Spermidine Exerts Protective Effects in Random-Pattern Skin Flap Survival in Rats: Possible Involvement of Inflammatory Cytokines, Nitric Oxide, and VEGF.

BACKGROUND: Distal necrosis and inflammation are two of the most common health consequences of random-pattern skin flaps survival (SFS). Anti-inflammatory effects of spermidine have been identified in various studies. On the other hand, considering the involvement of the nitric oxide molecule in the spermidine mode of action and also its role in skin tissue function, we analyzed the possible effects of spermidine on the SFS and also, potential involvement of nitrergic pathway and inflammatory cytokine in these phenomena. METHODS: Each rat was pretreated with either a vehicle (control) or various doses of spermidine (0.5, 1, 3, 5, 10 and 30 mg/kg) and then was executed a random-pattern skin flap paradigm. Also, spermidine at the dose of 5 mg/kg was selected and one group rats received spermidine 20 min prior to surgery and one additional dose 1 day after operation. Then, 7 days after operations, interleukin (IL)-6, tumor necrosis factor (TNF)-α, interferon-gamma (IFN-γ), and nitrite levels were inquired in the tissue samples by ELIZA kit. Vascular endothelial growth factor expression was assessed by DAPI staining and fluorescent microscopes. The concentrations of three polyamines, including spermidine, spermine, and cadaverine, were analyzed using HPLC. RESULTS: Pretreatment with spermidine 5 mg/kg improved SFS considerably in microscopic skin H&E staining analysis and decreased the percentage of necrotic area. Moreover, spermidine exerted promising anti-inflammatory effects via the modulation of nitric oxide and reducing inflammatory cytokines. CONCLUSIONS: Spermidine could improve skin flaps survival, probably through the nitrergic system and inflammation pathways. This preclinical study provides level III evidence for the potential therapeutic effects of spermidine on SFS in rats, based on the analysis of animal models. Further studies are needed to confirm these findings in clinical settings. LEVEL OF EVIDENCE III: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Nano-invasomes for simultaneous topical delivery of buprenorphine and bupivacaine for dermal analgesia.

Opioid and local anaesthetic receptors are abundantly concentrated in different layers of the skin. Therefore, simultaneous targeting of these receptors can produce more potent dermal anaesthesia. Herein, we developed lipid-based nanovesicles for the co-delivery of buprenorphine and bupivacaine to efficiently target skin-concentrated pain receptors. Invasomes incorporating two drugs were prepared by ethanol injection method. Subsequently, the size, zeta potential, encapsulation efficiency, morphology, and in-vitro drug release of vesicles were characterized. Ex-vivo penetration features of vesicles were then investigated by the franz diffusion cell on the full-thickness human skin. Wherein, it was demonstrated that invasomes penetrated the skin deeper and delivered bupivacaine more effectively than buprenorphine to the target site. The superiority of invasome penetration was further evidenced by the results of ex-vivo fluorescent dye tracking. Estimation of in-vivo pain responses by the tail-flick test revealed that compared with the liposomal group, the group receiving invasomal formulation and drug-free invasomal formulation (only containing menthol) displayed increased analgesia in the initial times of 5 and 10 min. Also, no signs of oedema or erythema were observed in the Daze test in any of the rats receiving the invasome formulation. Finally, ex-vivo and in-vivo assays demonstrated efficiency in delivering both drugs into deeper layers of skin and exposing them to the located pain receptors, which improves the time of onset and the analgesic effects. Hence, this formulation appears to be a promising candidate for tremendous development in the clinical setting.

Protective effect of pioglitazone on morphine-induced neuroinflammation in the rat lumbar spinal cord.

BACKGROUND: Morphine-induced tolerance is associated with the spinal neuroinflammation. The aim of this study was to explore the effects of oral administration of the pioglitazone, the peroxisome proliferator activated receptor gamma (PPAR-γ) agonist, on the morphine-induced neuroinflammation in the lumbar region of the male Wistar rat spinal cord. RESULTS: Co-administration of the pioglitazone with morphine not only attenuated morphine-induced tolerance, but also prevented the up-regulation of pro-inflammatory cytokines (tumor necrosis factor alpha, interleukin-1beta, and interleukin 6) and nuclear factor-kappa B activity. Administration of the GW-9662 antagonized the above mentioned effects of the pioglitazone. CONCLUSIONS: It is concluded that oral administration of the pioglitazone attenuates morphine-induced tolerance and the neuroinflammation in the lumbar region of the rat spinal cord. This action of the pioglitazone may be, at least in part, due to an interaction with the spinal pro-inflammatory cytokine expression and the nuclear factor-kappa B activity.

Gap junction blockers: a potential approach to attenuate morphine withdrawal symptoms.

BACKGROUND: The exact mechanisms of morphine-induced dependence and withdrawal symptoms remain unclear. In order to identify an agent that can prevent withdrawal syndrome, many studies have been performed. This study was aimed to evaluate the effect of gap junction blockers; carbenoxolone (CBX) or mefloquine (MFQ); on morphine withdrawal symptoms in male rat. Adult male Wistar rats (225 - 275 g) were selected randomly and divided into 10 groups. All groups underwent stereotaxic surgery and in order to induce dependency, morphine was administered subcutaneously) Sc) at an interval of 12 hours for nine continuous days. On the ninth day of the experiment, animals received vehicle or CBX (100, 400, 600 µg/10 µl/rat, icv) or MFQ (50, 100 and 200 µg/10 µl/rat, icv) after the last saline or morphine (Sc) injection. Morphine withdrawal symptoms were precipitated by naloxone hydrochloride 10 min after the treatments. The withdrawal signs including: jumping, rearing, genital grooming, abdomen writhing, wet dog shake and stool weight, were recorded for 60 minutes. RESULTS: Results showed that CBX and MFQ decreased all withdrawal signs; and the analysis indicated that they could attenuate the total withdrawal scores significantly. CONCLUSION: Taking together it is concluded that gap junction blockers prevented naloxone-precipitated withdrawal symptoms.

Dexamethasone and Citicoline Mitigate Cisplatin-Induced Peripheral Neuropathy: A Novel Experimental Study in Mice.

Objectives: Given the rising prevalence of cisplatin-induced peripheral neuropathy (CisIPN), investigations to alleviate its adverse effects are required. Oxidative stress and free radical development are essential pathways of CisIPN. Specifically, dexamethasone and citicoline are characterized by anti-inflammatory and antioxidant activities that might reduce CisIPN incidence and severity. The current study assessed the possible impacts of novel interventions, dexamethasone, and, citicoline on CisIPN. Materials and Methods: Seventy-two male mice were randomly allocated into nine groups (n: 8/each group). Different doses of dexamethasone (7.5, 15, 30 mg/kg, i.p.), citicoline (10, 20, 40 mg/kg, i.p.), and the combination (dexamethasone 7.5 mg/kg + citicoline 10 mg/kg, i.p.) were injected in the first three days and one day before receiving cisplatin (2 mg/kg, i.p.). The tail-flick method was used for assessing nociception. Besides, malondialdehyde (MDA), interleukin-1 beta (IL-1ß), tumor necrosis factor-α (TNF-α), total antioxidant capacity (TAC), and mice weight differences (ΔW) were measured. Results: Different doses of dexamethasone and citicoline enhanced latency time (p<0.05). Moreover, dexamethasone 15 mg/kg diminished the level of MDA and increased TAC (p<0.05) and in 30 mg/kg, MDA was reduced (p<0.05). Besides, 20 and 40 mg/kg of citicoline reduced MDA and elevated TAC (p<0.05), and 10 mg/kg merely reduced MDA (p<0.05). Dexamethasone in all doses declined IL-1ß and TNF-α levels, and citicoline only at 40 mg/kg lessened their levels (p<0.05). Interestingly, ΔW declined more in the dexamethasone and citicoline groups than the cisplatin group (p<0.05). Conclusion: Dexamethasone and citicoline attenuate CisIPN through anti-inflammatory activity, improving the antioxidant capacity, and inhibiting lipid peroxidation.

Ion-pair hollow fiber liquid-phase microextraction combined with capillary electrophoresis for the determination of biogenic amines in rat tissues.

Herein, the development of an ion-pair hollow fiber liquid-phase microextraction (IP-HF-LPME) procedure followed by capillary electrophoresis (CE) with indirect UV detection for the simultaneous extraction and determination of aliphatic biogenic amines including spermidine, spermine, and cadaverine in tissue samples of rat is described. In this study, tissue samples were firstly homogenized with a cold trichloroacetic acid solution then the developed method was applied to the supernatant resulting from tissue homogenization. Different CE separation and indirect UV detection conditions, as well as IP-HF-LPME extraction conditions were studied in detail and optimized. A 11 mmol L-1 imidazole solution containing 13 % (v/v) ethanol adjusted at pH 3.3 (by acetic acid) was the best running buffer for indirect UV detection of non-UV-absorbing amines. 1-Octanol in combination with salicylic acid, respectively as the membrane solvent and ion-pair reagent provided the highest extraction recovery for the studied amines. Method performances were established and good results of linearity, recovery, sensitivity, and repeatability were achieved for all the studied amines. Limits of detection were found to be between 2.8 and 4.5 ng mL-1 and limits of quantification were 15 ng mL-1 and the dynamic range was 15-2000 ng mL-1 for all three analytes.

Optimization of Influential Variables in the Development of Buprenorphine and Bupivacaine Loaded Invasome for Dermal Delivery.

Purpose: Hydrophilic drugs are extensively applied in clinical applications. Inadequate dermal penetration of these drugs is a great challenge. Incorporation of drugs into nano-carrier systems overcomes lower penetration drawbacks. Invasomes are novel nano-carrier systems which enhance transdermal penetration by using terpene and ethanol in their structures. buprenorphine and bupivacaine hydrochlorides are two potent analgesic drugs that are loaded simultaneously in the nano-invasome structure as opioid and non-opioid drugs. Methods: The full factorial experimental design was used for planning and estimating optimum formulations of invasome systems. Three influential factors like terpene type, terpene concentration and preparation method were comprehensively analyzed for achieving high encapsulation efficiency (EE) and optimum size. Results: The mean sizes of designed invasomes were in the range of 0.39-5.86 µm and high values of EE and loading capacity (LC) were reported as 98.77 and 19.75 for buprenorphine-loaded invasome, respectively. Zeta potential measurements confirmed that the obtained high value of EE might be as a result of reversible ionic interactions between positively charged drugs and negatively charged phospholipidic part of invasome structure. Another characterization of the prepared formulations was carried out by Fourier transform infrared (FTIR), X-ray diffraction (XRD) and dynamic light scattering (DLS) technique. Conclusion: The satisfactory obtained results of formulations encourage researchers to get optimum topical analgesic formulations with potent and rapid onset time properties required in invasive cutaneous procedures.

8-OH-DPAT prevents morphine-induced apoptosis in rat dorsal raphe nucleus: a possible mechanism for attenuating morphine tolerance.

BACKGROUND: Previously, we found that activation of serotonin 1A (5-HT1A) receptors in the dorsal raphe nucleus (DRN) decreased the development of tolerance to the analgesic effect of morphine. It has been indicated that tolerance to the analgesic effect of morphine is associated with apoptosis in the central nervous system. In this investigation we attempted to evaluate the effect of 8-OH-DPAT (8-hydroxy-2-[di-n-propylamino]tetralin), a specific 5-HT1A receptor agonist, on morphine-induced tolerance and apoptosis in rat DRN. METHODS: Nociception was assessed using a hotplate apparatus. The terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method was used to analyze apoptosis. RESULTS: Tolerance to the analgesic effect of morphine was complete by 10 days after morphine administration (5 mg/kg/d, i.p.), whereas a significant analgesic effect was observed through the 10th day in 8-OH-DPAT-treated animals. Furthermore, the results showed that the number of TUNEL positive cells had been increased in morphine-tolerant rats (control group: morphine, i.p. + saline, intra-DRN) in comparison with the saline-treated animals. The results also indicated that 8-OH-DPAT (2, 4, and 8 µg/rat/d) attenuated the number of apoptotic cells in the DRN in comparison with the control group. However, 8-OH-DPAT (8 µg/rat/d, intra-DRN) failed to reduce morphine-induced apoptosis in the presence of the 5-HT1A receptor antagonist, NAN-190 (6 µg/rat/d, intra-DRN). CONCLUSION: We found that intra-DRN injection of a specific 5-HT1A receptor agonist attenuated morphine-induced apoptosis in rat DRN, which may have a key role in morphine tolerance.

Central administration of minocycline and riluzole prevents morphine-induced tolerance in rats.

BACKGROUND: Long-term exposure to opiates induces tolerance to the analgesic effect. The neurobiological mechanism of this phenomenon is not completely clear. In this study, we evaluated the effects of central administration of minocycline (a tetracycline derivative) and riluzole (an antiglutamatergic drug) on morphine-induced tolerance in rats. METHODS: Groups of rats received daily morphine (10 mg/kg, IP) in combination with saline (10 microL/rat, intracerebroventricular [ICV]) or 1% Tween 80 (10 microL/rat, ICV) or minocycline (60, 120, and 240 microg/10 microL per rat, ICV) or riluzole (20, 40, 80 microg/10 microL per rat, ICV). Nociception was assessed using hotplate apparatus (55 degrees C +/- 0.5 degrees C). Hotplate latency was recorded when the rat licked its hindpaw. Baseline latencies were determined once per day for each rat, then morphine (10 mg/kg) was injected. After 20 min, the above-mentioned drugs were administered and postdrug latency was measured 10 min after the injection of drugs or vehicles. RESULTS: Results showed that ICV administration of minocycline and riluzole delayed morphine-induced tolerance. Morphine tolerance was complete after 8 days in the control groups but was complete in the groups treated with minocycline (120 microg/10 microL per rat) and riluzole (80 microg/10 microL per rat) on the 13th day. In addition, our results showed that minocycline and riluzole increased the total analgesic effect of morphine (area under the curve of the percentage of maximal possible effect values). CONCLUSION: The effects of minocycline on nitric oxide and the glutamatergic system and the effect of riluzole on the glutamate system are potentially important mechanisms in delaying morphine-induced tolerance.

Electrosprayed Nanosystems of Carbamazepine - PVP K30 for Enhancing Its Pharmacologic Effects.

This study was conducted to enhance the pharmacologic effect of carbamazepine (CBZ) (as a poorly water-soluble drug) by fabricating CBZ-PVP K30 nanobeads using an electrospraying technique. CBZ-PVP K30 nanosystems with various ratios (1:3 and 1:5) at total solution concentrations of 3% and 5% w/v were prepared. The solution concentration extremely affected the size of the samples; where, the nanobeads (mean diameter of 457.65 ± 113.72 nm and 1.16 ± 0.46 µm) were developed at low and high solution concentrations, respectively. DSC thermographs and PXRD patterns precisely showed CBZ amorphization in the electrosprayed nanosystems. Based on the FTIR spectrum of the electrosprayed samples, a feasible interaction between N-H/O-H group of CBZ and PVP carbonyl group was detected. The in-vitro release studies revealed that the electrosprayed nanosystems represent a comparable rapid dissolution rate with respect to the physical mixtures (PMs) and the pure drug. The in-vivo results in NMRI mice indicated that the electrosprayed nanoformulation (with the drug: polymer ratio of 1:5 at a total solution concentration of 5% (w/v)) prolonged seizure latency time and decreased mortality percent in strychnine (STR) induced seizure mice more efficiently than the PM. Our finding revealed that the electrospraying as a cost-benefit and one step technique could be effectively applied for improving the physicochemical characteristics and pharmacologic effect of CBZ.

Role of 5-HT(1A) and 5-HT(2) receptors of dorsal and median raphe nucleus in tolerance to morphine analgesia in rats.

Several studies indicate that central serotonergic neurons have important role in morphine analgesia and tolerance. The aim of this study was to investigate possible role of 5-HT(1A) and 5-HT(2) receptors in dorsal and median raphe nucleus on development of tolerance to analgesic effect of morphine using hot plate test. Chronic injection of 5-HT(1A) receptor agonist 8-OH-DPAT (8-hydroxy-2-[di-n-propylamino]tetralin) (2, 4 and 8 mug/rat/day) to dorsal raphe nucleus (DRN) delayed tolerance to morphine analgesia, whereas injection of the same doses of 8-OH-DPAT to the median raphe nucleus (MRN) did not alter tolerance to morphine. In addition, chronic administration of ketanserin (1.5, 3 and 6 mug/rat/day), as a 5-HT(2) receptors antagonist, in DRN and MRN did not produce any significant effect. We conclude that 5-HT(1A) receptors of DRN are involved in tolerance to antinociceptive effect of morphine. However, the exact mechanism of interaction between serotonergic and opioidergic systems is not clear and remains to be elucidated.

Pioglitazone prevents morphine antinociceptive tolerance via ameliorating neuroinflammation in rat cerebral cortex.

BACKGROUND: Opioid induced neuroinflammation is shown to be implicated in opioid analgesic tolerance development. In the present study the effect of pioglitazone on morphine-induced tolerance and neuroinflammation in the cerebral cortex of the rat was investigated. MATERIALS AND METHODS: Various groups of rats received morphine (10mg/kg; ip) and vehicle (po), or morphine (10mg/kg) and pioglitazone (20 or 40 mg/kg; po) once a day for 17 days. In order to determine the possible involvement of PPAR-γ in the pioglitazone effect, one group of rats received PPAR-γ antagonist, GW-9662 (2mg/kg; sc), and pioglitazone (40 mg/kg) and morphine once daily for 17 days. Nociception was assessed using a tail flick apparatus and the percentage of the maximal possible effect was calculated as well. On 18th day, 2h after the last morphine injection, the cerebral cortex of the animals were harvested and the tissue levels of tumour necrosis factor alpha, interleukin-1beta, interleukin-6, interleukin-10 and nuclear factor-kappa B activity were determined. RESULTS: Co-administration of pioglitazone (40 mg/kg) with morphine not only attenuated morphine-induced tolerance, but also prevented the up-regulation of pro-inflammatory cytokines (tumour necrosis factor alpha, interleukin-1beta, interleukin-6) and nuclear factor-kappa B activity in the rat cerebral cortex. Moreover, GW-9662 (2mg/kg) administration 30 min before pioglitazone, antagonized the above mentioned pioglitazone-induced effects. CONCLUSION: It is concluded that oral administration of pioglitazone attenuates morphine-induced tolerance. This effect of pioglitazone may be, at least in part, due to its anti-inflammatory property which suppressed the cortical pro-inflammatory cytokine and inhibited of nuclear factor-kappa B activity.

Standardization of Course Plan and Design of Objective Structured Field Examination (OSFE) for the Assessment of Pharm.D. Student's Community Pharmacy Clerkship Skills.

PURPOSE: This study was aimed to design Objective Structured Field Examination (OSFE) and also standardize the course plan of community pharmacy clerkship at Pharmacy Faculty of Tabriz University of Medical Sciences (Iran). METHODS: The study was composed of several stages including; evaluation of the old program, standardization and implementation of the new course plan, design and implementation of OSFE, and finally results evaluation. RESULTS: Lack of a fair final assessment protocol and proper organized educating system in various fields of community pharmacy clerkship skills were assigned as the main weaknesses of the old program. Educational priorities were determined and student's feedback was assessed to design the new curriculum consisting of sessions to fulfill a 60-hour training course. More than 70% of the students were satisfied and successfulness and efficiency of the new clerkship program was significantly greater than the old program (P<0.05). In addition, they believed that OSFE was a suitable testing method. CONCLUSION: The defined course plan was successfully improved different skills of the students and OSFE was concluded as a proper performance based assessment method. This is easily adoptable by pharmacy faculties to improve the educational outcomes of the clerkship course.

Pioglitazone prevents morphine antinociception tolerance and withdrawal symptoms in rats.

Long-term exposure to opiates induces tolerance to the analgesic effect and dependence. The purpose of the present study is to investigate the effects of pioglitazone, a peroxisome proliferator-activated receptors gamma (PPAR-γ) agonist, on the morphine-induced tolerance and dependence. Groups of rats received morphine in combination with a vehicle or pioglitazone (5, 10, 20, and 40 mg/kg) daily. Thirty minutes before pioglitazone (40 mg/kg), GW-9662, a selective PPAR-γ antagonist, (2 mg/kg) was administrated in order to evaluate the possible role of the PPAR-γ. Nociception was assessed by a tail flick apparatus, and the percentage of the maximal possible effect was calculated as well. For 9 days, rats received additive doses of morphine to induce dependence. Naloxone was administrated 2 h after the morphine last dose, and withdrawal symptoms were recorded for 45 min. Morphine administration to rats over a duration of 17 days resulted in the development of tolerance, whereas pioglitazone (40 mg/kg) delayed the day of the established tolerance for 15 days. Administration of pioglitazone also prevented morphine-induced 50 % effective dose (ED50) shift to the right in the dose-response curve and increased the global analgesic effect of morphine. In addition, pioglitazone decreased the total withdrawal score significantly, whereas GW-9662 significantly reversed the pioglitazone effects on the morphine tolerance and dependence. The prevention of the morphine-induced glia activation and the proinflammatory responses were the possible mechanisms for pioglitazone effect on delaying the morphine tolerance and attenuating the dependence.

Repeated central administration of selegiline attenuated morphine physical dependence in rat.

BACKGROUND: Long-term exposure to opiates induces physical dependence; however, the neurobiological mechanisms of this phenomenon are not completely clear. The purpose of this study was to evaluate the effects of systemic and intracerebroventricular (icv) administration of selegiline (a selective inhibitor of monoamine oxidase B) on the morphine withdrawal syndrome in rats. METHODS: To this aim, adult male Sprague Dawley rats were selected randomly, and then growing doses of morphine were administered subcutaneously at an interval of 12 h for nine days with the intention of inducing dependency. Nine days after, only the morning dose of morphine was administered, followed by systemic or central injection of saline or selegiline. Later, naloxone was injected after 30 min and withdrawal signs recorded for a period of 60 min. RESULTS: Results showed failure of systemic administration of selegiline in changing the withdrawal symptoms; nevertheless, icv injection attenuated the withdrawal signs significantly. CONCLUSION: In conclusion we found that central administration of selegiline attenuated morphine withdrawal symptoms.