Effects of Electromagnetic Pulses on the Effects of Electromagnetic Pulses on the Permeability of Blood-Brain-Barrier Permeability of Blood-Brain-Barrier

Gui-Rong Ding, Ph.D

Department of Radiaition Medicine, Fourth Military Medical University,

China

IntroductionIntroduction

EMP( Electromagnetic pulses) exposure resulted in EMP( Electromagnetic pulses) exposure resulted in various changes in morphology and function of nervous various changes in morphology and function of nervous system.system. BBB (Blood-Brain-Barrier) plays an important role in BBB (Blood-Brain-Barrier) plays an important role in maintaining homeostasis in brain, and BBB disruption can maintaining homeostasis in brain, and BBB disruption can initiate the development of brain injuries.initiate the development of brain injuries.

Our previous study indicate that membrane is one of Our previous study indicate that membrane is one of the sensitive targets of EMP.the sensitive targets of EMP.

Blood–Brain Barrier and the Tight Junction

Astrocytic Foot ProcessAstrocytic Foot Process

Tight junctionTight junction

EndotheliumEndothelium

Basal LaminaBasal Lamina

Our concernOur concern

EMP exposure EMP exposure BBB openningBBB openning??

Molecular mechanism?Molecular mechanism?

Material and MethodsMaterial and Methods

SD rats, SD rats, male, 6-8 weeks old ， 200±20g

Animals Animals

EMP Generator GTEM-Cell

Material and MethodsMaterial and Methods

Exposure apparatusExposure apparatus

EMP parametersEMP parametersFrequency band 0-1.5 GHzFrequency band 0-1.5 GHz

Half widthHalf width 100ns100ns

Pulses numberPulses number 50 50 , 100 100 and 200 200

Intensity Intensity 50 50 , 100, 200 and 400 kV/m100, 200 and 400 kV/m

Material and MethodsMaterial and Methods

BBB permeability detectionBBB permeability detection

Serial frozen section, whole coronal slices of brainSerial frozen section, whole coronal slices of brainEvans Blue (EB) tracer and fluorescent microscope Evans Blue (EB) tracer and fluorescent microscope Lanthanum nitrate Lanthanum nitrate tracertracer and transmission electron microscope and transmission electron microscope

Protein expressionProtein expression

Western blottingWestern blottingImmunofluorescence Immunofluorescence

Material and MethodsMaterial and Methods

200kV/m, 200pulses

ResultsEMP induced BBB opening (dose-effect)

100kV/m, 200pulses

50kV/m, 200 pulses

control

ResultsEMP induced BBB opening (dose-effect)

200kV/m, 50 pulses

200kV/m, 100pulses

200kV/m, 200pulses

Results

4h after exposure2h after exposure 6 h after exposure

200kV/m, 200pulses

EMP induced BBB opening (time-effect)

Results

control (×10000) control (×10000) 200kV/m 200pulses200kV/m 200pulses (×10000) (×10000)

EMP induced BBB opening

Changes of BBB tight junction related proteins Changes of BBB tight junction related proteins after EMP exposureafter EMP exposure

ControlControl

OccludinOccludin

Claudin-5Claudin-5

ZO-1ZO-1

3h after exposure3h after exposure ((200kV/m, 200pulses)200kV/m, 200pulses)

Protein expression of tight junction related Protein expression of tight junction related proteins after EMP exposureproteins after EMP exposure

ConCon

SOL CSK SOL CSK SOL CSK SOL CSKSOL CSK SOL CSK SOL CSK SOL CSK

0hr0hr 3hr3hr 6hr6hr

ZO-1ZO-1

OccludinOccludin 65kDa65kDa

220kDa220kDa

Claudin-5Claudin-5

ConCon 0hr0hr 3hr3hr 6hr6hr

OccludinOccludin 65kDa65kDa

22kDa22kDa

220kDa220kDaZO-1ZO-1

Membrane protein

CSK

SOL

Cellular fractionation

Conclusions

EMP exposure may lead to an increase in EMP exposure may lead to an increase in the permeability of BBB in rats. the permeability of BBB in rats.

The injury of BBB tight junction was The injury of BBB tight junction was involved in EMP-induced BBB permeability involved in EMP-induced BBB permeability increase. increase.

Ongoing workOngoing workPrimary cultured brain microvascular endothelial cells(BMEC)

Primary cultured brain astrocyte

In vitro BBB modelIn vitro BBB model

TranswellTranswellTranswellTranswell

Polyester-Polyester-multiple-multiple-membranemembrane

Polyester-Polyester-multiple-multiple-membranemembrane

TranswellTranswellTranswellTranswell

AstrocytesAstrocytesAstrocytesAstrocytes

Culture dishCulture dishCulture dishCulture dish Culture dishCulture dishCulture dishCulture dish

BMECBMECBMECBMEC

AstrocytesAstrocytesAstrocytesAstrocytes

TranswellTranswellTranswellTranswell

Blood Testicle BarrierBlood Testicle Barrier

1h

2h

control

6h

Effects of EMP (200kV/m, 200 pulses) on the Effects of EMP (200kV/m, 200 pulses) on the Permeability of Blood-Testicle -Barrier Permeability of Blood-Testicle -Barrier

Changes of BTB tight junction related protein-ZO-1 Changes of BTB tight junction related protein-ZO-1 after after EMP (200kV/m, 200 pulses)EMP (200kV/m, 200 pulses) exposureexposure

control 1h

2h 6h

Further research

To set up in vitro BBB modelTo set up in vitro BBB model

To identify the size of the To identify the size of the molecules that get though molecules that get though BBB after different parameters EMP exposureBBB after different parameters EMP exposure

To clarify the molecular mechanism of EMP-To clarify the molecular mechanism of EMP-induced BBB openinginduced BBB opening

Tight JunctionsTight Junctions（（ TJsTJs ））

BTBBTB

BRBBRB

BBBBBB In commonIn common

Barrier system

EMP EMP exposureexposure

Sen

sitive organs

Sen

sitive organs

By observing the permeability and changes of TJs By observing the permeability and changes of TJs in other in other ““Barrier systems”, to find out the common Barrier systems”, to find out the common mechanism of EMP-induced barrier injury.mechanism of EMP-induced barrier injury.

Further research