Extracellular Diffusion Parameters in the Rat Somatosensory Cortex during Recovery from Transient Global Ischemia/Hypoxia

Abstract

Changes in the extracellular space diffusion parameters during ischemia are well known, but information about changes during the postischemic period is lacking. Extracellular volume fraction (alpha) and tortuosity (lambda) were determined in the rat somatosensory cortex using the real-time iontophoretic method; diffusion-weighted magnetic resonance imaging was used to determine the apparent diffusion coefficient of water. Transient ischemia was induced by bilateral common carotid artery clamping for 10 or 15 mins and concomitant ventilation with 6% O(2) in N(2). In both ischemia groups, a negative DC shift accompanied by increased potassium levels occurred after 1 to 2 mins of ischemia and recovered to preischemic values within 3 to 5 mins of reperfusion. During ischemia of 10 mins duration, alpha typically decreased to 0.07+/-0.01, whereas lambda increased to 1.80+/-0.02. In this group, normal values of alpha=0.20+/-0.01 and lambda=1.55+/-0.01 were registered within 5 to 10 mins of reperfusion. After 15 mins of ischemia, alpha increased within 40 to 50 mins of reperfusion to 0.29+/-0.03 and remained at this level. Tortuosity (lambda) increased to 1.81+/-0.02 during ischemia, recovered within 5 to 10 mins of reperfusion, and was increased to 1.62+/-0.01 at the end of the experiment. The observed changes can affect the diffusion of ions, neurotransmitters, metabolic substances, and drugs in the nervous system.

Full text

Extracellular diffusion parameters in the rat
somatosensory cortex during recovery from
transient global ischemia/hypoxia
Norbert Zoremba
1
, Ales
˘Homola
2,3
, Karel S
˘lais
3
, Ivan Vor
ˇı
´s
˘ek
2,3
, Rolf Rossaint
1
,
Alfred Lehmenku
¨hler
4
and Eva Sykova
´
2,3
1
Department of Anaesthesiology, University Hospital RWTH Aachen, Aachen, Germany;
2
Department of
Neuroscience and Center for Cell Therapy and Tissue Repair, 2nd Medical Faculty, Prague, Czech Republic;
3
Department of Neuroscience, Institute of Experimental Medicine, Academy of Sciences of Czech Republic,
Prague, Czech Republic;
4
Center for Pain Therapy, St. Vincent Hospital, Du¨sseldorf, Germany
Changes in the extracellular space diffusion parameters during ischemia are well known, but
information about changes during the postischemic period is lacking. Extracellular volume fraction
(a) and tortuosity (k) were determined in the rat somatosensory cortex using the real-time
iontophoretic method; diffusion-weighted magnetic resonance imaging was used to determine the
apparent diffusion coefficient of water. Transient ischemia was induced by bilateral common carotid
artery clamping for 10 or 15 mins and concomitant ventilation with 6% O
2
in N
2
. In both ischemia
groups, a negative DC shift accompanied by increased potassium levels occurred after 1 to 2 mins
of ischemia and recovered to preischemic values within 3 to 5 mins of reperfusion. During ischemia
of 10 mins duration, atypically decreased to 0.07±0.01, whereas kincreased to 1.80±0.02. In this
group, normal values of a= 0.20±0.01 and k= 1.55±0.01 were registered within 5 to 10 mins of
reperfusion. After 15 mins of ischemia, aincreased within 40 to 50 mins of reperfusion to 0.29±0.03
and remained at this level. Tortuosity (k) increased to 1.81±0.02 during ischemia, recovered within 5
to 10 mins of reperfusion, and was increased to 1.62±0.01 at the end of the experiment. The
observed changes can affect the diffusion of ions, neurotransmitters, metabolic substances, and
drugs in the nervous system.
Journal of Cerebral Blood Flow & Metabolism advance online publication, 11 June 2008; doi:10.1038/jcbfm.2008.58
Keywords: diffusion; edema; extracellular space; ischemia; MRI; reperfusion
Introduction
The extracellular space (ECS) represents the
microenvironment of nerve cells and serves as an
important communication channel (Nicholson,
1979; Sykova
´, 1992). The movement of substances
in this microenvironment by diffusion is essential
for extrasynaptic or ‘volume’ transmission among
neurons, axons, and glia (Fuxe and Agnati, 1991;
Zoli et al, 1999; Nicholson and Sykova
´, 1998;
Sykova
´, 2004). Diffusion in the ECS is also essential
for the delivery of oxygen and glucose from the
vascular system to brain cells (Nicholson, 2001).
Normal brain function depends on a continuous
supply of oxygen and glucose to maintain the
extracellular/intracellular ionic distribution and to
enable synaptic as well as extrasynaptic transmis-
sion. Even short periods of ischemia result in a loss
of function and changes in the brain microenviron-
ment. It has been repeatedly shown in vivo (Van
Harreveld and Ochs, 1956; Hansen and Olsen, 1980;
Rice and Nicholson, 1991; Lundbaek and Hansen,
1992; Katayama et al, 1992; Sykova
´et al, 1994;
Sykova
´, 1997; Vor
ˇı
´s
˘ek and Sykova
´, 1997a,b) as well
as in vitro (Ames and Nesbett, 1983; Pe
´rez-Pinzo
´n
et al, 1995) that the ECS of the brain shrinks during
global ischemia. This shrinkage is caused by the
movement of water from the ECS into the cells,
which is accompanied by rapid cellular swelling.
Received 23 February 2008; revised 16 April 2008; accepted 13
May 2008
Correspondence: Dr N Zoremba, Department of Anesthesiology,
University Hospital RWTH Aachen, Pauwelsstrasse 30, D-52074
Aachen, Germany.
E-mail: nzoremba@ukaachen.de
This study was supported by the European Community project
HPMT-CT-2000-00187 and by the Grants AV0Z50390512 of the
Academy of Sciences of the Czech Republic, LC554 of the
Ministry of Education, Youth and Sports of the Czech Republic,
and 305/06/1316 of the Grant Agency of the Czech Republic.
Journal of Cerebral Blood Flow & Metabolism (2008), 1–9
&
2008 ISCBFM All rights reserved 0271-678X/08
$30.00
www.jcbfm.com

The reason for this water movement is an influx of
sodium and chloride ions across the cell mem-
branes. The persistent increase of intracellular
sodium chloride leads to a reversal of the sodium
chloride gradient such that the intracellular sodium
chloride concentration may exceed the extracellular
levels (Hansen, 1985; Somjen, 2002). Additionally,
the swelling of astrocytes under excitotoxic concen-
trations of glutamate in conjunction with potassium
uptake is well known (Somjen, 2002; Kimelberg,
2005). The ECS of the brain decreases to 5% to 6% of
the total tissue volume during anoxia (Vor
ˇı
´s
˘ek and
Sykova
´, 1997a,b), which is equivalent to a reduction
of 65% to 80% from preischemic values.
Diffusion of neuroactive substances in the ECS is
influenced by the width of the extracellular clefts,
presence of membranes, fine neuronal and glial
processes, macromolecules of the extracellular ma-
trix, charged molecules, and cellular uptake (Sykova
´
et al, 2000; Hrabetova and Nicholson, 2000; Sykova
´,
2004). In contrast to a free medium, diffusion in the
ECS can only be satisfactorily described by a
modified version of Fick’s law, if volume fraction,
tortuosity, and nonspecific uptake are taken into
account (Nicholson and Phillips, 1981; Nicholson,
1992). Volume fraction (a) is the proportion of the
tissue volume occupied by the ECS, whereas
tortuosity (l) describes the increased path length of
diffusing molecules in a complex medium. The
diffusion parameters of the ECS and their dynamic
changes can be determined using real-time ionto-
phoretic method. This method uses the iontophore-
tic application of tetramethylammonium (TMA
+
)
and TMA
+
-selective microelectrodes to record con-
centration–time profiles of TMA
+
in the ECS
(Nicholson and Phillips, 1981). Diffusion-weighted
magnetic resonance imaging (DW-MRI) is a non-
invasive method to determine the apparent diffu-
sion coefficient of water. It has been used to show a
shift of water between the intra- and extracellular
compartments after various types of brain injury
in animals as well as in humans (Le Bihan and
Basser, 1995).
The aim of the present study was to quantify the
changes in ECS diffusion parameters during recov-
ery from transient ischemia by TMA
+
-diffusion and
MRI measurements and to compare their time
courses. The data were correlated with DC-potential
recordings and measurements of extracellular po-
tassium levels. To the best of our knowledge, this is
the first evaluation of the diffusion parameters of the
ECS during recovery from transient ischemia in a
low cerebral blood flow model of bilateral carotid
artery occlusion.
Materials and methods
Animal Preparation
Three-month-old male Wistar rats (300 to 350 g) were
anesthetized by an intraperitoneal injection of urethane
(1.5 g/kg body weight; Sigma-Aldrich Chemie GmbH,
Steinheim, Germany), tracheotomized, relaxed with suxa-
methonium chloride (20 mg/kg per h; Lysthenon,
NycoMED Pharma, Vienna, Austria), and ventilated
mechanically with oxygen. Their body temperature was
maintained at 371C by a heating pad. Transient ischemia
was induced by bilateral common carotid arterial clamp-
ing for 10 mins (n= 5) or 15 mins (n= 6), and by reducing
the inspired oxygen concentration to 6% in 94% nitrogen.
After releasing the clamps, the animals were ventilated
with pure oxygen. The head of the rat was fixed in a
stereotaxic holder, and the somatosensory neocortex was
partially exposed by a burr hole that was 2 to 3 mm caudal
from the bregma and 3 to 4 mm lateral from the midline.
When the dura was removed, the surface of the brain was
continuously bathed in a warm solution (361Cto371C)
containing 1 mmol/L TMA, 150 mmol/L NaCl, and
3 mmol/L KCl. All measurements were done in the
somatosensory cortex at a depth of 1,200 to 1,500 mm from
the cortical surface (cortical layer V; Lehmenku
¨hler et al,
1993). For DW-MRI measurements (n= 6 in each group),
the animals were placed in a heated MR-compatible cradle
and their heads fitted in a built-in head holder.
The experiments were performed in accordance with
the European Communities Council Directive of 24
November 1986 (86/609/EEC). All efforts were made
to minimize both the suffering and the number of
animals used.
Measurement of Extracellular Space Diffusion
Parameters
The ECS diffusion parameters were studied by real-time
iontophoretic method, described in detail previously
(Nicholson and Phillips, 1981; Lehmenku
¨hler et al,
1993; Sykova
´et al, 1994). Briefly, an extracellular marker
that is restricted to the extracellular compartment is
used, such as tetramethylammonium ions (TMA
+
,
MW = 74.1 Da), to which cell membranes are relatively
impermeable. TMA
+
is administered into the ECS by
iontophoresis, and the concentration of TMA
+
measured
in the ECS using a TMA
+
-ion-selective microelectrode
(ISM) is inversely proportional to the ECS volume.
Double-barreled TMA
+
-ISMs were prepared by a proce-
dure described in detail previously (Sykova
´, 1992). The
tip of the ion-sensitive barrel was filled with a liquid ion
exchanger (Corning 477317); the rest of the barrel was
backfilled with 150 mmol/L TMA
+
chloride. The reference
barrel contained 150 mmol/L NaCl. The TMA
+
-ISMs were
calibrated in 0.01, 0.03, 0.1, 0.3, 1.0, 3.0, and 10.0 mmol/L
TMA
+
in a background of 3 mmol/L KCl and 150 mmol/L
NaCl. Calibration data were fitted to the Nikolsky equation
(Nicholson and Phillips, 1981). The shank of the ionto-
phoretic pipette was bent so that it could be aligned
parallel to that of the ISM and was backfilled with
150 mmol/L TMA
+
chloride. An electrode array was made
by gluing a TMA
+
-ISM to an iontophoretic micropipette
with a tip separation of 100 to 200 mm. The iontophoresis
parameters were + 20 nA bias current (continuously
applied to maintain a constant electrode transport
Diffusion in cortex after ischemia
N Zoremba et al
2
Journal of Cerebral Blood Flow & Metabolism (2008), 1 – 9

number), with a + 180 nA current step of 60 secs
duration, to generate the diffusion curve. TMA
+
was
administered at regular intervals of 5 mins. Before tissue
measurements, diffusion curves were first recorded
in 0.3% agar (Difco, Detroit, MI, USA) dissolved in a
solution containing 150 mmol/L NaCl, 3 mmol/L KCl, and
1 mmol/L TMACl. In agar, aand lare by definition set to 1
and nonspecific uptake k0to 0 (free-diffusion values). The
diffusion curves were analyzed to obtain the electrode
transport number (n) and free-TMA
+
-diffusion coefficient
(D) by curve-fitting, according to a diffusion equation
using the VOLTORO program (Nicholson and Phillips,
1981). Diffusion curves were then recorded in the
somatosensory cortex at depths of 1,200 to 1,500 mm.
Knowing nand D, the values of a,l, and k0can be obtained
from the recorded diffusion curves as described by
Nicholson and Phillips (1981).
Diffusion-Weighted Magnetic Resonance Imaging
The DW-MRI measurements were performed using an
experimental MR spectrometer BIOSPEC 4.7 T system
(Bruker, Ettlingen, Germany) equipped with a 200 mT/m
gradient system (190 ms rise time) and a homemade head
surface coil. We acquired a sequence of T
2
-weighted
sagittal images to position coronal slices. For DW
measurements, four coronal slices were selected (thick-
ness = 1.0 mm, interslice distance = 1.5 mm, field of view =
3.2 3.2 cm
2
, matrix size = 256 128). Diffusion weighting
serves to increase the contrast in T
2
-weighted images for
water diffusion. The b-factor denotes the strength of
diffusion weighting. Acquiring at least two DW images
with different b-factors allows for the determination of
the apparent diffusion coefficient of water (ADC
w
). The
DW images from each slice were acquired using a
stimulated echo sequence with the following parameters:
b-factors = 75, 499, 1,235, and 1,731 secs/mm
2
,D= 30 ms,
TE = 46 ms, TR = 1,200 ms. Diffusion weighting is accom-
plished by applying a gradient magnetic field; in our
measurements the gradient pointed along the rostrocaudal
direction, and, therefore, ADC
w
was measured in this
direction. Maps of ADC
w
were calculated using the linear
least-squares method and analyzed using ImageJ software
(W. Rasband, NIH, USA). The evaluated regions of interest
were positioned using a rat brain atlas (Paxinos and
Watson, 1998) and T
2
-weighted images in both the left and
right hemispheres. The minimal area of an individual
region of interest was 2.5 mm
2
. In each animal, we
analyzed four coronal slices from the interval between
0.1 mm frontal to bregma and 5.6 mm caudal to bregma.
The resulting eight values of ADC
w
(two regions of interest
per slice, four slices/rat) were averaged to obtain a single
representative value for comparison to other rats. The
reproducibility of the ADC
w
measurements was verified by
means of five diffusion phantoms placed on the top of a
rat’s head. The phantoms were made from glass tubes
(inner diameter = 2.3 mm, glass type: KS80; Ru
¨ckl Glass,
Nizbor, Czech Republic) filled with pure (99%) substances
having different diffusion coefficients. The substances
were 1-octanol, n-undecane (Sigma Aldrich, Steinheim,
Germany), isopropyl alcohol, n-butanol, and tert-butanol
(Penta, Prague, Czech Republic). The temperature of
the phantoms was maintained at a constant 371C. The
average diffusion coefficient for each compound
was determined at the same time as the experimental
measurements of each group of rats and compared with
the average diffusion coefficient of the same compound
measured in conjunction with the measurements of the
other groups of rats.
Measurement of DC Potentials and Extracellular
K
+
Concentrations
The registration of cortical DC potentials is a powerful
method to monitor the dynamics of sensory and cognitive
processing in the brain under normal conditions and in
the course of central nervous system disorders. DC
potentials from the cortical surface were recorded by
microelectrodes filled with 150 mmol/L NaCl, placed in
the cortex, and connected to a high impedance buffer
amplifier with Ag/AgCl wires. The common reference
electrode was positioned on the nasal bone (Lehmenku
¨hler
et al, 1999). The signal was amplified and transferred to a
PC using a Lab Trax acquisition system (World Precision
Instruments Inc., Sarasota, FL, USA). The extracellular
potassium concentration was measured by double-
barreled K
+
-sensitive microelectrodes, as described in
detail elsewhere (Sykova
´et al, 1994). Briefly, the tip of the
K
+
-selective barrel of the microelectrode was filled with
the liquid ion-exchanger Corning 477317 and back-filled
with 0.5 mol/L KCl, whereas the reference barrel con-
tained 150 mmol/L NaCl. Electrodes were calibrated in a
sequence of solutions containing 2, 4, 8, 16, 32, and
64 mmol/L KCl, with a background of either 151, 149, 145,
137, 121, or 89 mmol/L NaCl to keep the ionic strength
of the solution constant. The data were fitted to
Nikolsky equation to determine the electrode slope and
interference. Based on these electrode characteristics,
the measured voltage was converted to extracellular
concentrations.
Statistical Analysis
The results of the experiments are expressed as mean±
s.e.m. Statistical analysis of the differences within and
between groups was performed using a two-tailed
Mann–Whitney test (InStat; GraphPad Software, San Diego,
CA, USA). Values of P< 0.05 were considered significant.
Results
Extracellular Diffusion Parameters
The ECS diffusion parameters were recorded in
cortical layer IV or V (at depths of 1,200 to 1,500 mm)
of the somatosensory cortex before and after 10
or 15 mins of ischemia. The mean values of extra-
cellular volume fraction, a, and tortuosity, l, during
normoxia were similar in both ischemia groups
Diffusion in cortex after ischemia
N Zoremba et al
3
Journal of Cerebral Blood Flow & Metabolism (2008), 1– 9

(a= 0.19±0.01, l= 1.55±0.01 and a= 0.19±0.01,
l= 1.55±0.02) and comparable to the values found
in previous in vivo studies (Nicholson and Phillips,
1981; Lehmenku
¨hler et al, 1993; Vor
ˇı
´s
˘ek and
Sykova
´, 1997a,b; Mazel et al, 2002). During ische-
mia, adecreased to 0.07±0.01 in both groups,
whereas lincreased to 1.80±0.02 and 1.81±0.02
in the 10 and 15 mins ischemia groups, respectively.
After releasing the clamps and reoxygenation in the
rats subjected to 10 mins of ischemia, recovery to
preischemic values of a= 0.21±0.01 were found
after 15 to 20 mins of reperfusion; the values then
remained stable during the entire measurement
period of 90 mins. After ischemia of 15 mins
duration, arecovered within 10 to 15 mins of
reperfusion, but then increased substantially within
a further 30 to 40 mins up to 0.29±0.03 and
remained elevated during the postischemic mea-
surement period of 90 mins. This indicates an ECS
enlargement of 40% to 50% in the somatosensory
cortex of rats subjected to longer (15 mins) ischemia.
(see Figure 1, Table 1). The statistical difference
between the pre- and postischemic values of awas
extremely significant (0.19±0.01 versus 0.29±0.03,
P< 0.001). Typical diffusion curves recorded before
and 60 mins after ischemia of 10 or 15 mins are
shown in Figure 2.
In both groups, preischemic values of lwere
observed after 5 to 10 mins of reperfusion (10 mins
ischemia: l= 1.57±0.04; 15 mins ischemia:
l= 1.56±0.04). In the group subjected to 10 mins
ischemia, lremained stable at this level. In the
group subjected to 15 mins ischemia, a marginally
significant increase in lto 1.62±0.01 was found at
the end of our measurement period. The time
course of lafter ischemia in both groups is shown
in Figure 1.
Magnetic Resonance Imaging Measurements
Diffusion-weighted MRI measurements of
ADC
w
were performed bilaterally in the primary
somatosensory cortex and showed similar
preischemic values in both groups (597±14 versus
594±12 mm
2
/sec). In the group of 10 mins
ischemia, no significant changes in ADC
w
in the
postischemic period were found, compared with
preischemic values. In the animals exposed to
15 mins ischemia, a statistically significant increase
in ADC
w
to 665±15 mm
2
/sec was observed 60 mins
after ischemia. This elevated ADC
w
level remained
until the end of the measurement period, 120 mins
after ischemia (Table 2). Typical MRI images before
and after ischemia are shown in Figure 3. As the
carotid occlusion was performed outside the mag-
net, we did not measure ADC
w
values during
ischemia. However, it is known from previous
studies that global ischemia induces a rapid
decrease in ADC
w
(Fisher et al, 1995; Van der Toorn
et al, 1996).
DC Potentials and Extracellular Potassium
Concentrations
Before the induction of ischemia, extracellular
potassium concentrations of 3 mmol/L were found
in both groups. During 1 to 2 mins of ischemia, a
rapid increase up to 70 mmol/L was registered, and
the concentration remained stable at this level
during the ischemic period in both 10 and 15 mins
ischemia groups. After reoxygenation, the extracel-
lular potassium concentrations decreased within 2
to 3 mins to preischemic levels of 3 mmol/L. The DC
potential changed in a negative direction simulta-
neously with the increase in extracellular potas-
sium, followed by a small positive change. During
the ongoing ischemia the DC potential showed a
slight increase. Immediately after reopening of the
carotid arteries and ventilation with pure oxygen,
the DC potential showed a sharp negative shift, and
after the normalization of extracellular potassium
concentrations, DC potentials recovered to preis-
chemic levels. Typical measurements of extracellu-
lar potassium concentrations and DC potentials
during ischemia of 10 and 15 mins duration are
shown in Figure 4.
Discussion
Diffusion in the ECS is an important mode of
communication between brain cells, and many
acute pathologic processes in the CNS (i.e., hypoxia,
ischemia, seizures, and hypoglycaemia), accompa-
nied by cellular swelling, can affect the ADC
of neuroactive substances. We have previously
reported that ECS diffusion parameters are altered
not only during transient focal hypoxia/ischemia,
but also during recovery from this insult (Homola
et al, 2006). Although a substantial amount of data
obtained by DWI-MRI exist on extra- and intracel-
lular diffusion in areas affected by an ischemic/
hypoxic insult, the absolute values of the extra-
cellular diffusion parameters measured in the brain
cortex during recovery from hypoxia/ischemia have
not been available. Up to now, the only values of the
ECS diffusion parameters or values of ADC
w
obtained in the rat cortex under hypoxic/ischemic
conditions have been reported from experiments
using a terminal anoxia model (Van der Toorn et al,
1996; Lundbaek and Hansen, 1992; Sykova
´et al,
1994). In our study, we have evaluated the changes
in ECS diffusion parameters after transient global
hypoxia/ischemia. These parameters were studied
over the course of 90 mins after an ischemic/hypoxic
insult and were correlated with changes in
ADC
w
, DC potential, and extracellular potassium
concentration.
Cellular swelling during cerebral ischemia has
been described by several authors and is mainly a
consequence of massive ionic fluxes across cell
membranes, accompanied by the movement of
Diffusion in cortex after ischemia
N Zoremba et al
4
Journal of Cerebral Blood Flow & Metabolism (2008), 1 – 9

Figure 1 Time course of changes in the values of the extracellular space parameters (a,l) in the cortex of adult rats before, during,
and after ischemia of 10 or 15 mins duration, calculated from TMA
+
diffusion measurements. Data are shown as mean
values±s.e.m. and the number of animals as n. The duration of ischemia is marked by a time line. Upper graphs: average values of
extracellular volume fraction (a). After 10 mins ischemia, a quick recovery of aoccurs within 5 to 10 mins and the values remain
stable at this level. In the group subjected to 15 mins ischemia, aincreases extremely significantly above starting values after
40 mins of reperfusion (P< 0.001) and remains at this level until the end of the measurement period. *Postischemic values that are
significantly different from preischemic values (P< 0.05). Lower graphs: the time courses of tortuosity (l) initially showed no
difference between the two groups (P< 0.05). During ischemia, an elevated lrecovered to the starting values within 5 mins and
stayed at this level without any significant difference from preischemic values. In the group of 15 mins ischemia, lincreased
significantly at the end of the registration period.
Table 1 Values of extracellular volume fraction (a) and tortuosity (l) and nonspecific uptake (k0) before and after transient ischemia
of 10 or 15 mins duration
Before ischemia 30 mins after ischemia 60 mins after ischemia 90 mins after ischemia
Ischemia a= 0.19±0.01 a= 0.20±0.01 a= 0.20±0.01 a= 0.19±0.01
10 mins l= 1.55±0.01 l= 1.54±0.02 l= 1.55±0.02 l= 1.56±0.03
n=5 k0= 3.6 10
3
±0.8 10
3
k0= 3.3 10
3
±0.9 10
3
k0= 3.9 10
3
±1.0 10
3
k0= 4.1 10
3
±1.0 10
3
Ischemia a= 0.19±0.01 a= 0.26±0.01* a= 0.29±0.03* a= 0.30±0.03*
15 mins l= 1.55±0.02 l= 1.57±0.01 l= 1.60±0.02 l= 1.62±0.01*
n=6 k0= 3.6 10
3
±0.6 10
3
k0= 2.8 10
3
±0.2 10
3
k0= 3.6 10
3
±0.5 10
3
k0= 3.8 10
3
±0.4 10
3
Values of a,l, and k0are shown as mean values and s.e.m.; nrepresents the number of animals.
*Significant differences (two-tailed Mann–Whitney test, P< 0.05) in postischemic values when compared with values before ischemia.
Diffusion in cortex after ischemia
N Zoremba et al
5
Journal of Cerebral Blood Flow & Metabolism (2008), 1– 9

water, and develops concomitantly with ionic shifts
(McKnight and Leaf, 1977; Hansen and Olsen, 1980;
Sykova
´et al, 1994). The swelling occurs quickly
after the interruption of the energy supply, but never
before a rise in K
+
and changes in pH (Sykova
´et al,
1994). The dependence of ECS shrinkage because of
cellular swelling on ionic changes was also seen in
our experiments, and it was observed that a normal-
ization of elevated K
+
leads to a quick normalization
of ECS volume and tortuosity. The recovery in
extracellular potassium concentration and DC po-
tential indicates a sufficient supply of oxygen and
substrates after ischemia. During recovery from
longer lasting global ischemia, a significant increase
in the extracellular volume fraction adeveloped
within 60 mins, probably caused by an increase in
extracellular water content. Similar time courses of
ECS volume and tortuosity changes were observed
in the spinal cord during recovery from ischemia
(Sykova
´et al, 1994). The postischemic increase in a,
which was observed in this study, was promoted by
bilateral carotid occlusion and a reduction in
cerebral blood flow. It has been shown that hypoxia
of 30 mins duration without carotid occlusion
lead to a small decrease in a, followed by rapid
normalization during recovery (Zoremba et al,
2007). In contrast to these findings, hypoxia of
30 mins duration with unilateral carotid occlusion
results in a decrease in afollowed by an increase
during recovery to about 20% above the original
normoxic values (Homola et al, 2006). Based on the
results from our study, together with the results from
earlier studies, it could be suggested that in addition
to the degree and duration of hypoxia, cerebral
blood flow also influences the extent of damage. The
increase in ECS volume fraction after reperfusion
correlates well with changes in the ADC of brain
water. In our study, the values of ADC
w
obtained 60
and 90 mins after reperfusion were significantly
elevated, suggesting an increased amount of water
in the ECS where ADC
w
was reported to be higher,
compared with the intracellular compartment (Van
Zijl et al, 1991). A correction of orientational
dependence was not necessary because it is known
from previous studies that there is no significant
anisotropy in the cerebral cortex of the rat brain
(Vor
ˇı
´s
˘ek and Sykova
´, 1997a,b). In an earlier study,
full recovery of the diffusion constant of brain water
after 12 mins of incomplete global ischemia was
found (Davis et al, 1994). The results are comparable
Figure 2 Example of recorded diffusion curves and superimposed theoretical curve fittings as time–concentration plots before
(control) and 60 mins after ischemia (ischemia) of 10 or 15 mins duration. Total recording time was 80 secs for each diffusion curve.
After 8 secs, the main bias was elevated for 24 secs to 120 nA to apply TMA
+
by iontophoresis. Changes in the diffusion properties
after ischemia compared with preischemic conditions were found only in the group subjected to 15 mins ischemia.
Table 2 The apparent diffusion coefficient of water (ADC
w
) before and after 10 or 15 mins of ischemia was determined by magnetic
resonance imaging
ADC
w
(mm
2
/sec)
before ischemia
ADC
w
(mm
2
/sec)
60 mins after ischemia
ADC
w
(mm
2
/sec)
90 mins after ischemia
ADC
W
(mm
2
/sec)
120 mins after ischemia
Ischemia 10 mins (n= 6) 597±14 608±8 603±10 605±12
Ischemia 15 mins n= 6 594±12 665±15* 651±11* 647±13*
Data expressed as mean±s.e.m.; nrepresents the number of animals.
*Significant differences (two-tailed Mann–Whitney test, P< 0.05) between preischemic values and values after ischemia.
Diffusion in cortex after ischemia
N Zoremba et al
6
Journal of Cerebral Blood Flow & Metabolism (2008), 1 – 9

to the results in our 10 mins ischemia group. In
contrast to this earlier study, we also reduced the
oxygen content to 6% and therefore a more severe
ischemia resulted. These observations support the
hypothesis that a certain level of ischemia severity
has to be exceeded before cerebral edema develops.
It is well known that after ischemia, functional
damage of the blood–brain barrier occurs (Betz et al,
1989; Qiao et al, 2001) and the hypoxic-ischemic
insult initiates a series of events that lead to the
disruption of tight junctions and increased perme-
ability mediated by cytokines, vascular endothelial
growth factor, and nitric oxide (Ballabh et al, 2004).
Because of increased permeability, reperfusion may
lead to an early postischemic increase in cerebral
water content and to the formation of cortical edema
of ‘vasogenic’ origin (Papadopoulos et al, 2005).
These changes could also be caused by elevated
postischemic tissue osmolarity (Gisselson et al,
1992) or postischemic shrinkage of nerve cells. It is
evident that an ischemia of 10 mins duration is too
short to initiate a cascade of events that lead to the
disruption of the blood–brain barrier and the
development of vasogenic edema. This hypothesis
was supported by the different time courses of the
DC potential recovery between the two groups.
During ischemia, an increase in extracellular potas-
sium levels up to 70 mmol/L was registered,
whereas recovery in both groups was comparable
within a few minutes. In comparing the DC
potentials, a longer recovery period was necessary
in animals subjected to an ischemia of 15 mins.
These findings indicate that the cellular energy state
is affected in the same way in both groups, but that
the intercellular integrity because of neurotoxicity
was more affected in the group subjected to longer
ischemia. Additionally, the longer lasting extracel-
lular potassium increase could have direct effects or
modulate the influence of cytokines, vascular
endothelial growth factor, and nitric oxide on the
blood–brain barrier.
During reperfusion in the group with longer
ischemia, the extracellular volume fraction in-
creased within 40 to 50 mins and remained elevated
by about 40% to 50% above the normoxic values,
whereas the tortuosity was initially similar to
preischemic values, increasing significantly at the
end of our registration period. It could be expected
that the increase in ECS volume would facilitate
extracellular diffusion, but our results show a small
increase in tortuosity, indicating a diffusion hin-
drance and an effect on volume transmission. One
reason for this hindered diffusion could be released
Figure 3 Typical ADC
w
maps of a control rat brain and of a rat
brain 60 mins after ischemia of 15 mins duration. ADC
w
was
analyzed bilaterally in the primary somatosensory cortex. The
areas are outlined on the left part of the slices, and both images
are from the same coronal plane. The scale at the bottom of the
figure shows the relation between the intervals of ADC
w
values
and the colors used for visualization.
Figure 4 Time course of extracellular potassium concentrations
and DC potentials during an ischemia of 10 mins (left side) or
15 mins (right side) duration. The duration of ischemia is
marked by the shaded fields.
Diffusion in cortex after ischemia
N Zoremba et al
7
Journal of Cerebral Blood Flow & Metabolism (2008), 1– 9

macromolecules and fixed surface charges, which
affect free diffusion by charge-dependent bonding
or by van der Waals forces. Increased viscosity
impedes molecular movement and is affected by the
size and nature of the diffusing molecules and
results in their hydrodynamic interactions with
macromolecules and fixed charges and the bound-
aries that define pore structures. The change in
tortuosity, l, is influenced by many factors that
cannot be presently separated. These factors might
include membrane barriers, myelin sheaths, macro-
molecules, molecules with fixed negative surface
charges, ECS size, and pore geometry. As a result,
lcould be changed if certain pathways through the
ECS are either blocked off or opened up (Sykova
´
et al, 2000). Many studies have shown that land acan
change independently during the exposure of brain
slices to dextran (Hrabetova and Nicholson, 2000),
during X-irradiation (Sykova
´et al, 1996), or during
osmotic stress (Krizaj et al, 1996; Nicholson
and Sykova
´, 1998; Chen and Nicholson, 2000;
Kume-Kick et al, 2002).
We have shown changes in the extracellular
diffusion parameters that may affect the diffusion
of various substances (ions, neurotransmitters, meta-
bolites, and drugs) in the affected region. Our results
show that after ischemia of 15 mins, and most likely
after longer periods, the observed changes in the
ECS diffusion parameters are not reversible during
90 mins of reperfusion and result in long-lasting and
severe edema and perhaps permanent damage.
Studies using longer ischemic and postischemic
periods are needed to further clarify the crucial time
point between reversible and irreversible effects
induced by oxygen deficiency.
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