Clavulanic Acid Attenuating Effect on the Diabetic Neuropathic Pain in Rats.

Diabetic neuropathy is one of the most common complications of diabetes mellitus. Excess glutamate release and oxidative stress are hypothesized to be involved in the pathophysiology of diabetes-induced neuropathy. This study was designed to investigate the effect of clavulanic acid (CLAV), a competitive beta-lactamase inhibitor, on the streptozocin (STZ)-induced neuropathic pain and possible mechanisms in the spinal cord of rats. Male Wistar rats were divided into naive group; control group which got a single dose of STZ (50 mg/kg, i.p.), as a model of diabetic neuropathic pain; prophylactic groups: animals received CLAV (10, 20 and 40 mg/kg, i.p.) 1 week after STZ for 10 days; and therapeutic group: animals received 20 mg/kg CLAV, 21 days after STZ for 10 days. Study of pain behaviors was started on days 0, 7, 14, 21, 28, 35 and 42 after STZ. The expression of the glutamate transport 1 (GLT1), genes of oxidative stress including inducible nitric oxide synthase (iNOS), proinflammatory cytokine, tumor necrosis factor alpha (TNF-α), as well as genes involved in the apoptosis including bcl2, bcl2-associated x (bax) were measured in the spinal cord tissue by Real Time PCR, on day 42. On day 21 post injection of STZ, diabetic animals showed significant mechanical allodynia, cold allodynia and thermal hyperalgesia. CLAV in all doses of 10, 20 and 40 mg/kg reduced symptoms of allodynia and hyperalgesia, in both prophylactic and therapeutic regimens. While iNOS, TNF-α, bax/bcl2 were found significantly overexpressed in spinal cord of diabetic animals, their expression in animals received CLAV had been reduced. In contrast, GLT1 that had decreased in the spinal cord of diabetic animals, significantly increased in those received CLAV. CLAV was found a promising candidate for reliving neuropathic pain in diabetes mellitus. Such beneficial effect of CLAV could be, in part, attributed to the increased expression of GLT 1, inhibition of nitrosative stress, anti-inflammation, and inhibition of some apoptotic mediators followed by administration into diabetic animals.

Effect of the conditioned medium of mesenchymal stem cells on the expression levels of P2X4 and P2X7 purinergic receptors in the spinal cord of rats with neuropathic pain.

Recent studies have shown that mesenchymal stem cells (MSCs) and their conditioned medium (CM) have potential therapeutic effects in animal models of neuropathic pain (NP). However, the mechanisms underlying these effects are not fully understood. Because of the leading involvement of purinergic receptors in the pathogenesis of NP, this study aimed to investigate the effect of MSCs-CM on the expression levels of P2X4 and P2X7 receptors in a rat model of NP induced by chronic constriction injury (CCI) of the sciatic nerve. CM was prepared from the rats' bone marrow-derived MSCs culture. After that, NP rats were treated by intraperitoneal injection of CM, or Dulbecco's modified Eagle's medium (DMEM) 1 day before and 7 and 11 days after CCI surgery. The NP status was assessed in the treated animals using behavioral tests, including mechanical allodynia and thermal hyperalgesia, on days - 1, 3, 6, 9, 12, and 15 of the study. At the end of the study (Day 15), the animals were sacrificed, and the relative gene expression of P2X4 and P2X7 receptors were measured in the spinal cord using quantitative real-time PCR. The results demonstrated that in the CM-treated NP rats, mechanical allodynia and thermal hyperalgesia were significantly reduced compared with the DMEM-treated group. In addition, the expression levels of P2X4 and P2X7 receptors were noticeably prevented in the CM-treated group than the control group. These findings indicate that the antinociceptive effects of CM in the NP rats are partly mediated through preventing the upregulation of P2X4 and P2X7 receptors in the spinal cord.

The Effect of Preoperative Pentoxifylline on Postoperative Pain and Development of Secondary Hyperalgesia in Patients Undergoing Laparoscopic Appendectomy: A Randomized, Double-Blind, Placebo-Controlled Trial Study.

BACKGROUND: After surgery and loss of anesthetic effect, postoperative pain can annoy the patient and affect patient satisfaction with treatment. This study was aimed at evaluating the effect of preoperative pentoxifylline (PTX) on postoperative pain and development of secondary hyperalgesia in patients undergoing laparoscopic appendectomy (LA). METHODS: This randomized, double-blind, placebo-controlled clinical trial study was conducted on 91 eligible subjects with acute appendicitis referred to Shahid Beheshti hospital of Sabzevar, Iran, in 2018. The intervention and control groups were administered with a single oral dose of PTX (10 mg/kg) and placebo an hour before surgery, respectively. Postoperative pain was measured within 24 hours after surgery using a VAS, and the area of secondary hyperalgesia was measured 24 hours after surgery using the Stubhaug et al. method. RESULTS: The mean age of the subjects was 26.74 ± 9.99 years, and 57.14% were female. Pain intensity during rest was significantly greater in the control group as compared to the PTX group 24 hours after surgery (VAS scores 2.19 ± 0.49 and 3.13 ± 0.66, respectively; P < 0.001). Moreover, pain intensity during cough was substantially lower in the PTX group compared with the control group 24 hours after surgery (VAS scores 2.65 ± 1.90 and 4.10 ± 2.60, respectively; P = 0.003 in turn). The dynamic hyperalgesia was significantly greater in the control group as compared with the PTX group (3.80 ± 1.82 and 7.43 ± 2.38, respectively; P < 0.001). CONCLUSIONS: Findings suggest that oral administration of PTX 1 hour before surgery in patients undergoing LA can reduce postoperative pain in patients and prevent secondary hyperalgesia at a surgical site.

Inhibition of microglial activity alters spinal wide dynamic range neuron discharge and reduces microglial Toll-like receptor 4 expression in neuropathic rats.

It is believed that neuropathic pain results from aberrant neuronal discharges although some evidence suggests that the activation of glia cells contributes to pain after an injury to the nervous system. This study aimed to evaluate the role of microglial activation on the hyper-responsiveness of wide dynamic range neurons (WDR) and Toll-like receptor 4 (TLR4) expressions in a chronic constriction injury (CCI) model of neuropathic pain in rats. Adult male Wistar rats (230 ± 30 g) underwent surgery for induction of CCI neuropathy. Six days after surgery, administration of minocycline (10, 20, and 40 mg/kg, i.p.) was initiated and continued until day 14. After administration of the last dose of minocycline or saline, a behavioral test was conducted, then animals were sacrificed and lumbar segments of the spinal cord were collected for Western blot analysis of TLR4 expression. The electrophysiological properties of WDR neurons were investigated by single unit recordings in separate groups. The findings showed that after CCI, in parallel with thermal hyperalgesia, the expression of TLR4 in the spinal cord and the evoked response of the WDR neurons to electrical, mechanical, and thermal stimulation significantly increased. Post-injury administration of minocycline effectively decreased thermal hyperalgesia, TLR4 expression, and hyper-responsiveness of WDR neurons in CCI rats. The results of this study indicate that post-injury, repeated administration of minocycline attenuated neuropathic pain by suppressing microglia activation and reducing WDR neuron hyper-responsiveness. This study confirms that post-injury modulation of microglial activity is a new strategy for treating neuropathic pain.

Preemptive administration of mesenchymal stem cells-derived conditioned medium can attenuate the development of neuropathic pain in rats via downregulation of proinflammatory cytokines.

Neuropathic pain (NP) is a chronic condition characterized by persistent pain following nerve injury. It is a challenging clinical problem to manage due to limited treatment options. Mesenchymal stem cells (MSCs)-derived conditioned medium (CM) is a cell-free product that contains the secretome of MSCs and has been shown to have therapeutic potential in various inflammatory and degenerative disorders. Several animal studies have examined the antinociceptive effects of MSCs-CM on established neuropathic pain, but none have investigated the early prevention of neuropathic pain using MSCs-CM. Therefore, in this study, we tested whether preemptive administration of MSCs-CM could attenuate the development of NP in rats. To this end, NP was induced in Wistar rats using a chronic constriction injury (CCI) model (day 0), and then the animals were divided into four groups: Sham, CCI, CCI-Dulbecco's Modified Eagle Medium (DMEM), and CCI-CM. The CCI-CM group received 1 ml intraperitoneal administration of MSCs-CM on days - 1, 1, and 2, while the Sham, CCI, and CCI-DMEM groups received vehicle only (normal saline or DMEM). Mechanical withdrawal threshold and thermal withdrawal latency were assessed to evaluate pain sensitivities. In addition, the expression levels of proinflammatory cytokines (TNF-α and IL-1ß) in the spinal cord tissues were measured using quantitative real-time PCR (qRT-PCR). The results demonstrated that preemptive treatment with MSCs-CM can significantly attenuate the development of NP, as evidenced by improved mechanical withdrawal threshold and thermal withdrawal latency in the CCI-CM group compared to the CCI and CCI-DMEM groups. Furthermore, the relative gene expression of proinflammatory cytokines TNF-α and IL-1ß were significantly decreased in the spinal cord tissues of the CCI-CM group compared to the control groups. These findings suggest that preemptive administration of MSCs-CM can attenuate the development of NP in rats, partly due to the downregulation of proinflammatory cytokines.

Association between type 2 diabetes and branched chain amino acids (BCAA); a case-control study.

Background: Several amino acids and their derivatives have been implicated in insulin resistance (IR) and Type 2 Diabetes Mellitus (T2DM). This research sought to establish a relationship between the dietary levels of branched-chain amino acids (BCAA) and the risk of T2DM. Methods: This case-control study was carried out on 4200 participants consisting of 589 people with T2DM and 3611 non-diabetic aged 35 to 70 years residents in Sabzevar, Iran. Data on the economic-social, employment status, medical history, lifestyle, and sleep habits were collected via interview. The food frequency questionnaire (FFQ) was used to check the nutritional status. Participants' dietary BCAA consumption was estimated using Nutritionist IV software. Results: A significant negative association between the incidence of T2DM and the dietary levels of BCAAs after adjustment for age and sex (OR = 0.972, CI 95%:0.648-0.996, P = 0.022). The negative association remained significant after additional adjustments for body mass index (BMI) and physical activity (OR = 0.967, CI 95%: 0.943-0.992, P = 0.010). Interestingly, a positive association was found between T2DM and total BCAAs (OR = 1.067, CI 95%: 1.017-1.119, P = 0.008), Isoleucine (OR = 1.248, CI 95%: 1.043-1.494, P = 0.016), Leucine (OR = 1.165, CI 95%: 1.046-1.299, P = 0.006) and Valine (OR = 1.274, CI 95%: 1.088-1.492, P = 0.003) after further adjustment for calorie intake. Conclusions: Our results demonstrate branched-chain amino acids (BCAAs) including isoleucine, leucine, and valine are negatively associated with the incidence of type 2 diabetes (T2DM) after adjusting for age and sex, BMI, and physical activity. However, adjusting for calorie intake reversed the association between T2DM and BCAAs. These findings suggest that the association between BCAAs and T2DM may be influenced by calorie intake. Future longitudinal studies are warranted. Supplementary Information: The online version contains supplementary material available at 10.1007/s40200-023-01247-9.

The cytotoxicity and apoptotic effects of verbascoside on breast cancer 4T1 cell line.

BACKGROUND: Despite significant advancements in breast cancer therapy, novel drugs with lower side effects are still being demanded. In this regard, we investigated the anti-cancer features of verbascoside in 4 T1 mouse mammary tumor cell. METHODS: First, MTT assay was performed with various concentrations (ranging between 5 to 200 µM) of verbascoside and IC50 was calculated. Then the expression of Bax, Bcl-2, and caspase-3 was evaluated in treated 4 T1 cells. In addition, we investigated the expression of TLR4, MyD88, and NF-κB to ascertain the underlying mechanism of the anti-proliferative feature of verbascoside. Also, flow cytometry followed by double PI and Annexin V was conducted to confirm the apoptosis-inducing effect of verbascoside. RESULTS: Our results from MTT assay showed verbascoside inhibits proliferation of 4 T1 cancer cells (IC50 117 µM) while is safe for normal HEK293T cells. By qRT-PCR, we observed that verbascoside treatment (100, 117 and, 130 µM) increases the expression of caspase-3 and Bax while reduces the expression of Bcl-2. Also, verbascoside (100, 117 and, 130 µM) increased the expression of TLR4 only at 130 µM dose and the expression of MyD88 whereas reduced the expression of NF-κB at mRNA level. Flow cytometry analysis also confirmed verbascoside induces apoptosis in 4 T1 cells at 117 µM. CONCLUSION: Taken together, our data showed verbascoside is a safe natural compound for normal cells while has apoptosis-inducing feature through TLR4 axis on 4 T1 cells.

Evaluation of the GABAA Receptor Expression and the Effects of Muscimol on the Activity of Wide Dynamic Range Neurons Following Chronic Constriction Injury of Sciatic Nerve in Rats.

INTRODUCTION: The modality of γ-aminobutyric acid type a receptors (GABAA) controls dorsal horn neuronal excitability and inhibits sensory information. This study aimed to investigate the expression of the GABAA receptor and the effects of its agonist muscimol on Wide Dynamic Range (WDR) neuronal activity in the Chronic Constriction Injury (CCI) model of neuropathic pain. METHODS: Adult male Wistar rats weighing 200 to 250 g were used to induce CCI neuropathy. Fourteen days after surgery, muscimol (0.5, 1, and 2 mg/kg IP) was injected. Then, the behavioral tests were performed. After that, the animals were killed, and the lumbar segments of the spinal cords were collected for Western blot analysis of the GABAA receptor α1 subunit expression. The electrophysiological properties of WDR neurons were studied by single-unit recordings in separate groups 14 days after CCI. RESULTS: The outcomes indicated the development of thermal hyperalgesia and mechanical allodynia after neuropathy; nonetheless, the expression of the GABAA receptor α1 subunit did not change significantly. Moreover, the evoked responses of the WDR neurons to electrical, mechanical, and thermal stimuli increased considerably. Fourteen days after CCI, muscimol administration decreased thermal hyperalgesia, mechanical allodynia, and hyper-responsiveness of the WDR neurons in CCI rats. CONCLUSION: The modulation of the spinal GABAA receptors after nerve injury can offer further insights to design new therapeutic agents to reduce neuropathic pain symptoms.

Assessment of anti-nociceptive effect of allopurinol in a neuropathic pain model.

BACKGROUND: This study aimed to investigate the antinociceptive effect of allopurinol, a xanthine oxidase inhibitor, in the chronic constriction injury (CCI) to sciatic nerve rat model of neuropathic pain. METHODS: Allopurinol administration (30, 60, 90 mg/kg, i.p.) was started at the time of nerve injury, and given for 14 continuous days. Behavioural tests (von Frey filaments, acetone drop, hot plate) were conducted on days 0, 3, 7, 10 and 14. Reverse transcriptase polymerase chain reaction (RT-PCR) analysis was performed on the spinal cord of CCI animals on day 14. The contribution of adenosine (A) receptors was tested using the methylxanthine theophylline, a non-selective A receptor antagonist and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), a selective A1 receptor antagonist, administered 30 min before allopurinol on day 10. RESULTS: CCI of the sciatic nerve resulted in a persistent mechanical allodynia, cold allodynia, and heat hyperalgesia, together with increased iNOS, bax/bcl2, iba-1 and TNF-α expression in the lumbar spinal cord of animals. The highest-dose group of allopurinol (90 mg/kg) attenuated pain-like behaviors compared with the normal saline treated group, and this was accompanied by normalization of iNOS, bax/bcl2, caspase 3, iba-1 and TNF-α gene expression changes. DPCPX and theophylline reversed the thermal anti-hyperalgesic effect of allopurinol. In contrast, the mechanical anti-allodynic effect was only prevented by theophylline. CONCLUSION: Allopurinol through interacting with different aspects of neuropathic pain, via anti-oxidant effects, protection against neuroinflammation, and activating adenosine receptors, could be useful in the treatment of patients with neuropathic pain.

The effects of estradiol and testosterone on renal tissues oxidative after central injection of angiotensin II in female doca - salt treated rats.

Background Although numerous studies have proven that estrogen (Est) has a protective effect on the development of hypertension, more research needs to be done to show its detailed mechanism in a variety of hypertension. The important role of active oxygen species in blood pressure is well defined. We examined whether or not sex hormones change the growth of reactive oxygen species (ROS) in kidneys after central microinjection of angiotensin II (Ang II). Materials and methods Female Wistar rats, 8 weeks old (200 ± 10 g) were used in this study. The animal groups were (1) Sham, (2) Ovariectomy (OVX), (3) Sham-Hypertension (Sham-Hyper), (4) OVX-Hypertension (OVX-Hyper), (5) Sham-Hyper-Est, (6) OVX-Hyper-Est ; (7) Sham-Hyper-Testosterone (Tst) and (8) OVX-Hyper-Tst. Solutions of 1% NaCl and 0.1 KCl were used and desoxycorticostrone (doca-salt) was injected (45 mg/kg) 3 times a week in Hypertension groups. Estradiol and Tst (2 mg/kg and 5 mg/kg ; daily; subcutaneously) for 4 weeks. Ang II (50 µM, 5 µL) was microinjected by intracerebroventricular ( i.c.v.) infusion and malondialdehyde (MDA) and thiol in the kidneys were measured. Results MDA in the kidneys was increased by Ang II and doca-salt treatments. Both estradiol and Tst decreased the kidney's MDA. The level of thiol was higher in Hyper groups and reversed after treatment with estradiol and Tst. Conclusions Our findings suggest that central effect of Ang II on blood pressure and kidney disease is accompanied with increased levels of oxidative stress in the kidneys. Indeed sex hormones change the ROS level in the kidneys after central microinjection of Ang II. .

Protective effects of atorvastatin against morphine-induced tolerance and dependence in mice.

BACKGROUND: In this study, we evaluated the effects of atorvastatin, a lipid-lowering medication on morphine-induced tolerance and dependence in mice. METHODS: Tolerance was induced by subcutaneous administration of morphine (20mg/kg) to animals, twice a day for 9days. Atorvastatin was given at the doses of 5, 10 and 20mg/kg, 30min before each morphine administration, once daily for 9days. Hot plate test was employed to assess antinociceptive effect of morphine on days 1, 3, 5, 7 and 9. Dependence was evaluated by naloxone-precipitated withdrawal syndrome. We attempted to verify withdrawal regulation of induced nitric oxide synthase (iNOS), astroglia marker, glial fibrillary acidic protein (GFAP), ionized calcium-binding protein (Iba1), a microglia activation marker, a pro-inflammatory mediator, tumor necrosis alpha (TNF-α) and immune receptor, toll like receptor 4 (TLR-4) genes by real-time polymerase chain reaction (RT-PCR). Lipid peroxidation was estimated by assessing malondialdehyde (MDA) content in the spinal cord of animals. RESULTS: Tolerance to antinociceptive effects of morphine was observed on days 7 and 9. Decrease in morphine-induced antinociception was reversed by concomitant intraperitoneal administration of atorvastatin (10 and 20mg/kg). Atorvastatin (10 and 20mg/kg) mitigated naloxone-induced withdrawal parameters. Brain expression levels of TNF-α, GFAP, Iba1 and iNOS increased in morphine withdrawn animals which were attenuated by nine days treatment with atorvastatin. Increased MDA was also normalized in withdrawn animals received atorvastatin. CONCLUSION: Atorvastatin exhibits meaningful protective effects against both tolerance to antinociceptive effects of morphine and withdrawal-induced behavioral profile. Neuroprotective effects of atorvastatin is further supported via inhibition of glia activity and antioxidant effects.

Age-Related Differences in Neuropathic Pain Behavior and Spinal Microglial Activity after L5 Spinal Nerve Ligation in Male Rats.

INTRODUCTION: Several studies have reported the involvement of age-related changes in the development of neuropathic pain behaviors. However, limited data are available on the role of age in establishing and maintaining chronic neuropathic pain after peripheral nerve injury. METHODS: In the present study, we examined age-related neuropathic behavior among rats in 4 age groups: pups (4 weeks old; weight, 60-80 g), juvenile rats (6 weeks old; weight, 120-140 g), and mature rats (10-12 weeks old; weight, 200-250 g). Because the exact contribution of spinal microglia and its association with the development of neuropathic pain remains unknown, we also evaluated the expression of spinal Iba1, a microglial marker, by using western blotting before and 5 days after spinal nerve ligation (SNL) as well as after the daily IP administration of minocycline (30 mg/kg). RESULTS: Our results showed that SNL-induced mechanical allodynia but not thermal hyperalgesia in mature rats but not in pups (P<0.05 and P<0.01, respectively). The expression of spinal Iba1 in the juvenile rats was significantly lower than that in pups and mature rats (P<0.01). Moreover, administration of minocycline decreased the expression of spinal Iba1 in the pup rats more than in juvenile rats (P<0.001) and in the juvenile rats more than in the mature rats (P<0.05). CONCLUSION: These data suggest that the development of neuropathic behaviors and microglial activation after SNL could be age dependent.

Post-injury repeated administrations of minocycline improve the antinociceptive effect of morphine in chronic constriction injury model of neuropathic pain in rat.

It is confirmed that pharmacological attenuation of glial cells can alleviate neuropathic pain by lowering proinflammatory cytokine expression. The present study tries to confirm that post-injury administration of glia inhibitor, minocycline, can attenuate the neuropathic pain symptoms and improves the efficacy of morphine anti-nociception in chronic constriction injury (CCI). Male Wistar rats (230-270 g) underwent surgery for induction CCI model of neuropathy. For assessment of the thermal hyperalgesia and mechanical allodynia after CCI induction, morphine (2.5, 5, 7.5, 10 and 15 mg/kg; s.c.) and saline were administered on post-operative days (PODs) 0, 6 and 14. Hargreaves and Von-Frey tests were performed before and 30 min after morphine administration, respectively. The results showed significant decrease in antinociceptive effect of morphine on POD 6 compared to POD 0 only at the dose of 5 mg/kg. On the other hand, on POD 14 the antinociceptive effect of morphine (5, 7.5, 10 and 15 mg/kg) significantly decreased in comparison with POD 0. In another set of experiments, animals received minocycline (10, 20 and 40 mg/kg; i.p.) for eight days from POD 6 to 13 and then the antinociceptive effect of single dose of morphine 5 mg/kg was tested on POD 14. Behavioral tests showed that minocycline (40 mg/kg) could effectively attenuate the thermal hyperalgesia and mechanical allodynia on POD 13. Moreover, minocycline (40, 20 mg/kg) improved the anti-hyperalgesic, and minocycline (40 mg/kg) improved the anti-allodynic effects of morphine 5 mg/kg on POD 14. It seems that the reduction of antinociceptive effect of morphine after CCI may be mediated through glia activation. Modulation of glial activity by minocycline can attenuate CCI-induced neuropathic pain. It is also shown that repeated post-injury administration of minocycline improves the antinociceptive effect of morphine in neuropathic pain.

Alterations in blood pressure due to chronic exposure to natural sour gas leakage containing sulfur compounds.

In order to show the alteration of blood pressure in subjects chronically exposed to natural gas containing sulfur compounds, the present study was done. The blood pressure of 94 (43 males, 51 females) of healthy individuals living in the polluted area of Masjid-i-Sulaiman (Khozestan province, southwest of Iran) was measured. The non-parametric Sign test was applied in order to detect differences between the study subjects and the normal mean values according to the sex and age of subjects. Statistical analysis showed that the systolic blood pressure significantly decreased (Z=-2.74; P=0.0031) while the diastolic blood pressure (Z=+2.11; P=0.0174) and heart rate (Z=+3.62; P<0.001) significantly increased in individuals living in the contaminated areas compared with those of normal mean values. The systolic and diastolic blood pressure decreased and increased, respectively, in individuals chronically exposed to natural sour gas containing sulfur compounds.