Increased Metallothionein I/II Expression in Patients with Temporal Lobe Epilepsy

In the central nervous system, zinc is released along with glutamate during neurotransmission and, in excess, can promote neuronal death. Experimental studies have shown that metallothioneins I/II (MT-I/II), which chelate free zinc, can affect seizures and reduce neuronal death after status epilepticus. Our aim was to evaluate the expression of MT-I/II in the hippocampus of patients with temporal lobe epilepsy (TLE). Hippocampi from patients with pharmacoresistant mesial temporal lobe epilepsy (MTLE) and patients with TLE associated with tumor or dysplasia (TLE-TD) were evaluated for expression of MT-I/II, for the vesicular zinc levels, and for neuronal, astroglial, and microglial populations. Compared to control cases, MTLE group displayed widespread increase in MT-I/II expression, astrogliosis, microgliosis and reduced neuronal population. In TLE-TD, the same changes were observed, except that were mainly confined to fascia dentata. Increased vesicular zinc was observed only in the inner molecular layer of MTLE patients, when compared to control cases. Correlation and linear regression analyses indicated an association between increased MT-I/II and increased astrogliosis in TLE. MT-I/II levels did not correlate with any clinical variables, but MTLE patients with secondary generalized seizures (SGS) had less MT-I/II than MTLE patients without SGS. In conclusion, MT-I/II expression was increased in hippocampi from TLE patients and our data suggest that it is associated with astrogliosis and may be associated with different seizure spread patterns.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [2005/56447-7, 2009/53447-7, 2008/52657-5]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES

Níveis diferentes de MT-I/II entre pacientes com MTLE com ou sem crise generalizada: os níveis hipocampais de MT-I/II afetam o alastramento das crises, ou o alastramento das crises promove expressão diferencial de MT-I/II?

In the central nervous system, zinc is released along with glutamate during neurotransmission and, in excess, can promote neuronal death. Experimental studies have shown that metallothioneins I/II (MT-I/II), which chelate free zinc, can affect seizures and reduce neuronal death after status epilepticus. Our aim was to evaluate the expression of MT-I/II in the hippocampus of patients with temporal lobe epilepsy (TLE). Hippocampi from patients with pharmacoresistant mesial temporal lobe epilepsy (MTLE) were evaluated for expression of MT-I/II and for neuronal, astroglial, and microglial populations. Compared to control cases, MTLE group displayed widespread increase in MT-I/II expression, astrogliosis and reduced neuronal population. MT-I/II levels did not correlate with any clinical variables, but patients with secondary generalized seizures (SGS) had less MT-I/II than patients without SGS. In conclusion, MT-I/II expression was increased in hippocampi from MTLE patients and our data suggest that it may be associated with different seizure spread patterns

Manual Hippocampal Subfield Segmentation Using High-Field MRI: Impact of Different Subfields in Hippocampal Volume Loss of Temporal Lobe Epilepsy Patients

In patients with temporal lobe epilepsy (TLE), presurgical magnetic resonance imaging (MRI) often reveals hippocampal atrophy, while neuropathological assessment indicates the different types of hippocampal sclerosis (HS). Different HS types are not discriminated in MRI so far. We aimed to define the volume of each hippocampal subfield on MRI manually and to compare automatic and manual segmentations for the discrimination of HS types. The T2-weighted images from 14 formalin-fixed age-matched control hippocampi were obtained with 4.7T MRI to evaluate the volume of each subfield at the anatomical level of the hippocampal head, body, and tail. Formalin-fixed coronal sections at the level of the body of 14 control cases, as well as tissue samples from 24 TLE patients, were imaged with a similar high-resolution sequence at 3T. Presurgical three-dimensional (3D) T1-weighted images from TLE went through a FreeSurfer 6.0 hippocampal subfield automatic assessment. The manual delineation with the 4.7T MRI was identified using Luxol Fast Blue stained 10-μm-thin microscopy slides, collected at every millimeter. An additional section at the level of the body from controls and TLE cases was submitted to NeuN immunohistochemistry for neuronal density estimation. All TLE cases were classified according to the International League Against Epilepsy's (ILAE's) HS classification. Manual volumetry in controls revealed that the dentate gyrus (DG)+CA4 region, CA1, and subiculum accounted for almost 90% of the hippocampal volume. The manual 3T volumetry showed that all TLE patients with type 1 HS (TLE-HS1) had lower volumes for DG+CA4, CA2, and CA1, whereas those TLE patients with HS type 2 (TLE-HS2) had lower volumes only in CA1 (p ≤ 0.038). Neuronal cell densities always decreased in CA4, CA3, CA2, and CA1 of TLE-HS1 but only in CA1 of TLE-HS2 (p ≤ 0.003). In addition, TLE-HS2 had a higher volume (p = 0.016) and higher neuronal density (p < 0.001) than the TLE-HS1 in DG + CA4. Automatic segmentation failed to match the manual or histological findings and was unable to differentiate TLE-HS1 from TLE-HS2. Total hippocampal volume correlated with DG+CA4 and CA1 volumes and neuronal density. For the first time, we also identified subfield-specific pathology patterns in the manual evaluation of volumetric MRI scans, showing the importance of manual segmentation to assess subfield-specific pathology patterns

Correction: Increased Metallothionein I/II Expression in Patients with Temporal Lobe Epilepsy.

[This corrects the article DOI: 10.1371/journal.pone.0044709.]

HLA-DR immunopositive area in hippocampal subfields of Ctrl, MTLE and TLE-TD groups.

<p>Compared to Ctrl (white boxplots), TLE groups had increased HLA-DR immunoreactivity (showed as percentage of immunopositive area) in outer molecular layer (OML), granule cell layer (GCL), CA4, and CA1 subfields (p<0.001). MTLE (dark gray boxplots) had increased HLA-DR immunoreactivity in inner molecular layer (IML), subgranule zone (SGZ), hilus, CA3, CA2, prosubiculum (PRO) and subiculum (SUB) (p<0.01). In IML, MTLE also presented increased HLA-DR immunoreactivity when compared to TLE-TD (p<0.001). The * indicate difference from Ctrl and ^# difference from TLE-TD.</p

Subfields in the hippocampal formation under NeuN immunohistochemistry.

<p>In A can be seen: the granule cell layer of <i>fascia dentata</i> (GCL, composed by granular neurons) and the hilus (HIL, composed by several types of interneurons); pyramidal neuronal layers of the hippocampus (CA4-CA1); the subicular formation, composed by prosubiculum (PRO) and subiculum (SUB). In B, a higher magnification of the fascia dentate (marked as a black square in A), composed by subgranule zone (SGZ), granule cell layer (GCL), inner molecular layer (IML) and outer molecular layer (OML). Bar in A indicates 1 millimeter and in B indicates 50 micrometers.</p

MT-I/II immunopositive area in hippocampal subfields of Ctrl, MTLE and TLE-TD groups.

<p>Compared to Ctrl (white boxplots), TLE groups had higher MT-I/II immunopositive area (showed as percentage of immunopositive area) in outer molecular layer (OML), inner molecular layer (IML) and subgranule zone (SGZ) (p<0.01). MTLE (dark gray boxplots) had increased MT-I/II immunoreactivity in granule cell layer (GCL), hilus, CA4, CA3, CA2, CA1, prosubiculum (PRO) and subiculum (SUB) (p<0.05), compared to Ctrl, and also in CA1 when compared to TLE-TD (p<0.001). The * indicate difference from Ctrl and ^#difference from TLE-TD.</p

Representative images of MT-I/II staining in several hippocampal subfields.

<p>Almost all stained cells have astrocyte morphology (indicated by small arrows in A–F), while neurons remained unstained (white cells pointed by large arrows in B). Only in few cases from Ctrl (E and F) and in one region of one case of TLE were observed cells with neuron morphology (large arrows in E and F). No stained neuron presented the strong staining of astrocytes. In Ctrl, neuropil presented a weak staining (indicated by black circle in F). In TLE the neuropil staining level was heterogeneous, as can be seen in CA1 sections depicted in C and D (indicated by white circles). The representative images shown are from the <i>fascia dentate</i> (A), <i>subiculum</i> (B and E), CA1 (C and D) and <i>hilus</i> (F) of Ctrl (E and F) and TLE cases (A–D). Bar in F indicates 100 micrometers.</p