enkephalin--leucine-2-alanine and Brain-Ischemia

The introduction of therapeutic mild hypothermia after cardiac arrest allows the neuronal damage caused by global cerebral ischemia to be advantageously influenced for the first time. Currently, hypothermia is induced by external or internal cooling of the patient (forced hypothermia). However, this results in activation of counter-regulation mechanisms which could be possible risk factors for the patient. The aim of this article is to give a review of possible, but at present only experimental, methods which could allow the body temperature set point to be decreased pharmacologically (regulated hypothermia). Various classes of substances will be discussed based on their effect on thermoregulation and their performance in animal experiments on cerebral ischemia.

Topics: 8-Hydroxy-2-(di-n-propylamino)tetralin; Animals; Body Temperature; Body Temperature Regulation; Brain Ischemia; Enkephalin, Leucine-2-Alanine; Heart Arrest; Humans; Hypothermia, Induced; Neurotensin; Risk Factors; Serotonin Receptor Agonists

Topics: Animals; Apoptosis; Brain Ischemia; Enkephalin, Leucine-2-Alanine; ErbB Receptors; Infarction, Middle Cerebral Artery; Male; Phosphorylation; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Reperfusion Injury; Transcriptional Activation

This study aimed to investigate whether a selective delta opioid receptor agonist, [D-Ala2, D-Leu5]-Enkephalin (DADLE), regulates neurogenesis in the hippocampus of ischemic rats. Using an intracerebral cannula, rats were subjected to cerebral ischemia using the standard four-vessel occlusion. DADLE (2.5nmol), DADLE (2.5nmol) with naltrindole (NAL) (2.5nmol), or vehicle was administered at the onset of reperfusion. Bromodeoxyuridine (BrdU, 100mg/kg, intraperitoneal) was used to label newly formed cells from days 1 to 7 after ischemia. Immunohistochemistry was used to evaluate cell proliferation and apoptosis and differentiation 7days 28 days, respectively, after ischemia. Morris water maze test was conducted to test spatial learning and memory 23-27 days after ischemia. We found that DADLE treatment improved performance in the Morris water maze test, promoted proliferation and differentiation of newly formed neurons, and inhibited differentiation into astrocytes in a rat model of cerebral ischemia. Furthermore, the protective effects of DADLE were significantly reversed by co-administration of NAL (P<0.05), a highly potent and selective delta opioid receptor antagonist. Our findings suggest that DADLE promotes spatial cognitive function recovery and regulates neurogenesis after ischemia, which may provide a promising therapeutic strategy for cerebral ischemia.

Topics: Animals; Apoptosis; Brain; Brain Ischemia; Cell Differentiation; Cell Proliferation; Enkephalin, Leucine-2-Alanine; Male; Maze Learning; Neurogenesis; Neurons; Neuroprotective Agents; Rats, Sprague-Dawley; Receptors, Opioid, delta; Spatial Learning

To investigate the effect of delta opioid receptor agonist (D-Ala, D-Leu) enkephalin (DADLE) on the permanent focal cerebral ischemia in rats. Thirty four male Sprague-Dawley rats were assigned randomly into three groups: sham group (group Sham, n=10), artificial cerebrospinal fluid group (group ACSF, n=12), and DADLE group (group DADLE, n=12). Permanent middle cerebral artery occlusion was performed to induce permanent focal cerebral ischemia in rats. Then, the animals in group DADLE and group ACSF were treated with DADLE or ACSF by an intracerebroventricular injection at 45 min after ischemia. Neurologic deficit scores were assessed according to the Garcia criterion at 24 h after ischemia. Infarct volume was determined using the 2,3,5-triphenyltetrazolium chloride staining method. The histological analysis was used to evaluate the extent of cerebral injury. Compared with the control group, the Garcia scores were significantly higher (P=0.000) and the infarct volumes (P=0.018) were significantly smaller in the DADLE treatment group at 24 h after ischemia. These neurologic changes were closely correlated with the outcome of the infarct volumes. In addition, the histological examination showed more intact neurons in rats treated with DADLE than those treated with ACSF at 24 h after ischemia (P=0.000). DADLE by intracerebroventricular administration at 45 min after ischemia can improve neurologic outcome and mitigate cortical neuronal injury induced by permanent focal cerebral ischemia in rats.

Topics: Animals; Brain; Brain Ischemia; Enkephalin, Leucine-2-Alanine; Male; Neuroprotective Agents; Rats, Sprague-Dawley; Receptors, Opioid, delta

The delta opioid peptide [D-Ala2, D-Leu5]enkephalin (DADLE) plays a key role in neuronal protection against both hypoxic and ischemic conditions. However, the cellular mechanisms of action of DADLE under these conditions remain unclear.. Ischemia was simulated with perfusing the brain slices with glucose-free artificial cerebrospinal fluid. Apoptosis was examined using an in situ cell death detection kit and expressed as the percentage of positively labeled neurons relative to total number of neurons. PCR was performed by adding cDNA, 5 pm dNTP, 1 μL Taqase, and primers. PCR products were separated with electrophoresis, stained with ethidium bromide, and visualized under ultraviolet light.. To investigate the potential effects of DADLE in an ex vivo model of cerebral ischemia/reperfusion.. DADLE attenuated lactic dehydrogenase release and neuronal apoptosis in a concentration-dependent manner. The protective effects of DADLE were attenuated by representative selective delta2, but not delta1 opioid antagonists. Treatment with PD98059, a selective inhibitor of ERK kinase (MEK), also blocked the protective effect of DADLE as well as ERK phosphorylation induced by DADLE.. Endogenous opioid peptides could promote cell survival via delta2 opioid receptors, possibly through the downstream MEK-ERK pathway.

Topics: Animals; Apoptosis; Brain Ischemia; Cerebral Cortex; Disease Models, Animal; Dose-Response Relationship, Drug; Enkephalin, Leucine-2-Alanine; In Vitro Techniques; L-Lactate Dehydrogenase; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Reperfusion Injury

Preconditioning with selective delta opioid peptide [d-Ala2, d-Leu5] enkephalin (DADLE) provides ischemic tolerance following transient forebrain ischemia in rats. However, whether DADLE postconditioning retains its neuroprotective efficacy and the underlying molecular mechanism in ischemic brain is largely unknown. We investigated DADLE postconditioning protection of hippocampal CA1 neurons against transient forebrain ischemia. 6 days after being implanted with cannula at the right lateral ventricle, rats underwent 10 min of forebrain ischemia by four vessel occlusion. Hippocampal CA1 neuronal survival and degeneration were measured in the hippocampi of rats at 3 days after ischemia. The behavioral and cognitive improvements of DADLE treatment in rats were also evaluated on days 5-9 using open-field and Morris water maze tests. The results showed that DADLE at doses of 0.25 and 2.5 nmol, but not 25 nmol, could significantly protect CA1 neurons against ischemia/reperfusion injury. Co-administration with the delta-opioid receptor antagonist naltrindole or pretreatment with the Akt antagonist LY294002 completely abolished the DADLE postconditioning effect. Furthermore, DADLE postconditioning exhibited cognitive benefits in rats with transient forebrain ischemia. The study thus suggested a therapeutic opportunity of postconditioning neuroprotection by DADLE and also provided important information in understanding the mechanism of DADLE action in the ischemic brain.

Topics: Animals; Behavior, Animal; Brain Ischemia; Enkephalin, Leucine-2-Alanine; Hippocampus; Ischemic Postconditioning; Male; Neuroprotective Agents; Phosphatidylinositol 3-Kinases; Prosencephalon; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Signal Transduction

Delta opioid receptor (DOR) is essential for neuronal survival against hypoxic/ischemic damages. However, current understanding on how DOR activation affects astrocytic functions under ischemia remains incomplete. The present study investigated the astroglial responses to [d-Ala2, d-Leu5] enkephalin (DADLE) (a selective DOR agonist)-induced DOR activation after global cerebral ischemia. Adult male rats were preimplanted with intracerebral cannula and subjected to global ischemia for 10 min. The rats were divided into four groups: normal group (without any procedure), sham group (sham procedure with intracerebroventricular injection of ACSF), I/R group (ischemia procedure with intracerebroventricular injection of ACSF) and DAD-treated group (ischemia procedure with intracerebroventricular injection of DADLE). Hippocampal CA1 neuronal survival and activation of astrocytes were measured in the animals at 72 h post-ischemia. The distribution and phenotypes of p-Akt and active caspase-3 were also determined. The ischemic injury resulted in a significant neuronal loss and an increase in the dying astrocytes in the hippocampal CA1 region as compared with those in the sham animals (200.7±22.7/mm(2) vs. 6.6±3.1/mm(2), P<0.001). Improved neuronal survival in the DAD-treated animals was evident, which was accompanied by less dying astrocytes and enhanced astrocytes reaction with more active astrocytes than that in the I/R group (267.6±13.2/mm(2) vs. 157.0±18.1/mm(2), P<0.01) and a significantly increased immunoreactivity of p-Akt. However, the active caspase-3 positive cells were also evident in DAD-treated group (313.0±23.1/mm(2)) and significantly increased as compared with those of the sham group (159.0±15.8/mm(2), P<0.001) or I/R group (193.6±26.2/mm(2), P<0.01). Most of the active caspase-3-expressing cells were colabeled with glial fibrillary acidic protein (GFAP), an astrocytes marker. We conclude that the post-ischemic treatment with DADLE promotes beneficial astrocytes activation and induces astroglial apoptosis 72 h after reperfusion which may be involved in reducing their harmful effect to neurons survival.

Topics: Animals; Astrocytes; Brain Ischemia; Caspase 3; Enkephalin, Leucine-2-Alanine; Hippocampus; Immunohistochemistry; Male; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta

Delta opioid peptide [D-ala2,D-leU5]enkephalin (DADLE) induces hibernation in summer ground squirrels, and enhances preservation and survival of isolated or transplanted lungs and hearts. In the present study, we investigated the protective effect of DADLE in the central nervous system.. Adult Sprague-Dawley rats were pretreated with DADLE (4 mg/kg every 2 h x 4 injections, i.p.) or saline prior to unilateral occlusion of the middle cerebral artery (MCA). Daily behavioral tests revealed that ischemic animals treated with DADLE did not show any significant behavioral dysfunctions compared with saline-treated ischemic animals. Opioid antagonists only transiently inhibited the protective effect of DADLE, indicating the participation of non-opioid mechanisms in DADLE neuroprotection. Histological examination using triphenyltetrazolium chloride (TTC) revealed that brains from ischemic animals treated with DADLE, either alone or with adjuvant opioid blockers, exhibited almost completely intact striata. In contrast, brains from ischemic animals that received saline showed significant infarction in the lateral striatum. Analyses of apoptotic cell death revealed a significant increase in the p-53 mRNA expression in the striatum of ischemic animals that received saline, while those that received DADLE exhibited near normal striatal p-53 expression. This protective effect was accompanied by significant increments in protein levels of glial cell line-derived neurotrophic factor in the striatum of DADLE-treated ischemic animals.. These results indicate that DADLE protected against necrotic and apoptotic cell death processes associated with ischemia-reperfusion injury. The present study demonstrates that delta opioids are crucially involved in stroke, suggesting that the opioid system is important in the study of brain injury and protection.

Topics: Animals; Apoptosis; Brain; Brain Infarction; Brain Ischemia; Disease Models, Animal; Enkephalin, Leucine-2-Alanine; Glial Cell Line-Derived Neurotrophic Factor; Hibernation; Motor Activity; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tumor Suppressor Protein p53

We have recently shown that delta-opioid receptors (DORs) play an important role in neuroprotection from hypoxic injury via the regulation of extracellular signaling-regulated kinase (ERK) and cytochrome c release. Since ERK and cytochrome c are differentially involved in caspase signaling of oxidative injury that significantly contributes to neuronal damage in ischemia/reperfusion, we considered if DOR activation protects the ischemic brain by attenuating oxidative injury.. We observed that, in a model of cerebral ischemia with middle cerebral artery occlusion, DOR activation increased the activity of major antioxidant enzymes, glutathione peroxidase and superoxide dismutase, and decreased malondialdehyde and nitric oxide levels in the cortex exposed to cerebral ischemia/reperfusion. In addition, DOR activation reduced caspase 3 expression, though it did not significantly affect the increase in interleukin (IL)1beta and tumor necrosis factor (TNF)alpha expression at the same timepoint. PD98059, an inhibitor of mitogen-activated protein kinase (MAPK) extracellular signaling-regulated kinase kinase, accelerated animal death during ischemia/reperfusion.. DOR activation attenuates oxidative injury in the brain exposed to ischemia/reperfusion by enhancing antioxidant ability and inhibiting caspase activity, which provides novel insights into the mechanism of DOR neuroprotection.

Topics: Animals; Brain Ischemia; Caspase 3; Enkephalin, Leucine-2-Alanine; Gene Expression Regulation; Glutathione Peroxidase; Hippocampus; Interleukin-1beta; Male; Malondialdehyde; Models, Biological; Nitric Oxide; Oxidative Stress; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Reperfusion Injury; RNA, Messenger; Superoxide Dismutase; Tumor Necrosis Factor-alpha

It has been reported that delta opioid agonists can have neuroprotective efficacy in the central nervous system. This study was conducted to test the hypothesis that a delta opioid receptor (DOR) agonist, [D-Ala2, D-Leu5] enkephalin (DADLE), can improve neuron survival against experimental forebrain ischemia in rats. Using male rats (n=125), intraperitoneal injection of DADLE (0, 0.25, 1, 4, 16 mg kg-1) was performed 30 min before ischemia. Ten minutes interval forebrain ischemia was provided by the bilateral carotid occlusion combined with hypotension (35 mm Hg) under isoflurane (1.5%) anesthesia. All animals were neurologically and histologically evaluated after a recovery period of 1 week. As histological evaluation, percentages of ischemic neurons in the CA1, CA3, dentate gyrus (DG) were measured. During the recovery period, 27 rats died because of apparent upper airway obstruction, seizure, or unidentified causes. There were no differences in the motor activity score among the groups. Ten minutes forebrain ischemia induced approximately 75, 20, and 10% neuronal death in the CA1, CA3, and DG, respectively. Any doses of DADLE did not attenuate neuronal injury in the hippocampus after ischemia. Pre-ischemic treatment of DORs agonism with DADLE did not provide any neuroprotection to the hippocampus in rats subjected to forebrain ischemia.

Topics: Analgesics, Opioid; Animals; Brain Ischemia; Cell Survival; Cerebral Infarction; Dentate Gyrus; Dose-Response Relationship, Drug; Enkephalin, Leucine-2-Alanine; Hippocampus; Male; Nerve Degeneration; Neuroprotective Agents; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta; Treatment Failure

Cerebral ischemic insult, mainly induced by cardiovascular disease, is one of the most severe neurological diseases in clinical. There's mounting evidence showing that delta opioid agonist [D-Ala2, D-Leu5] enkephalin (DADLE) has a tissue-protective effect. However, whether this property is effective to prevent neuronal death induced by forebrain ischemia is not clear. This study was aimed to investigate whether intracerebroventricular (ICV) administration of DADLE has a neuroprotective effect against forebrain ischemia in rats. We found in our study that administration of DADLE 45 min before forebrain ischemia had significant protective effect against CA1 neuronal lose. Further more, we found that DADLE had a dose-dependent protection for improving behavioral retardation revealed by Morris water maze and motor score test, while naltrindole, the antagonist of delta opioid receptor, partially abolished neuroprotective effect of DADLE, which implicated that both opioid and non-opioid systems are involved in ischemic insults and neuroprotection.

Topics: Animals; Behavior, Animal; Brain Ischemia; Cell Death; Dose-Response Relationship, Drug; Enkephalin, Leucine-2-Alanine; Injections, Intraventricular; Male; Naltrexone; Narcotic Antagonists; Neurons; Neuroprotective Agents; Prosencephalon; Rats; Rats, Sprague-Dawley

It has been reported that delta-opioid (DOP) receptor agonists may be neuroprotective in the central nervous system. However, the DOP agonist [d-Ala(2), d-Leu(5)]enkephalin (DADLE) does not produce neuroprotection in severe forebrain ischaemia. The aim of this study was to examine the effects of DADLE on hippocampal neurone survival against less severe forebrain ischaemia.. Intraperitoneal injection of DADLE (0 or 16 mg kg(-1)) in male Sprague-Dawley rats was performed 30 min before ischaemia. Severe (10 min), moderate (8 min), or mild (6 min) forebrain ischaemia was produced by bilateral carotid occlusion combined with hypotension (35 mm Hg) under isoflurane (1.5%) anaesthesia. Naltrindole (10 mg kg(-1)) (DOP antagonist) was administered 30 min before DADLE in order to confirm DOP receptor activation in the neuroprotective efficacy of DADLE. Naltrindole alone was also administered 30 min before ischaemia to examine endogenous DOP agonism as a self-protecting mechanism against ischaemia. All animals were evaluated neurologically and histologically after a 1 week recovery period.. DADLE improved neurone survival in hippocampal CA3 and dentate gyrus (DG) sectors. CA1 neurones were not protected against moderate and mild ischaemia. Naltrindole abolished DADLE neuroprotection in the CA3 and DG after both moderate and mild ischaemia. Interestingly, regardless of co-administration of DADLE, naltrindole significantly worsened neuronal injury in the CA1 region after mild ischaemia.. These results suggest that DADLE provides limited neuroprotection to relatively ischaemia-resistant regions but not to selectively vulnerable regions. This was probably mediated by DOP stimulation. Pre-ischaemic treatment with a DOP antagonist, regardless of co-administration of DADLE, worsened neuronal damage at the selectively vulnerable regions only after mild forebrain ischaemia. These data suggest that DOP activation with endogenous DOP ligand may be involved in self-protecting ischaemia-sensitive regions of the brain.

Topics: Animals; Brain Ischemia; Cell Survival; Drug Evaluation, Preclinical; Enkephalin, Leucine-2-Alanine; Hippocampus; Male; Naltrexone; Narcotic Antagonists; Neurons; Neuroprotective Agents; Prosencephalon; Rats; Rats, Sprague-Dawley; Receptors, Opioid, delta