Induction of an in vitro reversible hypometabolism through chitosan-based nanoparticles

Objective: Chitosan-based nanoparticles (NPs) were prepared to promote intracellular sustained delivery of the synthetic delta opioid D-Ala(2)-D-Leu(5)-enkephalin (DADLE), prolonging peptide activity and inducing a safe and reversible hypometabolic state.

Materials and methods: NPs were prepared by combining ionotropic gelation and ultrasonication treatment. NP uptake studies and the effects of encapsulated DADLE on HeLa cells proliferation were tested by transmission electron microscopy (TEM) analysis, by immuno-fluorescence and immuno-cytochemistry.

Results: DADLE-loaded NPs are produced with suitable characteristics, a satisfactory process yield (55.4%?±?2.4%) and encapsulation efficiency (64.6%?±?2.1%). NPs are effective in inducing a hypometabolic stasis at a 10^?4?M DADLE concentration. Moreover, as seen from the immunofluorescence study, the effect persists through the recovery period (72?h). Indeed, NPs labelled by anti-enkephalin antibody inside cell nucleus reassert that the in vivo release of the peptide can be prolonged with respect to the case of free peptide supply.

Conclusion: The nanoparticulate drug delivery system described seems to be effective in inducing and prolonging a sort of hibernation-like state in the cells.     

Convergent effects of oxytocin and a delta-opioid agonist in the bed nuclei of the stria terminalis of the peripartum rat

In the bed nuclei of the stria terminalis (BST), opioids may suppress the facilitatory effect of oxytocin on its own release pre-partum. In vitro electrophysiological recording showed that in virgin, late-pregnant, and lactating rats, a δ-opioid agonist inhibited a high proportion of BST neurons, many of which were also oxytocin responsive. Response magnitude did not differ significantly between groups, suggesting that the postulated pre-partum increase in opioid tone does not involve postsynaptic changes in neuronal sensitivity.     

Effect of opiates on the release of cholecystokinin from in vitro hypothalamus and frontal cortex of Zucker lean (Fa/−) and obese (fa/fa) rats

Opiates, morphine and [d-Ala²-d-Leu⁵]-enkephalin (DADLE), inhibited the K⁺-stimulated release of cholecystokinin (CCK) from the hypothalamus of both Zucker obese (fa/fa) and lean (Fa/−) rats, in vitro. Morphine and DADLE did not inhibit the K⁺-stimulated release of CCK from frontal cortex from either strain. The opiates did not affect basal efflux of CCK and their effects were all blocked by equimolar concentrations of naloxone. These studies indicate a regional specificity for the effect of opiates on CCK release, and may provide evidence for a cellular mechanism by which endogenous opiates modulate feeding behavior.     

The thalamic nucleus submedius and ventrolateral orbital cortex are involved in nociceptive modulation: A novel pain modulation pathway

Recently, a series of studies have given rise to and provided evidence for the hypothesis that the nucleus submedius (Sm) in the medial thalamus is involved in modulation of nociception. The Sm, ventrolateral orbital cortex (VLO) and the periaqueductal gray (PAG) constitute a pain modulatory pathway, activation of which leads to activation of the PAG–brainstem descending inhibitory system and depression of the nociceptive inputs in the spinal cord and trigeminal nucleus. Other studies have indicated that the Sm–VLO–PAG pathway plays an important role in the analgesia induced by electroacupuncture stimulation of the acupuncture point (acupoint) for exciting small diameter fiber (A-δ and C group) afferents. Opioid peptides, serotonin, dopamine, glutamate and their related receptors are involved in Sm- and/or VLO-mediated descending antinociception, and a GABAergic disinhibitory mechanism participates in mediating the antinociception induced by activation of μ-opioid receptors, serotonin 1_A receptors, and dopamine D₂-like receptors. This review describes these findings, which provide important new insights into the roles of the thalamus and cerebral cortex in descending pain modulation.     

Assessment of the delta opioid agonist DADLE in a rat model of lethal hemorrhage treated by emergency preservation and resuscitation

Emergency preservation and resuscitation (EPR) is a new approach for resuscitation of exsanguination cardiac arrest (CA) victims. EPR uses a cold aortic flush to induce deep hypothermic preservation during no-flow to buy time for transport and damage control surgery, followed by resuscitation with cardiopulmonary bypass (CPB). We reported previously that 20–60 min EPR in rats was associated with intact outcome, while 75 min EPR resulted in high mortality and neurological impairment in survivors. The delta opioid agonist DADLE ([d-Ala(2),d-Leu(5)]-enkephalin) was shown previously to be protective against ischemia-reperfusion injury in multiple organs, including brain. We hypothesized that DADLE could augment neurological outcome after EPR in rats. After rapid lethal hemorrhage, EPR was initiated by perfusion with ice-cold crystalloid to induce hypothermia (15 °C). After 75 min EPR, resuscitation was attempted with CPB. After randomization, three groups were studied (n = 10 per group): DADLE 0 mg/kg (D0), 4 mg/kg (D4) or 10 mg/kg (D10) added to the flush and during reperfusion. Survival, overall performance category (OPC; 1 = normal, 5 = death), neurological deficit score (NDS; 0–10% normal, 100% = max deficit), and histological damage score (HDS) were assessed in survivors on day 3. In D0 group, 2/10 rats survived, while in D4 and D10 groups, 4/10 and 5/10 rats survived, respectively (p = NS). Survival time (h) was 26.7 ± 28.2 in D0, 36.3 ± 31.9 in D4 and 47.1 ± 30.3 in D10 groups, respectively (p = 0.3). OPC, NDS and HDS were not significantly different between groups. In conclusion, DADLE failed to confer benefit on functional or histological outcome in our model of prolonged rat EPR.     

The effect of in vivo ethanol consumption on cyclic AMP and δ-opioid receptors in mouse striatum

In this study the effect of in vivo ethanol consumption on cyclic AMP (cAMP) and [

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-Ala²,

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-Leu⁵]enkephalin (DADLE) inhibition of forskolin-stimulated cAMP production was examined in mouse striatum. Effects of ethanol on striatal δ-opioid receptor (DOR) density and mRNA were also examined. Mice had unlimited access to 7% (v/v) ethanol alone or water for 1 or 7 days and were then sacrificed and striatum removed for analysis. There was no difference in basal cAMP formation between water and ethanol-treated mouse striatum following 7 day treatment, and a small, but statistically significant increase in basal cAMP in the ethanol group following 1 day treatment. Both 1 day and 7 day ethanol treatment did not significantly alter the percentage increase in cAMP following treatment with 10 μM forskolin. There was a significant effect of ethanol treatment on the maximum inhibitory effect of DADLE on forskolin-stimulated cAMP formation following both 1 and 7 day ethanol treatment. The DADLE IC₅₀ was unaffected by ethanol treatment. Saturation binding studies ([³H]Deltorphin II) indicated no effect of ethanol on B_max or K_d in striatum. Similarly, no difference between water and ethanol-treated was observed for DOR mRNA in striatum. These data indicate that ethanol consumption can alter opioid regulation of cAMP formation. However, this effect is not related to changes in any δ-opioid receptor parameters that were examined.     

δ阿片受体激动剂DADLE对脓毒症大鼠急性肺损伤的保护作用

目的 探讨δ阿片受体激动剂DADLE对脓毒症性急性肺损伤(ALI)的保护作用.方法 采用盲肠结扎穿孔法(CLP)制作脓毒症急性肺损伤模型,将模型随机分为ALI组、假手术组(SHAM组)、糖皮质激素(GC)治疗组和DADLE治疗组.分别于建模3、6和12h后开腹抽血行动脉血气分析,ELISA法检测血浆中肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)和核因子-κB(NF-κB)的含量,观察与比较各组肺组织病理改变.结果 DADLE组中PaCO2、PaO2、HCO2-和BE与GC组相比较差异无统计学意义(P＞0.05),但均高于ALI组(P＜0.05);GC组和DADLE组血浆中TNF-α、IL-6和NF-κB水平均明显低于ALI组(P＜0.01);DADLE组和GC组肺组织病理学变化均较ALI组轻.结论 DADLE对脓毒症大鼠的肺组织有明确的保护作用,具有抑制炎性细胞因子释放作用,其抗炎作用与GC相似.     

Effects of morphine and D-Ala, D-Leu enkephalin on the electrical activity of supraoptic neurosecretory cells in vitro

Experiments were performed to investigate the effects of morphine and [D-Ala2, D-Leu5]enkephalin on supraoptic cells in hypothalamic slices in vitro. To ensure the presence of a steady background activity, the cells were recorded with glutamate-filled glass microelectrodes and the level of activity was controlled by selecting a suitable retaining current (0.1-9.8 nA). Under these conditions, supraoptic cells showed either the non-phasic (65%) or phasic (35%) firing pattern previously associated with oxytocin or vasopressin cells, respectively. During perifusion of the slice with morphine (10 microM), 10 out of 17 non-phasic supraoptic cells were profoundly inhibited, five cells showed no response and the remaining 2 cells were excited. Similarly with [D-Ala2, D-Leu5]enkephalin (10 microM), 11 out of 15 non-phasic cells were inhibited, 3 cells showed no response and 1 cell was excited. The inhibition produced by morphine or [D-Ala2, D-Leu5]enkephalin could be reversed by concomitant application of naloxone (10 microM). In contrast to the profound effects seen in the non-phasic cells, only 1 out of 13 phasic cells tested with either morphine or [D-Ala2, D-Leu5]enkephalin was inhibited. The remaining 12 phasic cells showed no change in either their overall firing rate or pattern of activity during opiate perifusion. These results provide further evidence that, in addition to their inhibitory effects within the posterior pituitary, opiates can directly suppress the electrical activity of magnocellular neurosecretory cells at the level of the hypothalamus. However, the absence of an opiate effect on the phasic cells might suggest that the action of opioid peptides within the hypothalamus would be exerted predominantly on the oxytocin, rather than the vasopressin cells.     

DADLE对大鼠急性全脑缺血再灌注后海马区神经元ERK信号通路和Caspase-3表达的影响

目的：观察8-阿片受体激动剂DADLE（[D—Ala2,D—Leu5]enkephali）对大鼠急性全脑缺血再灌注后海马区神经元ERK信号通路和Caspase一3表达的影响。方法：将50只sD大鼠随机分为5组：假手术组（Sham）、模型组（I／R）、DADLE处理组（根据不同剂量可分为2mg／kg[A]、3mg／kg[B]、5mg／kg[C]o采用改良的二血管阻断加低血压法建立全脑缺血再灌注模型。于缺血15min后经颈外静脉注射DADLE并于再灌注120min后处死。开颅取其新鲜海马组织,采用western blot检测海马组织Caspase-3的表达,以及采用免疫组织化学法检测非磷酸化ERK与磷酸化ERK（P—ERK）的表达状况。结果：Sham组ERK和P—ERK蛋白表达水平显著低于其他各组（P〈0．05）,DADLE处理组与I／R组相比,其ERK和P—ERK蛋白的表达量明显上调（P〈0．05）。海马组织Caspase-3蛋白表达I／R组较Sham组表达明显上调（P〈O．05）,而DADLE处理组与IR组比较,海马组织Caspase-3蛋白表达明显下降（P〈0．05）。结论：DADLE对大鼠急性全脑缺血再灌注后海马区神经元有保护作用,说明DADLE可通过上调ERK信号通路以及抑制Caspase-3的表达而起到保护脑组织的作用。     

δ阿片受体激动剂DADLE对脓毒症大鼠血清HMGB1表达的影响及其作用的研究

目的观察δ阿片受体激动剂DADLE对脓毒症大鼠血清HMGB1水平及器官功能的影响。方法 96只SD大鼠分为假手术组(SO)、脓毒症组(SEP)、DADLE组[制模后腹腔内注射DADLE(5 mg/kg)],各32只。盲肠结扎穿孔(CLP)建立脓毒症模型,制模后6、12、18、24 h各取8只处死取材。ELISA法检测血清HMGB1水平及24 h ALT、AST、BUN、Cr、CK及PaO2的变化。结果 HMGB1水平在SEP组、DADLE组12 h 18 h及24 h均较SO组高(P<0.05),DADLE组则显著低于SEP组(P<0.05)。24 h时DADLE组血ALT、AST、BUN、Cr、CK均较SEP组明显改善,PaO2明显升高(P<0.05),且各指标变化与HMGB1有显著相关性(P<0.05)。结论 DADLE抑制HMGB1释放,对脓毒症大鼠重要脏器功能有保护作用。