Akt Inhibitor Perifosine Prevents Epileptogenesis in a Rat Model of Temporal Lobe Epilepsy

Accumulating data have revealed that abnormal activity of the mTOR (mammalian target of rapamycin) pathway plays an important role in epileptogenesis triggered by various factors. We previously reported that pretreatment with perifosine, an inhibitor of Akt (also called protein kinase B), abolishes the rapamycin-induced paradoxical increase of S6 phosphorylation in a rat model induced by kainic acid (KA). Since Akt is an upstream target in the mTOR signaling pathway, we set out to determine whether perifosine has a preventive effect on epileptogenesis. Here, we explored the effect of perifosine on the model of temporal epilepsy induced by KA in rats and found that pretreatment with perifosine had no effect on the severity or duration of the KA-induced status epilepticus. However, perifosine almost completely inhibited the activation of p-Akt and p-S6 both acutely and chronically following the KA-induced status epilepticus. Perifosine pretreatment suppressed the KA-induced neuronal death and mossy fiber sprouting. The frequency of spontaneous seizures was markedly decreased in rats pretreated with perifosine. Accordingly, rats pretreated with perifosine showed mild impairment in cognitive functions. Collectively, this study provides novel evidence in a KA seizure model that perifosine may be a potential drug for use in anti-epileptogenic therapy.     

Pentylenetetrazole-induced seizures cause acute, but not chronic, mTOR pathway activation in rat

Purpose: The mammalian target of rapamycin (mTOR) pathway has been implicated in contributing to progressive epileptogenesis in models of chronic epilepsy. Conversely, seizures themselves may directly cause acute activation of the mTOR pathway. To isolate the direct effects of seizures on the mTOR pathway, the time course and mechanisms of mTOR activation were investigated with acute seizures induced by pentylenetetrazole (PTZ), which does not lead to chronic epilepsy. Methods: Western blot analysis was used to assay the phosphorylation of Akt and S6, as measures of activation of the phosphoinositide 3‐kinase (PI3K)/Akt and mTOR pathways, respectively, at various time points after PTZ‐induced seizures in rats. The ability of wortmannin, a PI3K inhibitor, to inhibit PTZ seizure–induced activation of the mTOR pathway was tested. Key Findings: PTZ‐induced seizures produced an immediate, transient mTOR activation lasting several hours, but no later, more chronic activation over days to weeks. This acute stimulation of the mTOR pathway by PTZ‐induced seizures was mediated by upstream PI3K/Akt pathway activation and was blocked by a PI3K inhibitor. Significance: Compared with models of chronic epilepsy that exhibit biphasic (acute and chronic) mTOR pathway activation, PTZ‐induced seizures produce only acute, but not chronic, mTOR activation. These results in the PTZ seizure model highlight potential differences in the involvement of the mTOR pathway between self‐limited seizures and progressive epileptogenesis. These findings also suggest a potential therapeutic role of PI3K inhibitors in epilepsy.     

Inhibition of mammalian target of rapamycin reduces epileptogenesis and blood-brain barrier leakage but not microglia activation

Purpose: Previous studies have shown that inhibition of the mammalian target of rapamycin (mTOR) pathway with rapamycin prevents epileptogenesis after pharmacologically induced status epilepticus (SE) in rat models of temporal lobe epilepsy. Because rapamycin is also known for its immunosuppressant properties we hypothesized that one of the mechanisms by which it exerts this effect could be via suppression of brain inflammation, a process that has been suggested to play a major role in the development and progression of epilepsy. Methods: Rats were treated with rapamycin or vehicle once daily for 7 days (6 mg/kg/day, i.p.) starting 4 h after the induction of SE, which was evoked by electrical stimulation of the angular bundle. Hereafter rapamycin was administered every other day until rats were sacrificed, 6 weeks after SE. Video‐electroencephalography was used to monitor the occurrence of seizures. Neuronal death, synaptic reorganization, and microglia and astrocyte activation were assessed by immunohistologic staining. Fluorescein was administered to quantify blood–brain barrier leakage. Key Findings: Rapamycin treatment did not alter SE severity and duration compared to vehicle treatment rats. Rapamycin‐treated rats developed hardly (n = 9) or no (n = 3) seizures during the 6‐week treatment, whereas vehicle‐treated rats showed a progressive increase of seizures starting 1 week after SE (mean 8 ± 2 seizures per day during the sixth week). Cell loss and sprouting that normally occur after SE were prominent but on average significantly less in rapamycin‐treated rats versus vehicle‐treated rats. Nevertheless, various inflammation markers (CD11b/c and CD68) were dramatically upregulated and not significantly different between post‐SE groups. Of interest, blood–brain barrier leakage was barely detected in the rapamycin‐treated group, whereas it was prominent in the vehicle‐treated group. Significance: mTOR inhibition led to strong reduction of seizure development despite the presence of microglia activation, suggesting that effects of rapamycin on seizure development are not due to a control of inflammation. Whether the effects on blood–brain barrier leakage in rapamycin‐treated rats are a consequence of seizure suppressing properties of the drug, or contribute to a real antiepileptogenic effect still needs to be determined.     

Rapamycin has age‐, treatment paradigm‐, and model‐specific anticonvulsant effects and modulates neuropeptide Y expression in rats

Purpose: Rapamycin (RAP) has certain antiepileptogenic features. However, it is unclear whether these effects can be explained by the anticonvulsant action of RAP, which has not been studied. To address this question, we tested potential anticonvulsant effects of RAP in immature and adult rats using different seizure models and treatment paradigms. In addition, we studied changes in the expression of neuropeptide Y (NPY) induced by RAP, which may serve as an indirect target of the RAP action. Methods: A complex approach was adopted to evaluate the anticonvulsant potential of RAP: We used flurothyl‐, pentylenetetrazole (PTZ)–, N‐methyl‐d ‐aspartate (NMDA)–, and kainic acid (KA)–induced seizures to test the effects of RAP using different pretreatment protocols in immature and adult rats. We also evaluated expression of NPY within the primary motor cortex, hippocampal CA1, and dentate gyrus (DG) after different pretreatments with RAP in immature rats. Key Findings: We found the following: (1) RAP administered with short‐term pretreatment paradigms has a weak anticonvulsant potential in the seizure models with compromised inhibition. (2) Lack of RAP efficacy correlates with decreased NPY expression in the cortex, CA1, and DG. Specifically in immature rats, a single dose of RAP (3 mg/kg) 4 or 24 h before seizure testing had anticonvulsant effects against PTZ‐induced seizures. In the flurothyl seizure model only the 4‐h pretreatment with RAP was anticonvulsant in the both age groups. Short‐term pretreatments with RAP had no effects against NMDA‐ and KA‐induced seizures tested in immature rats. Long‐term pretreatments with RAP over 8 days did not show beneficial effect in all tested seizure models in developing rats. Moreover, the long‐term pretreatment with RAP had a slight proconvulsant effect on KA‐induced seizures. In immature rats, any lack of anticonvulsant effect (including proconvulsant effect of multiple doses of RAP) was associated with downregulation of NPY expression in the cortex and DG. In immature animals, after a single dose of RAP with 24 h delay, we found a decrease of NPY expression in DG, and CA1 as well. Significance: Our data show weak age‐, treatment paradigm‐, and model‐specific anticonvulsant effects of RAP as well as loss of those effects after long‐term RAP pretreatment associated with downregulation of NPY expression. These findings suggest that RAP is a poor anticonvulsant and may have beneficial effects only against epileptogenesis. In addition, our data present new insights into mechanisms of RAP action on seizures indicating a possible connection between mammalian target of rapamycin (mTOR) signaling and NPY system.     

Coactivation of GABA receptors inhibits the JNK3 apoptotic pathway via disassembly of GluR6‐PSD‐95‐MLK3 signaling module in KA‐induced seizure

Purpose: Past work has demonstrated that kainic acid (KA)–induced seizures could cause the enhancement of excitation and lead to neuronal death in rat hippocampus. To counteract such an imbalance between excitation and inhibition, we designed experiments by activating the inhibitory γ‐aminobutyric acid (GABA) receptor to investigate whether such activation suppresses the excitatory glutamate signaling induced by KA and to elucidate the underlying molecular mechanisms. Methods: Muscimol coapplied with baclofen was intraperitoneally administrated to the rats 40 min before KA injection by intracerebroventricular infusion. Subsequently we used a series of methods including immunoprecipitation, immunoblotting, histologic analysis, and immunohistochemistry to analyze the interaction, expression, and phosphorylation of relevant proteins as well as the survival of the CA1/CA3 pyramidal neurons. Results: Coadministration of muscimol and baclofen exerted neuroprotection against neuron death induced by KA; inhibited the increased assembly of the GluR6‐PSD‐95‐MLK3 module induced by KA; and suppressed the activation of MLK3, MKK7, and JNK3. Discussion: Taken together, we demonstrate that coactivation of the inhibitory GABA receptors can attenuate the excitatory JNK3 apoptotic signaling pathway via inhibiting the increased assembly of the GluR6‐PSD‐95‐MLK3 signaling module induced by KA. This provides a new insight into the therapeutic approach to epileptic seizure.     

Lean and obese Zucker rats exhibit different patterns of p70s6 kinase regulation in the tibialis anterior muscle in response to high‐force muscle contraction

Increased phosphorylation of the 70‐kDa ribosomal S6 kinase (p70S6k) signaling is strongly correlated with the degree of muscle adaptation following exercise. Herein we compare the phosphorylation of p70S6k, Akt, and mammalian target of rapamycin (mTOR) in the tibialis anterior (TA) muscles of lean and obese Zucker rats following a bout of eccentric exercise. Exercise increased p70S6k (Thr389) phosphorylation immediately after (33.3 ± 7.2%) and during [1 h (24.0 ± 14.9%) and 3 h (24.6 ± 11.3%)] recovery in the lean TA and at 3 h (33.5 ± 8.0%) in the obese TA Zucker rats. mTOR (Ser2448) phosphorylation was elevated in the lean TA immediately after exercise (96.5 ± 40.3%) but remained unaltered in the obese TA. Exercise increased Akt (Thr308) and Akt (Ser473) phosphorylation in the lean but not the obese TA. These results suggest that insulin resistance is associated with alterations in the ability of muscle to activate p70S6k signaling following an acute bout of exercise. Muscle Nerve 39: 503–511 2009     

阿司匹林通过抑制PI3K/Akt/mTOR 通路治疗癫痫 大鼠反复自发发作

目的 研究阿司匹林治疗癫痫大鼠反复自发发作的分子信号通路.方法 80只SD大鼠,75只随机分为5组,分别为(1)对照组(C组);(2)癫痫组(S组):匹鲁卡品诱导癫痫;(3)癫痫+慢病毒转染组(ST组):海马注射慢病毒转染shRNA[沉默磷脂酰肌醇激酶(PI3K)的p85亚基];(4)癫痫+阿司匹林组(SA组):腹腔注射阿司匹林20mg/kg;(5)癫痫+雷帕霉素组(SR组):腹腔注射雷帕霉素6mg/kg.剩余5只大鼠分入验证组,用于慢病毒转染的验证.建模后2周,检测各组大鼠癫痫发作的时间和频率,以及海马内环氧化酶-2(COX-2)、白介素1β(IL-1β)、PI3K的p85亚基(p85)、蛋白激酶B(Akt)、核糖体蛋白S6激酶(p70S6K)和微管相关蛋白2(MAP2)的表达水平.利用免疫组化染色,检测p70S6K在各组大鼠海马内的表达量.结果 (1)S组癫痫发作的时间和发作频率均显著高于SA、SR和ST组(P<0.05),而3个干预组间差异无统计学意义(P>0.05);(2)S组COX-2、IL-1β、p85、Akt、p70S6K和MAP2蛋白水平均显著高于C组(P<0.05);ST和SR组p85、Akt、p70S6K、MAP2蛋白水平均显著低于S组(P<0.05);SA组COX-2、IL-1β、p85、Akt、p70S6K、MAP2蛋白水平均显著低于S组(P<0.05);SA组的p85、Akt、p70S6K、MAP2蛋白水平与SR组差异无统计学意义(P>0.05).结论 阿司匹林可能通过抑制PI3K/Akt/mTOR通路和MAP2的表达来治疗癫痫大鼠反复自发发作.     

Histone deacetylase inhibition inhibits brachial plexus avulsion‐induced neuropathic pain

Introduction: Neuropathic pain induced by brachial plexus avulsion (BPA) is a pathological condition. We hypothesized that inhibition of histone deacetylase (HDAC) could suppress BPA‐induced neuropathic pain through inhibition of transient reception potential (TRP) overexpression and protein kinase B (Akt)‐mediated mammalian target of rapamycin (mTOR) activation. Methods: We generated a rat BPA model; administered HDAC inhibitor tricostatin A (TSA) for 7 days postsurgery; and assessed the effects on HDAC expression, Akt phosphorylation, neuroinflammation, and mTOR activation. Results: TSA treatment alleviated BPA‐induced mechanical hyperalgesia, suppressed Akt phosphorylation, and increased HDAC. We found suppressed proinflammatory cytokine levels, TRPV1 and TRPM8 expression, and mTOR activity in TSA‐treated BPA rats. Discussion: Our results suggest that altered HDAC and Akt signaling are involved in BPA‐induced neuropathic pain and that inhibition of HDAC could be an effective therapeutic approach in reducing neuropathic pain. Muscle Nerve 58 : 434–440, 2018     

Acetaldehyde promotes rapamycin-dependent activation of p70(S6K) and glucose uptake despite inhibition of Akt and mTOR in dopaminergic SH-SY5Y human neuroblastoma cells

Alcohol intake is one of the important lifestyle factors for the risk of insulin resistance and type 2 diabetes. Acetaldehyde, the major ethanol metabolite which is far more reactive than ethanol, has been postulated to participate in alcohol-induced tissue injury although its direct impact on insulin signaling is unclear. This study was designed to examine the effect of acetaldehyde on glucose uptake and insulin signaling in human dopaminergic SH-SY5Y cells. Akt, mammalian target of rapamycin (mTOR), ribosomal-S6 kinase (p70(S6K)), the eukaryotic translation initiation factor 4E (eIF4E)-binding protein 1 (4E-BP1) and insulin receptor substrate (IRS)-2 were evaluated by Western blot analysis. Glucose uptake and apoptosis were measured using [(3)H]-2-deoxyglucose uptake and caspase-3 assay, respectively. Short-term exposure (12 h) of acetaldehyde (150 muM) facilitated glucose uptake in a rapamycin-dependent manner without affecting apoptosis, IRS-2 expression and insulin-stimulated glucose uptake in SH-SY5Y cells. Acetaldehyde suppressed basal and insulin-stimulated Akt phosphorylation without affecting total Akt expression. Acetaldehyde inhibited mTOR phosphorylation without affecting total mTOR and insulin-elicited response on mTOR phosphorylation. Rapamycin, which inhibits mTOR leading to inactivation of p70(S6K), did not affect acetaldehyde-induced inhibition on phosphorylation of Akt and mTOR. Interestingly, acetaldehyde enhanced p70(S6K) activation and depressed 4E-BP1 phosphorylation, the effect of which was blunted and exaggerated, respectively, by rapamycin. Collectively, these data suggested that acetaldehyde did not adversely affect glucose uptake despite inhibition of insulin signaling cascade at the levels of Akt and mTOR, possibly due to presence of certain mechanism(s) responsible for enhanced p70(S6K) phosphorylation.     

Seizures in the intrahippocampal kainic acid epilepsy model: characterization using long‐term video‐EEG monitoring in the rat

Objective – Intrahippocampal injection of kainic acid (KA) in rats evokes a status epilepticus (SE) and leads to spontaneous seizures. However to date, precise electroencephalographic (EEG) and clinical characterization of spontaneous seizures in this epilepsy model using long‐term video‐EEG monitoring has not been performed. Materials and Methods – Rats were implanted with bipolar hippocampal depth electrodes and a cannula for the injection of KA (0.4 μg/0.2 μl) in the right hippocampus. Video‐EEG monitoring was used to determine habitual parameters of spontaneous seizures such as seizure frequency, severity, progression and day–night rhythms. Results – Spontaneous seizures were detected in all rats with 13 out of 15 animals displaying seizures during the first eight weeks after SE. A considerable fraction (35%) of the spontaneous seizures did not generalize secondarily. Seizure frequency was quite variable and the majority of the KA‐treated animals had less than one seizure per day. A circadian rhythm was observed in all rats that showed sufficient seizures per day. Conclusions – This study shows that the characteristics of spontaneous seizures in the intrahippocampal KA model display many similarities to other SE models and human temporal lobe epilepsy.     

Kainic Acid–Induced Limbic Seizures in Cats: Some Reflections on Sleep–Epilepsy Interactions

Sleep-epilepsy interactions were investigated in a model of temporal lobe seizures induced in cats by intra-amygdaloid kainic acid (KA) microinjections. We found that limbic status epilepticus disrupted sleep for 2 or 3 days after injection. Sleep, in turn, modulated the frequency of interictal discharges. However, such modulation was variable depending on the time elapsed since KA injection. For this and other reasons (such as the occurrence of subclinical seizures during paradoxical sleep), we postulate a dual effect--facilitatory or inhibitory--of paradoxical sleep on limbic epilepsy. A role in seizure induction for bulbopontine structures is proposed on the basis of seizure precipitation during phasic paradoxical sleep. Propagated limbic seizures and paradoxical sleep without atonia displayed similar behavioral patterns. This fact and the possibility that a seizure may substitute for paradoxical sleep, lead us to think that limbic seizures and paradoxical sleep subserve similar functions. One of them might be the elimination of a potentially neurotoxic endogenous product.     

Amyloid‐β interrupts the PI3K‐Akt‐mTOR signaling pathway that could be involved in brain‐derived neurotrophic factor‐induced Arc expression in rat cortical neurons

The deposition of amyloid‐β (Aβ) contributes to the pathogenesis of Alzheimer's disease. Even at low levels, Aβ may interfere with various signaling cascades critical for the synaptic plasticity that underlies learning and memory. Brain‐derived neurotrophic factor (BDNF) is well known to be capable of inducing the synthesis of activity‐regulated cytoskeleton‐associated protein (Arc), which plays a fundamental role in modulating synaptic plasticity. Our recent study has demonstrated that treatment of fibrillar Aβ at a nonlethal level was sufficient to impair BDNF‐induced Arc expression in cultured rat cortical neurons. In this study, BDNF treatment alone induced the activation of the phosphatidylinositol 3‐kinase‐Akt‐mammlian target of rapamycin (PI3K‐Akt‐mTOR) signaling pathway, the phosphorylation of eukaryotic initiation factor 4E binding protein (4EBP1) and p70 ribosomal S6 kinase (p70S6K), the dephosphorylation of eukaryotic elongation factor 2 (eEF2), and the expression of Arc. Interrupting the PI3K‐Akt‐mTOR signaling pathway by inhibitors prevented the effects of BDNF, indicating the involvement of this pathway in BDNF‐induced 4EBP1 phosphorylation, p70S6K phosphorylation, eEF2 dephosphorylation, and Arc expression. Nonlethal Aβ pretreatment partially blocked these effects of BDNF. Double‐ immunofluorescent staining in rat cortical neurons further confirmed the coexistence of eEF2 dephosphorylation and Arc expression following BDNF treatment regardless of the presence of Aβ. These results reveal that, in cultured rat cortical neurons, Aβ interrupts the PI3K‐Akt‐mTOR signaling pathway that could be involved in BDNF‐induced Arc expression. Moreover, this study also provides the first evidence that there is a close correlation between BDNF‐induced eEF2 dephosphorylation and BDNF‐induced Arc expression. © 2009 Wiley‐Liss, Inc.     

N w‐Propyl‐l‐arginine (L‐NPA) reduces status epilepticus and early epileptogenic events in a mouse model of epilepsy: behavioural,EEG and immunohistochemical analyses

We investigated the anticonvulsant and neurobiological effects of a highly selective neuronal nitric oxide synthase (nNOS) inhibitor, N ^w‐propyl‐l ‐arginine (L‐NPA), on kainic acid (KA)‐induced status epilepticus (SE) and early epileptogenesis in C57BL/6J mice. SE was induced with 20 mg/kg KA (i.p.) and seizures terminated after 2 h with diazepam (10 mg/kg, i.p). L‐NPA (20 mg/kg, i.p.) or vehicle was administered 30 min before KA. Behavioural seizure severity was scored using a modified Racine score and electrographic seizure was recorded using an implantable telemetry device. Neuronal activity, activity‐dependent synaptogenesis and reactive gliosis were quantified immunohistochemically, using c‐Fos, synaptophysin and microglial and astrocytic markers. L‐NPA treatment reduced the severity and duration of convulsive motor seizures, the power of electroencephalogram in the gamma band, and the frequency of epileptiform spikes during SE. It also reduced c‐Fos expression in dentate granule cells at 2 h post‐KA, and reduced the overall rate of epileptiform spiking (by 2‐ to 2.5‐fold) in the first 7 days after KA administration. Furthermore, treatment with L‐NPA suppressed both hippocampal gliosis and activity‐dependent synaptogenesis in the outer and middle molecular layers of the dentate gyrus in the early phase of epileptogenesis (72 h post‐KA). These results suggest that nNOS facilitates seizure generation during SE and may be important for the neurobiological changes associated with the development of chronic epilepsy, especially in the early stages of epileptogenesis. As such, it might represent a novel target for disease modification in epilepsy.     

米诺环素对红藻氨酸诱导的癫痫大鼠海马组织凋亡相关蛋白表达的影响

目的:观察癫发作后不同时间海马组织凋亡相关蛋白表达水平的变化,探讨米诺环素(MT)减轻癫所致的海马神经元损伤的机制。方法:杏仁核立体定向注射红藻氨酸(KA)建立大鼠癫模型,随机分为治疗(MT)组和非治疗(KA)组,以杏仁核注射生理盐水为空白对照(SC)组。用免疫印迹法检测caspase-3裂解片段及Bcl-2和Bcl-xl表达水平的变化。结果:与KA组相比,MT组caspase-3裂解水平明显降低(P<0.05)并接近SC组;Bcl-2在癫发作终止后2h开始上调,24h达高峰并持续到72h。Bcl-xl上调不明显。结论:米诺环素能减少caspase-3的裂解,抑制线粒体凋亡通路激活,从而减轻癫发作对神经元的损伤。这一作用与抗凋亡因子Bcl-2的上调有关,提示Bcl-2可能是癫潜在的治疗靶点。     

Role of Akt and mammalian target of rapamycin in functional outcome after concussive brain injury in mice

Akt (protein kinase B) and mammalian target of rapamycin (mTOR) have been implicated in the pathogenesis of cell death and cognitive outcome after cerebral contusion in mice; however, a role for Akt/mTOR in concussive brain injury has not been well characterized. In a mouse closed head injury (CHI) concussion traumatic brain injury (TBI) model, phosphorylation of Akt (p-Akt), mTOR (p-mTOR), and S6RP (p-S6RP) was increased by 24 hours in cortical and hippocampal brain homogenates (P<0.05 versus sham for each), and p-S6RP was robustly induced in IBA-1+ microglia and glial fibrillary acidic protein-positive (GFAP+) astrocytes. Pretreatment with inhibitors of Akt or mTOR individually by the intracerebroventricular route reduced phosphorylation of their respective direct substrates FOXO1 (P<0.05) or S6RP (P<0.05) after CHI, confirming the activity of inhibitors. Rapamycin pretreatment significantly worsened hidden platform (P<0.01) and probe trial (P<0.05) performance in CHI mice. Intracerebroventricular administration of necrostatin-1 (Nec-1) before CHI increased hippocampal Akt and S6RP phosphorylation and improved place learning (probe trials, P<0.001 versus vehicle), whereas co-administration of rapamycin or Akt inhibitor with Nec-1 eliminated improved probe trial performance. These data suggest a beneficial role for Akt/mTOR signaling after concussion TBI independent of cell death that may contribute to improved outcome by Nec-1.     

Chronic bilateral stimulation of the anterior thalamus of kainate-treated rats increases seizure frequency

PURPOSE: Electrical stimulation of the anterior nucleus of the thalamus (ANT) is receiving increased attention as a novel means of controlling intractable epilepsy, and has entered human clinical trial. Animal data supporting the anticonvulsant benefit of ANT stimulation, however, has been obtained from acute chemoconvulsant models of epilepsy rather than models of chronic epilepsy with spontaneous seizures. It is unknown whether ANT stimulation is effective in models of chronic epilepsy. METHODS: Bilateral ANT stimulation was evaluated in rats with chronic epilepsy following acute status epilepticus (SE) produced by systemic kainic acid (KA) administration. The evolution of epilepsy following KA SE and the effects of ANT stimulation were monitored by continuous video-EEG. RESULTS: Following KA SE, most rats have 2-8 seizures per day, and the average seizure rate increases over time, doubling over the course of 14 weeks. Behavioral seizure severity, after the initial development of epilepsy, remains stable. Seizure frequency during ANT stimulation was 2.5 times the baseline seizure frequency. In some cases stimulation triggered seizures were observed. The effects of stimulation were specific to the ANT. Stimulation applied to electrodes placed outside the ANT did not significantly worsen seizure frequency. CONCLUSIONS: ANT stimulation exacerbated seizure frequency in rats with chronic epilepsy following kainate status epilepticus.     

Repetitive stretch suppresses denervation‐induced atrophy of soleus muscle in rats

This study was conducted to examine whether stretch‐related mechanical loading on skeletal muscle can suppress denervation‐induced muscle atrophy, and if so, to depict the underlying molecular mechanism. Denervated rat soleus muscle was repetitively stretched (every 5 s for 15 min/day) for 2 weeks. Histochemical analysis showed that the cross‐sectional area of denervated soleus muscle fibers with repetitive stretching was significantly larger than that of control denervated muscle (P < 0.05). We then examined the involvement of the Akt/mammalian target of the rapamycin (mTOR) cascade in the suppressive effects of repetitive stretching on muscle atrophy. Repetitive stretching significantly increased the Akt, p70S6K, and 4E‐BP1 phosphorylation in denervated soleus muscle compared to controls (P < 0.05). Furthermore, repetitive stretching‐induced suppression of muscle atrophy was fully inhibited by rapamycin, a potent inhibitor of mTOR. These results indicate that denervation‐induced muscle atrophy is significantly suppressed by stretch‐related mechanical loading of the muscle through upregulation of the Akt/mTOR signal pathway. Muscle Nerve, 2009     

Role of AMP‐Activated Protein Kinase Activators in Antiproliferative Multi‐Drug Pituitary Tumour Therapies: Effects of Combined Treatments with Compounds Affecting the mTOR‐p70S6 Kinase Axis in Cultured Pituitary Tumour Cells

AMP‐activated protein kinase (AMPK) is activated under conditions that deplete cellular ATP levels and elevate AMP levels. We have recently shown that AMPK can represent a valid target for improving the medical treatment of growth hormone (GH)‐secreting pituitary adenomas and the effects of its activation or inhibition in pituitary tumour cells are worthy of further characterisation. We aimed to determine whether AMPK may have a role in combined antiproliferative therapies based on multiple drugs targeting cell anabolic functions at different levels in pituitary tumour cells to overcome the risk of cell growth escape phenomena. Accordingly, we tried to determine whether a rationale exists in combining compounds activating AMPK with compounds targeting the phosphatidylinositol‐3‐kinase (PI3K)/Akt/mTOR/p70S6K signalling pathway. AMPK down‐regulation by specific small‐interfering RNAs confirmed that activated AMPK had a role in restraining growth of GH3 cells. Hence, we compared the effects of compounds directly targeting the mTOR‐p70S6K axis, namely the mTOR inhibitor rapamycin and the p70S6K inhibitor PF‐4708671, with the effects of the AMPK activator 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) on cell signalling and cell growth, in rat pituitary GH3 cells. AICAR was able to reduce growth factor‐induced p70S6K activity, as shown by the decrease of phospho‐p70S6K levels. However, it was far less effective than rapamycin and PF‐4708671. We observed significant differences between the growth inhibitory effects of the three compounds in GH3 and GH1 cells. Interestingly, PF‐4708671 was devoid of any effect. AICAR was at least as effective as rapamycin and the co‐treatment was more effective than single treatments. AICAR induced apoptosis of GH3 cells, whereas rapamycin caused preferentially a decrease of cell proliferation. Finally, AICAR and rapamycin differed in their actions on growth factor‐induced extracellular signal regulated kinase 1/2 phosphorylation. In conclusion, the results of the present study suggest the increased efficacy of combined antiproliferative therapies, including rapamycin analogues and AMPK activators in GH‐secreting pituitary tumours, as a result of complementary and only partially overlapping mechanisms of action.