influence of gsm and umts on the blood brain barrier in vitro · influence of gsm and umts on the...
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Influence of GSM and UMTS on theBlood Brain Barrier in vitro
– additional resultsIntl. Workshop on long term effects, München, 11.-12. Okt. 2007
Dr. rer. nat. Helmut FrankeKlinik und Poliklinik für Neurologie
Universitätsklinikum Münster
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In vitro-Experiments on exposure to RF-fields of mobile telecommunication
C. Blood brain barrier
- BBB in vitro (rat brain endothelial cell cultures) - GSM 1800 exposure - UMTS exposure• differential gene expression (genechip arrays)• selection of BBB related candidates• verification of diff. gene expression (qRT-PCR)
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goals• Influence of RF-EMF on endothelial cellsof the BBB ?
• reduction of BBB towards an in-vitro model• investigations on cellular level• identification of potential EMF-targets onmolecular level
• no hypotheses on pathophysiological issues
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overview: project parts• establishment and characterization of RBEC cultures as BBB in-vitro model
• design of exposure unit• exposure of RBEC and isolation of RNA• gene expression analysis• qRT-PCR verification of gene regulation
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Endothelial cells of the cerebral capillaries form the permeability barrier
The Blood-Brain Barrier (BBB) •maintenance homeostasis of the CNS•essential for proper brain function•control of substance flow between brain tissue and circulating blood•controlled import of nutrients into the CNS•protection against toxins
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Rat brain capillary endothelial cells (RBEC)
isolation of endothelial cells
sowing and culturingcortices
homogenizing
1st enzymatic digest
isolation of capillary vessels
2nd enzymatic digest RBEC monolayer
removal of meninges
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characterization of RBEC• + squamous morphology• + von-Willbrand-Factor-VIII• + vimentin• + tight junction proteins: ZO-1, occludin• - smooth muscle actin, GFAP, CD11b• ---> minimal cell contamination
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overview: project parts• establishment and characterization of RBEC cultures as BBB in-vitro model
• design of exposure unit• exposure of RBEC and isolation of RNA• gene expression analysis• qRT-PCR verification of gene regulation
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assembly of radial waveguide
Ø 82
Ø 100
52
5
32
3
R192
R115.0Ø55
Ø82.0
• 6 petridishes• 40 cm diameter, 9 cmheight
• sample holder centrespetridishes
• temperature probe • field probe
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GSM-Exposure setup:
• amplifier• signal generator• wave guides• fiberoptic temperature probes
• incubator
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overview: project parts• establishment and characterization of RBEC cultures as BBB in-vitro model
• design of exposure unit• exposure of RBEC and isolation of RNA• gene expression analysis• qRT-PCR verification of gene regulation
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exposure groups: GSM1800 / UMTS @0.4-8.0 W/kg (72h)
5+2 GSM 5+2 GSM 5+2 GSM 5+2 GSM5+2 UMTS 5+2 UMTS 5+2 UMTS 5+2 UMTS
0.4 1.0 3.0 8.0
sham sham sham sham
t
t ttt
t t t
biological replicates for gene chip analysis:
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temperature control groups: 38°C / 40°C (72h)
5+2 (38°C) 5+2 (40°C)ΣΣΣΣ = 70 Chip Arrays
38°C 40°C
37°C 37°Ctt
t t
biological replicates:
max. temperature @ 8W/kg: 38°C
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RNA isolation protocol • lysis of RBEC immediately after termination of exposure (< 5 min.)
• RNA isolation from RBEC: Qiagen RNeasy Micro-Kitstore samples @ -70°C
• quality control: Agilent Bioanalyzer• RNA-conc: min. 1µg/µL
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overview: project parts• establishment and characterization of RBEC cultures as BBB in-vitro model
• design of exposure unit• exposure of RBEC and isolation of RNA• gene expression analysis• qRT-PCR verification of gene regulation
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Affymetrix GeneChip® Rat Genome 230 2.0 Array
•data tables showing the signal intensities of the various probe sets•28000 genes on the chip!(=31099 probe sets)
31099 probe sets
source: http://privatewww.essex.ac.uk/~harry/images/affymetrix-genechip-hgu133p.gif
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18,663filtering of ‚absent calls‘
•MAS 5.0 (Microarray Suite, Affymetrix)•normalization of signal levels•of 31099 probe sets on the chip, 18663 could be detected reliably („present“ calls in ≥ 3 of 5 chips per experimental group)
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14,287
filtering of genes with fold-change < 1.4
•of 18663 present probe sets,14287 showed at least 1.4x change in gene expression compared to sham exposed RBEC
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filtering by t-test vs. temperature controls
11,488 GSM8,900 UMTS
•of 14287 differentially expressed probe sets, differential expression of 11488 (GSM) or 8900 (UMTS) was not merely dueto temperature increase(p < 0.05).•manual selection of genes related to BBB
38°C 40°C37°C 37°Ctt
t t
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360 GSM231 UMTS
Discriminatory Genes Analysis: SAM (Significance Analysis of Microarrays)
1W/kg vs. 3W/kg
• GSM1800: of 11488 genes, 360 genes were identified by SAM as differentially expressed between 1 W/kg group and 3 W/kg•UMTS: 231 of 8900 genes•parameter: no false positives
1.0 W/kg 3.0 W/kg
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overview: project parts• establishment and characterization of RBEC cultures as BBB in-vitro model
• design of exposure unit• exposure of RBEC and isolation of RNA• gene expression analysis• qRT-PCR verification of gene regulation
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Selection of genes for qRT-PCR validation
•SELECTION CRITERIA:•BBB-Genes: 2x regulation•SAM analysis 1W/kg vs. 3W/kg: 3x regulation •other genes: 5x regulation
68 genes GSM61 genes UMTS
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Selected genes for qRT-PCR validation
•SELECTION CRITERIA:•BBB-Genes: 2x regulation•SAM analysis 1W/kg vs. 3W/kg: 3x regulation •other genes: 5x regulation•available for TaqMan® Low Densitiy Arrays
47 genes GSM45 genes UMTS
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selection of genes with BBB relevance(regulation to be validated)• Agtr1a (angitensin II receptor; vasoconstriction)• Col5a3 (procollagen Vα3; ECM)• Sdc2 (syndecan 2; monocyte migrat. brain endot.)• Slc33a1 (acetyl-CoA transporter)• Abcc8 (ATP binding cassette transp.: MRP)• Slc19a1 (folate & drug transporter)• Cldn1 (claudin1, ZO-1 & Occl. associated TJ-prot.)• Mmp2 (matrix metalloproteinase, involved in BBB regulation)
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samples for qRT-PCR analysis
5+5 GSM5+5 UMTS
biological replicates for qRT-PCR:
0.4 1.0 3.0 8.0t
shamt
t t t
shamtshamt shamt
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quantitative real time RT-PCR with TaqMan® Low Density Arrays
• reverse transcription of RNA to cDNA• 200 ng cDNA in 100 µL per channel• PCR reaction volume ~ 1 µL• 2x 47 genes (UMTS and GSM)• comparative quantification: ∆∆Ct-method• 7680 individual qRT-PCR reactions
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amplification plot for 384 qRT-PCR reactions (one TLDA)
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Ct(target gene) - Ct(endogenous control) = ∆Ct
∆Ct(exposed) - ∆Ct(sham) = ∆∆Ct
RQ = 2-∆∆Ct
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� establishment of an isolation method for RBEC� characterazation of RBEC� installation of exposure device and determination of field parameters� exposure of RBEC � RNA isolation� chip-arrays for differential gene expression� bioinformatic evaluation of gene-chip data� qRT-PCR experiments / evaluation of PCR data • identification of potential protein targets
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• Dept. Neurology Dr. Helmut Franke, Prof. Dr. P. Young Dir: Prof. E.B. Ringelstein, University Hospital Münster
• Chair f. Electromagnetic Theory Dr. Joachim Streckert, Dr. Andreas Bitz Dir: Prof. V. Hansen, BU Wuppertal
• IGF(Integrated Functional Genomics)Dr. Kurt Sieberns, Heike Stegemann, Hendrik Lahl, Dr. Martin EisenacherIZKF, University Hospital Münster
• Miltenyi BiotecDr. Corinna Scholz, Dr. Jan Schäferkordt
• Federal Office for Radiation Protection Dr. Monika Asmuß
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thank you