Quantification of Neuronal Dendritic Spine Density and Lengths of Apical and Basal Dendrites in Temporal Lobe Structures Using Golgi-Cox Staining.

The objective of this chapter is to provide an overview of the methods used to investigate the connectivity and structure of the nervous system. These methods allow neuronal cells to be categorized according to their location, shape, and connections to other cells. The Golgi-Cox staining gives a thorough picture of all significant neuronal structures found in the brain that may be distinguished from one another. The most significant characteristic is its three-dimensional integrity since all neuronal structures may be followed continuously from one part to the next. Successions of sections of the brain's neurons are seen with the Golgi stain. The Golgi method is used to serially segment chosen brain parts, and the resulting neurons are produced from those sections.

Quantification of Neuroinflammatory Markers in Blood, Cerebrospinal Fluid, and Resected Brain Samples Obtained from Patients.

Cytokines have the potential to be the ideal biomarkers to track the onset and progression of immune-mediated diseases, study the development of novel therapeutic strategies, and they can serve as outcome parameters due to their crucial role in the regulation of immune and inflammatory responses. It is vital to keep track of the entire cytokine spectrum due to the complex interactions, pleiotropic effects, and redundancy in the cytokine network. The multiplex immunoassay (MIA) is, therefore, the best method for achieving that goal. This chapter addresses the key methodological processes of this technique, such as sample preparation, antibody coupling to beads, and assay procedure.

Epigenetics of neurological diseases.

Higher-order DNA structure and gene expression are governed by epigenetic processes like DNA methylation and histone modifications. Abnormal epigenetic mechanisms are known to contribute to the emergence of numerous diseases, including cancer. Historically, the chromatin abnormalities were only considered to be limited to discrete DNA sequences and were thought to be associated with rare genetic syndrome however, recent discoveries have pointed to genome-wide level changes in the epigenetic machinery which has contributed to a better knowledge of the mechanisms underlying developmental and degenerative neuronal problems associated with diseases such as Parkinson's disease, Huntington's disease, Epilepsy, Multiple sclerosis, etc. In the given chapter we describe the epigenetic alterations seen in various neurological disorders and further discuss the influence of these epigenetic changes on developing novel therapies.

Correlation of age at seizure onset with GABAA receptor subunit and chloride Co-transporter configuration in Focal cortical dysplasia (FCD).

Focal cortical dysplasia (FCD) represents a group of malformations of cortical development, which are speculated to be related to early developmental defects in the cerebral cortex. According to dysmature cerebral development hypothesis of FCD altered GABAA receptor function is known to contribute to abnormal neuronal network. Here, we studied the possible association between age at seizure onset in FCD with the subunit configuration of GABAA receptors in resected brain specimens obtained from patients with FCD. We observed a significantly higher ratio of α4/α1 subunit-containing GABAA receptors in patients with early onset (EO) FCD as compared to those with late onset (LO) FCD as is seen during the course of development where α4-containing GABAA receptors expression is high as compared to α1-containing GABAA receptors expression. Likewise, the influx to efflux chloride co-transporter expression of NKCC1/KCC2 was also increased in patients with EO FCD as seen during brain development. In addition, we observed that the ratio of GABA/Glutamate neurotransmitters was lower in patients with EO FCD as compared to that in patients with LO FCD. Our findings suggest altered configuration of GABAA receptors in FCD which could be contributing to aberrant depolarizing GABAergic activity. In particular, we observed a correlation of age at seizure onset in FCD with subunit configuration of GABAA receptors, levels of NKCC1/KCC2 and the ratio of GABA/Glutamate neurotransmitters such that the patients with EO FCD exhibited a more critically modulated GABAergic network.

Differential regulation of excitatory synaptic transmission in the hippocampus and anterior temporal lobe by cyclin dependent kinase 5 (Cdk5) in mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS).

Mesial temporal lobe epilepsy with hippocamapal sclerosis (MTLE-HS) is the most common form of drug resistant epilepsy (DRE). MTLE-HS is a distributed network disorder comprising of not only the hippocampus, but other anatomically related extrahippocampal regions. Excitatory synaptic transmission is differentially regulated in the hippocampal and extra-hippocampal regions of patients with MTLE-HS, but its mechanism not understood. Cyclin-dependent kinase 5 (Cdk5) is known to regulate synaptic transmission and plasticity through up-regulation of NMDA receptors by phosphorylating NR2Asubunits. The present study is designed to investigate whether Cdk5 differentially regulates the excitatory synaptic transmission in the hippocampus and anterior temporal lobe (ATL) samples obtained from patients of MTLE-HS. We have measured the Cdk5 kinase activity and the protein levels of Cdk5, p-Cdk5, p35/p25, NR2A, pNR2A in the hippocampal and ATL samples obtained from patients with MTLE-HS. We have also determined the effect of roscovitine, a Cdk5 antagonist, on spontaneous excitatory postsynaptic currents (EPSCs) recorded from the hippocampal and ATL using patch-clamp technique. We observed significant increase in the expression of Cdk5, p-Cdk5, p35/p25, NR2A, pNR2A in the ATL samples as compared to the hippocampal samples. Cdk5 activity was significantly higher in ATL samples as compared to the hippocampal samples. Magnitude of reduction in the frequency of EPSCs by roscovitine in the ATL samples was higher than that in the hippocampal samples. Our studies suggest that Cdk5 differentially regulates excitatory synaptic activity in the hippocampal and ATL region of patients with MTLE-HS.

Basilar Invagination and Atlantoaxial Dislocation: Reduction, Deformity Correction and Realignment Using the DCER (Distraction, Compression, Extension, and Reduction) Technique With Customized Instrumentation and Implants.

OBJECTIVE: The technique of distraction, compression, extension, and reduction (DCER) is effective to reduce, realign, and relieve cranio-spinal compression through posterior only approach. METHODS: Study included all patients with atlantoaxial dislocation and basilar invagination (BI) with occipitalized C1 arch. Study techniques included Nurick grading, computed tomography scan to study atlanto-dental interval, BI, hyper-lordosis, and neck tilt. Sagittal inclination (SI), coronal inclination (CI), cranio-cervical tilt, presence of pseudo-joints, and anomalous vertebral artery were also noted. Patients underwent DCER with/without joint remodeling or extra-articular distraction (EAD) based on the SI being <100°, 100°-160°, or >160° respectively. In cases with pseudo-joints, joint remodeling was performed in type I and EAD in type II. Customized 'bullet shaped' PSC spacers (n=124) and prototype of the universal craniovertebral junction reducer (UCVJR, n=36) were useful. RESULTS: A total of 148 patients with average age 27.25±17.43 years, ranging from 3 to 71 years (87 males) were operated. Nurick's grading improved from 3.14±1.872 to 1.22±1.17 (p<0.0001). Fifty-two percent of total joints (n=154/296 joints) were either type I (19%)/type II (33%) pseudo-j oints. All traditional indices such as Chamberlein line, McRae line, atlanto-dental interval, and Ranawat line improved (p

Upregulation of breast cancer resistance protein and major vault protein in drug resistant epilepsy.

PURPOSE: Identifying factors involved in the development of drug resistant epilepsy (DRE) remains a challenge. Candidate gene studies have shown modulation of resistance to drugs by various multidrug resistance proteins in DRE. However the resistance to drugs in DRE could be more complex and multifactorial involving molecules in different pharmacokinetic processes. In this study for the first time we have analyzed the relative expression of four molecules with different drug resistance mechanisms in two most common DRE pathologies, mesial temporal lobe epilepsy (MTLE) and focal cortical dysplasia (FCD) with respect to each other and also with different non-epileptic controls. METHODS: Brain tissues resected from MTLE (n=16) and FCD type I and II (n=12) patients who had undergone surgery were analysed for mRNA levels of multidrug resistance-associated protein 1(MRP1), major vault protein (MVP), breast cancer resistance protein (BCRP), and one drug metabolising enzyme (UGT1A4) as compared to non-epileptic controls which were tissues resected from tumor periphery (n=6) and autopsy tissues (n=4) by quantitative PCR. RESULTS: We found significant upregulation of MVP and BCRP whereas MRP1 and UGT1A4 were unaltered in both pathologies. While upregulation of BCRP was significantly higher in MTLE (9.34±0.45; p<0.05), upregulation of MVP was significantly higher in FCD (2.94±0.65; p<0.01). CONCLUSION: We propose that upregulation of BCRP and MVP is associated with MTLE and FCD and these molecules not only may have the potential to predict pathology specific phenotypes but may also have therapeutic potential as adjunct treatment in these pathologies.

Role of inflammation and its miRNA based regulation in epilepsy: Implications for therapy.

There is a need to develop innovative therapeutic strategies to counteract epilepsy, a common disabling neurological disorder. Despite the recent advent of additional antiepileptic drugs and respective surgery, the treatment of epilepsy remains a major challenge. The available therapies are largely based on symptoms, and these approaches do not affect the underlying disease processes and are also associated frequently with severe side effects. This is mainly because of the lack of well-defined targets in epilepsy. The discovery that inflammatory mediators significantly contribute to the onset and recurrence of seizures in experimental seizure models, as well as the presence of inflammatory molecules in human epileptogenic tissue, highlights the possibility of targeting specific inflammation related pathways to control seizures that are otherwise resistant to the available AEDs. Emerging studies suggest that miRNAs have a significant role in regulating inflammatory pathways shown to be involved in epilepsy. These miRNAs can possibly be used as novel therapeutic targets in the treatment of epilepsy as well as serve as diagnostic biomarkers of epileptogenesis. This review highlights the immunological features underlying the pathogenesis of epileptic seizures and the possible miRNA mediated approaches for drug resistant epilepsies that modulate the immune-mediated pathogenesis.

Initial experience of using high field strength intraoperative MRI for neurosurgical procedures.

We report our initial experience to optimize neurosurgical procedures using high field strength intraoperative magnetic resonance imaging (IOMRI) in 300 consecutive patients as high field strength IOMRI rapidly becomes the standard of care for neurosurgical procedures. Three sequential groups (groups A, B, C; n=100 each) were compared with respect to time management, complications and technical difficulties to assess improvement in these parameters with experience. We observed a reduction in the number of technical difficulties (p<0.001), time to induction (p<0.001) and total anesthesia time (p=0.007) in sequential groups. IOMRI was performed for neuronavigation guidance (n=252) and intraoperative validation of extent of resection (EOR; n=67). Performing IOMRI increased the EOR over and beyond the primary surgical attempt in 20.5% (29/141) and 18% (11/61) of patients undergoing glioma and pituitary surgery, respectively. Overall, EOR improved in 59.7% of patients undergoing IOMRI (40/67). Intraoperative tractography and real time navigation using re-uploaded IOMRI images (accounting for brain shift) helps in intraoperative planning to reduce complications. IOMRI is an asset to neurosurgeons, helping to augment the EOR, especially in glioma and pituitary surgery, with no significant increase in morbidity to the patient.

Ten years' experience in the management of spinal intramedullary tumors in a single institution.

We retrospectively reviewed the outcomes of 195 patients with intramedullary tumors who underwent surgery between January 2001 and December 2010 at a single institution. The symptomatology, neurological and neuroradiological findings, operative details, perioperative and postoperative complications, histopathological data and follow-up examinations of the 137 (70.2%) males and 58 (29.7%) females were studied and analyzed. Epidermoid was the most common intramedullary tumour in children (23%), whereas in adults, ependymomas were more common (46%). Ependymomas were more amenable to resection (total excision in 57.7% and near-total excision in 39.4%) as compared to astrocytomas (total excision in 29%; near total excision in 60.5%). At the final clinical follow-up, 24 patients (16.4%) had improved in McCormick grade, 112 patients (76.7%) remained unchanged and 11 patients (7.5%) had worsened. Complete removal of the lesion is the primary goal of surgery. We conclude that the strongest predictor of functional outcome was the preoperative neurological condition, beyond the histological differentiation of the intramedullary tumor.

Outcome following surgery for ophthalmic segment aneurysms.

Ophthalmic segment aneurysms account for about 5% of all intracranial aneurysms. Anatomical complexity of the paraclinoid region makes surgical management of aneurysms arising from the ophthalmic segment challenging. This study was carried out to assess the presenting features, complications and outcomes after surgical treatment of ophthalmic segment aneurysms. The authors retrospectively analysed the clinical records of patients with ophthalmic aneurysms treated at our Institute from January 2001 to September 2008, which constituted about 9% (78/850) of all intracranial aneurysms. Of the 78 ophthalmic segment aneurysms, six patients (8%) had giant aneurysms and 19 (24%) patients had multiple aneurysms. Fifty-six patients underwent microsurgery, with direct clipping in most. The mean age was 42 years (range 12-75 years) and the mean follow-up was 8 months (range, 2-93 months). A good outcome was achieved in 46 (83%) patients (Glasgow Outcome Scale [GOS] score 4-5) and 17% had a poor outcome (GOS score 1-3) at last follow-up. The overall complication rate was 21% (12/56), most of which were transient complications, with 3.5% (2/56) mortality. Direct microsurgical clipping remains our preferred treatment approach, whenever possible, for ophthalmic segment aneurysms. This surgery has an acceptable complication rate and leads to a good outcome in more than 80% of patients with ophthalmic aneurysms. Use of modern microsurgical instrumentation and endovascular adjuncts can further reduce the surgical morbidity associated with these vascular lesions.

Expression of CD34 as a novel marker for glioneuronal lesions associated with chronic intractable epilepsy.

The spectrum of glioneuronal lesions underlying intractable epilepsies includes malformative pathologies like focal cortical dysplasia (FCD); and neoplastic lesions like gangliogliomas (GG) and dysembryoplastic neuroepithelial tumours (DNT). These may occur either singly or as dual lesions, having simultaneous presence of both elements. Currently, the relationship between the malformative and neoplastic glioneuronal lesions is poorly understood. Recently, CD34, a stem cell marker transiently expressed during early neurulation, has been identified in these tumours. This study was undertaken to (i) evaluate the role of CD34 as a diagnostic marker for glioneuronal lesions of epilepsy, namely, GG, DNT and FCD, and (ii) attempt to define the relationship among these lesions, using CD34 as a marker. Tissues resected from 47 patients with intractable epilepsy due to glioneuronal lesions (GG, FCD, DNT) were studied. These were evaluated for CD34 expression, using immunohistochemistry. Dysplastic or atypically differentiated neural precursors which could not be identified on routine haematoxylin and eosin (H&E) staining were highlighted by CD34 immunostaining. The pattern of immunostaining was diffuse in GGs, unlike FCDs, wherein cells were present singly or in small clusters. However, cases of DNT and control tissue were largely CD34-immunonegative. Based on these findings, we propose a possible common origin of GG and FCD, from a bipotent precursor that undergoes abnormal glioneuronal development, while DNTs possibly have a different origin. The CD34-immunoreactive cells represent dysplastic or undifferentiated neural precursors, which may signify a valuable marker for the diagnostic evaluation of neoplastic and/or malformative pathologies in patients with intractable epilepsy.

Primary lumbosacral Wilms tumor associated with diastematomyelia and occult spinal dysraphism. A report of a rare case and a short review of literature.

BACKGROUND: The occurrence of an extrarenal Wilms tumor in the lumbosacral region is an extremely uncommon condition. CASE REPORT: We report a case of Wilms tumor in the lumbosacral region that was associated with diastematomyelia and occult spina bifida. An 18-month-old girl presented with a swelling over the lower back with a tuft of hair on it, which she had had since birth. Imaging of the spine revealed spina bifida, bony diastematomyelia, and tethered cord. Excision of the bony spur and detethering of the cord was done. After a year, she had recurrence of swelling at the same site, weakness of both lower limbs, and incontinence of bladder and bowel. Excision of the mass and bony spur and detethering of the spinal cord were done. Histopathological examination showed features of a Wilms tumor.