Thermal Nociceptive Thresholds Research Articles

Effect of fentanyl on thermal and mechanical nociceptive thresholds in horses and estimation of anti-nociceptive plasma concentration

There are few investigations relating anti-nociception to plasma concentrations of fentanyl in horses. The study objective was to evaluate analgesic efficacy and duration in horses and determine the minimum anti-nociceptive plasma concentrations. Eight horses were treated with saline (P) and fentanyl (F2.5=2.5μg/kg; F5=5μg/kg; F10=10μg/kg) given IV over 5min, with a wash-out period of 10 days. To evaluate thermal (°C) and mechanical (N) nociceptive threshold single stimulations were applied prior to (baseline) and 10, 30, 60, 90, 120, 180, 240, 300, 360, 420, 540min and 22.5h after treatment. Plasma fentanyl concentrations were measured at specific time points. Locomotor activity, heart rate, respiratory rate and gastrointestinal sounds were recorded. Two-way repeated measures ANOVA and pairwise comparisons were used for data analysis (P<0.05).With treatment F10, there was a significant increase in thermal threshold above baseline (47.2ö4.1°C) at t10 (53.7ö4.2°C) and t30 (52.1ö5.6°C), whereas mechanical threshold increased considerably above baseline (3.7ö1.3N) only at t10 (6.6ö3.6N). Estimated mean minimum anti-nociceptive plasma concentration determined by thermal stimulation was 6.1–6.8ng/mL. Dose-dependent increased locomotion occurred, but no significant changes in heart rate, respiratory rate and gastrointestinal sounds were observed. Fentanyl IV at 10μg/kg produced anti-nociception for 10–30min and fentanyl plasma concentrations of ≥6.1–6.8ng/mL appear necessary to induce thermal anti-nociception. Dose-dependent increased locomotion was the main side effect observed.

MicroRNA-1224 Splicing CircularRNA-Filip1l in an Ago2-Dependent Manner Regulates Chronic Inflammatory Pain via Targeting Ubr5.

Dysfunctions of gene transcription and translation in the nociceptive pathways play the critical role in development and maintenance of chronic pain. Circular RNAs (circRNAs) are emerging as new players in regulation of gene expression, but whether and how circRNAs are involved in chronic pain remain elusive. We showed here that complete Freund's adjuvant-induced chronic inflammation pain significantly increased circRNA-Filip1l (filamin A interacting protein 1-like) expression in spinal neurons of mice. Blockage of this increase attenuated complete Freund's adjuvant-induced nociceptive behaviors, and overexpression of spinal circRNA-Filip1l in naive mice mimicked the nociceptive behaviors as evidenced by decreased thermal and mechanical nociceptive threshold. Furthermore, we found that mature circRNA-Filip1l expression was negatively regulated by miRNA-1224 via binding and splicing of precursor of circRNA-Filip1l (pre-circRNA-Filip1l) in the Argonaute-2 (Ago2)-dependent manner. Increase of spinal circRNA-Filip1l expression resulted from the decrease of miRNA-1224 expression under chronic inflammation pain state. miRNA-1224 knockdown or Ago2 overexpression induced nociceptive behaviors in naive mice, which was prevented by the knockdown of spinal circRNA-Filip1l. Finally, we demonstrated that a ubiquitin protein ligase E3 component n-recognin 5 (Ubr5), validated as a target of circRNA-Filip1l, plays a pivotal role in regulation of nociception by spinal circRNA-Filip1l. These data suggest that miRNA-1224-mediated and Ago2-dependent modulation of spinal circRNA-Filip1l expression regulates nociception via targeting Ubr5, revealing a novel epigenetic mechanism of interaction between miRNA and circRNA in chronic inflammation pain.SIGNIFICANCE STATEMENT circRNAs are emerging as new players in regulation of gene expression. Here, we found that the increase of circRNA-Filip1l mediated by miRNA-1224 in an Ago2-dependent way in the spinal cord is involved in regulation of nociception via targeting Ubr5 Our study reveals a novel epigenetic mechanism of interaction between miRNA and circRNA in chronic inflammation pain.

Evaluation of ameliorative effect of sodium nitrate in experimental model of streptozotocin-induced diabetic neuropathy in male rats.

Diabetes induces sensory symptoms of neuropathy as positive (hyperalgesia), negative (hypoalgesia), or both. In the present study, fifty male Wistar rats were allocated to five groups: control, control+nitrate, diabetes, diabetes+insulin, and diabetes+nitrate. Thirty days after diabetes confirmation, insulin (2-4 U/day) was injected subcutaneously in diabetes+insulin group and nitrate (100 mg/l) was added into drinking water of the control+nitrate and diabetes+nitrate groups for a period of 2 months. In order to assess the mechanical and thermal algesia, tail immersion, hot plate, and von Frey tests were performed. The serum insulin levels were determined with insulin ELISA Kit. Serum level of NOx was determined by the Griess method. Both thermal and mechanical nociceptive thresholds showed a significant decrease (p<0.05) which was followed by a significant increase (p<0.01) in the thermal nociceptive threshold in the diabetes group. Chronic nitrate or insulin treatment led to a significant decrease (p<0.01) in blood glucose levels, as well as a significant (p<0.05) increase in the body weight and serum NOx. Moreover, nitrate treatment significantly increased serum insulin levels (p<0.001) compared to the other groups. Chronic nitrate treatment modified the thermal and mechanical sensitivities in diabetic animals.

The pharmacokinetics and pharmacodynamics of intravenous hydromorphone in horses

ObjectiveDescribe the pharmacokinetics and pharmacodynamics of intravenous hydromorphone in healthy horses. Study designMasked, randomized, cross-over, Latin square design. AnimalsA group of eight healthy adult horses MethodsHorses were administered each of four treatments with an 8 day washout. Treatments groups included intravenous hydromorphone 0.02 mg kg–1 (LD), 0.04 mg kg–1 (MD), 0.08 mg kg–1 (HD) and saline (P). Blood samples for hydromorphone analysis were obtained for 24 hours after treatment. Plasma hydromorphone was quantified and pharmacokinetic parameters were determined using non-compartmental analysis. Pharmacodynamic data collected for 24 hours after treatment included thermal nociceptive threshold, heart rate (HR), respiratory rate (fR) and rectal temperature, and analyzed using mixed-effects linear models. ResultsMean (± standard deviation) hydromorphone terminal half-life (t1/2), systemic clearance and apparent volume of distribution at steady state (Vdss) were 18.1 ± 18.6, 34.0 ± 12.8, and 41.3 ± 32.5 minutes, 66.6 ± 5.3, 550.0 ± 76.4, and 92.7 ± 13.9 mL kg–1 minute–1, and 1118 ± 369, 1460 ± 325 and 2242 ± 950 mL kg–1 for treatments LD, MD and HD, respectively. Thermal threshold increased significantly compared to baseline for all treatments for up to 12 hours. HR was elevated above baseline in treatments LD, MD and HD, extending to 30, 15 and 105 minutes after treatment, respectively. Respiratory rate was elevated above baseline in treatments MD and HD from 30 to 195 minutes and from 45 to 480 minutes after treatment, respectively. Temperature was elevated above baseline in treatment HD until 255 minutes after treatment. ConclusionsHydromorphone exhibited a short t1/2, rapid clearance and large Vdss in horses. It also provided a dose-dependent increase in thermal threshold with associated increases in HR, fR and rectal temperature. Clinical relevanceHydromorphone 0.04 mg kg–1 provided clinically relevant thermal antinociception with minimal adverse effects.

Pain-like behaviors and local mechanisms involved in the nociception experimentally induced by Latrodectus curacaviensis spider venom

The present study was undertaken to characterize the behavioral manifestations of nociception and the local mechanisms involved with the nociceptive response elicited by Latrodectus curacaviensis venom (LCV) in mice. After the intraplantar LCV inoculation, spontaneous nociception, mechanical and thermal nociceptive thresholds, motor performance, edema and cytokine levels were evaluated using von Frey filaments, hot/cold plate, rota-rod, plethismometer and ELISA, respectively. Analysis of LCV was performed by SDS-PAGE and chromatography. Intraplantar injection of LCV (1–100 ng/paw) induced intense and heat-sensitive spontaneous nociception, mediated by serotonin and bradykinin receptors, TRPV1 channels, as well as by transient local inflammation. LCV (0.1–10 ng/paw) induced mechanical allodynia, which was reduced by the local pretreatment with H1 receptor or TRPV1 antagonists. Corroborating the TRPV1 involvement, in thermal nociception assays, LCV induced a similar response to that of capsaicin, a TRPV1 agonist, facilitating the response to noxious hot stimuli and inhibiting the response to cold noxious stimulation. LCV promoted mast cell degranulation, increased IL-1β paw levels, but did not produce a relevant edematogenic effect. Analysis of LCV components showed a predominance of high molecular weight proteins. This work provides the first mechanistic hypothesis to explain the local pain induced by LCV, the most frequent clinical symptom of human envenomation.

Bone marrow-derived mesenchymal stem/stromal cells reverse the sensorial diabetic neuropathy via modulation of spinal neuroinflammatory cascades

BackgroundDiabetic neuropathy (DN) is a frequent and debilitating manifestation of diabetes mellitus, to which there are no effective therapeutic approaches. Mesenchymal stem/stromal cells (MSC) have a great potential for the treatment of this syndrome, possibly through regenerative actions on peripheral nerves. Here, we evaluated the therapeutic effects of MSC on spinal neuroinflammation, as well as on ultrastructural aspects of the peripheral nerve in DN-associated sensorial dysfunction.MethodsC57Bl/6 mice were treated with bone marrow-derived MSC (1 × 106), conditioned medium from MSC cultures (CM-MSC) or vehicle by endovenous route following the onset of streptozotocin (STZ)-induced diabetes. Paw mechanical and thermal nociceptive thresholds were evaluated by using von Frey filaments and Hargreaves test, respectively. Morphological and morphometric analysis of the sciatic nerve was performed by light microscopy and transmission electron microscopy. Mediators and markers of neuroinflammation in the spinal cord were measured by radioimmunoassay, real-time PCR, and immunofluorescence analyses.ResultsDiabetic mice presented behavioral signs of sensory neuropathy, mechanical allodynia, and heat hypoalgesia, which were completely reversed by a single administration of MSC or CM-MSC. The ultrastructural analysis of the sciatic nerve showed that diabetic mice exhibited morphological and morphometric alterations, considered hallmarks of DN, such as degenerative changes in axons and myelin sheath, and reduced area and density of unmyelinated fibers. In MSC-treated mice, these structural alterations were markedly less commonly observed and/or less pronounced. Moreover, MSC transplantation inhibited multiple parameters of spinal neuroinflammation found in diabetic mice, causing the reduction of activated astrocytes and microglia, oxidative stress signals, galectin-3, IL-1β, and TNF-α production. Conversely, MSC increased the levels of anti-inflammatory cytokines, IL-10, and TGF-β.ConclusionsThe present study described the modulatory effects of MSC on spinal cord neuroinflammation in diabetic mice, suggesting new mechanisms by which MSC can improve DN.

Upregulation of P2X2 and P2X3 receptors in rats with hyperalgesia induced by heroin withdrawal.

Drug dependence and withdrawal syndrome induced by abrupt cessation of opioid administration remain a severe obstacle in the clinical treatment of chronic pain and opioid drug addiction. One of the key symptoms during opioid withdrawal is hyperalgesia. The mechanism of opioid withdrawal-induced hyperalgesia remains unclear. P2X2 and P2X3 receptors, members of P2X receptor subunits, act as the integrator of multiple forms of noxious stimuli and play an important role in nociception transduction of chronic neuropathic and inflammatory pain. The process of P2X2 and P2X3 receptor antagonism inhibits inflammatory hyperalgesia, involving the spinal opioid system. However, the role of P2X receptors involved in opioid withdrawal-induced hyperalgesia has seldom been discussed. To explore the role of P2X2 and P2X3 receptors in the opioid-induced hyperalgesia, heroin self-administration rats were adopted, and the thermal and mechanical nociceptive thresholds were evaluated using the paw withdrawal test after abstinence from heroin for 8 days. In addition, the expressions of P2X2 and P2X3 receptors in dorsal root ganglia were analyzed by immunofluorescence. The results showed that after 8 days of abstinence, heroin self-administration rats showed thermal hyperalgesia and mechanical allodynia. Meanwhile, the expressions of the P2X2 and P2X3 receptors in dorsal root ganglia were increased. These results suggest that upregulation of P2X2 and P2X3 receptors might partially play a role in heroin withdrawal-induced hyperalgesia.

Effects of three forms of local anesthesia on perioperative fentanyl-induced hyperalgesia.

Both local infiltration analgesia (LIA) and nerve block are common analgesic modalities for pain relief after surgery. The aim of the current study was to investigate the effects of those two modalities on pain behavior and the expression of pro-inflammatory cytokines such as interleukin (IL)-1β and IL-6 and tumor necrosis factor-α (TNF-α) in the spinal cord and dorsal root ganglion (DRG) in a rat model of perioperative fentanyl induced hyperalgesia. Rats were injected with fentanyl (60 μg/kg) 4 times and received a plantar incision after the second injection or they received pre-incision LIA and sciatic nerve block (SNB) or post-incision LIA with levobupivacaine (0.5%, 0.2 mL). Mechanical and thermal nociceptive thresholds were assessed using the tail pressure test and paw withdrawal test on the day before drug injection, 1 and 4 hours after injection, and 1-7 days later. The lumbar spinal cord and dorsal root ganglia were collected from rats in each group to measure IL-1β, IL-6, and TNF-α on the day before drug injection, 4 hours after injection, and 1, 3, 5, and 7 days later. Fentanyl and an incision induced a significantly delayed mechanical hyperalgesia in the tail and thermal hyperalgesia in both hind paws and up-regulation of pro-inflammatory cytokines in the spinal cord and dorsal root ganglia. Rats treated with pre-incision LIA and SNB or post-incision LIA had alleviated hyperalgesia and significantly reduced levels of IL-1β, IL-6, and TNF-α compared to the control group. LIA and SNB partly prevented perioperative fentanyl-induced hyperalgesia and up-regulation of pro-inflammatory cytokines in the spinal cord and dorsal root ganglia.

The imidazoline I2 receptor agonist 2-BFI attenuates hypersensitivity and spinal neuroinflammation in a rat model of neuropathic pain

Chronic pain is a large, unmet public health problem. Recent studies have demonstrated the importance of neuroinflammation in the establishment and maintenance of chronic pain. However, pharmacotherapies that reduce neuroinflammation have not been successfully developed to treat chronic pain thus far. Several preclinical studies have established imidazoline I2 receptor (I2R) agonists as novel candidates for chronic pain therapies, and while some I2R ligands appear to modulate neuroinflammation in certain scenarios, whether they exert anti-neuroinflammatory effects in models of chronic pain is unknown. This study examined the effects of the prototypical I2R agonist 2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-BFI) on hypersensitivity and neuroinflammation induced by chronic constriction injury (CCI), a neuropathic pain model in rats. In CCI rats, twice-daily treatment with 10 mg/kg 2-BFI for seven days consistently increased mechanical and thermal nociception thresholds, reduced GFAP and Iba-1 levels in the dorsal horn of the spinal cord, and reduced levels of TNF-α relative to saline treatment. These results were recapitulated in primary mouse cortical astrocyte cultures. Incubation with 2-BFI attenuated GFAP expression and supernatant TNF-α levels in LPS-stimulated cultures. These results suggest that I2R agonists such as 2-BFI may reduce neuroinflammation which may partially account for their antinociceptive effects.

Neurotoxic effect of oxaliplatin: Comparison with its oxalate-free analogue cis-[PtII(1R,2R-DACH)(3-acetoxy-1,1-cyclobutanedicarboxylato)] (LLC-1402) in mice

Studies suggest that oxalate is involved in the development oxaliplatin-induced peripheral sensory neuropathy (OPSN). This study aimed to compare the neurotoxic effects of oxaliplatin with its oxalate-free cytotoxic analogue cis-[PtII(1R,2R-DACH)(3-acetoxy-1,1-cyclobutanedicarboxylato)] (LLC-1402) in mice. Oxaliplatin and LLC-1402 were intravenously injected in male Swiss mice with a total of nine injections. Oxalate was intraperitoneally injected in other animals. The development of OPSN was evaluated using mechanical and thermal sensitivity tests. Dorsal root ganglia of the mice were removed to evaluate c-Fos, ATF3 and iNOS expression and a sample of blood was collected for leukocyte count and hepatic and renal biochemical function tests. Oxaliplatin and LLC-1402 decreased the mechanical and thermal nociceptive threshold, whilst oxalate lead to a partial and later increase in the mechanical sensitivity (P<0.05). c-Fos, ATF3 and iNOS expressions were increased in neuronal cells during and after the end of the injections in animals treated with oxaliplatin and LLC-1402 (P<0.05), even though oxaliplatin lead to an earlier increase. Only c-Fos expression was elevated during the period of injections in the oxalate group (P<0.05), but this expression reduced after the end of the treatment. c-Fos expression was also shown in glial satellite cells only in the oxaliplatin-treated animals. Oxaliplatin and LLC-1402 reduced leukocyte count (P<0.05), but did not change renal and liver functions. In conclusion, oxalate may contribute to an earlier development of peripheral sensory neuropathy. However, the antitumor cytotoxic mechanism of oxaliplatin seems to be the main responsible by its neurotoxic effect.

Phosphatidylcholine attenuated docetaxel-induced peripheral neurotoxicity in rats

Docetaxel is a taxane chemotherapeutic agent used in the treatment of breast cancer, prostate cancer and gastric cancer, but several side effects such as peripheral neurotoxicity could occur. The present study was designed to investigate the therapeutic potential of phosphatidylcholine (PC) on docetaxel-induced peripheral neurotoxicity. Rats were randomly divided into three groups and treated for 4 weeks. Behavioral tests were conducted to measure the effects of PC on docetaxel-induced decreases in mechanical & thermal nociceptive threshold. Biochemical tests were conducted to measure the level of oxidative stress on sciatic nerve. Histopathological and immunohistochemical experiments were also conducted to assess neuronal damage and glial activation. PC treatment significantly attenuated docetaxel-induced changes in mechanical & thermal nociceptive response latencies. PC decreased oxidative stress in sciatic nerve by increasing antioxidant levels (glutathione, glutathione peroxidase and superoxide dismutase activity). In immunohistochemical evaluation, PC treatment ameliorated docetaxel-induced neuronal damage and microglial activation in the sciatic nerve and spinal cord. Thus, PC showed protective effects against docetaxel-induced peripheral neurotoxicity. These effects may be attributed to its antioxidant properties and modulation of microglia.

Increased Hyperalgesia and Proinflammatory Cytokines in the Spinal Cord and Dorsal Root Ganglion After Surgery and/or Fentanyl Administration in Rats.

BACKGROUND:Perioperative fentanyl has been reported to induce hyperalgesia and increase postoperative pain. In this study, we tried to investigate behavioral hyperalgesia, the expression of proinflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α), and the activation of microglia in the spinal cord and dorsal root ganglion (DRG) in a rat model of surgical plantar incision with or without perioperative fentanyl.METHODS:Four groups of rats (n = 32 for each group) were subcutaneously injected with fentanyl at 60 μg/kg or normal saline for 4 times with 15-minute intervals. Plantar incisions were made to rats in 2 groups after the second drug injection. Mechanical and thermal nociceptive thresholds were assessed by the tail pressure test and paw withdrawal test on the day before, at 1, 2, 3, 4 hours, and on the days 1–7 after drug injection. The lumbar spinal cord, bilateral DRG, and cerebrospinal fluid of 4 rats in each group were collected to measure IL-1β, IL-6, and TNF-α on the day before, at the fourth hour, and on the days 1, 3, 5, and 7 after drug injection. The lumbar spinal cord and bilateral DRG were removed to detect the ionized calcium-binding adapter molecule 1 on the day before and on the days 1 and 7 after drug injection.RESULTS:Rats injected with normal saline only demonstrated no significant mechanical or thermal hyperalgesia or any increases of IL-1β, IL-6, and TNF-α in the spinal cord or DRG. However, injection of fentanyl induced analgesia within as early as 4 hours and a significant delayed tail mechanical and bilateral plantar thermal hyperalgesia after injections lasting for 2 days, while surgical plantar incision induced a significant mechanical and thermal hyperalgesia lasting for 1–4 days. The combination of fentanyl and incision further aggravated the hyperalgesia and prolonged the duration of hyperalgesia. The fentanyl or surgical incision upregulated the expression of IL-1β, IL-6, and TNF-α in the spinal cord and bilateral DRG for more than 7 days and increase of ionized calcium-binding adapter molecule 1 in the spinal cord. The combination of fentanyl and incision resulted in higher increase of IL-1β, IL-6, and TNF-α in the spinal cord and bilateral DRG.CONCLUSIONS:The surgical plantar incision with or without perioperative fentanyl induced significant mechanical and thermal hyperalgesia, an increased expression of IL-1β, IL-6, TNF-α in the spinal cord and DRG, and activation of microglia in the spinal cord.

A prospective study of breed differences in the thermal pain sensitivity of dogs

ObjectiveTo compare the thermal nociceptive thresholds (TNTs) of dogs from three working/sport breeds. Study designExperimental prospective study. AnimalsThirty healthy adult dogs of mixed age, sex and reproductive status, 10 of each of three breeds: Harrier Hound, Greyhound and New Zealand Huntaway. MethodsOn one day of each week for 4 weeks, unrestrained dogs were tested six times. TNTs were measured using a remotely activated device comprising a thermode attached to the thoracic limb, controlled by a microprocessor attached to the animal. Latency to exhibit behaviour indicative of nociception after initiation of heating and the temperature of the thermode at the point of behavioural response were measured. Linear mixed-effects models were fitted to the data, with dog included as a random effect, initial thermode temperature as a covariate and day, week and breed as fixed effects. ResultsInitial thermode temperature significantly affected dogs’ latency to respond (p < 0.001). Breed had a significant effect on both latency to respond and response temperature. Huntaways took longer to respond than Harriers or Greyhounds. For example, when the initial thermode temperature was 30 °C, Huntaways took 39.0 seconds to respond compared with 35.8 seconds for Harriers and 36.8 seconds for Greyhounds. Huntaways also responded at higher temperatures (mean±standard deviation: Huntaways 49.7±1.3 °C, Harriers 48.4±1.6 °C and Greyhounds 48.7±1.6 °C). Conclusions and clinical relevanceHuntaways appeared to be less sensitive to thermal pain than the other breeds. Such information could be used by researchers and clinicians to better understand the generalizability of data gathered from a specific breed to the wider canine population or to tailor more effective pharmacological approaches to pain management in dogs.

A novel method for investigating the importance of visual feedback on somatosensation and bodily-self perception

Abstract Aims Visual feedback is hypothesized to play an important role in the phantom limb condition. In this study we attempt to create an illusory experimental model of phantom limb wherein this condition is simulated by removing the visual input from the upper limb in a group of intact participants. The aim of the study is to investigate the role of visual feedback on somatosensation, nociception and bodily-self perception. Methods Using a novel mixed reality (MR) system, the visual feedback of the left hand is removed in order to visually simulate a left hand amputation on 30 healthy participants (15 females). Using a within-subject design, three conditions are created: visual amputation condition (MR with no visual input); visual condition (MR with normal vision); and a baseline condition (no MR). Thermal detection and nociceptive thresholds using method of limits are measured. Proprioception of the visually amputated hand is investigated by probing the felt hand position on a proximal-distal axis from the body. Using a questionnaire the effects of the missing visual feedback on bodily self is assessed. Results There was a clear drift in proprioception of the left hand in the proximal direction between the control and visual amputation condition (p &lt;0.001). A decrease in cold detection was also significant between the control and visual amputation condition (p &lt; 0.001). Finally, questions on perceptual experiences indicated that the observed proprioceptive retraction of the visually amputated hand was also felt by the participants. Conclusions Missing visual feedback greatly influences the perception of the visually amputated arm underlining the importance of visual feedback. The observed proprioceptive retraction of the hand resembles the telescoping perceptions often reported by phantom limb patients. The novel method developed for this study, is a new tool to investigate the influence of visual feedback on the relationship of bodily-self and chronic pain.

Neurovascular unit alteration in somatosensory cortex and enhancement of thermal nociception induced by amphetamine involves central AT1 receptor activation.

The use of psychostimulants, such as amphetamine (Amph), is associated with inflammatory processes, involving glia and vasculature alterations. Brain Angiotensin II (Ang II), through AT1 -receptors (AT1 -R), modulates neurotransmission and plays a crucial role in inflammatory responses in brain vasculature and glia. Our aim for the present work was to evaluate the role of AT1 -R in long-term alterations induced by repeated exposure to Amph. Astrocyte reactivity, neuronal survival and brain microvascular network were analysed at the somatosensory cortex. Thermal nociception was evaluated as a physiological outcome of this brain area. Male Wistar rats (250-320g) were administered with AT1 -R antagonist Candesartan/vehicle (3mg/kg p.o., days 1-5) and Amph/saline (2.5mg/kg i.p., days 6-10). The four experimental groups were: Veh-Sal, CV-Sal, Veh-Amph, CV-Amph. On day 17, the animals were sacrificed and their brains were processed for Nissl staining and immunohistochemistry against glial fibrillary acidic protein (GFAP) and von Willebrand factor. In another group of animals, thermal nociception was evaluated using hot plate test, in the four experimental groups, on day 17. Data were analysed with two-way anova followed by Bonferroni test. Our results indicate that Amph exposure induces an increase in: neuronal apoptosis, astrocyte reactivity and microvascular network, evaluated as an augmented occupied area by vessels, branching points and their tortuosity. Moreover, Amph exposure decreased the thermal nociception threshold. Pretreatment with the AT1 -R blocker prevented the described alterations induced by this psychostimulant. The decreased thermal nociception and the structural changes in somatosensory cortex could be considered as extended neuroadaptative responses to Amph, involving AT1 -R activation.

Sedative and antinociceptive effects of different combinations of detomidine and methadone in standing horses

ObjectiveTo evaluate intravenous (IV) detomidine with methadone in horses to identify a combination which provides sedation and antinociception without adverse effects. Study designRandomized, placebo-controlled, blinded, crossover. AnimalsA group of eight adult healthy horses aged (mean ± standard deviation) 7 ± 2 years and 372 ± 27 kg. MethodsA total of six treatments were administered IV: saline (SAL); detomidine (5 μg kg−1; DET); methadone (0.2 mg kg−1; MET) alone or combined with detomidine [2.5 (MLD), 5 (MMD) or 10 (MHD) μg kg−1]. Thermal, mechanical and electrical nociceptive thresholds were measured, and sedation, head height above ground (HHAG), cardiopulmonary variables and intestinal motility were evaluated at 5, 15, 30, 45, 60, 75, 90, 120 and 180 minutes. Normal data were analyzed by mixed-model analysis of variance and non-normal by Kruskal–Wallis (p < 0.05). ResultsNociceptive thresholds in horses administered methadone with the higher doses of detomidine (MMD, MHD) were increased above baseline to a greater degree and for longer duration (MMD: 15–30 minutes, MHD: 30–60 minutes) than in horses administered low dose with methadone or detomidine alone (MLD, DET: 5–15 minutes). No increases in nociceptive thresholds were recorded in SAL or MET. Compared with baseline, HHAG was lower for 30 minutes in MMD and DET, and for 45 minutes in MHD. No significant sedation was observed in SAL, MET or MLD. Intestinal motility was reduced for 75 minutes in MHD and for 30 minutes in all other treatments. ConclusionsMethadone (0.2 mg kg−1) potentiated the antinociception produced by detomidine (5 μg kg−1), with minimal sedative effects. Clinical relevanceDetomidine (5 μg kg−1) with methadone (0.2 mg kg−1) produced antinociception without the adverse effects of higher doses of detomidine.

A Novel Approach for the Control of Inflammatory Pain: Prostaglandin E2 Complexation by Randomly Methylated β-Cyclodextrins.

Inhibitors of cyclooxygenase, which block the formation of prostaglandin (PG) E2, are the standard treatment of inflammatory pain. These drugs, however, have serious gastrointestinal, renal, and cardiovascular side effects that limit their clinical use. Cyclodextrins are neutral glucose oligomers that form a hydrophilic outer and a hydrophobic interior cavity used to carry hydrophilic substances. Methyl-β-cyclodextrins are used currently in several drugs as enhancers and also to deliver PGs. We therefore hypothesized that randomly methylated β-cyclodextrins (RAMEB) could be used for pain treatment. An in silico screening for important inflammatory mediators (eg, PGE2, substance P, bradykinin, and calcitonin gene-related peptide) was performed to predict the probability of these molecules binding to RAMEB. Thereafter, a comprehensive in vitro study investigated the complexation affinity of the best target toward RAMEB or its RAMEB-fraction L (FL) using capillary electrophoresis.Wistar rats were injected intraplantarly with complete Freund's adjuvant (CFA) for 96 hours to induce inflammatory hyperalgesia. Subsequently, rats were treated intraplantarly or intravenously either with RAMEB or RAMEB FL and compared with the respective controls. Parecoxib was used as positive control. Mechanical (paw pressure threshold, PPT) and thermal (paw withdrawal latency) nociceptive thresholds were determined before injection and at the indicated time points thereafter. Paw tissue was collected after treatments, and PGE2 and PGD2 contents were measured. Analysis of variance was used for data analysis followed by appropriate post hoc comparisons. In silico screening indicated that PGE2, with the highest affinity, was the best candidate for RAMEB binding. Likewise, in capillary electrophoresis experiments, RAMEB had a high affinity to form inclusion complexes with the PGE2 (stability constant [K], 360 1/M; 95% confidence interval [C]: 347.58-372.42 M). Local treatment with RAMEB alleviated CFA-induced mechanical (PPT: 76.25 g; 95% CI: 56.24-96.25 g) and thermal hyperalgesia (PPT: 8.50 seconds; 95% CI: 6.76-10.23 seconds). Moreover, a systemic administration of RAMEB decreased CFA-induced mechanical (PPT: 126.66 g; 95% CI: 114.54-138.77 g) and thermal hyperalgesia (paw withdrawal latency: 11.47 seconds; 95% CI: 9.26-13.68 seconds). RAMEB FL resulted in greater in vitro PGE2-binding capacity and decreased PG content as well as hyperalgesia in vivo to a similar extent. Motor activity of the rats was not altered by RAMEB or RAMEB FL. Capture of PGs by cyclodextrins could be a novel and innovative tool for the treatment of inflammatory pain and bypassing some unwanted side effects of cyclooxygenase inhibitors.

Relationship between the melanocortin-1 receptor (MC1R) variant R306ter and physiological responses to mechanical or thermal stimuli in Labrador Retriever dogs

ObjectiveVariants in the MC1R gene have been associated with red hair color and sensitivity to pain in humans. The study objective was to determine if a relationship exists between MC1R genotype and physiological thermal or mechanical nociceptive thresholds in Labrador Retriever dogs. Study designProspective experimental study. AnimalsThirty-four Labrador Retriever dogs were included in the study following public requests for volunteers. Owner consent was obtained and owners verified that their dog was apparently not experiencing pain and had not been treated for pain during the previous 14 days. The study was approved by the Institutional Animal Care and Use Committee. MethodsNociceptive thresholds were determined from a mean of three thermal and five mechanical replications using commercially available algometers. Each dog was genotyped for the previously described MC1R variant (R306ter). Data were analyzed using one-way anova with post hoc comparisons using Tukey’s test (p < 0.05). ResultsThirteen dogs were homozygous wild-type (WT/WT), nine were heterozygous (WT/R306ter), and eight were homozygous variant (R306ter/R306ter) genotype. Four dogs could not be genotyped. A significant difference (p = 0.04) in mechanical nociceptive thresholds was identified between dogs with the WT/WT genotype (12.1±2.1 N) and those with the WT/R306ter genotype (9.2±2.4 N). ConclusionA difference in mechanical, but not thermal, nociceptive threshold was observed between wild-type and heterozygous MC1R variants. Differences in nociceptive thresholds between homozygous R306ter variants and other genotypes for MC1R were not observed. Clinical relevanceCompared with the wild-type MC1R genotype, nociceptive sensitivity to mechanical force in dogs with a single variant R306ter allele may be greater. However, in contrast to the reported association between homozygous MC1R variants (associated with red hair color) and nociception in humans, we found no evidence of a similar relationship in dogs with the homozygous variant genotype.

Histamine H4 receptor agonist-induced relief from painful peripheral neuropathy is mediated by inhibition of spinal neuroinflammation and oxidative stress.

Neuropathic pain is under-treated, with a detrimental effect on quality of life, partly because of low treatment efficacy, but also because pathophysiological mechanisms are not fully elucidated. To clarify the pathobiology of neuropathic pain, we studied the contribution of neuroinflammation and oxidative stress in a model of peripheral neuropathy. We also assessed an innovative treatment for neuropathic pain by investigating the effects of histamine H4 receptor ligands in this model. A peripheral mononeuropathy was induced in mice, by spared nerve injury (SNI). Neuroinflammation and oxidative stress parameters were evaluated by spectrophotometry. The mechanical (von Frey test) and thermal (plantar test) nociceptive thresholds were evaluated. SNI mice showed increased expression of the pro-inflammatory cytokines IL-1ß and TNF-α, decreased antioxidant enzyme Mn-containing SOD (MnSOD), increased levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG), an indicator of oxidative DNA damage, and of PARP, nuclear enzyme activated upon DNA damage. Intrathecal administration of VUF 8430 (H4 receptor agonist) reversed the mechanical and thermal allodynia and was associated with decreased expression of IL-1ß, TNF-α, 8-OHdG and PARP and with restoration of MnSOD activity in the spinal cord and sciatic nerve. These effects were prevented by JNJ 10191584 (H4 receptor antagonist). In the SNI mouse model of neuropathic pain, neuronal H4 receptor stimulation counteracts hyperalgesia and reduces neuroinflammation and oxidative stress in the spinal cord and sciatic nerve. Targeting both oxidative stress and pro-neuroinflammatory pathways through H4 receptor-mediated mechanisms could have promising therapeutic potential for neuropathic pain management.

Lentiviral vector-driven inhibition of 5-HT synthesis in B3 bulbo-spinal serotonergic projections – Consequences on nociception, inflammatory and neuropathic pain in rats

Although it is well established that bulbo-spinal serotonergic projections contribute to pain control mechanisms, whether they exert anti- or pro-nociceptive modulations is still a matter of debate. In order to reappraise the role of 5-HT in descending controls, we used RNA interference to selectively inhibit 5-HT synthesis in B3 neurons and assess resulting changes in nociception.Rats were injected into the bulbar B3 group with a recombinant lentiviral vector, LV-shTPH2, encoding RNA interfering with tryptophan hydroxylase 2 expression. Together with the long term disappearance of this enzyme in the whole rostro-caudal extent of B3 group, 5-HT was markedly depleted selectively in the dorsal horn at all levels of the spinal cord. In contrast, immunolabeling of the 5-HT transporter was unaffected by LV-shTPH2 injection, indicating the preservation of serotonergic fibers integrity.Whereas mechanical and thermal nociceptive thresholds were unchanged by 5-HT depletion, marked reductions in intraplantar formalin (but not carrageenin)-evoked nocifensive responses, and, in contrast, significant increases in mechanical and thermal hyperalgesia evoked by sciatic nerve ligation were noted in LV-shTPH2-injected rats versus controls. Parallel changes in c-Fos immunolabeling within the dorsal horn confirmed that bulbo-spinal serotonergic projections modulate pain signaling under these various conditions.These results suggest that serotonergic neurons of the B3 group are only moderately concerned, if any, by acute nociception but exert modulatory influences under pain sensitizing conditions. The opposite changes in formalin injected- versus sciatic nerve ligated rats might be related to the implication of different receptors in 5-HT-mediated modulation of inflammatory versus neuropathic pain.