Left Hind Paw Research Articles

Adverse effect of rheumatoid arthritis on male Wistar rat’s fertility: protective role of Costus extract

Rheumatoid arthritis (RA) is a systemic autoimmune complaint. Advanced treatments resort to the traditional herbal therapy. The aim of this study is to assess the protective effect of Costus extract on the fertility of male rats with Freund's adjuvant-induced rheumatoid arthritis. Thirty male adult Wistar rats (190-200 g) were divided into six groups. They were subdivided into three groups; group I was the control group that received distilled water, and groups II and III received two various doses of Costus extract (200 and 400 mg/kg, respectively) for 60 days. Another three groups were subjected to RA induction via Freund's adjuvant. Rats were injected a dose of 0.1 ml of Freund's complete adjuvant (FCA) in the planter area of the left hind paw and then subdivided into 3 groups. Group I of RA-induced rats were given distilled water. The other two groups were given orally (200 and 400 mg/kg dosage of extract, respectively) from the 2nd day of RA induction for 60 days. Sex organ relative weight, sperm concentration assay, testicular histopathology and immunohistochemistry of androgen receptors, TNF α, and BAX protein were determined. The results showed that RA caused a significant decrease in the relative weight of sex organs and sperm count, which were relatively improved by doses of Costus (200, 400 mg/kg). RA induction caused testicular degeneration which markedly enhanced with Costus treatment as shown in histopathological sections. RA caused a reduction in %IHC of androgen receptors and increased expression level of both TNF α and BAX protein. Using IHC, it was revealed that RA caused a reduction in the expression level of androgen receptors and an increase in the expression of both TNF α and BAX protein. We can conclude that Costus speciosus had a potentially valuable role in improving fertility disorders caused by RA.

Influence of Intragastric Administration of Traditional Japanese Medicine, Ninjin'Yoeito, on Cerebral Blood Flow via Muscarinic Acetylcholine Receptors.

Ninjin'yoeito (NYT) is a traditional medicine that has been used for mitigating physical frailty, such as fatigue and anorexia, as well as for cognitive dysfunction. Maintenance of adequate cerebral blood flow (CBF) is important for preventing cognitive dysfunction. The present study aimed to examine the effect of NYT on CBF. Male C57BL/6 J mice were anesthetized with urethane and were artificially ventilated. We measured CBF in the neocortex with laser-speckle contrast imaging for 10 min before administration and 60 min after administration. We administered NYT solution (0.25, 0.5, 1, and 2 g/kg) or vehicle (distilled water; DW) over 5 min via an intragastric catheter. We surgically transected the vagus nerve to investigate its contribution as a neural pathway and intraperitoneally injected atropine to block muscarinic acetylcholine receptors. Finally, we tested the CBF response to cutaneous brushing stimulation applied to the left hind paw (30 sec). CBF decreased after DW administration, starting from 30 min onward, whereas CBF did not change after NYT. The averaged CBF change following DW administration differed from that following NYT (1 g/kg) but not from those following the other doses of NYT. Arterial pressure was not affected by either solution. CBF after NYT (1 g/kg) was not affected by vagotomy but was lower following additional atropine. In response to brushing stimulation, CBF in the right (contralateral) parietal cortex increased. The magnitude of CBF increase following NYT was greater than that following DW. These results suggest that NYT prevents CBF decrease via cholinergic activation independent of vagal activity and enhances the CBF response to somatosensory stimulation.

Ononis spinosa alleviated capsaicin-induced mechanical allodynia in a rat model through transient receptor potential vanilloid 1 modulation

BackgroundTransient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel implicated in pain sensation in response to heat, protons, and capsaicin (CAPS). It is well established that TRPV1 is involved in mechanical allodynia. This study investigates the effect of Ononis spinosa (Fabaceae) in CAPS-induced mechanical allodynia and its mechanism of action.MethodsMechanical allodynia was induced by the intraplantar (ipl) injection of 40 µg CAPS into the left hind paw of male Wistar rats. Animals received an ipl injection of 100 µg O. spinosa methanolic leaf extract or 2.5% diclofenac sodium 20 minutes before CAPS injection. Paw withdrawal threshold (PWT) was measured using von Frey filament 30, 90, and 150 minutes after CAPS injection. A molecular docking tool, AutoDock 4.2, was used to study the binding energies and intermolecular interactions between O. spinosa constituents and TRPV1 receptor.ResultsThe ipsilateral ipl injection of O. spinosa before CAPS injection increased PWT in rats at all time points. O. spinosa decreased mechanical allodynia by 5.35-fold compared to a 3.59-fold decrease produced by diclofenac sodium. The ipsilateral pretreatment with TRPV1 antagonist (300 µg 4-[3-Chloro-2-pyridinyl]-N-[4-[1,1-dimethylethyl] phenyl]-1-piperazinecarboxamide [BCTC]) as well as the β2-adrenoreceptor antagonist (150 µg butoxamine) attenuated the action of O. spinosa. Depending on molecular docking results, the activity of the extract could be attributed to the bindings of campesterol, stigmasterol, and ononin compounds to TRPV1.ConclusionsO. spinosa alleviated CAPS-induced mechanical allodynia through 2 mechanisms the direct modulation of TRPV1 and the involvement of β2 adrenoreceptor signaling.

Stereological Study of Changes of GABA-Immunoreactive Neurons in Spinal Dorsal Horn of SNI Rats.

Objective To observe the changes in the mechanical withdrawal threshold (MWT) and the proportion of GABA-immunoreactive neurons in spinal dorsal horn (SDH) of the spared nerve injury (SNI) rat model. Methods Thirty-six healthy male SD rats were randomly divided into a sham-operated group (group D, n = 18) and an SNI group (group S, n = 18). The left sciatic nerve trunk and three branches were exposed, two of which, known as tibial and the peroneal nerve, were ligated and cut off. The sural nerve was preserved to build the SNI model in group S. The left sciatic nerve trunk and three branches were only exposed in group D. MWT tests were performed on the medial and lateral sides of the rats' left hindpaw 1 day before surgery and at 7th, 14th, and 28th day after surgery. Results In group S, compared with the baseline measured 1 day before surgery, MWT on the medial and lateral sides of the rats' left hindpaw decreased significantly on the 7th, 14th, and 28th days after surgery (P < 0.05), while in group D, there was no statistically significant difference (P > 0.05). Compared with right SDH, there were not statistically significant reductions in the proportions of GABAergic neurons of left SDH on 7th and 28th day after SNI (P > 0.05); however, the proportion of GABAergic neurons in left SDH significantly decreased, compared with that in right side on 14th day after SNI (P < 0.05). On the same way, the proportions of GABAergic neurons on 7th, 14th, and 28th day after surgery were not statistically different (P > 0.05) in group D. Conclusion The SNI model could reduce the proportion of GABA-immunoreactive neurons in the rat's spinal dorsal horn on the nerve-injured side, and this change was lasting, which might be related to the transformation of the GABA-immunoreactive neurons.

Evaluation of the Antinociceptive, Antiallodynic, Antihyperalgesic and Anti-Inflammatory Effect of Polyalthic Acid.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are very commonly used, but their adverse effects warrant investigating new therapeutic alternatives. Polyalthic acid, a labdane-type diterpenoid, is known to produce gastroprotection, tracheal smooth muscle relaxation, and antitumoral, antiparasitic and antibacterial activity. This study aimed to evaluate the antinociceptive, antiallodynic, antihyperalgesic and anti-inflammatory effect of polyalthic acid on rats. Moreover, the effectiveness of treating hyperalgesia with a combination of polyalthic acid and naproxen was analyzed, as well as the type of drug–drug interaction involved. Nociception was examined by injecting 1% formalin into the right hind paw and thermal hyperalgesia and inflammation by injecting a 1% carrageenan solution into the left hind paw of rats. Allodynia was assessed on an L5/L6 spinal nerve ligation model. Polyalthic acid generated significant antinociceptive (56–320 mg/kg), antiallodynic (100–562 mg/kg), and antihyperalgesic and anti-inflammatory (10–178 mg/kg) effects. Antinociception mechanisms were explored by pretreating the rats with naltrexone, ODQ and methiothepin, finding the effect blocked by the former two compounds, which indicates the participation of opioid receptors and guanylate cyclase. An isobolographic analysis suggests synergism between polyalthic acid and naproxen in the combined treatment of hyperalgesia.

Mesenchymal stem cell derived extracellular vesicles as treatment for osteoarthritis in a rat high fat diet groove model

Purpose: Mesenchymal stromal/stem cells (MSC) hold a great promise to control the imbalance between anabolic and catabolic processes in an OA joint due to their immunomodulatory and regenerative capacities. Recently, intra-articular injections with human MSC entered the clinical trials as a novel, low invasive and relatively cheap therapy for OA. Although, the results of this novel treatment are promising, it remains unclear how MSC exert their beneficial effects in inflammation and tissue (cartilage) repair. Increasing evidence suggests that the therapeutic efficacy of MSC depends on paracrine signaling. Recently we have shown that MSC-derived extracellular vesicles (MSC-EVs) are an important component of this signaling and have a major contribution to cartilage regeneration and inhibition of TNF-alpha stimulated inflammatory responses in chondrocytes in vitro. Extracellular vesicles (EVs) are secreted by all cell types and range in size from 40 - 100 nm (exosomes) and from 100 -1000 nm (microvesicles). They increasingly appear to play an important role in intercellular communication by transportation of important molecules (peptides, mRNA, miRNA, enzymes). MSC-derived EVs have a great potential to serve as a safer cell free therapy for OA. The purpose of this study was to investigate if MSC-derived EVs can inhibit inflammation and promote cartilage repair to a similar extent as MSCs in a rat high fat diet groove model of OA. Methods: 36 male Wistar-han (Crl:WI(Han) rats were fed a high fat diet for 24 weeks, starting at an age of 12 weeks old. In week 12 of the study the rats received unilateral surgery where local cartilage damage was induced at the right knee joint by making five longitudinal grooves on the femoral condyles. In week 13 the rats were randomized into 3 groups and given an intraarticular injection with either PBS, 2 million human bone marrow derived MSCs or EVs derived from 2 million MSCs. The extracellular vesicles group received 5 of these doses with 5 days interval. Before, during and after the injections pain behavior was monitored by dynamic weight bearing and via measuring the left and right hind paw withdrawal responses to the application of von Frey hairs. uCT scans were made at week 0 and week 24, and used to determine the number of osteophytes and cartilage mineralization in the tibia. Additionally, tibial subchondral plate thickness, trabecular bone thickness, and trabecular bone volume fraction (total volume/trabecular bone volume) were measured using ImageJ 1.47v. At endpoint animals were sacrificed and the knee joints were harvested. Joint tissue is currently under preparation for histological staining to determine the OARSI histopathological score for rats. Data is represented as mean +/- standard deviation (SD) and the difference between the means in the three groups was statistically tested using one way ANOVA. Results: Results from the von Frey tests showed a clear decrease in 50% withdrawal threshold in the right hind paw, which was expected as the right side was the grooved limb. However, no difference between the three treatment groups in either left or right paw was seen. The mean osteophyte number in the PBS group was 4.7 (+/- 0.65) versus 3.6 (+/-2.15) in the MSC group and 4.3 (+/-1.8) in the MSC-EV group (figure 1A). In the MSC and MSC-EV treatment groups the variation in osteophyte number and total osteophyte diameter between the animals was large compared to the PBS group. Cartilage mineralization were observed in 9 of 12 animals in the PBS and MSC group and in 5 of 12 animals in the MSC-EV group. Both the number and total diameter was lowest in the MSC-EV group, but there was no significant difference as compared with control groups (Figure 1B). Other parameters measured on uCT like bone volume fraction, subchondral plate and trabecular bone thickness were similar between the 3 groups. Results from the OARSI histopathological score for rats is currently under preparation. Conclusions: Preliminary results from this in vivo study show no significant differences between PBS, MSC or MSC-EV treatment on uCT data or von Frey test. However, we do observe a large spread in osteophyte score and total diameter in the MSC and MSC-EV treated groups, which could be a sign that individual animals are responding differently to the treatments. Data from the OARSI scoring will help us determine if the response really is variable for MSC and MSC-EV treatment groups. This would be in accordance with observations made in humans, as many clinical trials that test MSC-based therapies report a substantial nonresponder group. In summary, although osteophytes are one of the hallmarks of osteoarthritis, we feel a complete picture of the joint damage including cartilage and synovium is needed to draw conclusions on the effect of the treatments in this study.

Piroxicam reduces acute and chronic pain response in type 1 diabetic rats.

Pain associated with various underlying pathologies is a major cause of morbidity and diminished life quality in diabetic patients. Effective control of pain requires the use of analgesics with the best efficacy and with minimal side effects. Therefore, our aim in this study was to investigate the effects of non-steroidal anti-inflammatory drugs (NSAIDs) on pain in diabetic rats. In this study, we investigated the analgesic effects of drugs belonging to three different classes of NSAIDs in a rat model of diabetes. Four diabetic groups received normal saline, diclofenac, piroxicam and ketorolac, respectively, and four non-diabetic groups received normal saline, diclofenac, piroxicam and ketorolac. Type 1 diabetes was induced in rats by a single injection of streptozotocin (60mg/kgbw). Formalin (50µL of 2.5%) nociception assay was used to examine the effect of treatment with diclofenac, piroxicam and ketorolac on acute and chronic pain in healthy and diabetic rats. Piroxicam showed significant analgesic effects both in the acute phase of pain (5-10min after injection of formalin into the left hind paw), and in the chronic phase (20-60min after formalin injection) in healthy as well as diabetic rats. Diclofenac and ketorolac also reduced pain scores in healthy rats. However, these two drugs failed to diminish pain in diabetic rats. Our data point for better efficacy of piroxicam in controlling pain in diabetes.

Resistance of Food-Maintained Operant Responding to Mechanical Punishment in Rats: Further Evidence for Weak "Affective/Motivational Pain" in Rat Models of Inflammatory and Neuropathic Pain.

Clinically relevant chronic pain is often associated with functional impairment and behavioral depression as an “affective/motivational” sign of pain; however preclinical animal models of inflammatory and neuropathic pain often produce weak evidence of impaired function. We hypothesized that hindpaw mechanical stimulation produced by a requirement to rear on a textured “NOX” plate would punish operant responding in rats treated with intraplantar complete Freund’s adjuvant (CFA, a model of inflammatory pain) or the chemotherapeutic paclitaxel (PTX, a model of neuropathic pain) and produce sustained pain-related depression of operant behavior. Male Sprague–Dawley rats were trained under a progressive-ratio (PR) schedule of food-maintained operant responding, then treated with CFA (100 µL in left hindpaw), PTX (2.0 mg/kg IP on alternate days for four total injections; 6.6 mg/kg IV on alternate days for three total injections), or saline vehicle. PR break points and mechanical thresholds for paw withdrawal from von Frey filaments were then tracked for 28 days. Subsequently, rats were tested with the opioid receptor antagonist naltrexone to assess latent sensitization and with the kappa opioid receptor (KOR) agonist U69593 to assess KOR function. CFA produced significant mechanical hypersensitivity for 3 weeks but decreased PR breakpoints for only 1 day. Both IP and IV PTX produced mechanical hypersensitivity for at least three weeks; however, only IV PTX decreased PR breakpoints, and this decrease was not alleviated by morphine. After recovery, naltrexone reinstated mechanical hypersensitivity in CFA- but not PTX-treated rats, and it did not reinstate depression of breakpoints in any group. U69593 dose-dependently decreased PR breakpoints in all groups with no difference between control vs. CFA/PTX groups. These results suggest that rearing on a textured NOX plate was not sufficient to punish operant responding in CFA- and PTX-treated rats despite the presence of sustained mechanical hypersensitivity. The rapid recovery of operant responding could not be attributed to latent sensitization, KOR downregulation, or behavioral tolerance. These results extend the range of conditions under which putative chronic pain manipulations produce weak evidence for depression of operant responding as a sign of the “affective/motivational” component of pain in rats.

Methadone effectively attenuates acute and long-term consequences of neonatal repetitive procedural pain in a rat model.

Painful procedures in early life cause acute pain and can alter pain processing at a spinal level lasting into adulthood. Current methods of analgesia seem unable to prevent both acute and long-term hypersensitivity associated with neonatal pain. The current study aims to prevent acute and long-term hypersensitivity associated with neonatal procedural pain using methadone analgesia in rat pups. Sprague-Dawley rat pups received either methadone (1 mg/kg) or saline prior to repetitive needle pricks into the left hind paw from the day of birth (postnatal day (P)0) to P7. Control littermates received a tactile stimulus. Mechanical sensitivity was assessed during the neonatal period (P0-P7), from weaning to adulthood (3-7 weeks) and following surgical re-injury of the same dermatome in adulthood. Methadone administration completely reversed acute hypersensitivity from P0 to P7. In addition, neonatal methadone analgesia prevented prolonged hypersensitivity after re-injury in adulthood, without affecting sensitivity from weaning to adulthood. The current study shows that neonatal methadone analgesia can attenuate acute as well as long-term hypersensitivity associated with neonatal procedural pain in a rat model. Methadone treatment attenuates acute and long-term hypersensitivity associated with neonatal pain in a rat model. Clinical effectiveness studies are urgently warranted to assess acute and long-term analgesic effectivity of methadone.

Galanin Receptor 2 Is Involved in Galanin-Induced Analgesic Effect by Activating PKC and CaMKII in the Nucleus Accumbens of Inflammatory Pain Rats.

It has been reported that galanin has an analgesic effect via activating galanin receptors (GALRs). This study focused on the involvement of GALR2 in the galanin-induced analgesic effect and its signaling mechanism in the nucleus accumbens (NAc) of inflammatory rats. Animal models were established through injecting carrageenan into the plantar of rats’ left hind paw. The results showed that GALR2 antagonist M871 weakened partially the galanin-induced increases in hind paw withdrawal latency (HWL) to thermal stimulation and hind paw withdrawal threshold (HWT) to mechanical stimulation in NAc of inflammatory rats. Moreover, the GALR2 agonist M1145 prolonged the HWL and HWT, while M871 blocked the M1145-induced increases in HWL and HWT. Western blotting showed that the phosphorylation of calcium/calmodulin-dependent protein kinase II (p-CaMKII) and protein kinase C (p-PKC) in NAc were upregulated after carrageenan injection, while p-PKC and p-CaMKII were downregulated after intra-NAc administration of M871. Furthermore, the CaMKII inhibitor KN93 and PKC inhibitor GO6983 attenuated M1145-induced increases in HWL and HWT in NAc of rats with inflammatory pain. These results prove that GALR2 is involved in the galanin-induced analgesic effect by activating CaMKII and PKC in NAc of inflammatory pain rats, implying that GALR2 agonists probably are potent therapeutic options for inflammatory pain.

Cymbopogon citratus mitigates neurobehavioral impairments via inhibition of oxidative stress and inflammatory changes in mice submitted to complete Freund adjuvant

Cymbopogon citratus aqueous leaf extract (CYC) otherwise known as lemongrass tea is used for achy joints (rheumatism) and central nervous system disorders in ethnomedicine. This study was designed to investigate the effects of CYC on neurobehavioral, oxidative, and inflammatory changes produced by complete Freund adjuvant (CFA) in male Swiss mice. The mice were allotted into 6 groups (n = 6). The animals in group 1 received saline (control), group 2 also had saline (CFA-control), groups 3–5 received CYC (50, 100, or 200 mg/kg), whereas group 6 were given celecoxib (20 mg/kg) orally for 14 consecutive days. Mice in groups 2–6 also received 0.1 mL injection of CFA (10 mg/mL) into the left hind paw 30 min earlier on day 1. The paw volumes were measured on days 0, 7, and 14. Neurobehavioral changes were evaluated on day 14. Thereafter, the left hind paw tissues were processed for estimation of malondialdehyde, nitrite, glutathione, tumor necrosis factor-alpha, and interleukin-6 contents. The increase in paw volume and weight produced by CFA was reduced by CYC (p < 0.05). CYC attenuated postural instability, anxiety, depression, memory deficits, and nociceptive responses in CFA-mice. The CFA-induced increases in malondialdehyde and pro-inflammatory cytokines accompanied by decreased glutathione contents in mouse hind paw were attenuated by CYC (p < 0.05). The findings that CYC reduces inflammatory edema, neurological deficits, nociception, biomarkers of oxidative stress, and release of inflammatory cytokines in CFA-treated mice further supports its acclaimed benefit in arthritic pain in ethnomedicine.

Antiarthritic Potential of Calotropis procera Leaf Fractions in FCA-Induced Arthritic Rats: Involvement of Cellular Inflammatory Mediators and Other Biomarkers

Calotropis procera (commonly known as Swallow wort) is described in the Ayurvedic literature for the treatment of inflammation and arthritic disorders. Therefore, in the present work, the antiarthritic activity of potential fractions of Swallow wort leaf was evaluated and compared with standards (indomethacin and ibuprofen). This study was designed in Wistar rats for the investigation of antiarthritic activity and acute toxicity of Swallow wort. Arthritis was induced in Wistar rats by injecting 0.1 mL of Freund’s complete adjuvant (FCA) on the 1st and 7th days subcutaneously into the subplantar region of the left hind paw. Evaluation of our experimental findings suggested that antiarthritic activity of methanol fraction of Swallow wort (MFCP) was greater than ethyl acetate fraction of Swallow wort (EAFCP), equal to standard ibuprofen, and slightly lower than standard indomethacin. MFCP significantly reduced paw edema on the 17th, 21st, 24th, and 28th days. It also showed significant effect (p &lt; 0.01) on arthritic score, paw withdrawal latency, and body weight. The inhibition of serum lysosomal enzymes and proinflammatory cytokines along with improvement of radiographic features of hind legs was also recorded with MFCP. Finally, it was concluded that MFCP can be a feasible therapeutic candidate for the treatment of inflammatory arthritis.

Pharmacological screening of ethanolic extract of Pithecellobium dulce for antiarthritic activity in Rats

Arthritis is an inflammatory joint disorder in which cartilage of the joint is gradually lost and categorized by swelling of joints, pain, and loss of function. The present study was conducted to explore the antiarthritic activity of ethanolic extract of Pithecellobium dulce against formaldehyde induced at sub plantar region of the left hind paw during days 1 and 3 of the study period. The changes observed in paw diameter during the study period, various biochemical, and haematological parameters were monitored. The Radiographic analysis and histopathology significantly improved after treatment with test extract P. dulce (250 mg/kg, b.w., p.o. ) as compared to the standard treatment with indomethacin (10 mg/kg, b.w., p.o. ).The results of the current investigation concluded that ethanolic extract of P. dulce possesses significant anti-arthritic activity against formaldehyde induced arthritis model, justifying its therapeutic role in arthritic conditions. The observed antiarthritic activity may be due to the presence of phytoconstituents such as alkaloids and flavonoids. P. dulce significantly suppressed the paw oedema in formaldehyde models ( P &lt;0.001). The Histopathological and radiographic studies of joints also showed a protective effect of P. dulce .

Evaluation of efficacy of Ocimum sanctum, Azadirachta indica, and their combination in comparison with aspirin on acute inflammatory pain

Background: Aspirin, a potent drug used for acute inflammatory pain, is also associated with various adverse effects, whereas some Indian herbs such as Ocimum sanctum and Azadirachta indica are known to have efficacy against such pain with less serious adverse effects. Aims and Objectives: This study aims to evaluate efficacy of Ocimum sanctum, Azadirachta indica, and their combination in acute inflammatory pain in comparison with aspirin. Materials and Methods: Wistar albino rats of either sex (150–250 g) were divided into five groups with six rats in each group. To induce inflammation, formalin (2.5%, 0.1 ml) was injected into subplantar region of the left hind paw of rats. The study groups were administered orally with distilled water (3 ml), aspirin (200 mg/kg), O. sanctum (400 mg/kg), A. indica (500 mg/kg), and their combination (combination of O. sanctum and A. indica having 400 mg/kg O. sanctum + 500 mg/kg A. indica) half an hour before administration of formalin. Effect of test drugs was assessed by the formalin test, where rats were observed for a period of 40 min to record any response after formalin injection. Two responses were observed ~ time spent in paw licking and number of paw lifting in early/neurogenic and late/inflammatory phases. Results (mean ± SD) were analyzed using one-way ANOVA test followed by Tukey’s HSD test. Results: A. indica had produced a comparable effect to aspirin on inflammatory phase of pain. Conclusion: A. indica could be used as an add-on therapy to conventional drugs for acute inflammatory pain. However, clinical studies on humans would be required to test its accurate efficacy.

Antinociceptive potential of methanol leaf extracts of Cissampelos parreira (Linn), Lantana camara (Linn) and Ocimum gratissimum (African basil)

The effects of Cissampelos pareira (Linn), Lantana camara (Linn) and Ocimum gratissimum (African basil) leaf extracts on pain have not been biologically determined despite their frequent traditional use in pain management. The present study evaluated the effects of methanol leaf extracts of these three plants on formalin-induced pain in Swiss albino mice. Leaves of C. pareira, L. camara and O. gratissimum were harvested, cleaned, shade dried, crushed, extracted in absolute methanol and concentrated to dryness. The quantitative phytochemical screening of the three plant extracts was first carried out. Then, the pain assay tests constituted eight groups of five mice each: normal control group, positive control group, negative control group and experimental groups of 50, 100, 150, 200 and 250mg/kg bw extracts. The animals were administered with various treatments thirty minutes before induction of pain through injection of 0.01ml of 2.5% formalin solution into the sub-plantar region of the left hind paw. Paracetamol at the dose of 50mg/kg bw and 5% dimethyl sulfoxide were used as the positive and negative controls respectively. The plant extracts were administered intraperitoneally and orally. Data was analyzed using one-way analysis of variance and unpaired t-test. Phytochemical screening on separate extracts of C. pareira, L. camara and O. gratissimum revealed fatty acids, phenols, flavonoids and terpenoids. The different dosages of methanol leave extracts of C. pareira, L. camara and O. gratissimum reduced pain significantly (p˃0.05) in mice. The significant reduction of pain was associated with fatty acids, phenols, flavonoids and terpenoids revealed in the plant extracts.

Research of low molecular fraction of C. albicans fungus cells by the ELISA in subcutaneous administration

The purpose of this work is a study of low molecular weight fraction with C. albicans protein concentrations of 1, 2, 3, and 4 mg/ml by antibody titers in subcutaneous administration. A low molecular fraction of C. albicans proteins in concentration 1, 2, 3, 4, and 5 mg/ml has been studied in white mice. Mice have subcutaneously injected 0.2 ml of the test fraction into the upper part of the right hind paw. After 14 days, 0.2 ml of test fractions were injected repeatedly into the upper part of the left hind paw. 1 month for one group and 3 months for the second group of experimental animals after the second injection intraperitoneal infection of the animals was carried out. After 14 days, determination of the protective functions of the animal body by titer of C. albicans specific antibodies has been performed by enzyme-linked immunoassay. Studies have shown that the low molecular weight fraction of C. albicans fungi cells antigens at the double subcutaneous injection of 0.2 ml does not provide the activation of immune mechanisms. The low molecular weight fraction of C. albicans proteins at subcutaneous administration does not activate the body's defense mechanisms in preventing candidiasis.

Phospholipase Cβ3 Expressed in Mouse DRGs is Involved in Inflammatory and Postoperative Pain.

BackgroundPrevious studies suggested that phospholipase Cβ3 (PLCβ3), which is a common downstream component in the signaling cascade, plays an important role in peripheral mechanisms of perception including nociception. However, detailed profiles of PLCβ3-expressing dorsal root ganglion (DRG) neurons and involvement of PLCβ3 in inflammatory and postoperative pain have not been fully investigated.PurposeWe evaluated neurochemical char0acteristics of PLCβ3-expressing DRG neurons in mice and then we examined the effects of selective knockdown of PLCβ3 expression in DRGs on inflammatory and postoperative pain.MethodsMale C57BL/6-strain mice were used. For the inflammatory model, each mouse received subcutaneous injection of complete Freund’s adjuvant (CFA) in the left hindpaw. For the postoperative pain model, a plantar incision was made in the left hindpaw. PLCβ3 antisense oligodeoxynucleotide or PLCβ3 mismatch oligodeoxynucleotide was intrathecally administered once a day for three consecutive days in each model. The time courses of thermal hyperalgesia and mechanical hyperalgesia were investigated. Changes in PLCβ3 protein levels in DRGs were evaluated by Western blotting.ResultsImmunohistochemical analysis showed that high proportion of the PLCβ3-positive profiles were biotinylated isolectin B4-positive or transient receptor potential vanilloid subfamily 1-positive. PLCβ3 protein level in DRGs during CFA-induced inflammation was comparable to that at baseline. Intrathecal administration of PLCβ3 antisense oligodeoxynucleotide, which significantly suppressed PLCβ3 expression in DRGs, did not affect pain thresholds in normal conditions but inhibited CFA-induced thermal and mechanical hyperalgesia both at the early and late phases compared to that in mismatch oligodeoxynucleotide-treated mice. Intrathecal administration of PLCβ3 antisense oligodeoxynucleotide also inhibited surgical incision-induced thermal and mechanical hyperalgesia.ConclusionOur results uncover a unique role of PLCβ3 in the development and maintenance of inflammatory pain induced by CFA application and in those of surgical incision-induced pain, although PLCβ3 does not play a major role in thermal nociception or mechanical nociception in normal conditions.

Local injury and systemic infection in infants alter later nociception and pain affect during early life and adulthood

Newborns in intensive care are regularly exposed to minor painful procedures at developmental time points when noxious stimulation would be normally absent. Pain from these interventions is inconsistently treated and often exists concurrently with systemic infection, a common comorbidity of prematurity. Our understanding of the independent and combined effects of early painful experiences and infection on pain response is incomplete. The main goals of this research therefore were to understand how pain and infection experienced early in life influence future nociceptive and affective responses to painful stimuli. Rat pups were infected with E-coli on postnatal day 2 (PN2) and had left hind paw injury with carrageenan on PN3. Standard thermal tests for acute pain, formalin tests for inflammatory pain, and conditioned place aversion testing were performed at different ages to assess the nociceptive and affective components of the pain response. Early E-coli infection and early inflammatory injury with carrageenan both independently increased pain scores following hind paw reinjury with formalin on PN8, with effects persisting into adulthood in the carrageenan exposed group. When experienced concurrently, early E-coli infection and carrageenan exposure also increased conditioned aversion to pain in adults. Effect of sex was significant only in formalin testing, with males showing higher pain scores in infancy and females showing higher pain scores as adults. These findings demonstrate that infection experienced early in life can alter both the nociceptive and affective components of the pain response and that there is a cumulative effect of local and systemic pro-inflammatory processes on the aversive component of pain.

Effect of moxibustion on expression of autophagy related molecules in synovial tissues of rheumatoid arthritis rats

To observe the effect of moxibustion on expression of autophagy related gene(Atg), serine/threonine protein kinase-uncoordinated 51 like kinase-1 (ULK1), Beclin1 and microtubule associated proteins light chain 3 (LC3) and ultrastructure of synovium in rheumatoid arthritis (RA) rats, so as to explore its mechanisms underlying improvement of RA. Forty SD rats were randomly divided into normal control, RA model, moxibustion, cigarette-roasting and medication groups (n=8 rats in each group). The RA model was established by keeping the rats in wind, cold and wet environment for 12 h, once a day for 20 days and subcutaneous injection of Freund's adjuvant complete into the sole of the left hind paw. Moxibustion was applied to the left "Zusanli" (ST36) for 20 min, once a day for 15 days. Rats of the cigarette-roasting group was treated by ignited cigarettes instead of moxa strips. Rats of the medication group was treated by gavage of Tripterygium wilfordii polyglycoside tablet suspension (0.8 mg/100 g) once a day for 15 days. The rats' paw volume of the left hindlimb was measured by using a water-based paw plethysmometer. The synovial tissue of the left plantar joint was harvested at the end of experiments for observing changes of the ultrastructure with transmission electron microscope, and the expression of ULK1, Atg3, Atg5, and Atg12 mRNAs was detected with quantitative real-time PCR and the expression of LC3-Ⅱ and Beclin-1 proteins were detected with Western blot. Following modeling, the paw volume of the left hindlimb was significantly increased (P<0.01), while the expression levels of Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins of the synovial tissue were notably down-regulated in the model group relevant to the normal control group (P<0.01). The increase of the paw volume in the moxibustion and medication groups and the down-regulation of synovial Atg3, Atg12 and ULK1 mRNAs in the 3 intervention groups, and Atg5 mRNA , and LC3-Ⅱ and Beclin-1 proteins in both moxibustion and medication groups were considerably suppressed (P<0.01, P<0.05). The therapeutic effect of moxibustion was apparently superior to that of cigarette-roasting in down-regulating the paw volume, and up-regulating the expression levels of Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins (P<0.05, P<0.01), and notably weaker than that of medication in up-regulating Atg3 and ULK1 mRNAs (P<0.01), but was comparable to that of medication in up-regulating the expression levels of Atg5 and Atg12 mRNAs, LC3-Ⅱand Beclin-1 proteins (P＞0.05). Results of the ultrastructural observation showed an obvious injury of synovial cells, such as unclear and incomplete nuclear membrane, chromatin condensation, swollen mitochondria with broken crests, cavitation-like degeneration of cytoplasma, and appearance of autophagosomes and lysosomes in the model group, which was relatively milder in the 3 intervention groups. Moxibustion can reduce the paw edema and inflammatory injury of the plantar synovial tissue in RA rats, which may be related to its effects in up-regulating Atg3, Atg5, Atg12 and ULK1 mRNAs, and LC3-Ⅱ and Beclin-1 proteins to enhance the cellular autophagy. The therapeutic effect of moxibustion is obviously superior to that of cigarette-roasting and medication in relieving swelling.

FUNCTIONAL ANALYSIS OF GAIT WITH INSOLE USE IN DIABETIC FEMALE RATS: A CROSS-SECTIONAL STUDY

ABSTRACT Introduction: Diabetes mellitus is a chronic disease that is characterized by causing damage to the peripheral nervous system, generating sensory and motor changes. Objective: This study aims at analyzing the impact of the use of different orthotic insoles on the gait of diabetic female rats. Methods: Twenty-six female Wistar rats were randomly divided into the Control and Diabetic groups. The mechanical sensitivity test was performed manually on the surface of the animals' hind paws using the von Frey test. The functional evaluation was carried out on an adapted platform where the animals were stimulated to walk in order to capture images of the ventral region for measurements of the right and left hind paws. After the images were collected they were processed using Kinovea software version 0.8.27 to assess: stride distance, time, speed and acceleration. Results: There was a reduction in the weight of the animals in the Diabetic Group (p = 0.0018), associated with hyperglycemia (p = &lt;0.0001), and a decrease in mechanical sensitivity as compared to the Control Group (p = 0.0372). Gait analysis showed a reduction in stride speed (p = 0.0482) and acceleration (p = 0.0149), with the silicone orthosis in the Diabetic Group. Conclusions: The silicone orthosis demonstrated a reduction in stride speed and acceleration, without compromising the other variables in the diabetic rats. The other insoles showed no functional difference between groups. Even though the animals showed a change in sensitivity at the end of 28 days of DM induction, this time does not appear to have been able to develop extensive changes in the rats' gait function. Level of evidence II; Therapeutic studies - Investigating the Results of Treatment.