Paediatric onset of multiple sclerosis: Analysis of chemokine and cytokine levels in the context of the early clinical course.

BACKGROUND: Inflammatory activity in children with paediatric onset multiple sclerosis (POMS) is higher than that in adults with MS. Chemokine/cytokine profiling in children may provide new insights into the disease pathogenesis and clinical course. The levels of chemokines/cytokines and their roles in POMS remain largely unknown. OBJECTIVE: To identify the possible utility of chemokines/cytokines in children with POMS, we analysed their levels at the time of disease diagnosis and in the context of subsequent clinical relapse. METHODS: CC and CXC motif ligand chemokines (CCL2, CXCL8, CXCL10, and CXCL13), interleukin (IL)-4, IL-17A, interferon gamma and B cell-activating factor in the blood and cerebrospinal fluid (CSF) of 34 POMS patients and 20 age-related controls were measured using Luminex multiplex bead and enzyme-linked immunosorbent assay techniques. Nonparametric tests were used for statistical analyses. RESULTS: The CSF levels of CXCL8 (p = 0.002), CXCL10 (p = 0.001), and CXCL13 (p<0.0001) were higher in POMS than in controls; CXCL10 and CXCL13 correlated with pleocytosis and oligoclonal bands. A subsequent clinical relapse occurred in 17/34 of the children; the median time from the diagnosis of POMS was 6 months (range, 2-64 months). The follow-up period of patients who did not experience a clinical relapse was significantly longer than the time to first relapse (p = 0.003). The initial CCL2 level was lower in relapsing than in non-relapsing patients (p = 0.063) and correlated negatively with the CSF/serum albumin ratio and positively with the time to relapse (p<0.04). CONCLUSIONS: Elevated CSF levels of CXL10 and CXCL13 in children with POMS at the time of disease diagnosis reflect inflammatory activity and suggest the involvement of adaptive immunity; elevated CXCL8 levels further indicate the involvement of innate immunity. An initial low CSF level of CCL2 may be associated with an unfavourable early MS course.

Modeling of Brain Tissue Heating Caused by Direct Cortical Stimulation for Assessing the Risk of Thermal Damage.

This paper aims to employ the numerical simulations to assess the risk of cellular damage during the application of a novel paradigm of electrical stimulation mapping (ESM) used in neurosurgery. The core principle of the paradigm is the use of short, high-intensity and high-frequency stimulation pulses. We developed a complex numerical model and performed coupled electro-thermal transient simulations. The model was optimized by incorporating ESM electrodes' resistance obtained during multiple intraoperative measurements and validated by comparing them with the results of temperature distribution measurement acquired by thermal imaging. The risk of heat-induced cellular damage was assessed by applying the Arrhenius equation integral on the computed time-dependent spatial distribution of temperature in the brain tissue. Our results suggest that the impact of the temperature increase during our novel ESM paradigm is thermally non-destructive. The presented simulation results match the previously published thermographic measurement and histopathological examination of the stimulated brain tissue and confirm the safety of the novel ESM.

Intraoperative Thermography of the Electrical Stimulation Mapping: A Safety Control Study.

A standard procedure for continuous intraoperative monitoring of the integrity of the corticospinal tracts by eliciting muscle responses is the electric stimulation mapping (ESM). However, standard ESM protocols are ineffective in 20% of young children. We have developed a novel, highly efficient paradigm consisting of short-time burst (30 ms) of high frequency (500 Hz) and high peak current (≤100 mA), which may cause local tissue overheating. The presented safety control study was therefore designed. The infrared thermography camera captured to-be-resected cortex of 13 patients in vivo during ESM. Thermograms were image processed to reveal discrete ESM thermal effect of currents from 10 to 100 mA. Peak 100 mA currents induced a maximal increase in temperature of 3.1 °C, 1.23±0.72 °C in average. The warming correlated with stimulating electrode resistance ( ). The measurement uncertainty was estimated ± 1.01 ºC for the most skeptical conditions. The histopathological evaluation of stimulated tissue (performed in all cases) did not show any destructive changes. Our study demonstrates the ability of the thermographic camera to measure the discrete thermal effect of the ESM. The results provide evidence for the safety of the proposed protocol for full range currents with minimal risk of brain tissue damage.

Promoter-Specific Hypomethylation Correlates with IL-1ß Overexpression in Tuberous Sclerosis Complex (TSC).

In tuberous sclerosis complex (TSC), overexpression of numerous genes associated with inflammation has been observed. Among different proinflammatory cytokines, interleukin-1ß (IL-1ß) has been shown to be significantly involved in epileptogenesis and maintenance of seizures. Recent evidence indicates that IL-1ß gene expression can be regulated by DNA methylation of its promoter. In the present study, we hypothesized that hypomethylation in the promoter region of the IL-1ß gene may underlie its overexpression observed in TSC brain tissue. Bisulfite sequencing was used to study the methylation status of the promoter region of the IL-1ß gene in TSC and control samples. We identified hypomethylation in the promoter region of the IL-1ß gene in TSC samples. IL-1ß is overexpressed in tubers, and gene expression is correlated with promoter hypomethylation at CpG and non-CpG sites. Our results provide the first evidence of epigenetic modulation of the IL-1ß signaling in TSC. Thus, strategies that target epigenetic alterations could offer new therapeutic avenues to control the persistent activation of interleukin-1ß-mediated inflammatory signaling in TSC brain.

Expression of microRNAs miR21, miR146a, and miR155 in tuberous sclerosis complex cortical tubers and their regulation in human astrocytes and SEGA-derived cell cultures.

Tuberous sclerosis complex (TSC) is a genetic disease presenting with multiple neurological symptoms including epilepsy, mental retardation, and autism. Abnormal activation of various inflammatory pathways has been observed in astrocytes in brain lesions associated with TSC. Increasing evidence supports the involvement of microRNAs in the regulation of astrocyte-mediated inflammatory response. To study the role of inflammation-related microRNAs in TSC, we employed real-time PCR and in situ hybridization to characterize the expression of miR21, miR146a, and miR155 in TSC lesions (cortical tubers and subependymal giant cell astrocytomas, SEGAs). We observed an increased expression of miR21, miR146a, and miR155 in TSC tubers compared with control and perituberal brain tissue. Expression was localized in dysmorphic neurons, giant cells, and reactive astrocytes and positively correlated with IL-1ß expression. In addition, cultured human astrocytes and SEGA-derived cell cultures were used to study the regulation of the expression of these miRNAs in response to the proinflammatory cytokine IL-1ß and to evaluate the effects of overexpression or knockdown of miR21, miR146a, and miR155 on inflammatory signaling. IL-1ß stimulation of cultured glial cells strongly induced intracellular miR21, miR146a, and miR155 expression, as well as miR146a extracellular release. IL-1ß signaling was differentially modulated by overexpression of miR155 or miR146a, which resulted in pro- or anti-inflammatory effects, respectively. This study provides supportive evidence that inflammation-related microRNAs play a role in TSC. In particular, miR146a and miR155 appear to be key players in the regulation of astrocyte-mediated inflammatory response, with miR146a as most interesting anti-inflammatory therapeutic candidate.

The relationships between quantitative MR parameters in hippocampus in healthy subjects and patients with temporal lobe epilepsy.

We introduce a new magnetic resonance (MR) method based on a pixel-by-pixel image processing to examine relationships between metabolic and structural processes in the pathologic hippocampus. The method was tested for lateralization of the epileptogenic zone in patients with temporal lobe epilepsy (TLE). Twenty patients with drug-resistant TLE and fifteen healthy controls were examined at 3T. The measurement protocol contained T2-weighted MR images, spectroscopic imaging, diffusion tensor imaging and T2 relaxometry. Correlations between quantitative MR parameters were calculated on a pixel-by-pixel basis using the CORIMA program which enables automated pixel identification in the normal tissue according to control data. All MR parameters changed in the anteroposterior direction in the hippocampus and correlation patterns and their slopes differed between patients and controls. Combinations of T2 relaxation times with metabolite values represent the best biomarkers of the epileptogenic zone. Correlations with mean diffusivity did not provide sufficiently accurate results due to diffusion image distortions. Quantitative MR analysis non-invasively provides a detailed description of hippocampal pathology and may represent complementary tool to the standard clinical protocol. However, the automated processing should be carefully monitored in order to avoid possible errors caused by MR artifacts.

Distinctive MRI features of the epileptogenic zone in children with tuberous sclerosis.

OBJECTIVE: Localization of the epileptogenic zone (EZ) is challenging in children with tuberous sclerosis complex (TSC). We sought to ascertain whether brain MRI could identify the EZ in TSC patients independent of the clinical and diagnostic data. METHODS: Presurgical MRI's of 34 children with TSC who underwent epilepsy surgery at Miami Children's Hospital were retrospectively reevaluated by experts blinded to all other data. Changes typical of TSC (tubers, calcifications, cystic changes) and abnormalities of the perituberal cortex typical of focal cortical dysplasia (FCD) (increased cortical thickness, abnormal gyration, transmantle change, gray/white matter junction blurring) were identified and their localization was compared with the resection site. Sensitivity, specificity and accuracy of individual MRI features to localize the EZ were determined and statistically compared between postoperatively seizure-free and non-seizure-free patients as well as clusters of features typical of FCD and TSC. RESULTS: MRI alone correctly localized the resection cavity in all 19 postoperatively seizure-free patients and 12 of 15 non-seizure-free subjects. Sensitivity, specificity and accuracy of MRI features typical of FCD to localize EZ (90%, 96% and 96%, respectively) were superior to those typical of TCS (79%, 75% and 75%, p<0.0001). Increased cortical thickness and abnormal gyral formation outside tubers occurred only in the resection site. Resection sites were better predicted by MRI in seizure-free than in non-seizure-free patients. CONCLUSION: Thorough MRI evaluation identifies the EZ in a significant proportion of TSC patients. Epileptogenic regions were mostly characterized by "FCD-like" changes outside cortical tubers. The findings may have important practical consequences for surgical planning in TSC.

The diffusion parameters of the extracellular space are altered in focal cortical dysplasias.

Most hypotheses concerning the mechanisms underlying seizure activity in focal cortical dysplasia (FCD) are based on alterations in synaptic transmission and glial dysfunction. However, neurons may also communicate by extrasynaptic transmission, which was recently found to affect epileptiform activity under experimental conditions and which is mediated by the diffusion of neuroactive substances in the extracellular space (ECS). The ECS diffusion parameters were therefore determined using the real-time iontophoretic method in human neocortical tissue samples obtained from surgically treated epileptic patients. The obtained values of the extracellular space volume fraction and tortuosity were then correlated with the histologicaly assessed type of cortical malformation (FCD type I or II). While the extracellular volume remained unchanged (FCD I) or larger (FCD II) than in normal/control tissue, tortuosity was significantly increased in both types of dysplasia, indicating the presence of additional diffusion barriers and compromised diffusion, which might be another factor contributing to the epileptogenicity of FCD.

Incomplete resection of focal cortical dysplasia is the main predictor of poor postsurgical outcome.

BACKGROUND: Focal cortical dysplasia (FCD) is recognized as the major cause of focal intractable epilepsy in childhood. Various factors influencing postsurgical seizure outcome in pediatric patients with FCD have been reported. OBJECTIVE: To analyze different variables in relation to seizure outcome in order to identify prognostic factors for selection of pediatric patients with FCD for epilepsy surgery. METHODS: A cohort of 149 patients with histologically confirmed mild malformations of cortical development or FCD with at least 2 years of postoperative follow-up was retrospectively studied; 113 subjects had at least 5 years of postoperative follow-up. Twenty-eight clinical, EEG, MRI, neuropsychological, surgical, and histopathologic parameters were evaluated. RESULTS: The only significant predictor of surgical success was completeness of surgical resection, defined as complete removal of the structural MRI lesion (if present) and the cortical region exhibiting prominent ictal and interictal abnormalities on intracranial EEG. Unfavorable surgical outcomes are mostly caused by overlap of dysplastic and eloquent cortical regions. There were nonsignificant trends toward better outcomes in patients with normal intelligence, after hemispherectomy and with FCD type II. Other factors such as age at seizure onset, duration of epilepsy, seizure frequency, associated pathologies including hippocampal sclerosis, extent of EEG and MRI abnormalities, as well as extent and localization of resections did not influence outcome. Twenty-five percent of patients changed Engel's class of seizure outcome after the second postoperative year. CONCLUSIONS: The ability to define and fully excise the entire region of dysplastic cortex is the most powerful variable influencing outcome in pediatric patients with focal cortical dysplasia.

Use of 3D geometry modeling of osteochondrosis-like iatrogenic lesions as a template for press-and-fit scaffold seeded with mesenchymal stem cells.

Computed tomography (CT) is an effective diagnostic modality for three-dimensional imaging of bone structures, including the geometry of their defects. The aim of the study was to create and optimize 3D geometrical and real plastic models of the distal femoral component of the knee with joint surface defects. Input data included CT images of stifle joints in twenty miniature pigs with iatrogenic osteochondrosis-like lesions in medial femoral condyle of the left knee. The animals were examined eight and sixteen weeks after surgery. Philips MX 8000 MX and View workstation were used for scanning parallel plane cross section slices and Cartesian discrete volume creation. On the average, 100 slices were performed in each stifle joint. Slice matrices size was 512 x 512 with slice thickness of 1 mm. Pixel (voxel) size in the slice plane was 0.5 mm (with average accuracy of +/-0.5 mm and typical volume size 512 x 512 x 100 voxels). Three-dimensional processing of CT data and 3D geometrical modelling, using interactive computer graphic system MediTools formerly developed here, consisted of tissue segmentation (raster based method combination and 5 % of manual correction), vectorization by the marching-cubes method, smoothing and decimation. Stifle- joint CT images of three individuals of different body size (small, medium and large) were selected to make the real plastic models of their distal femurs from plaster composite using rapid prototyping technology of Zcorporation. Accuracy of the modeling was +/- 0.5 mm. The real plastic models of distal femurs can be used as a template for developing custom made press and fit scaffold implants seeded with mesenchymal stem cells that might be subsequently implanted into iatrogenic joint surface defects for articular cartilage-repair enhancement.

[Computer modeling correlation between the entering wound and the final position of the metallic intraocular foreign body]. / Korelace vstupní rány a výsledného ulozeni kovového nitroocního telesa pomocí pocítacového modelování.

AIM: The aim of this study was to establish the correlation coefficient between given parameters of the entering wound and final position of the metallic intraocular foreign body. MATERIAL AND METHODS: Fifty patients (50 eyes) with a penetrating injury of the eye and present metallic intraocular foreign body were included in this study. Clinically found data of the entering wound and final position of the intraocular foreign body (IFB) as well were transformed with a computer model into the three-dimensional parameters. Both parameters were statistically evaluated by means of correlation analysis, and correlation coefficient and determination coefficient were calculated. RESULTS: The extent of correlation between two variables is called correlation coefficient. The coefficient values range between -1 to +1. The closer is the calculated value to ranges (to -1 or to +1) the more are the two variables more correlated. The coefficient of determination values range from 0 to +1. The closer the results are to +1, the better the model describes the dependence between the two variables. The results obtained by means of correlation analysis were for the correlation coefficient 0.454, and for the coefficient of determination 0.6411 respectively. CONCLUSION: Results of the correlation analysis show that the knowledge of coordinates of the entering wound has no prediction value for final position of the foreign body in the eye. These two variables are not correlated and so the accurate final position of the foreign body cannot be predicted. The final position of the intraocular metallic foreign body is probably influenced by biophysical factors not included in this study.

Densities of parvalbumin-immunoreactive neurons in non-malformed hippocampal sclerosis-temporal neocortex and in cortical dysplasias.

The changes in density of inhibitory parvalbumin-immunoreactive interneurons were quantitatively studied by immunohistochemistry in a series of human neocortical samples comprising the spectrum of malformations of cortical development (MCD) encountered in epilepsy surgery and the non-malformed hippocampal sclerosis-temporal neocortex in patients with refractory temporal lobe epilepsy. The highest relative density of parvalbumin-immunoreactive cells was obtained in the control samples (n = 21). The number of parvalbumin-immunoreactive neurons was significantly decreased in non-malformed hippocampal sclerosis-temporal neocortex (n = 73, 80.5% of control values). In a proportion of the latter samples as well as in two controls we observed patchy regions of absence of parvalbumin staining. The total counts of parvalbumin-immunoreactive cells in all the categories of MCD - "mild MCD" (n = 25), focal cortical dysplasia type I (n = 19) and type II (n = 15) - were decreased representing 72.4%, 55.0% and 12.2% of control values, respectively. Significantly different parvalbumin-immunoreactive cell densities were demonstrated between the focal cortical dysplasia types IIA and IIB. In "mild MCD", we observed a more pronounced decrease of parvalbumin-immunoreactive cells in the infragranular layers. No significant differences were revealed between the temporal and extratemporal examples of analogous MCD types. This study provides evidence for reduction of inhibitory parvalbumin-immunoreactive interneurons in the epileptic neocortex affected by MCD as well as in morphologically unaffected epileptic temporal neocortex, thus representing a possible mechanism for their epileptogenicity.

[Microscopic disorders of cortical development of the brain and its etiopathogenic importance for detection in patients with temporal epilepsy associated with hippocampal sclerosis]. / Mikroskopické poruchy kortikálního vývoje mozku a etiopatogenetický význam jejich detekce u pacientu s temporální epilepsií pri skleróze hipokampu.

Hippocampal sclerosis represents a common structural basis of temporal lobe epilepsy. However, the etiological factors and mechanisms leading to its development still remain unexplained. In our study, we present neuropathological findings in the resected hippocampus and the pole of the temporal lobe in 15 patients with hippocampal sclerosis. "Initial precipitating injuries" that are thought to cause the development of hippocampal sclerosis (febrile seizures in early childhood, head injury or meningoencephalitis) were present in the history of 12 patients. In the remaining 3 cases, no predisposing factors were found. Attention was paid to the histopathological identification of disturbed neuronal migration and differentiation in the temporal lobe. These defects were observed in 7 cases; in three of these, no predisposing factors were stated in the patients' histories. We suggest that in these cases, hippocampal sclerosis arises due to previously undetected disorders of cortical development. A latent neocortical malformation may also contribute to the development of hippocampal sclerosis in patients with an initial precipitating injury in anamnensis. Histopathological examination of resected epileptic brain tissue can provide insights into the individual pathogenesis of epileptic disorders, especially by the detection of microscopic disorders of cortical development.

An animal model of nonconvulsive status epilepticus: a contribution to clinical controversies.

PURPOSE: To characterize electroencephalographic and behavioral effects as well as electrophysiologic and morphologic consequences of a subconvulsive dose of pilocarpine in lithium chloride-pretreated rats. METHODS: Pilocarpine (15 mg/kg) was administered intraperitoneally to adult rats pretreated with lithium chloride (3 mEq/kg, i.p.). Behavior was observed for 2 h and videotaped in three consecutive sessions. At the same time, EEG was recorded from the sensorimotor cortex and the dorsal hippocampus. Threshold intensities of currents necessary to elicit hippocampal afterdischarges were determined 24 h and 1 week after the pilocarpine administration. The brains were histologically examined 1 week after pilocarpine administration using Nissl stain. RESULTS: Pilocarpine induced time-limited nonconvulsive status epilepticus (NCSE). Epileptic EEG activity concurrent with prominent behavioral features was observed both in the neocortex and, predominantly, in the hippocampus. No changes in afterdischarge thresholds were observed in the dorsal hippocampus 24 h and 1 week after NCSE. One week after NCSE, seizure-related brain damage was found mainly in the motor neocortical fields. CONCLUSIONS: Pilocarpine-induced NCSE in rats strongly resembles a short-term human complex partial status epilepticus. Our animal model is suitable for studying the possible adverse effects of prolonged nonconvulsive seizures.

Nonconvulsive status epilepticus in rats: impaired responsiveness to exteroceptive stimuli.

An animal model of human complex partial status epilepticus induced by lithium chloride and pilocarpine administration was developed in our laboratory. The objective of the study was to provide a detailed analysis of both ictal and postictal behavior and to quantify seizure-related morphological damage. In order to determine the animal's responsiveness to either visual or olfactory stimuli, adult male rats were submitted to the following behavioral paradigms: the object response test, the social interaction test, and the elevated plus-maze test. The rotorod test was used to evaluate motor performance. Two weeks after status epilepticus, brains were morphologically examined and quantification of the brain damage was performed. Profound impairment of behavior as well as responsiveness to exteroceptive stimuli correlated with the occurrence of epileptic EEG activity. When the epileptic EEG activity ceased, responsiveness of the pilocarpine-treated animals was renewed. However, remarkable morphological damage persisted in the cortical regions two weeks later. This experimental study provides support for the clinical evidence that even nonconvulsive epileptic activity may cause brain damage. We suggest that the model can be used for the study of both functional and morphological consequences of prolonged nonconvulsive seizures.

Age-dependent phenytoin effects on cortical stimulation in rats.

The effects of phenytoin on threshold intensities of stimulation were studied in cortical epileptic afterdischarges (ADs) in 12-day-old and adult rats with implanted electrodes. Stimulation of the sensorimotor cortical area induced movements directly related to the stimulation as well as EEG afterdischarges (ADs) of the spike-and-wave type and of the limbic type. Rat pups exhibited lower thresholds for stimulation-bound movements and spike-and-wave ADs than adult animals. On the contrary, the limbic type of ADs was elicited with lower current intensity in adult than in immature rats. Phenytoin increased the threshold for stimulation-related movements only in adult rats, whereas threshold intensities for spike-and-wave ADs were increased and thresholds for limbic type of ADs remained uninfluenced in both age groups. The age-dependent effect on stimulation-related movements might be due to a maturation of connectivity in the motor system or to developmental changes in the voltage-gated sodium channels as the main target of phenytoin action.