Gender issues in antiepileptogenic treatments.

Disease modification of epilepsy refers to the alleviation of epileptogenesis or comorbidities after genetic or acquired epileptogenic brain insults. There are currently 30 proof-of-concept experimental pharmacologic studies that have demonstrated some beneficial disease-modifying effects. None of these studies, however, has yet passed from the laboratory to the clinic. The International League Against Epilepsy and American Epilepsy Society working groups on antiepileptogenic (AEG) therapies recently released recommendations for conducting preclinical AEG studies, taking into account many of the critiques raised by previous study designs. One of the issues relates to the lack of analysis of AEG efficacy in both sexes. A review of the literature reveals that most of the preclinical studies have been performed using male rodents, whereas clinical study cohorts include both males and females. Therefore, it is important to determine whether sex differences should be taken into account to a greater extent than they have been historically at different phases of experimental studies. Here we address the following questions based on analysis of available experimental AEG studies: (a) whether sex differences should be considered when searching for novel AEG targets, (b) how sex differences can affect the preclinical AEG study designs and analysis of outcome measures, and (c) what factors should be considered when examining the effect of sex on outcome of clinical AEG trials or the clinical use of AEGs.

Bepridil decreases Aß and calcium levels in the thalamus after middle cerebral artery occlusion in rats.

Alzheimer's disease (AD) and cerebral ischaemia share similar features in terms of altered amyloid precursor protein (APP) processing and ß-amyloid (Aß) accumulation. We have previously shown that Aß and calcium deposition, and ß-secretase activity, are robustly increased in the ipsilateral thalamus after transient middle cerebral artery occlusion (MCAO) in rats. Here, we investigated whether the non-selective calcium channel blocker bepridil, which also inhibits ß-secretase cleavage of APP, affects thalamic accumulation of Aß and calcium and in turn influences functional recovery in rats subjected to MCAO. A 27-day bepridil treatment (50 mg/kg, p.o.) initiated 2 days after MCAO significantly decreased the levels of soluble Aß40, Aß42 and calcium in the ipsilateral thalamus, as compared with vehicle-treated MCAO rats. Expression of seladin-1/DHCR24 protein, which is a potential protective factor against neuronal damage, was decreased at both mRNA and protein levels in the ipsilateral thalamus of MCAO rats. Conversely, bepridil treatment restored seladin-1/DHCR24 expression in the ipsilateral thalamus. Bepridil treatment did not significantly affect heme oxygenase-1- or NAD(P)H quinone oxidoreductase-1-mediated oxidative stress or inflammatory responses in the ipsilateral thalamus of MCAO rats. Finally, bepridil treatment mitigated MCAO-induced alterations in APP processing in the ipsilateral thalamus and improved contralateral forelimb use in MCAO rats. These findings suggest that bepridil is a plausible therapeutic candidate in AD or stroke owing to its multifunctional role in key cellular events that are relevant for the pathogenesis of these diseases.

Experimental approaches to study functional recovery following cerebral ischemia.

Valid experimental models and behavioral tests are indispensable for the development of therapies for stroke. The translational failure with neuroprotective drugs has forced us to look for alternative approaches. Restorative therapies aiming to facilitate the recovery process by pharmacotherapy or cell-based therapy have emerged as promising options. Here we describe the most common stroke models used in cell-based therapy studies with particular emphasis on their inherent complications, which may affect behavioral outcome. Loss of body weight, stress, hyperthermia, immunodepression, and infections particularly after severe transient middle cerebral artery occlusion (filament model) are recognized as possible confounders to impair performance in certain behavioral tasks and bias the treatment effects. Inherent limitations of stroke models should be carefully considered when planning experiments to ensure translation of behavioral data to the clinic.

Common data elements and data management: Remedy to cure underpowered preclinical studies.

Lack of translation of data obtained in preclinical trials to clinic has kindled researchers to develop new methodologies to increase the power and reproducibility of preclinical studies. One approach relates to harmonization of data collection and analysis, and has been used for a long time in clinical studies testing anti-seizure drugs. EPITARGET is a European Union FP7-funded research consortium composed of 18 partners from 9 countries. Its main research objective is to identify biomarkers and develop treatments for epileptogenesis. As the first step of harmonization of procedures between laboratories, EPITARGET established working groups for designing project-tailored common data elements (CDEs) and case report forms (CRFs) to be used in data collection and analysis. Eight major modules of CRFs were developed, presenting >1000 data points for each animal. EPITARGET presents the first single-project effort for harmonization of preclinical data collection and analysis in epilepsy research. EPITARGET is also anticipating the future challenges and requirements in a larger-scale preclinical harmonization of epilepsy studies, including training, data management expertise, cost, location, data safety and continuity of data repositories during and after funding period, and incentives motivating for the use of CDEs.

KB-R7943, an inhibitor of the reverse Na(+)/Ca(2+) exchanger, does not modify secondary pathology in the thalamus following focal cerebral stroke in rats.

Remote areas connected to cortical infarcts, such as the thalamus, are affected by stroke due to delayed retrograde degeneration of afferent connections. This is temporally associated with the accumulation of ß-amyloid (Aß) and calcium. Here we tested a hypothesis that prevention of excessive Ca(2+) influx into the axoplasm via the reverse Na(+)/Ca(2+) exchanger (NCX) would provide axonal protection and eventually lessen the Aß and calcium load in the thalamus. We found that chronic treatment with a specific inhibitor of the reverse NCX, KB-R7943 (30mg/kg once daily, 27 days) after middle cerebral artery occlusion did not prevent atypical secondary pathology in the thalamus or improve functional outcome. The present data do not support a role for reverse NCX activity in the complex pathology within the thalamus after cerebral ischemia.

Non-selective calcium channel blocker bepridil decreases secondary pathology in mice after photothrombotic cortical lesion.

Experimental studies have identified a complex link between neurodegeneration, ß-amyloid (Aß) and calcium homeostasis. Here we asked whether early phase ß-amyloid pathology in transgenic hAPPSL mice exaggerates the ischemic lesion and remote secondary pathology in the thalamus, and whether a non-selective calcium channel blocker reduces these pathologies. Transgenic hAPPSL (n = 33) and non-transgenic (n = 30) male mice (4-5 months) were subjected to unilateral cortical photothrombosis and treated with the non-selective calcium channel blocker bepridil (50 mg/kg, p.o., once a day) or vehicle for 28 days, starting administration 2 days after the operation. Animals were then perfused for histological analysis of infarct size, Aß and calcium accumulation in the thalamus. Cortical photothrombosis resulted in a small infarct, which was associated with atypical Aß and calcium accumulation in the ipsilateral thalamus. Transgenic mice had significantly smaller infarct volumes than non-transgenic littermates (P<0.05) and ischemia-induced rodent Aß accumulation in the thalamus was lower in transgenic mice compared to non-transgenic mice (P<0.01). Bepridil decreased calcium load in the thalamus (P<0.01). The present data suggest less pronounced primary and secondary pathology in hAPPSL transgenic mice after ischemic cortical injury. Bepridil particularly decreased calcium pathology in the thalamus following ischemia.

Lack of secondary pathology in the thalamus after focal cerebral ischemia in nonhuman primates.

Remote regions such as the thalamus undergo secondary degeneration after cerebral ischemia. In rodents, the pathology in the thalamus is characterized by a robust inflammatory reaction, ß-amyloid (Aß) accumulation and calcification. Here we studied whether nonhuman primates subjected to middle cerebral artery occlusion (MCAO) display a similar pathology. Common marmosets (n=4) were subjected to transient MCAO for 3 h. Two sham-operated animals served as controls. All animals underwent MRI examination (T2) on postoperative day 7 to assess the location of the infarct. After a 45-day follow-up period, the animals were perfused for histology to evaluate ß-amyloid and calcium load in the peri-infarct regions and the thalamus. There was no Aß or calcium staining in the sham-operated marmosets. The contralateral hemisphere was devoid of Aß and calcium staining in MCAO animals, except calcium staining in one animal. In the ipsilateral cortex, patchy groups of Aß-positive cells were observed. Occasional calcium staining was observed in the peri-infarct regions, lesion core, and remote regions such as the substantia nigra. The most important, the thalamus was devoid of any sign of Aß and calcium aggregation in MCAO animals. Staining for glial fibrillary acidic protein (GFAP) showed marked astrogliosis in the ipsilateral cortex and thalamus. In conclusion, our preliminary study in marmosets did not identify Aß and calcium pathology in the thalamus following cerebral ischemia as shown in rodents.

Chronic ibuprofen treatment does not affect the secondary pathology in the thalamus or improve behavioral outcome in middle cerebral artery occlusion rats.

Anti-inflammatory drug ibuprofen decreases the ß-amyloid (Aß) deposition and associated inflammation in transgenic Alzheimer disease mice. Based on this, we studied whether ibuprofen could modulate the secondary pathology described in the thalamus of middle cerebral artery occlusion (MCAO) rats. Our hypothesis was that ibuprofen could decrease inflammatory reaction and Aß load in the thalamus of MCAO rats, which in turn is reflected in improved behavioral outcome. Forty male Wistar rats (250-340 g) were subjected to sham-operation or transient occlusion of the right middle cerebral artery (120 min). Ibuprofen (4 0mg/kg/day, per os) was administrated for 27 days beginning the treatment on post-operative day 2. MCAO controls were given vehicle. Sensorimotor impairment was assessed using the limb-placing, tapered ledged beam-walking and cylinder tests during the follow-up. The rats were perfused for histology on postoperative day 29. Histological data showed that ibuprofen did not affect Aß or calcium load in the thalamus of MCAO rats. In addition, behavioral tests did not show significant difference between vehicle- and ibuprofen-treated MCAO rats. The present data do not support the idea that ibuprofen reduces the secondary Aß/calcium pathology in the thalamus or associated sensorimotor impairment following cerebral ischemia.