Alexandre Rainha Campos

Establishment of primary cultures of human brain microvascular endothelial cells to provide an in vitro cellular model of the blood-brain barrier

We describe a method for generating primary cultures of human brain microvascular endothelial cells (HBMVECs). HBMVECs are derived from microvessels isolated from temporal tissue removed during operative treatment of epilepsy. The tissue is mechanically fragmented and size filtered using polyester meshes. The resulting microvessel fragments are placed onto type I collagen-coated flasks to allow HBMVECs to migrate and proliferate. The overall process takes less than 3 h and does not require specialized equipment or enzymatic processes. HBMVECs are typically cultured for approximately 1 month until confluent. Cultures are highly pure (∼97% endothelial cells; ∼3% pericytes), are reproducible, and show characteristic brain endothelial markers (von Willebrand factor, glucose transporter-1) and robust expression of tight and adherens junction proteins as well as caveolin-1 and efflux protein P-glycoprotein. Monolayers of HBMVECs show characteristically high transendothelial electric resistance and have proven useful in multiple functional studies for in vitro modeling of the human blood-brain barrier.

Simple and complex dysembryoplastic neuroepithelial tumors (DNT) variants: clinical profile, MRI, and histopathology

IntroductionDysembryoplastic neuroepithelial tumors (DNTs) are long-term epilepsy associated tumors subdivided into simple and complex variants. The purpose of this study was to relate different DNT components identified on magnetic resonance imaging (MRI) to histopathological features and to test the hypothesis that glial nodules as a histopathological feature of complex variants induce an occasional glioma misdiagnosis.MethodsClinical, MRI, and histopathologic features of DNTs operated between 1988 and 2008 were reviewed.ResultsFrom a total of 61 DNTs, 48 simple and 13 complex variants were identified. Multiple or single pseudocysts in a cortical/subcortical location with small cysts sometimes separated from the tumor represented the glioneuronal element and were found in all DNTs. FLAIR hyperintense tissue was found between pseudocysts but—in neocortical DNTs—also circumscript in deeper tumor parts. Calcification and hemorrhages in this location occurred in four of 13 complex variants, and one of these patients was also the only one with tumor growth. Patients with complex variants had earlier seizure onset, and complex variants were more often located outside the temporal lobe. Although complex variants represented a higher diagnostic challenge, misdiagnoses also occurred in simple variants. One of five of DNTs showed contrast enhancement, which varied on follow-up studies with enhancing parts becoming nonenhancing and vice versa.ConclusionThe glioneuronal element is readily identifiable on MRI and should be considered to support the DNT diagnosis. Complex DNT variants have a different clinical profile and a more variable histopathological and MRI appearance; however, misdiagnoses occasionally also occur in simple variants.

The basal temporal approach for mesial temporal surgery: sparing the Meyer loop with navigated diffusion tensor tractography.

BACKGROUND: Visual field defects are a common side effect after mesial temporal resections such as selective amygdalohippocampectomy (SelAH). OBJECTIVE: To present a method of diffusion tensor tractography (DTT) of the Meyer loop for preoperative planning of the surgical approach for SelAH and for intraoperative visualization on a navigation-guided operating microscope. METHODS: Twelve patients were selected for SelAH to treat mesial temporal lobe epilepsy. All received preoperative MRI with diffusion tensor imaging sequences. The Meyer loop was determined and reconstructed as an object with DTT. Images were utilized for preoperative planning in which a safe approach not affecting the Meyer loop was specified. A navigation-guided operating microscope was used for image-guided surgery. RESULTS: DTT was a reliable method for visualization of the Meyer loop. Reconstruction of the Meyer loop had a direct impact on the approach planning. In all 12 cases, the optic tract could only be spared using a basal approach. Ten patients underwent SelAH by the subtemporal approach, and 2 underwent SelAH by the transcortical approach through the inferior temporal gyrus. During the critical early phase of the operation image guidance remained accurate until entry into the ventricle. Nine of 12 patients had no postoperative field deficits (75%). Three patients (25%) experienced peripheral incomplete quadrantanopia. CONCLUSION: DTT and intraoperative visualization of the Meyer loop is a helpful tool for preoperative planning and during surgery to find a safe trajectory to mesial temporal structures while avoiding the optic radiation. This technique in combination with a basal approach seems to be a promising strategy to prevent postoperative visual field deficits in most patients.

Rosette-forming glioneuronal tumor: pathology case report.

OBJECTIVE Rosette-forming glioneuronal tumor is a newly described mixed glial and neuronal tumor. We describe two cases and review the literature to better characterize this entity. METHODS Patients were surgically treated, and tumors were diagnosed by light microscopy and immunohistochemistry using the avidin-biotin complex method. PubMed was searched for previously reported cases. RESULTS Patient 1 was a 38-year-old woman who presented with headaches and no neurological abnormality. Magnetic resonance imaging showed a solid mass in the fourth ventricle. Subtotal excision of the mass caused transient gait ataxia. Patient 2 was a 51-year-old woman with dizziness who fell and sustained head trauma. Magnetic resonance imaging revealed a right paramedian cerebellar cystic and nodular mass and a separate nodule in the vermis, which were excised gross totally with no morbidity. Microscopic examination showed neuroepithelial tumors composed of neurocytic cells focally forming well-defined rosettes that were immunopositive for neuronal markers and of elongated, glial fibrillary acidic protein-immunoreactive astrocytes. No histological anaplasia was present. Both patients were well 18 and 8 months after surgery, respectively. Eighteen rosette-forming glioneuronal tumors were identified with the literature search. CONCLUSION These are tumors of young adulthood (range, 12-59 yr) usually in or close to the fourth ventricle. Histologically, they are low-grade, although multiple foci or local extension may prevent total excision and account for some recurrences. On imaging, they are cystic, solid, or both, with minimal perilesional edema or mass effect. They are composed of neurocytic and glial elements, probably arising from a common progenitor in the subependymal plate, and need to be differentiated from a variety of glioneuronal tumors.

Tricellulin expression in brain endothelial and neural cells.

Tricellulin is a tight junction (TJ) protein, which is not only concentrated at tricellular contacts but also present at bicellular contacts between epithelial tissues. We scrutinized the brain for tricellulin expression in endothelial and neural cells by using real-time polymerase chain reaction, Western blot and immunohistochemical and immunocytochemical analysis of cultured brain cells and paraffin sections of brain. Tricellulin mRNA was detected in primary cultures and in a cell line of human brain microvascular endothelial cells. Protein expression was confirmed by Western blot and immunofluorescence analysis, which further highlighted the localization of tricellulin in the cell membrane at tricellular and along bicellular contacts, and in the nucleus and perinuclear region. Compared with the well-studied TJ protein, zonula occludens-1, tricellulin expression was less marked at the cell membrane but more evident in the nuclear and perinuclear regions. The presence of tricellulin in cultured endothelial cells was corroborated by immunohistochemical and immunofluorescence staining in brain blood vessels, where it was colocalized with another TJ protein, claudin-5. Tricellulin mRNA was detected in neurons and astrocytes, whereas protein expression was observed in astrocytes but not in neurons, as shown by immunofluorescence analysis. This study reveals the presence and subcellular distribution of tricellulin in brain endothelial cells, both in vitro and in situ and its colocalization with other relevant TJ proteins. Moreover, it demonstrates the expression of the protein in astrocytes opening new avenues for future research to establish the biological significance of tricellulin expression in glial cells.

Antiepileptic drugs management and long-term seizure outcome in post surgical mesial temporal lobe epilepsy with hippocampal sclerosis.

Surgery is the treatment of choice for refractory temporal lobe epilepsies, but unexpected seizure recurrences occur and the AEDs management strategy may be an implicated factor. We evaluated the AEDs managements role in the outcome of post surgical epilepsy patients with hippocampal sclerosis (HS). Epileptic patients submitted to amigdalohippocampectomy due to HS in Engel class IA 12 months after surgery were selected. The following variables were studied: age, gender, time of post-surgical follow-up, present Engel class, number of antiepileptic AEDs before surgery and at the time of the interview, AED changes after surgery (stopped, increased, decreased, maintained), timing for AED changes after surgery and seizure recurrences. Sixty-seven consecutive patients were studied (mean time of follow-up of 4.9 ± 2.8 years). Among these, 46.3% were tapering AEDs, 38.8% had not changed and 14.9% had increased AEDs. The global recurrence rate was 32.8%. Recurrence rates for patients tapering and not tapering AEDs were similar (34.2% and 31%, respectively). Fifteen patients tapered AEDs before 2 years and 20 at or 2 years after surgery, with similar recurrence rates (33% and 30%, respectively). All patients who recurred due to AED tapering and 66.7% of the patients who recurred with no AED reduction resumed the Engel class I. This study suggests that in HS patients submitted to AHE who are seizure free during the first postsurgical year, AEDs tapering is achieved in a substantial percentage of patients. Tapering AEDs, independently of its timing, will induce seizure recurrence in about a third of patients. However, patients relapsing after tapering AEDs regain control after resuming therapy.

Deep Brain Stimulation for Refractory Cocaine Dependence

Refractory cocaine dependence (RCD) is a severe condition that includes motivational and behavioral disturbances for which there are no specific treatments. A dysfunction of the brainrewarding circuitry (1,2), which includes the nucleus accumbens (Acc), the bed nucleus of the stria terminalis (BNST), the anterior limb of the internal capsule, and the medial forebrain bundle, is thought to underlie RCD. These structures have been implicated in other disorders, such as obsessive-compulsive disorder. For cases of refractory obsessive-compulsive disorder, deep brain stimulation (DBS) emerged as a viable new therapeutic approach (3–11). More recent articles have also shown benefits of DBS for patients with nicotine (12–14), ethanol (15–18), and heroin (19–21) dependence. Although the ideal target is controversial, updated human Acc segmentation based on stereotactic anatomy and magnetic resonance imaging probabilistic tractography (22–25) prompted us to elect its posterior-medial part (Acc shell) plus the neighboring BNST to optimize DBS results. Our main objective was to evaluate the clinical efficacy of DBS in the treatment of RCD. Overall tolerability and safety profiles were also assessed. The present case was subject to a pilot study with a longitudinal double-blind crossover randomized control for 30 months including three phases, as follows:

Intracerebral amyloidoma: case report and review of the literature.

Intracerebral amyloidoma (ICA) is a type of monoclonal immunoglobulin deposition disease (MIDD) which is accompanied by an overexpression and fibrillary assembly of monoclonal light chains, ultimately leading to nodular deposits of light chains in the form of amyloid light chain (AL-amyloid). The diagnosis is made by the histological demonstration of intracerebral masses harboring the classical staining and birefringence features of amyloid. We aim to report a case of ICA and review histological features of previous cases. A 51-year-old man with epilepsy and cognitive decline was admitted for epileptic seizures. A brain magnetic resonance imaging (MRI) disclosed periventricular enhancing lesions, hypointense on T1 and heterogeneous on T2-weighted images. A brain stereotactic biopsy was performed. The neuropathological examination revealed several congophilic nodules, allowing the diagnosis of ICA. The immunohistochemical study was positive for transthyretin (TTR), and both lambda and kappa immunoglobulin light chains. No inflammatory infiltrates were seen. Although a plasma cell clone may play a major role in the etiopathogeny of ICA, plasma cells were scarce or even absent when reviewing histological reports. ICA has a poorly understood patgogenesis. ICA may simulate malignant neoplasms, hence the need for a definite histological diagnosis.

Surgical control of limbic encephalitis associated with LGI1 antibodies.

Limbic encephalitis with LGI1 antibodies may cause drugresistant temporal lobe epilepsy. We report a case of a young man with progressive drug-resistant focal epilepsy, hyperhidrosis, and memory impairment associated with a left mesial temporal lesion. Epilepsy surgery was performed with the provisional diagnosis of cortical dysplasia or tumour. A neuropathological study following amygdalohippocampectomy revealed limbic encephalitis and LGI1 antibodies were identified in the serum. Two and a half years after surgery, the patient remains seizurefree without medication, with normal memory and without hyperhidrosis. Although immunosuppression is the first-line therapy for autoimmune limbic encephalitis, this case suggests that, in selected cases, a lasting response can be achieved with surgery.

Long-term and late seizure outcome after surgery for temporal lobe epilepsy

AimAlthough surgery for temporal lobe epilepsy (TLE) harbours a good prognosis, post-operative seizures may occur. Long-term, postoperative seizure follow-ups are rare but necessary to properly define outcome.MethodsLongitudinal, long-term, post-operative seizure follow-up in TLE patients with outcome analysed using Engel’s classification. Three groups were considered according to the type of resection: isolated amygdalohippocampectomy (IAH), further divided into anterior and complete, AH plus focal neocortical resections (AH + FR) and focal neocortical resections (FR).ResultsEighty-nine patients were enrolled (61 in the IAH group, 24 in the AH + FR group, and four in the FR group), with a mean follow-up time of 46.7 months. For the three groups together, 90.9% and 86.7% of the patients were in Engel class I for six months and five years, respectively. Kaplan-Meir analysis of the IAH and AH + FR groups showed that, while 82.2% of patients of the IAH group tended to remain in class I within 84 months after surgery, 86.7% of the AH + FR group tended to remain in class I within 12 months. Kaplan-Meier analysis of the IAH sub-groups showed that more patients (91.0%) with anterior resection tended to remain in class I, although for a longer period of time (36 months), compared to those with complete resection (84.0% of patients and 12 months, respectively). For the IFR group, only three patients were in Engel class I for long-term follow-up.ConclusionsHigh rates of seizure freedom were obtained and stably maintained for years. The reasons for better long-term prognosis of the anterior IAH group are so far unclear, the IFR group was too small to draw any conclusive data.