Region Of Hind Paw Research Articles

Quantitative orbital tightening for pain assessment using machine learning with DeepLabCut

Pain assessment in animal models is essential for understanding mechanisms underlying pathological pain and developing effective pain medicine. The grimace scale (GS), facial expression features in pain such as orbital tightening (OT), is a valuable measure for assessing pain in animal models. However, the classical grimace scale for pain assessment is labor-intensive, subject to subjectivity and inconsistency, and is not a quantitative measure. In the present study, we utilized machine learning with DeepLabCut to annotate the superior and inferior eyelid margins and the medial and lateral canthus of the eyes in animals’ video images. Based on the annotation, we quantified the eyelid distance and palpebral fissure width of the animals’ eyes so that the degree of OT in animals with pain could be measured and described quantitatively. We established criteria for the inclusion and exclusion of the annotated images for quantifying OT, and validated our quantitative grimace scale (qGS) in the mice with pain caused by capsaicin injections in the orofacial or hindpaw regions, the Nav1.8-ChR2 mice following orofacial noxious stimulation with laser light, and the oxaliplatin-treated mice following tactile stimulation with a von Frey filament. We showed that both the eyelid distance and the palpebral fissure width were shortened significantly in the animals in pain compared to the control animals without nociceptive stimulation. Collectively, the present study has established a quantitative orbital tightening for pain assessment in mice using DeepLabCut, providing a new tool for pain assessment in preclinical studies with mice.

Peripheral neural mechanism of visceral pain and acupoint sensitization in 2, 4, 6-trinitrobenzene sulfonic acid-induced colitis model mice.

To explore the neural mechanism of visceral pain and related somatic (acupoints) sensitization by using in vivo calcium imaging of dorsal root ganglia (DRG) neurons. Eight BALB/c mice were randomly divided into control and model groups, with 4 mice in each group. The colitis model was induced by colorectal perfusion of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS) once daily for 7 days. Mice of the control group received colorectal perfusion of normal saline once daily for 7 days. The location and area of the somatic neurogenic inflammation (cutaneous exudation of Evans blue ［EB］) of the 2 groups of mice were observed after intravenous injection of EB. For pain behavioral tests, sixteen C57BL/6J mice were randomly divided into control and model groups, with 8 mice in each group, and a Von Frey filament was used to stimulate the referred somatic reactive regions in colitis mice, and the number of avoidance and paw withdraw reaction within 10 tests was recorded. For in vivo DRG calcium imaging tests, 24 Pirt-GCaMP6s transgenic mice were randomly and equally divided into control group and colitis model group. The responses of the neurons in L6 or L4 DRG to colorectal distension (CRD), lower back brushing, or mechanical stimulation at the hindpaw were observed using confocal fluorescence microscope. Compared with the control group, the area of EB exudation spot in the hindpaw and lower back regions was increased in the colitis model group (P<0.05), and the avoidance or paw withdraw numbers induced by Von Frey stimulation at the lower back and hindpaw were increased (P<0.01, P<0.05), indicating that colitis induced regional skin (acupoints) sensitization in the lower back and hindpaw regions. Compared with the control group, the percentage of L6 DRG neurons activated by 60 mm Hg CRD in the colitis model mice were apparently increased (P<0.01), the activated neurons mainly involved the medium-sized DRG neurons (P<0.01). In Pirt-GCaMP6s transgenic mice, following brushing the skin of the receptive field (lower back) of L6 DRG neurons, the fluorescence intensity of the brushing-activated DRG neurons and small, medium and large-sized neurons were significantly higher in the colitis model group than those in the control group (P<0.001, P<0.01, P<0.05). After brushing and clamping the skin of the right hindpaw (receptive field of L4 DRG neurons), the percentages of the activated L4 DRG neurons were obviously higher in the colitis model group than those in the control group (P<0.01, P<0.05), while there were no significant changes in the proportion of small, medium and large-sized neurons between the control and colitis model groups. Colitis may lead to body surface sensitization at the same and adjacent neuro-segments as well as to an increase of the number and activity of the responsive lumbar DRG neurons, among which the L6 DRG neurons at the same neuro-segment as the rectum colon showed an increase in the number of responders and intensity of calcium fluorescence signal while L4 DRG neurons at the level adjacent to the rectum colon showed an increase in the number of responders, suggesting that there may be different mechanisms of peripheral neural sensitization.

Identification of brain areas in mice with peak neural activity across the acute and persistent phases of post-traumatic headache.

Post-traumatic headache is very common after a mild traumatic brain injury. Post-traumatic headache may persist for months to years after an injury in a substantial proportion of people. The pathophysiology underlying post-traumatic headache remains unknown but is likely distinct from other headache disorders. Identification of brain areas activated in acute and persistent phases of post-traumatic headache can provide insights into the underlying circuits mediating headache pain. We used an animal model of mild traumatic brain injury-induced post-traumatic headache and c-fos immunohistochemistry to identify brain regions with peak activity levels across the acute and persistent phases of post-traumatic headache. Male and female C57BL/6 J mice were briefly anesthetized and subjected to a sham procedure or a weight drop closed-head mild traumatic brain injury . Cutaneous allodynia was assessed in the periorbital and hindpaw regions using von Frey filaments. Immunohistochemical c-fos based neural activity mapping was then performed on sections from whole brain across the development of post-traumatic headache (i.e. peak of the acute phase at 2 days post- mild traumatic brain injury), start of the persistent phase (i.e. >14 days post-mild traumatic brain injury) or after provocation with stress (bright light). Brain areas with consistent and peak levels of c-fos expression across mild traumatic brain injury induced post-traumatic headache were identified and included for further analysis. Following mild traumatic brain injury, periorbital and hindpaw allodynia was observed in both male and female mice. This allodynia was transient and subsided within the first 14 days post-mild traumatic brain injury and is representative of acute post-traumatic headache. After this acute post-traumatic headache phase, exposure of mild traumatic brain injury mice to a bright light stress reinstated periorbital and hindpaw allodynia for several hours - indicative of the development of persistent post-traumatic headache. Acute post-traumatic headache was coincident with an increase in neuronal c-fos labeling in the spinal nucleus of the trigeminal caudalis, primary somatosensory cortex, and the nucleus accumbens. Neuronal activation returned to baseline levels by the persistent post-traumatic headache phase in the spinal nucleus of the trigeminal caudalis and primary somatosensory cortex but remained elevated in the nucleus accumbens. In the persistent post-traumatic headache phase, coincident with allodynia observed following bright light stress, we observed bright light stress-induced c-fos neural activation in the spinal nucleus of the trigeminal caudalis, primary somatosensory cortex, and nucleus accumbens. Examination of mild traumatic brain injury-induced changes in peak c-fos expression revealed brain regions with significantly increased neural activity across the acute and persistent phases of post-traumatic headache. Our findings suggest mild traumatic brain injury-induced post-traumatic headache produces neural activation along pain relevant pathways at time-points matching post-traumatic headache-like pain behaviors. These observations suggest that the spinal nucleus of the trigeminal caudalis, primary somatosensory cortex, and nucleus accumbens may contribute to both the induction and maintenance of post-traumatic headache.

The anti-inflammatory effects of minocycline on lipopolysaccharide-induced paw oedema in rats: a histopathological and molecular study.

Minocycline is a semi-synthetic antimicrobial agent with claimed anti-inflammatory properties reported from different experimental models. This study was aimed to evaluate the anti-inflammatory effects of minocycline, compared to the actions of two common anti-inflammatory agents, on lipopolysaccharide (LPS)-induced paw oedema through some clinical, histopathological, haematological and molecular analyses. Forty-eight rats were divided into eight groups (n = 6). In control group (Ctrl), each animal was injected with normal saline into its sub-plantar region of hind paw. In groups 2-7, hind paw oedema was induced by injection of LPS. One hour before injections, groups 1 (Ctrl) and 2 (LPS) were treated orally with distilled water, 3 and 4 with methylprednisolone (Pred) and meloxicam (Melo) and 5-7 with minocycline in doses of 50, 150 and 450mg/kg (M50, M150 and M450, respectively). The 8th group (MC) was given minocycline (150mg/kg) orally and normal saline was injected into sub-plantar region. Paw swelling and body temperature were assessed at 0, 2, 4, 6 and 24h post-injections. At 24h, samples of blood and liver, kidney, spleen and hind paw tissues were taken for haematological and histopathological examinations. Some samples of the paw were also obtained for molecular analysis of some inflammatory-related cytokines at mRNA level. Paw swelling and body temperature increased in all LPS-injected groups 2h post-injection. In LPS group, they remained significantly increased up to 24h; however, these parameters decreased to normal in Pred, Melo and all minocycline groups. The histological findings showed mild-to-moderate signs of inflammation in tissue samples of groups 2-6, but not in group M450. Additionally, gene expression of pro-inflammatory cytokines (IL-1β and IL-6) increased significantly in LPS group compared to other groups. In conclusion, this study supports the role of minocycline as an anti-inflammatory agent with effects comparable to those of meloxicam and methylprednisolone.

Pain-related neuronal ensembles in the primary somatosensory cortex contribute to hyperalgesia and anxiety

The mechanism by which acute pain or itch information at the periphery is processed in the primary somatosensory cortex (S1) remains unclear. To elucidate this, we used a viral-mediated targeted-recombination-in-active population system to target S1 neuronal ensembles that are active during pain or itch sensations. We induced the expression of excitatory or inhibitory designer receptors exclusively activated by designer drugs in pain- or itch-related S1 neurons. We identified neuronal populations in mice that regulate the sensory components of pain and itch in the S1 hind paw region. Notably, the neuronal circuit between pain-related S1 neurons and the parafascicular nucleus contributed to hyperalgesia and anxiety-like behavior. We propose that S1 plays an essential role in sensory and affective responses to noxious stimuli, such as pain.

Comparison of Anti-Inflammatory Efficacy of Ocimum Sanctum, Azadirachta Indica and their Combination with Aspirin in Chemically Induced Acute Inflammation.

Scope and Objective:Ocimum sanctum and Azadirachta indicaare known to be safe and effective anti-inflammatory agents in ayurveda. So, this study was planned to evaluate and compare anti-inflammatory activity of Ocimum sanctum, Azadirachta indica and combination of Ocimum sanctum + Azadirachta indica (COA) with Aspirin on acute inflammation in rats and also to assess mechanism behind their anti-inflammatory action. Materials and Methods: Wistar albino rats of either sex (150-250 g) were divided into 5 groups with six rats in each group. To induce inflammation, formalin (2.5%, 0.1 ml) was injected into sub-plantar region of left hind paw of rats. The study groups were administered orally with distilled water (3 ml), Aspirin (200 mg/kg), Ocimum sanctum (400 mg/kg), Azadirachta indica (500 mg/kg) and COA (400 mg/kgOcimum sanctum+500 mg/kgAzadirachta indica) half an hour before the formalin challenge. Effect of test drugs on acute inflammation was assessed by rat paw oedema test&amp;mechanism behind their action was assessed using histopathological examination. Results:In rat paw oedema test, Ocimum sanctum, Azadirachta indica and COA groups showed significant reductionin oedema as compared to control; Azadirachta indica and COA groupsalso showed comparable effect to Aspirin group. In histopathological examination, Aspirin, Azadirachta indica and COA groups caused significant reduction in vasodilation, oedema, infiltration and margination of neutrophils while Ocimum sanctum group only caused significant reduction in oedema. Conclusion: This study revealedthat all test groups have significant anti-inflammatory efficacy;Azadirachta indica and COA also have comparable efficacy to Aspirin.

CGRP monoclonal antibody prevents the loss of diffuse noxious inhibitory controls (DNIC) in a mouse model of post-traumatic headache.

Determine the role of calcitonin-gene related peptide in promoting post-traumatic headache and dysregulation of central pain modulation induced by mild traumatic brain injury in mice. Mild traumatic brain injury was induced in lightly anesthetized male C57BL/6J mice by a weight drop onto a closed and unfixed skull, which allowed free head rotation after the impact. We first determined possible alterations in the diffuse noxious inhibitory controls, a measure of net descending pain inhibition called conditioned pain modulation in humans at day 2 following mild traumatic brain injury. Diffuse noxious inhibitory control was assessed as the latency to a thermally induced tail-flick that served as the test stimulus in the presence of right forepaw capsaicin injection that provided the conditioning stimulus. Post-traumatic headache-like behaviors were assessed by the development of cutaneous allodynia in the periorbital and hindpaw regions after mild traumatic brain injury. We then determined if intraperitoneal fremanezumab, an anti-calcitonin-gene related peptide monoclonal antibody or vehicle administered 2 h after sham or mild traumatic brain injury induction could alter cutaneous allodynia or diffuse noxious inhibitory control responses on day 2 post mild traumatic brain injury. In naïve and sham mice, capsaicin injection into the forepaw elevated the latency to tail-flick, reflecting the antinociceptive diffuse noxious inhibitory control response. Periorbital and hindpaw cutaneous allodynia, as well as a loss of diffuse noxious inhibitory control, was observed in mice 2 days after mild traumatic brain injury. Systemic treatment with fremanezumab blocked mild traumatic brain injury-induced cutaneous allodynia and prevented the loss of diffuse noxious inhibitory controls in mice subjected to a mild traumatic brain injury. Sequestration of calcitonin-gene related peptide in the initial stages following mild traumatic brain injury blocked the acute allodynia that may reflect mild traumatic brain injury-related post-traumatic headache and, additionally, prevented the loss of net descending inhibition within central pain modulation pathways. As loss of conditioned pain modulation has been linked to multiple persistent pain conditions, dysregulation of descending modulatory pathways may contribute to the persistence of post-traumatic headache. Additionally, evaluation of the conditioned pain modulation/diffuse noxious inhibitory controls response may serve as a biomarker of vulnerability for chronic/persistent pain. These findings suggest that early anti-calcitonin-gene related peptide intervention has the potential to be effective both for the treatment of mild traumatic brain injury-induced post-traumatic headache, as well as inhibiting mechanisms that may promote post-traumatic headache persistence.

Gabapentin reduces painful bladder hypersensitivity in rats with lipopolysaccharide-induced chronic cystitis.

Although interstitial cystitis/bladder pain syndrome (IC/BPS) is a chronic condition causing bladder pain and urinary symptoms, effective treatments have not been established. The aim of this study was to adapt a chronic cystitis model in rats using lipopolysaccharide (LPS), which reflects IC/BPS pathology, and characterize the model's histological and behavioral effects. Furthermore, we investigated the effect of an α2δ subunit ligand, gabapentin (GBP), on bladder hypersensitivity of rats with chronic cystitis. Cystitis models were created by repeated intravesical injections of LPS. In the histological examination, the LPS‐injected group had greater inflammatory response, fibrosis, and abnormally thick re‐epithelialization. In the LPS‐injected group, LPS prompted hyperalgesia in both the lower abdomen and hind paw regions after day 1 of the first injection compared with the saline‐injected controls, without any recovery for 21 days at least. During cystometry, the LPS‐injected group showed bladder hyperactivity at all times. Systemic administration of GBP reduced cystitis‐related pain due to chronic inflammation and reduced the increased frequency of voiding in the LPS‐injected group. These results suggest that repeated intravesical injections of LPS induce long‐lasting bladder inflammation, pain, and overactivity in rats, while GBP is effective in the management of those symptoms in this chronic cystitis model. The current study identifies a relatively simple method to develop an animal model for chronic cystitis and provides evidence that GBP may be an effective treatment option for patients with IC/BPS.

Ubrogepant does not induce latent sensitization in a preclinical model of medication overuse headache.

BackgroundUbrogepant, a small-molecule calcitonin gene-related peptide receptor antagonist, was recently approved as an oral medication for the acute treatment of migraine. This study aimed to determine whether ubrogepant shows efficacy in a preclinical model of migraine-like pain and whether repeated oral administration of ubrogepant induces latent sensitization relevant to medication overuse headache in rats.MethodsA “two-hit” priming model of medication overuse headache was used. Female Sprague-Dawley rats received six oral doses of sumatriptan 10 mg/kg over 2 weeks to induce latent sensitization (i.e. “priming”). Cutaneous allodynia was measured periodically over 20 days in the periorbital and hindpaw regions using von Frey filaments. The rats were then subjected to a 1-hour bright light stress challenge on two consecutive days. At the start of the second bright light stress exposure, oral sumatriptan 10 mg/kg, oral ubrogepant 25, 50, or 100 mg/kg, or vehicle was administered; thereafter, cephalic and hindpaw sensory thresholds were monitored hourly over 5 hours to determine the efficacy of ubrogepant in reversing bright light stress-induced cutaneous allodynia. A dose of ubrogepant effective in the medication overuse headache model (100 mg/kg) was then selected to determine if repeated administration would produce latent sensitization. Rats were administered six oral doses of ubrogepant 100 mg/kg, sumatriptan 10 mg/kg (positive control), or vehicle over 2 weeks, and cutaneous allodynia was evaluated regularly. Testing continued until mechanosensitivity returned to baseline levels. Rats were then challenged with bright light stress on days 20 and 21, and periorbital and hindpaw cutaneous allodynia was measured. On days 28 to 32, the same groups received a nitric oxide donor (sodium nitroprusside 3 mg/kg, i.p.), and cutaneous allodynia was assessed hourly over 5 hours.ResultsSumatriptan elicited cutaneous allodynia in both cephalic and hindpaw regions; cutaneous allodynia resolved to baseline levels after cessation of drug administration (14 days). Sumatriptan priming resulted in generalized and delayed cutaneous allodynia, evoked by either bright light stress (day 21) or nitric oxide donor (day 28). Ubrogepant dose-dependently blocked both stress- and nitric oxide donor-induced cephalic and hindpaw allodynia in the sumatriptan-induced medication overuse headache model with a 50% effective dose of ∼50 mg/kg. Unlike sumatriptan, ubrogepant 100 mg/kg in repeated effective doses did not produce cutaneous allodynia or latent sensitization.ConclusionsBoth ubrogepant and sumatriptan demonstrated efficacy as acute medications for stress- and nitric oxide donor-evoked cephalic allodynia in a preclinical model of medication overuse headache, consistent with their clinical efficacy in the acute treatment of migraine. However, in contrast to sumatriptan, repeated treatment with ubrogepant did not induce cutaneous allodynia or latent sensitization. These studies suggest ubrogepant may offer an effective acute treatment of migraine without risk of medication overuse headache.Trial Registration Number: Not applicable

Evaluation of LY573144 (lasmiditan) in a preclinical model of medication overuse headache.

BackgroundMedication overuse is a significant issue that complicates the treatment of headache disorders. The most effective medications for the acute treatment of migraine all have the capacity to induce medication overuse headache (MOH). Novel acute migraine-specific treatments are being developed. However, because the mechanism(s) underlying medication overuse headache are not well understood, it is difficult to predict whether any particular acute medication will induce MOH in susceptible individuals. LY573144 (lasmiditan), a 5-HT1F receptor agonist, has recently been shown to be effective in the acute treatment of migraine in phase 3 trials. The aim of this study is to determine whether frequent administration of lasmiditan induces behaviors consistent with MOH in a pre-clinical rat model.MethodsSprague Dawley rats were administered six doses of lasmiditan (10 mg/kg), sumatriptan (10 mg/kg), or sterile water orally over 2 weeks and cutaneous allodynia was evaluated regularly in the periorbital and hindpaw regions using von Frey filaments. Testing continued until mechanosensitivity returned to baseline levels. Rats were then submitted to bright light stress (BLS) or nitric oxide (NO) donor administration and were again evaluated for cutaneous allodynia in the periorbital and hindpaw regions hourly for 5 hours.ResultsBoth lasmiditan and sumatriptan exhibited comparable levels of drug-induced cutaneous allodynia in both the periorbital and hindpaw regions, which resolved after cessation of drug administration. Both lasmiditan and sumatriptan pre-treatment resulted in cutaneous allodynia that was evoked by either BLS or NO donor.ConclusionsIn a pre-clinical rat model of MOH, oral lasmiditan, like sumatriptan, induced acute transient cutaneous allodynia in the periorbital and hindpaw regions that after resolution could be re-evoked by putative migraine triggers. These results suggest that lasmiditan has the capacity to induce MOH through persistent latent peripheral and central sensitization mechanisms.

Monitoring of Stimulus Evoked Murine Somatosensory Cortex Hemodynamic Activity With Volumetric Multi-Spectral Optoacoustic Tomography.

Sensory stimulation is an attractive paradigm for studying brain activity using various optical-, ultrasound- and MRI-based functional neuroimaging methods. Optoacoustics has been recently suggested as a powerful new tool for scalable mapping of multiple hemodynamic parameters with rich contrast and previously unachievable spatio-temporal resolution. Yet, its utility for studying the processing of peripheral inputs at the whole brain level has so far not been quantified. We employed volumetric multi-spectral optoacoustic tomography (vMSOT) to non-invasively monitor the HbO, HbR, and HbT dynamics across the mouse somatosensory cortex evoked by electrical paw stimuli. We show that elevated contralateral activation is preserved in the HbO map (invisible to MRI) under isoflurane anesthesia. Brain activation is shown to be predominantly confined to the somatosensory cortex, with strongest activation in the hindpaw region of the contralateral sensorimotor cortex. Furthermore, vMSOT detected the presence of an initial dip in the contralateral hindpaw region in the delta HbO channel. Sensorimotor cortical activity was identified over all other regions in HbT and HbO but not in HbR. Pearson’s correlation mapping enabled localizing the response to the sensorimotor cortex further highlighting the ability of vMSOT to bridge over imaging performance deficiencies of other functional neuroimaging modalities.

ANTI-INFLAMMATORY AND ANALGESIC ACTIVITY OF CURCUMIN WITH DIFFERENT NSAIDS

The objective of this study was to investigate the anti-inflammatory and analgesic activity of curcumin with combination of different drugs using carrageenan induced paw edema model and writhing test in mice. Nimesulide and tenoxicam were administered orally at different doses of 2.5, 5.0 and 7.5 mg/kg and the lower doses of these drugs were also administered orally with curcumin 60 mg/kg. After, 1 hour 0.1 ml of carrageenan (i.e. 10 mg/ml) was injected into the sub-plantar region of right hind paw of the Wistar Albino rats (150-200g) and paw volume was measured after 30 mins 1 hr, 2 hr and 3 hr using a Plethysmometer. In analgesic activity, the drug ketorolac was used with combination of curcumin and number of writhes induced by glacial acetic acid administration in mice were observed. Administration of carrageenan produced significant edema in the hind paw of rats. The edema was found to be significantly inhibited by combination of curcumin 60 mg/kg and the lower doses of nimesulide &amp; tenoxicam in different time interval and having comparable effects as of higher doses of these drugs. Similar results were also observed in analgesic activity. From this study it can be concluded that curcumin having synergistic effect when administered with combination different anti-inflammatory and analgesic agents.

EVALUATION OF ANTIDIABETIC DRUG ALOGLIPTIN FOR THE TREATMENT OF INFLAMMATION IN RATS

Objective: The present study was planned to evaluate the Alogliptin (Anti diabetic drug) for the treatment of inflammation in experimental models in rats. Methods: Total of 5 groups of wister rats of either sex weighing 180- 220 g, selected for the study of 2 animal model were kept at ambient temperature of 28±2°C and relative humidity of 45 to 55% with a 12:12 h light/dark cycle. The animals were fasted for 12 h before commencing the experiment with water ad libitum. Fasting was continued till completion of the experiment. Group A was served as normal toxicant control treated with toxicant Carrageenan (model 1) and Histamine (model 2), group B with Ibuprofen (40 mg/Kg p.o.) served as standard, groups C, D and E administered with Alogliptin (low, medium and high doses p.o) respectively in each model. The Groups B, C, D and E were administered with 0.1 ml of 1% w/v of carrageenan in model 1,Histamine in model 2 into sub plantar region of right hind paw of rats 1 h after the administration of Ibuprofen/ Alogliptin. Immediately thereafter the oedema volumes of the injected paws were measured plethysmographically at prefixed time intervals. Results: The Alogliptin with three selected doses i.e. 1, 2 and 3 mg/kg/day have exhibited a significant reduction in paw oedema volume at 4th h in carrageenan 36.92%, 51.49%, 65.46% and histamine 27.41%, 48.24%, 69.07% respectively. Ibuprofen (40 mg/kg) was used as standard reference thus standard drug has exhibited time dependent reduction in oedema volume. Conclusion: The results of recent studies suggest that dipeptidyl-peptidase-4 inhibitors (Alogliptin) have anti inflammatory effect on experimental models in rats. Peer Review History: Received 7 June 2018; Revised 21 June; Accepted 10 July, Available online 15 July 2018 UJPR follows the most transparent and toughest ‘Advanced OPEN peer review’ system. The identity of the authors and, reviewers will be known to each other. This transparent process will help to eradicate any possible malicious/purposeful interference by any person (publishing staff, reviewer, editor, author, etc) during peer review. As a result of this unique system, all reviewers will get their due recognition and respect, once their names are published in the papers. We expect that, by publishing peer review reports with published papers, will be helpful to many authors for drafting their article according to the specifications. Auhors will remove any error of their article and they will improve their article(s) according to the previous reports displayed with published article(s). The main purpose of it is ‘to improve the quality of a candidate manuscript’. Our reviewers check the ‘strength and weakness of a manuscript honestly’. There will increase in the perfection, and transparency. Received file: Reviewer's Comments: Average Peer review marks at initial stage: 5.5/10 Average Peer review marks at publication stage: 9.5/10 Reviewer(s) detail: Prof. Dr. Syamsudin Abdillah, Pancasila University, Indonesia, syamsudin.abdillah@gmail.com Omnia Momtaz Al-Fakharany, Tanta, University, Eygpt, amina.elfakharany@pharm.tanta.edu.eg Similar Articles: EVALUATION OF METHANOLIC EXTRACT OF EUPHORBIA NERIIFOLIA STEM BARK ON BLOOD SUGAR LEVELS, SERUM AND TISSUE LIPIDS IN A PRECLINICAL MODEL PLASMA FERRITIN AND HEPCIDIN LEVELS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS

Pharmacological and biochemical studies on protective effects of mangiferin and its interaction with nitric oxide (NO) modulators in adjuvant-induced changes in arthritic parameters, inflammatory, and oxidative biomarkers in rats.

Current study was designed to evaluate protective effect of mangiferin and its interaction with low dose of nitric oxide (NO) modulators in complete Freund's adjuvant (CFA) inoculated rats. Male wistar rats (200-300g, n = 8 per group) were used in the study. On day ''0'' of study arthritis was induced in rats by injecting 0.2ml CFA in sub-planter region of right hind paw of animals. Treatment with methotrexate (5mg/kg), mangiferin (10-30mg/kg) alone and in combination with NO modulators was given (i.p.) from days 14 to 28. After 28days, blood and joint synovial fluid was collected for biochemical analysis and rat paws were excised to estimate MDA and SOD in tissue (paw) homogenates. CFA inoculation significantly increases (1) arthritic index, (2) ankle diameter, (3) paw volume, and (4) serum TNF-α, IL-6, IL-1β, and synovial TNF-α levels (p < 0.001). The serum Th1 (IFN-γ) and Th2 (IL-4) cytokine levels, MDA levels in rat paw tissue homogenates and serum NF-κB levels were also found significantly increased. Significant decrease in serum IL-10 levels and SOD activity was found after CFA inoculation. These CFA-induced arthritic changes, cytokine profile, and oxidative stress markers were significantly reversed by mangiferin (10-30mg/kg) treatment alone and in combination with L-arginine and L-NAME nitric oxide modulators (p < 0.05). Treatment with methotrexate (5mg/kg) also significantly reversed these adjuvant changes (p < 0.05). However, effect of methotrexate was less marked as compared to mangiferin (30mg/kg) alone and in combination with L-NAME (10mg/kg), but was comparable or slightly better than mangiferin (10 and 20mg/kg). Thus, on the basis of our findings, we can suggest that interaction of mangiferin with nitric oxide modulators may have therapeutic value for chronic inflammatory disease such as RA.

THERAPEUTIC ACTIVITY OF POLYHERBAL FORMULATION AGAINST PASSIVE ANAPHYLAXIS BY USING RAT MESENTERIC MAST CELLS

Anaphylaxis is the result of alteration in mast cell physiology and is responsible for the various physiological changes which are observed during anaphylaxis. The symptoms of anaphylaxis are due to the release of the histamine from the mast cells and it results mainly due to the degranulation of the mast cells. Antianaphylactic activity of polyherbal formulation against passive anaphylaxis was evaluated with the help of sheep serum induced passive anaphylaxis model and passive paw anaphylaxis model. Sheep serum induced passive anaphylaxis model was carried out by wistar rats. The selected rats were administered intraperitoneally with serum of active anaphylactic rats. After 48 h all the rats are administered intraperitoneally with 1ml of sheep serum. The rats which are pre-treated with prednisolone (10 mg), polyherbal formulation (250 mg) were analyzed for the mast cell degranulation. Passive paw anaphylaxis model was carried out by subcutaneous administration of 0.1 ml serum of active anaphylactic rats into the plantar region of left hind paw of rats. After 24 h the left hind paw of rats was administered subcutaneously with 0.1 ml of egg albumin. The rats which are pre-treated with prednisolone (10 mg), polyherbal formulation (250 mg) were analyzed for the increase in paw volume. Antianaphylactic activity of polyherbal formulation on the mesenteric mast cells of the rat may be possibly due to the membrane stabilizing potential.

Anti-inflammatory activity and toxicological effect of Securidaca longepedunculata (Fresen.) root bark extract in albino rats

This study investigated the anti-inflammatory potential and toxicological effect of Securidaca longepedunculata root bark methanol extract in formalin-induced paw oedema in albino rats. Thirty-five male albino rats divided into 7 groups of five rats each (groups 1-7) were used for the study. Paw oedema was induced in groups 1-5 by injection of 0.1ml of 2% formalin in the sub planter region of left hind paw. Three of the induced groups (1-3) were treated with 100, 200 and 300mg/kg of the 70% methanol extract of S. longepedunculata respectively. The fourth group was treated with indomethacin while the fifth group was the positive control. Groups 6 and 7 served as the drug and normal control respectively. Treatment with various doses of S. longepedunculata resulted in marked decrease of paw oedema after the six days of treatment. Rats treated with 100 and 300mg/kg of the extract had the highest (93.01%) and least (77.25%) percentage inhibition respectively. Dose-dependent increase in levels of AST, ALT and GGT in rats treated with the extract coupled with mild hepatocellular vacuolations observed in the histology of the liver was indicative of liver impairment. Elevated level of white blood cells, eosinophils and lymphocytes in the treated groups are suggestive of their roles in combating inflammation. The root extract of S. longepedunculata showed anti-inflammatory activity in rats and can therefore be a good alternative drug in the management of inflammatory diseases.

Pharmacological studies on the anti-inflammatory and immunomodulatory role of pentoxifylline and its interaction with nitric oxide (NO) in experimental arthritis in rats.

Present study was designed to evaluate protective effects of pentoxifylline and its potentiation with low dose of nitric oxide (NO) modulators in adjuvant-induced experimental arthritis in rats. Wistar rats (200-300g, n=8 per group) of both sexes were used in the study. On day "0" experimental arthritis was induced by injecting 0.2ml of Complete Freund's adjuvant (CFA) in sub-planter region of right hind paw of animals. Pentoxifylline treatment alone and in combination with NO modulators was given (i.p.) from day 14 to 28. Various arthritic parameters were recorded and blood and joint synovial fluid was collectedfor biochemical analysis. CFA inoculation significantly increases (1) arthritic index (2) ankle diameter (3) paw volume (4) histopathology score (5) serum TNF-α, IL-6, IL-1β and synovial TNF-α levels (p<0.001) (6) serum Th1 and Th2 cytokine levels g) MDA levels in rat paw tissue homogenates (7) serum NF-κB levels. Significant decrease in serum IL-10 levels and SOD activity was observed in rats after CFA inoculation. Decrease in body weight and suppressed general quality of life of CFA inoculated rats was also observed. These CFA-induced arthritic changes were significantly reversed by pentoxifylline alone and in combination with low dose of NO modulators (p<0.05). These results are suggestive of protective effects of pentoxifylline and its potentiation in combination with low dose of NO modulators. These results may provide new pharmacological therapy for management of rheumatoid arthritis (RA).

Low-Dose Cannabinoid Type 2 Receptor Agonist Attenuates Tolerance to Repeated Morphine Administration via Regulating μ-Opioid Receptor Expression in Walker 256 Tumor-Bearing Rats

Morphine is widely used in patients with moderate and severe cancer pain, whereas the development of drug tolerance remains a major problem associated with opioid use. Previous studies have shown that cannabinoid type 2 (CB2) receptor agonists induce morphine analgesia, attenuate morphine tolerance in normal and neuropathic pain animals, induce transcription of the μ-opioid receptor (MOR) gene in Jurkat T cells, and increase morphine analgesia in cancer pain animals. However, no studies of the effects of CB2 receptor agonists on morphine tolerance in cancer pain have been performed. Therefore, we investigated the effect of repeated intrathecal (IT) injection of the low-dose CB2 receptor agonist AM1241 on the development of morphine tolerance in walker 256 tumor-bearing rats. We also tested the influence of the CB2 receptor agonist AM1241 on MOR protein and messenger ribonucleic acid (mRNA) expression in the rat spinal cord and dorsal root ganglia (DRG). Walker 256 cells were implanted into the plantar region of each rat's right hindpaw. Tumor-bearing rats received IT injection of the CB2 receptor agonist AM1241 or antagonist AM630 with or without morphine subcutaneously twice daily for 8 days. Rats receiving drug vehicle only served as the control group. Mechanical paw withdrawal threshold and thermal paw withdrawal latency were assessed by a von Frey test and hot plate test 30 minutes after drug administration every day. MOR protein and mRNA expression in the spinal cord and DRG were detected after the last day (day 8) of drug administration via Western blot and real-time reverse transcription polymerase chain reaction. The data were analyzed via analysis of variance followed by Student t test with Bonferroni correction for multiple comparisons. Repeated morphine treatments reduced the mechanical withdrawal threshold and thermal latency. Coadministration of a nonanalgetic dose of the CB2 receptor agonist AM1241 with morphine significantly inhibited the development of morphine tolerance and increased the MOR protein expression in the spinal cord and DRG and mRNA expression in the spinal cord in tumor-bearing rats. Our findings indicate that IT injection of a nonanalgetic dose of a CB2 receptor agonist increased the analgesia effect and alleviated tolerance to morphine in tumor-bearing rats, potentially by regulating MOR expression in the spinal cord and DRG. This receptor may be a new target for prevention of the development of opioid tolerance in cancer pain.

Anaesthetic efficacy of unilamellar and multilamellar liposomal formulations of articaine in inflamed and uninflamed tissue

We compared the efficacy of articaine encapsulated in multilamellar and unilamellar liposomes with that of articaine with epinephrine, after infiltration into inflamed and uninflamed tissue in rats. We encapsulated 4% articaine in multilamellar (articaine:multi) and unilamellar (articaine:uni) liposomes and compared them with 4% articaine with 1:100 000 epinephrine (articaine:epinephrine), in inflamed (plantar incision into the hind paw) and uninflamed (infraorbital nerve block) tissue in rats. Anaesthetic formulations (0.1ml) were injected near the right infraorbital foramen in uninflamed tissue, where success and duration of anaesthesia were assessed by pinching the upper lip every 5minutes. For inflamed tissue the anaesthetic formulations (0.1ml) were injected laterally into a surgical wound made 24hours earlier in the plantar region of the rat's right hind paw. The degree of anaesthesia was assessed by application of forces laterally to the wound with electronic von Frey filaments. Articaine:uni resulted in less successful anaesthesia than both articaine:multi (p=1.1x10-5) and articaine:epinephrine (p=4.3x10-8) in uninflamed tissue, but there were no differences in duration or success of anaesthesia between articaine:epinephrine and articaine:multi. In inflamed tissue articaine:epinephrine gave significantly more effective anaesthesia for longer than articaine:uni (p=2.3x10-6), and articaine:epinephrine (p=1.8x10-6) formulations, which did not differ from each other. Multilamellar liposomal articaine could be an option for local anaesthesia in uninflamed tissues. However, articaine with epinephrine gave better results than liposomal formulations in inflamed tissue.

Study of anti-inflammatory effect of simvastatin in rats

Background: Inflammation is a complex reaction in tissues that consists mainly of response of blood vessels and leukocytes. Simvastatin is a hypolipedemic drug belonging to the class of statins. Statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in cholesterol synthesis, are widely prescribed for the treatment of hyperlipedemia. This study was conducted to evaluate the anti-inflammatory activity of simvastatin in albino rats using digital plethysmometer. Methods: Male wistar albino rats weighing between 200-250 gm were selected for the study and rats were randomly divided into groups (control, standard, and test). The anti-inflammatory activity was evaluated by using carrageenan induced paw edema volume by using digital plethysmometer. Control group rats were administered 0.2 ml of normal saline, whereas, test group rats were administered simvastatin 40 mg and standard group diclofenac 50mg as single dose half an hour before injecting 0.1 ml of 1% carrageenan to the sub-plantar region of hind paw of rat. The paw edema of each rat was measured at 0 hour and after 3 hours. Results: At a dose of 40 mg Simvastatin showed anti-inflammatory effect which is statically highly significant. Conclusions: However, the above preclinical experiments only give us an idea about the anti-inflammatory activity, but large scale clinical trials are necessary for final assessment.