hyothermia in stroke 1

Elucidation of neuroprotective effect of hypothermia in

stroke: It’s underlying mechanism

Dr. Manik Mathur

MBBS, Dipcard (U.K)

PhD Student

Division of stroke

University of Nottingham

Contents

• Introduction

• Pathogenesis

Cytokines

Blood-Brain Barrier

• Hypothesis and Aims

• Hypothermia and its neuroprotective effects

• Material-Methods

• Results

• Conclusion

• Future Aspects

Introduction

• Stroke is defined as rapidly developing clinical signs of focal ( or global )disturbance of cerebral function , with symptoms lasting 24 h or longer ,or leading to death , with no apparent cause other than of vascular origin.

• Every year an estimated 152,000 people in the UK have a stroke.

• There are around 1.2 million stroke survivors in the UK.

• Stroke costs the UK over £8.2 billion every year.

• Stroke can affect anyone, no matter what their age and sex. Arounda quarter of all strokes happen to people under the age of 65, and around400 children (0-18) have a stroke each year.

Types of Stroke

Pathogenesis

Cytokines

• These are many number of substances that are secreted by specific cells of

the immune system

• They are a category of signaling molecules that are used extensively in

cellular communication.

• They are proteins, peptides, or glycoproteins

Tumor Necrosis Factor- α

• It is a cytokine involved in systemic inflammation and stimulates acute phase reaction.

• It is produced chiefly by activated macrophages and CD4+ lymphocytes, Natural Killer cells.

• A local increase in its concentration results in cardinal signs of Inflammation to occur: heat, swelling, redness, pain and loss of function.

• TNF-alpha expression after stroke stimulates expression of tissue factor and adhesion molecules for leukocytes, release of interleukin-1 (IL-1), nitric oxide, reduction of tissue-plasminogen activator.

Interleukin-1beta(IL-1β)

• Also known as Catabolin.

• It is a member of interleukin 1 cytokine family.

• It is produced by activated macrophages as a proprotein, which is proteolytically processed to its active form by caspase 1.

• It is an important mediator of inflammatory response, and is involved in a variety of cellular activities, including cell proliferation, differentiation, and

apoptosis.

Blood-Brain Barrier(BBB)

The BBB is a separation of circulating blood from the brain extracellular fluid

(BECF) in the central nervous system (CNS). It occurs along all capillaries and

consists of tight junctions around the capillaries that do not exist in normal

circulation.

Hypothesis and Aims

The aim of the project is to comparatively investigate whether intra-ischaemic

and post-ischaemic hypothermia(35 C) can effectively prevent oxygen-glucose

deprivation-mediated BBB damage using a triple culture model of human BBB.

It is also to assess whether the regulations of prominent pro-inflammatory

cytokines, namely IL-1β and TNF-α may be involved in the putative beneficial

effects of hypothermia.

Hypothermia(HT)

• Mild 32–35 C (90–95 F)

• Moderate, 28–32 C (82–90 F)

• Severe, 20–28 C (68–82 F)

• Profound at less than 20 C (68 F)

Neuroprotective Effects of HT

• The Neuroprotective effects of HT has been long recognized, but use for the therapy of neuronal injuries was abandoned due to severe side effects such as cardiac arrhythmia, shivering , infections and coagulation disorders.

• In past decade it has come to be recognized that even mild or moderate therapeutic modulation of temperature may substantially avert brain damage caused by ischemia in both experimental stroke and neural injuries.

• Lowers inflammatory cytokines levels

• Maintains BBB integrity and function

• Changes in membrane permiability leading to increased cytosolic Na+ and H+ levels

• Inhibition of transcription and translation

Material-Methods and Results

Cell culture

• Human brain microvascular endothelial cells(HBMEC)

• Human Astrocytes(HA)

• Human Pericytes(HP)

In vitro model of the blood-brain barrier

Inserts in well plates

• Dual culture model

• Culturing HBMEC and HA

• HP cultured in fresh 12-well plates.

Blood-brain barrier integrity

• Blood-brain barrier integrity was

assessed by measuring

transendothelial electrical

resistance (TEER; Ωcm2).

• TEER was measured using STX

electrodes and an EVOM

resistance meter .

• To measure the membrane permeability , Evan’s blue albumin(EBA) and

sodium fluorescein (NaF) dyes were used.

• inserts fresh 12-well plates containing Hank’s Balanced Salt Solution

(HBSS)

• HBSS + Dye in luminal chamber.

• Samples were taken after 1 hour of incubation at 37 C and the concentration of dye measured using a multiscan plate reader.

*denotes P<0.05 v/s controls

†denotes P<0.05 v/s 4hours OGD

#denotes P<0.05 v/s 20hours OGD

Trans Endothelial Electrical Resistance , (n=11)

Samples with HT show better outcome then OGD only samples

0

20

40

60

80

100

120

140

controls 4 h ogd 4h ogd+ ht 20h ogd 20h ogd + ht 4h ogd+ 20ht

TEER

Ὠcm

2

**

*, †*, †, #

*,†

*denotes P<0.05 v/s controls

+denotes P<0.05 v/s 4 hours OGD + HT

denotes P<0.05 v/s 4 hours OGD

$denotes P<0.05 v/s 20 hours OGD + HT

Evan’s Blue Dye, (n=6)

Decreased EBA levels in HT samples when compared to OGD samples

signifies less membrane damage with HT

0

5

10

15

20

25

controls 4 h ogd 4h ogd+ ht 20h ogd 20h ogd + ht

4h ogd+ 20ht

EB

A c

lea

red

vo

l.6

0m

in/μ

l

**,♯

*,+

*,+*,+,$

*denotes P<0.05 v/s controls

+denotes P<0.05 v/s 4 hours OGD

$denotes P<0.05 v/s 20 hours OGD

denotes P<0.05 v/s 4 hours OGD + HT

Sodium Fluorescence, (n=5)

Decreased Naf levels in HT samples when compared to OGD samples signifies

less membrane damage with HT

0

10

20

30

40

50

60

70

80

controls 4 h ogd 4h ogd+ ht 20h ogd 20h ogd + ht 4h ogd+ 20ht

Nafcle

are

d v

ol.6

0m

in/μ

l

**

*,♯

+,♯,$

+,$

ELISA

• Enzyme-linked immunosorbent assay (ELISA) is a test that uses antibodies

and color change to identify a substance

• Superanatants are collected from cell flasks after the respective treatments.

• 300μl of superanatant used.

• Cytokines activities measured using specific antigen-anibody kit.

Hypothermia decreases TNF-α conc. In both 20 & 4 hours samples.

*denotes P<0.05 v/s controls

+denotes P<0.05 v/s 4hours OGD

#denotes P<0.05 v/s 4hours OGD +HT

$denotes P<0.05 v/s 20 hours OGD

Tumor necrosis factor-α (n=6), HBMEC only

0

2

4

6

8

10

12

14

16

controls 4 hours OGD 4 hours OGD + HT

20 hpurs OGD

20 hours OGD + HT

4 hours OGD + 20 HT

Hu

man

TN

F-α

co

nc.(

pg

/ml/

mg

)

*,♯

*,+,♯,$

++,$

TNF-α conc. decreased in HT samples

Tumor Necrosis Factor- α (n=2) , Human Pericytes only

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

controls 4 hours 4 h + Ht 20 hours 20 h + ht 4 h+ 20 HT

TN

F –

αc

on

c(p

g/m

l/m

g)

*denotes p<0.05 v/s controls

#denotes p<0.05 v/s 4 hours OGD

+denotes p<0.05 v/s 4 hours OGD + HT

$denotes p<0.05 v/s 20 hours OGD

%denotes p<0.05 v/s 4 hours OGD + 20 HT

Interleukin-1β (n=6), HBMEC only

HT decreased IL-1β levels with 20 hours samples.

0

2

4

6

8

10

12

14

controls 4 hours OGD 4 hours OGD + HT 20 hours OGD 20 hours OGD + HT

4 hours OGD + 20 HT

Hu

man

IL-1β

con

c.(p

g/m

l/m

g)

*

*,+,$,%

*,+,$

#

#

*denotes P<0.05 v/s controls

#denotes P<0.05 v/s 4 hours OGD

+denotes P<0.05 v/s 20 hours OGD

$denotes P<0.05 v/s 4 hours OGD + HT

Interleukin-1β (n=6) Human Pericytes only

HT decreased IL-1β levels with 20 hours samples

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

controls 4 hours OGD 4 hours OGD + HT

20hours OGD 20hours OGD + HT

4hours OGD + 20 HT

Hu

am IL

-1β

con

c(p

g/m

l/m

g)

*,#

*,#,+

*,#,+

$

*denotes p<0.05 v/s controls

Interleukin-1β (n=5), Hypothermia only samples (-OGD)

Samples with HT(4&20 hours) have less IL-1β conc. when compared controls.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

control 4ht 20ht

**

IL-1β levels in human pericytes with only HT(-OGD)

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

control 4ht 20ht

Hu

ma

n IL

-1β

co

nc.(

pg

/ml/

mg

)

*denotes p<0.05 v/s controls

+denotes p<0.05 v/s 4hours hypothermia

Human Astrocytes

IL-1β levels significantly decreased in 4 & 20 HT only samples without OGD

0

0.5

1

1.5

2

2.5

control 4ht 20ht

Hu

ma

n IL

-1β

co

nc.(

pg

/ml/

mg

)

*,+

*

Conclusion

• It is increasingly recognized that inflammation cascade is a key element in

progression of stroke.

• Induced, moderate HT may have a role in clinical setting.

• HT has significantly reduced cytokines levels both intra and post ischemic

treatments with/out OGD.

• It has been beneficial in reducing BBB damage by maintaining integrity and

function.

• Much more is required to evaluate the benefits of HT in future.

Future Aspects /On going research

• Many more experiments to be conducted on HT

• Oxidative stress levels under HT treatments

• Explore other tight junctions such as Claudin -5, occludin

• For DNA defects under apoptosis(TUNNEL)

• F-Actin scans

• MMP 2 & 9 activities

Sincere Thanks :Prof : Ulvi Bayraktutan

&

Kirtiman srivastava

Beili Shao

Kamini Vashisht

Zuraidah Abdulah

Any Questions?