J Neuropathol Exp Neurol Copyright Ó 2013 by the American Association of Neuropathologists, Inc.

Vol. 72, No. 11 November 2013 pp. 1029Y1042

ORIGINAL ARTICLE

Neurotrophins in Mesial Temporal Lobe Epilepsy With and Without Psychiatric Comorbidities Ludmyla Kandratavicius, PhD, Mariana Raquel Monteiro, MSc, Joao Alberto Assirati, Jr., MD, PhD, Carlos Gilberto Carlotti, Jr., MD, PhD, Jaime Eduardo Hallak, MD, PhD, and Joao Pereira Leite, MD, PhD

Abstract

From the Department of Neurosciences and Behavior, School of Medicine of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil (LK, MRM, JEH, JPL); Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), University of Sao Paulo, Sao Paulo, Brazil (LK, JEH, JPL); National Institute of Science and Technology in Translational Medicine (INCT-TM-CNPq), Brazil (JEH); Department of Surgery, School of Medicine of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil (JAA Jr., CGC Jr.). Send correspondence and reprint requests to: Joao Pereira Leite, MD, PhD, Department of Neurosciences and Behavior, School of Medicine of Ribeirao Preto, University of Sao Paulo, Av Bandeirantes 3900, CEP 14049-900, Ribeirao Preto, Sao Paulo, Brazil; E-mail: [email protected] This work was supported by the Fundacao de Apoio a Pesquisa do Estado de Sao Paulo-Fapesp (07/56721-7 to Ludmyla Kandratavicius, CInAPCe Project 05/56447-7 to Joao Pereira Leite) and Conselho Nacional de Desenvolvimento Cientifico e Tecnologico-CNPq. The authors declare that they have no conflict of interest. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal’s Web site (www.jneuropath.com).

Key Words: Brain-derived neurotrophic factor, Depression, Nerve growth factor, Neurotrophin, Psychiatric comorbidity, Psychosis, Temporal lobe epilepsy.

INTRODUCTION Temporal lobe epilepsy (TLE) is the most common cause of intractable epilepsy in adults. The mesial subtype (MTLE) usually shows hippocampal sclerosis (HS), neuronal loss, gliosis, and mossy fiber sprouting (MFS) (1Y5). Psychiatric comorbidities are very frequent in TLE patients (6Y11), although the precise nature of this association is still a matter of debate (12, 13). We have recently shown neuropathologic data suggesting that there is a structural basis for psychiatric manifestations in MTLE patients, such as increased MFS in patients with a history of major depression and decreased MFS in interictal psychosis (4). Neurotrophins (NTs) are low-molecular-weight growth factors that act as extracellular ligands and affect differentiation, maintenance, and survival of cells. Among these endogenous proteins, nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin 3 (NT3) were shown to promote MFS in different animal and in vitro models of TLE (14Y16). Neurotrophins are upregulated in the dentate gyrus of TLE patients (17), and in animal models, seizure activity results in a transient increase of NGF and BDNF in hippocampal and neocortical neurons (14, 18Y21). Although unaltered in the kindling model (15), NT3 expression is increased in the subchronic phase of the kainate model (22). Moreover, prolonged NT3 intracerebral infusion triggers sprouting and might play an antiepileptogenic role (15). In major depression and schizophrenia, decreased NT expression has been documented (23Y27), whereas antidepressant and antipsychotic treatment might restore NT plasma levels (23, 28, 29). Interestingly, chronic treatment with antidepressants, antipsychotics, and benzodiazepine in naive rats does not alter BDNF or NGF hippocampal levels (30, 31). Although growth factor involvement in TLE and neuropsychiatric disorders seems unequivocal, the detailed expression patterns and functions of NTs within the human hippocampal formation remain largely unknown. Here, we characterized NT

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Despite the strong association between epilepsy and psychiatric comorbidities, data on clinicopathologic correlations are scant. We previously reported differential mossy fiber sprouting (MFS) in mesial temporal lobe epilepsy (MTLE) patients with psychosis (MTLE + P) and major depression (MTLE + D). Because neurotrophins (NTs) can promote MFS, here, we investigated MFS, neuronal density and immunoreactivity for the NT nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and neurotrophin 3 (NT3) in hippocampi of 14 MTLE patients without a psychiatric history, 13 MTLE + D, 13 MTLE + P, and 10 control necropsies. Mossy fiber sprouting correlated with granular layer NGF immunoreactivity and seizure frequency. Patients with secondarily generalized seizures exhibited less NGF immunoreactivity versus patients with complex partial seizures. There was greater NT immunoreactivity in MTLE versus control groups but lesser NT immunoreactivity in MTLE + P versus MTLE patients; these findings correlated with neuropsychologic scores. Patients with MTLE + D taking fluoxetine showed greater BDNF immunoreactivity than those not taking fluoxetine; MTLE + P patients taking haloperidol had decreased neuronal density and immunoreactivity for NGF and BDNF in specific subfields versus those not taking haloperidol. There were no differences in NT3 immunoreactivity among the groups. These findings support a close association between MFS and NT expression in the hippocampi of

MTLE patients and suggest that distinct structural and neurochemical milieu may contribute to the genesis or maintenance of psychiatric comorbidities in MTLE.

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TABLE. Demographic and Clinical Data MTLE

MTLE + D

MTLE + P

Controls

Statistics

8 6 9 5 5 4 0 3.0 T 2.3 10.2 T 6.4 6 8 12.7 T 10.1 6 6 2 12 1 1 3 11 9 5 82.2 T 7.9 6.2 T 3.0 36.1 T 4.7 25.9 T 6.4 7 7

5 8 8 5 3 3 2 4.3 T 5.5 13.0 T 8.1 1 12 17.2 T 15.6 7 4 2 11 2 0 7 6 7 6 84.3 T 9.6 5.1 T 3.6 37.1 T 5.4 24.1 T 8.9 8 5

7 6 7 6 5 1 1 6.8 T 8.4 13.8 T 8.4 4 9 11.7 T 10.1 5 6 2 13 0 0 2 11 3 10 77.2 T 7.3 4.2 T 2.6 38.0 T 6.0 24.1 T 8.6 6 7

4 6 na na na na na na na na na na na na na na na na na na na na na na 48.1 T 18.9 na 6 4

No difference No difference No difference

No difference No difference No difference tr No difference No difference

No difference

No difference No difference tr No difference No difference No difference No difference No difference

Values are indicated as mean T SD when applicable. There was a statistical trend (tr: 0.05 Q p e 0.07), suggesting a higher proportion of MTLE + D patients with SGS than MTLE and a higher proportion of MTLE + P patients with worse performance in nonverbal memory tasks than MTLE. CPS, complex partial seizure; HS, hippocampal sclerosis; IPI, initial precipitant injury; MTLE, mesial temporal lobe epilepsy; MTLE + D, MTLE patients with a diagnosis of major depression; MTLE + P, MTLE patients with interictal psychosis; na, not applicable; SGS, secondarily generalized seizures.

immunoreactivity in subfields of the mesial temporal structures, including the hippocampus, subicular complex, and entorhinal cortex in a new cohort of MTLE patients with and without comorbid major depression and psychosis. We also investigated the possible correlation between NT immunoreactivity and MFS and further clinical characteristics.

MATERIALS AND METHODS Patients We analyzed the hippocampal formation from MTLE specimens freshly collected in the operating room and nonepileptic controls from necropsy, collected between 3.5 and 6 hours after death. A less than 24-hour postmortem time limit allows comparison of necropsy tissue with freshly collected surgical specimens for their protein levels, cell morphology, and tissue integrity (32, 33). Tissue collection and processing were conducted according to protocol approved by our institution’s research ethics board. Mesial temporal lobe epilepsy specimens were derived from 40 MTLE patients who underwent a standard en bloc anterior temporal resection (including 3Y4 cm of the hippocampus) for medically intractable seizures. All had clinical

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neuropathologic confirmation of HS. They were divided into 3 groups: 14 MTLE patients without any history of psychiatric disorder (MTLE group); 13 MTLE patients with interictal psychosis (MTLE + P group); and 13 MTLE patients with a diagnosis of major depression (MTLE + D group). According to Blu¨mcke HS categories (34), 17 patients had severe HS, 16 classical HS, 5 CA1 HS, and 2 CA4 HS. No differences were seen among MTLE groups with respect to HS categories (Table, Supplemental Digital Content 1, http://links.lww.com/NEN/A508). For comparison, 10 nonepi leptic control hippocampi from necropsies were processed and analyzed in the same manner as the surgical cases. Underlying diseases causing death were cardiomyopathy, pulmonary infarct, or renal-hepatic failure, with no history of hypoxic episodes before death, seizures, or neurologic diseases. Furthermore, there was no evidence of pathologic abnormalities of the mesial temporal structures on postmortem examination. Mesial temporal lobe epilepsy and control specimens were collected between 1996 and 2006. Clinical characteristics of all groups are shown in the Table.

Clinical Features of MTLE Patients All patients were referred for presurgical assessment because of drug-resistant seizures, as defined by Berg (35). Patients Ó 2013 American Association of Neuropathologists, Inc.

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Male (n) Female (n) IPI present (n) IPI absent (n) Febrile seizure present in IPI (n) Febrile seizure absent in IPI (n) Febrile seizure unknown (n) Age of first seizure, years Age when seizures became recurrent or age of onset, years Seizure type: CPS (n) Seizure type: SGS (n) Seizure frequency (monthly) Right HS (n) Left HS (n) Bilateral HS (n) Right handedness (n) Left handedness (n) Bilateral handedness (n) Memory in verbal tasks: average or above (n) Memory in verbal tasks: below average (n) Memory in nonverbal tasks: average or above (n) Memory in nonverbal tasks: below average (n) Full-scale IQ Years at school Age at surgery (or at death for controls), years Duration of epilepsy, years Collected side: right (n) Collected side: left (n)

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Tissue Collection and Immunohistochemical Processing Specimens were segmented into 1-cm blocks transversely oriented to the hippocampal long axis. Blocks were placed in neoTimm fixative solution (0.1% sodium sulfide [Sigma, St. Louis, MO] in Millonig buffered glutaraldehyde [Vetec, Rio de Janeiro, Brazil]) or buffered paraformaldehyde (Sigma). After 48 to 96 hours, the specimens were processed for neo-Timm histochemistry and hematoxylin and eosin (Laborclin, Pinhais, Brazil) staining or paraffin embedded for immunohistochemistry. Cryostat sections were mounted on chrome-alum gelatincoated slides and air-dried for neo-Timm staining (2). Slides were processed in batches containing at least 1 MTLE and 2 control slides. Slides were immersed in a physical developer maintained at 26-C in a darkroom. Developer consisted of 10 mL of a 50% gum arabic (Sigma) solution, 30 mL of an aqueous solution of 1.3 mol/L citric acid (Merck, Darmstadt, Germany), 0.9 mol/L sodium citrate (Merck), 90 mL of an aqueous solution of 0.5 mol/L hydroquinone (Merck), and 1.5 mL of a 17% silver nitrate solution (Merck). Light sections were developed for 40 minutes and dark sections for 50 minutes. Slides were washed in distilled water for 5 minutes and running tap water for 10 minutes. They were subsequently air-dried, ethanol dehydrated, xylene cleared, and coverslipped. Immunohistochemistry was performed with antibodies that identified immunoreactivity for NeuN, a nuclear protein found in the nuclei of mature neurons (1:1000 dilution; Chemicon-Millipore, Billerica, MA) and for the NTs: neurotrophic growth factor (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), BDNF (1:30 dilution; ChemiconMillipore), and NT3 (1:30 dilution; Chemicon-Millipore). According to the manufacturers and selected references, these antibodies show no cross-reactivity with other NTs and/or are capable of neutralizing the biologic activity of its own isoform only (38Y40). Their Western blot profile in human tissue is shown in Figure, Supplemental Digital Content 2, http://links.lww.com/NEN/A509. Briefly, paraffin-embedded MTLE and control hippocampi were processed together for each antibody as described in Kandratavicius et al (5), with overnight incubation at room temperature and developed simultaneously for 10 minutes in 0.05% 3,3¶-diaminobenzidine tetrahydrochloride (Pierce, Rockford, IL) and 0.01% H2O2 (Merck). After sufficient colorization, reaction was halted by washing in several rinses of distilled water, dehydrated through graded ethanol to xylene (Merck), and coverslipped with Krystalon (EM Science, Gibbstown, NJ). Adjacent sections were hematoxylin and eosin stained and examined for tissue integrity. Control sections without the primary antisera did not reveal staining (data not shown).

Cell Count and Neo-Timm Quantification Mesial temporal lobe epilepsy and control hippocampi were compared for neuronal density and neo-Timm staining. Neuronal counting was performed based on Lorente de No classification (41), including fascia dentata granular and subgranular cells, polymorphic hilar neurons (limited to a region between stratum granulosum and CA4 pyramidal cells, being at least 50 Km from the stratum granulosum and 100 Km

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were evaluated at the Ribeirao Preto Epilepsy Surgery Program using standardized protocols approved by the institution’s ethics committee, and written consent was obtained from each patient. Presurgical investigation at the Epilepsy Monitoring Unit included detailed clinical history, neurologic examination, interictal and ictal scalp/sphenoidal electroencephalography, neuropsychology evaluation, and intracarotid amobarbital memory and language procedure whenever deemed clinically necessary. Mesial temporal lobe epilepsy was defined following Engel criteria (36): 1) seizure semiology consistent with MTLE, usually with epigastric/autonomic/psychic auras, followed by complex partial seizures (CPSs); 2) presurgical investigation confirming seizure onset zone in the temporal lobe; 3) anterior and mesial temporal interictal spikes on electroencephalography; 4) no lesions other than unilateral or bilateral hippocampal atrophy on high-resolution magnetic resonance imaging scans (reduced hippocampal dimensions and increased T2 signal); 5) clinical histopathologic examination compatible with HS; and 6) no evidence of dual pathology identifiable by any of the assessment methods described (clinical, electrophysiology, neuroimaging, and histopathology). Exclusion criteria were focal neurologic abnormalities on physical examination, generalized or extratemporal electroencephalography spikes, and marked cognitive impairment indicating dysfunction beyond the temporal regions. Information regarding antecedent of an initial precipitant injury, febrile seizures, seizure types, drug regimen, and estimated monthly frequency (within the 2 years before surgery) were retrospectively collected from medical records for each patient. Psychiatric evaluations were conducted in all MTLE patients. Each diagnosis of major depression was independently established during the presurgical evaluation by 2 psychiatrists with experience in psychiatric disorders associated with epilepsy using the guidelines of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition. Once a consensus on the classification of psychotic syndromes associated with epilepsy is lacking, and neither Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, nor International Classification of Disease, 10th Revision, has addressed this issue specifically, the diagnosis of psychosis associated with MTLE was established according to Sachdev (37), that is, 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 paranoid ideas that might become systematized, ideas of influence, and auditory hallucinations often of a menacing quality. The points of difference are common religious coloring of the paranoid ideas, tendency of the affect to remain warm and appropriate, and no typical deterioration to the hebephrenic state (9). Patients had no history of psychiatric disorders (before seizure onset) or of substance dependence at any time. Global IQ was calculated after neuropsychologic tests (complete WAIS-III or WAIS-R protocol).

NTs in MTLE With Psychiatric Comorbidities

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from CA4), as well as pyramidal cells in CA4 (the portion of Ammon horn that permeates the inner part of the dentate gyrus), CA3, CA2, CA1, prosubiculum, subiculum, parasubiculum, and entorhinal cortex layer III. Cell densities (neurons per cubic millimeter) were estimated in 8-Km NeuNstained slices at 400 magnification with a morphometric

grid methodology using Abercrombie correction (42), as previously described and well established in the literature for surgical hippocampal fragments (1, 3Y5, 43Y47). We also performed measurements of granular layer dispersion using NeuN-stained sections and the straight line tool of ImageJ analysis system (National Institutes of Health, USA, public

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FIGURE 1. Neuronal density in human hippocampal formation subfields. (A, B) NeuN-stained hippocampal formation in a mesial temporal lobe epilepsy (MTLE) patient (A) and a control (B). There is marked neuronal loss in the MTLE dentate gyrus, Ammon, horn and Sommer sector, as well as granular layer (GL) dispersion. (C) Neuronal density values from MTLE (black bars), MTLE + major depression (MTLE + D) (gray bars), MTLE + psychosis (MTLE + P) (light gray bars), and from nonepileptic controls (white bars) are indicated as mean T SD. **Significant difference between epileptics and the control group (p G 0.001). Neuronal loss was observed in GL, hilus, CA4, CA1, prosubiculum, and entorhinal cortex. There is also a statistical trend (tr: 0.05 Q p e 0.07) to decreased neuronal density in MTLE + P CA2 versus control. ENT, entorhinal cortex; HIL, hilus; PAR, parasubiculum; PRO, prosubiculum; SUB, subiculum. Scale bar = (A, B) 2 mm.

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domain). The bottom was considered as the farthest granular neuron in the subgranular zone and the top the farthest granular neuron invading the molecular layer. Mossy fiber sprouting was evaluated in neoTimmYstained sections in the hilus, granular layer, and inner molecular layer, always in the superior blade of the dentate gyrus. Each subfield of each specimen had 3 different samples measured and averaged for statistical analysis. Measurements of gray value and length were estimated using ImageJ, as previously described (4). In brief, length measurements of sprouting consisted of the distance from the outer border of the granular layer to the visible limit of the sprouted mossy fibers within the molecular layer, including the sprouted mossy fibers that eventually invaded the outer molecular layer (measurement referred as visible sprouting in the molecular layer). Images were collected and digitized with a high-resolution CCD monochrome camera attached to an Olympus microscope. This method was used to obtain digitized images of neo-

NTs in MTLE With Psychiatric Comorbidities

Timm and anti-NeuN, -NGF, -BDNF, and -NT3Ystained slides. Uniform luminance was maintained and checked every 10 measurements using an optical density standard and a gray value scale ranging from 0 (white) to 255 (black).

Semiquantitative Analysis of Immunohistochemistry

Data Analysis Data were analyzed using the statistical program PAWS (version 18.0) and SigmaPlot (version 11.0). Groups were compared using analysis of variance (ANOVA one-way, with Bonferroni post hoc test) or unpaired t test for variables with normal distribution and Kruskal-Wallis one-way ANOVA on ranks (with Dunn post hoc test) or Mann-Whitney U rank sum test for variables without normal distribution. Fisher Exact test was applied for comparison of relative frequencies of clinical variables between groups. Other statistical tests included Pearson correlation analyses and analysis of covariance. Statistical significance was set at p G 0.05, and values were presented as mean T SD.

RESULTS FIGURE 2. Mossy fiber sprouting in human dentate gyrus. (AYD) Representative images from mesial temporal lobe epilepsy (MTLE) (A), MTLE + major depression (MTLE + D) (B), MTLE + psychosis (MTLE + P) (C), and control (D) groups, showing aberrant sprouting in the molecular layer of the dentate gyrus in the MTLE groups. (E) Increased gray value measurements in MTLE and MTLE + D versus control in GL (* p G 0.05), IML (** p G 0.001), and OML (tr: 0.05 Q p e 0.07). In the IML, the gray value in the MTLE + P group is lower than that in the MTLE and MTLE + D groups (# p G 0.05). (F) Length measurements show greater sprouting in MTLE groups versus the control group (** p G 0.001) and significant differences among the 3 MTLE groups (## p G 0.001). Values from MTLE (black bars), MTLE + D (gray bars), MTLE + P (light gray bars), and from nonepileptic controls (white bars) are indicated as mean T SD. GL, granular layer; H, hilus; IML, inner molecular layer; OML, outer molecular layer. Scale bar = (AYD) 100 Km.

Clinical Profiles The 4 patients groups did not show significant differences in sex, age, or collected side (Table). Clinical variables such as the presence of an initial precipitant injury and febrile seizures, age of first seizure and seizure onset, seizure frequency and epilepsy duration, HS side, handedness, IQ, years at school, and performance in verbal memory tests were homogeneously distributed among MTLE groups. The MTLE + D patients exhibited a trend to increased proportion of patients with secondarily generalized seizures versus patients without psychiatric comorbidities (0.05 Q p e 0.07), and MTLE + P patients exhibited a trend to worse performance in nonverbal memory tasks. All epileptic patients were on antiepileptic drugs (carbamazepine, oxcarbazepine, phenobarbital, and/or phenytoin). In addition, patients were also taking benzodiazepines (MTLE

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Slides adjacent to those examined for neuronal density were analyzed for NT immunoreactivity. In brief, all digitized images taken in 20 magnification were analyzed with ImageJ software, following the same criteria: 1) the software identifies the gray value distribution of a subfield’s digital image (total area for each subfield analyzed = 313.7  235.3 Km, which corresponds, e.g. to approximately one-third of an Ammon horn subfield); 2) the immunoreactive area, that is,positively stained pixels, is selected, limited to a threshold range; and 3) the threshold range is presettled based on control group sections to exclude the low-intensity gray value of background staining from the analysis. A similar approach was used by our group elsewhere (48). Results for granular layer included granular cell layer per se and proximal molecular layer. Analyses were conducted by one investigator (Ludmyla Kandratavicius), blinded to hippocampal pathology and group classification.

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group, 8 of 14; MTLE + D, 10 of 13; MTLE + P group, 10 of 13), fluoxetine (MTLE + D, 4 of 13), and haloperidol (MTLE + P group, 10 of 13). No differences in neuronal density, MFS, or NT immunoreactivity were seen between patients taking or not taking benzodiazepines. No differences in neuropsychologic tests between patients taking or not taking benzodiazepines, fluoxetine, or haloperidol were seen.

Neuropathologic Characterization: Neuronal Density and MFS Evaluation of epileptogenic and control hippocampal formation (Fig. 1A, B) showed reduced neuron density in the

granular layer, hilus, CA4, CA1, and prosubiculum of all MTLE groups when compared with control (Fig. 1C). In addition, there was a significant neuron density reduction in the entorhinal cortex of MTLE patients with major depression and interictal psychosis versus controls and a trend to decreased neuronal density in CA2 of MTLE + P specimens. The MTLE groups exhibited increased granular layer width when compared with that in controls (ANOVA F3,47 = 8.31, p G 0.0001), with no statistically significant differences between MTLE groups (ANOVA F2,37 = 0.20, p = 0.817) (Table, Supplemental Digital Content 1, http://links.lww.com/NEN/A508). The MFS gray value and length were increased in MTLE groups versus

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FIGURE 3. Nerve growth factor (NGF) expression in the human hippocampal formation. (AYT) There is greater expression in mesial temporal lobe epilepsy (MTLE) granular cells (A, B), in hypertrophic hilar cells (C, D), and in pyramidal cells in Ammon horn (EYL), subicular complex (MYR), and entorhinal cortex (S, T). Cells with glial profiles (arrows) can also be seen particularly in CA1 and prosubiculum of MTLE specimens. ENT, entorhinal cortex; GL, granular layer; HIL, hilus; PAR, parasubiculum; PRO, prosubiculum; SUB, subiculum. Scale bar = (AYT) 50 Km.

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NGF: r = +0.42, p = 0.017; parasubiculum NGF: r = +0.58, p = 0.001). No differences in NGF immunoreactivity were seen in MTLE + D patients taking fluoxetine versus those not taking fluoxetine. The MTLE + P patients taking haloperidol showed less NGF immunoreactivity in parasubiculum (2,022.31 T 410.6  102 Km2 vs 3,303.65 T 138.2  102 Km2, t10 = j2.85, p = 0.019). Moreover, when we analyzed NGF expression considering seizure type, we found that patients with secondarily generalized seizures exhibited decreased values versus those with CPS in the hilus (1,640.12 T 693.4  102 Km2 vs 2,249.85 T 816.2  102 Km2, t37 = 2.20, p = 0.03), CA2 (2,482.40 T 747.9  102 Km2 vs 3,316.06 T 1,194.1  102 Km2, t34 = 2.17, p = 0.04), and subiculum (2,571.59 T

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those in control (Fig. 2). Moreover, MTLE + P specimens exhibited decreased MFS gray value in the molecular layer when compared with those in MTLE and MTLE + D groups. Sprouting length was also decreased in the MTLE + P group. Specimens from the MTLE + D group showed increased MFS length versus those in other MTLE groups (Fig. 2F). Interestingly, neuronal density correlated inversely with seizure frequency in CA4 (r = j0.41, p = 0.045), CA2 (r = j0.50, p = 0.021), prosubiculum (r = j0.38, p = 0.043), subiculum (r = j0.37, p = 0.039), and parasubiculum (r = j0.45, p = 0.015), whereas molecular layer gray value of MFS correlated positively with seizure frequency (r = +0.46, p = 0.014). Also, granular cell dispersion in the superior blade of the dentate gyrus correlated inversely with CA4 neuronal density (r = j0.49, p = 0.023), suggesting that dispersion could be related to loss of granular cells original targets. Within the MTLE + D group, no differences in MFS or neuronal density were observed between patients taking fluoxetine or not. The MTLE + P patients taking haloperidol had decreased neuronal density when compared with those not taking haloperidol in CA3 (9.2 T 2.9  103 neurons/mm3 vs 26.0 T 2.7  103 neurons/mm3, t8 = j7.54, p = 0.002) and subiculum (12.1 T 7.5  103 neurons/mm3 vs 31.0 T 7.1  103 neurons/mm3, t11 = j3.22, p = 0.012).

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NT Immunoreactivity Strong NGF immunoreactivity was detected in all hippocampal formation subfields (Fig. 3). In MTLE specimens, increased NGF expression was detected in hypertrophic hilar cells and pyramidal neurons in the Ammon horn, subicular complex, and entorhinal cortex. Glia-like cells were also evident in the MTLE Sommer sector (CA1 and prosubiculum; Fig. 3K, M). Nerve growth factor immunoreactivity in the granular layer (Fig. 4AYD) was increased in MTLE and MTLE + D groups versus the control group (Fig. 4F). Among epileptic groups, specimens from the MTLE + D group showed greater NGF immunoreactivity, and specimens from the MTLE + P group had less immunoreactivity (Fig. 4AYC). Other hippocampal subfields with decreased NGF immunoreactivity in the MTLE + P group were the hilus, CA2, CA1, prosubiculum, subiculum, and parasubiculum (Fig. 4F). Nerve growth factor immunoreactivity in CA3 of epileptic patients correlated positively with granular cell dispersion in the superior (r = +0.47, p = 0.027) and inferior blade (r = +0.45, p = 0.036), indicating that the increased trophic tone from CA3 to the dentate gyrus might support the reverberatory excitatory activity between CA3, mossy cells, and granule cells, and possibly reinforced by MFS (49). Moreover, NGF immunoreactivity in the granular layer correlated with MFS gray value (r = +0.54, p = 0.008) and length (r = +0.51, p = 0.014) and with seizure frequency (r = +0.40, p = 0.016). Nerve growth factor immunoreactivity correlated positively with neuronal density in CA1 (r = +0.39, p = 0.038) and prosubiculum (r = +0.42, p = 0.042). In addition, NGF immunoreactivity in the granular layer (r = +0.44, p = 0.008), subiculum (r = +0.34, p = 0.047), and parasubiculum (r = +0.47, p = 0.009) correlated positively with global IQ and with neuropsychologic nonverbal scores in the same regions (granular layer NGF: r = +0.44, p = 0.011; subiculum

FIGURE 4. Nerve growth factor (NGF) expression in mesial temporal lobe epilepsy (MTLE) specimens with and without psychiatric comorbidities and in nonepileptic controls. (AYD) MTLE + major depression (MTLE + D) granular layer (B) exhibited greater NGF expression versus MTLE (A), MTLE + psychosis (MTLE + P) (C), and control (D) groups. (E) NGFimmunoreactive area values were higher in epileptic groups versus controls (** p G 0.001; * p G 0.05), and there were differences among MTLE groups (## p G 0.001; # p G 0.05). In CA2 and CA1, there was a trend (tr: 0.05 Q p e 0.07) to decreased NGF expression in MTLE + D versus MTLE. Values from MTLE (black bars), MTLE + D (gray bars), MTLE + P (light gray bars), and from nonepileptic controls (white bars) are indicated as mean T SD. Scale bar = (AYD) 50 Km.

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666.7  102 Km2 vs 3,694.60 T 1,190.3  102 Km2, t38 = 3.52, p = 0.02). Brain-derived neurotrophic factor immunoreactivity in neurons was detected in all subfields of the hippocampal formation (Fig. 5). Glia-like cells were also immunostained, particularly in MTLE dentate gyrus and Ammon horn (Fig. 5AYK). Comparing all groups, the granular layer was the subfield with major differences among them (Fig. 6AYD). The MTLE and MTLE + D showed increased BDNFimmunoreactive area versus that of the control group (Fig. 6E); and MTLE + P granular layer BDNF immunoreactivity was

less than that of MTLE (Fig. 6E). In the hilus, BDNF in MTLE + P was less than that of controls, and in CA1, greater BDNF immunoreactivity area was seen for MTLE and MTLE + D (Fig. 6E). Brain-derived neurotrophic factor immunoreactivity correlated positively with neuronal density of epileptic patients in the hilus (r = +0.65, p = 0.001), CA3 (r = +0.56, p = 0.045), and subiculum (r = +0.41, p = 0.035), but not with MFS or with dispersion in the granular layer. Brain-derived neurotrophic factor immunoreactivity in the granular layer (r = +0.36, p = 0.037) and subiculum (r = +0.42, p = 0.022) also correlated positively with global IQ, but not

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FIGURE 5. Brain-derived neurotrophic factor (BDNF) expression in the human hippocampal formation. (AYT) There is greater immunoreactivity in mesial temporal lobe epilepsy (MTLE) granular cells and in cells with glial profiles, for example, in dentate gyrus and Ammon horn (arrows, A, C, K, M). ENT, entorhinal cortex; GL, granular layer; HIL, hilus; PAR, parasubiculum; PRO, prosubiculum; SUB, subiculum. Scale bar = (AYT) 50 Km.

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NTs, Epilepsy, and Psychopathologic States Neurotrophins are part of a set of molecules that can influence MFS development. Indeed, a positive correlation between MFS progression and increased NGF concentration, but not increased BDNF concentration, was demonstrated in the kainate model of epilepsy (22), which is similar to the correlations we found in this MTLE series. In particular, a study on transgenic mice suggests that BDNF is not essential to MFS because hippocampal slices of a BDNF-deficient (-/-) mouse display MFS as well as the wild type (51). With respect to NT3, a negative correlation with MFS was found, in

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with other neuropsychologic scores. Brain-derived neurotrophic factor immunoreactivity correlated inversely with age of seizure onset in the hilus (r = j0.40, p = 0.032) and CA3 (r = j0.51, p = 0.020). Regarding MTLE + D patients taking fluoxetine, greater BDNF immunoreactivity in CA1 was seen when compared with those not taking fluoxetine (3,920.77 T 161.8  102 Km2 vs 2,529.46 T 451.4  102 Km2, t9 = +4.11, p = 0.009). In contrast, MTLE + P patients taking haloperidol showed decreased BDNF immunoreactivity in the hilus (1,038.60 T 440.6  102 Km2 vs 1,840.75 T 207.3  102 Km2, t11 = j2.96, p = 0.016) and CA4 (1,451.76 T 638.9  102 Km2 vs 3,100.54 T 284.0  102 Km2, t11 = j4.22, p = 0.002) versus those not taking haloperidol. Neurotrophin 3 expression was detected in neurons and glia, as well as in blood vessels through all hippocampal formation subfields (Fig. 7AYT). No differences in NT3immunoreactive area (Fig. 7U) or in the qualitative assessment of how strong NT3 staining was in blood vessels (data not shown) were found among groups in any subfield. Neurotrophin 3 and MFS correlated inversely in CA4 (r = j0.64, p = 0.003), CA1 (r = j0.58, p = 0.005), subiculum (r = j0.52, p = 0.008), and entorhinal cortex (r = j0.59, p = 0.013). No differences in NT3 expression between patients taking or not fluoxetine or haloperidol were seen. Given the differences in neuronal densities (Fig. 1), an important question was whether the statistical differences in NT immunoreactivity for the 4 groups could be accounted for by changes in neuronal densities. Therefore, we performed an analysis of covariance comparing groups and neuronal densities with NGF, BDNF, and NT3-immunoractive area. Differential BDNF expression in the hilus, where a positive correlation was seen between neuronal density and BDNF expression, lost its statistical significance after neuron count correction. Differences between groups in other subfields and for other NTs remained significant after cell count correction, indicating that the 4 patient categories showed significant differences in NT expression levels that were not influenced by changes in neuronal densities.

NTs in MTLE With Psychiatric Comorbidities

DISCUSSION An extensive number of neuropathologic studies have been done on hippocampi from MTLE patients, whereas fewer have examined extrahippocampal tissue (50). Postmortem studies with specimens from patients with schizophrenia and major depression have also shown several abnormalities in the hippocampal formation (12, 13). Despite the strong association between epilepsy and psychiatric comorbidities, neuropathologic data are scant. In another series of patients, we previously reported decreased MFS in MTLE patients with psychosis and increased MFS in MTLE patients with major depression (4); this result was confirmed in the present series. Another important replicated finding was decreased neuronal density in the entorhinal cortex of MTLE patients with psychiatric comorbidities (4). More severe neuron loss in the entorhinal cortex would contribute to disrupted communication between the hippocampal formation and neocortical and limbic sites, which are known to have a profound effect in modulation of the psychopathologic state (12).

FIGURE 6. Brain-derived neurotrophic factor (BDNF) expression in mesial temporal lobe epilepsy (MTLE) specimens with and without psychiatric comorbidities and in nonepileptic controls. (AYD) In MTLE (A) and MTLE + major depression (MTLE + D) (B), there is greater BDNF expression in the granular layer versus MTLE + psychosis (MTLE + P) (C) and control (D) groups. (E) BDNF-immunoreactive area values are higher in the dentate gyrus and CA1 of epileptic groups and less in the hilus of MTLE + P versus controls (** p G 0.001; * p G 0.05). The only subfield with differences among epileptic groups was the granular layer, where MTLE + P specimens showed decreased BDNF expression versus MTLE without psychiatric comorbidities (# p G 0.05). Values from MTLE (black bars), MTLE + D (gray bars), MTLE + P (light gray bars), and from nonepileptic controls (white bars) are indicated as mean T SD. Scale bar = (AYD) 50 Km.

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relatives (61) and in healthy subjects injected with ketamine (62). Diminished hippocampal NGF and BDNF immunoreactivity might be related to the lower cognitive performance trend seen in the MTLE + P group because NGF and BDNF, but not NT3 (63), are required for optimal cholinergic neurotransmission (64). In turn, low cholinergic function results in poor memory performance, as seen in schizophrenia and Alzheimer disease (65, 66). In accordance with our findings of positive correlations between NGF immunoreactivity and IQ and nonverbal memory scores, it has been shown in rodents that increases in hippocampal and neocortical NGF reverse deficits in learning and memory in spatial navigation and object recognition tasks (67). More importantly, in conditions of glutamatergic hypofunction (as it occurs in schizophrenia and animal models of schizophrenia based on N-methyl-d-aspartate antagonism [68Y70]), there is a decrease in BDNF expression (71), as we found in the MTLE + P group versus the MTLE group. Likewise, rats injected with ketamine show decreased MFS induced by electroconvulsive seizures and decreased BDNF expression (72). Low BDNF levels have been observed in the plasma of schizophrenic patients (25, 73, 74) and seem independent of medication despite the high variability between the studies (75). Other studies have also shown association between BDNF polymorphisms and schizophrenia and cognitive deficits (76Y79), but not in cases of febrile seizures (80) or TLE (81). Decreased BDNF expression has been described in postmortem hippocampus of schizophrenic patients (24, 27), and, in contrast, there are also reports of increased BDNF expression (82, 83). The 2 latter studies speculate that the results would be related to a deficient BDNF secretion leading to intraneuronal BDNF accumulation. In view of reciprocal BDNF/glutamate modulation (84), normalization of BDNF levels in psychosis would counteract glutamatergic hypofunction. Based on the increased BDNF expression found in epilepsy, a comorbid psychotic state would display milder symptoms than in schizophrenia, as is indeed depicted in interictal psychosis (9).

Drugs and NT Expression Dose-dependent fluoxetine-induced neuroprotection has been described in animal models of epilepsy, ischemia, and Parkinson disease (85Y87). In our series, we did not observe differences in neuronal density but found increased BDNF immunoreactivity in CA1, the same region reported as protected, and with increased BDNF levels in fluoxetinetreated ischemic gerbils (85). A similar result has also been found in the human dentate gyrus (88). Interestingly, the CA1 region is one of the most affected subfields by neuronal loss in MTLE. In our series, most BDNF immunoreactivity detected in CA1 was in glial cells, and it has been shown that astrocytes are particularly affected by fluoxetine and paroxetine (but not tricyclics) treatment and respond with BDNF upregulation (89).

FIGURE 7. Neurotrophin 3 (NT3) expression in mesial temporal lobe epilepsy (MTLE) specimens with and without psychiatric comorbidities and in nonepileptic controls. (AYT) There was faint to moderate NT3 expression in neuronal and glial cells and faint to strong staining in blood vessels (arrows) in all groups and subfields of the hippocampal formation. (U) No differences in NT3immunoreactive area were found among groups. ENT, entorhinal cortex; GL, granular layer; HIL, hilus; PAR, parasubiculum; PRO, prosubiculum; SUB, subiculum. Scale bar = (AYT) 50 Km. Ó 2013 American Association of Neuropathologists, Inc.

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contrast to animal model results (22), and measurements of NT3 mRNA in the granular layer in epileptic patients (52). It has been suggested that NT3 can trigger sprouting and inhibit epileptogenesis (15), and we hypothesize that, in human MTLE, where a seizure-prone circuit is already established, decreased NT3 input to the dentate gyrus (e.g. from the entorhinal cortex) would contribute in the chronic phase to sustain hyperexcitability and to halt further progression of sprouting. Indeed, we found a positive correlation between seizure frequency, MFS, and granular layer NGF, in agreement with earlier work in animal models (53, 54). Neurotrophin 3 expression in non-neuronal tissue such as blood vessels has been verified by others, and one of the proposed mechanisms would result in increased nitric oxide production via endothelial nitric oxide synthase (55). Although increased neuronal nitric oxide production in MTLE may be inferred (56), we did not identify qualitative differences in NT3 expression in hippocampal blood vessels between control and epileptic patients, suggesting that NT3-mediated endothelial nitric oxide production is unaltered. The association between seizure facilitation and increased NGF expression has been shown in animal models of epilepsy (14, 53) and is in agreement with our result of positive correlation between seizure frequency and NGF expression in the granular layer. On the other hand, we found that patients with secondarily generalized seizures exhibited decreased NGF expression in the hilus, CA2, and subiculum versus patients with CPS. In a recent study, Alapirtti et al (57) compared blood samples of 3 TLE patients with secondarily generalized seizures and 11 patients with CPS, suggesting that the more severe the seizure type (i.e. secondarily generalized seizures), the stronger the inflammatory response after an acute seizure. In our series, a molecule related to seizure facilitation was found downregulated in patients with more severe seizures;however, when NGF is not able to interact with their receptors, seizure development is halted (58). Inasmuch as we found distinct hippocampal subfields related to seizure frequency and seizure type, differential modulation and action of NGF are feasible. Future studies with NTs receptors will be able to clarify this finding. Despite NGF upregulation found in most hippocampal formation subfields of patients with epilepsy, specimens with MTLE and psychosis showed decreased NGF immunoreactivity in the granular layer, hilus, CA2, CA1, and all subicular complex when compared with MTLE without psychiatric comorbidities. An equivalent result was seen for BDNF immunoreactivity in the granular layer. There is a significant and stable association between schizophrenia and memory impairment (59), although its basis is unknown (60). Most studies comprise verbal memory deficits, but poor performance of auditory memory tasks and visuospatial delayed recognition have also been reported in patients with schizophrenia and their

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The effects of typical and atypical antipsychotics on cell proliferation and apoptosis have been investigated repeatedly, with controversial results. Haloperidol treatment in animal models has been shown to increase hippocampal neurogenesis (90), or to have no effect (91), to promote survival of hippocampal stem cells (92) and to induce apoptosis in cortical neurons (93). In our series, significant neuronal loss was seen in CA3 and subiculum of MTLE + P patients taking haloperidol versus those not taking haloperidol in the same group, in agreement with evidence that antipsychotics can reduce brain tissue volume (94). Our results also showed decreased BDNF and NGF hippocampal expression in MTLE + P patients taking haloperidol, in accordance with animal model studies showing decreased BDNF hippocampal levels (95) and NGF cortical levels (96).

CONCLUSIONS

ACKNOWLEDGMENTS The authors thank Renata Caldo Scandiuzzi, Renato Meirelles e Silva, and Jose Eduardo Peixoto-Santos for the excellent technical support in neo-Timm histochemistry, panoramic NeuN image acquisition, and Western blot, respectively. REFERENCES 1. Mathern GW, Babb TL, Pretorius JK, et al. Reactive synaptogenesis and neuron densities for neuropeptide Y, somatostatin, and glutamate decarboxylase immunoreactivity in the epileptogenic human fascia dentata. J Neurosci 1995;15:3990Y4004 2. TL, Kupfer WR, Pretorius JK, et al. Synaptic reorganization by mossy fibers in human epileptic fascia dentata. Neuroscience 1991;42:351Y63

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It is important to acknowledge some limitations inherent to our findings in this study. Even with a relatively small sample size, the present results suggest that further studies exploring MTLE and related comorbidities are worthwhile. Although we could not perform stereologic counts because of limited tissue source from surgery, our neuron density numbers are in agreement with recent hippocampal stereologic counts performed in MTLE specimens (97). In fact, because all MTLE surgical specimens were freshly collected and submitted to identical processing, differences among them are particularly relevant. In summary, the present results provide the first demonstration of differential NT expression in human MTLE hippocampal formation with and without psychiatric comorbidities, supporting the close association between MFS and NTs. It also indicates that the use of haloperidol in MTLE might relate to increased neuronal loss and decreased NT expression. Our results are in agreement with most studies done with postmortem major depression, schizophrenia specimens, and animal models that have independently provided important pathophysiologic hallmarks. The relatively high prevalence of psychiatric symptoms in MTLE patients suggests mechanisms and/or substrates shared in these conditions. Clearly, different psychopathologic states in MTLE rely on distinct structural and neurochemical milieu. In the static concept of chronic MTLE, we are not able to define which exact variable might contribute to the genesis or to the maintenance of a particular psychiatric comorbidity, but it is hoped that future research on the morphologic and biochemical abnormalities in this scenario will delineate the molecules that may become targets for new treatments.

3. Babb TL, Brown WJ, Pretorius J, et al. Temporal lobe volumetric cell densities in temporal lobe epilepsy. Epilepsia 1984;25:729Y40 4. Kandratavicius L, Hallak JE, Young LT, et al. Differential aberrant sprouting in temporal lobe epilepsy with psychiatric co-morbidities. Psychiatry Res 2012;195:144Y50 5. Kandratavicius L, Rosa-Neto P, Monteiro MR, et al. Distinct increased metabotropic glutamate receptor type 5 (mGluR5) in temporal lobe epilepsy with and without hippocampal sclerosis. Hippocampus 2013: Epub Ahead of Print; doi: 10.1002/hipo.22160 6. Sundram F, Cannon M, Doherty CP, et al. Neuroanatomical correlates of psychosis in temporal lobe epilepsy: Voxel-based morphometry study. Br J Psychiatry 2010;197:482Y92 7. Taylor DC. Factors influencing the occurrence of schizophrenia-like psychosis in patients with temporal lobe epilepsy. Psychol Med 1975;5: 249Y54 8. Roberts GW, Done DJ, Bruton C, et al. A ‘‘mock up’’ of schizophrenia: Temporal lobe epilepsy and schizophrenia-like psychosis. Biol Psychiatry 1990;28:127Y43 9. Beard AW, Slater E. The schizophrenic-like psychoses of epilepsy. Proc R Soc Med 1962;55:311Y16 10. Kanner AM. Is major depression a neurologic disorder with psychiatric symptoms? Epilepsy Behav 2004;5:636Y44 11. Craddock N, Owen MJ. Molecular genetics and the relationship between epilepsy and psychosis. Br J Psychiatry 2010;197:75Y76 12. Kandratavicius L, Lopes-Aguiar C, Bueno-Junior LS, et al. Psychiatric comorbidities in temporal lobe epilepsy: Possible relationships between psychotic disorders and involvement of limbic circuits. Rev Bras Psiq 2012;34:454Y66 13. Kandratavicius L, Ruggiero RN, Hallak JE, et al. Pathophysiology of mood disorders in temporal lobe epilepsy. Rev Bras Psiq 2012;34: S233Y59 14. Adams B, Sazgar M, Osehobo P, et al. Nerve growth factor accelerates seizure development, enhances mossy fiber sprouting, and attenuates seizure-induced decreases in neuronal density in the kindling model of epilepsy. J Neurosci 1997;17:5288Y96 15. Xu B, Michalski B, Racine RJ, et al. Continuous infusion of neurotrophin-3 triggers sprouting, decreases the levels of TrkA and TrkC, and inhibits epileptogenesis and activity-dependent axonal growth in adult rats. Neuroscience 2002;115:1295Y308 16. Scharfman HE, Goodman JH, Sollas AL. Actions of brain-derived neurotrophic factor in slices from rats with spontaneous seizures and mossy fiber sprouting in the dentate gyrus. J Neurosci 1999;19: 5619Y31 17. Mathern GW, Babb TL, Micevych PE, et al. Granule cell mRNA levels for BDNF, NGF, and NT-3 correlate with neuron losses or supragranular mossy fiber sprouting in the chronically damaged and epileptic human hippocampus. Mol Chem Neuropathol 1997;30:53Y76 18. Isackson PJ, Huntsman MM, Murray KD, et al. BDNF mRNA expression is increased in adult rat forebrain after limbic seizures: Temporal patterns of induction distinct from NGF. Neuron 1991;6:937Y48 19. Binder DK, Croll SD, Gall CM, et al. BDNF and epilepsy: Too much of a good thing? Trends Neurosci 2001;24:47Y53 20. Kokaia Z, Kelly ME, Elmer E, et al. Seizure-induced differential expression of messenger RNAs for neurotrophins and their receptors in genetically fast and slow kindling rats. Neuroscience 1996;75:197Y207 21. Vezzani A, Ravizza T, Moneta D, et al. Brain-derived neurotrophic factor immunoreactivity in the limbic system of rats after acute seizures and during spontaneous convulsions: Temporal evolution of changes as compared to neuropeptide Y. Neuroscience 1999;90: 1445Y61 22. Shetty AK, Zaman V, Shetty GA. Hippocampal neurotrophin levels in a kainate model of temporal lobe epilepsy: A lack of correlation between brain-derived neurotrophic factor content and progression of aberrant dentate mossy fiber sprouting. J Neurochem 2003; 87:147Y59 23. Shimizu E, Hashimoto K, Okamura N, et al. Alterations of serum levels of brain-derived neurotrophic factor (BDNF) in depressed patients with or without antidepressants. Biol Psychiatry 2003;54:70Y75 24. Durany N, Michel T, Zochling R, et al. Brain-derived neurotrophic factor and neurotrophin 3 in schizophrenic psychoses. Schizophr Res 2001;52:79Y86

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51. Bender R, Heimrich B, Meyer M, et al. Hippocampal mossy fiber sprouting is not impaired in brain-derived neurotrophic factor-deficient mice. Exp Brain Res 1998;120:399Y402 52. Mathern GW, Babb TL, Leite JP, et al. The pathogenic and progressive features of chronic human hippocampal epilepsy. Epilepsy Res 1996;26:151Y61 53. Gall CM, Isackson PJ. Limbic seizures increase neuronal production of messenger RNA for nerve growth factor. Science 1989;245: 758Y61 54. Rocamora N, Palacios JM, Mengod G. Limbic seizures induce a differential regulation of the expression of nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3, in the rat hippocampus. Brain Res Mol Brain Res 1992;13:27Y33 55. Meuchel LW, Thompson MA, Cassivi SD, et al. Neurotrophins induce nitric oxide generation in human pulmonary artery endothelial cells. Cardiovasc Res 2011;91:668Y76 56. Leite JP, Chimelli L, Terra-Bustamante VC, et al. Loss and sprouting of nitric oxide synthase neurons in the human epileptic hippocampus. Epilepsia 2002;43(Suppl 5):235Y42 57. Alapirtti T, Waris M, Fallah M, et al. C-reactive protein and seizures in focal epilepsy: A video-electroencephalographic study. Epilepsia 2012;53:790Y96 58. Rashid K, Van der Zee CE, Ross GM, et al. A nerve growth factor peptide retards seizure development and inhibits neuronal sprouting in a rat model of epilepsy. Proc Natl Acad Sci U S A 1995;92: 9495Y99 59. Aleman A, Hijman R, de Haan EH, et al. Memory impairment in schizophrenia: A meta-analysis. Am J Psychiatry 1999;156:1358Y66 60. Harrison PJ. The neuropathology of schizophrenia. A critical review of the data and their interpretation. Brain 1999;122(Pt 4):593Y624 61. Robles O, Blaxton T, Adami H, et al. Nonverbal delayed recognition in the relatives of schizophrenia patients with or without schizophrenia spectrum. Biol Psychiatry 2008;63:498Y504 62. Newcomer JW, Farber NB, Jevtovic-Todorovic V, et al. Ketamineinduced NMDA receptor hypofunction as a model of memory impairment and psychosis. Neuropsychopharmacology 1999;20:106Y18 63. da Penha Berzaghi M, Cooper J, Castren E, et al. Cholinergic regulation of brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) but not neurotrophin-3 (NT-3) mRNA levels in the developing rat hippocampus. J Neurosci 1993;13:3818Y26 64. Knipper M, da Penha Berzaghi M, Blochl A, et al. Positive feedback between acetylcholine and the neurotrophins nerve growth factor and brain-derived neurotrophic factor in the rat hippocampus. Eur J Neurosci 1994;6:668Y71 65. Buckingham SD, Jones AK, Brown LA, et al. Nicotinic acetylcholine receptor signalling: Roles in Alzheimer’s disease and amyloid neuroprotection. Pharmacol Rev 2009;61:39Y61 66. D’Hoedt D, Bertrand D. Nicotinic acetylcholine receptors: An overview on drug discovery. Expert Opin Ther Targets 2009;13:395Y411 67. Rispoli V, Marra R, Costa N, et al. Choline pivaloyl ester enhances brain expression of both nerve growth factor and high-affinity receptor TrkA, and reverses memory and cognitive deficits, in rats with excitotoxic lesion of nucleus basalis magnocellularis. Behav Brain Res 2008; 190:22Y32 68. Coyle JT. Glutamate and schizophrenia: Beyond the dopamine hypothesis. Cell Mol Neurobiol 2006;26:365Y84 69. Javitt DC, Zukin SR. Recent advances in the phencyclidine model of schizophrenia. Am J Psychiatry 1991;148:1301Y8 70. Rujescu D, Bender A, Keck M, et al. A pharmacological model for psychosis based on N-methyl-d-aspartate receptor hypofunction: Molecular, cellular, functional and behavioral abnormalities. Biol Psychiatry 2006;59:721Y29 71. Castren E, da Penha Berzaghi M, Lindholm D, et al. Differential effects of MK-801 on brain-derived neurotrophic factor mRNA levels in different regions of the rat brain. Exp Neurol 1993;122:244Y52 72. Chen AC, Shin KH, Duman RS, et al. ECS-Induced mossy fiber sprouting and BDNF expression are attenuated by ketamine pretreatment. J Ect 2001;17:27Y32 73. Grillo RW, Ottoni GL, Leke R, et al. Reduced serum BDNF levels in schizophrenic patients on clozapine or typical antipsychotics. J Psychiatr Res 2007;41:31Y35

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1041

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25. Toyooka K, Asama K, Watanabe Y, et al. Decreased levels of brain-derived neurotrophic factor in serum of chronic schizophrenic patients. Psychiatry Res 2002;110:249Y57 26. Vargas HE, Gama CS, Andreazza AC, et al. Decreased serum neurotrophin 3 in chronically medicated schizophrenic males. Neurosci Lett 2008; 440:197Y201 27. Knable MB, Barci BM, Webster MJ, et al. Molecular abnormalities of the hippocampus in severe psychiatric illness: Postmortem findings from the Stanley Neuropathology Consortium. Mol Psychiatry 2004;9: 609Y20 28. Pedrini M, Chendo I, Grande I, et al. Serum brain-derived neurotrophic factor and clozapine daily dose in patients with schizophrenia: A positive correlation. Neurosci Lett 2011;491:207Y10 29. Gama CS, Andreazza AC, Kunz M, et al. Serum levels of brain-derived neurotrophic factor in patients with schizophrenia and bipolar disorder. Neurosci Lett 2007;420:45Y48 30. Balu DT, Hoshaw BA, Malberg JE, et al. Differential regulation of central BDNF protein levels by antidepressant and non-antidepressant drug treatments. Brain Res 2008;1211:37Y43 31. Valvassori SS, Stertz L, Andreazza AC, et al. Lack of effect of antipsychotics on BNDF and NGF levels in hippocampus of Wistar rats. Metab Brain Dis 2008;23:213Y19 32. Gittins R, Harrison PJ. Neuronal density, size and shape in the human anterior cingulate cortex: A comparison of Nissl and NeuN staining. Brain Res Bull 2004;63:155Y60 33. Stan AD, Ghose S, Gao XM, et al. Human postmortem tissue: What quality markers matter? Brain Res 2006;1123:1Y11 34. Blumcke I, Coras R, Miyata H, et al. Defining clinico-neuropathological subtypes of mesial temporal lobe epilepsy with hippocampal sclerosis. Brain Pathol 2012;22:402Y11 35. Berg AT. Identification of pharmacoresistant epilepsy. Neurol Clin 2009; 27:1003Y13 36. Engel J Jr. Surgery for seizures. N Engl J Med 1996;334:647Y52 37. Sachdev P. Schizophrenia-like psychosis and epilepsy: The status of the association. Am J Psychiatry 1998;155:325Y36 38. Zochodne DW, Cheng C. Neurotrophins and other growth factors in the regenerative milieu of proximal nerve stump tips. J Anat 2000;196: 279Y83 39. Bechade C, Mallecourt C, Sedel F, et al. Motoneuron-derived neurotrophin-3 is a survival factor for PAX2-expressing spinal interneurons. J Neurosci 2002;22:8779Y84 40. Calinescu AA, Liu T, Wang MM, et al. Transsynaptic activityYdependent regulation of axon branching and neurotrophin expression in vivo. J Neurosci 2011;31:12708Y15 41. Lorente de No R. Studies on the structure of the cerebral cortex II. Continuation of study of the ammonic system. J Psychol Neurol 1934;46: 113Y77 42. Abercrombie M. Estimation of nuclear population from microtome sections. Anat Rec 1946;94:239Y47 43. Mathern GW, Adelson PD, Cahan LD, et al. Hippocampal neuron damage in human epilepsy: Meyer’s hypothesis revisited. Prog Brain Res 2002;135:237Y51 44. Mathern GW, Pretorius JK, Mendoza D, et al. Hippocampal N-methyld-aspartate receptor subunit mRNA levels in temporal lobe epilepsy patients. Ann Neurol 1999;46:343Y58 45. Mathern GW, Mendoza D, Lozada A, et al. Hippocampal GABA and glutamate transporter immunoreactivity in patients with temporal lobe epilepsy. Neurology 1999;52:453Y72 46. Mathern GW, Pretorius JK, Kornblum HI, et al. Human hippocampal AMPA and NMDA mRNA levels in temporal lobe epilepsy patients. Brain 1997;120:1937Y59 47. Mathern GW, Babb TL, Vickrey BG, et al. The clinical-pathogenic mechanisms of hippocampal neuron loss and surgical outcomes in temporal lobe epilepsy. Brain 1995;118:105Y18 48. Peixoto-Santos JE, Galvis-Alonso OY, Velasco TR, et al. Increased metallothionein I/II expression in patients with temporal lobe epilepsy. Plos One 2012;7:e44709 49. Scharfman HE. The CA3 ‘‘backprojection’’ to the dentate gyrus. Prog Brain Res 2007;163:627Y37 50. Thom M. Hippocampal sclerosis: Progress since Sommer. Brain Pathol 2009;19:565Y72

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86. 87. 88. 89. 90. 91. 92. 93. 94. 95.

96.

97.

hippocampal CA1 region induced by transient ischemia. Exp Neurol 2007;204:748Y58 Jin Y, Lim CM, Kim SW, et al. Fluoxetine attenuates kainic acidYinduced neuronal cell death in the mouse hippocampus. Brain Res 2009;1281: 108Y16 Chung YC, Kim SR, Park JY, et al. Fluoxetine prevents MPTP-induced loss of dopaminergic neurons by inhibiting microglial activation. Neuropharmacology 2011;60:963Y74 Chen B, Dowlatshahi D, MacQueen GM, et al. Increased hippocampal BDNF immunoreactivity in subjects treated with antidepressant medication. Biol Psychiatry 2001;50:260Y65 Allaman I, Fiumelli H, Magistretti PJ, et al. Fluoxetine regulates the expression of neurotrophic/growth factors and glucose metabolism in astrocytes. Psychopharmacology (Berl) 2011;216:75Y84 Dawirs RR, Hildebrandt K, Teuchert-Noodt G. Adult treatment with haloperidol increases dentate granule cell proliferation in the gerbil hippocampus. J Neural Transm 1998;105:317Y27 Halim ND, Weickert CS, McClintock BW, et al. Effects of chronic haloperidol and clozapine treatment on neurogenesis in the adult rat hippocampus. Neuropsychopharmacology 2004;29:1063Y69 Keilhoff G, Grecksch G, Bernstein HG, et al. Risperidone and haloperidol promote survival of stem cells in the rat hippocampus. Eur Arch Psychiatry Clin Neurosci 2010;260:151Y62 Pillai A, Dhandapani KM, Pillai BA, et al. Erythropoietin prevents haloperidol treatment-induced neuronal apoptosis through regulation of BDNF. Neuropsychopharmacology 2008;33:1942Y51 Moncrieff J, Leo J. A systematic review of the effects of antipsychotic drugs on brain volume. Psychol Med 2010;40:1409Y22 Angelucci F, Mathe AA, Aloe L. Brain-derived neurotrophic factor and tyrosine kinase receptor TrkB in rat brain are significantly altered after haloperidol and risperidone administration. J Neurosci Res 2000;60: 783Y94 Terry AV Jr, Gearhart DA, Warner S, et al. Protracted effects of chronic oral haloperidol and risperidone on nerve growth factor, cholinergic neurons, and spatial reference learning in rats. Neuroscience 2007;150:413Y24 Alonso-Nanclares L, Kastanauskaite A, Rodriguez JR, et al. A stereological study of synapse number in the epileptic human hippocampus. Front Neuroanat 2011;5:8

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74. Pirildar S, Gonul AS, Taneli F, et al. Low serum levels of brain-derived neurotrophic factor in patients with schizophrenia do not elevate after antipsychotic treatment. Prog Neuropsychopharmacol Biol Psychiatry 2004;28:709Y13 75. Green MJ, Matheson SL, Shepherd A, et al. Brain-derived neurotrophic factor levels in schizophrenia: A systematic review with meta-analysis. Mol Psychiatry 2011;16:960Y72 76. Egan MF, Kojima M, Callicott JH, et al. The BDNF val66met polymorphism affects activity-dependent secretion of BDNF and human memory and hippocampal function. Cell 2003;112:257Y69 77. Numata S, Ueno S, Iga J, et al. Brain-derived neurotrophic factor (BDNF) Val66Met polymorphism in schizophrenia is associated with age at onset and symptoms. Neurosci Lett 2006;401:1Y5 78. Gratacos M, Gonzalez JR, Mercader JM, et al. Brain-derived neurotrophic factor Val66Met and psychiatric disorders: Meta-analysis of case-control studies confirm association to substance-related disorders, eating disorders, and schizophrenia. Biol Psychiatry 2007;61:911Y22 79. Forlenza OV, Diniz BS, Teixeira AL, et al. Effect of brain-derived neurotrophic factor Val66Met polymorphism and serum levels on the progression of mild cognitive impairment. World J Biol Psychiatry 2010; 11:774Y80 80. Chou IC, Tsai CH, Lee CC, et al. Brain-derived neurotrophic factor (BDNF) Val66Met polymorphisms in febrile seizures. Epilepsy Res 2004;60:27Y29 81. Bragatti JA, Schenkel LC, Torres CM, et al. No major clinical impact of Val66Met BDNF gene polymorphism on temporal lobe epilepsy. Epilepsy Res 2010;88:108Y11 82. Iritani S, Niizato K, Nawa H, et al. Immunohistochemical study of brain-derived neurotrophic factor and its receptor, TrkB, in the hippocampal formation of schizophrenic brains. Prog Neuropsychopharmacol Biol Psychiatry 2003;27:801Y7 83. Takahashi M, Shirakawa O, Toyooka K, et al. Abnormal expression of brain-derived neurotrophic factor and its receptor in the corticolimbic system of schizophrenic patients. Mol Psychiatry 2000;5: 293Y300 84. Mattson MP. Glutamate and neurotrophic factors in neuronal plasticity and disease. Ann N Y Acad Sci 2008;1144:97Y112 85. Kim do H, Li H, Yoo KY, et al. Effects of fluoxetine on ischemic cells and expressions in BDNF and some antioxidants in the gerbil

Neurotrophins in mesial temporal lobe epilepsy with and without psychiatric comorbidities.

Despite the strong association between epilepsy and psychiatric comorbidities, data on clinicopathologic correlations are scant. We previously reporte...
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