Adenosine A1R/A3R (Adenosine A1 and A3 Receptor) Agonist AST-004 Reduces Brain Infarction in a Nonhuman Primate Model of Stroke.

BACKGROUND AND PURPOSE: Treatment with A1R/A3R (adenosine A1 and A3 receptor) agonists in rodent models of acute ischemic stroke results in significantly reduced lesion volume, indicating activation of adenosine A1R or A3R is cerebroprotective. However, dosing and timing required for cerebroprotection has yet to be established, and whether adenosine A1R/A3R activation will lead to cerebroprotection in a gyrencephalic species has yet to be determined. METHODS: The current study used clinical study intervention timelines in a nonhuman primate model of transient, 4-hour middle cerebral artery occlusion to investigate a potential cerebroprotective effect of the dual adenosine A1R/A3R agonist AST-004. Bolus and then 22 hours intravenous infusion of AST-004 was initiated 2 hours after transient middle cerebral artery occlusion. Primary outcome measures included lesion volume, lesion growth kinetics, penumbra volume as well as initial pharmacokinetic-pharmacodynamic relationships measured up to 5 days after transient middle cerebral artery occlusion. Secondary outcome measures included physiological parameters and neurological function. RESULTS: Administration of AST-004 resulted in rapid and statistically significant decreases in lesion growth rate and total lesion volume. In addition, penumbra volume decline over time was significantly less under AST-004 treatment compared with vehicle treatment. These changes correlated with unbound AST-004 concentrations in the plasma and cerebrospinal fluid as well as estimated brain A1R and A3R occupancy. No relevant changes in physiological parameters were observed during AST-004 treatment. CONCLUSIONS: These findings suggest that administration of AST-004 and combined A1R/A3R agonism in the brain are efficacious pharmacological interventions in acute ischemic stroke and warrant further clinical evaluation.

Pharmacological modulation of brain activation to non-noxious stimulation in a cynomolgus macaque model of peripheral nerve injury.

In vivo neuroimaging could be utilized as a noninvasive tool for elaborating the CNS mechanism of chronic pain and for elaborating mechanisms of potential analgesic therapeutics. A model of unilateral peripheral neuropathy was developed in the cynomolgus macaque, a species that is phylogenetically close to humans. Nerve entrapment was induced by placing a 4 mm length of polyvinyl cuff around the left common sciatic nerve. Prior to nerve injury, stimulation of the foot with a range of non-noxious von Frey filaments (1, 4, 8, 15, and 26 g) did not evoke brain activation as observed with functional magnetic resonance imaging (fMRI). Two weeks after injury, stimulation of the ipsilateral foot with non-noxious filaments activated the contralateral insula/secondary somatosensory cortex (Ins/SII) and anterior cingulate cortex (ACC). By contrast, no activation was observed with stimulation of the contralateral foot. Robust bilateral activation of thalamus was observed three to five weeks after nerve injury. Treatment with the clinical analgesic pregabalin reduced evoked activation of Ins/SII, thalamus and ACC whereas treatment with the NK1 receptor antagonist aprepitant reduced activation of the ipsilateral (left) thalamus. Twelve to 13 weeks after nerve injury, treatment with pregabalin reduced evoked activation of all regions of interest (ROI). By contrast, brain activation persisted in most ROI, except the ACC, following aprepitant treatment. Activation of the contralateral Ins/SII and bilateral thalamus was observed six months after nerve injury and pregabalin treatment suppressed activation of these nuclei. The current findings demonstrated persistent changes in CNS neurons following nerve injury as suggested by activation with non-painful mechanical stimulation. Furthermore, it was possible to functionally distinguish between a clinically efficacious analgesic drug, pregabalin, from a drug that has not demonstrated significant clinical analgesic efficacy, aprepitant. In vivo neuroimaging in the current nonhuman model could enhance translatability.

Pain-related behavior and brain activation in cynomolgus macaques with naturally occurring endometriosis.

STUDY QUESTION: Can pain be objectively assessed in macaques with naturally occurring endometriosis? SUMMARY ANSWER: Behavioral, pharmacological and in vivo brain imaging findings indicate that pain can be quantified in macaques with endometriosis. WHAT IS KNOWN ALREADY: Endometriosis is characterized by abdominopelvic hypersensitity. The mechanism by which endometriosis evokes pain is largely unknown, as currently available analgesics offer limited pain relief. Thus, there is a need for both greater understanding of the in vivo mechanism of endometriosis-associated pain and better methods of testing novel therapeutics. STUDY DESIGN, SIZE, DURATION: Pain-related behavior and brain activation were assessed in five cynomolgus macaques with endometriosis. Three healthy female macaques served as controls. PARTICIPANTS/MATERIALS, SETTING, METHODS: Abdominopelvic sensitivity to force was assessed with an algometer. Activation of brain areas using block design force stimulation and the effects of a single dose of the analgesic drug morphine and 2-month treatment with the progestin dienogest on brain activation were observed via functional magnetic resonance imaging. MAIN RESULTS AND THE ROLE OF CHANCE: Pain response thresholds in macaques with endometriosis were significantly less than that of healthy macaques (P = 0.0003). In addition, non-noxious force activated the insula and thalamus, which was reduced with morphine and 2-month dienogest treatment. LIMITATIONS, REASONS FOR CAUTION: The specific role of cysts, such as peritoneal cysts, in endometriosis pain was not explored. While non-noxious stimulation activated the insula and thalamus, macaques were sedated during fMRI scans. Current findings need further confirmation in a larger cohort. WIDER IMPLICATIONS OF THE FINDINGS: The current study demonstrated central sensitization and related pain behavior in macaques with naturally occurring endometriosis. Altered functioning of the central nervous system could be the focus of future mechanistic studies and for the development of novel therapeutics. STUDY FUNDING/COMPETING INTEREST(S): Supported by a grant from the Shizuoka Industrial Foundation. All authors are employees of Hamamatsu Pharma Research, Inc.

Continuous infusion of substance P into rat striatum alleviates nociceptive behavior via phosphorylation of extracellular signal-regulated kinase 1/2.

Intraplantar injection of 0.4% formalin into the rat hind paw leads to a biphasic nociceptive response; an 'acute' phase (0-15 min) and 'tonic' phase (16-120 min), which is accompanied by significant phosphorylation of extracellular signal-regulated kinase (ERK)1/2 in the contralateral striatum at 120 min post-formalin injection. To uncover a possible relationship between the slow-onset substance P (SP) release and increased ERK1/2 phosphorylation in the striatum, continuous infusion of SP into the striatum by reverse microdialysis (0.4 µg/mL in microdialysis fiber, 1 µL/min) was performed to mimic volume neurotransmission of SP. Continuous infusion for 3 h of SP reduced the duration of 'tonic' phase nociception, and this SP effect was mediated by neurokinin 1 (NK1) receptors since pre-treatment with NK1 receptor antagonist CP96345 (10 µM) blocked the effect of SP infusion. However, formalin-induced 'tonic' phase nociception was significantly prolonged following acute injection of the MAP/ERK kinase 1/2 inhibitor PD0325901 (100 pmol) by microinjection. The coinfusion of SP and PD0325901 significantly increased the 'tonic' phase of nociception. These data demonstrate that volume transmission of striatal SP triggered by peripheral nociceptive stimulation does not lead to pain facilitation but a significant decrease of tonic nociception by the activation of the SP-NK1 receptor-ERK1/2 system. Noxious stimulation induces a slow-onset substance P (SP) release as a volume transmitter, activating extra-synaptic NK1 receptors, and evokes phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. The SP-NK1-ERK1/2 system in the striatum decreases tonic nociception.

Potential sex differences in activation of pain-related brain regions in nonhuman primates with a unilateral spinal nerve ligation.

The lack of truly robust analgesics for chronic pain is owed, in part, to the lack of an animal model that reflects the clinical pain state and of a mechanism-based, objective neurological indicator of pain. The present study examined stimulus-evoked brain activation with functional magnetic resonance imaging in male and female cynomolgus macaques following unilateral L7 spinal nerve ligation and the effects of clinical analgesics pregabalin, duloxetine, and morphine on brain activation in these macaques. A modified straight leg raise test was used to assess pain severity in awake animals and to evoke regional brain activation in anesthetized animals. The potential effects of clinical analgesics on both awake pain behavior and regional brain activation were examined. Following spinal nerve ligation, both male and female macaques showed significantly decreased ipsilateral straight leg raise thresholds, suggesting the presence of radicular-like pain. Morphine treatment increased straight leg raise thresholds in both males and females whereas duloxetine and pregabalin did not. In male macaques, the ipsilateral straight leg raise activated contralateral insular and somatosensory cortex (Ins/SII), and thalamus. In female macaques, the ipsilateral leg raise activated cingulate cortex and contralateral insular and somatosensory cortex. Straight leg raises of the contralateral, unligated leg did not evoke brain activation. Morphine reduced activation in all brain regions in both male and female macaques. In males, neither pregabalin nor duloxetine decreased brain activation compared with vehicle treatment. In females, however, pregabalin and duloxetine decreased the activation of cingulate cortex compared with vehicle treatment. The current findings suggest a differential activation of brain areas depending on sex following a peripheral nerve injury. Differential brain activation observed in this study could underlie qualitative sexual dimorphism in clinical chronic pain perception and responses to analgesics. Future pain management approaches for neuropathic pain will need to consider potential sex differences in pain mechanism and treatment efficacy.

Regional brain activation during rectal distention and attenuation with alosetron in a nonhuman primate model of irritable bowel syndrome.

Greater understanding of the mechanism that mediates visceral pain and hypersensitivity associated with irritable bowel syndrome (IBS) would facilitate the development of effective therapeutics to manage these symptoms. An objective marker associated with the underlying mechanisms of visceral pain and hypersensitivity could be used to guide therapeutic development. The current study examined brain activation evoked by rectal distention with functional magnetic resonance imaging (fMRI) in a cynomolgus macaque model of visceral hypersensitivity. Male, cynomolgus macaques underwent five four-week treatments of dextran sodium sulfate (DSS)-distilled water (DW), which induced mild-moderate colitis with remission during each treatment cycle. Balloon rectal distention (RD) was performed under anesthesia 14 weeks after the final DSS-DW treatment. Colonoscopy confirmed the absence of colitis prior to the start of RD. In naïve, untreated macaques, 10, 20 and 30 ml RD did not evoke brain activation. However, insular cortex/somatosensory II cortex and cerebellum were significantly activated in DSS-treated macaques at 20 and 30 ml rectal distention. Intra-rectal pressure after DSS treatment was not significantly different from that of naïve, untreated macaques, indicating lack of alteration of rectal functioning following DSS-treatment. Treatment with 5-HT3 receptor antagonist alosetron (p.o.) reduced distension-evoked brain activation and decreased intra-rectal pressure. The current findings demonstrated activation of brain regions to RD following DSS treatments which was not present in naïve macaques, suggesting visceral hypersensitivity. Brain activation in turn was reduced by alosetron, which could underlie the analgesic effect alosetron in IBS patients.

A longitudinal comparison in cynomolgus macaques of the effect of brimonidine on optic nerve neuropathy using diffusion tensor imaging magnetic resonance imaging and spectral domain optical coherence tomography.

Early detection of optic neuropathy is crucial for initiating treatment that could delay or prevent visual field loss. Preclinical studies have advanced a number of potential neuroprotective strategies to prevent retinal ganglion cell (RGC) degeneration, but none have successfully completed clinical trials. One issue related to the lack of preclinical to clinical translation is the lack of preclinical morphometric assessments that could be used to track neuroprotection, as well as neurodegeneration, over time within the same animal. Thus, to assess whether clinically used morphometric assessments can identify neuroprotection of RGC, the current study compared optic nerve fractional anisotropy (FA) obtained with diffusion tensor imaging (DTI) and retinal nerve fiber layer (RNFL) thickness measured with spectral domain optical coherence tomography (SD-OCT) to observe not only the early progression of RGC axonal degeneration but to also discern which imaging modality identifies signs of neuroprotection during treatment with the alpha-adrenoceptor agonist brimonidine. Elevated and sustained intraocular pressure (IOP) was observed following laser photocoagulation of the trabecular meshwork in one eye of nonhuman primates (NHP). Either brimonidine (0.1%) or control treatment was instilled twice daily for two months. In control-treated eyes, increased IOP, increased vertical cup-to-disc (C/D), reduced rim-to-disc (R/D) ratio, decreased RNFL thickness and decreased FA were observed. While IOP remained elevated during the course of the study, brimonidine tended to delay the progression of RNFL thinning. However, in the same animal, optic nerve FA did not appear to decline. Brimonidine treatment did not affect other measures of RGC axonal degeneration. The current findings demonstrate that early progression of optic neuropathy can be tracked over time in a nonhuman primate model of ocular hypertension using either DTI or SD-OCT. Furthermore, the delayed changes to RNFL thickness and FA appear to be a neuroprotective effect of brimonidine independent of its effect on IOP.

Exploratory clinical characterization of experimentally-induced ulcerative colitis nonhuman primates.

A limitation of currently used preclinical models of colitis is that disease and treatment assessment methods differ from clinically used methods. Thus, a modified Mayo score and an endoscopic index (EI) were developed for use in cynomolgus macaques with 0.25% dextran sulfate sodium (DSS)-induced ulcerative colitis. Macaques were treated with water with DSS for two weeks followed by water without DSS for two weeks. Disease activity was classified according to a modified Mayo score: stool consistency, rectal bleeding, colonoscopy examination and global assessment. Findings on colonoscopy were further graded according the Rachmilewitz EI. To demonstrate the sensitivity of the modified Mayo score and EI to therapeutic intervention, macaques were treated with the anti-inflammatory steroid prednisolone followed eight weeks later by the integrin antibody vedolizumab. Before DSS treatment, normal stool consistency and no rectal bleeding were observed. Colonoscopy demonstrated no mucosal abnormalities. Following the first DSS treatment, Mayo score and EI indicated signs of mild colitis. Following subsequent DSS treatments, mild to moderate colitis emerged with each DSS treatment and reduced signs of colitis were observed 2 weeks after DSS treatment termination. Prednisolone treatment during DSS treatment suppressed the emergence of colitis. Vedolizumab reduced signs of colitis during DSS treatment and further reduced signs of colitis that persisted after termination of DSS treatment. The current study demonstrated the potential of utilizing clinical outcome measures to assess experimentally-induced colitis in the macaque. Furthermore, signs of colitis, as assessed with the current methods, were reduced following therapeutic treatment. The current findings suggest that clinically relevant outcome measures in the macaque model of ulcerative colitis could be used to test novel treatments.

Pharmacologic Modulation of Noxious Stimulus-evoked Brain Activation in Cynomolgus Macaques Observed with Functional Neuroimaging.

Maintaining effective analgesia during invasive procedures performed under general anesthesia is important for minimizing postoperative complications and ensuring satisfactory patient wellbeing and recovery. While patients under deep sedation may demonstrate an apparent lack of response to noxious stimulation, areas of the brain related to pain perception may still be activated. Thus, these patients may still experience pain during invasive procedures. The current study used anesthetized or sedated cynomolgus macaques and functional magnetic resonance imaging (fMRI) to assess the activation of the parts of the brain involved in pain perception during the application of peripheral noxious stimuli. Noxious pressure applied to the foot resulted in the bilateral activation of secondary somatosensory cortex (SII) and insular cortex (Ins), which are both involved in pain perception, in macaques under either propofol or pentobarbital sedation. No activation of SII/Ins was observed in macaques treated with either isoflurane or a combination of medetomidine, midazolam, and butorphanol. No movement or other reflexes were observed in response to noxious pressure during stimulation under anesthesia or sedation. The current findings show that despite the lack of visible behavioral symptoms of pain during anesthesia or sedation, brain activation suggests the presence of pain depending on the anesthetic agent used. These data suggest that fMRI could be used to noninvasively assess pain and to confirm the analgesic efficacy of currently used anesthetics. By assessing analgesic efficacy, researchers may refine their experiments, and design protocols that improve analgesia under anesthesia.

Antinociceptive effects of the marine snail peptides conantokin-G and conotoxin MVIIA alone and in combination in rat models of pain.

There are a number of neurologically active ion channel blocking peptides derived from cone snail venom, such as conantokin-G and omega-conotoxin MVIIA. Conantokin-G inhibits NMDA receptors containing the NR2B subunit whereas omega-conotoxin MVIIA blocks N-type Ca(2+) channels. Separately, these peptides induce antinociceptive effects in pre-clinical pain models following intrathecal injection. In the current study, the efficacies of these peptides were determined separately and in combination by intrathecal injection into rats with a spinal nerve ligation, in rats with a spinal cord compression injury and in the formalin test. Separately, both conantokin-G and omega-conotoxin MVIIA dose-dependently attenuated nociceptive responses in all of these models. However, at high antinociceptive doses for both formalin and nerve injury models, omega-conotoxin MVIIA evoked untoward side effects. Using isobolographic analysis, the combination of sub-antinociceptive doses of peptides demonstrated additive antinociception in rats with a nerve ligation and in the formalin test, without apparent adverse side effects. In a model of neuropathic spinal cord injury pain, which is clinically difficult to treat, the combination of conantokin-G and omega-conotoxin MVIIA resulted in robust synergistic antinociception. These data suggest that a combination of these peptides may be analgesic across diverse clinical pains with limited untoward side effects, and particularly potent for reducing spinal cord injury pain.

Distinguishing analgesic drugs from non-analgesic drugs based on brain activation in macaques with oxaliplatin-induced neuropathic pain.

The antineoplastic agent oxaliplatin is a first-line treatment for colorectal cancer. However, neuropathic pain, characterized by hypersensitivity to cold, emerges soon after treatment. In severe instances, dose reduction or curtailing treatment may be necessary. While a number of potential treatments for oxaliplatin-induced neuropathic pain have been proposed based on preclinical findings, few have demonstrated efficacy in randomized, placebo-controlled clinical studies. This failure could be related, in part, to the use of rodents as the primary preclinical species, as there are a number of distinctions in pain-related mechanisms between rodents and humans. Also, an indicator of preclinical pharmacological efficacy less subjective than behavioral endpoints that is translatable to clinical usage is lacking. Three days after oxaliplatin treatment in Macaca fascicularis, a significantly reduced response latency to cold (10 °C) water was observed, indicating cold hypersensitivity. Cold-evoked bilateral activation of the secondary somatosensory (SII) and insular (Ins) cortex was observed with functional magnetic resonance imaging. Duloxetine alleviated cold hypersensitivity and significantly attenuated activation in both SII and Ins. By contrast, neither clinically used analgesics pregabalin nor tramadol affected cold hypersensitivity and cold-evoked activation of SII and Ins. The current findings suggest that suppressing SII and Ins activation leads to antinociception, and, therefore, could be used as a non-behavioral indicator of analgesic efficacy in patients with oxaliplatin-induced neuropathic pain.

Spinal subarachnoid adrenal medullary transplants reduce hind paw swelling and peripheral nerve transport following formalin injection in rats.

Previous studies have demonstrated that adrenal medullary chromaffin cells transplanted into the spinal subarachnoid space significantly reduced pain-related behavior following hind paw plantar formalin injection in rats. The data suggests a centrally mediated antinociceptive mechanism. The spinal transplants may have effects on sciatic nerve function as well. To address this, the current study examined the effects of spinal adrenal transplants on hind paw edema and the anterograde transport of substance P (SP) that occur following formalin injection. Robust formalin-evoked edema, as well as hind paw flinching, was observed in striated muscle control-transplanted rats, which were not observed in adrenal-transplanted rats. To visualize transport of SP, the sciatic nerve was ligated ipsilateral to formalin injection and the nerve was processed 48 h later for immunocytochemistry. A significant formalin-induced accumulation of SP immunoreactivity (IR) was observed proximal to the ligation in control-transplanted rats. In contrast, there was significantly less SP IR observed from nerve of adrenal-transplanted rats, suggesting a diminution of anterograde axoplasmic transport by adrenal transplants. The change in SP IR may have been due to an alteration of transport due to formalin injection, thus, transport was visualized by the accumulation of growth-associated protein 43 (GAP43) at the ligation site. Formalin injection did not significantly increase proximal accumulation of GAP43 IR, indicating that formalin does not increase anterograde transport. Surprisingly, however, adrenal transplants significantly diminished GAP43 IR accumulation compared to control-transplanted rats. These data demonstrate that spinal adrenal transplants can attenuate the formalin-evoked response by modulating primary afferent responses.

Prolonged nociceptive responses to hind paw formalin injection in rats with a spinal cord injury.

Unilateral lesioning of the spinal dorsal horn with the excitotoxin quisqualic acid (QUIS) leads to robust degeneration of dorsal horn grey matter, and robust pain-related symptoms, such as cutaneous hypersensitivity, persist long after injury. A possible mechanism that underlies the pain-related symptoms is the disruption of dorsal horn inhibitory neuron function, leading to decreased inhibition of nociceptive neurons. Five percent formalin was injected into the hind paw of rats with either a QUIS lesion or sham lesion. Both QUIS- and sham-lesioned rats displayed bi-phasic hind paw flinches following formalin injection, but a prolonged response was observed in QUIS-lesioned rats. The expression of the immediate-early gene product Fos in the dorsal horn ipsilateral to formalin injection was similar between QUIS- and sham-lesioned rats. In QUIS-lesioned rats, however, there was a marked absence of dorsal horn neurons, particularly GABAergic neurons, compared to sham-lesioned rats. The prolonged nociceptive response observed with a unilateral QUIS lesion may be due to generalized changes in dorsal horn neuron function including a loss of inhibitory neuron function.

Gaps in Understanding Mechanism and Lack of Treatments: Potential Use of a Nonhuman Primate Model of Oxaliplatin-Induced Neuropathic Pain.

The antineoplastic agent oxaliplatin induces an acute hypersensitivity evoked by cold that has been suggested to be due to sensitized central and peripheral neurons. Rodent-based preclinical studies have suggested numerous treatments for the alleviation of oxaliplatin-induced neuropathic pain, but few have demonstrated robust clinical efficacy. One issue is that current understanding of the pathophysiology of oxaliplatin-induced neuropathic pain is primarily based on rodent models, which might not entirely recapitulate the clinical pathophysiology. In addition, there is currently no objective physiological marker for pain that could be utilized to objectively indicate treatment efficacy. Nonhuman primates are phylogenetically and neuroanatomically similar to humans; thus, disease mechanism in nonhuman primates could reflect that of clinical oxaliplatin-induced neuropathy. Cold-activated pain-related brain areas in oxaliplatin-treated macaques were attenuated with duloxetine, the only drug that has demonstrated clinical efficacy for chemotherapy-induced neuropathic pain. By contrast, drugs that have not demonstrated clinical efficacy in oxaliplatin-induced neuropathic pain did not reduce brain activation. Thus, a nonhuman primate model could greatly enhance understanding of clinical pathophysiology beyond what has been obtained with rodent models and, furthermore, brain activation could serve as an objective marker of pain and therapeutic efficacy.

Pain-Related Behavior and Brain Activation in a Cynomolgus Macaque Model of Postoperative Pain.

BACKGROUND: Inadequate postoperative pain management could lead to persistent pain and this is, in part, due to incomplete understanding of the mechanism of postoperative pain. Currently available rodent models may have limited translatability to clinical postoperative pain. Thus, a preclinical model of postoperative pain was developed in the cynomolgus macaque, a species that is phylogenetically closer to humans than rodents. METHOD: The presence of pressure hypersensitivity was assessed with non-noxious pressure applied proximally and distally (approximately 10 cm) to an abdominal incision in macaques. The effect of the opioid morphine (intramuscular, i.m.), the nonsteroidal anti-inflammatory drug diclofenac (i.m.) and the anticonvulsant pregabalin (i.m.) on pressure hypersensitivity was evaluated one and two days following surgery. Brain activation during non-noxious pressure stimulation was observed with functional magnetic resonance imaging. RESULTS: Hypersensitivity to non-noxious pressure applied proximally and distally (approximately 10 cm) to the incision was observed, lasting for up to seven days and three days, respectively, following surgery. Postoperative pressure hypersensitivity was attenuated with morphine but not with either diclofenac or pregabalin. Bilateral activation of the insular cortex and cingulate cortex was observed during non-noxious pressure stimulation proximal to the incision, which was attenuated with morphine. By contrast, pregabalin reduced only cingulate cortex activation. CONCLUSION: The lack of antinociceptive efficacy of pregabalin on postoperative pain could be due to the incomplete suppression of pressure-evoked brain activation. It is speculated that incomplete postoperative pain relief observed in general could be due to residual or persistent activity of key pain nuclei such as the insular cortex. The current macaque model could be used for further elaborating the mechanism of postoperative pain as well as confirming the efficacy of potential treatments for the management of postoperative pain.

FK506 reduces the severity of cutaneous hypersensitivity in rats with a spinal cord contusion.

Spinal cord injury (SCI) leads to persistent pain as well as motor dysfunction, both of which lack effective therapeutics. The immunosuppressant FK506 (tacrolimus) has been shown to improve behavioral outcome following SCI in rats. Just prior to a mid-thoracic spinal cord contusion injury, rats were injected with either vehicle or FK506 and treatment was continued through the duration of the experiment. Vehicle-treated rats developed significant and long-lasting hind paw hypersensitivity to innocuous mechanical stimulation, noxious heat and cooling stimuli. In contrast, FK506 treatment reduced the duration of both mechanical and cold hypersensitivity. Neither treated groups demonstrated an improvement in locomotor function. Thus, some SCI-induced pain is mediated by an FK506-sensitive mechanism. The data also suggest that motor and sensory dysfunctions resulting from SCI are mediated by distinct mechanisms, requiring the use of multiple therapeutic interventions.

Expansion of formalin-evoked Fos-immunoreactivity in rats with a spinal cord injury.

Peripheral tissue injury as well as spinal cord injury (SCI) may lead to sensitization of dorsal horn neurons and alterations in nociceptive processing. Thus, peripheral injuries experienced by SCI patients, even if not initially perceived, could result in a persistent and widespread activation of dorsal horn neurons and emerge as chronic pain with interventive repair or modest recovery from SCI. To visualize the spinal neuron response to peripheral tissue injury following complete SCI in rats, the neural transcription factor Fos was quantitated in the spinal cord. Two weeks following either a complete transection of the spinal cord at the level of T8 or a sham surgery (laminectomy), rats were injected with formalin into the left hind paw. Sham-operated rats demonstrated biphasic hind paw pain-related behavior following formalin injection, but transected rats displayed fewer behaviors in the second (tonic) phase. Stereological analysis of the sham group revealed that the extent of formalin-induced Fos expression was within the lumbar dorsal horn, with numerous Fos-like immunoreactive profiles in the ipsilateral dorsal horn and some contralateral immunoreactive profiles. In contrast, the level of Fos-like immunoreactivity in the transected group was significantly elevated and expanded in range compared to the sham group, with increases observed in the normal laminar distribution regions, as well as multi-segmentally through sacral levels and increases in the contralateral dorsal horn segments. The data demonstrate that widespread activation of spinal, especially dorsal horn, neurons following peripheral insult can occur in the injured spinal cord, despite reduced pain responsiveness, and suggests that exaggerated pain may emerge as spinal recovery or repair progresses.

Behavioral characterization and effect of clinical drugs in a rat model of pain following spinal cord compression.

Chronic pain symptoms, including spontaneous unevoked pain and evoked cutaneous hypersensitivity, appear following spinal cord injury (SCI). A reliable preclinical model is needed to develop effective analgesic treatments for these symptoms. A previously described rat model of SCI pain was modified and behaviorally characterized and used to test clinically available drugs. A segment of the mid-thoracic spinal cord was compressed for 60 s with a micro-vascular clip. The sensitivity of the hind paws to noxious heat (Hargreaves test), innocuous tactile (von Frey filaments), and cooling (acetone) stimuli were determined once per week beginning 1 week following spinal compression. Spinal cord compression led to long lasting hypersensitivity to stimuli, lasting for at least 12 weeks post-surgery. Systemic baclofen, gabapentin, tramadol, and morphine dose-dependently attenuated tactile hypersensitivity. No effect on tactile hypersensitivity was observed with amitriptyline, carbamazepine, rofecoxib, and diazepam. Baclofen and morphine also dose-dependently ameliorated heat hypersensitivity. In contrast, no effect on heat hypersensitivity was observed with amitriptyline, carbamazepine, diazepam and gabapentin. The current data suggest that the model can potentially differentiate those drugs with analgesic efficacy from those that do not have efficacy in SCI pain. Thus, the model may be useful for rapid screening and clinical translation of promising SCI pain therapies.

Altered antinociceptive efficacy of tramadol over time in rats with painful peripheral neuropathy.

Pain due to peripheral nerve injury or disease is a dynamic process, such that the mechanism that underlies it alters over time. Tramadol has been reported to be analgesic in clinical neuropathic pain, with varying levels of efficacy due to a patient population that has had neuropathic pain for a wide range of time. In order to address and examine the issue, the antinociceptive efficacy of tramadol over time was tested in rats with a chronic constriction injury (CCI) of the left sciatic nerve. Rats developed a robust hind paw hypersensitivity to innocuous mechanical stimulation ipsilateral to CCI surgery. Subcutaneous injection of tramadol in rats two weeks after CCI surgery dose-dependently attenuated mechanical hypersensitivity, which was abolished with the mu-opioid receptor antagonist naloxone but not the alpha(2)-adrenoceptor antagonist yohimbine. Systemic tramadol also attenuated mechanical hypersensitivity four weeks after CCI surgery, but the efficacy significantly diminished at this time point. In addition, the effect of tramadol at this later time point could be reduced with yohimbine as well as naloxone. These data demonstrate that the efficacy of tramadol depends in part on the duration of nerve injury-evoked nociception, and that its antinociceptive mechanism changes over time. Alteration in antinociceptive mechanism over time may explain the inconsistency in efficacy of this and other analgesic drugs in chronic pain patients.

Enhancement of morphine antinociception with the peptide N-methyl-D-aspartate receptor antagonist [Ser1]-histogranin in the rat formalin test.

Opiates may be used to attenuate chronic pain, but long-term use is complicated by the possible increase in pain over time, escalating dose requirements, and untoward side effects. Adjuncts such as ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, may be added to opiates to provide more consistent analgesia. However, the unwanted motor side effects of NMDA receptor antagonists prevent their widespread clinical usage. In the current study, an analogue of the naturally-derived peptide histogranin, [Ser(1)]histogranin (SHG), an NMDA receptor modulator without adverse side effects like those in current clinical use, was evaluated for its potential to enhance the antinociceptive effect of intrathecal morphine in the rat formalin test. Intrathecal injection of a combination of SHG and morphine resulted in significantly reduced hind paw flinching compared with morphine alone in the first and second phases. The effective dose of SHG used in the combination had no efficacy when tested alone. These results were similar to the increased efficacy that was obtained with a combination of ketamine and morphine. Thus, enhancement of opiate efficacy is possible using a novel peptide NMDA receptor modulator with a potentially improved safety profile.