A different look on the pathophysiology
Gerd Wasser
wasser2010

wasser2010
In most of the cases
there is only a small
vessel involved in
brain strokes.



wasser2010
A small vessel or some
capillaries are occluded.
Cells in the spot of
impact survive app. 8
min then undergo
necrosis with release of
potassium.
This potassium
overload depolarizes the
neighboring tissue.



Neurons do not have
access to capillaries and
do not get glucose
delivered by the serum.
They are supplied by
astrocytes with lactate.
This explains the high
dependency on oxygen
supply.
waßer2010©


ATP deficiency causes MPT (Mitochondrial
Permeability Transition), rupture of the MOM,
and the release of cytochrome c out of the intermembrane space.
wasser2010


The ATP-dependent Potassium channel (KATP) is
a potassium selective ion channel. Normal
intracellular ATP concentrations inhibit the
channel.
Consequently it is opened if ATP
concentrations decline.
Wasser09©


The wave of potassium released into the
interstitium depolarizes the surrounding cells,
creating the penumbra.
From now on the Penumbra decides the loss of
function and the patient’s fate.
wasser2010

Since the clinical symptoms of brain strokes rely
on the penumbra expansion and not on the loss
of function in the small primary necrotic area,
most of the symptoms will disappear after
repolarisation.
waßer2010©
Fig. 3. Microglia are rapidly activated after local BBB disruption
A. Nimmerjahn et al., Science 308, 1314 -1318 (2005)
Published by AAAS
ATP Content in Focal Ischemia
3
nmol/kg
2.5
2
1.5
MCA cortex ATP
1
Penumbra ATP
0.5
0
Control
60-min ischemia
60-min reperfusion
24-h reperfusion
FRANK A. WELSCH: Regional Expression of Immediate-Early Genes and Heat-Shock Genes after Cerebral Ischmia,
Annals of the New York Academy of Sciences,Vol 723, pp 318-327
wasser2010
ATP Content in Focal Ischemia
3
The spot of primary
impact will undergo
necrosis,
nmol/kg
2.5
2
1.5
1
0.5
MCA cortex ATP
Penumbra ATP
0
whereas the penumbra
will develop controlled
destruction by apoptosis.
FRANK A. WELSCH: Regional Expression of Immediate-Early Genes
and Heat-Shock Genes after Cerebral Ischmia, Annals of the New York
wasser2010

One factor leading to resistance to ischemia is
the elevated reducing power of glycogen
present within astrocytes but not in neurons.
Glycogen regulation and functional role in mouse white matter. A. M Brown, S. B. Tekkok, and B. R Ransom (2003)
J. Physiol. 549, 501-512
waßer2010©

The ATP content after occlusion and
reperfusion does not reflect the metabolism of
surviving neurons but is picturing metabolic
active glial cells.
waßer2010©

wasser2010
Consecutively more
and more cells
depolarize widening
the area of the
Penumbra (perifocal
edema).

wasser2010
Finally it comes to a
balance between cells
being capable of
maintaining their ion
channels and
depolarized cells.
12
Lactate in Focal Ischemia
10
nmol/kg
8
MCA cortex Lactate
6
Penumbra Lactate
4
2
0
Control
60-min
ischemia
60-min
reperfusion
24-h
reperfusion
FRANK A. WELSCH: Regional Expression of Immediate-Early Genes and Heat-Shock Genes after Cerebral Ischmia, Annals of the New York Academy of Sciences,Vol 723, pp
318-327
wasser2010
12
Lactate in Focal Ischemia
Lactate
10
Lactate content in MCA
area and penumbra
results from astrocytes
glucose consumption.
nmol/kg
8
6
MCA cortex Lactate
Penumbra Lactate
4
2
Lactate normally is
transmitted to neurons
to fuel energy demand.
0
wasser2010


wasser2010
The occluded vessel is
not perfused, but
vessels and capillaries
of the penumbra are
not occluded.
Hence they can
deliver nutrients and
oxygen.

wasser2010
But since the cells
inside the edema are
depolarized even the
supply of oxygen and
glucose does not
restart metabolism.
80
Penumbra
Water
Body Water in % of Body Weight
70
60
% of total weight
The water content of
the penumbra is
often called vasogen.
And there have been
recommendations to
use hypertonic glucose
or mannose solutions
to remove it.
50
baby
40
female
75
male
30
60
50
20
40
40
35
30
10
30
20 20
16 16
5 4 4
0
total
intra cellular extracellular intravascular insterstitial
wasser2010
80
Body Water in % of Body Weight
70
% of total weight
60
50
40
baby
female
75
male
30
60
50
20
40
40
30
10
35
30
20
20
16
5
0
total
intra cellular
extracellular
wasser2010
4
16
4
intravascular
insterstitial
80
Penumbra
Water
Body Water in % of Body Weight
70
60
% of total weight
Since the BBB (BrainBlood-Barrier)
formed by unique
cerebral capillaries is
mostly still intact,
the larger part of the
edema water is
derived from
intracellular space.
50
baby
40
female
75
male
30
60
50
20
40
40
35
30
10
30
20 20
16 16
5 4 4
0
total
intra cellular extracellular intravascular insterstitial
wasser2010


wasser2010
CT tries to reopen the
occluded vessel. In
case of success oxygen
enriched blood will
perfuse the area
behind the former
occlusion.
This area is
endangered to get
damaged by RI
(Reperfusion Injury)


wasser2010
Since there is no
metabolism in the
area of PENUMBRA,
the situation is
unchanged.
Scarring will start in
the wake of ongoing
inflammation.


wasser2010
In animal trials the
survival time of the
cells in the
PENUMBRA was 48
hours.
Then scarring will
start in the wake of
progressive apoptosis.

Dy is in the area undergoing apoptosis
sufficiently high to sustain prolonged ATP
production (24-48 h) and protein import.
Yulia Kushnareva, Donald D. Newmeyer
Bioenergetics and Cell Death, Volume 1201 Mitochondrial Research in Tranaslational Medicine, pp. 50-57, July 2010
wasser2010

wasser2010
For starting the
glycolysis one mol
glucose requires two
mols of ATP ( first one
to create glucose-6phosphate, the second
one for
transformation into
fructose-1,6diphosphate).

We showed some
twenty years ago that
full blood in ozone
treated patients
contains two fold
more ATP than
controls even three
weeks after the last
treatment.

wasser2010
Extra corporeal
treatment of blood
cells with ozone gas is
mandatory because
due to chemical laws
not a single molecule
of ozone can enter the
vasculature.
R
R
+
→
R
R
R-C-C=C-C-R
+
O3
→
wasser2010
R-C-O-O-O-C-C-R
R
R
+
→
R
R-C-C=C-C-R
+
O3
wasser2010
→
R
R-C-O-O-O-C-C-R


Ozone gas perfused through full blood will
react in msec with blood cell membranes.
The oxygen (ozone gas contains at least 97 %
oxygen) will not react.
wasser2010
Ozone Therapy
By means of the
glutathioneperoxidase-reductase
and the pentose shunt
the cells gain ATP
via glycolysis.
Since ozone therapy is
not an oxygen
delivering therapy it
does not induce RI
(Reperfusion Injury).
wasser2010
wasser20003
Wasser09©

Inflammation is the reaction to the disturbance
of the metabolic body balance.
wasser2010

Stages of Inflammation

1.) Stage of edema
2.) Invasion of WBC and macrophages
3.) Activation of fibroblasts / micro glial cells

This is the only way our body can respond with!


wasser2010
Ozone and
Inflammation
If we reduce the first
stage of inflammation,
the other following
stages are abrogated.
Wasser09©


wasser2010
RBCs deliver ATP to
the penumbra area
through still perfused
capillaries and
vessels.
The edema effectively
shrinks while ion
channels again restart
working.

wasser2010
From clinical
experience we know
this procedure will
take only 10 min of
time.

wasser2010
After 48 hours of
stroke onset in
Conventional treatment,
Scarring affects the
spot of primary impact
and the penumbra
zone.


wasser2010
In contrast to the
extended scarring
under conventional
treatment
ozone treated patients
develop small
scarring reflecting the
zone of first impact.
The scarring of the Penumbra defines the extent of loss
of function after stroke.

Last not least comparison of MRIs after stroke in
conventional and ozone treatment:
wasser2010
wasser2010

With the extracorporeal ozone treatment we have
a powerful tool at hand to treat inflammation
not only in the penumbra area, but also in
inflammation supported diseases like tumors.
wasser2010

Thank you very much for your
patience and attention.
wasser2010
News Focus
CANCER IMMUNOLOGY:
Cancer's Bulwark Against Immune Attack: MDS Cells
Jean Marx
First noticed in the 1970s, myeloid-derived suppressor cells appear to play
a key role in sustaining tumors; new methods of overcoming them are
being tested
Science 11 January 2008:
Vol. 319. no. 5860, pp. 154 - 156
DOI: 10.1126/science.319.5860.154
wasser2010


By means of the glutathione-peroxidase-reductase
and the pentose shunt the cells gain ATP via
glycolysis.
Since ozone therapy is not an oxygen delivering
therapy it does not induce RI (Reperfusion
Injury).
wasser2010
Additional information.
wasser2010
DG0’ = -25,1 kJ mol-1
;
waßer2010©
+
NAD
+
NADH + H
Lactate
Pyruvate
waßer2010©



MOMP (Mitochondrial Outer Membrane
Permeabilisation) requires ATP for the
apoptotic pathway otherwise necrosis occurs.
Yulia Kushnareva, Donald D. Newmeyer
Bioenergetics and Cell Death, Volume 1201 Mitochondrial Research in
Tranaslational Medicine, pp. 50-57, July 2010

Temporary (3h) and reversible lowering of ATP
levels beyond a threshold value (by about 30%)
committed cells to undergo apoptosis.
wasser2010

The partial loss of ATP for a longer time (6h) or
a short-term nearly complete ATP depletion
resulted in necrosis.

The released cytochrome c can re-enter
mitochondria, and its pool is initially
sufficiently abundant to sustain respiration.
wasser2010
waßer2010©

MOM is impermeable for proteins, and this
protein barrier is essential for cell survival.

The decline of ATP to nearly zero levels can
easily be explained. The elevation of lactate
raises many questions. One might be touched
to assume the elevated lactate levels after 60
min of ischemia and 60 min and 24 hours of
reperfusion, respectively, might indicate a
survival of neurons with basic metabolic
activity.
waßer2010©

One factor leading to resistance is the elevated
reducing power of glycogen present within
astrocytes but not in neurons.
waßer2010©


Cortical astrocytes exhibit a relative resistance
to NAD(H) catabolism induced by O2- and
glucose deprivation.
Annals of the New York Academy of Sciences, Vol. 1147, Fiskum et al.:
Oxidative Stress Promotes Mitochondrial Metabolic Failure.
waßer2010©


Lactate uptake from extracellular fluid shows
that astrocytes have faster influx and transport
capacity than neurons.
Annals of the New York Academy of Sciences, Vol. 1147, Gerald A. Dienel and
Nanxy F. Cruz: Imaging Brain Activation, Simple Pictures of Complex Biology.
waßer2010©

Lactate transfer from a single gap junction
coupled astrocyte to another exceeds astrocyteto- neuron lactate shuttle.
waßer2010©

In animal trials fluerescent positiv proteins
belonging to mitochondria were by 66% higher
in filipodia of astrocytes than in the surounding
neuronal elements.
waßer2010©


The spot of primary impact will undergo
necrosis,
whereas the penumbra will develop controlled
destruction by apoptosis.
wasser2010
12

10
Lactate in Focal Ischemia
The spot of primary impact will undergo
necrosis,
nmol/kg
8
6
whereas the penumbra will develop controlled
destruction by apoptosis.
MCA cortex Lactate
Penumbra Lactate
4
2
0
Control
60-min
ischemia
60-min
reperfusion
wasser2010
24-h
reperfusion

In neurons glucolytic ATP is preferred for
transport of glutamate to synaptical vesicles.
waßer2010©
Brain energetics in the limelight.
Science 22 January 1999:
Vol. 283. no. 5401, pp. 496 - 497
DOI: 10.1126/science.283.5401.496NEUROSCIENCE:
Energy on Demand
Pierre J. Magistretti, Luc Pellerin, Douglas L. Rothman, Robert G. Shulman*



Kinases use ATP as substrate to induce
conformational changes in downstream enzymes.
If we use a kinase first and then ozone
treatment, there is no effect on the penumbra.
We are endangered to overdose the kinase.
wasser2010
wasser2010
wasser2010

MOM is permeable to small molecules,
containing nonselective channels formed by the
family of mitochondrial porins, also known as
voltage dependent anion channel proteins
(VDAC).
wasser2010

However the MOM is impermeable for
proteins.
wasser2010

Ca2+ overload and oxidative stress, conditions in
RI, neurodegeneration, and toxic stress pave the
way for MPT-dependent cell death.
wasser2010

Cyclophilin D deficient cells fail to undergo
necrosis, but show typical signs of apoptosis.
wasser2010

Thapsigargin is an inhibitor of the ER Ca2+
pumps, leading to mitochondrial Ca2+
overload.
wasser2010


Na+/Ca2+ exchanger (NCX) by expelling 3 Na+
against 1 Ca2+ uses the electrochemical gradient
of Na+ .
It has low affinity but high capacity moving
thousands of Ca2+ ions per second.
wasser2010



PMCA (Plasma membrane Ca2+ ATPase) has high
affinity, but exerts low capacity, and deals with
lower Ca2+ concentrations. It is ATP powered.
Since the transport is electrogenic, depolarization
might reverse the exchanger’s direction.
This may occur in excitotoxity.
wasser2010

Cytochrome c upon release out of the
mitochondrial intermembrane space activates
caspases in the induction of apoptosis.
wasser2010
Regional Metabolite Levels in Focal Ischemia
FRANK A. WELSCH: Regional Expression of Im m ediate-Early Genes and Heat-Shock Genes after Cerebral Ischm ia, Annals of the New
York Academ y of Sciences,Vol 723, pp 318-327
12
10
8
nmol/kg
MCA cortex ATP
Penumbra ATP
6
MCA cortex Lactate
Penumbra Lactate
4
ATP
2
Lactate
0
Control
60-min ischemia
60-min reperfusion
waßer2010©
24-h reperfusion
Regional Metabolite Levels in Focal Ischemia
3
2,5
nmol/kg
2
MCA cortex ATP
1,5
Penumbra ATP
1
0,5
0
Control
60-min ischemia
60-min reperfusion
24-h reperfusion
FRANK A. WELSCH: Regional Expression of Immediate-Early Genes and Heat-Shock Genes after Cerebral Ischmia, Annals of
the New York Academy of Sciences,Vol 723, pp 318-327
waßer2010©


Letm1 (EF hand-containing trans membrane
protein 1) is one of the elusive Ca2+-transport
proteins.
Letm 1 catalyzes slow uptake of Ca2+ into
mitochondria at submicromolar concentrations
in exchange for H+ ions.
Nicolas Demaurex and Damon Poburko: A Revolving Door for Calcium,
Science 2 October 2009,Vol. 326. no. 5949, pp. 57-58
wasser2010


MIM contains a Ca2+ uniporter, a Na+/Ca2+ exchanger, and a Ca2+/H+ -exchanger.
The uniporter mediates rapid uptake of Ca2+
driven by the negative membrane potential,
Whereas the exchangers extrude Ca2+ from the
mitochondria.
wasser2010




Anti- apoptotic proteins:
Bcl-2 and Bcl-xL
Pro-apoptotic proteins:
Bax and Bak
wasser2010

Cytochrome c triggers caspase-9 activation by
binding to Apaf-1 to form a procaspase-9activating heterodimeric protein complex
named the apoptosome.
wasser2010

The apoptosome proteolitically activates the
executioner procaspase-3 to induce apoptosis.
wasser2010

Mitochondria contain the caspase independent
death effectors AIF and EndoG, which reside in
the mitochondrial intermembrane space.
wasser2010

Staurosporine or TNF mediated changes n
intracellular pH activates Bax, a pro-apoptotic
Bcl-2 family protein.
wasser2010


AIF trans located to the nucleus through the
cytosol induces chromatin condensation and
DNA fragmentation.
EndoG causes oligonucleosomal DNA
fragmentation without caspase activation.
wasser2010

Bcl-2 and Bcl-xL are predominantly located in
the MOM, maintaining mitochondrial
membrane integrity during apoptotic stress.
wasser2010


The enzyme Dicer cuts out of dsRNA 21-28
nucleotide long RNAs which are named
siRNAs.
This short, double strand then are built in into
a protein complex RISC (RNA-induced
silencing complex).
wasser2010



RISC with the help of siRNA docks on to
mRNAs.
Since the RISC complex exhibits RNA-helicase
and nuclease activities, the mRNA gets
disentangled and dissect.
And since the mRNA is now unprotected,
intracellular nucleases degrade it soon.
wasser2010

Cardiolipin is obligatory required for the
activity of proteins involved in energy
transduction, such as ANT and Complexes III
and IV of the ETC.
wasser2010

Oxidation of Cardiolipin causes cytochrome c
detachment form the MIM and compromises
electron shuttling from Complex III to IV.
wasser2010

China did express the Yin-Yang Theory:
Wasser09©
Despite Optimal Myocardial Reperfusion due to Reperfusion Injury
25
20
15
10
5
0
death directly related to acute MI
incidence of cardiac failure over 5 ys
Derek, M. and al.: Myocardial Reperfusion Injury, N. Engl. Med. 2007; 357; 1121-1135
wasser2010
wasser2010


The water content of the penumbra is often
called vasogen.
And there have been recommendations to use
hypertonic glucose or mannose solutions to
remove it.
wasser2010