ESTUDO DAS RELAÇÕES ENTRE POPULAÇÕES CELULARES, EXPRESSÃO DE AQUAPORINA-4 E SULFATO DE CONDROITINA COM O TEMPO DE RELAXAMENTO E A TAXA DE TRANSFERÊNCIA DE MAGNETIZAÇÃO NO HIPOCAMPO DE PACIENTES COM EPILEPSIA DO LOBO TEMPORAL FARMACORRESISTENTE

Rationale: Drug resistant temporal lobe epilepsy is often associated with hippocampal sclerosis. Histological evaluation reveals differential neuronal loss, gliosis and changes in molecules associated with water homeostasis, such as aquaporin 4 and chondroitin sulfate. Magnetic resonance imaging in these cases often

Metallothioneins (MTs) are low molecular weight, cysteinenriched proteins that bound Zn 2+ and cadmium. They can be found in various tissues, in four isoforms [30]. Isoforms I, II and III are found in the central nervous system (CNS), where the isoforms I and II are expressed in astrocytes and the isoform III is expressed only in neurons [31], [32]. MTs participate in Zn 2+ homeostasis, scavenging ROS in the brain [33] and stimulate the expression of several neurotrophic and antiinflamatory factors [34]. Studies on rodent models of TLE have shown that MT expression is increased in the hippocampal formation shortly after seizures [35], [36] and that high levels of MTs I and II are associated with reduced neuronal death after seizure-induced damage [37], [36], [38]. However, some studies with neuronal MT (MT-III) indicate that MTs could also contribute to neuronal death in some circumstances [39], [29].
Since MT-I/II levels may be associated with neuron survival after seizures, we hypothesize that MT-I/II expression is altered in TLE and can be associated with the preservation of neuronal density in the hippocampus of TLE patients. Therefore, in this study we evaluated the immunoexpression of MT-I/II and its correlation with hippocampal neuron density in hippocampi of patients with chronic TLE.

Patients and clinical data
Patients with drug-resistant epilepsy were evaluated at the University of São Paulo Epilepsy Surgical Centre in Ribeirão Preto (Brazil), according to standard protocols published elsewhere [40]. The presurgical evaluation protocol included interviews for epilepsy history, neurological examination, EEG recording, video-EEG assessment, T1-and T2-weighted MRI, ictal and interictal single-photon emission computed tomography (SPECT) scans and neuropsychological tests. Drug resistance was defined according to previous published literature [41].
TLE patients were divided in two groups: (i) mesial TLE (MTLE) and (ii) TLE associated with extrahippocampal tumor or dysplasia (TLE-TD). MTLE group (n = 69) were patients with hippocampal atrophy or with normal hippocampal volume at MRI without other lesions associated with TLE. TLE-TD (n = 17) were TLE patients with tumor or cortical dysplasia in temporal lobe structures other than the hippocampus. From all TLE-TD patients, 4 had non-Taylor focal cortical dysplasia and the remaining had tumors. The tumors observed were grade I ganglioglioma (n = 3), grade I dysembryoplastic neuroepithelial tumor (n = 3), hamartoma (n = 3), teratoma (n = 2), grade III astrocytoma (n = 1) and angioma (n = 1).
For comparison purposes in the neuropathology studies, autopsy controls (Ctrl, n = 19) were obtained from autopsy cases without history of neurological diseases, with no sign of CNS pathologies in post mortem pathological evaluation and no history of hypoxic episodes during agony. Post mortem time (i.e., time between death and hippocampal fixation) was of 5.1561.43 hours, ranging from 3.16 to 9 hours. The causes of death were pulmonary insuficiency (n = 6), cardiomyopathy (n = 3), cardiogenic shock (n = 2), sepsis (n = 3), hepatic failure (n = 3), acute lymphoblastic leukemia (n = 1) and gastric adenocarcinoma (n = 1).
Medical records of all evaluated patients were assessed for clinical data analysis. The clinical variables investigated were age at death and cause of death for Ctrl patients and age at surgery, epilepsy duration, age at the first recurrent seizure, seizure frequency per month, presence of secondary generalized seizures, and neuropathological evaluation for TLE patients. This study followed the principles of the Declaration of Helsinki, was registered in Brazilian's Health Ministry and was approved by the Research Ethics Committee of the Hospital das Clínicas, where this study was performed (process HCRP 2634/2008). Written informed consent was obtained from all patients used in this study, and the Research Ethics Committee also approved the Consent Term. Tissue from autopsy cases came from a Brain Bank approved by the Research Ethics Committee of Hospital das Clínicas (process HCRP 9370/2003).

Tissue collection and histological techniques
Hippocampi from surgery or autopsy were cut in coronal sections and placed in 10% (vol/vol) buffered formaldehyde for one week, followed by paraffin embedding. Immunohistochemistry was performed in 8 mm sections at the level of hippocampal body for evaluation of neuronal, astroglial and activated microglial populations and for MT-I/II expression with antibodies against, respectively, NeuN, GFAP, HLA-DR and MT-I/II. The sections were submitted to endogenous peroxidase blocking with 4.5% H 2 O 2 in 50 mM phosphate-saline buffer (PSB) pH 7.4, for 15 minutes, followed by microwave antigenic retrieval in 10 mM sodium citrate buffer pH 6.0 (for GFAP) or 50 mM Tris-HCl pH 9.6 (for NeuN, HLA-DR and MT-I/II). After achieving room temperature, the sections went through blocking free aldehyde groups with Tris-glycine 0.1 M pH 7.4 for 45 minutes, followed by blocking buffer with 5% defatted milk and 15% goat serum (#S-1000, Vector) in Triton buffer (PTB, 20 mM phosphate +0.45 M NaCl, pH 7.4, with 0.3% Triton X-100) for four hours. The sections were then incubated with primary antibodies in blocking buffer for 16 hours. We used primary monoclonal antibodies raised in mouse anti-human GFAP (clone 6F2, #M0761, Dako), anti-murine NeuN (clone A60, #MAB377, Chemicon), anti-human HLA-DR (clone TAL.1B5, #M0746, Dako) and anti-equine MT-I/II (clone E9, #M0639, Dako), diluted in blocking buffer at concentrations of 1:500, 1:500, 1:100 and 1:500, respectively. The primary antibodies were detected using biotinylated rabbit anti-murine IgG (#E0354, Dako), at 1:200 dilution in blocking buffer, for one hour, followed by revelation with avidin-biotin-peroxidase system (Vectastain Elite ABC kit, #PK6100, Vector) and diaminobenzidine as chromogen (DAB, #34001, Pierce Biotechnology). The development times in DAB solution were 12 minutes for HLA-DR, 10.5 minutes for NeuN and 8 minutes for MT-I/II and GFAP. In order to assure that the different times of fixation of autopsy hippocampi and surgical tissue were comparable, an additional experiment was performed with temporal cortical tissue from one TLE patient. Briefly, a cortical sample was removed during surgery, sectioned in 5 fragments which were kept at room temperature for 1, 2, 4, 6 and 8 hours before immersion-fixation in 10% buffered formaldehyde. Sections of these cortical fragments with different prefixation times were mounted on slides and processed in the same manner as the surgical and autopsy hippocampi.
Vesicular Zn 2+ was evaluated in a subset of cases by neo-Timm histochemistry [19]. Briefly, a fresh hippocampal section was placed in buffered fixative solution (4% glutaraldehyde and 0.1% sodium sulfite) at 4uC for one week, followed by water removal with 20% buffered saccarose for one day. The fragment was dried and frozen in cryostat. Thirty mm sections were utilized for neo-Timm technique, according to previously published protocols [19], [42], [43].

Immunofluorescence
Colocalization of MT-I/II with neuronal and astroglial markers was performed with the same protocol described above. Endogenous peroxidase blocking and the revelation procedure were omitted. Primary antibodies were raised in mouse anti-equine for MT-I/II (clone E9, #M0639, Dako), in rabbit anti-cow for GFAP (#Z0334, Dako) and anti-human for MAP2 (#sc-20172, Santa Cruz Biotechnology). Sections were submitted to MT-I/II plus GFAP or MT-I/II plus MAP2 incubation, with antibodies diluted in blocking buffer at 1:100 for MT-I/II, 1:1000 for GFAP and 1:50 for MAP2, for 20 hours. The primary antibodies were detected using goat anti-mouse IgG conjugated with Alexa Fluor 488 (#A11001, Molecular Probes) and goat anti-rabbit IgG conjugated with Texas Red (#T2767, Molecular Probes), diluted in blocking buffer, at 1:300 each, for 2 hours. Following incubation, the sections were submitted to Hoechst 33342 staining (#H1399, Molecular Probes) for 4 minutes, and were mounted in Fluoromount-G (#17984-25, EMS). With this procedure, GFAP and MAP2 were observed in red, MT-I/II in green and cell nucleus in blue. All images were captured in Leica SP5 confocal microscope.

Histological analysis
Images of all hippocampal regions were obtained with a video monochrome charge-coupled device camera (CCD; Hamamatsu Photonics Model 2400, Japan) attached to an Olympus microscope (Model BX60, Melville, NY), and captured, averaged, and digitized using a frame grabber (Scion Corporation, Frederick, MD) on a Macintosh computer (Model G3, Cupertino, CA). Illumination exposure was uniformly maintained and regularly checked using optical density standards (Kodak, Rochester, NY) in order to prevent any distortion of measurements (immunopositive area, gray level) between the samples. After captured, the image was analyzed using image system software (ImageJ, version 1.37c).
Quantification of the immunohistochemistry was performed with threshold tool, with the investigator blind to the group allocation. After the selection of the region of interest (ROI), the software calculated the immunopositive area by counting all pixels with gray intensity equal or superior to the threshold of staining. A complete protocol for threshold tool can be found at rsbweb.nih.gov/ij/docs/examples/stained-sections/index.html. The threshold was defined for each protein evaluated, based on the mean immunopositivity of all control cases. The evaluated regions were the fascia dentata (outer molecular layer, inner molecular layer, granule cell layer, subgranular zone), the hilus and the stratus piramidale of CA4, CA3, CA2, CA1, prosubiculum and subiculum ( Figure 1). The characterization of hippocampal regions was based on the Lorente de Nó's classification [44]. Results were shown as percentage of immunopositive area/total area. Additionally, neuronal density was evaluated in the NeuN stained sections. Neuronal count was processed in ImageJ 1.37c software with a 5206 magnification for granule cell layer and 2606 for pyramidal neurons of CA4, CA3, CA2, CA1, prosubiculum and subiculum. Neuronal densities were estimated with the correction of Abercrombie [45], which permits to estimate the neuronal density through mathematical method, and the results were shown as thousands of cells per cubic millimeter.
Quantification of neo-Timm sections was done by measurement of mean gray value, which varied from 0 to 255, of the hippocampal regions in ImageJ software. The evaluated regions comprised outer molecular layer, inner molecular layer, granule cell layer, subgranule zone and hilus/CA4.

Statistical analysis
Statistics were carried out in SigmaStat 3.1 software for all tests except for simple regression models, which were performed with SPSS 20. Tests for normality and homogeneity of variances were performed to define data distribution. For parametric variables, One Way ANOVA with Bonferroni post hoc or t-test was performed. For the non-parametric variables, Kruskal-Wallis with Dunn post hoc or Mann-Whitney tests were used. Fisher's exact test was performed to evaluate categorical data. Correlation between MT expression and cellular populations was performed using the Spearman's test, when n#30, or Pearson's test, for n.30. Multiple linear regressions were used to define associations between age, neuronal and astroglial populations over MT-I/II expression. All results were considered significant at p,0.05.

Clinical data
The clinical characteristics of study participants are summarized in Table 1. The mean age at evaluation was significantly lower in TLE-TD group than Ctrl and MTLE groups (Kruskal-Wallis, p = 0.001). Epilepsy duration was lower in TLE-TD group than in MTLE group (Mann-Whitney, p = 0.002). Recurrent seizures onset (t-test, p = 0.651), minimal seizure frequency in a month (Mann-Whitney, p = 0.397) and frequency of secondary generalized seizures per month (Mann-Whitney, p = 0.557) were similar in MTLE and TLE-TD groups. Fisher's exact test showed that the prevalence of secondary generalized seizures was similar between MTLE and TLE-TD (p = 1.0).

Changes in immunoreactivity in different fixation times
Quantification of MT-I/II, NeuN, GFAP and HLA-DR immunostaining in sections of cortical fragment in different fixation times revealed that a delay on fixation time was not associated with a decrease of immunoreactivity for all antibodies evaluated ( Figure S1).

Neuronal density
NeuN immunopositive cells ( Figure 2) were counted to estimate the neuronal density in the hippocampal subfields. The quantification studies (Figure 3) revealed reduced neuronal density in granule cell layer (Kruskal-Wallis, p,0.001), CA4 (Kruskal-Wallis, p,0.001), CA1 (Kruskal-Wallis, p,0.001) and prosubiculum (ANOVA, p,0.001) of the MTLE group, when compared to Ctrl and TLE-TD groups. In CA2 subfield, the neuronal densities of MTLE and TLE-TD groups were reduced when compared to Ctrl (ANOVA, p,0.001). In CA3, MTLE and TLE-TD had reduced neuronal density when compare to each other and to the Ctrl group (ANOVA, p,0.001). No differences in neuronal density were found in the subiculum (ANOVA, p = 0.08). All hippocampal regions of MTLE group showed reduced NeuN immunopositive area when compared with Ctrl, in agreement with neuron density measurements (Data not shown).

Reactive astroglial population
GFAP immunopositive area, shown in Figures 2 and 5, indicated increased GFAP immnunoreactivity labeling in the outer and inner molecular layers, granule cell layer, subgranule zone, hilus and CA4 of MTLE and TLE-TD, when compared to Ctrl (ANOVA for granule cell layer and Kruskal-Wallis for the remaining regions, p,0.001). In CA2, Sommer sector (CA1 and prosubiculum) and the subiculum, there was increased GFAP immnunoreactivity labeling of the MTLE group, when compared to Ctrl and TLE-TD (Kruskal-Wallis, p,0.001). Increased reactive astrogliosis was also observed in CA3 of MTLE (Kruskal-Wallis, p,0.001), when compared to Ctrl.

Metallothionein I/II immunoreactivity
MT-I/II staining revealed both cellular and neuropil staining ( Figure 7A-F). MT-I/II-positive cells had astrocyte morphology, with small round soma and radial processes ( Figure 7A-D). The staining was present in nucleus, cytoplasm and the proximal portion of the cytoplasmic processes. In two individuals of the Ctrl group and in one MTLE patient, some cells with neuronal morphology and size were also stained for MT-I/II ( Figure 7E, F). No microglia-like cells were stained for MT-I/II. Neuropil staining showed a granular pattern in all hippocampal subfields ( Figure 7A-F). Confocal microscopy confirmed the expression of MT-I/II in astrocytes by GFAP-positive labeling (Figure 8). A comparison between MT-I/II expression in Ctrl, TLE-TD and MTLE groups is shown in Figure 9.
Higher MT-I/II immunoreactivity area ( Figure 10) was observed in both TLE groups, when compared to Ctrl group. The increase in MT-I/II immunoreactivity area observed in TLE was due to an increased number of MT-I/II-positive cells and to increased neuropil staining. MTLE group showed increased immunopositive area when compared to Ctrl in granule cell layer (Kruskal-Wallis, p = 0.028), hilus (Kruskal-Wallis, p,0.001), CA3

MT-I/II immunoreactivity and seizures
In MTLE group, patients without secondary generalized seizures (SGS) had increased MT-I/II immunopositivity, when compared with patients with SGS, in the inner molecular layer (ttest, p = 0.037), granule cell layer (t-test, p = 0.018), subgranule zone (t-test, p = 0.004), CA2 (Mann-Whitney, p = 0.039) and CA1 (t-test, p = 0.043) (Figure 11). No differences in neuronal, astroglial or microglial populations were observed between MTLE patients with or without SGS. In TLE-TD patients, no differences in hippocampal MT-I/II immunopositivity, neuronal, astroglial or microglial populations were observed between patients with and without SGS. Frequency of seizures did not correlate with MT-I/ II immunopositivity in all hippocampal subfields.

Correlations between MT-I/II immunoreactivity, cellular populations and vesicular Zn 2+
Considering all TLE patients, correlation analysis revealed that MT-I/II immunoreactivity correlated with GFAP immunoreactivity in CA4 (r = 0.312; p = 0.012; n = 65), CA2 (r = 0.275; p = 0.038; n = 57) and CA1 (r = 0.319; p = 0.004; n = 78) and with NeuN in CA1 (r = 20.241; p = 0.034; n = 78). No correlation was found between MT-I/II immunoreactivity and HLA-DR immunoreactivity or neo-Timm staining. In CA4, multiple linear regression revealed a trend to association between MT-I/II expression and GFAP immunopositivity (r = 0.347; p = 0.061, with p = 0.753 for NeuN, p = 0.02 for GFAP and p = 0.111 for age; n = 53). In CA2, multiple regression model revealed that MT expression was significantly explained by GFAP and age (r = 0.574; p,0.001, with p = 0.533 for NeuN, p = 0.018 for GFAP, p,0.001 for age; n = 55). In CA1, MT-I/II expression has a trend to be explained by increased GFAP immunoreactivity (r = 0.364 ; p = 0.015, with p = 0.817 for NeuN, p = 0.069 for GFAP and p = 0.107 for age; n = 77). In summary, in some hippocampal subfields (CA4, CA2, and CA1) there was a positive correlation between MT-I/II immunoreactivity and GFAP immunoreactivity. Different regressions models did not provided a best fit for any of the variables evaluated.
In TLE-TD, there was a positive correlation between NeuN and MT-I/II expression in CA4 (r = 0.543; p = 0.0353; n = 15). No correlation was observed between MT-I/II expression and GFAP, HLA-DR area or neo-Timm density in TLE-TD. Multiple linear

Discussion
In the present study, we found an increased MT-I/II expression in all hippocampal subfields of MTLE patients and in the fascia dentata of patients with TLE-TD. In MTLE patients, MT-I/II expression correlated with astroglial population but not with neuronal population. In TLE-TD group, MT-I/II expression correlated positively with neuronal population only in CA4. In the CNS, MT-I/II are expressed mainly by astrocytes [46] and, when the tissue suffers an injury, increased MT-I/II expression is observed in astrocytes and microglias [46], [32]. In our study, an increased expression of MT-I/II was observed in astrocytes and  occasionally in neurons of autopsy and TLE patients. Confocal microscopy in our TLE patients corroborated the finding that MT-I/II are expressed by astrocytes. We also observed an increased expression of MT-I/II in the neuropil of TLE patients. Studies in tissue obtained from animal models of CNS injury have shown that increased MT-I/II expression in the neuropil is most likely the result of higher release of MT-I/II from the astrocytes [47], [48]. Therefore, our data support the notion that MT-I/II changes are essentially related to astroglial population.
Gliosis is a common finding in TLE [21], [22], [23], [24] and is associated with the degree of neuronal death [22], [49], [23], [24]. Similarly with MT-I/II expression, gliosis was more intense and widespread in MTLE than in TLE-TD groups. Furthermore, correlations between the degree of astrogliosis and the expression of MT-I/II observed in TLE patients indicate that MT-I/II expression in TLE is a phenomenon associated with the astrogliosis and, consequently, with the degree of tissue damage. In agreement with this hypothesis, an association between the severity of tissue damage and the increase in MT-I/II expression has been reported in mice subjected to soman-induced SE [35].
Studies in rodents with kainic acid-induced SE showed an association between MT-I/II expression and neuronal survival. Transgenic mice over-expressing MT-I/II have reduced neuronal death, compared to wild type animals [38]. In addition, mice with reduced MT-I/II expression [36] or in knockouts for MT-I/II [37] had increased neuronal death following SE, compared to wild type mice. In our study, MT-I/II expression correlated positively with neuronal population only in CA4 of TLE-TD patients. In MTLE group, where neuronal death and MT-I/II expression are more pronounced, no correlation between neuronal death and MT-I/II was observed. These findings contradict the hypothesis that an increased MT-I/II expression could be related with   neuronal survival. Different mechanisms contribute to neuronal death that occurs in the hippocampus of MTLE and TLE-TD patients. In TLE-TD patients, evidence has been shown that neuronal death is a consequence of the recurrent seizures [50]. Although neuronal death in MTLE can also be caused by recurrent seizures [50], the bulk of neuronal death is rather a consequence of an initial precipitating insult (IPI), which usually occurs several years before the epilepsy onset [50], [51]. The neuronal death is also severe in MTLE, often resulting in hippocampal sclerosis, while TLE-TD patients generally have preserved neuronal density [18], [52]. In addition, data indicate that hippocampal atrophy may be determined by a strong genetic predisposition and occur in individuals who never had seizures [53]. Therefore, it is possible that the differential increase in MT-I/II expression in TLE-TD and MTLE is also the result of the different mechanisms associated with neuronal death in such epileptic syndromes.
According to other studies, MTs could also be responsible to neuronal damage and death following SE. In mice knockout for ZnT3, a protein responsible to stock Zn 2+ in synaptic vesicles, SE increases damage in CA1 [39], [28], [29] and other cerebral regions [29], when compared to wild type mice. In these knockout mice lacking vesicular Zn 2+ , damage in CA1 can be prevented by chelating extracellular Zn 2+ [28], [29] or by knocking out MT-III gene [39], [29]. However, knocking out MT-III gene in mice with [54] or without vesicular Zn 2+ [29] increases damage in CA3 after SE. Since all studies that associated MT-I/II with neuronal survival after SE studied mainly the CA3 region, where MT-III is also known to protect from damage [54], [29], one could argue that, in CA1 and other hippocampal regions, MT-I/II could cause damage, similarly to MT-III. We did not find any positive association between increased MT-I/II expression and reduced neuronal population in all hippocampal subfields. Furthermore, mice with reduced levels of MT-I/II [36] have increased damage in CA1 after SE. It is known that MT-I/II binds Zn 2+ more  strongly that MT-III [55], [56]. These observations make us believe that MT-I/II do not contribute to the neuronal damage observed in the hippocampus of TLE patients. Further studies must be performed to better address this issue. Several developmental studies have indicated that MT-I/II levels increase with the age, [57], [58], [59], [60], [61], [62], [63], [64]. On the other hand, reduced MT-I/II expression has also been reported in the adult rat brain when compared to young brain [65], and no differences were observed in aged and adult brain specimens of rat [66] and calf [67]. Also, it is already known that longer epilepsy duration can increase the neuronal death observed in hippocampal sclerosis and is associated with the neuronal death in non-sclerosis cases [50]. Therefore, we must also account for age and epilepsy duration as factors for the changes observed in MT-I/II expression. We did not see relation between epilepsy duration and MT expression in our multivariate analysis. However, in some regions, age at evaluation was significantly associated with MT-I/II expression. For example, in CA2 of all TLE patients and in CA4 of TLE-TD age at evaluation predicted MT-I/II expression. Although our findings indicate that age can contribute to the increased MT-I/II expression observed in TLE, the pathological changes associated to the epileptic condition (i.e., gliosis and neuronal death) are still the main factors related to the increased MT-I/II expression in the hippocampus of TLE patients.
Reorganization of vesicular Zn 2+ in the hippocampus is often observed in TLE [19], [20], and Zn 2+ can trigger MT-I/II expression [59]. Then, it is also important to consider the effect of the Zn 2+ pool over MT-I/II expression. In agreement with other studies [68], [69], [19], we only observed significant increase in vesicular Zn 2+ in the inner molecular layer of MTLE patients. No correlation was observed between MT-I/II expression and vesicular Zn 2+ in our TLE cases. This does not exclude an association between MT-I/II expression and Zn 2+ , provided that only 10% of all Zn 2+ in the brain is located in vesicles [70], [7] and only a small fraction of the Zn 2+ released during neurotransmission will reach the astrocytes to induce MT-I/II expression.
Data have shown that the increased MT-I/II immunoreactivity observed in animal models of TLE can also be a factor associated with the seizure generation process. Transgenic mice overexpressing MT-I, have increased seizure duration, a tendency to reduced latency, but similar number of seizures after kainic acid administration [38]. Since MT-I/II act chelating free Zn 2+ [31], [14] and Zn 2+ chelation increases tissue excitability and facilitates seizure generation [71], excessive MT-I/II levels can reduce free Zn 2+ in the synaptic cleft, increasing neuronal excitability and affecting seizure generation. Our data showed a similar frequency of seizure between MTLE and TLE-TD patients. In agreement with previous studies, we found no correlation between seizure frequency and MT-I/II expression in TLE [38].
In MTLE, we found increased levels of MT-I/II expression in patients without SGS, when compared with those with SGS. This could indicate that MT-I/II expression is associated with different seizure spread patterns from the epileptogenic hippocampus to other brain regions. It is important to point out that no difference in neurons or glial cells was observed between MTLE with and without SGS. Studies from different groups also observed no association between changes in the hippocampus and SGS [72], [73], [74]. These observations suggest that the increased MT-I/II expression in patients without SGS is not an effect of gliosis, but it is independently associated with SGS. Further studies with animal models of TLE should evaluate more closely the relationship between MT-I/II expression and seizure susceptibility.
The differential pattern of increase in MT-I/II expression in MTLE and TLE-TD patients may also be associated with the site of seizure generation. Seizures are known to induce MT-I/II expression in the epileptic hippocampus [75]. In MTLE patients, where MT-I/II increase was widespread, most focal seizures are generated within the hippocampus [76]. In TLE-TD, the seizures are generally generated in the cerebral cortex surrounding the tumor or in the cortical dysplasia and hence propagate to the hippocampus [18], [77], [78]. The main area of input entry in the hippocampus is the molecular layer of the fascia dentata [10], where increased MT-I/II expression was observed in the TLE-TD patients of our study.
Some limitations of our study must be pointed out. So far, studies about MT-I/II expression in animal models of TLE only evaluated the acute period following SE. Considering that our study was performed in patients with chronic epilepsy, it is difficult to establish comparisons between human and animal data. Besides, the reduced number of patients in the TLE-TD group can be the reason why only in one hippocampal subfields the neuronal density correlated with MT-I/II expression. The lack of correlation between seizure frequency and MT-I/II expression does not exclude an association between seizures and MT-I/II expression. Other seizure characteristics, such as seizure duration and time between the last seizure and the surgery, could better correlate with MT-I/II expression than isolated seizure frequency.
Finally, our study may have translational implications in the future. The role of MTs in antiinflamatory response, neurotrophic factor expression, and protection against ROS and heavy metals make those proteins interesting for clinical applications. Studies have shown that EmtinB, a syntethic peptide that mimics the actions of MTs, attenuates kainic acid-induced seizures and protects neurons from excitotoxic death [34]. Further studies with EmtinB and MTs in acute and chronic models of epilepsy might assess, in more detail, the role of these proteins in neuronal survival and seizure susceptibility.
In summary, our data indicate that increased MT-I/II expression is a plastic alteration of chronic TLE, primarily related to the astrogliosis, a common finding in chronic TLE. In opposition to other studies, MT-I/II expression was not associated with significant neuronal survival in TLE. Nevertheless, our findings suggest that increased MT-I/II expression may contribute to the control of the brain hyperexcitability.

INTRODUCTION
Zinc (Zn 2+ ) is an important modulator of glutamatergic transmission in the central nervous system (CNS). 1-3 Zn 2+ is concentrated in presynaptic vesicles, along with glutamate, and released during normal neurotransmission. [4][5][6][7][8] Hippocampal neurons are specially rich in vesicular Zn 2+ , particularly in the axonal boutons of granule cells, CA3 and CA1 pyramidal cells and prosubicular neurons. [5][6][7]9,10 In temporal lobe epilepsy (TLE), one of the most frequent drug-resistant epilepsies in adults, the hippocampus is associated with seizure generation. 11,12 The intense neuronal activity during seizures can release high amounts of Zn 2+ in the synaptic cleft, 13,14 promoting reactive oxygen species (ROS) production, 15 which can ultimately lead to hippocampal neuronal death. [13][14][15][16][17] In fact, studies in hippocampi from TLE patients who underwent epilepsy surgery have shown neuronal loss, [18][19][20] increased glial reaction [21][22][23][24] and reorganization of mossy fibers axon collaterals into the inner molecular layer of the granule cell dendrites. 19,25 This synaptic reorganization of Zn 2+enriched terminals has been hypothesized to contribute to synchronous firing and epileptiform activity. 19 Metallothioneins (MTs) are low molecular weight, cystein-enriched proteins that bound Zn 2+ and cadmium. They can be found in various tissues, in four isoforms. 26 Isoforms I, II and III are found in the central nervous system (CNS), where the isoforms I and II are coexpressed in astrocytes and the isoform III is expressed in neurons. 27,28 MTs participate in Zn 2+ homeostasis, scavenging ROS in the brain 29 and stimulate the expression of several neurotrophic and antiinflamatory factors. 30 Studies on rodent models of TLE have shown that MT expression is increased in the hippocampal formation shortly after seizures 31,32 and that high levels of MTs I and II are associated with reduced neuronal death after seizure-induced damage. [32][33][34] Since MT-I/II levels may be associated with neuron survival after seizures, we hypothesize that MT-I/II expression is altered in TLE and can be associated with the preservation of neuronal density in the hippocampus of TLE patients. Therefore, in this study we evaluated the immunoexpression of MT-I/II and its correlation with hippocampal neuron density in hippocampi of patients with chronic TLE.

Patients and clinical data
Patients with drug-resistant epilepsy were evaluated at the University of São Paulo Epilepsy Surgical Centre in Ribeirão Preto (Brazil), according to standard protocols published elsewhere. 35 MTLE patients (n=69) were patients with hippocampal atrophy or with normal hippocampal volume at MRI without other lesions associated with TLE.
For comparison purposes in the neuropathology studies, autopsy controls (Ctrl, n=20) were obtained from autopsy cases without history of neurological diseases, with no sign of CNS pathologies in post mortem pathological evaluation, and with less than 10 hours post mortem.
Medical records of all evaluated patients were assessed for clinical data analysis. The clinical variables investigated were age at death and cause of death for Ctrl patients and age at surgery, epilepsy duration, age at the first recurrent seizure, seizure frequency per month, presence of secondary generalized seizures, and neuropathological evaluation for MTLE patients. This study followed the principles of the Declaration of Helsinki, was registered in Brazilian's Health Ministry and was approved by our local ethics committee (processes HCRP 9370/2003 and HCRP 2634/2008).

Tissue collection and immunohistochemistry
Hippocampi from surgery or autopsy were cut in coronal sections and placed in 10% (vol/vol) buffered formaldehyde for one week, followed by paraffin embedding. Immunohistochemistry was performed in 8 μm sections at the level of hippocampal body for evaluation of neuronal and astroglial populations and for MT-I/II expression with antibodies against, respectively, NeuN, GFAP and MT-I/II. The sections were submitted to endogenous peroxidase blocking with 4.5% H 2 O 2 in 50 mM phosphate-saline buffer (PSB) pH 7.4, for 15 minutes, followed by microwave antigenic retrieval in 10mM sodium citrate buffer pH 6.0 (for GFAP) or 50 mM Tris-HCl pH 9.6 (for NeuN and MT-I/II). After achieving room temperature, the sections went through blocking free aldehyde groups with Tris-glycine 0.1 M pH 7.4 for 45 minutes, followed by blocking buffer with 5% defatted milk and 15% goat serum (# S-1000, Vector) in Triton buffer (PTB, 20mM phosphate + 0.45M NaCl, pH 7.4, with 0.3% Triton X-100) for four hours. The sections were then incubated with primary antibodies in blocking buffer for 16 hours. We used primary monoclonal antibodies raised in mouse anti-human GFAP (clone 6F2, #M0761, Dako), anti-murine NeuN (clone A60, #MAB377, Chemicon) and anti-equine MT-I/II (clone E9, #M0639, Dako), diluted in blocking buffer at concentrations of 1:500. The primary antibodies were detected using biotinylated rabbit anti-murine IgG (#E0354, Dako), at 1:200 dilution in blocking buffer, for one hour, followed by revelation with avidin-biotin-peroxidase system (Vectastain Elite ABC kit, #PK6100, Vector) and diaminobenzidine as chromogen (DAB, #34001, Pierce Biotechnology). The development times in DAB solution were 10.5 minutes for NeuN and 8 minutes for MT-I/II and GFAP.

Immunohistochemistry analysis
Images of all hippocampal regions were obtained with a video monochrome charge-coupled device camera (CCD; Hamamatsu Photonics Model 2400, Japan) attached to an Olympus microscope (Model BX60, Melville, NY), and captured, averaged, and digitized using a frame grabber (Scion Corporation, Frederick, MD) on a Macintosh computer (Model G3, Cupertino, CA). Illuminance was uniformly maintained and regularly checked using optical density standards (Kodak, Rochester, NY). After captured, the image was analyzed using image system software (ImageJ, version 1.37c). Quantification of the immunohistochemistry was performed with threshold tool, with the investigator blind to the group allocation. After the selection of the region of interest (ROI), the software calculated the immunopositive area by counting all pixels with gray intensity equal or superior to the threshold of staining. A complete protocol for threshold tool can be found at rsbweb.nih.gov/ij/docs/ examples/stained-sections/index.html. The threshold was defined for each protein evaluated, based on the mean immunopositivity of all control cases. The evaluated regions were outer molecular layer (OML), inner molecular layer (IML), granule cell layer (GCL), subgranular zone (SGZ), the hilus (HIL) and the stratus piramidale of CA4, CA3, CA2, CA1, prosubiculum (PRO) and subiculum (SUB). The characterization of hippocampal regions was based on the Lorente de Nó's classification. 36 Results were shown as percentage of immunopositive area/total area.
Additionally, neuronal density was evaluated in the NeuN stained sections. Neuronal count was processed in ImageJ 1.37c software with a 520x magnification for granule cell layer and 260x for pyramidal neurons of CA4, CA3, CA2, CA1, prosubiculum and subiculum. Neuronal densities were estimated with the correction of Abercrombie 37 , which permits to estimate the neuronal density through mathematical method, and the results were shown as thousands of cells per cubic millimeter.

Statistical analysis
Statistics were carried out in SigmaStat 3.1 software. Tests for normality and homogeneity of variances were performed to define data distribution. For parametric variables, t-test was performed and, for the non-parametric variables, Mann-Whitney test was used. Correlation between MT expression and clinical variables was performed using Pearson's test. All results were considered significant at p<0.05.

Clinical data
The clinical characteristics of study participants are summarized in Table 1. Patients with MTLE and Ctrl patients have the same age (p=0.175). Epilepsy duration was 25±10 years, and the age at onset was 13±1. MTLE patients had seven seizures by month, being one of those a secondary generalized seizure (SGS).

Immunohistochemistry evaluation
Neuronal density, estimated with the count of NeuN positive cells, was reduced in the granule cell layer, CA4, CA3, CA2, CA1, prosubiculum and subiculum of the MTLE group, when compared to Ctrl (p < 0.03). Increased astrogliosis was observed in all hippocampal regions of MTLE, compared to Ctrl (p < 0.001). MT-I/II expression, observed in astrocyte-like cells, was increased in all hippocampal subfields evaluated (p < 0.032). For further details, see Table 2.

Tissue alterations and seizures
In MTLE group, patients without SGS had increased MT-I/II immunopositivity, when compared with patients with SGS, in the inner molecular layer (p=0.037), granule cell layer (p=0.018), subgranule zone (p=0.004), CA2 (p=0.039) and CA1 (p<0.043). A trend to increased MT-I/II immunopositivity was observed in the outer molecular layer (p=0.072), CA4 (p=0.076) and subiculum (p=0.068). No differences in neuronal or astroglial populations were observed between MTLE patients with or without SGS. Frequency of seizures did not correlate with NeuN, GFAP or MT-I/II in all hippocampal subfields.

DISCUSSION
In the present study, we found an increased MT-I/II expression in all hippocampal subfields of MTLE patients. In the CNS, MT-I/II are expressed mainly by astrocytes 38 and, when the tissue suffers an injury, increased MT-I/II expression is observed in astrocytes and microglias. 28,38 We also observed that higher degree of MT-I/II expression was observed in regions with higher astrogliosis. Increased glial population is a common finding in TLE [21][22][23][24] and is associated with the degree of neuronal death. [22][23][24]39 In our study, an increased expression of MT-I/II was observed in astrocytes and in a few neurons of some patients. Thus, our data support the notion that MT-I/II changes are essentially related to astroglial population.
Studies in rodents with kainic acid-induced SE showed an association between MT-I/II expression and neuronal protection. Transgenic mice over-expressing MT-I/II have reduced neuronal death, compared to wild type animals. 34 In addition, mice with reduced MT-I/II expression 32 or knockouts for MT-I/II 33 had increased neuronal death following SE, compared to wild type mice. In our study, however, the higher MT-I/II expression was observed in regions with lower neuronal density, indicating that MT-I/II was not associated with neuronal survival. In agreement with our data, an association between the severity of tissue damage and the increase in MT-I/II expression has been reported in mice subjected to soman-induced status epilepticus (SE). 31 Data have shown that the increased MT-I/II immunoreactivity observed in animal models of TLE can also be a factor associated with the seizure generation process. Transgenic mice over-expressing MT-I, have increased seizure duration, a tendency to reduced latency, but similar number of seizures after KA administration. 34 Since MT-I/II act chelating free Zn 2+14,27 and Zn 2+ chelation increases tissue excitability and facilitates seizure generation 40 , excessive MT-I/II levels can reduce free Zn 2+ in the synaptic cleft, increasing neuronal excitability and affecting seizure generation. We found no correlation between seizure frequency and MT-I/II expression in TLE. 34 In MTLE, we found increased levels of MT-I/II in patients without SGS, when compared with those with SGS. This could indicate that MT-I/II is associated with different seizure spread patterns from the epileptogenic hippocampus to other brain regions. It is important to point out that no difference in neurons or glial cells was observed between MTLE with and without SGS. Studies from different groups also observed no association between changes in the hippocampus and SGS. [41][42][43] All those observations suggest that the increased MT-I/II expression in patients without SGS is not an effect of gliosis, but it is independently associated with SGS. Further studies with animal models of TLE must evaluate more closely the relationship between MT-I/II expression and seizure susceptibility.
Some limitations of our study must be pointed out. So far, studies about MT-I/II expression in animal models of TLE only evaluated the acute period following SE. Considering that our study was performed in patients with chronic epilepsy, it is difficult to establish comparisons between human and animal data. The lack of correlation between seizure frequency and MT-I/II expression does not exclude an association between seizures and MT-I/II expression. Other seizure characteristics, such as seizure duration and time between the last seizure and the surgery, could better correlate with MT-I/II expression than isolate seizure frequency.
Finally, our study may have translational implications in the future. The role of MTs in antiinflamatory response, neurotrophic factor expression, and protection against ROS and heavy metals make those proteins interesting for clinical applications. Studies have shown that EmtinB, a syntethic peptide that mimics the actions of MTs, attenuates KA-induced seizures and protects neurons from excitotoxic death. 30 Further studies with EmtinB and MTs should be done to evaluate the role of these proteins in neuronal survival and seizure susceptibility.
In summary, our data indicate that increased MT-I/II expression is a plastic alteration of chronic TLE, primarily related to the astrogliosis, a common finding in chronic TLE. Our findings suggest that increase MT-I/II expression may contribute to the control of the brain hyperexcitability.

Introduction
TLE is the most frequent focal epilepsy in adults and is often drug resistant [1]. HS is a common pathological finding in TLE patients [2], observed in up to 48% of cases [3]. The most common patterns are classic and severe HS. Classic HS is characterized by intense neuronal loss in CA1 and CA4, moderate loss in granule cell layer and CA3 and preservation of CA2 and subiculum [4][5][6][7]. In severe HS, there is intense neuronal loss in all Ammon's horn [3,8]. More rare forms of HS are CA1 and endfolium HS [3,8], in which neuronal loss is confined to CA1 and CA4, respectively. In 19 % of TLE cases, however, no significant neuronal loss is observed in the hippocampal subfields [8].
MRI can detect most cases of HS by showing increased signal in T2-weighted images and reduced hippocampal volume in T1-weighted images [9][10][11]. However, MRI may be negative in atypical HS, such as endfolium sclerosis [12], and 5-10 % of the cases with normal hippocampal volume can have significant neuronal loss through several hippocampal subfields [13]. What could maintain the hippocampal volume in these TLE cases with neuronal loss remains to be defined. Since different TLE substrates could be associated with differences in antiepileptic drug response and post-surgery seizure outcome [8,14,15], to understand the pathological substrates associated with different MRI patterns can be important for a better treatment of these patients.
Although neuron population is an important contributor to MRI hippocampal volume in HS [11], the importance of glial cells is less clear. And since astrocytes can affect the extracellular space volume with the production of components of ECM, such as CSPG, it would also be important to understand how the ECM contributes to hippocampal volume. In this study, we evaluate the association between the hippocampal volume, measured by MRI, and the neuronal and astroglial population, as well as the content of ECM chondroitin sulfate, in the hippocampus of patients with drug-resistant TLE. We found evidence that the histological variables that contributed the most to hippocampal volume were the neuron density and levels of chondroitin sulfate in CA1 subfield.

Patients and clinical data
Patients with drug-resistant epilepsy were evaluated at the University of São Paulo Epilepsy Surgical Centre in Ribeirão Preto (Brazil), according to standard protocols published elsewhere [16,17]. Inclusion criteria were diagnosis of TLE not involving tumor, dysplasia or cortical malformations; age between 18 and 70 years; and digital 3D T1-weighted MRI at 1.5 T scanning.
For definition of histopathological changes, TLE hippocampi were compared to control hippocampi (n = 19), obtained from autopsy cases without history of neurological diseases, with no sign of brain pathologies in post mortem pathological evaluation, and with less than 10 hours post mortem.
Medical records of all patients were assessed for clinical data analysis. The clinical variables investigated were age at death and cause of death for control and age at surgery, epilepsy duration, age at epilepsy onset, seizure frequency, presence of IPI, and age at IPI for TLE patients. This study was registered in Brazilian's Health Ministry and was approved by our local ethics committee (processes HCRP 9370/2003 and HCRP 2634/2008). TLE patients were further divided, according to hippocampal volume in: 1) Normal volume (NV), patients with MRI hippocampal volume above 2.5 cm 3 (n = 22); and 2) Hippocampal atrophy (HA), patients with MRI hippocampal volume equal or bellow 2.5 cm 3 (n = 45). This classification was adapted from a previous study from our institution [18]. Hippocampal boundaries were defined according to previous studies [19,20]. The hippocampal delineation was made manually in coronal slices and corrections, when needed, were made in sagittal and axial slices. After delineation the program calculates the hippocampal volume. The volumetry was made in a blind way regarding the presurgical qualitative evaluation.

Immunohistochemistry
Hippocampi from surgery or autopsy were cut in coronal sections and placed in 10 % buffered formaldehyde for one week, followed by paraffin embedding. Following previously described protocols [21,22], immunohistochemistry were performed in 8 μm sections from hippocampal body with antibodies anti-NeuN, anti-GFAP, and anti-CS-56 for evaluation of, respectively, neuronal population, astroglial population, and for the levels of CSPG. Briefly, the sections were submitted to endogenous peroxidase blocking, followed by microwave antigenic retrieval and blocking solution. The sections were then incubated with primary antibodies in blocking solution for 16 hours. We used primary monoclonal antibodies raised in mouse anti-human GFAP (clone 6F2, M0761, Dako), anti-mouse NeuN (clone A60, MAB377, Chemicon) and anti-chicken CS-56 (clone CS-56, C8035, Sigma), at concentrations of, respectively, 1:500, 1:500 and 1:100 (antibody:blocking solution). The primary antibodies were detected using biotinylated rabbit antimouse IgG (E0354, Dako), at 1:200 dilution in blocking buffer, for one hour, followed by amplification with avidin-biotin-peroxidase system (Vectastain Elite ABC kit, #PK6100, Vector) and revelation with diaminobenzidine as chromogen (#34001, Pierce Biotechnology).

Immunohistochemistry analysis
Images of all hippocampal regions were obtained with a video monochrome camera (CCD; Hamamatsu Photonics Model 2400, Japan) attached to an Olympus microscope (Model BX60, USA), and captured, averaged, and digitized using the software Scion (Scion Corporation, USA) on a Macintosh computer (Model G3, USA). Luminance was uniformly maintained and regularly checked using optical density standards (Kodak, USA).
Immunohistochemistry quantification was made with ImageJ software (version 1.45s).
Quantification of immunohistochemistries for GFAP and CS-56was performed with threshold tool, following previous published protocols [22], with the investigator blind to the groups. The threshold was defined for each protein evaluated, according to the pattern of staining observed in control cases. The evaluated regions were: GCL of fascia dentate, hilus, and the stratus piramidale of CA4, CA3, CA2, CA1, prosubiculum and subiculum. The characterization of hippocampal regions was based on the Lorente de Nó's classification [23]. Results were shown as immunopositive area, in squared micrometers.
Additionally, neuronal density was evaluated in the NeuN stained sections. Neuronal count was processed in ImageJ software at 200x magnification for GCL and pyramidal neurons of CA4, CA3, CA2, CA1, prosubiculum and subiculum. Neuronal densities were estimated with the correction of Abercrombie [24], and the results were shown as thousands of cells per cubic millimeter. Using the recently reviewed Wyler's score for HS [8], we further subdivided our patients in 5 groups: No HS (no significant neuronal loss; n = 2), CA4 HS (neuronal loss in CA4 only; n = 1), CA1 HS (neuronal loss in CA1 only; n = 8), classic HS (with the standard neuronal loss; n = 10) and severe HS (severe neuronal loss in all hippocampus; n = 29). Since the Wyler's classification requires several hippocampal subfields, only some of the patients could be correctly graded.

Statistical analysis
Statistics were carried out in SigmaStat 3.1 software. For parametric variables, t-test or One Way ANOVA with Bonferroni post hoc test were performed. For the non-parametric variables, Mann-Whitney test or Kruskal-Wallis with Dunn post hoc test were performed. Correlations between histopathological variables and the hippocampal volumes were performed using the Pearson's test. All results were considered significant at p < 0.05.

Clinical Data
The clinical data of TLE patients and control cases are summarized in Table 1. Patients with TLE and controls had similar ages at evaluation (p = 0.164). In TLE, both groups showed similar epilepsy duration (p = 0.297), age at recurrence (p = 0.623), seizure frequency (p = 0.992), IPI frequency (p = 0.991), and age at IPI occurrence (p = 0.645).

Hippocampal Volumetry
The ipsilateral hippocampus (i.e., the hippocampus surgically removed) had volume ranging from 2.562 to 3.704 cm 3 in NV, and 1.338 to 2.498 cm 3 in HA (Fig 1). The contralateral hippocampus ranged from 2.926 to 4.827 cm 3 in NV, and 2.135 to 3.748 cm 3 in HA (Fig 1). The ipsilateral and contralateral hippocampi from NV had higher volume than the respective hippocampi from HA (p < 0.001). Qualitative analysis of MRI, made by experienced radiologists, revealed reduction of the hippocampal volume and increased T2 signal in the ipsilateral hippocampus of most patients ( Table 2).

Histological evaluation
Neuronal cell density, evaluated in sections submitted to immunohistochemistry for NeuN (Fig 2 A-B), was reduced in both TLE groups in the GLC, CA4, CA3, CA1 and prosubiculum, when compared to controls (p ≤ 0.001). Astrogliosis, evaluated as GFAP immunopositive area (Fig 2 C-D), was observed in the GCL, hilus, CA4, CA3, CA2, CA1, prosubiculum and subiculum of TLE patients, when compared to controls (p < 0.001). The immunopositive area of CSPG, measured in sections submitted to immunohistochemistry for CS-56 antigen (Fig 2 E-F), was increased in the GCL, CA3, CA2 and CA1 of NV group (p ≤ 0.043), and in the prosubiculum and subiculum of both TLE groups (p ≤ 0.006), when compared to controls. No difference was observed between HA and NV in relation to neuronal cell density, GFAP or CS-56 immunopositive areas.
Although the No HS and CA4 HS groups were only observed in NV cases, there was no significant difference between the groups NV and HA regarding Wyler's revised score (Exact test, p = 0.179; Table 3).
To evaluate the relative contributions of neuron density in CA1 and prosubiculum and the CA1immunopositive areas for CS-56 and GFAP to hippocampal volume we performed multiple linear regression. According to the model, the higher the neuron density and CA1 levels of chondroitin sulfate in CA1, the greater the hippocampal volume (R = 0.619; p < 0.001, with p = 0.001 for neuron density and p = 0.03 for CSPG). The prosubiculum neuron density and the CA1 immunopositive area for GFAP did not predict hippocampal volume (p = 0.996 and p = 0.843, respectively).

Discussion
In this study, we evaluated the association between the MRI hippocampal volume and histopathological variables in patients with drug-resistant TLE. Our aim was to evaluate which tissue changes observed in the hippocampus of TLE patients could contribute to the hippocampal volume measured with MRI. We found that neuron density and levels of CSPG in CA1 subfield independently correlated with hippocampal volume.
However, most studies used qualitative data to report such association and there are few studies showing quantitative evidence. Studies using neuronal/glial ratios revealed that the severity of pathological changes (i.e., neuronal loss and gliosis) is associated with the degree of hippocampal volume reduction [12,32]. In agreement with our study, they found that CA1 cell changes were the main contributors for hippocampal atrophy on MRI [12]. Only one study using similar methods showed that neuronal density in GCL, but not in CA1, correlated with hippocampal volume [11].
Astrogliosis is observed through all hippocampal subfields in TLE [12,22]. In MRI studies, increased astrogliosis is commonly associated with increased T2 signal [11]. Regarding MRI volumetric studies, gliosis was inversely correlated with hippocampal volume [12,32]. Similarly, our data indicate a negative correlation between GFAP immunoreactivity in CA1 and hippocampal volume. However, it is well known that gliosis is a reactive change of glial cells in response to tissue damage [36]. Therefore, most likely the CA1 neuron loss found in patients with TLE result in reactive gliosis and reduced hippocampal volume. This conclusion seems to be the most plausible provided that when we included both neuron loss and gliosis in our regression model, gliosis was excluded as an important contributor to hippocampal volume.
In the brain, extracellular matrix accounts for up to 20% of the tissue and is rich in chondroitin sulfate proteoglycans [37][38][39][40]. Increased levels of CSPG were observed in patients with TLE [41]. We also observed an increased chondroitin sulfate levels in our TLE patients. Studies with MRI indicated that increased CSPG expression is associated with changes in water diffusion [42]. However, it is not known if increased CSPG could affect hippocampal volume. In our study, higher levels of CSPG in CA1 correlated with higher hippocampal volume in MRI. Both our quantitative and qualitative evaluations of the neuronal population showed that patients with normal volume on MRI could have cell loss as high as patients with severe hippocampal atrophy.
We believe that the levels of chondroitin sulfate are important to maintain the hippocampal volume in cases that, otherwise, would have hippocampal atrophy, as observed by the severe neuronal loss.
Volumetric studies also point the Sommer sector (CA1 subfield and prosubiculum) as the most determinant region to hippocampal volume [12,27]. We found that the combination of neuron density and the levels of CSPG in CA1 explain more than 38 % of hippocampal volume. The multiple regression models also excluded the astroglial population effect over hippocampal volume, thus indicating that the astrogliosis follows the severity of the neuronal death, as discussed earlier.
The reader should know that for the extracellular matrix evaluations, we used the broadspectrum antibody anti-CS-56 epitope to quantify most chondroitin sulfate proteins. However, brain ECM is also constituted by hyaluronic acid. Therefore, we did not evaluate the contribution of hyaluronic acid to the hippocampal volume.

Conclusion
The present study showed that the CA1 subfield is critical to hippocampal volume in TLE.
Our data indicate that neuron density and levels of the extracellular matrix molecule CSPG in CA1 correlate positively with hippocampal volume, whereas the reactive astrocyte population correlates negatively. Linear regressions further confirmed that the combination of neuron population and levels of CSPG in the CA1 subfield are important contributors for hippocampal volume in TLE.

Patients
We analyzed the hippocampal formation from MTLE specimens freshly collected in the operating room and non-epileptic controls from necropsy, collected between 4-9 hours after death. A  Table 1.

Clinical features of MTLE patients
All patients were referred for pre-surgical assessment due to drug-resistant seizures as defined in the literature ). Patients were evaluated at the Ribeirao Preto Epilepsy Surgery Program using standardized protocols approved by the institution's Ethics Committee and a written consent form was obtained from each patient. Pre-surgical investigation at the Epilepsy Monitoring Unit included detailed clinical history, neurological examination, interictal and ictal scalp/sphenoidal electroencephalography (EEG), neuropsychology evaluation, and intracarotid amobarbital memory and language procedure whenever deemed clinically necessary.
Definition of MTLE followed Engel's criteria   2014)), the diagnosis of psychosis associated with MTLE was established according to Sachdev (1998), meaning that patients with interictal psychosis did not experience the following: psychotic disorder temporally associated with seizures, changes in antiepileptic medications, epileptic status, delirium, and psychosis for paradoxical normalization. This group was defined by a prolonged psychotic state that was not related to the epileptic seizures. Typically, the psychotic states closely resemble schizophrenia, with