The Traditional Chinese Medicine MLC901 inhibits inflammation processes after focal cerebral ischemia.

Inflammation is considered as a major contributor to brain injury following cerebral ischemia. The therapeutic potential of both MLC601/MLC901, which are herbal extract preparations derived from Chinese Medicine, has been reported both in advanced stroke clinical trials and also in animal and cellular models. The aim of this study was to investigate the effects of MLC901 on the different steps of post-ischemic inflammation in focal ischemia in mice. In vivo injury was induced by 60 minutes of middle cerebral artery occlusion (MCAO) followed by reperfusion. MLC901 was administered in post-treatment 90 min after the onset of ischemia and once a day during reperfusion. MLC901 treatment resulted in a reduction in infarct volume, a decrease of Blood Brain Barrier leakage and brain swelling, an improvement in neurological scores and a reduction of mortality rate at 24 hours after MCAO. These beneficial effects of MLC901 were accompanied by an inhibition of astrocytes and microglia/macrophage activation, a drastically decreased neutrophil invasion into the ischemic brain as well as by a negative regulation of pro-inflammatory mediator expression (cytokines, chemokines, matrix metalloproteinases). MLC901 significantly inhibited the expression of Prx6 as well as the transcriptional activity of NFκB and the activation of Toll-like receptor 4 (TLR4) signaling, an important pathway in the immune response in the ischemic brain. MLC901 effects on the neuroinflammation cascade induced by cerebral ischemia probably contribute, in a very significant way, in its potential therapeutic value.

Positive effects of the traditional Chinese medicine MLC901 in cognitive tasks.

MLC901 (NurAiDII) is used as a treatment for stroke patients. It has been shown that MLC901 improves motor and cognitive recovery in ischemic and traumatic brain-injured rodents. The present study seeks to delineate cognitive effects induced by MLC901 in normal, noninjured mice. To this end, the behaviors of vehicle- and MLC901-treated C57BL/6 mice in hippocampus-dependent (passive avoidance, Morris water maze) and hippocampus-independent (novel object recognition) cognitive tasks are compared. The potential influence of the compound on the anxiety level and nycthemeral rhythm of mice is also assessed. In addition, the long-term effects of MLC901 on hippocampal neurogenesis are measured. The results clearly demonstrate that MLC901 promotes extinction in passive avoidance and reversal learning in the Morris water maze and improves the performance of mice in novel object recognition. In parallel, this study shows the long-term proneurogenesis effects of MLC901 that result in the increase in the number of mature neurons in the hippocampus. If these observations can be extended to humans, then MLC901 could represent a promising therapeutic strategy.

In vitro and in vivo regulation of synaptogenesis by the novel antidepressant spadin.

BACKGROUND AND PURPOSE: We have described a novel antidepressant peptide, spadin, that acts by blocking the TWIK-related-potassium channel, type 1 (TREK-1). Here, we examined possible mechanisms of action of spadin at both molecular and cellular levels. EXPERIMENTAL APPROACHES: Effects of spadin were measured in primary cultures of neurons or tissues from mice injected i.v. with spadin. Western blots, qPCR, histochemical and electrophysiological techniques were used. KEY RESULTS: In vitro, spadin increased neuronal membrane potential and activated both the MAPK and PI3K signalling pathways, in a time- and concentration-dependent manner. The latter pathway was involved in the protective effect of spadin against staurosporine-induced apoptosis. Also, spadin enhanced both mRNA expression and protein of two markers of synaptogenesis, the post-synaptic density protein of 95 kDalton (PSD-95) and synapsin. We confirmed these effects on synaptogenesis by the observation that spadin treatment significantly increased the proportion of mature spines in cortical neurons. Finally, in vivo injections of spadin led to a rapid increase in both mRNA expression and protein level of brain-derived neurotrophic factor (BDNF) in the hippocampus, confirming the antidepressant action of the peptide. We argue for a new role of spadin in synaptogenesis as both PSD-95 and synapsin mRNA expression and protein levels were further enhanced in the hippocampus, following treatment in vivo with the peptide. CONCLUSIONS AND IMPLICATIONS: These findings provide new mechanisms of action for the rapidly acting antidepressant peptide spadin by stimulating expression of BDNF and synaptic proteins, both in vitro and in vivo.

Production, in Pichia pastoris, of a recombinant monomeric mapacalcine, a protein with anti-ischemic properties.

Mapacalcine is a small homodimeric protein of 19 kDa with 9 disulfide bridges extracted from the Cliona vastifica sponge (Red Sea). It selectively blocks a calcium current insensitive to most calcium blockers. Specific receptors for mapacalcine have been described in a variety of tissues such as brain, smooth muscle, liver, and kidney. Previous works achieved on hepatocytes and nervous cells demonstrated that this protein selectively blocks a calcium influx triggered by an ischemia/reperfusion (I/R) shock and efficiently protects cells from death after I/R. The aim of this work was to produce the recombinant mapacalcine in the yeast Pichia pastoris. Mass spectrometry, light scattering analysis and biological characterization demonstrated that the recombinant mapacalcine obtained was a monomeric form with 4 disulfide bridges which retains the biological activity of the natural protein.

Targeting two-pore domain K(+) channels TREK-1 and TASK-3 for the treatment of depression: a new therapeutic concept.

Depression is a disease that is particularly frequent, affecting up to 20% of the population in Western countries. The origins of this pathology involve multiple genes as well as environmental and developmental factors leading to a disorder that remains difficult to treat. Several therapies for depression have been developed and these mainly target monoamine neurotransmitters. However, these treatments are not only associated with numerous adverse effects, but they are also ineffective for more than one-third of patients. Therefore, the need to develop new concepts to treat depression is crucial. Recently, studies using knockout mouse models have provided evidence for a crucial role of two members of the two-pore domain potassium channel (K2P ) family, tandem P-domain weak inward rectifying K(+) (TWIK)-related K(+) channel 1 (TREK-1) and TWIK-related acid-sensitive K(+) channel 3 (TASK-3) in the pathophysiology of depression. It is believed that TREK-1 and TASK-3 antagonists could lead to the development of new antidepressants. Herein, we describe the discovery of spadin, a natural peptide released from the maturation of the neurotensin receptor-3 (also known as sortilin), which specifically blocks the activity of the TREK-1 channel and displays particular antidepressant properties, with a rapid onset of action and the absence of adverse effects. The development of such molecules may open a new era in the field of psychiatry.

MLC901, a Traditional Chinese Medicine induces neuroprotective and neuroregenerative benefits after traumatic brain injury in rats.

Traumatic brain injury (TBI) is a frequent and clinically highly heterogeneous neurological disorder with large socioeconomic consequences. NeuroAid (MLC601 and MLC901), a Traditional Medicine used in China for patients after stroke has been previously reported to induce neuroprotection and neuroplasticity. This study was designed to evaluate the neuroprotective and neuroregenerative effects of MLC901 in a rat model of TBI. TBI was induced by a moderate lateral fluid percussion applied to the right parietal cortex. MLC901 was injected intraperitoneally at 2h post-TBI, and then administered in drinking water at a concentration of 10mg/ml until sacrifice of the animals. The cognitive deficits induced by TBI were followed by using the "what-where-when" task, which allows the measurement of episodic-like memory. MLC901 treatment decreased brain lesions induced by TBI. It prevented the serum increase of S-100 beta (S100B) and neuron-specific enolase (NSE), which may be markers to predict the neurologic outcome in human patients with TBI. MLC901 reduced the infarct volume when injected up to 2h post-TBI, prevented edema formation and assisted its resolution, probably via the regulation of aquaporin 4. These positive MLC901 effects were associated with an upregulation of vascular endothelial growth factor (VEGF) as well as an increase of endogenous hippocampal neurogenesis and gliogenesis around the lesion. Furthermore, MLC901 reduced cognitive deficits induced by TBI. Rats subjected to TBI displayed a suppression of temporal order memory, which was restored by MLC901. This work provides evidence that MLC901 has neuroprotective and neurorestorative actions, which lead to an improvement in the recovery of cognitive functions in a model of traumatic brain injury.

NeuroAiD: properties for neuroprotection and neurorepair.

BACKGROUND: Treatments for stroke and other brain injuries are limited. NeuroAiD has been shown to be beneficial in clinical studies. We reviewed the pharmacological effects of NeuroAiD on the normal and ischemic brain and neurons. METHODS: In vivo and in vitro experiments using mouse model of stroke (focal ischemia), rat model of cardiac arrest (global ischemia) and cortical neurons in culture were reviewed and summarized. RESULTS: NeuroAiD improved survival, attenuated infarct size, improved functional recovery in the model of focal ischemia, and protected neurons against glutamate-induced injury. Furthermore, it enhanced cognitive recovery by reducing hippocampal CA1 cell degeneration, DNA fragmentation, Bax expression and ma-londialdehyde release in the model of global ischemia. Activation of the Akt survival pathway and opening of KATP channels may contribute to the neuroprotective properties of NeuroAiD. NeuroAiD increased BDNF expression and induced proliferation of cells which differentiate and mature into neurons. It enhanced rosette formation of human embryonic stem cells. NeuroAiD-treated embryonic cortical neurons developed into neurons with longer neurites, denser outgrowths and networks, and more synaptic release sites. CONCLUSIONS: NeuroAiD demonstrated both neuroprotective and neuroregenerative properties in rodent models of focal and global ischemia and in cortical cell cultures. These properties would be important for developing a treatment strategy in reducing the long-term disability of stroke, cardiac arrest and other brain injuries.

Activation of ATP-sensitive potassium channels as an element of the neuroprotective effects of the Traditional Chinese Medicine MLC901 against oxygen glucose deprivation.

NeuroAid (MLC601 and MLC901), a Traditional Medicine used in China for patients after stroke has been reported in preclinical models of ischemia to induce neuroprotection and neuroplasticity. This work shows the effects of MLC901 on an in vitro model of oxygen glucose deprivation (OGD). MLC901 prevents neuronal death induced by 120 min OGD and decreases the exaggerated Ca²âº entry in mature cortical neurons exposed to 120 min OGD. The neuroprotective effect of MLC901 is associated with a large hyperpolarization of ∼20 mV which is antagonized by glibenclamide, the specific inhibitor of K(ATP) channels. In addition MLC901 strengthens the activation of K(ATP) channels. MLC901 has been directly shown to act as an activator of K(ATP) channels as potent as the classical K(ATP) channel opener. The capacity of MLC901 to produce a large hyperpolarization, particularly in neurons that have suffered from energy deprivation probably plays an important role in the neuroprotective effects of this traditional medicine that comes in addition to its previously demonstrated neuroregenerative properties.

Relative contribution of SKCa and TREK1 channels in purinergic and nitrergic neuromuscular transmission in the rat colon.

Purinergic and nitrergic neurotransmission predominantly mediate inhibitory neuromuscular transmission in the rat colon. We studied the sensitivity of both purinergic and nitrergic pathways to spadin, a TWIK-related potassium channel 1 (TREK1) inhibitor, apamin, a small-conductance calcium-activated potassium channel blocker and 1H-[1,2,4]oxadiazolo[4,3-α]quinoxalin-1-one (ODQ), a specific inhibitor of soluble guanylate cyclase. TREK1 expression was detected by RT-PCR in the rat colon. Patch-clamp experiments were performed on cells expressing hTREK1 channels. Spadin (1 µM) reduced currents 1) in basal conditions 2) activated by stretch, and 3) with arachidonic acid (AA; 10 µM). l-Methionine (1 mM) or l-cysteine (1 mM) did not modify currents activated by AA. Microelectrode and muscle bath studies were performed on rat colon samples. l-Methionine (2 mM), apamin (1 µM), ODQ (10 µM), and N(ω)-nitro-l-arginine (l-NNA; 1 mM) depolarized smooth muscle cells and increased motility. These effects were not observed with spadin (1 µM). Purinergic and nitrergic inhibitory junction potentials (IJP) were studied by incubating the tissue with l-NNA (1 mM) or MRS2500 (1 µM). Both purinergic and nitrergic IJP were unaffected by spadin. Apamin reduced both IJP with a different potency and maximal effect for each. ODQ concentration dependently abolished nitrergic IJP without affecting purinergic IJP. Similar effects were observed in hyperpolarizations induced by sodium nitroprusside (1 µM) and nitrergic relaxations induced by electrical stimulation. We propose a pharmacological approach to characterize the pathways and function of purinergic and nitrergic neurotransmission. Nitrergic neurotransmission, which is mediated by cyclic guanosine monophosphate, is insensitive to spadin, an effective TREK1 channel inhibitor. Both purinergic and nitrergic neurotransmission are inhibited by apamin but with different relative sensitivity.

Spadin as a new antidepressant: absence of TREK-1-related side effects.

Despite several decades of research, current antidepressant (AD) treatments remain of a limited efficacy justifying the need to find new drugs. These drugs have to be more efficacious, more rapid and display lesser side effects. Using rodent models, we recently identified spadin as a new antidepressant molecule that acts more quickly than classical ADs, working within 4 days to get same effects obtained with other ADs after 21 days. Spadin blocks TREK-1 K(2P) potassium channels that are considered as new targets for ADs. Deletion of the TREK-1 channel is known to increase sensitivity to pain, seizures and ischemia. Thus blocking these channels could result in deleterious side effects. In this study we showed that spadin did not interfere with other TREK-1 controlled functions such as pain, epilepsy and ischemia. We also demonstrated that spadin was unable to inhibit currents generated by TREK-2, TRAAK, TASK and TRESK four other K2P channels. More importantly, spadin did not induce cardiac dysfunctions, did not block I(Kr) and I(Ks) and did not modify the systolic pressure or cardiac pulses. After a three week treatment spadin remained an efficacious AD and did not modify the infarct size in brain following focal ischemia. Finally, we showed that kainate induced seizures and glycemia were not modified by spadin treatments. These data, together with those previously published reinforce the idea that spadin represents a good candidate for a new generation of ADs. This article is part of a Special Issue entitled 'Anxiety and Depression'.

MLC901, a traditional Chinese medicine protects the brain against global ischemia.

Global ischemia leads to damage in the hippocampal CA1 region and is associated with behavioral deficits. NeuroAid (MLC601 and MLC901), a Traditional Chinese Medicine is used in China for patients after stroke. We have investigated here the effects of MLC901 on brain injury and deficits after global ischemia in the rat. Global ischemia induced by four-vessel occlusion resulted in degeneration of CA1 neurons. MLC901 (0.074 mg/ml) prevented both necrosis and apoptosis of neurons up to 3 h after ischemia. These positive MLC901 effects were associated with a decrease in Bax expression and in levels of the lipid peroxidation product malondialdehyde. Using the PI3-kinase inhibitor LY294002 we also demonstrated the critical role of the Akt pathway in MLC901-mediated neuroprotection. MLC901 enhanced neurogenesis. Furthermore, MLC901 improved functional recovery of rats after global ischemia as assessed by the Morris water maze. In this test MLC901 reduced the increase in escape latency and in swim distance induced by ischemia. MLC901 also improved post-ischemic grip strength. If observations made with rats can be extended to humans, then MLC901 will represent a novel therapeutic strategy after cardiac arrest with a clinically interesting time window of protection.

Neuroprotective and neuroproliferative activities of NeuroAid (MLC601, MLC901), a Chinese medicine, in vitro and in vivo.

Although stroke remains a leading cause of death and adult disability, numerous recent failures in clinical stroke trials have led to some pessimism in the field. Interestingly, NeuroAid (MLC601), a traditional medicine, particularly used in China, South East Asia and Middle East has been reported to have beneficial effects in patients, particularly in post-stroke complications. Here, we demonstrate in a rodent model of focal ischemia that NeuroAid II (MLC901) pre- and post-treatments up to 3 h after stroke improve survival, protect the brain from the ischemic injury and drastically decrease functional deficits. MLC601 and MLC901 also prevent neuronal death in an in vitro model of excitotoxicity using primary cultures of cortical neurons exposed to glutamate. In addition, MLC601/MLC901 treatments were shown to induce neurogenesis in rodent and human cells, promote cell proliferation as well as neurite outgrowth and stimulate the development of a dense axonal and dendritic network. MLC601 and MLC901 clearly represent a very interesting strategy for stroke treatment at different stages of the disease.

Dietary supplementation of alpha-linolenic acid in an enriched rapeseed oil diet protects from stroke.

Populations of Western countries are severely deficient in omega-3 intake, both in the form of alpha-linolenic acid (ALA) and the Long Chain derivatives (LC-n-3), Eicosa-Pentaenoic-Acid and Docosa-Hexaenoic-Acid. Omega-3 insufficiency is a risk factor for cardiovascular and cerebral diseases such as coronary heart disease and stroke. Stroke is a major cause of mortality and morbidity, and induces a significant socioeconomic cost and a marked increase in patient/family burden. To date, preventive treatments and neuroprotective drugs identified in preclinical studies failed in clinical trials, in part because of an inability to tolerate drugs at neuroprotective concentrations. Therefore testing alternative protective strategies, such as functional foods/nutraceuticals, are of considerable interest. We have previously demonstrated that a single injection of ALA reduced ischemic damage by limiting glutamate-mediated neuronal death, whereas repeated injections displayed additive protective benefits as a result of increased neurogenesis, synaptogenesis and neurotrophin expression. Because intravenous injections are not a suitable long-term strategy in humans, the present study investigated the effect of ALA supplementation by an experimental diet containing rapeseed oil (RSO, a rich source of ALA) as the only source of lipids for stroke prevention. We tested several experimental diets which included 5, 10, and 20% RSO-enriched diet and feeding paradigms (fresh diet was provided once or twice a week for 4 or 6 weeks). Our results showed that ALA supplemented diets are more sensitive to lipid peroxidation than a regular chow diet. Because the diet affected feeding behavior and animal growth, we defined concrete guidelines to investigate the effect of omega-3 supplementation on neuropathology. Among the different sets of experiments, animals fed with 10% and 20% RSO-enriched diet displayed a reduced mortality rate, infarct size and increased probability of spontaneous reperfusion in the post-ischemic period. In addition, a drastic reduction of lipid peroxidation levels was observed in the ischemic brain of RSO-fed animals. Overall, our findings provide new insights into the potential of employing rapeseed oil as a functional food/nutraceutical aiding in stroke prevention and protection.

Concomitant transitory up-regulation of X-linked inhibitor of apoptosis protein (XIAP) and the heterogeneous nuclear ribonucleoprotein C1-C2 in surviving cells during neuronal apoptosis.

Although cap-dependent translation initiation is the prevalent mode of ribosome binding to mRNAs in eukaryotes, some mRNAs exhibit the ability to bypass the requirement for the cap structure. The translation of X-chromosome-linked inhibitor of apoptosis protein (XIAP) mRNA is controlled by an internal ribosome entry site (IRES) element, which requires the interaction of the heterogeneous nuclear ribonucleoprotein C1-C2 (hnRNP-C1/C2). We analyze, at the protein level, the time course and distribution of XIAP and hnRNP-C1/C2 upon ischemia in mice or staurosporine (STP)-induced apoptosis in HT22 cells. Both ischemia and STP induced a parallel upregulation of XIAP and hnRNP-C1/C2 protein levels in the penumbra and in HT22 cells. These results suggest that the increased levels of hnRNP C1/C2 may modulate XIAP translation, probably by interacting with the XIAP-IRES. The up-regulation of hnRNP-C1/C2 may foster the synthesis of XIAP as a protective pathway by which neurons try to counteract the initial deleterious effects of apoptosis.

Alpha-linolenic acid and riluzole treatment confer cerebral protection and improve survival after focal brain ischemia.

We investigated here the effects of alpha-linolenic acid and riluzole, both activators of the 2P-domain K+ channel family TREK/TRAAK, in a model of focal ischemia clinically relevant to stroke, not only assessing neuronal protection, but also long term survival. Moreover, all the drug treatments were initiated post-ischemia. Mice were subjected to transient middle cerebral artery occlusion (1 h) and reperfusion according to the intraluminal filament model. Drugs were injected into the jugular vein according to three protocols: (i) a single dose of 4 mg/kg riluzole or 500 nmol/kg alpha-linolenic acid at different reperfusion time; (ii) a three-day therapy (a single dose of 2 mg/kg riluzole and 250 nmol/kg alpha-linolenic acid given 1-2, 48 and 72 h after reperfusion); (iii) a three-week therapy (a single dose of 2 mg/kg riluzole and 250 nmol/kg alpha-linolenic acid given once a week during three weeks after reperfusion. A combined treatment with 2mg/kg riluzole+250 nmol/kg alpha-linolenic acid injected 2 h after reperfusion was also tested. A single dose of riluzole (4 mg/kg) or alpha-linolenic acid (500 nmol/kg) injected up to 3 h after reperfusion reduced drastically the stroke volume by 75% and 86%, respectively. Neurological deficits 24 h after ischemia were significantly improved by alpha-linolenic acid500 or riluzole4 with a neurological score of 1.8 as compared with 2.5 observed in vehicle-treated mice. Alpha-linolenic acid- and riluzole treatment were associated with a reduction in cytopathological features of cell injury, including DNA fragmentation and Bax expression in the cortex and the caudate putamen. With regard to the survival rate at 30 days, the best protections were obtained with the alpha-linolenic acid-injection in the three-week therapy as well as with a single dose of the combined treatment (2 mg/kg riluzole+250 nmol/kg alpha-linolenic acid). Palmitic acid, a saturated fatty acid that does not activate the 2P-domain K-channel TREK/TRAAK family, did not provide any neuroprotection. Taken together, these data suggest that the TREK/TRAAK K-channel family may be a promising target for neuroprotection, and that riluzole and alpha-linolenic acid could be of therapeutic value against focal ischemia/reperfusion injury to the brain.

TREK-1, a K+ channel involved in neuroprotection and general anesthesia.

TREK-1 is a two-pore-domain background potassium channel expressed throughout the central nervous system. It is opened by polyunsaturated fatty acids and lysophospholipids. It is inhibited by neurotransmitters that produce an increase in intracellular cAMP and by those that activate the Gq protein pathway. TREK-1 is also activated by volatile anesthetics and has been suggested to be an important target in the action of these drugs. Using mice with a disrupted TREK-1 gene, we now show that TREK-1 has an important role in neuroprotection against epilepsy and brain and spinal chord ischemia. Trek1-/- mice display an increased sensitivity to ischemia and epilepsy. Neuroprotection by polyunsaturated fatty acids, which is impressive in Trek1+/+ mice, disappears in Trek1-/- mice indicating a central role of TREK-1 in this process. Trek1-/- mice are also resistant to anesthesia by volatile anesthetics. TREK-1 emerges as a potential innovative target for developing new therapeutic agents for neurology and anesthesiology.

Polyunsaturated fatty acids induce ischemic and epileptic tolerance.

The findings reported in this work show that pretreatment with polyunsaturated fatty acids, particularly linolenic acid, present in vegetable oils, can provide a potent tolerance against neurodegeneration in two models of neuronal death-generating treatments such as kainic acid injection and global ischemia. Rats were injected i.v. with 500 nmol/kg of linolenic acid as long as 3 days prior to 6 min global ischemia or received an injection of linolenic acid as long as 3 days prior to a dose of 7.5 mg/kg kainic acid. Neuronal degeneration, assessed by analysis of neuronal density on Cresyl Violet-stained hippocampal sections, was significantly reduced in linolenic acid-treated rats (94-85% of cell survival in the ischemic model and 99-79% of cell survival in the epileptic model in respective CA1 and CA3 subfields). The neuroprotection observed following the injection of linolenic acid 3 days prior to induction of a severe ischemic or epileptic challenge was associated with the induction of the neuroprotective HSP70 heat shock protein within the time window of protection. The injection of 500 nmol/kg of linolenic acid induced a maximal HSP70 expression of 387% at 72 h. In contrast, the overexpression of one well-known protein inducer of neuronal cell death, Bax, which is induced by both ischemic and kainic acid-induced epileptic insults, was prevented by linolenic acid in the 3-day window of protection. These results strengthen the idea of an interesting potential therapeutical value of polyunsaturated fatty acids in neuronal protection.

Intermittent ischemia reduces warm hypoxia-reoxygenation-induced JNK(1)/SAPK(1) activation and apoptosis in rat hepatocytes.

Prolonged liver ischemia followed by reperfusion (I/R) causes functional and structural damage to liver cells, resulting in necrosis and apoptosis. c-jun N-terminal kinase 1/stress-activated protein kinase 1 (JNK(1)/SAPK(1)) is activated during I/R and participates in the onset of the apoptosis program. Excessive blood loss during surgery can hinder postoperative recovery. Intermittent portal triad clamping (PTC) is better tolerated than prolonged continuous ischemia. This study was designed to demonstrate that intermittent ischemia could improve postischemic survival rates by a decrease of JNK(1)/SAPK(1) and caspase 3 activation, which were involved in the apoptosis process. Rats were subjected to intermittent 1-hour ischemia (15-minute ischemia/5-minute reperfusion, 4 times), followed by 220-minute reperfusion, or to continuous ischemia (1 hour), followed by 240-minute reperfusion. Mortality rates were assessed on day 7. Serum aspartate transaminase (AST), alanine transaminase (ALT), and lactate dehydrogenase levels (LDH) were measured 6 hours after ischemia. This study was completed in primary cultured isolated rat hepatocytes, subjected to the same continuous or intermittent hypoxic conditions. The activation status of JNK(1)/SAPK(1) was evaluated by immunoprecipitation or Western blotting experiments. Apoptosis was assessed by measuring caspase activation and by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick end labeling (TUNEL) reaction. Eighty percent of the intermittent-ischemia group was alive 7 days after surgery and serum enzyme levels were significantly decreased. Intermittent hypoxia or ischemia did not lead to JNK(1)/SAPK(1) activation, but at least 3 hypoxia-reoxygenation (H/R) sets were necessary to inhibit kinase activation. Consequently, caspase 3 activation and apoptosis were dramatically reduced. Intermittent ischemia is a powerful, protective way to reduce I/R damage of the liver, by reduction of JNK(1)/SAPK(1) activation associated with a down-regulation of caspase 3 activity, which leads to inhibition of hepatocyte apoptosis.

Activation of the nuclear factor-kappaB is a key event in brain tolerance.

The transcription factor nuclear factor-kappaB (NFkappaB) is an ubiquitously expressed inducible regulator of a broad range of genes and plays a pivotal role in cell death and survival pathways. Three models of brain tolerance (ischemic, epileptic, and polyunsaturated fatty acid-induced preconditioning), known to confer resistance to neurons against ischemia or status epilepticus, were used to determine whether NFkappaB mediated the late preconditioning. A sublethal 3 min ischemia, a dose of 5 mg/kg kainic acid (KA5) or 500 nmol of linolenic acid (LIN500) led to a rapid increase of NFkappaB DNA-binding activity and nuclear translocation of p65 and p50 subunits of NFkappaB in neurons. Pretreatment with the NFkappaB inhibitor diethyldithiocarbamate or kappaB decoy DNA blocked the increased DNA-binding activity and the nuclear translocation of NFkappaB and abolished the neuroprotective effects of different delayed preconditionings against severe ischemia or epilepsy. The inhibition of NFkappaB observed in rats preconditioned with 3 min ischemia, KA5 or LIN500 treatments compared with ischemic or epileptic controls was correlated with the prevention of the inducible degradation of the inhibitory protein IkappaBalpha. Preconditioning probably inhibits the activation of NFkappaB by interfering with a pathway that leads to the direct transcriptional activation of IkappaBalpha by NFkappaB itself. The present work provides evidence that activation of NFkappaB is a crucial step in the signal transduction pathway that underlies the development of brain tolerance and may open new strategies in the prevention of cerebral diseases, such as ischemia or epilepsy.

Hyperpolarization-activated cyclic nucleotide-gated channel 1 is a molecular determinant of the cardiac pacemaker current I(f).

The pacemaker current I(f) of the sinoatrial node (SAN) is a major determinant of cardiac diastolic depolarization and plays a key role in controlling heart rate and its modulation by neurotransmitters. Substantial expression of two different mRNAs (HCN4, HCN1) of the family of pacemaker channels (HCN) is found in rabbit SAN, suggesting that the native channels may be formed by different isoforms. Here we report the cloning and heterologous expression of HCN1 from rabbit SAN and its specific localization in pacemaker myocytes. rbHCN1 is an 822-amino acid protein that, in human embryonic kidney 293 cells, displayed electrophysiological properties similar to those of I(f), suggesting that HCN1 can form a pacemaker channel. The presence of HCN1 in the SAN myocytes but not in nearby heart regions, and the electrophysiological properties of the channels formed by it, suggest that HCN1 plays a central and specific role in the formation of SAN pacemaker currents.