore alternate therapeutic methods to target the early- to mid-phase of injury to wholly alleviate NP. PURPOSE: Furthering our previous findings on inhibiting MMP-2 expression by FA in mid- and late- phase following cSCI in rats, we hypothesized that FA will methylate and suppress MMP-9 expression during the early- phase, day 1, 3, 7 post cSCI and mid- phase (day 18 post cSCI), in comparison with MMP-2 expression during mid- and the late-phase of cSCI. METHODS: Adult male Sprague Dawley rats (250-270g) underwent cSCI, using a NYU impactor, with 12.5 gm/cm injury. The spinal cord-injured animals were treated intraperitoneally (i.p.) with a standardized dose of FA (80 µg/kg body weight) on day 1, 2, 3, prior to cSCI, followed by daily injection up to 14 or 17 days post-cSCI in different experiments. Animals were euthanized on day 1, 3, 7 post cSCI (early- phase), day 18 post cSCI (mid- phase), and day 42 post cSCI (late-phase) and the epicenter region of injured spinal cord were harvested for MMP-9 and MMP-2 expression analysis by Western blots technique. RESULTS: i) During early-phase on day 1, 3, and 7, the quantitation displayed no statistical significance in MMP-9 expression, between water- and FA- injected rats. ii) On day 18 post-cSCI, FA significantly modulates the expression of MMP-9 (p = 0.043) iii) Comparing results with MMP-2 expression and inhibition, FA significantly modulates the expression of MMP-2 on day 18 post cSCI (FA- and water-injected rats (p = 0.003). iv) In addition, FA significantly modulates the expression of MMP-2 on day 42 post-cSCI comparing FA- and water- injected rat groups (p = 0.034). CONCLUSION: We report that FA administration results in alleviating cSCI-induced NP by inhibiting MMP-9 in the proposed mid- phase of cSCI. However, FA administration resulted in MMP-2 decline during both mid- through late- phase following cSCI. Our study elucidates a new phase of cSCI, the mid-phase. We conclude that further investigation on discovering and quantifying the nature of the mid- phase of SCI injury is needed.

Long-Term Surface Electrode Impedance Recordings Associated with Gliosis for a Closed-Loop Neurostimulation Device.

BACKGROUND: Closed-loop neurostimulation is a novel alternative therapy for medically intractable focal epilepsy for patients who are not candidates for surgical resection of a seizure focus. Electrodes for this system can be implanted either within the brain parenchyma or in the subdural space. The electrodes then serve the dual role of detecting seizures and delivering an electrical signal aimed at aborting seizure activity. The Responsive Neurostimulation (RNS®) system (Neuropace, Mountain View, CA, USA) is an FDA-approved implantable device designed for this purpose. OBJECTIVE: One of the challenges of the brain machine interface devices is the potential for implanted neurostimulator devices to induce progressive gliosis, apart from that associated with the minimal trauma at implantation. Gliosis has the potential to alter impedances over time, thereby affecting the clinical efficacy of these devices, and also poses a challenge to the prospects of in vivo repositioning of depth electrodes. We present a clinical case with 3-year follow-up and pathology. METHODS: Single-case, retrospective review within a randomized trial with specific minimum follow-up and impedance measurements. RESULTS: Impedance changes in the surface electrode over time were observed. Surgical pathological findings revealed significant gliosis in the leptomeninges of the cortices. CONCLUSION: We report, for the first time, long-term impedance recordings from a surface electrode associated with pathologic findings of gliosis at the Neuropace device-tissue interface in a patient who was enrolled in the multicenter RNS System Pivotal Clinical Investigation. Further study is required to elucidate the temporal relationship of pathological findings over time. Impedance changes were more complex than can be explained by a progressive or transient pathological mechanism. Further effort is required to elucidate the relationship between impedance change and seizure event capture.

Folic Acid Modulates Matrix Metalloproteinase-2 Expression, Alleviates Neuropathic Pain, and Improves Functional Recovery in Spinal Cord-Injured Rats.

BACKGROUND: The molecular underpinnings of spinal cord injury (SCI) associated with neuropathic pain (NP) are unknown. Recent studies have demonstrated that matrix metalloproteinases (MMPs) such as MMP2 play a critical role in inducing NP following SCI. Promoter methylation of MMPs is known to suppress their transcription and reduce NP. In this context, it has been shown in rodents that folic acid (FA), an FDA approved dietary supplement and key methyl donor in the central nervous system (CNS), increases axonal regeneration and repair of injured CNS in part via methylation. PURPOSE: Based on above observations, in this study, we test whether FA could decrease MMP2 expression and thereby decrease SCI-induced NP. METHODS: Sprague-Dawley male rats weighing 250-270 g received contusion spinal cord injuries (cSCIs) with a custom spinal cord impactor device that drops a 10 g weight from a height of 12.5 mm. The injured rats received either i.p. injections of FA (80 µg/kg) or water (control) 3 days prior and 17 days post-cSCI (mid phase) or for 3 days pre-cSCI and 14 days post-cSCI ending on the 42nd day of cSCI (late phase). The functional neurological deficits due to cSCI were then assessed by Basso, Beattie, and Bresnahan (BBB) scores either on post-impaction days 0 through 18 post-cSCI (mid phase) or on days 0, 2, 7, 14, 21, 28, 35, and 42 (late phase). Baseline measurements were taken the day before starting treatments. Thermal hyperalgesia (TH) testing for pain was performed on 4 days pre-cSCI (baseline data) and on days 18, 21, 28, 35, and 42 post-cSCI. Following TH testing, animals were euthanized and spinal cords harvested for MMP-2 expression analysis. RESULT: The FA-treated groups showed higher BBB scores during mid phase (day 18) and in late phase (day 42) of injury compared to controls, suggesting enhanced functional recovery. There is a transient decline in TH in animals from the FA-treated group compared to controls when tested on days 18, 21, 28, and 35, indicative of a decrease in NP. However, when tested 25 days after stopping FA administration on day 42 of cSCI, no significant difference in TH was observed between FA-treated and control animals. Western blot analysis of the injured spinal cord from FA-treated animals showed significant decline in MMP2 expression compared to spinal cord samples from water-treated controls. CONCLUSION: Together, these data suggest that FA could alleviate NP and improve functional recovery post-SCI, possibly by reducing the expression of MMP2. Further studies will open up a novel and easy natural therapy, ideal for clinical translation with minimal side effects, for managing SCI-induced NP. Such studies might also throw light on a possible epigenetic mechanism in FA-induced recovery after SCI.

Translational Relevance of Swine Models of Spinal Cord Injury.

Spinal cord injury (SCI) is a physically and psychologically devastating clinical condition. The typical treatment regimens of decompressive surgery and rehabilitation therapy still leave many patients with permanent disability. The development of new therapies and devices can be accelerated if relevant translational animal models are more effectively used in pre-clinical stages. Swine is a highly relevant model for SCI research, especially with respect to spine and spinal cord anatomy, spine vasculature, immune responses to injury, and functional assessments. Several spine injury models have recently been developed for swine and are beginning to be used to evaluate new therapies. Swine models of SCI offer tremendous advantages for efficient translation of pre-clinical discoveries and the development of new therapies and devices. Future swine models will also be enhanced by advances in gene-editing technology to further elucidate the complex pathophysiology associated with SCI and provide a means to engineer specific spinal pathologies.

Implication of Hypothalamus in Alleviating Spinal Cord Injury-Induced Neuropathic Pain.

Neuropathic pain (NP) is common among spinal cord injury (SCI) patients, and there remain clinical difficulties in treating NP due to the lack of understanding of underlying mechanisms. Extracellular proteins, such as matrix metalloproteinase and ß-catenin, have been shown to be activated in the spinal cord regions following an injury, and may play a key role in contributing to NP states. While these extracellular proteins have been used as therapeutic targets in the spinal cord, there has also been evidence of up-regulation in the hypothalamus following a SCI. We hypothesize that the hypothalamus is involved in regulating NP following a SCI, and hence should be researched further to determine if it is a viable target for future therapeutic treatments.

Role of Matrix Metalloproteinases 2 in Spinal Cord Injury-Induced Neuropathic Pain.

Neuropathic pain (NP) affects approximately 4 million people in the United States with spinal cord injury (SCI) being a common cause. Matrix metalloproteinases (MMPs) play an integral role in mediating inflammatory responses, cellular signaling, cell migration, extracellular matrix degradation and tissue remodeling and repair. As such, they are major components in the pathogenesis of secondary injury within the central nervous system. Other gene regulatory pathways, specifically MAPK/extracellular signaling-regulated kinase (ERK) and Wnt/ß-catenin, are also believed to participate in secondary injury likely intersect. The study aims to examine the MMP-2 signaling pathway associated with ERK and Wnt/ß-catenin activity during contusion SCI (cSCI)-induced NP in a rat model. This is an experimental study investigating the implication of MMP-2 in SCI-induced NP and its association with the cellular and molecular changes in the interactions between extracellular signaling kinase and ß-catenin. Adult Sprague-Dawley rats received cSCI injury by NYU impactor by dropping 10 g weight from a height of 12.5 mm. Locomotor functional recovery of injured rats was measured on post cSCI day 1, and weekly thereafter for 6 weeks using Basso, Beattie and Bresnahan scores. Thermal hyperalgesia (TH) testing was performed on days 21, 28, 35 and 42 post cSCI. The expression and/or activity of MMP-2, ß-catenin and ERK were studied following harvest of spinal cord tissues between 3 and 6 weeks post cSCI. All experiments were funded by the department of Neurological Surgery at the University of Wisconsin, School of Medicine and Public Health having no conflict of interest. MMP-2 and ß-catenin expression were elevated and gradually increased from days 21 to 42 compared to sham-operated rats and injured rats that did not exhibit TH. The expression of phosphorylated ERK (phospho-ERK) increased on day 21 but returned to baseline levels on day 42 whereas total ERK levels remained relatively unchanged and constant. Chronic NP is associated with changes in the expression of MMP-2, ß-catenin and ERK. Our data suggest that the transient upregulation of phospho-ERK is involved in the initial upregulation of both ß-catenin and MMP-2 following cSCI-induced NP states.

Pathogenesis of spinal cord injury induced edema and neuropathic pain: expression of multiple isoforms of wnk1.

BACKGROUND: Neuropathic pain (NP) is a common occurrence following spinal cord injury (SCI). Identification of specific molecular pathways that are involved in pain syndromes has become a major priority in current SCI research. We have investigated the role of a cation-dependent chloride transporter, Cl-regulatory protein Na(+)-K(+)-Cl(-) 1 (NKCC1), phosphorylation profile of NKCC1 and its specific involvement in neuropathic pain following contusion SCI (cSCI) using a rat model. Administration of the NKCC1 inhibitor bumetanide (BU) increases the mean hindpaw withdrawal latency time (WLT), thermal hyperalgesia (TH) following cSCI. These results demonstrate implication of NKCC1 co-transporter and BUin SCI-induced neuropathic pain. The with-no-lysine (K)-1 (WNK1) kinase has been shown to be an important regulator of NKCC1 phosphorylation in many systems, including nocioception. Mutations in a neuronal-specific exon of WNK1 (HSN2) was identified in patients that have hereditary sensory neuropathy type II (HSANII) also implicates WNK1 in nocioception, such that these patients have loss of perception to pain, touch and heat. In our ongoing research we proposed two studies utilizing our contusion SCI (cSCI) NP model of rat. PURPOSE: Study 1 aimed at NKCC1 expression and activity is up-regulated following cSCI in the early edema and chronic neuropathic pain phases. Study 2 aimed at identifying the expression profile of alternatively spliced WNK1 isoforms in animals exhibiting thermal hyperalgesia (TH) following cSCI. METHODS: Adult male Sprague Dawley rats (275-300 g) following laminectomy received cSCI at T9 with the NYU impactor-device II by dropping 10 g weight from the height of 12.5 mm. Control rats obtained laminectomy but no impaction. Following injury, functional recovery was assessed by BBB locomotor scores on day 1, 7, 14, 21, 35, and 42 and development of thermal hyperalgesia on day 21, 28, 35, and 42 day of injury by monitoring hind paw withdraw latency time (WLT) in seconds compared with the baseline data before injury. RESULTS: Increased NKCC1 may explain observed increase in magnetic resonance imaging (MRI) T2, exhibiting NKCC1 localization in neurons. This data supports NKCC1's role in the pathogenesis of acute and chronic phases of injury, namely spinal cord edema and chronic phase neuropathic pain. NKCC1 dependent chloride influx requires the phosphorylation at specific residues. Probing for the HSN2 exon of WNK1 reveals two key findings: i) the HSN2 exon is found in alternatively spliced neuronal isoforms found at 250 kDa and 230 kDa; ii) the 250 kDa isoform is found only in tissue that is injured. CONCLUSIONS: This data implicates the NKCC1/WNK1/WNK1HSN2 involvement in post-injury response that contributes to the development of neuropathic pain. Targeting this system may have therapeutic benefit.

Thermal hyperalgesia assessment for rats after spinal cord injury: developing a valid and useful pain index.

BACKGROUND CONTEXT: Ongoing research to understand the mechanism behind pain is heavily dependent on animal testing. However, unlike humans, animal subjects cannot directly communicate with researchers to express the degree of pain they are experiencing. Therefore, measuring the presence of pain in animal studies is based on behavioral tests. The use of arbitrary values for determining the presence of pain in animal studies is an oversimplification of a complex and cortically dependent process. PURPOSE: The purpose of the present study was to identify a statistically supported latency time indicator that can be used as an accurate index for hyperalgesia to thermal stimuli in Sprague-Dawley rats subjected to T9 contusive spinal cord injury (SCI). STUDY DESIGN: A statistical analysis of latency of withdrawal from stimulus-mediated spinal reflex in 979 Sprague-Dawley rats that had been subjected to a T9 contusive SCI was performed. METHODS: This is a retrospective review of a large research database derived from a series of studies performed evaluating thermal hyperalgesia in rats after SCI. Sprague-Dawley rats underwent a T9 contusive SCI and were tested for withdrawal latency from a heat stimulus. Assessment was done preinjury and on Postinjury Days 21, 28, 35, and 42 of the chronic phase of injury via a plantar withdrawal test. RESULTS: The baseline test results of the 979 rats showed a significant resemblance to the normal distribution. The observed change in withdrawal showed mean latency drops of 0.42 second (standard error of the mean [SEM], 0.18; p=.026), 0.57 second (SEM, 0.19; p=.004), 0.63 second (SEM, 0.19; p=.002), and 0.69 second (SEM, 0.19; p=.0003). The standard deviation from the mean at all four postsurgical assessments was between 2.8 and 2.9 seconds. CONCLUSIONS: Interpretation of withdrawal latency times as a marker for thermal hyperalgesia must be based on an appreciation for the normal distribution of pain scores. Recognizing that withdrawal latency is normally distributed both before and after injury allows for rational assignment of animals to groups designated as hyperalgesic and nonhyperalgesic. Two point nine seconds faster than the mean latency time is a statistically reliable indicator of thermal hyperalgesia in Sprague-Dawley rats subjected to contusive SCI. Repeated testing of animals to establish the presence or absence of thermal hyperalgesia beyond 21 days is not necessary in the absence of intervention.

Persistent phosphorylation of NKCC1 and WNK1 in the epicenter of the spinal cord following contusion injury.

BACKGROUND CONTEXT: NKCC1 regulates neuronal homeostasis of chloride ions and mediates GABAergic activities in nociceptive processing. WNK1 is an upstream regulator of NKCC1 and acts via SPAK (STE20/SPS1-related proline/alanine-rich kinase) and oxidative stress-responsive kinase 1. NKCC1 activity has been shown to be important in edema formation and nociception following spinal cord injury (SCI). PURPOSE: To determine the role of NKCC1 and WNK1 in spinal cord tissues in the acute and chronic phases following contusional SCI. STUDY DESIGN: An experimental study investigating the phosphorylation profile of an important Cl-regulatory protein Na+-K+-Cl- cotransporter 1 (NKCC1) and its regulatory-kinase WNK1 (kinase with-no-lysine). METHODS: Sprague-Dawley rats underwent a contusive SCI at T9. The epicenter spinal cord tissues were harvested at Days 1, 3, and 7 for acute phase of injury or Days 35 and 42 in the chronic phase of injury. Western blot was used to compare phosphorylated levels of both NKCC1 and WNK1 in injured tissues compared with those of sham. RESULTS: A sustained increase in phosphorylation of NKCC1 and WNK1 was detected in the lesion epicenter in spinal cord during both acute and chronic phases following SCI. CONCLUSIONS: These results suggest that persistent activation of NKCC1 and WNK1 may play an important role in SCI.

Novel target for Spinal Cord Injury Neuropathic Pain.

In the world today, millions of people suffer from spinal cord injury (SCI) with little known effective clinical therapy. Neuropathic pain (NP) is often the result of SCI, making clinical treatment difficult. Even though key mediators in the development of NP have been discovered, the pathogenesis is still unclear. Some of the key mediators in the formation of NP include the inflammatory process, cannabinoid receptors, matrix metalloproteases, and their tissue inhibitors. Animal models have shown promising results with these mediators, yet the clinical models are still unsuccessful. One such study focusing on matrix metalloproteases (MMPs) has produced encouraging results. The relationship between MMPs and their tissue inhibitors (TIMPs) plays a significant role in the pathogenesis and recovery of SCI and the CNS. Key factors that lead to the functional consequences of MMP activity are cellular localization, tissue distribution, and temporal pattern of MMP expression. Controlling the MMP activity and expression are transcription factors, signaling molecules, and inhibition by TIMPs. Studies saying that MMPs can be seen as contributors of tissue damage and as contributors in the repair mechanisms have provided a need to reexamine their roles after acute and chronic process like traumatic SCI and NP, respectively. In this review, we focus on novel findings related to NP mediators like cannabinoid receptors, MMPs, and TIMPs. We will also review current clinical findings; stressing areas that show great therapeutic potential.

Gene-based therapy of Parkinson's Disease: Translation from animal model to human clinical trial employing convection enhanced delivery.

The existing treatment of Parkinson's disease (PD) is directed towards substituting dopamine loss with either dopamine replacement therapy or pharmacological therapies aimed at increasing dopamine at the synapse level. Emerging viable alternatives include the use of cell-based and gene-based therapeutics. In this review, we discuss efforts in developing in vitro and in vivo models and their translation to human clinical trials for gene-based therapy of this distressing and prevalent neurodegenerative disorder. Given the mismatch between expectations from preclinical data and results of human pivotal trials, drug delivery has been identified as the key emerging area for translational research due to limitation of limited efficacy. The chief highlights of the current topic include use of improved delivery methods of gene-based therapeutic agents. Convection-enhanced delivery (CED), an advanced infusion technique with demonstrated utility in ex vivo and in vivo animal models has recently been adopted for PD gene-based therapy trials. Several preclinical studies suggest that magnetic resonance imaging (MRI)-guided navigation for accurately targeting and real time monitoring viral vector delivery (rCED) in future clinical trials involving detection of gene expression and restoration of dopaminergic function loss using pro-drug approach will greatly enhance these PD treatments.

Emerging role of WNK1 in pathologic central nervous system signaling.

WNK1 (with no lysine (K)) is a widely expressed serine/threonine protein kinase. The role of this kinase was first described in the kidney where it dynamically controls ion channels that regulate changes in cell volume. WNK1, through intermediates oxidative stress-responsive kinase-1 (OSR1) and STE20/SPS1-related proline/alanine-rich kinase (SPAK), phosphorylates the inwardly directed Na(+)-K+-Cl(-)--cotransporter 1 (NKCC1) and the outwardly directed K(+)-Cl(-)-cotransporter 2 (KCC2), activating and deactivating these channels, respectively. WNK1, NKCC1 and KCC2 are also expressed in the central nervous system (CNS). Growing evidence implicates WNK1 playing a critical role in pathologic nervous system signaling where changes in intracellular ion concentration in response to γ-aminobutyric-acid (GABA) can activate otherwise silent pathways. This review will focus on current research about WNK1, its downstream effectors and role in GABA signaling. Future perspectives include investigating WNK1 expression in the CNS after spinal cord injury (SCI), where altered neuronal signaling could underlie pathological states such as neuropathic pain (NP).

Novel targets for Spinal Cord Injury related neuropathic pain.

Millions of people suffer from spinal cord injury (SCI) with little known effective clinical therapy. Neuropathic pain (NP) is often accompanied with SCI, making clinical treatment challenging. Even though the key mediators in the development of NP have been discovered, the pathogenesis is still unclear. Some of the key mediators in the sustenance of NP include the inflammatory processes, cannabinoid receptors, matrix metalloproteases, and their tissue inhibitors. Animal models have shown promising results with modulation of these mediators, yet the clinical models have been unsuccessful. One such study with matrix metalloproteases (MMPs) has yielded encouraging results. The relationship between MMPs and their tissue inhibitors (TIMPs) plays a significant role in the pathogenesis and recovery of SCI and the CNS. Key factors that lead to the functional consequences of MMP activity are cellular localization, tissue distribution, and temporal pattern of MMP expression. Studies concluding that MMPs can be seen as contributors of tissue damage and as contributors in the repair mechanisms have provided a need to reexamine their roles after acute and chronic neuropathic pain.

Cannabinoid subtype-2 receptors modulate the antihyperalgesic effect of WIN 55,212-2 in rats with neuropathic spinal cord injury pain.

BACKGROUND CONTEXT: There is increasing evidence for a role of the cannabinoid (CB) system in the development of neuropathic pain (NP) after spinal cord injury (SCI). The nonspecific CB1 and CB2 receptor agonists, WIN 55, 212-2 (WIN), have previously been shown to alleviate both mechanical and thermal hyperalgesia (TH) after peripheral nerve injury. PURPOSE: The present study was designed to identify the CB receptors involved in the antihyperalgesic effect of WIN by using selective antagonists for CB1 and CB2 receptors. STUDY DESIGN: This is an in vivo and behavioral study using a moderate T9 contusion SCI. After injury, TH of the hind paws was measured on postinjury days 21 through 42. METHODS: Sprague-Dawley rats underwent a contusion SCI using the Multicenter Animal Spinal Cord Injury Study (MASCIS) weight-drop impactor, which induced a moderate T9 SCI. Only animals showing consistent plantar stepping and consistent forelimb and hind limb coordination (Basso, Beattie, and Bresnahan score=15) were tested for TH. Animals exhibiting decreased withdrawal latency time, indicating TH, on or before Day 42, were selected for pharmacological intervention. Animals not exhibiting TH did not receive pharmacological intervention and were sacrificed. Rats underwent hind paw testing before any drug administration (after injury), 45 minutes after selective CB antagonist (AM 251 or AM 630) administration (postantagonist) and again 45 minutes after WIN administration (post-WIN). There were a total of seven treatment groups: saline vehicle control; Dimethyl sulfoxide (DMSO) vehicle control; low-dose WIN (0.2 mg/kg); and high-dose WIN (2.0 mg/kg); AM 251 (3 mg/kg) and AM 630 (1 mg/kg) were given subcutaneously in a total volume of 0.5 mL. Followed by intraperitoneal injection of WIN after each antagonist, sham-operated rats repeated pharmacological intervention used with treatment Groups 5 and 6. RESULTS: Thermal hyperalgesia was significantly ameliorated in a dose-dependent manner with systemically administered WIN. Cannabinoid receptor Type 1 antagonist AM 251 pretreatment did not affect the antihyperalgesic effect of WIN. By contrast, pretreatment with the CB2 receptor antagonist AM 630 significantly attenuated the effect of WIN. CONCLUSION: Taken together, these results suggest a role of the CB2 receptor in modulating SCI-induced TH. Selective activation of the CB2 receptor could potentially lead to analgesic effects on NP while avoiding psychotropic side effects in patients with SCI.

In Vivo application of RNAi to study pain.

Chronic pain is associated with several disease conditions. The inadequacy of current analgesics to treat chronic pain is the result of a lack of understanding of the mechanisms that mediate pain. RNA interference has emerged in recent years as a new way to evaluate the roles of molecules involved in the pain response. Selective knockout of proteins has proven to be a powerful technique for target validation, but has been limited as a potential therapeutic due to short-lived responses induced by RNAi. The short responses of RNAi illustrate the need for better delivery techniques, which is being addressed by current work to induce RNAi through the cell's natural mechanisms. In order to gain a better understanding of chronic pain, it will be necessary to evaluate the pain molecules that are expressed as part of an injury induced pain response, which can be modeled by contusion spinal cord injury. RNAi will prove to be an important technique in this work. The present minireview will summarize the work that has been done using RNAi in vivo to study pain and discuss future directions for the use of RNAi to study chronic pain.

Antihyperalgesic effects of vanilloid-1 and bradykinin-1 receptor antagonists following spinal cord injury in rats.

OBJECT: The authors previously discovered that genes for the bradykinin-1 (B1) receptor and the transient receptor potential vanilloid subtype 1 (TRPV1) were overexpressed in animals exhibiting thermal hyperalgesia (TH) following spinal cord injury (SCI). They now report the effect of TRPV1 (AMG9810) and B1 (Lys-[Des-Arg9,Leu8]-bradykinin) antagonists on TH in animals following SCI. METHODS: The rats were subjected to contusion SCI and then divided into groups in which TH did or did not develop. The animals from both groups were given either AMG9810, Lys-(Des-Arg9,Leu8)-bradykinin, or the drug-specific vehicle (control groups). Animals were tested for TH preinjury and at regular intervals after SCI by using the hindlimb withdrawal latency test. CONCLUSIONS: The administration of AMG9810 likely improves TH as a result of a generalized analgesic effect, whereas the effect of Lys-(Des-Arg9,Leu8)-bradykinin appears more specific to the reversal of TH. This information has potential usefulness in the development of treatment strategies for post-SCI neuropathic pain.

B1 and TRPV-1 receptor genes and their relationship to hyperalgesia following spinal cord injury.

STUDY DESIGN: Laboratory investigation of pain behavior following spinal cord injury. OBJECTIVE: To explore changes in the spinal cord expression of nociceptive genes following spinal cord injury (SCI) as they relate to the manifestation of pain behavior in rats. SUMMARY OF BACKGROUND DATA: Neuropathic pain following SCI is common, disabling, and largely untreatable. In peripheral nerve injury models, bradykinin B1 and vanilloid 1 (TRPV-1) receptor activity is associated with neuropathic pain behavior. We sought to examine the role of these gene products in SCI-mediated pain. METHODS: Rats were subjected to SCI using the MASCIS impactor. Animals were tested preinjury and at regular intervals postinjury for the appearance of thermal hyperalgesia using a hind limb withdrawal latency test. The expression of B1 and TRPV-1 genes was assessed using real-time polymerase chain reaction. Immunohistochemistry was used to localize the B1 and TRPV-1 receptors within the spinal cord. RESULTS: Greater than twofold increases in the expression of the B1 and TRPV-1 genes were detected in the injured region of the spinal cord in animals exhibiting hyperalgesia compared with animals with SCI that did not display hyperalgesia. Immunohistochemical staining revealed that both receptor types were largely localized to the dorsal horn. Staining for TRPV-1 receptors decreased while that for B1 receptors increased in all of the injured animals when compared with sham-operated controls. CONCLUSION: B1 and TRPV-1 receptor genes are overexpressed in the injured spinal cord of animals manifesting thermal hyperalgesia following SCI compared with similarly injured animals without hyperalgesia. This finding is consistent with past work regarding the role of these receptors in nociception and indicates that ongoing modifiable processes are occurring in the spinal cord that lead to clinical pain syndromes.

Changes in spinal cord injury-induced gene expression in rat are strain-dependent.

BACKGROUND AND CONTEXT: The functional recovery of animals subject to experimental spinal cord injury (SCI) is dependent on the injury model as well as the species and strain of animal used. Previous studies have shown differences in rates and degree of recovery between rats of different strains. PURPOSE: We sought to explore the hypothesis that differences in gene expression are associated with differences in functional recovery. STUDY DESIGN/SETTING: Laboratory study involving cohorts of three different strains of rat. METHODS: We used the Impactor device to produce identical spinal cord contusion injuries in groups of Long Evans, Sprague-Dawley, and Lewis rats (10 each). The functional recovery of animals was assessed using the Basso, Beattie, and Bresnahan rating scale. Six weeks after injury, rats were killed and the spinal cords were harvested for deoxyribonucleic acid microarray analysis. Changes in gene expression compared with intraspecies controls (3 each) were assessed at the region of injury and at a rostral segment of the spinal cord. Selected genes were also studied with real-time polymerase chain reaction. RESULTS: We found that different strains tended to exhibit different patterns of functional recovery. There were differences between the strains in terms of gene expression. CONCLUSIONS: These results emphasize the importance of testing novel therapies for SCI in a variety of animal species before introduction into human trials. Further research into the influence of several gene products on functional recovery is needed.