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11MEDICAL SCIENCES
MORFOLOGICAL CHANGES IN EPILEPTIC FOCUS OF BRAIN TISSUE FROM PATIENTS WITH
DRUG-RESISTENT EPILEPSY
Rjabceva S.N.,
PhD, Institute of Physiology of the NAS of Belarus, Minsk
Siamionik I.A,,
PhD, Institute of Physiology of the NAS of Belarus, Minsk
Derevianko M.A.,,
PhD, Institute of Physiology of the NAS of Belarus, Minsk
Terechov V.S.,,
PhD, Republican Scientific and Practical Center for Neurology and Neurosurgery, Minsk
Yermachenka V.A.,
PhD, City Clinical Pathological Bureau, Minsk
Abstract:
The article presents structural and functional cell changes in epileptic focus of brain tissue from adult patients with drug-resistant epilepsy. A focal cortical dysplasia different types was found in most cases. A focal cortical dysplasia was combined with hippocampal sclerosis in 14.3%, with arteriovenous malformation - in 14.3% of our cases. Epileptogenic focus of patient's brain with drug-resistant epilepsy was characterized by "mosaic" glial changes with foci of "active" and "non-active" gliosis and focal or diffuse neuronal damages. An "active" gliosis was detected as a local focus of astroglial and microglial activation and proliferation. A change in cellular density of astrocytes was associated with an increase in microglial proliferation.
Keywords: drug-resistant epilepsy, histological characteristic, epileptogenic focus, active and non-active gliosis.
Introduction. Epilepsy is a disease that characterized by recurrent spontaneous partial or general seizures [1,p.174]. Current theories of epileptogenesis include neuronal dysfunction and damage as a reason of seizures development. However, in epileptic focus was also found astroglial and microglial activation and proliferation. Most antiepileptic drugs target neuronal mechanisms. But about one-third of patients with epilepsy have resistant to pharmacotherapy [4,p.1]. Focal cortical dysplasia (FCD) is frequently associated with drug-resistant epilepsy. FCD is specific malformation of the cortical development that characterized by dys-lamination of cortex and abnormal neuronal cell morphology [2,p.288-294;3,p.1-2]. The aim of this study was to analyze neuronal and glial cell histological changes in epileptic focus of brain tissue from patient with refractory epilepsy.
Materials and methods. We analyzed morphological changes of cortex from seven patients who underwent surgical resection of the epileptogenic region for treatment of drug-resistant epilepsy. Epileptogenic focus of each patient was identified by magnetic resonance imaging and was resected by surgeon. The brain tissues specimens were fixed in 10% 0.1 M phosphate-buffered formalin (12-14 hours) and paraffin embedded. Serial sections of cortical tissue with a thickness of 4 ^m were made using SLEE microtome and were analyzed using hematoxylin and eosin as routine stain and immunohistochemistry (IHC) assay with antibody: NeuN (FNab05669, dilution - 1:2000, FineTest, China) as a neuron marker, GFAP (glial fibrillary acidic protein, FNab03428, dilution - 1:4000, FineTtest, China) as a astrocyte marker and IBA-1 (ionized calcium binding adaptor molecule 1, MABN92, dilution - 1:800, Millipore, USA) for microglial cells identify. Immuno-histochemistry assay was performed according to IHC
protocols of source antibody recommendation. Antigen detection was carried out using the mouse/rabbit Un-oVueTMHRP/DAB Detection System (UMR1000PD, Diagnostic BioSystems, The Netherlands). Antigen demasking was performed in a water bath at temperature of 960C during 20 minutes. Cortical specimens were incubated one hour with primary antibody and one hour with detection system at temperature 370C. A micrograph of brain tissue was done by using an Optec BK5000 microscope (Optec, China). Count glial cell was done in 8-10 non-overlapping fields at high power magnification (x400, HPF). The area of one field was 66585.8 ^m2. Glial cell number was counted using the morphometric program ImageJ (USA) and its application "Multi-point Tool", then assess of cell density (units / mm2) in each case. Statistical morphometric data are presented as median (Me) and percentiles (25%-75%).
Results. We studied the cortical tissue from seven patients (4 females and 3 males). Patient age ranged from 21 to 37 years; the mean age was 27.8±1.2 years. Cortical specimens were resected from temporal lobe (4/66.7%) and frontotemporal part (3/33.3%) of the brain cortex. The site of resected tissue ranged from 0.5 to 1.8 cm; the mean tissue site was 0.86±0.6 cm.
Histologically, FCD type Ib with laminar (tangential) dyslamination and the presence of hypertrophic neurons in second and sixth cortex layers was revealed in temporal lobe cortex specimens from three (42.9%) patients. Hypertrophic neurons expressed NeuN and characterized by large cell body with homogenous cytoplasm (without Nissl substance) and centrally positioned hypochromic nucleus. Focal cortical dysplasia type IIb with columnar and laminar (radial and tangential) dyslamination and balloon and dysmorphic neurons were found in cortex specimens from three
(42.9%) patients. Balloon neurons (BNs) were present in all cortex cell layers with increased density of this neurons type in white matter and gray-white matter transition. Balloon neurons were identified as large cells with homogenous eosinophilic cytoplasm (without Nissl substance) and eccentric hypochromic nuclei with nucleoli. BNs expressed NeuN (weak) and GFAP (strong). Dysmorphic neurons (DNs) were large cells with a Nissl substance clumped in cytoplasm (baso-philic masses displaced towards the cell membrane) and with eccentric nuclei. DNs expressed NeuN. Hip-pocampal sclerosis without FCD was detected in cortical specimens from one (14.2%) patient. Among them, in brain tissue from one patient, FCD type Ib was combined with hippocampal sclerosis (FCD type IIIa), and from another - with arteriovenous malformation of the brain (FCD type IIIc) [2,p.292].
Glial sclerosis was detected in all cortical specimens. Foci of "active" and "non-active" gliosis were found in analyzed brain tissue of all patients. The "active" gliosis was identified as a foci of increased astrocytes density compare with all around brain tissue. Astrocytes in foci of "active" gliosis have large cell body, large number and longer dendrites, and characterized by strong GFAP-expression. In "active" gliosis foci, the median of astrocytes count in HPF was 11 (10;13) cells, and the cell density was 163.85 (148.96;193.64) cells/mm2. In part of "non-active" gliosis, the median of astrocytes count in HPF was 5 (3;6), and the cell density was 74.48 (44.69;89.37) cells/mm2.
Foci of microglial activation and proliferation were also found in all cortical brain tissue. In foci of microglia activation, the median of cell count in HPF was 9 (6;13), the cell density was 134.06 (89.37;193.64) cells/mm2. In other areas, the median of microglial cells count in HPF was 6 (4;9), and cell density - 89.37 (59.58;134.06) cells/mm2. Three type of microglial cells were detected in foci of activation: ameboid, ramified and activated microglia. Ameboid type was found rare and characterized by a small round cell with short branches. Ramified microglia have a small cellular body and numerous branching processes
and possess little cytoplasm. Activated microglia have large cell body with thickened and retraction of branches.
Middle relationship between increased astrocytes density and increase microglial cell density was detected using Spearman's rank correlation (rs=0,54). The relationship between the cell density in activated and non-activated glial foci of cortex was determined using a non-parametric Mann-Withey test. Statistical processing of morphometric data revealed significant differences in astrocytes density of (p = 0.000) and microglia cells density (p = 0.0038) in activated glial foci compare with non-activated glial foci of brain tissue.
Conclusion. This study revealed structural and functional changes in cortex brain tissue of adult patients with drug-resistant epilepsy. We found that patients with drug-resistant epilepsy have combined cellular changes in the epileptogenic focus of the cortex, that are characterized by dyslamination of cerebral cortex and neuronal and glial cell changes. Neurons transformed to balloon, hypertrophic and dystrophic cells. Glial cell changes were characterized as foci of increased glial (astrocytes and microglia) cell density with activation of microglial cells and astrocytes.
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