The mechanisms involved in the pathogenesis of epilepsy, a chronic neurological disorder that affects approximately 1 percent of the world population, are not well understood [1][2][3] . Using a mouse model of epilepsy, we show that seizures induce elevated expression of vascular cell adhesion molecules and enhanced leukocyte rolling and arrest in brain vessels mediated by the leukocyte mucin P-selectin glycoprotein ligand-1 (PSGL-1) and leukocyte integrins α4β1 and αLβ2. Inhibition of leukocytevascular interactions either with blocking antibodies, or in mice genetically deficient in functional PSGL-1, dramatically reduced seizures. Treatment with blocking antibodies following acute seizures prevented the development of epilepsy. Neutrophil depletion also inhibited acute seizure induction and chronic spontaneous recurrent seizures. Blood-brain barrier (BBB) leakage, which is known to enhance neuronal excitability, was induced by acute seizure activity but was prevented by blockade of leukocyte-vascular adhesion, suggesting a pathogenetic link between leukocyte-vascular interactions, BBB damage and seizure generation. Consistent with potential leukocyte involvement in the human, leukocytes were more abundant in brains of epileptics than of controls. Our results Correspondence should be addressed to: P.F.F (E-mail: paolo.fabene@univr.it) or G.C. (E-mail: gabriela.constantin@univr.it). Author contribution G.N.M., D.B., A.C., L.Z., F.S. performed epilepsy experiments, telemetry and open field behavior. M.M., B.R., L.O., S.B., S.A., performed intravital microscopy, in vivo staining for adhesion molecules, adhesion assays and contributed to the obtainment of behavioral data. A.O. provided the human samples. F.M., A.C. and F.O. performed immunohistochemistry on human and animal samples. P.M., E.N. and A.S provided MRI expertise. J.W.H., L.X., J.B.L., R.P.M provided vital reagents and mice. E.C.B contributed experimental suggestions, reagents and assistance with writing. P.F.F and G.C. designed the study, analyzed the data and wrote the paper NIH Public Access suggest leukocyte-endothelial interaction as a potential target for the prevention and treatment of epilepsy.Experimental data from animal models as well as human evidence indicate that seizures can lead to neuronal damage and cognitive impairement 2, 3 . However, the molecular mechanisms leading to seizures and epilepsy are not well understood. Recent data suggests that inflammation may play a role in the pathogenesis of epilepsy 4, 5 . For instance, elevation in inflammatory cytokines are seen in the central nervous system (CNS) and plasma in experimental models of seizures and in clinical cases of epilepsy 4, 5 . Moreover, CNS inflammation is associated with breakdown in the blood-brain barrier (BBB), and BBB leakage has been implicated both in the induction of seizures and in the progression to epilepsy 6-9 . Leukocyte recruitment is a hallmark of and a point of therapeutic intervention in tissue inflammation 10,11 , but a role for leukocyte-endothelia...

In refractory temporal lobe epilepsy, seizures often arise from a shrunken hippocampus exhibiting a pattern of selective neuron loss called "classic hippocampal sclerosis." No single experimental injury has reproduced this specific pathology, suggesting that hippocampal atrophy might be a progressive "endstage" pathology resulting from years of spontaneous seizures. We posed the alternate hypothesis that classic hippocampal sclerosis results from a single excitatory event that has never been successfully modeled experimentally because convulsive status epilepticus, the insult most commonly used to produce epileptogenic brain injury, is too severe and necessarily terminated before the hippocampus receives the needed duration of excitation. We tested this hypothesis by producing prolonged hippocampal excitation in awake rats without causing convulsive status epilepticus. Two daily 30-minute episodes of perforant pathway stimulation in Sprague-Dawley rats increased granule cell paired-pulse inhibition, decreased epileptiform afterdischarge durations during 8 hours of subsequent stimulation, and prevented convulsive status epilepticus. Similarly, one 8-hour episode of reduced-intensity stimulation in Long-Evans rats, which are relatively resistant to developing status epilepticus, produced hippocampal discharges without causing status epilepticus. Both paradigms immediately produced the extensive neuronal injury that defines classic hippocampal sclerosis, without giving any clinical indication during the insult that an injury was being inflicted. Spontaneous hippocampal-onset seizures began 16-25 days post-injury, before hippocampal atrophy developed, as demonstrated by sequential magnetic resonance imaging. These results indicate that classic hippocampal sclerosis is uniquely produced by a single episode of clinically "cryptic" excitation. Epileptogenic insults may often involve prolonged excitation that goes undetected at the time of injury.

Under certain experimental conditions, neurotrophic factors may reduce epileptogenesis. We have previously reported that local, intrahippocampal supplementation of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) increases neurogenesis, reduces neuronal loss, and reduces the occurrence of spontaneous seizures in a model of damage-associated epilepsy. Here, we asked if these possibly anti-epileptogenic effects might involve anti-inflammatory mechanisms. Thus, we used a Herpes-based vector to supplement FGF-2 and BDNF in rat hippocampus after pilocarpine-induced status epilepticus that established an epileptogenic lesion. This model causes intense neuroinflammation, especially in the phase that precedes the occurrence of spontaneous seizures. The supplementation of FGF-2 and BDNF attenuated various parameters of inflammation, including astrocytosis, microcytosis and IL-1β expression. The effect appeared to be most prominent on IL-1β, whose expression was almost completely prevented. Further studies will be needed to elucidate the molecular mechanism(s) for these effects, and for that on IL-1β in particular. Nonetheless, the concept that neurotrophic factors affect neuroinflammation in vivo may be highly relevant for the understanding of the epileptogenic process.

In obesity, an inflammatory process of the adipose tissue has been hypothesized; however, direct evidence for a tissue lesion is still lacking. Macrophage infiltration in the adipose tissue of obese individuals seems to be proven, but other alterations of the tissue have not been demonstrated. Moreover, in humans it has not been clarified whether inflammation is an early characteristic of obesity, because no data from obese children are available. In the present study, we assessed the inflammatory involvement of the adipose tissue and identified the elementary "inflammatory" lesion in a group of obese children. The study of children gives us the chance to investigate adipose tissue during early phases of obesity. In all the obese subjects, ultramicroscopic analysis of the adipose tissue demonstrated inflammatory involvement, and the extent of the lesions seemed to depend on the SD score of body mass index. The elementary lesion is a microgranuloma, with fragments of adipocytes, that evolves to fibrosis. Macrophages (and less frequently, lymphocytes or granulocytes) were found in perivascular positions. The lesions were not found in nonobese children. Our study proved that an "inflammatory" process exists in the adipose tissue of obese children, confirming previous findings in animals and obese adults and demonstrating that it is an early alteration in humans. However, the accumulation of macrophages was just one of the components of the inflammatory lesion, which also involved adipocyte degeneration, fibrosis, and, to a lesser extent, granulocyte/lymphocyte accumulation. The finding of fragments of adipocytes in the elementary lesion suggests that, at the beginning of the process, adipocytes may degenerate and that the materials generated by this process can recruit macrophages and other leukocytes. These preliminary results suggest that additional studies should be designed to clarify the cause of adipocyte fragility in obese children.

Purpose: The purpose of this research was to assess in vivo by dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) the antiangiogenic effect of SU6668, an oral, small molecule inhibitor of the angiogenic receptor tyrosine kinases vascular endothelial growth factor receptor 2 (Flk-1/KDR), platelet-derived growth factor receptor, and fibroblast growth factor receptor 1.Experimental Design: A s.c. tumor model of HT29 human colon carcinoma in athymic mice was used. DCE-MRI with a macromolecular contrast agent was used to measure transendothelial permeability and fractional plasma volume, accepted surrogate markers of tumor angiogenesis. CD31 immunohistochemical staining was used for assessing microvessels density and vessels area. Experiments were performed after 24 h, and 3, 7, and 14 days of treatment.Results: DCE-MRI clearly detected the early effect (after 24 h of treatment) of SU6668 on tumor vasculature as a 51% and 26% decrease in the average vessel permeability measured in the tumor rim and core (respectively). A substantial decrease was also observed in average fractional plasma volume in the rim (59%) and core (35%) of the tumor. Histological results confirmed magnetic resonance imaging findings. After 3, 7, and 14 days of treatment, postcontrast magnetic resonant images presented a thin strip of strongly enhanced tissue at the tumor periphery; histology examination showed that this hyperenhanced ring corresponded to strongly vascularized tissue adjacent but external to the tumor. Histology also revealed a strong decrease in the thickness of peripheral viable tissue, with a greatly reduced vessel count. SU6668 greatly inhibited tumor growth, with 60% inhibition at 14 days of treatment.Conclusions: DCE-MRI detected in vivo the antiangiogenic efficacy of SU6668.

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Purpose: To compare two dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) techniques in terms of their ability in assessing the early antiangiogenic effect of SU11248, a novel selective multitargeted tyrosine kinase inhibitor, that exhibits direct antitumor and antiangiogenic activity via inhibition of the receptor tyrosine kinases platelet-derived growth factor receptor, vascular endothelial growth factor receptor, KIT, and FLT3. Experimental Design: A s.c. tumor model of HT29 human colon carcinoma in athymic mice was used. Two DCE-MRI techniques were used based, respectively, on macromolecular [Gddiethylenetriaminepentaacetic acid (DTPA)-albumin] and low molecular weight (Gd-DTPA) contrast agents. The first technique provided a quantitative measurement of transendothelial permeability and fractional plasma volume, accepted surrogate markers of tumor angiogenesis. With the second technique, we quantified the initial area under the concentration-time curve, which gives information related to tumor perfusion and vascular permeability. Experiments were done before and 24 hours after a single dose administration of SU11248. Results:The early antiangiogenic effect of SU11248 was detected by DCE-MRI with macromolecular contrast agent as a 42% decrease in vascular permeability measured in the tumor rim. The effect was also detected by DCE-MRI done with Gd-DTPA as a 31% decrease in the initial area under the concentration-time curve. Histologic slices showed a statistically significant difference in mean vessel density between the treated and control groups. Conclusions: The early antiangiogenic activity of SU11248 was detected in vivo by DCE-MRI techniques using either macromolecular or low molecular weight contrast agents. Because DCE-MRI techniques with low molecular weight contrast agents can be used in clinical studies, these results could be relevant for the design of clinical trials based on new paradigms.