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.

Evaluation of brain activation related to resting pain using functional magnetic resonance imaging in cynomolgus macaques undergoing knee surgery.

Purpose: Functional magnetic resonance imaging (fMRI) visualizes hemodynamic responses associated with brain and spinal cord activation. Various types of pain have been objectively assessed using fMRI as considerable brain activations. This study aimed to develop a pain model in cynomolgus macaques undergoing knee surgery and confirm brain activation due to resting pain after knee surgery. Methods: An osteochondral graft surgery on the femoral condyle in the unilateral knee was performed on four cynomolgus macaques (Macaca fascicularis). Resting pain was evaluated as changes in brain fMRI findings with a 3.0-T MRI scanner preoperatively, postoperatively, and after postoperative administration of morphine. In the fMRI analysis, Z-values >1.96 were considered statistically significant. Results: Brain activation without stimulation after surgery in the cingulate cortex (3.09) and insular cortex (3.06) on the opposite side of the surgery was significantly greater than that before surgery (1.05 and 1.03, respectively) according to fMRI. After the administration of morphine, activation due to resting pain decreased in the cingulate cortex (1.38) and insular cortex (1.21). Conclusion: Osteochondral graft surgery on the femoral condyle can lead to postoperative resting pain. fMRI can reveal activation in pain-related brain areas and evaluate resting pain due to knee surgery.

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.

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.

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.

Brain Activation in a Cynomolgus Macaque Model of Chymopapain-Induced Discogenic Low Back Pain: A Preliminary Study.

INTRODUCTION: There is currently a lack of translatable, preclinical models of low back pain (LBP). Chymopapain, a proteolytic enzyme used to treat lumbar intervertebral disc (IVD) herniation, could induce discogenic LBP. The current study developed a behavioral model of discogenic LBP in nonhuman primates. Significant brain activation is observed in clinical LBP. Thus, the current study also sought to define brain activation over time in a macaque with discogenic LBP. METHODS: Responses to pressure applied to the back at L4/L5 were measured in eight adult male Macaca fasciculata using a pressure algometer. The nucleus pulpous of the IVD between L4 and L5 was aspirated and chymopapain (1 mg/mL) was injected under fluoroscopic guidance (n = 2). In two macaques, the nucleus pulpous was only aspirated. Brain activation in response to pressure applied to the lower back was assessed using a 3.0T magnetic resonance imaging scanner in four macaques before and 1, 3, 9, and 14 days after treatment. RESULTS: The mean (±SD) response pressure before treatment was 1.4 ± 0.1 kg. One day after chymopapain treatment, the response pressure decreased to 0.6 ± 0.05 kg (P < 0.01), suggestive of pressure hypersensitivity. Over time, the pressure thresholds following chymopapain treatment gradually returned to normal. Following aspiration only, the response pressure was 1.4 ± 0.05 kg, which was not significantly different from the uninjured controls. There was activation of the secondary somatosensory cortex and insular cortex one and three days after chymopapain treatment; there was no activation following aspiration only. CONCLUSIONS: Enzymatic treatment of the nucleus pulpous leads to acute LBP and pressure-evoked activation in pain-related brain areas. The current model of discogenic LBP parallels clinical LBP and could be used to further elaborate the mechanism of acute LBP.

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.

Antinociceptive effect of clinical analgesics in a nonhuman primate model of knee osteoarthritis.

A number of potential analgesic pharmacotherapies developed in preclinical osteoarthritis animal models have failed clinical trials. A possible basis for the lack of translation of preclinical findings to clinical efficacy is the use of a preclinical species that is distinct from that of humans. The current study tested clinical analgesics in a nonhuman primate model of knee osteoarthritis. Following a medial meniscectomy, the animals developed a robust ipsilateral reduction in knee pressure threshold (hyperalgesia) and an ipsilateral reduction in weight bearing (resting pain). The serotonin-noradrenalin reuptake inhibitor duloxetine and opioid morphine increased ipsilateral pressure threshold and weight bearing. By contrast, the anticonvulsant pregabalin did not affect either pressure hyperalgesia or resting pain. The current findings in the nonhuman primate model of osteoarthritis parallel clinical findings, in that duloxetine and opioids are used in the management of osteoarthritis pain whereas pregabalin is not. The current findings also suggest the possible differentiation of pharmacotherapeutics in a nonhuman primate model, of distinguishing potential clinically useful analgesics for the management of osteoarthritic pain from those that are not.

Pharmacological comparison of a nonhuman primate and a rat model of oxaliplatin-induced neuropathic cold hypersensitivity.

Oxaliplatin is a first-line treatment for colorectal cancer. However, shortly following treatment, cold-evoked hypersensitivity appears in the extremities and over time, the pain is such that oxaliplatin dosing may need to be markedly reduced or even terminated. There is currently a lack of efficacious treatments for oxaliplatin-induced peripheral neuropathy, which is due in part to the difficulty in translating findings obtained from preclinical rodent models of chemotherapy-induced peripheral neuropathy. Nonhuman primates (NHP) are phylogenetically closer to humans than rodents and may show drug responses that parallel those of humans. A significant decrease in tail withdrawal latency to 10°C water ("cold hypersensitivity") was observed beginning 3 days after intravenous infusion of oxaliplatin (5 mg/kg) in Macaca fascicularis. A single treatment of duloxetine (30 mg/kg, p.o.) ameliorated oxaliplatin-induced cold hypersensitivity, whereas pregabalin (30 mg/kg, p.o.) and tramadol (30 mg/kg, p.o.) did not. By contrast, in rats, no significant cold hypersensitivity, or increased responsiveness to acetone applied to the hind paws, was observed 3 days after the first injection of oxaliplatin (5 mg/kg, i.p., once per day, two injections). Therefore, rats were tested after six treatments of oxaliplatin, 17 days after the first treatment. All analgesics (30 mg/kg, p.o.) significantly ameliorated cold hypersensitivity in rats. The activity of analgesics in the oxaliplatin-treated macaques parallel clinical findings. The current results indicate that the NHP could serve as a bridge species to improve translatability of preclinical findings into clinically useful treatments for oxaliplatin-induced peripheral neuropathy.

Characterization of a novel thrombotic middle cerebral artery occlusion model in monkeys that exhibits progressive hypoperfusion and robust cortical infarction.

In an attempt to establish a thrombotic middle cerebral artery (MCA) occlusion model using cynomolgus monkeys, we measured the blood flow in the main MCA tract and cerebral cortex, brain damage, and neurological deficits, and compared them with those of mechanical MCA occlusion model. Thrombotic occlusion was induced photochemically by green light application on the MCA following rose bengal treatment; mechanical occlusion was induced by MCA clipping for 3h. Patency of the main MCA tract showed two patterns in the thrombotic model: permanent occlusion or cyclical flow reduction (CFR). Regional cerebral blood flow (rCBF) decreased during occlusion followed by post-ischemic hyperperfusion in the clipping model, whereas rCBF reduction expanded time-dependently in the thrombotic occlusion model. Brain infarction and neurological scores in the thrombotic occlusion model were significantly larger than those in the clipping occlusion model. In histological assessment, microthrombi containing myeloperoxidase- and fibrinogen-positive cells were observed in the cortex following the thrombotic but not clipping occlusion. These results collectively suggest that this thrombotic MCA occlusion model, because it shows impairment of cerebral microcirculation, could provide a vital platform for understanding progressive ischemia as well as for evaluating potential therapeutic drugs.

Synthesis and evaluation of [11C]FR194921 as a nonxanthine-type PET tracer for adenosine A1 receptors in the brain.

This report describes the synthesis of [11C]2-(1-methyl-4-piperidinyl)-6-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-3(2H)-pyridazinone ([11C]FR194921), a highly selective, nonxanthine-type adenosine A(1) receptor antagonist, used in brain imaging in rats and conscious monkeys as a potential novel PET tracer. [11C]FR194921 was successfully synthesized in 19 min after [11C]CH3I formation. The radiochemical yield was 38+/-3%; and radioactivity was 4.1+/-0.4 GBq, calculated from end of synthesis; radiochemical purity was higher than 99%; and the specific radioactivity was 25.0+/-8.1 GBq micromol(-1) (n=5). In a rat experiment, the distribution of [11C]FR194921 was higher in the hippocampus, striatum and cerebellum regions. This accumulation was significantly decreased by approximately 50% by pretreatment with 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), an adenosine A1 receptor antagonist, which indicated specific binding of the radioligand to adenosine A1 receptors. In conscious monkey PET experiments, [11C]FR194921 accumulated in several regions of the brain, especially in the occipital cortex, thalamus and striatum. These results suggest that [11C]FR194921 can be used as an agent for imaging adenosine A1 receptors in vivo by positron emission tomography (PET).