Nrf2 Mediates Effect of Resveratrol in Ischemia-reperfusion Injury.

Ischemia-Reperfusion Injury (IRI) is a paradoxical phenomenon where removing the source of injury can cause additional damage. Ischemia reduces ATP production and intracellular pH, reducing oxidative reactions, increasing lactic acid release, and activating anaerobic metabolism. Reperfusion restores aerobic respiration and increases ROS production, leading to malfunction of transmembrane transport, activation of proteases, DNA dissolution, and protein denaturation, leading to apoptotic cell death. Nrf2 is a transcription factor that regulates cellular inflammation and oxidative responses. It is activated by oxidants and electrophiles and enhances detoxifying enzyme expression, maintaining redox homeostasis. It also activates ARE, which activates several ARE-regulated genes that favor cell survival by exhibiting resistance to oxidants and electrophiles. Nrf2 regulates the antioxidant defense system by producing phase II and antioxidant defense enzymes, including HO-1, NQO-1, gglutamylcysteine synthetase, and rate-limiting enzymes for glutathione synthesis. Nrf2 protects mitochondria from damage and supports mitochondrial function in stress conditions. Resveratrol is a stilbene-based compound with a wide variety of health benefits for humans, including antioxidant, anticarcinogenic, antitumor, and estrogenic/antiestrogenic. Resveratrol protects against IRI through several signaling pathways, including the Nrf2/ARE pathway. Here, we review the studies that investigated the mechanisms of resveratrol protection against IRI through modulation of the Nrf2 signaling pathway.

Targeting the NF-E2-related factor 2 pathway for overcoming leukemia.

Leukemia is cancer of the body's blood-forming tissues, including the bone marrow and the lymphatic system. There are many types of leukemia that some of them occur in children and the others are more common in adults. Currently, there are many different chemotherapy agents for leukemia while chemoresistance increases the survival of the leukemic cells. One of the main reasons of chemoresistance, is a transcription factor called Nuclear factor erythroid 2-Related Factor 2 (NRF2). An increase in NRF2 expression in leukemic cells which are being treated with chemotherapy agents, can increase the survival of these cells in the presence of therapeutics. Accordingly, the inhibition of NRF2 by different methods as a cotreatment with classical chemotherapy agents, can be a promising procedure in leukemia treatment. In this study we focus on the association of NRF2 and leukemia and targeting it as a new therapeutic method in leukemia treatment.

Polychlorinated biphenyls and thyroid function: a scoping review.

OBJECTIVE: Numerous evidence indicates the association between polychlorinated biphenyls (PCBs), an endocrine disrupter, with thyroid hormone disruption, contradictory findings also exist. Herein, we tried to address this question by performing a scoping review. CONTENT: The search was performed on PubMed, Scopus, Web of Science, and Google Scholar databases from 2010 onwards. Animal studies on PCBs' effect on thyroid function were searched. The SYRCLE's RoB scale assessed the risk of bias. I2 and Q tests are used for investigating heterogeneity. A random-effects model with the pooled standard means difference (SMD) and 95 % confidence interval (CI) was performed for the TSH, TT4, TT3, and FT4 outcomes using Comprehensive Meta-Analyses (CMA) Software version 3. Also, we conducted subgroup analyses based on the different types of PCB. The initial search identified 1,279 publications from the main databases 26 of them fulfilled our eligibility criteria for the study, and then five studies among selected studies had sufficient data for analysis. Meta-analysis of data revealed that Aroclor 1260 (SDM: -0.47, 95 % CI: -0.92, -0.01, p=0.044) and PCB 126 (SDM: 0.17, 95 % CI: -0.40, 0.75, p=0.559) significantly increased TSH concentration in the exposed groups vs. the control groups. Related to the effects of PCBs on the TT4, our findings indicated a significant reduction the TT4 concentration of animals exposed to Aroclor 1260 (SDM: -5.62, 95 % CI: -8.30, -2.94, p=0.0001), PCB 118 (SDM: -6.24, 95 % CI: -7.76, -4.72, p=0.0001), PCB 126 (SDM: -1.81, 95 % CI: -2.90, -0.71, p=0.001), and PCB 153 (SDM: -1.32, 95 % CI: -2.29, -0.35, p=0.007) vs. the controls. Our meta-analysis indicated a significant increase in TT3 concentration following exposure to PCB 118 and PCB 153 (SDM: -0.89, 95 % CI: -1.36, -0.42, p=0.0001, and SDM: -1.45, 95 % CI: -2.15, -0.75, p=0.0001, respectively). Aroclor 1254 and PCB 126 significantly decreased TT3 concentration (SDM: 1.25, 95 % CI: 0.29, 2.21, p=0.01 and SDM: 3.33, 95 % CI: 2.49, 4.18, p=0.0001, respectively). PCB 126 significantly decreased FT4 in the exposed groups vs. the control groups (SDM: -7.80, 95 % CI: -11.51, -5.35, p=0.0001). SUMMARY: Our findings showed an association between PCBs exposure and hypothyroidism in rodents, fish, and chicken embryos. OUTLOOK: Regarding to the most evidence of hypothyroidism effects of PCBs in animal species, it is necessary to consider large cohort studies to address the association between PCBs exposure and thyroid function impairment in humans.

The Role of Resveratrol in Aging and Senescence: A Focus on Molecular Mechanisms.

Resveratrol (Res), a polyphenol found in red wine, has been shown to decelerate aging, the progressive loss of physiological integrity and cellular senescence, characterized by the inability to progress through the cell cycle. No successful clinical trials have yet to be completed in humans on dose limitations. Yet, the potent anti-aging and anti-senescence efficacy of Res has been documented in several in vivo animal models. In this review, we highlight the molecular mechanisms of Res efficacy in anti-aging disorders, such as diabetes, neurodegenerative disorders, eye diseases, and cardiovascular diseases.

Protective effects of curcumin and its analogues via the Nrf2 pathway in metabolic syndrome.

Metabolic Syndrome (MetS) refers to a set of medical conditions including insulin resistance, central obesity, atherogenic dyslipidemia, and hypertension. Due to these dysregulations, if not treated, MetS could increase the risk of CVA, CVD, and diabetes. As described by WHO, CVD is the leading cause of mortality in the world which motivates researchers to investigate the management of its risk factors, especially MetS. It is reported that oxidative stress secondary to the abundant generation of free radicals oxygen species (ROS) and the ensuing altered redox status play an important role as a mediator in MetS. As a result, using new antioxidant agents with higher bioavailability has been proposed as an efficient treatment. Curcumin (a polyphenol of the diarylheptanoids class), which is used as a traditional medicine for various diseases including cardiovascular diseases and diabetes, is characterized by its antioxidant properties which, at least in part, are mediated via the activation of the Nrf2/ARE signaling pathway. Nrf2 is a transcription factor that plays a key role in regulating internal defense systems and increases antioxidant levels to decrease oxidative damage and cell apoptosis. Nrf2 expression and stability are enhanced by curcumin, leading to a higher rate of Nrf2 migration to the cell nucleus to regulate ARE gene expression, thus protecting cells against oxidative stress. In this article, we provide a comprehensive review of the molecular effect of curcumin and its derivatives via Nrf2 regulation in several conditions, such as diabetes, hypertension, dyslipidemia, and obesity.

Protective Effects of Chrysin on Hippocampal Damage Induced by Chlorpyrifos in Adult Rats.

OBJECTIVE: This study aimed to evaluate the possible effects of chlorpyrifos on the rat hippocampus and evaluate whether these effects can be decreased with chrysin co-administration in an animal model. METHODS: Male Wistar rats were randomly divided into 5 groups; Control (C), Chlorpyrifos (CPF), Chlorpyrifos + Chrysin (12.5 mg/kg) (CPF + CH1), Chlorpyrifos + Chrysin (25 mg/kg) (CPF + CH2), Chlorpyrifos + Chrysin (50 mg/kg) (CPF + CH3). After 45 days, hippocampus tissues were evaluated by biochemical and histopathological tests. RESULTS: Biochemical findings indicated that CPF and CPF plus CH administration could not significantly change SOD activity, and MAD, GSH, and NO levels in the hippocampus tissue of animals versus controls. Histopathological findings of the toxic effects of CPF on hippocampus tissue as evidenced by inflammatory cell infiltration, degeneration/necrosis, and mild hyperemia. CH could ameliorate these histopathological changes in a dose-dependent manner. CONCLUSION: In conclusion, CH was effective against histopathological damage induced by CPF in the hippocampus through modulating inflammation and apoptosis.

Pharmaceutical toxicity is a common pattern of inpatient acute poisonings in Birjand City, East of Iran.

Information on the pattern of acute poisonings in hospitals of Birjand city, Iran, is limited. This study aimed to address this knowledge gap by examining the admissions in a major poisoning center in eastern Iran. This cross-sectional study included patients admitted to the Imam Reza Hospital in Birjand over 12 months. Medical records of the poisoned patients were reviewed, and the study variables were used for data analysis. During the study period, 534 cases of acute poisonings were evaluated. The patient's ages ranged from 12 to 84 years, with a high rate of poisonings between 15 and 35 years. The female predominance in poisoning cases was 52.1%. Most cases of poisonings occurred in spring, and the common route of exposure was oral (93.1%). The incidence of poisoning in married couples, uneducated patients, and residents of urban areas was 56.5%, 90.1%, and 74.6%, respectively. Patients with a previous medical history experienced addiction and psychiatric disorders. Intentional poisoning accounted for 23.4% of acute poisoning cases referred to the hospital in the current study. The main groups of toxicants were pharmaceutical products (48.1%), narcotics (25.8%), chemical products (10.1%), envenomation (7.1%), and alcohol (1.7%). The mean hospital stay was 2.5 ± 3.0 days, and the final treatment outcome was complete recovery, except for one patient intoxicated by warfarin and alprazolam. Our results indicate that the high toxicity cases were related to pharmaceutical product and opioids abuse, especially methadone (8.4%), alprazolam (7.9%), clonazepam (7.5%), and acetaminophen (9.9%) taken orally and more commonly happened at home. Due to the high rate of deliberate poisonings, especially among young adults and students, monitoring drug distribution and exceptional attention to mental health should be seriously considered by national health authorities to prevent suicide attempts.

Toxic Mechanisms of Five Heavy Metals: Mercury, Lead, Chromium, Cadmium, and Arsenic.

The industrial activities of the last century have caused massive increases in human exposure to heavy metals. Mercury, lead, chromium, cadmium, and arsenic have been the most common heavy metals that induced human poisonings. Here, we reviewed the mechanistic action of these heavy metals according to the available animal and human studies. Acute or chronic poisonings may occur following exposure through water, air, and food. Bioaccumulation of these heavy metals leads to a diversity of toxic effects on a variety of body tissues and organs. Heavy metals disrupt cellular events including growth, proliferation, differentiation, damage-repairing processes, and apoptosis. Comparison of the mechanisms of action reveals similar pathways for these metals to induce toxicity including ROS generation, weakening of the antioxidant defense, enzyme inactivation, and oxidative stress. On the other hand, some of them have selective binding to specific macromolecules. The interaction of lead with aminolevulinic acid dehydratase and ferrochelatase is within this context. Reactions of other heavy metals with certain proteins were discussed as well. Some toxic metals including chromium, cadmium, and arsenic cause genomic instability. Defects in DNA repair following the induction of oxidative stress and DNA damage by the three metals have been considered as the cause of their carcinogenicity. Even with the current knowledge of hazards of heavy metals, the incidence of poisoning remains considerable and requires preventive and effective treatment. The application of chelation therapy for the management of metal poisoning could be another aspect of heavy metals to be reviewed in the future.

Health risk assessment of Cd, Cr, Cu, Ni and Pb in the muscle, liver and gizzard of hen's marketed in East of Iran.

The present study aimed to determine the average concentration of some metals, including cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni) and lead (Pb) in the chicken, hen's liver, and gizzard in the east of Iran. Estimated daily intake (EDI), target hazard quotient (THQ), hazard index (HI) and carcinogenic risk (CR) were calculated. In this cross-sectional study, fifty one samples including chicken, hen's liver and gizzard were obtained from Birjand, Iran. Measurement of Cd, Cr, Cu, Ni, and Pb was carried out by using an Inductively Coupled Plasma-Optic Emission Spectroscopy (ICP-OES). All of the measured metals were detected in 100 % of the samples. The metals had a different distribution pattern. The highest concentration of Cd and Cu was in the liver samples while the Cr and Ni had the highest levels in the chicken. Pb concentration was at the highest level in the gizzard. The least amount of Cr, Ni, and Pb was found in the liver while Cu had the least content in the muscle. EDI had an acceptable level, but the highest daily intake of all studied metals was through muscle. Cr had the highest THQ and it was more than one in the meat. HI in chicken was more than one. Liver and gizzard of hens had a neglectable HI. CR was neglectable in the case of both Cd and Pb, but it was considerable for Cr and Ni. The consumption of chicken in both adults and children may pose a significant health risk for consumers.

The association between diazinon exposure and dyslipidemia occurrence: a systematic and meta-analysis study.

The effects of diazinon (DZN), an organophosphate pesticide, on lipid profiles have been extensively reported. However, controversy on this issue persists. Here, we performed a systematic and meta-analysis study to investigate the association between DZN exposure and dyslipidemia in rodents and fish species. This systematic review was prepared according to the PRISMA guidelines. Main databases, including Google Scholar, Scopus, PubMed, Ovid MEDLINE, and Web of Science, were systematically searched through March 4, 2020. The risk of bias was evaluated with the SYRCLE's RoB tool. Once all articles were assessed for scientific quality, a random-effects model was applied to perform a pooled analysis. I2 and Q test were used to assess the heterogeneity between articles, and Forest plots, indicating point and pooled estimates, were drawn. Twenty-eight articles were included; between them, 13 publications were selected for meta-analysis. Random-effects meta-analysis showed low heterogeneity between the articles. A pooled analysis indicated that DZN significantly increased total cholesterol levels (95% CI: 0.86-3.79; Z = 3.10; p = 0.002), triglyceride (95% CI: 0.38-3.22; Z = 2.48; p = 0.09), low-density lipoprotein cholesterol (95% CI: 0.25-2.85; Z = 2.34; p = 0.7) in the DZN vs. control groups. In addition, DZN significantly decreased high-density lipoprotein cholesterol (95% CI: - 2.92, - 0.42; Z = 2.62; p = 0.07) in the DZN vs. control groups. No publication bias was observed. Our findings suggest that DZN induces dyslipidemia in rodents and fish species in a dose-dependent manner.

The effect of diazinon on blood glucose homeostasis: a systematic and meta-analysis study.

Though evidence exists on the association between diazinon (DZN), an organophosphate pesticide, with hyperglycemia, contrasting reports also exist. Herein, we performed a systematic and meta-analysis study to address this issue. A systematic search was conducted in PubMed, Ovid Medline, Google Scholar, Scopus, and Web of Science up to April 5, 2020, searching for animal studies (rodents and fish) that assessed the impact of DZN on blood glucose concentration. The risk of bias was assessed by the SYRCLE's RoB scale. Once each article's quality was assessed, a random-effects meta-regression was used to pool the data into a meta-analysis. Heterogeneity between the studies was evaluated with the I square and Q test. Random-effect meta-analysis of 19 studies (I2 = 90.5%, p < 0.001) indicated low heterogeneity between the studies. DZN significantly increased blood glucose levels in the exposed versus control groups (95% CI: 2.46-4.94; Z = 5.86; p < 0.001). Subgroup analysis indicated that the effect of high-dose (3.40 (95% CI: 2.03-4.76)) DZN on changes in blood glucose was more pronounced than in the low dose (4.83 (95% CI: 1.56-8.11)). It was also ascertained that the blood glucose level was significantly higher in females (3.55 (95% CI: 2.21-4.89)) versus males (4.87 (95% CI: 0.20-9.55)) exposed to DZN. No publication bias was observed. Sensitivity analysis showed the robustness of the (standardized mean differences: 3.26-4.03). Our findings establish an association between DZN exposure and hyperglycemia in rodents and fish, which is both dose- and gender-dependent.

Drinking water heavy metal toxicity and chronic kidney diseases: a systematic review.

Heavy metals in drinking water can threat human health and may induce several diseases. The association between heavy metals exposure and chronic kidney disease (CKD) has been indicated by few epidemiological studies. We conducted a systematic review of the epidemiologic publications of the association between exposure to heavy metals through drinking water and CKD. Keywords related to heavy metals and kidney diseases on MeSH were identified and searched in PubMed, Google Scholar, Scopus, Ovid-Medline and Web of Science until July 2020. 14 publications met our inclusion criteria and included in the current review. The included articles were conducted on the association between arsenic, cadmium, lead and chromium in drinking water and CKD. Our study could not find strong evidence between heavy exposure to through drinking water and CKD, except for arsenic. The negative association was found between arsenic and lead and glomerular filtration rate (eGFR). The positive correlation was observed between cadmium exposure and urinary N-acetyl-ß-d-glucosaminidase (NAG) concentrations, and also arsenic and chromium exposure and kidney injury molecule (KIM-1). Assessment of studies showed an association between arsenic, cadmium, lead and chromium and albuminuria and proteinuria, without CKD outcomes. Current systematic study showed few evidence for exposure to arsenic, cadmium, lead and chromium through drinking water and incidence of kidney problems. However, more epidemiological studies are required to confirm this association.

Electrochemical determination of atypical antipsychotic drug quetiapine using nano-molecularly imprinted polymer modified carbon paste electrode.

In this research, the advantages of molecularly imprinted polymer (MIP) materials have been used to develop a new electrochemical sensor for determination of quetiapine (QTP) drug. MIP nanoparticles were synthesized by precipitation polymerization method and used as QTP recognition elements in the composition of modified carbon paste electrode (CPE) for selective and sensitive assay of this drug. Cyclic voltammetry (CV) and square wave voltammetry (SWV) techniques were used for electrochemical analysis. Some parameters affecting the sensor performance were optimized and under optimal conditions, the proposed sensor showed linear responses with QTP concentration in the range of 1.6 × 10-8 to 2.5 × 10-6 M (R2 = 0.9964). The limits of detection (LOD) and quantification (LOQ) were calculated 5.04 × 10-9 M and 1.68 × 10-8 M respectively. Also, the amounts of %RSD for evaluation of repeatability and reproducibility of the proposed sensors were respectively obtained 2.19 and 3.02%. The method was successfully applied to determination of QTP in its pharmaceutical formulation and human urine samples.

Evaluation of organochlorine pesticide (OCP) residues in meat and edible organs, Iran.

Organochlorine pesticides (OCPs) are a more persistent and lipophilic group of insecticides. The present study aims to evaluate the residues of OCPs in meat, liver, and kidney of the cattle and sheep. A total of 54 samples were obtained from butcheries and local markets in Birjand, east of Iran. The residual contents of aldrin; dieldrin; dichlorodiphenyltrichloroethane; and its metabolites (DDTs), endosulfan isomers, endrin, hexachlorocyclohexane isomers (HCHs), heptachlor, heptachlor epoxide, chlordans, methoxychlor, and hexachlorobenzene (HCB), were analyzed in all samples. OCP residues were extracted by using the QuEChERS technique and quantified by gas chromatography-mass spectrometry (GC-MS). All of the analyzed OCPs were lower than the detection limit. The findings showed that meat and edible organs marketed in this region enjoyed a good status in terms of the residues of OCP. Monitoring of pesticide residues in meat and edible organs is necessary from the public health point of view.

Dietary Intake of Cadmium, Chromium, Copper, Nickel, and Lead through the Consumption of Meat, Liver, and Kidney and Assessment of Human Health Risk in Birjand, Southeast of Iran.

This study aimed to evaluate the mean concentration of cadmium (Cd), chromium (Cr), copper (Cu), nickel (Ni), and lead (Pb) in the meat and offal of cow and sheep. Also, the estimated daily intake (EDI) and health risk of these metals were calculated. Analysis of metals was undertaken by the use of an inductively coupled plasma-optic emission spectroscopy (ICP-OES). All samples were contaminated with all metals. Principal component analysis (PCA) showed a clear differentiation of cow and sheep in both the kidney and liver samples. In the liver and kidney, level of Cd, Cu, and Pb were positively correlated. The highest target hazard quotients (THQs) were calculated for Pb. Cd level in cow kidney had the highest carcinogenic rate (CR). Although, hazard index (HI) was lower than one, consumption of muscle especially in children should be noticed in both national and international consumers due to higher level of HI.

Peroxisomal dysfunctions cause lysosomal storage and axonal Kv1 channel redistribution in peripheral neuropathy.

Impairment of peripheral nerve function is frequent in neurometabolic diseases, but mechanistically not well understood. Here, we report a novel disease mechanism and the finding that glial lipid metabolism is critical for axon function, independent of myelin itself. Surprisingly, nerves of Schwann cell-specific Pex5 mutant mice were unaltered regarding axon numbers, axonal calibers, and myelin sheath thickness by electron microscopy. In search for a molecular mechanism, we revealed enhanced abundance and internodal expression of axonal membrane proteins normally restricted to juxtaparanodal lipid-rafts. Gangliosides were altered and enriched within an expanded lysosomal compartment of paranodal loops. We revealed the same pathological features in a mouse model of human Adrenomyeloneuropathy, preceding disease-onset by one year. Thus, peroxisomal dysfunction causes secondary failure of local lysosomes, thereby impairing the turnover of gangliosides in myelin. This reveals a new aspect of axon-glia interactions, with Schwann cell lipid metabolism regulating the anchorage of juxtaparanodal Kv1-channels.

Estradiol attenuates spinal cord injury-induced pain by suppressing microglial activation in thalamic VPL nuclei of rats.

In our previous study we showed that central pain syndrome (CPS) induced by electrolytic injury caused in the unilateral spinothalamic tract (STT) is a concomitant of glial alteration at the site of injury. Here, we investigated the activity of glial cells in thalamic ventral posterolateral nuclei (VPL) and their contribution to CPS. We also examined whether post-injury administration of a pharmacological dose of estradiol can attenuate CPS and associated molecular changes. Based on the results,in the ipsilateral VPL the microglial phenotype switched o hyperactive mode and Iba1 expression was increased significantly on days 21 and 28 post-injury. The same feature was observed in contralateral VPL on day 28 (P<.05). These changes were strongly correlated with the onset of CPS (r(2)=0.670). STT injury did not induce significant astroglial response in both ipsilateral and contralateral VPL. Estradiol attenuated bilateral mechanical hypersensitivity 14 days after STT lesion (P<.05). Estradiol also suppressed microglial activation in the VPL. Taken together, these findings indicate that selective STT lesion induces bilateral microglia activation in VPL which might contribute to mechanical hypersensitivity. Furthermore, a pharmacological dose of estradiol reduces central pain possibly via suppression of glial activity in VPL region.

Role of microglia and astrocyte in central pain syndrome following electrolytic lesion at the spinothalamic tract in rats.

Central pain syndrome (CPS) is a debilitating state and one of the consequences of spinal cord injury in patients. Many pathophysiological aspects of CPS are not well documented. Spinal glia activation has been identified as a key factor in the sensory component of chronic pain. In this study, the role of glial subtypes in the process of CPS induced by unilateral electrolytic lesion of spinothalamic tract (STT) is investigated. Male rats received a laminectomy at T8-T9 and then unilateral electrolytic lesion centered on the STT. Thermal and mechanical thresholds as well as locomotor function were measured on days 0, 3, 7, 14, 21, and 28 post-injuries by tail flick, von Frey filament, and open field tests, respectively. To investigate the spinal glial activation following denervation in STT-lesioned groups, Iba1 and GFAP were detected by immunohistochemistry and Western blotting at the same time points. Data showed that STT lesion significantly decreased thermal pain at day 3 in comparison with sham groups. Significant bilateral allodynia appeared in hind paws at day 14 after spinal cord injury and continued to day 28 (P < 0.05). Additionally, electrolytic spinal lesion attenuated locomotor function of injured animals after 7 days (P < 0.05). In both histological assessments and Western blotting, Iba1 increased at days 3 and 7 while increased GFAP occurred from day 14 to 28 after lesion. It appears that microglial activation is important in the early stages of pain development and astrocytic activation occurs later. These events may lead to behavioral outcomes especially central neuropathic pain.

Enhancement of Antinociception by Co-administrations of Nefopam, Morphine, and Nimesulide in a Rat Model of Neuropathic Pain.

BACKGROUND: Neuropathic pain is a chronic pain due to disorder in the peripheral or central nervous system with different pathophysiological mechanisms. Current treatments are not effective. Analgesic drugs combined can reduce pain intensity and side effects. Here, we studied the analgesic effect of nimesulide, nefopam, and morphine with different mechanisms of action alone and in combination with other drugs in chronic constriction injury (CCI) model of neuropathic pain. METHODS: Male Wistar rats (n = 8) weighing 150-200 g were divided into 3 different groups: 1- Saline-treated CCI group, 2- Saline-treated sham group, and 3- Drug-treated CCI groups. Nimesulide (1.25, 2.5, and 5 mg/kg), nefopam (10, 20, and 30 mg/kg), and morphine (1, 3, and 5 mg/kg) were injected 30 minutes before surgery and continued daily to day 14 post-ligation. In the combination strategy, a nonanalgesic dose of drugs was used in combination such as nefopam + morphine, nefopam + nimesulide, and nimesulide + morphine. Von Frey filaments for mechanical allodynia and acetone test for cold allodynia were, respectively, used as pain behavioral tests. Experiments were performed on day 0 (before surgery) and days 1, 3, 5, 7, 10, and 14 post injury. RESULTS: Nefopam (30 mg/kg) and nimesulide (5 mg/kg) blocked mechanical and thermal allodynia; the analgesic effects of morphine (5 mg/kg) lasted for 7 days. Allodynia was completely inhibited in combination with nonanalgesic doses of nefopam (10 mg/kg), nimesulide (1.25 mg/kg), and morphine (3 mg/kg). CONCLUSIONS: It seems that analgesic drugs used in combination, could effectively reduce pain behavior with reduced adverse effects.