Time-Dependent Changes in the Serum Levels of Neurobiochemical Factors After Acute Methadone Overdose in Adolescent Male Rat.

Acute methadone toxicity is a major public health concern which has adverse effects on brain tissue and results in recurrent or delayed respiratory arrest. Our study aimed to investigate the time-dependent changes in several serum biochemical markers of brain damage, spatial working memory, and the brain tissue following acute methadone overdose. Adolescent male rats underwent an intraperitoneal (i.p.) injection of 15 mg/kg methadone. In case of apnea occurrence, resuscitation was performed by a ventilatory pump and administrating naloxone (2 mg/kg; i.p.). The animals were classified into groups of treated rats; methadone and naloxone-Apnea (M/N-Apnea), M/N-Sedate, Methadone, Naloxone, and control (saline) groups. The serum levels of S100B, neuron-specific enolase (NSE), myelin basic protein factors, and (Lactate/Pyruvate) L/P ratio were evaluated at the time-points of 6, 24, and 48 h (h). We found that the alterations of S100B and L/P ratio were considerable in the M/N-Apnea and Methadone groups from the early hours post-methadone overdose, while NSE serum levels elevation was observed only in M/N-Apnea group with a delay at 48 h. Further, we assessed the spatial working memory (Y-maze test), morphological changes, and neuronal loss. The impaired spontaneous alternation behavior was detected in the M/N-Apnea groups on days 5 and 10 post-methadone overdose. The morphological changes of neurons and the neuronal loss were detectable in the CA1, striatum, and cerebellum regions, which were pronounced in both M/N-Apnea and Methadone groups. Together, our findings suggest that alterations in the serum levels of S100B and NSE factors as well as L/P ratio could be induced by methadone overdose with the presence or absence of apnea before the memory impairment and tissue injury in adolescent male rats.

Methadone-induced encephalopathy: a case series and literature review.

BACKGROUND: Accidental ingestion or consumption of supra-therapeutic doses of methadone can result in neurological sequelae in humans. We aimed to determine the neurological deficits of methadone-poisoned patients admitted to a referral poisoning hospital using brain magnetic resonance (MR) and diffusion weighted (DW) imaging. METHODS: In this retrospective study, brain MRIs of the patients admitted to our referral center due to methadone intoxication were reviewed. Methadone intoxication was confirmed based on history, congruent clinical presentation, and confirmatory urine analysis. Each patient had an MRI with Echo planar T1, T2, FLAIR, and DWI and apparent deficient coefficient (ADC) sequences without contrast media. Abnormalities were recorded and categorized based on their anatomic location and sequence. RESULTS: Ten patients with abnormal MRI findings were identified. Eight had acute- and two had delayed-onset encephalopathy. Imaging findings included bilateral confluent or patchy T2 and FLAIR high signal intensity in cerebral white matter, cerebellar involvement, and bilateral occipito-parietal cortex diffusion restriction in DWI. Internal capsule involvement was identified in two patients while abnormality in globus pallidus and head of caudate nuclei were reported in another. Bilateral cerebral symmetrical confluent white matter signal abnormality with sparing of subcortical U-fibers on T2 and FLAIR sequences were observed in both patients with delayed-onset encephalopathy. CONCLUSIONS: Acute- and delayed-onset encephalopathies are two rare adverse events detected in methadone-intoxicated patients. Brain MRI findings can be helpful in detection of methadone-induced encephalopathy.

Delay-Dependent Impairments in Memory and Motor Functions After Acute Methadone Overdose in Rats.

Methadone is used as a substitution drug for the treatment of opioid dependence and chronic pain. Despite its widespread use and availability, there is a serious concern with respect to the relative safety of methadone. The purpose of this study was to characterize how acute methadone overdose affects the cognitive and motor performance of naïve healthy rats. The methadone overdose was induced by administering an acute toxic dose of methadone (15 mg/kg; ip; the equivalent dose of 80% of LD50) to adolescent rats. Resuscitation using a ventilator pump along with a single dose of naloxone (2 mg/kg; ip) was administered following the occurrence of apnea. The animals which were successfully resuscitated divided randomly into three apnea groups that evaluated either on day 1, 5, or 10 post-resuscitation (M/N-Day 1, M/N-Day 5, and M/N-Day 10 groups) in the Y-maze and novel object memory recognition tasks as well as pole and rotarod tests. The data revealed that a single toxic dose of methadone had an adverse effect on spontaneous behavior. In addition, Recognition memory impairment was observed in the M/N-Day 1, 5, and 10 groups after methadone-induced apnea. Further, descending time in the M/N-Day 5 group increased significantly in comparison with its respective Saline control group. The overall results indicate that acute methadone-overdose-induced apnea produced delay-dependent cognitive and motor impairment. We suggest that methadone poisoning should be considered as a possible cause of delayed neurological disorders, which might be transient, in some types of memory or motor performance in naïve healthy rats.

The role of potassium BK channels in anticonvulsant effect of cannabidiol in pentylenetetrazole and maximal electroshock models of seizure in mice.

Cannabidiol is a nonpsychoactive member of phytocannabinoids that produces various pharmacological effects that are not mediated through putative CB1/CB2 cannabinoid receptors and their related effectors. In this study, we examined the effect of the i.c.v. administration of potassium BK channel blocker paxilline alone and in combination with cannabidiol in protection against pentylenetetrazol (PTZ)- and maximal electroshock (MES)-induced seizure in mice. In the PTZ-induced seizure model, i.c.v. administration of cannabidiol caused a significant increase in seizure threshold compared with the control group. Moreover, while i.c.v. administration of various doses of paxilline did not produce significant change in the PTZ-induced seizure threshold in mice, coadministration of cannabidiol and paxilline attenuated the antiseizure effect of cannabidiol in PTZ-induced tonic seizures. In the MES model of seizure, both cannabidiol and paxilline per se produced significant increase in percent protection against electroshock-induced seizure. However, coadministration of cannabidiol and paxilline did not produce significant interaction in their antiseizure effect in the MES test. The results of the present study showed a protective effect of cannabidiol in both PTZ and MES models of seizure. These results suggested a BK channel-mediated antiseizure action of cannabidiol in PTZ model of seizure. However, such an interaction might not exist in MES-induced convulsion.

Kindling-induced learning deficiency and possible cellular and molecular involved mechanisms.

Hippocampus learning disturbance is a major symptom of patients with seizure, hence hippocampal dysfunction has essential role in worsening the disease. Hippocampal formation includes neurons and myelinated fibers that are necessary for acquisition and consolidation of memory, long-term potentiation and learning activity. The exact mechanism by which seizure can decrease memory and learning activity of hippocampus remains unknown. In the present study, electrical kindling-induced learning deficit in rats was evaluated by Morris water maze (MWM) test. The hippocampus was removed and changes in neurons and myelin sheaths around hippocampal fibers were investigated using histological and immunohistochemical methods. Demyelination was assessed by luxol fast blue staining, and immunohistological staining of myelin-binding protein (MBP). The TUNEL assay was used for evaluation of neuronal apoptosis and the glial fibriliary acetic protein (GFAP) was used for assessment of inflammatory reaction. The results indicated that electrical kindling of hippocampus could induce deficiency in spatial learning and memory as compared to control group. In addition, electrical kindling caused damage to the myelin sheath around hippocampal fibers and produced vast demyelination. Furthermore, an increase in the number of apoptotic cells in hippocampal slices was observed. In addition, inflammatory response was higher in kindled animals as compared to the control group. The results suggested that the decrease in learning and memory in kindled animals is likely due to demyelination and augmentation in apoptosis rate accompanied by inflammatory reaction in hippocampal neurons of kindled rats.

L-type calcium channel mediates anticonvulsant effect of cannabinoids in acute and chronic murine models of seizure.

The anticonvulsant activities of cannabinoid compounds have been shown in various models of seizure and epilepsy. At least, part of antiseizure effects of cannabinoid compounds is mediated through calcium (Ca(2+)) channels. The L-type Ca(2+) channels have been shown to be important in various epilepsy models. However, there is no data regarding the role of L-type Ca(2+) channels in protective action of cannabinoids on acute and chronic models of seizure. In this study, the effects of cannabinoid compounds and L-type Ca(2+) channels blockers, either alone or in combination were investigated using acute model of pentylenetetrazole (PTZ)-induced seizure in mice and chronic model electrical kindling of amygdala in rats. Pretreatment of mice with both cannabinoid CB1 receptor agonist arachidonyl-2'-chloroethylamide (ACEA) and endocannabinoid degradating enzyme inhibitor cyclohexylcarbamic acid 3'-carbamoyl-biphenyl-3-yl ester (URB597) produced a protective effect against PTZ-induced seizure. Administration of various doses of the two L-type Ca(2+) channel blockers verapamil and diltiazem did not alter PTZ-induced seizure threshold. However, co-administration of verapamil and either ACEA or URB597 attenuated the protective effect of cannabinoid compounds against PTZ-induced seizure. Also, pretreatment of mice with diltiazem blocked the anticonvulsant activity of both ACEA and URB597. Moreover, (R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate (WIN55,212-2), the non-selective cannabinoid CB1 and CB2 receptor agonist showed anticonvulsant effect in amygdala-kindled rats. However, co-administration of WIN55,212-2 and verapamil attenuated the protective properties of WIN55,212-2. Our results showed that the anticonvulsant activity of cannabinoid compounds is mediated, at least in part, by L-type Ca(2+) channels in these two models of convulsion and epilepsy.

Modulation of anticonvulsant effects of cannabinoid compounds by GABA-A receptor agonist in acute pentylenetetrazole model of seizure in rat.

Cannabinoid system plays an important role in controlling neuronal excitability and brain function. On the other hand, modulation of gamma-aminobutyric acid (GABA) transmission is one of the initial strategies for the treatment of seizure. The aim of the present study was to evaluate possible interaction between cannabinoidergic and GABAergic systems in pentylenetetrazole (PTZ)-induced acute seizure in rat. Drugs were administered by intracerebroventricular (i.c.v.) administration 20 min before a single intraperitoneal (i.p.) injection of PTZ and the latency to the first generalized tonic-clonic seizure was measured. Both the cannabinoid receptor agonist WIN55212-2 (10, 30, 50 and 100 µg/rat) and the GABA-A receptor agonist isoguvacine (IGN; 10, 30 and 50 µg/rat) significantly increased the latency of seizure occurrence. Moreover, the fatty acid amide hydrolase inhibitor URB597 showed no anticonvulsive effect while the monoacyl glycerol lipase (MAGL) inhibitor URB602 (10, 50 and 100 µg/rat) protected rats against PTZ-induced seizure. Moreover, co-administration of IGN and cannabinoid compounds attenuated the anticonvulsant action of both WIN55212-2 and IGN in this model of seizure. Our data suggests that exogenous cannabinoid WIN55212-2 and MAGL inhibitor URB602 imply their antiseizure action in part through common brain receptorial system. Moreover, the antagonistic interaction of cannabinoids and IGN in protection against PTZ-induced seizure could suggest the involvement of GABAergic system in their anticonvulsant action.

Effects of electrolytic lesion of dorsolateral periaqueductal gray on analgesic response of morphine microinjected into the nucleus cuneiformis in rat.

The periaqueductal gray (PAG) and nucleus cuneiformis (CnF), like the rostral ventromedial medulla, have functional roles in descending pain-inhibitory pathway related to morphine antinociception. There is not any evidence concerning the role of different regions of the PAG on antinociceptive effect of morphine administered into the CnF in pain modulatory system. In the present study, we investigate whether electrolytic lesion of dorsolateral periaqueductal gray (dl-PAG) influence the analgesic effect of morphine microinjected into the CnF. 71 adult male Wistar rats weighting 230-280 g cannulated bilaterally into the CnF, concurrently lesion of dl-PAG was done. The tail-flick and formalin tests were performed to measure pain and antinociceptive effect of morphine microinjected into the CnF (2.5 microg/0.3 microl saline per side). The tail-flick latency was measured at 15, 30, 45, 60 and 75 min following morphine microinjection. In formalin test, pain behavior was recorded for 60 min in early (0-5 min) and late (15-60 min) phases after formalin injection. Each rat was given a subcutaneous 50-microl injection of formalin 2.5% into plantar surface of hind paw following morphine administration. The results showed that dl-PAG lesion attenuated the effect of morphine microinjected into the CnF both in tail-flick and formalin tests while dl-PAG lesion solely did not alter basal pain behavior as compared to control group. In conclusion, our results suggest the existence of a direct or indirect projection from CnF to the dl-PAG at least at the level of the morphine antinociception in pain modulation.