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1.  Gene expression profiling and endothelin in acute experimental pancreatitis 
AIM: To analyze gene expression profiles in an experimental pancreatitis and provide functional reversal of hypersensitivity with candidate gene endothelin-1 antagonists.
METHODS: Dibutyltin dichloride (DBTC) is a chemical used as a polyvinyl carbonate stabilizer/catalyzer, biocide in agriculture, antifouling agent in paint and fabric. DBTC induces an acute pancreatitis flare through generation of reactive oxygen species. Lewis-inbred rats received a single i.v. injection with either DBTC or vehicle. Spinal cord and dorsal root ganglia (DRG) were taken at the peak of inflammation and processed for transcriptional profiling with a cDNA microarray biased for rat brain-specific genes. In a second study, groups of animals with DBTC-induced pancreatitis were treated with endothelin (ET) receptor antagonists [ET-A (BQ123) and ET-B BQ788)]. Spontaneous pain related mechanical and thermal hypersensitivity were measured. Immunohistochemical analysis was performed using anti-ET-A and ET-B antibodies on sections from pancreatic tissues and DRG of the T10-12 spinal segments.
RESULTS: Animals developed acute pancreatic inflammation persisting 7-10 d as confirmed by pathological studies (edema in parenchyma, loss of pancreatic architecture and islets, infiltration of inflammatory cells, neutrophil and mononuclear cells, degeneration, vacuolization and necrosis of acinar cells) and the pain-related behaviors (cutaneous secondary mechanical and thermal hypersensitivity). Gene expression profile was different in the spinal cord from animals with pancreatitis compared to the vehicle control group. Over 260 up-regulated and 60 down-regulated unique genes could be classified into 8 functional gene families: circulatory/acute phase/immunomodulatory; extracellular matrix; structural; channel/receptor/transporter; signaling transduction; transcription/translation-related; antioxidants/chaperones/heat shock; pancreatic and other enzymes. ET-1 was among the 52 candidate genes up-regulated greater than 2-fold in animals with pancreatic inflammation and visceral pain-related behavior. Treatments with the ET-A (BQ123) and ET-B (BQ-788) antagonists revealed significant protection against inflammatory pain related mechanical and thermal hypersensitivity behaviors in animals with pancreatitis (P < 0.05). Open field spontaneous behavioral activity (at baseline, day 6 and 30 min after drug treatments (BQ123, BQ788) showed overall stable activity levels indicating that the drugs produced no undesirable effects on normal exploratory behaviors, except for a trend toward reduction of the active time and increase in resting time at the highest dose (300 μmol/L). Immunocytochemical localization revealed that expression of ET-A and ET-B receptors increased in DRG from animals with pancreatitis. Endothelin receptor localization was combined in dual staining with neuronal marker NeuN, and glia marker, glial fibrillary acidic protein. ET-A was expressed in the cell bodies and occasional nuclei of DRG neurons in naïve animals. However, phenotypic expression of ET-A receptor was greatly increased in neurons of all sizes in animals with pancreatitis. Similarly, ET-B receptor was localized in neurons and in the satellite glia, as well as in the Schwann cell glial myelin sheaths surrounding the axons passing through the DRG.
CONCLUSION: Endothelin-receptor antagonists protect against inflammatory pain responses without interfering with normal exploratory behaviors. Candidate genes can serve as future biomarkers for diagnosis and/or targeted gene therapy.
PMCID: PMC3436040  PMID: 22969188
Gene expression; Endothelin receptors; Pancreatitis; Pain; Dibutyltin dichloride; Hypersensitivity; Hyperalgesia
2.  Reversal of Inflammatory and Non-Inflammatory Visceral Pain by Central or Peripheral Actions of Sumatriptan 
Gastroenterology  2008;135(4):1369-1378.
Background and Aims
Sumatriptan is used specifically to relieve headache pain. The possible efficacy of sumatriptan was investigated in two models of visceral pain.
Pancreatic inflammation was induced by intravenous injection of dibutyltin dichloride. Non-inflammatory irritable bowel syndrome was induced by intracolonic instillation of sodium butyrate. The effects of systemic sumatriptan on referred hypersensitivity were tested in both models. Effects of sumatriptan within the rostral ventromedial medulla (RVM), a site of descending modulation of visceral pain, was determined by (a) testing the effects of RVM administration of 5HT1B/D antagonists on systemic sumatriptan action and (b) determining whether RVM application of sumatriptan reproduced the actions of systemic drug administration.
Systemic sumatriptan elicited a dose- and time-related blockade of referred hypersensitivity in both models that was blocked by systemic administration of either 5HT1B or 5HT1D antagonists. Sumatriptan administered into the RVM similarly produced dose- and time-related blockade of referred hypersensitivity in both visceral pain models. This was blocked by local microinjection of the 5HT1B antagonist, but not the 5HT1D antagonist. Microinjection of 5HT1B or 5HT1D antagonists into the RVM did not block the effects of systemic sumatriptan.
Our findings suggest that sumatriptan suppresses either inflammatory or non-inflammatory visceral pain, most likely through peripheral 5HT1B/1D receptors. Actions at 5HT1B receptors within the RVM offer an additional potential site of action for the modulation of visceral pain by triptans. These studies offer new insights into the development of strategies which may improve therapy of visceral pain conditions using already available medications.
PMCID: PMC4028637  PMID: 18694754
Pain  2006;129(0):35-45.
Opioids can induce hyperalgesia in humans and in animals. Mechanisms of opiate-induced hyperalgesia and possibly of spinal antinociceptive tolerance may be linked to pronociceptive adaptations occurring at multiple levels of the nervous system including activation of descending facilitatory influences from the brainstem, spinal neuroplasticity, and changes in primary afferent fibers. Here, the role of NK-1 receptor-expressing cells in the spinal dorsal horn in morphine-induced hyperalgesia and spinal antinociceptive tolerance was assessed by ablating these cells with intrathecal injection of SP-saporin (SP-SAP). Ablation of NK-1 receptor expressing cells prevented (a) morphine-induced thermal and mechanical hypersensitivity, (b) increased touch-evoked spinal FOS expression, (c) upregulation of spinal dynorphin content and (d) the rightward displacement of the spinal morphine antinociceptive dose-response curve (i.e., tolerance). Morphine-induced hyperalgesia and antinociceptive tolerance were also blocked by spinal administration of ondansetron, a serotonergic receptor antagonist. Thus, NK-1 receptor expressing neurons play a critical role in sustained morphine-induced neuroplastic changes which underlie spinal excitability reflected as thermal and tactile hypersensitivity to peripheral stimuli, and to reduced antinociceptive actions of spinal morphine (i.e., antinociceptive tolerance). Ablation of these cells likely eliminates the ascending limb of a spinal-bulbospinal loop that engages descending facilitation and elicits subsequent spinal neuroplasticity. The data may provide a basis for understanding mechanisms of prolonged pain which can occur in the absence of tissue injury.
PMCID: PMC4028682  PMID: 17123731
Opioid-induced hyperalgesia; descending facilitation; spinal plasticity; spinal tolerance; NK-1 receptors; projection cells
4.  Descending Facilitatory Pathways From the Rostroventromedial Medulla Mediate Naloxone-precipitated Withdrawal in Morphine-Dependent Rats 
Opioids produce analgesic effects and extended use can produce physical dependence in both humans and animals. Dependence to opiates can be demonstrated by either termination of drug administration or through precipitation of the withdrawal syndrome by opiate antagonists. Key features of the opiate withdrawal syndrome include hyperalgesia, anxiety and autonomic signs such as diarrhea. The rostral ventromedial medulla (RVM) plays an important role in the modulation of pain and for this reason, may influence withdrawal-induced hyperalgesia. The mechanisms that drive opiate withdrawal-induced hyperalgesia have not been elucidated. Here, rats made dependent upon morphine received naloxone to precipitate withdrawal. RVM microinjection of lidocaine, kynurenic acid (excitatory amino acid antagonist) or YM022 (CCK2 receptor antagonist) blocked withdrawal-induced hyperalgesia. Additionally, these treatments reduced both somatic and autonomic signs of naloxone-induced withdrawal. Spinal application of ondansetron, a 5HT3 receptor antagonist thought to ultimately be engaged by descending pain facilitatory drive, also blocked hyperalgesia and somatic and autonomic features of the withdrawal syndrome. These results indicate that the RVM plays a critical role in mediating components of opioid withdrawal that may contribute to opioid dependence.
Manipulations targeting these descending pathways from the RVM may diminish the consequences of prolonged opioid administration-induced dependence and be useful adjunct strategies in reducing the risk of opioid addiction.
PMCID: PMC4028695  PMID: 21354865
naloxone-induced withdrawal; hyperalgesia; RVM; descending facilitation; morphine dependence
NeuroImage  2008;44(1):23-34.
Abdominal pain is a major reason patients seek medical attention yet relatively little is known about neuronal pathways relaying visceral pain. We have previously characterized pathways transmitting information to the brain about visceral pain. Visceral pain arises from second order neurons in lamina X surrounding the spinal cord central canal. Some of the brain regions of interest receiving axonal terminations directly from lamina X were examined in the present study using enhanced functional magnetic resonance imaging (fMRI) before and one week after induction of a rat pancreatitis model with persistent inflammation and behavioral signs of increased nociception. Analysis of imaging data demonstrates an increase in MRI signal for all the regions of interest selected including the rostral ventromedial medulla, dorsal raphe, periaqueductal grey, medial thalamus, and central amygdala as predicted by the anatomical data, as well as increases in the lateral thalamus, cingulate/retrosplenial and parietal cortex. Occipital cortex was not activated above threshold in any condition and served as a negative control. Morphine attenuated the MRI signal, and the morphine effect was antagonized by naloxone in lower brainstem sites. These data confirm activation of these specific regions of interest known as integration sites for nociceptive information important in behavioral, affective, emotional and autonomic responses to ongoing noxious visceral activation.
PMCID: PMC2593090  PMID: 18722538
morphine; visceral pain; nociception; central pain; pancreatitis
6.  Intrathecal Gabapentin Enhances the Analgesic Effects of Subtherapeutic Dose Morphine in a Rat Experimental Pancreatitis Model 
Anesthesiology  2004;101(3):759-765.
Morphine sulfate has long been used for analgesia, but clinical applications can be limited by side effects, tolerance, and potential for addiction at therapeutic doses. An agent that produces therapeutic analgesia when coadministered with low-dose morphine could have important clinical uses. The anticonvulsant agent gabapentin has been identified as having antihyperalgesic properties acting on the α2δ1 subunit of N-type voltage-activated calcium channels on dorsal root ganglia neurons. In this study, intrathecal gabapentin, which by itself is ineffective when administered spinally, was combined with low-dose morphine and tested in an acute bradykinin-induced pancreatitis model in rats.
An intrathecal catheter was surgically inserted into the subarachnoid space of male Sprague-Dawley rats. A laparotomy was performed for ligation and cannulation of the bile-pancreatic duct. Rats were pretreated intrathecally with artificial cerebrospinal fluid, gabapentin, morphine, or combined gabapentin and morphine 30 min before bradykinin injection into the bile-pancreatic duct. Spontaneous behavioral activity (cage crossing, rearing, and hind limb extension) was monitored before drug injection (baseline) and after bradykinin injection into the bile-pancreatic duct to assess visceral pain.
Spinal pretreatment with up to 300 μg gabapentin alone was not effective in reducing hind limb extension in this model, but did restore some cage crossing and rearing behaviors. Spinal treatment with low-dose morphine reduced hind limb extension only. Spinal pretreatment with combined gabapentin and subtherapeutic doses of morphine sulfate resulted in restoration of all spontaneous behaviors to surgical baseline levels including elimination of hind limb extension.
Combined spinal administration of gabapentin and low doses of morphine significantly reduces pain-related behaviors in this acute rat pancreatitis model, whereas these agents were ineffective when used alone in this dose range. These data suggest that the α2δ1 subunit of the N-type voltage-activated Ca2+ channels is involved in transmission of this visceral pain, likely through effects on primary afferent endings in the spinal cord. Thus, gabapentin may be an effective adjuvant to initial low dose spinal opioid therapy for clinical management of visceral pain.
PMCID: PMC2770328  PMID: 15329602

Results 1-6 (6)