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  Clinical Study Expression analysis and clinical correlation of aquaporin 1 and 4genes in human hippocampal sclerosis N. Bebek a,b, ⇑ , Ö. Özdemir b , M. Sayitoglu b , Ö. Hatırnaz b , B. Baykan a,b , C. Gürses a , A. Sencer c , A. Karasu c,1 ,E. Tüzün a,b , I. Üzün d,e , S. Akat d , N. Cine f  , G. Sargin Kurt a , M. Imer c , U. Ozbek b , A. Canbolat c , A. Gökyigit a a Neurology Department, Istanbul Faculty of Medicine, Millet cad., 34390 Capa, Istanbul, Turkey b Institute for Experimental Medicine, Istanbul University, Istanbul, Turkey c Neurosurgery Department, Istanbul University, Istanbul, Turkey d Council of Forensic Medicine, Ministry of Justice, Istanbul, Turkey e Forensic Medicine Department, Faculty of Medicine, Mediterranean University, Antalya, Turkey f  Genetics Department, Kocaeli Medical Faculty, Kocaeli, Turkey a r t i c l e i n f o  Article history: Received 2 July 2012Accepted 2 December 2012 Keywords: AquaporinsEpilepsyEpileptogenesisGene expressionHuman hippocampusMesial temporal sclerosis a b s t r a c t Mesial temporal sclerosis (MTS) is the most frequent cause of drug resistant symptomatic partial epi-lepsy.Themechanismandgeneticbackgroundofthisuniquepathologyarenotwellunderstood.Aquapo-rins(AQP)areregulatorsofwaterhomeostasisinthebrainandareexpressedinthehumanhippocampus.WeexploredtheroleofAQPgenesinthepathogeneticmechanismsofMTSthroughanevaluationofgeneexpression in surgically removed human brain tissue. We analyzed  AQP1  and  4  mRNA levels by quanti-tative real-time polymerase chain reaction and normalized to  ABL  and  cyclophilin  genes, followed byimmunohistochemistry for  AQP4.  Relative expressions were calculated according to the delta Ct methodand the results were compared using the Mann-Whitney Utest. Brain specimens of 23 patients with epi-lepsy who had undergone surgery for MTS and seven control autopsy specimens were investigated. Clin-ical findings were concordant with previous studies and 61% of the patients were seizure-free in thepostoperative period.  AQP1  and  4  gene expression levels did not differ between MTS patients and controlgroups. Immunofluorescence analysis of   AQP4  supported the expression results, showing no difference.Previous studies have reported contradictory results about the expression levels of   AQP   in MTS. To ourknowledge, only one study has suggested upregulation whereas the other indicated downregulation of perivascular  AQP4 . Our study did not support these findings and may rule out the involvement of AQPin human MTS.   2013 Elsevier Ltd. All rights reserved. 1. Introduction Mesial temporal sclerosis (MTS), also known as hippocampalsclerosis (HS), is the most frequent underlying pathology in drugresistantepilepsy.Theepileptogenicfocusmainlyinvolvesthehip-pocampal formation, which shows typical neuropathological fea-tures. 1 The anatomical and physiological structure of thehippocampus, particularly the large pyramidal cells in the CA1 re-gion, is sensitive to external factors such as hypoxia during devel-opment. Therefore, early childhood risk factors causing neuronaldamage are thought to initiate the process of epileptogenesis pos-sibly in association with genetic factors. 2,3 A significant relation-ship between complicated febrile seizures (FS) and MTS has beenreported. 4,5 A number of molecular studies on human brain tissueinMTShave beenperformed 6,7 withmanygeneexpressionstudiescarried out in animal models and using human biopsy speci-mens. 8,9 However, the mechanism and genetic background of thisunique pathology is currently not elucidated.Aquaporins(AQP),thoughttobetheregulatorsofwaterhomeo-stasis in the brain, are expressed in the human hippocampus. Theyare transmembrane molecules and facilitate the movement of water across cellular compartments. 10  AQP4  normally displays ahigher distribution on the perivascular end feet membranes of astrocytes, andasignificantincreaseinperivascular  AQP4  hasbeenobserved in the CA1 region of sclerotic hippocampi, 11 but the nat-ure of this change – either genetic or acquired – is not known. It iswell-known that  AQP4  is important in the complex pathways of water and ion homeostasis. It has been hypothesized that  AQP4  in-creasesexcitabilitybypotassium(K + )buffermodification,interleu-kin secretion enhancement, and calcium-induced release of  0967-5868/$ - see front matter    2013 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +90 212 53 43 93; fax: +90 212 533 43 93. E-mail address: (N. Bebek). 1 In memory of Dr. Aykut Karasu, a dear and invaluable colleague who wasdedicated to epilepsy surgery. He oriented us to investigate the role of AQPs inhippocampal sclerosis. Unfortunately, he passed away before the termination of theproject.  Journal of Clinical Neuroscience 20 (2013) 1564–1570 Contents lists available at SciVerse ScienceDirect  Journal of Clinical Neuroscience journal homepage:  glutamate. 12,13  AQP4  is expressed by astrocytes in the scleroticCA1, and is furthermore positively correlated with glial fibrillaryacidic protein. Moreover, an increased and highly differentiateddistribution pattern of   AQP4  is associated with changes in theexpression of dystrophin and dystrophin associated protein com-plex genes. Astrocytes in the sclerotic hippocampus may directlyaffectexcitabilitybyalterationofwaterhomeostasisandK + buffer-ing due to the redistribution of   AQP4  transporters. 14 In MTS, theT2-weighted MRI diffusion coefficient of the hippocampus is highwhichis thought to be due to the presence of increased water flowin the hippocampus. 11 Therefore we aimed to investigate theexpression levels of aquaporins in the mesial temporal lobe tissueof patients with drug resistant epilepsy.  AQP4  is the predominant water channel in the brain and is con-centrated in astrocytic foot processes at the blood–brain bar-rier. 12,15 Defects in other aquaporin genes are known to berelated to some human diseases. 16 Aquaporin-1 (  AQP1 ), alsoknown as channel-forming integral membrane protein, was thefirst protein shown to function as a molecular water channel.  AQP1  is abundant in the choroid plexus. Scarcity or absence of   AQP1  has been reported in a few patients, resulting in restrictedability to concentrate the urine. 17  AQP2  and  3  are most importantin kidney function. Autoimmunity against  AQP4  is found in Devic’sdisease. A better understanding of these molecules and possiblemanipulation of their structures could help prevent brain edemadue to brain tumor or infarction. 18 In the field of epilepsy, information about the effects of   AQP   islimited.Onestudyshowed  AQP1 and  AQP4 expressioninastrocyteslocated in the hippocampal tissues, but only  AQP4  expression wasfound to be increased in perivascular astrocytes. 11 Conversely, Eidet al. showed a loss of perivascular  AQP4  in epilepsy patients . 16  Jamali et al. reported elevated  AQP1  in microarray studies on hip-pocampaltissues,butthesefindingswerenotverifiedinanexpres-sion analysis. 6 Our aim was to explore the role of the relevant  AQP   genes,namely  AQP1  and  4 , in the pathogenetic mechanisms of MTSthrough evaluation of the gene expression in surgically removedhuman brain tissue. 2. Material and methods Brain tissue was surgically removed from 23 patients with epi-lepsywhohadundergonesurgeryduetoMTS,andfromsevencon-trol autopsy specimens.  2.1. Brain tissue samples 2.1.1. Mesial temporal sclerosis samples All patients were investigated presurgically using a detailedstandard protocol including seizure semeiology, neurologicalexamination, neuropsychological testing, neuroimaging, and longterm non-invasive video-electroencephalogram (EEG) monitoring.Allpatientsmetthecriteriafordrugresistantmesialtemporallobeepilepsy. 19–21 The study was approved by the local Ethics Commit-tee. All patients were informed about the procedure and writteninformed consent was obtained. Twenty-three patients with con-cordant findings in their presurgical evaluation underwent selec-tive amigdalohippocampectomy (SAH) with or without anteriortemporal lobectomy (ATL), carried out by an experienced neuro-surgeon, and hippocampal tissue was removed (Table 1). A limitednumber of temporal polectomies were performed, just enough toidentifythe dura of the middle fossa and gain access to the tempo-ral horn. During resection, biopsies were taken from the head andbody of the hippocampus for examination as describedpreviously. 22,23  2.1.2. Control hippocampal samples Thecontrolgroupwascomprisedofautopsysamples.Autopsieswere performed within 24hours of death. Only one of the autopsycontrols was female and the average age was 50.1±standarddevi-ation(SD) 19.3(range, 21–73years). Thepatientshadnohistoryof brain disease and suffered from sudden death without associatedbrain damage. The cause of death was cardiovascular disease inthree patients, with penetrating injury, intoxication, hanging, andembolismdue to fracture of the femur the causes in the remainingfour patients. One additional patient who had died at age 1 due togastroenteritis was excluded for better uniformity of age. Entirehippocampi were removedat autopsy fromseven patients in asso-ciation with The Council of Forensic Medicine. This part of thestudy was approved by The Education and Research Commissionof The Council of Forensic Medicine.All surgical and autopsy tissue samples were frozen immedi-ately after removal in liquid nitrogen and stored at  80  C.  2.1.3. RNA isolation and cDNA synthesis The hippocampal tissues were homogenized in QIAzol Lysis Re-agent (Qiagen, Venlo, Netherlands). Total RNA was isolated byRNeasy Lipid Tissue Kit (Qiagen). RNA samples were treated usingDNAse (1U/ l g). RNA quality and quantity were checked withNanodrop 1000 (Thermo Fisher Scientific, Waltham, MA, USA).1 l gof totalRNAwasusedforcDNAsynthesisusingrandomhexa-mers(RocheDiagnostics, Mannheim,Germany)andMoloneyMur-ineLeukemiaVirusreversetranscriptase(ThermoFisherScientific)according to the protocol of the manufacturer.  2.2. Analysis of gene expression by real time quantitative reversetranscription polymerase chain reaction We analyzed  AQP1  and  4  besides two reference genes (  ABL  and cyclophilin [CYBP] ) by real time quantitative reverse transcriptionpolymerase chain reaction (PCR) which was carried out on theLight Cycler Instrument 480, with the LightCyler 480 Fast StartSYBR Green I Master Kit (Roche Diagnostics). The PCR conditionswere prepared according to the instructions of the manufacturer;5pmol of primers and 200ng of cDNA were used in each runand each sample was studied in duplicate. The specificity of theproduct amplification was confirmed by melting curve analysesand agarose gel electrophoresis. The PCR program was as follows:initial denaturation at 95  C for 7 minutes, amplification for 5s at95  C, 10s at 56–60  C and 10s at 72  C for 45 cycles, and meltingcurve for 15s at 60  C for one cycle. The suitable reference geneswere selected by GeNorm version 3.4 (GeNorm, Amel Ardennes,Belgium), as describedby Vandesompele et al. 24 The two most sta-ble genes were selected (  ABL  and  CYBP  ) for normalization. Relativeexpressions were calculated according to the delta Ct method,based on the mathematical model described by Livak et al. 25 REST2005 (Beta V1.9.9, Corbett Life Science, Germany) and the Statisti-calPackagefortheSocialSciences(SPSS,Chicago, IL, USA)softwarewere used. 26 Relative expression results of control and MTS tissues werecompared using the Mann-Whitney U(MWU) test. The associationbetween expression values and clinical variables (such as sex, age,age at seizure onset, FS, seizure frequency, age at surgery, diseaseduration from onset to surgery, and outcome) were also evaluatedusing the MWU test. 6,25 A  p  value of <0.05 was regarded as statis-tically significant.  2.3. Immunofluorescence for aquaporin 4 expression Five paraffin-embedded sections were obtained from both theMTS patients (n=5) and the autopsy specimens (n=5). The sam-ples were deparaffinized and the antigens retrieved, as described N. Bebek et al./Journal of Clinical Neuroscience 20 (2013) 1564–1570  1565  elsewhere. 27 Tissue sections were serially incubated with 10% goatserum for 1hour, rabbit anti-human  AQP4  Ab (1:400, Santa CruzBiotechnology, Santa Cruz, CA, USA) overnight at 4  C and second-ary Alexa-Fluor 594-conjugated anti-rabbit antibody (1:1000,Invitrogen, Carlsbad, CA, USA). Additional sections only incubatedwith the secondary antibody were used as negative controls. Thesections were investigated with a fluorescence microscope (Zeiss,Thornwood, NY, USA) for  AQP4  expression. Images of five fieldswere randomly selected and the number of   AQP4  positive cells ineachfieldwasrecorded.Theaveragenumberofcellsobtainedfrom125 fields (5 patients  5 sections  5 fields) was compared be-tween the MTS and control groups using Student’s  t  -test. A  p  valueof <0.05 was regarded as statistically significant. 3. Results The clinical characteristics of the patients are summarized inTable 1.  3.1. Clinical findings of mesial temporal sclerosis patients Seizure semeiology was concordant with mesial temporal lobesrcin, including rising epigastric sensations, staring, unrespon-siveness, and oral and manual automatisms. Seven patients hadexperienced secondarily generalized seizures at least once. All pa-tients met the drug resistant epilepsy criteria. 21 All had used morethan two first- and second-line appropriate antiepileptic drugcombinationsatthemaximumtolerabledoseandallofthemexpe-rienced failure of these antiepileptic drug therapies, and experi-enced seizures more than once a month causing disability intheir daily activities. Interictal and ictal EEG findings were consis-tent withneuroradiological and clinical features regardingthesideof MTS in all patients but Patient 17. In this patient, invasive EEGmonitoring was performed with two-sided depth electrodes and15 seizures were recorded before surgery.  3.2. Postoperative outcome TwelvepatientsunderwentSAHand11patientsunderwentATL (Table 1). The mean age at the time of surgery was 28.5years  Table 1 Clinical features, surgical procedure, prognostic evaluation and gene expression levels of patients with mesial temporal sclerosis No.  AQP1 expression  AQP4 expressionSex FebrileseizureClinicalhistoryFamily history Age atseizureonset(years)SurgicalprocedureAge atepilepsysurgery(years)Duration fromseizure onset tosurgery (years)Outcome-EngelclassificationP1  ; ;  F FS Left HPafter firstFSParkinsonism ingrandfather12 Right SAH 28 16 IBP2  ; ;;;;  M CFS PD athomeFS in five siblings andthree nephews, three of these siblings died8 Left SAH 47 39 IAP3    ;;;;  F CFS None None 11 Left SAH 32 21 IAP4    ;  M CFS HT Epilepsy in cousin, FS insister7 Left SAH 15 8 IIP5  ;    M None None Epilepsy in grandfather,FS in cousin22 Left SAH 28 7 IAP6  ;    M None None None 20 Left SAH 29 9 IIP7  ; ;  M None HT-LOC None 15 Right SAH 31 16 IAP8  ; ;;  M CFS DD,meningitisEpilepsy in grandmotherat 65 years old7 LeftAH+ATL 24 17 III or IVP9  ;    F None HT-LOC Far consanguinity 12 RightAH+ATL 39 27 IAP10  ;;    F FS PCOS Consanguinity, epilepsyin sister, twin7 Left SAH 28 21 IIP11    F CFS Deliveryat homeFS in uncle 22 RightAH+ATL 32 10 IAP12  ; ;  F None Oral afts Consanguinity, Behçetdisease15 LeftAH+ATL 19 4 III or IVP13  ; ;;  F None HT FS in daughter 34 Left SAH 63 29 IAP14  ;; ;;  F None HT None 10m LeftAH+ATL 25 24 IAP15  ; ;;  F None None None 19 LeftAH+ATL 34 15 IAP16    ;  F None None None 14 RightAH+ATL 38 24 IAP17  ; ;;  M None None Psychiatric disease intwo uncles10 Left SAH 45 35 III or IVP18  ; ;  M None Meningitis Cousin died due toepilepsy8m LeftAH+ATL 32 31 IIP19  ; ;  F CFS HT FS in father 7 LeftAH+ATL 27 20 IAP20  ; ;;;  M FS HT-LOC None 7 LeftAH+ATL 19 9 IBP21    F CFS None None 8 Right SAH 25 17 IAP22  ; ;  F FS DD FS in daughter 17 LeftAH+ATL 21 4 IAP23  ;    M FS None Consanguinity 20 Left SAH 27 7 IAAH=Amygdalohippocampectomy, ATL=anterior temporal lobectomy, CFS=complicated febrile seizure, DD=difficult delivey, FS=febrile seizure, F=female, HT=headtrauma, HP=hemiparesis, LOC=loss of consciousness, M=male, M=months, PCOS=polycystic ovary syndrome, PD=preterm delivery, SAH=selective amygdalohippo-campectomy,   / ;  =1-fold increased/decreased from mean,   / ;;  =10-fold increased/decreased from mean,   / ;;;  =100-fold increased/decreased mean,   / ;;;;  =1000-fold increased/decreased from mean.1566  N. Bebek et al./Journal of Clinical Neuroscience 20 (2013) 1564–1570  (range, 15–63years). The mean time elapsed between seizure on-set and surgery was 17years (range, 4–39years). The mean post-operative follow-up period was 4.2years (range, 1–8years).Fourteenpatientswere seizure-free(60.9%, Engel Class IA). Seizurefrequency did not change in three patients (Engel Class III or IV).Three patients experienced seizures after cessation of antiepilepticdrugs.  3.3. Tissue samples Thestandardpathologicalexaminationofthesurgicalspecimenwas carried out for all MTS patient samples and clear evidence of hippocampal sclerosis, such as diffuse neuronal loss, reactive glio-sis, granulation tissue, and corpora amilacea, was detected in allsamples.  3.4. Expression analysis The relative expression levels of   AQP1  and  AQP4  did not differbetween the MTS patients and the control group (Fig. 1). Therewere no significant association between clinical features and  AQP1  or  AQP4  expression (Fig. 2). However, it was notable thattwo patients (Patients 2 and 3) showing markedly decreased  AQP4  relative expression levels had distinct clustering in all  AQP4 expression–clinical comparisons. They were all over 30years of age, with a history of FS. They were seizure-free in the postopera-tive period. The time from seizure onset to epilepsy surgery wasover 17years.As expected, a significant association between  AQP4  and  AQP1 genes was found using the Spearman rho correlation (  p  =0.002).  3.5. Immunofluorescence for aquaporin 4 results All examined MTS brain samples displayed cells expressing  AQP4 , whereas control sections incubated only with secondaryantibody did not yield any staining. In both MTS and control brainsections,  AQP4  was mainly distributed in the cell surface and cyto-plasm, as described previously. 28,29 The average number (±SD) of   AQP4  positive cells for MTS and control groups was 32.9±6.4and 33.5±7.2, respectively, without statistical significance beingreached. 4. Discussion AQP functions as the primary water-selective channels in theplasma membranes of many cells and  AQP1  and  AQP4  are the pre-dominant AQP proteins in the brain. Previous reports have pre-sented contradictory results about the expression levels of   AQP  genes in MTS. To our knowledge, only one study suggests perivas-cular upregulation of   AQP4  whereas another single study revealeddownregulation. To our knowledge, our study is the third to ana-lyze AQP4 expression in human MTS. Although no statistically sig-nificantdifferencewasfoundbetweentheMTSpatientandcontroltissues,ageneraldecreasewasobservedintheexpressionlevelsof the  AQP1  and  AQP4  genes. Furthermore, two patients with similarcharacteristics showed very low expression levels in both genes.The clinical and laboratory characteristics of our MTS group re-flected the typical findings of MTS in the literature with regards toage, past medical and family history, FS, MRI and EEG find-ings. 4,30,31 Furthermore, seizure types, seizure frequency, responseto therapy, neuropsychological and psychiatric comorbidities, andpostoperative follow-up characteristics confirmed the commonclinical features of the disease, thus supportingthe conclusionthatthis was a reliable study group. However, our control group issmall due to the difficulty in obtaining human control tissue.Inour study,  AQP1  expressionlevels in MTS tissue didnot differfromthe control tissue, but five out of 23 (21.7%) MTS patients ex-pressed  AQP1  at slightly higher levels compared to controls.Although there was no statistical significance (  p  =0.09), the  AQP1 expression level was higher in MTS patients with a history of headtrauma than those with MTS who had not experienced head trau-ma. The microarray data in the study by Jamali et al. have shownan increased expression level of   AQP1 , but quantitative real-timePCR data were not consistent and the gene was excluded fromfur-ther analyses. 6 Lee et al. used the U133A GeneChip (Affymetrix,Santa Clara, CA, USA), which has oligonucleotide reporter se-quences for nine AQP genes (  AQP1  to  AQP9 ), and expression levelsshowed only  AQP1  and  AQP4  in the CA1 region of the hippocam-pus. 11 Similar to our results, two  AQP1  transcripts, though present,were not significantly elevated in MTS specimens. These two stud-ies were similar to our results. We also showed that no significantassociation between clinical features  AQP1  expression levels.However, our  AQP4  expression results contradict the report of Lee et al. 11 Lee et al. found  AQP4  to be significantly elevated inMTS specimens. In our study, relative expression levels of   AQP4 did not differ significantly between MTS patients and the controlgroup, and this finding was further supported by immunofluo-rescence experiments. Seven of 23 (30.4%) patients expressedthe  AQP4  gene at a slightly higher level compared to controls.However, 70% of patients showed up to 3.32 fold decreasedAQP4 expression levels compared to control samples, and eightof 23 patients showed a pronounced decrease in the  AQP4 expression compared to the mean expression value of MTS pa-tients as seen in Table 1 and Fig. 1b. We noted two exceptional MTS patients who showed a very prominent decrease in  AQP4 relative expression levels; they were both over 30years old,had a history of complicated FS, and a family history of FS caus-ing death in three siblings. These two patients were seizure freeafter 2.5 and 4years, although there was a long delay from sei-zure onset to epilepsy surgery. These findings could be explained Fig. 1.  (a) Aquaporin 1  (AQP1)  and (b) aquaporin 4  (AQP4)  relative expression levels between mesial temporal sclerosis patients and autopsy controls. N. Bebek et al./Journal of Clinical Neuroscience 20 (2013) 1564–1570  1567
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