1Bio-Imaging Lab, University Center Antwerp, University of Antwerp, 2020 Antwerp, Belgium; and 2Division of General Physiology, Department of Biology, University of Oslo, N-0316 Oslo, Norway
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ABSTRACT |
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Linden, Annemie Van der, Marleen Verhoye, and Göran E. Nilsson. Does Anoxia Induce Cell Swelling in Carp Brains? In Vivo MRI Measurements in Crucian Carp and Common Carp. J. Neurophysiol. 85: 125-133, 2001. Although both common and crucian carp survived 2 h of anoxia at 18°C, the response of their brains to anoxia was quite different and indicative of the fact that the crucian carp is anoxia tolerant while the common carp is not. Using in vivo T2 and diffusion-weighted magnetic resonance imaging (MRI), we studied anoxia induced changes in brain volume, free water content (T2), and water homeostasis (water diffusion coefficient). The anoxic crucian carp showed no signs of brain swelling or changes in brain water homeostasis even after 24 h except for the optic lobes, where cellular edema was indicated. The entire common carp brain suffered from cellular edema, net water gain, and a volume increase (by 6.5%) that proceeded during 100 min normoxic recovery (by 10%). The common carp recovered from this insult, proving that the changes were reversible and suggesting that the oversized brain cavity allows brain swelling during energy deficiency without a resultant increase in intracranial pressure and global ischemia. It is tempting to suggest that this is a function of the large brain cavity seen in many ectothermic vertebrates.
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INTRODUCTION |
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Most vertebrate brains are
irreversibly damaged by only a few minutes of anoxia. A well-documented
event in the anoxic brain is cell swelling, i.e., an increase in
intracellular volume and a decrease in extracellular volume. Cell
volume regulation is highly dependent on ATP consuming ion pumps,
primarily the Na/K ATPase, and the fall in ATP levels that rapidly
occurs in anoxia/ischemia is thought to be the primary cause of cell
swelling in the brain (Cervos-Navarro and Ferszt 1989;
Go and Baethmann 1982
; Hossmann 1976
;
Kohno et al. 1995
; Schuier and Hossmann
1980
). In mammals, the anoxic cell swelling causes an increase
in the intracranial pressure, and when this pressure exceeds the blood
pressure, blood flow to the brain will stop (a state called global
ischemia). At this stage, the brain cannot be saved even if the
organism is reoxygenated.
However, a few vertebrates readily survive prolonged episodes of
anoxia. The best-studied examples of such anoxia tolerant vertebrates
are some North American freshwater turtles (genera Trachemys
and Chrysemys) and the crucian carp (Carassius
carassius). These animals, which can survive 1 or 2 days of anoxia
at room temperature, are able to maintain their brain ATP levels during anoxia by combining an increased rate of anaerobic (glycolytic) ATP
production with a depressed rate of energy use (Lutz and Nilsson 1997). Still, the possibility that these animals avoid cerebral cell swelling has so far not been examined. The fact that they maintain
the neural energy charge suggests that they are able to avoid cell
swelling. However, like in many fish, the brain of the crucian carp is
imbedded in a soft mass of a jelly-like tissue called meninx, and the
cranial room surrounding the brain is much larger than the brain
volume. Thus there is a possibility that the crucian carp can let its
brain swell in anoxia, thereby reducing its costs for cell-volume
regulation. On the other hand, if the crucian carp brain does not swell
in anoxia, then this would be another strong indication of a functional
link between cell swelling and cellular energy charge. Such a
conclusion could be backed up by comparative measurements on a related,
but anoxia-intolerant, fish; the obvious choice of species being the
common carp (Cyprinus carpio), which can only survive 1-2 h
of anoxia at room temperature during which time a continuous fall in
brain ATP levels is seen (Van den Thillart and Van Waarde
1991
; Van Ginneken et al. 1996
).
Nuclear magnetic resonance imaging (MRI) has become much used to monitor changes in brain water balance during situations such as anoxia and ischemia. MRI-based determinations of the water apparent diffusion coefficient (ADC) and water content (from T2 weighted images) provide noninvasive measures of shifts in extracellular and intracellular water volumes and changes in brain water content (see also METHODS). Moreover, MRI can be used to monitor changes in the size of the brain during anoxia.
The present study aims at using MRI to examine the effects of 2, 5, and 24 h of anoxia on the size, water content, and extracellular/intracellular water volumes in crucian carp brain and to make comparative measurements in the related but much less anoxia tolerant common carp submitted to 2 h anoxia.
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METHODS |
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Animal handling
Common carp (C. carpio) were obtained from the fish hatchery at the Agricultural University of Wageningen, The Netherlands, and crucian carp (Carassius carassius) were obtained from Van Stallen fish supplier (Antwerp, Belgium). They were raised at the University of Antwerp at a temperature of 18°C in softened Antwerp City tap water (Ca2+ 0.875 mmol/l, Mg2+ 0.145 mmol/l, and pH 7.0-8.0). Carp were daily fed ad libitum with Pond sticks (Tetrapond, Henkel) and excess food was removed 15 min after feeding. The water in all the aquaria was filtered with trickling filters and water quality was checked twice a week with Visicolor Test Kits (Macherey-Nagel, Düren) for ammonia, nitrite, and nitrate.
Crucian carp and common carp (both species weighing 40-60 g, ca 12 mm body length) were immobilized by adding MS 222 (ethyl meta aminobenzoate metanesulfonic acid salt 98%, Sigma) to the water (0.011% final concentration, pH controlled) and mounted in an open flow through system fixed in a PVC pipe which fitted in the bore of the magnet of the MR instrument (Figs. 1 and 2). Water containing MS222 was pumped from an aquarium to a narrow high water column to allow a rapid equilibration either with air or N2 (Fig. 1). The fish received this water (500 ml/min) through a tube fixed in its mouth. Water oxygen concentration and temperature (18 ± 1°C) were monitored on-line with an OXY 325-B probe from WTW connected to a PC with Lab Windows software. During the experiment, the temperature of the water was kept at 18°C. The body temperature of the fish was also monitored by a termistor positioned on the fish surface. A closed water-circulation system connected with a water bath was placed in the immediate vicinity of the belly of the fish, submerged in the surrounding water and used to adjust the temperature (Fig. 1).
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Fish (n = 5) were subsequently submitted to normoxia (air bubbling) and 2 h anoxia (nitrogen bubbling) and allowed to recover under normoxia for 100 min (air bubbling to reach a 100% oxygen saturation = 9 mg O2/ml). For crucian carp, longer anoxia exposure (5 and 24 h, n = 5, n = 5) was accomplished by keeping the fish in a closed water-filled reservoir (10 l) that was bubbled with N2 until the registered oxygen concentration was 0 mg/ml. The vial was then closed without inclusion of gas bubbles, and the fish remained there for the desired period. This set-up did not allow an initial normoxic MRI measurement.
Magnetic resonance imaging (MRI)
In the reported study, 1H MRI is used as a sensitive and noninvasive tool to monitor changes in tissue water balance. The method is based on localizing the concentration and relaxation properties of 1H nuclei, primarily in water, resulting in a digital image of the spatial distribution of 1H nuclei in the object. To that end, a sample (e.g., an animal) is placed in a strong magnetic field while applying a sequence of radio frequency pulses and magnetic field gradients. By choosing an appropriate pulse sequence, the intensity of the MR image can be made to reflect one or more of several MRI parameters. Such parameters, in turn, are sensitive to the physicochemical environment of the nuclei and provide important information on the biological environment of the 1H nuclei.
For example, the T2 parameter depends on the
ratio of free-to-bound water, the hemodynamics and the oxygenation
status of the tissue. The impact of the latter two is most important on the T*2 parameter and is then more specifically
referred to as the blood-oxygenation-level-dependent (BOLD) contrast.
In this study, the T2 MRI parameter was monitored to determine changes in the ratio free-to-bound water or water content
in different brain areas taking into account potential hemodynamic
changes. The rationale behind this is the demonstrated linear
correlation between T2 and the water content (as
measured from dry weight-wet weight ratios) of several tissues (e.g.,
Herfkens et al. 1981, 1983
; Kundel et al.
1986
) and in infarcted rat brain (Kato et al.
1985
).
Other MRI sequences allow measuring the dynamics of tissue water such
as the molecular mobility or the molecular diffusion of water
(Le Bihan et al. 1991). Water in tissues has a diffusion coefficient that is two to three times less than that of free water
(Cooper et al. 1974
). This is largely explained by the
high viscosity of bulk water in tissues due to the presence of large molecules such as proteins in the intracellular spaces. Tissues with
different viscosity's or different balance between intra- and
extracellular water might thus present different diffusion coefficients, which are the source of contrast in diffusion images. Changes in ADC of different brain areas have been determined under normoxic and anoxic circumstances, and such changes have been experimentally proven to be directly linked to changes in the balance
between intra and extracellular water volumes. Different types of edema
were distinguished on the basis of ADC values obtained in vivo
(Ebisu et al. 1993
). Evidence for a correlation between ADC changes and changes in cell volume or in fractional volume of the
extracellular space exists from studies on hyponatremic rat brain
(Sevick et al. 1992
), on ischemic rat brain
(Benveniste et al. 1992
), and on
N-methyl-D-aspartate (NMDA)-induced rat brain injury (Verheul et al. 1994
). Moreover, ADC values of
brain tissue, obtained in vivo, seem to match perfectly with the
calculated weighted average of the diffusion coefficient of the
intracellular compartment (1.43 × 10
4
mm2/s), as determined in vitro by Van Zijl
et al. (1990)
, and the extracellular compartment (3.25 × 10
3
mm2/s, equal to that of free water)
(Sevick et al. 1992
; Verheul et al.
1994
).
MRI protocol
MRI was accomplished by mounting the fish such that the head of the fish fitted in a custom-made RF headphone antenna of 40 mm diam (Fig. 2). The entire set up was inserted in the bore of a horizontal 7 Tesla magnet of a SMIS MRI system (Guildford, UK).
Subsequently T*2-weighted gradient echo (GE) images
(TE/TR = 12 ms/500 ms, acquisition matrix 128 × 128), different
T2-weighted spin echo (SE) images (TE/TR
=18/2,000, 34/2,000, 50/2,000 ms), and different diffusion-weighted
spin echo (DW) images (TE/TR = 50/2,000 ms) were obtained of the brain
of the fish. The diffusion-sensitizing gradient pulse was applied in
the X direction with a duration () of 10 ms, a
diffusion-gradient separation time (
) of 15 ms, a diffusion gradient
ramp time of 1 ms, and b values of
(b0x = 0, b1x = 2.993, b2x = 6.736) × 108 s/m2. The FOV was 40 mm, spectral width 25 kHz, two averages were taken and 12 consecutive
slices of 1 mm thick were made, the acquisition matrix for the SE
images was 256 × 128, with the image matrix in both GE and SE
images being 256 × 256. The GE images took 3 min while the SE
images took 8 min 40 s. The entire imaging set, starting
with GE MR images, followed by three T2-weighted SE MR images and two diffusion- weighted SE MR images took about 50 min and
was repeated continuously.
The GE T*2-weighted images, which showed a visible darkening when the blood perfusing the brain became anoxic (increased amount of paramagnetic deoxy Hb), served to control appropriate vascular circulation, heart function, and gill perfusion of the fish during the experiment. They also support the accuracy of the T2 interpretations pointing either to water balance changes or hemodynamic changes.
MRI processing
All image processing was done using IDL (Interactive Data
Language) software. For each time point, the T2
values were calculated on a pixel-by-pixel basis. The signal
intensities of the three SE images were fitted to time according the
T2-relaxation process of the spin system
determined by an exponential decay of the signal in time
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From the diffusion-weighted images, we calculated diffusion maps,
images in which the gray level of each pixel represents the apparent
diffusion coefficients (ADC). On a pixel-by pixel basis, we fitted the
signal intensities of the DW images (SDWi) to
the corresponding b values (bix)
following an exponential decay (Le Bihan et al. 1991)
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The ADCmeas measured in a small water-filled tube
placed in the vicinity of the fish head was used to obtain brain
temperature-independent ADC data, and this for reasons of comparison.
The measured mean ADCmeas values were scaled to
an ADC-value corresponding to a temperature T = 20°C
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Data processing
In vivo MRI allowed the on-line follow-up of changes in the same
animal as a function of time. To take advantage of this characteristic and to rule out individual differences, data were expressed as changes
in ADC or T2, relative to the ADC or
T2 value obtained during normoxia of the same
animal:
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RESULTS |
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General observations
Figure 3 shows a set of high-resolution MR images obtained on anesthetized carps. The consecutive slices (1 mm thick) were obtained in a horizontal plane and highlight different areas in the brain of the common carp (Fig. 3A) and the crucian carp (Fig. 3B). These images display anatomical species differences. In particular, the optic lobes are much broader in the common carp than in the crucian carp. The total brain volumes were significantly different for same sized fish (P < 0.001): the crucian carp brain volume was 230 ± 23 mm3, which is 40% smaller than that of the common carp (384 ± 14 mm3).
The ADC values of normoxic brain tissue in common carp (399 ± 74 × 1012
m2/s) were significantly smaller than those of
crucian carp (mean of all brain regions 487 ± 60 × 10
12
m2/s; P < 0.001; mean of all
regions except cerebellum 510 ± 35 × 10
12
m2/s; P < 0.001). With exception
of the cerebellum, all brain regions displayed this difference. In both
species, the cerebellum had a lower ADC, which was not significantly
different between the crucian carp (395 ± 34 × 10
12
m2/s) and the common carp (332 ± 84 × 10
12
m2/s). The generally higher ADC values in crucian
carp suggest a higher molecular mobility of water, which might reflect
a higher extracellular volume and/or a smaller intracellular volume in the crucian carp brain.
Finally, the mean T2 value of normoxic brain tissue of common carp (31 ± 5 ms) was significantly smaller (P < 0.001) than that of the crucian carp (46 ± 4 ms); this might point to differences in the ratio free-to-bound water or hemodynamic differences between crucian and common carp brains.
Exposure of common carp to 2 h of anoxia
Of the 11 carp submitted to 2 h of anoxia at 18°C, only 1 could not be reanimated in MS-222-free water after the anoxic episode. Figure 4 shows relative changes in ADC induced by anoxia (as compared with the initial normoxic values), indicative for dynamic changes in intra- and extracellular water volume in a context of brain anoxia.
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A significant and consistent drop in ADC values was observed after 2 h of anoxia in all investigated brain regions of common carp (P < 0.001 for hypothalamus; P < 0.01 for optic lobe and telencephalon; P < 0.05 for vagal lobes and cerebellum, Fig. 4). This drop varied from 31 ± 10% within 1 h to 53 ± 8% within 2 h and might demonstrate an anoxia-induced decrease in the extracellular volume and/or an increase in the intracellular volume, consistent with cell swelling. No significant differences were seen between the different brain regions in their response to anoxia. On 40 min of reoxygenation, normoxic ADC values were not yet attained (57 ± 16% of control values). Even after 100 min of reoxygenation, the ADC values were still significantly lower than the initial normoxic values in the optic lobes (P < 0.01), hypothalamus (P < 0.01) and vagal lobes (P < 0.05), while the telencephalon and cerebellum displayed fully recovered ADC values.
On 1 and 2 h of anoxia, no significant changes were observed in the T2 values of the different brain regions, except for the telencephalon which displayed a significant T2 drop (P < 0.05; Fig. 5). During reoxygenation, the relative T2 values appeared to increase during 40 and 100 min of normoxic recovery, but this was not statistically significantly. However, when pooling the entire data set obtained from different brain regions, significant differences were found between the initial normoxic T2 brain values (58 ± 10) ms and the T2 brain values obtained at 40 min (68 ± 15 ms, or 118 ± 15% increase; P < 0.001) and 100 min (67 ± 7 ms, or 122 ± 19% increase; P < 0.01) of normoxic recovery. Such an increase in T2 values on normoxic recovery can be interpreted as an increase in the ratio free-to-bound water or/and the occurrence of hemodynamic changes (e.g., increase in oxyHb concentration).
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Figure 6 shows the total brain volumes of common carp (n = 5) during normoxia, 2 h of anoxia and 100 min of subsequent reoxygenation. The brain volumes were, respectively, 384 ± 14, 409 ± 18, and 422 ± 18 mm3. The data obtained during anoxia and normoxic recovery were significantly different (P < 0.05) from the initial normoxic brain volume values. These data show that the brain of a common carp swells on anoxia (volume increasing by ca 6.5%) and that swelling proceeds during subsequent normoxic recovery up to a volume 10% larger than the initial. This volume increase is also indicated on the in vivo high-resolution T2-weighted MR images (Fig. 7) by the concomitant disappearance of the ventricles on the images, which reflects a possible collapse of these structures.
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Exposure of crucian carp to 2 h of anoxia
All crucian carp included survived the anoxic exposure (2 h at 18°C) and recovered completely from anesthesia.
On anoxic exposure, there were no significant changes in the ADC values
except for the optic lobes, which showed a significant 35% ADC drop
(normoxia = 508 ± 40 × 1012
m2/s, 2 h of anoxia = 329 ± 34 × 10
12
m2/s; P < 0.001; Fig.
8). This might indicate the occurrence of cell swelling in the optic lobes that seemed completely reversible since the initial normoxic ADC values were attained during the reoxygenation period. No significant changes were seen in other brain
regions during reoxygenation.
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After 2 h of anoxia, all brain regions showed a significant drop in T2 (P < 0.01 for optic lobes and hypothalamus; P < 0.05 for telencephalon, vagal lobes, and cerebellum). The T2 values recovered completely on reoxygenation (Fig. 9). The reason for the T2 fall during anoxia and the subsequent T2 increase on normoxic recovery was most likely the effect of the deoxygenation and subsequent oxygenation of hemoglobin on the T2 and T*2 (BOLD MRI contrast). According to these data, there could not be a net increase in the ratio free-to-bound water after 2 h of anoxia in crucian carp brain since no significant increase in T2 occurred in comparison to the initial normoxic values.
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The brain volume of the crucian carp was 230 ± 23, 232 ± 23, and 228 ± 24 mm3, respectively, during normoxia, 2 h of anoxia and subsequent 100 min normoxic recovery (Fig. 6). This lack of any significant volume change demonstrated that the crucian carp maintains its brain volume under anoxic conditions.
Exposure of crucian carp to 24 h of anoxia
While all crucian carp tolerated 5 h of anoxia, 24 h of anoxia occasionally ended in mortality or strongly weakened animals (which were not included in this study).
Figure 10 shows that the optic lobes are the only region displaying significantly lowered ADC values, probably reflecting cell swelling, during the entire experimental anoxia period (1-24 h; P < 0.05 for 1 h, P < 0.001 for 2, 5, and 24 h of anoxia). The cerebellum showed significantly lowered ADC values at 5 and 24 h of anoxia (P < 0.01). On subsequent 100 min of normoxic recovery, the ADC values increased, reaching control values in the cerebellum while the ADC values in the optic lobes were not yet completely restored at that time (P < 0.001). The vagal lobes, the hypothalamus, and the telencephalon never showed any significant changes in ADC, not even after 24 h of anoxia, suggesting a regional heterogeneity in the avoidance of cell swelling during anoxia.
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The crucian carp displayed a significant decrease in T2 values during 1-24 h of anoxia (Fig. 11). This was most likely a reflection of contrast drops resulting from the deoxygenation of the entire hemoglobin pool in the blood perfusing the anoxic brain. On normoxic recovery, the T2 values never exceeded the normoxic values; this makes it unlikely that anoxia induced an increase in free water even after 24 h of anoxia. On the contrary, the T2 values obtained after 24 h of anoxia and on normoxic recovery after extended anoxia (24 h) remained significantly lower than the expected normoxic values in the optic lobe (P < 0.05) and in the cerebellum (P < 0.05). In the other regions, T2 stayed lower but not significantly (P = 0.07 for vagal lobe; P = 0.06 for hypothalamus; P = 0.09 for telencephalon). It is likely that this trend of decreased T2 values in the entire brain after 24 h anoxia and on normoxic recovery reflects major hemodynamic changes (increase in cerebral blood volume or Hb concentration).
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Brain volumes of crucian carp measured after 24 h anoxia (230 ± 5 mm3) were remarkably similar to the normoxic data obtained on other individuals (230 ± 23 mm3). This shows that even on 24 h anoxia, the crucian carp brain still maintains its original volume.
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DISCUSSION |
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Brain swelling in common carp
Although both carp species survived 2 h of anoxia at 18°C, the presently measured responses of their brains to anoxia were quite different and indicative of the fact that the crucian carp is anoxia tolerant while the common carp is not. Two hours of anoxia is on the limit of what the common carp can endure, while crucian carp generally survives 24 h of anoxia at the present temperature. Thus while the crucian carp maintained its brain volume even after 24 h of anoxia, the common carp brain displayed a considerable volume increase already after 2 h of anoxia. This swelling proceeded on reoxygenation, ultimately resulting in a 10% increase in the brain volume (Fig. 6). The reason that the common carp recovered uneventfully from this insult is probably related to the presence of an oversized brain cavity, allowing a significant brain swelling without an increase in intracranial pressure.
Anoxia or ischemia induced brain swelling is the result of an increased
water content of the brain tissue. This is accomplished by the
consecutive occurrence of cytotoxic edema and vasogenic edema and
eventually also interstitial edema (Cervos-Navarro and Ferszt
1989; Go and Baethmann 1982
; Hossmann
1976
). Cytotoxic or cellular edema is the result of cerebral
energy depletion and is characterized by cell swelling and a
concomitant reduction in the extracellular fluid space. Cellular edema
is a reversible process if short-lived, if not, vasogenic edema
develops. The latter is characterized by increased brain water and
sodium content and an expanded extracellular space. The extracellular
fluid volume is increased by edema fluid, a plasma filtrate containing
plasma proteins. In vasogenic edema, there is an increased permeability of capillary endothelial cells to macromolecules (Cervos-Navarro and Ferszt 1989
; Fishman 1980
; Go and
Baethmann 1982
).
Since common carp showed a significant increase in brain volume on
anoxia and even during normoxic recovery, we expected edema to occur in
its brain during the anoxic challenge and subsequent normoxic recovery.
Indeed in anoxic common carp, significant drops in the ADC values were
observed in all the investigated brain regions. The ADC decrease
proceeded as anoxia continued from 1 (by 31 ± 10%) to 2 h
(by 53 ± 8%). This strongly suggests that the carp brain
suffered from cellular edema, characterized by cell swelling, probably
as a result of the documented energy depletion during anoxia
(Van Ginneken et al. 1996).
As soon as the common carp regained access to oxygen, a concomitant increase in ADC and T2 was observed, although a regional heterogeneity in the response to anoxia was indicated. After 100 min of normoxic recovery, the optic lobes, hypothalamus, and telencephalon ended up with ADC values that were still significantly lower than the initial normoxic values, while the relative T2 values stayed higher than the initial normoxic values (only significant if data of all regions were pooled). This indicates that even after 100 min of normoxic recovery, the optic lobes, vagal lobes, and hypothalamus are still affected by cellular edema (decreased ADC). The increased T2 values on the other hand might point to a general increase in the ratio free-to-bound water (potential increase in free water) provided no hemodynamic changes occur between normoxia and normoxic recovery from anoxia.
The telencephalon and the cerebellum on the other hand displayed complete recovery of the ADC values after 100 min of recovery, potentially reflecting an increased ability to regulate cell volume as soon as oxygen was provided and oxidative ATP production could be resumed. However, these regions still displayed increased T2 values. A potential explanation for the concomitant ADC and T2 increases on recovery would be the occurrence of vasogenic edema which is characterized, as outlined in the preceding text, by increased extracellular fluid (increased ADC). However, since the ADC values still did not significantly exceed the initial normoxic values, we assume that the increase in free water (increased T2) occurred both intra- and extracellulary.
Another type of edema that could be taken into account is interstitial edema accompanied by ventricular swelling. This was, however, never observed. On the contrary, the ventricles tended to become less delineated on normoxic recovery reflecting ventricle collapse rather than swelling (see Fig. 7).
ADC drops, comparable in size to that of the common carp, has been
observed in mammals during experimentally induced brain ischemia. A
50% ADC decrease was observed in ischemic rat brain on middle cerebral
artery occlusion but already within the first 15 min (Busza et
al. 1991; Mintorovitch et al. 1991
). In cat
brain, an ADC decrease of typically 30 and 40% has been observed
within the first 20 min following middle cerebral artery occlusion
(Moseley et al. 1991
, 1993
). The common carp displays
falling ATP levels in brain during anoxia (Van Ginneken et al.
1996
), and the main reasons why common carp survives anoxia
much longer than mammals are probably the low temperature (18°C),
slowing down the loss of brain ATP, and the relatively high ability of
the common carp to produce ATP anaerobically. In contrast to the
crucian carp, however, the common carp does not display a noticeable
metabolic depression during anoxia and is unable to produce ATP through ethanol producing glycolysis (van Ginneken et al. 1996
).
Thus even if the common carp cannot, like the crucian carp, maintain its brain ATP levels during anoxia, it still shows a relatively slow
loss of ATP compared with mammals.
Hold off of brain swelling in the crucian carp
The hold off of any sings of volume increase in the crucian
carp's brain even after 24 h of anoxia fits entirely with the demonstrated hold off of any signs of edema. Moreover the crucian carp
did not show any anoxia induced decrease in ADC in brain except for the
optic lobes, which suffered from an ADC drop during anoxic exposure
that, however, was entirely reversible as concluded from the ADC and
the T2 data on normoxic recovery. Interestingly, Johansson et al. (1997) showed that anoxia made the
optic system (retina and optic lobes) virtually completely unresponsive
to light stimuli, suggesting that the sense of vision is shut down during anoxia to save energy. It is possible that a less strict volume
regulation of the brain region involved in processing visual information (i.e., the optic lobes) is another facet of this
energy-saving strategy.
Using extracellular K+ electrodes inserted into
the optic lobes, it was demonstrated that crucian carp were able to
maintain their extracellular [K+] below 3 mM
for up to 6 h of anoxia. After an initial increase in
extracellular [K+] from a normoxic level of
1.7 ± 0.1 to 2.4 ± 0.2 mM during the first 60 min of
anoxia, no further changes were seen (Nilsson et al.
1993). In the light of the present results, the slight increase
in extracellular [K+] seen in the optic lobes
of anoxic crucian carp could be primarily related to a decrease in the
extracellular water volume rather than a net release of
K+ from brain cells. Thus the amount of
K+ present in the extracellular space may
actually be unchanged during anoxia.
The anoxia induced drop in T2 values displayed by
the crucian carp brain illustrates the phenomenon of BOLD MRI. During
anoxia, the entire hemoglobin pool should become deoxygenated.
Deoxygenated hemoglobin (Hbdeoxy) is paramagnetic and causes the MRI
signal intensity to drop in T2- and
T*2- weighted images (Ogawa et al.
1990; Prielmeier et al. 1994
; Thulborn et
al. 1982
; Van Zijl et al. 1998
). This signal
intensity drop was immediately reversed as the fish regained access to
oxygen and the hemoglobin became oxygenated (oxyHb is diamagnetic and displays no effect on the MR image contrast). The fact that this T2 drop was only significant in the crucian carp
and not in the common carp might be explained by a concomitant
T2 increase in the common carp due to an increase
in the ratio free-to-bound water, superimposed on the
T2 drop caused by deoxygenation of Hb. However,
another explanation could be that a significantly large BOLD drop
during anoxia reflects a higher concentration of Hb or a larger
cerebral blood volume in the crucian carp compared with common carp.
This large BOLD drop could then also be related to the earlier
demonstrated doubling of the cerebral perfusion in the crucian carp
during anoxia (Nilsson et al. 1994
).
The T2 values obtained after extended anoxic
exposure (24 h) or on normoxic recovery after 24 h of anoxia
remained lower than those initially obtained in normoxia; this could
reflect an increased cerebral blood volume or Hb content.
Houston et al. (1996) and Kakuta et al.
(1992)
demonstrated that both Hb and hematocrit increase in
goldfish and common carp during prolonged hypoxia; this provides a
plausible explanation for our T2 data.
Yoshikawa et al. (1995)
on the other hand demonstrated
hypoxia increased cerebral blood flow (by 70%) and cerebral blood mass
(by 10%) in common carp. If similar physiological changes occur in the crucian carp, then this could also contribute to the observed fall in
T2 values.
Differences in brain tissue characteristics correlated with anoxia tolerance
A likely explanation for the generally higher ADC value of the
crucian carp is a higher extracellular volume or a smaller intracellular volume as compared with the common carp. This could be of
functional significance with regard to anoxia tolerance. During anoxia
or hypoxia, a larger extracellular volume fraction (EVF) should
attenuate a rise in interstitial concentrations of transmitters such as
glutamate and glycine and thus reduce the risk of a tonic activation of
NMDA receptors. A maybe more important advantage of a large EVF would
be an attenuation of a rise in extracellular
[K+] that could threaten to depolarize the
brain (McBain et al. 1990). In other words, a large EVF
could be an advantage during anoxic or ischemic situations. When
comparing the adult mammalian brain with the brain of neonates, also a
difference in the EVF is found: 10-20% of the whole brain is
extracellular fluid in the adult, while this value is 15-25% for
neonates. Neonates are more anoxia resistant than adults (Hansen
1985
). However, although in mammals there appears to be a
species difference in normoxic ADC values, from 600 to 700 × 10
12
m2/s for rats, 800 to 1,000 × 10
12 m2/s in cats, to well over
1,000 to 2,000 × 10
12
m2/s in humans (Knight et al.
1991
; Mosely et al. 1990
; Warach et al.
1992
), there is no great difference in brain anoxia tolerance between these species.
The T2 values of brain tissue of common carp were significantly smaller than those of the crucian carp. Together with the higher ADC data, this shows that the crucian carp has a larger EVF and subsequently probably a higher free water content.
Impact of anesthesia and temperature on the MRI measurements
All experiments were done under MS 222 anesthesia. The main reason
for the use of an anesthetic was to immobilize the animals and to
anesthetize the common carp to which anoxia is likely to be stressful.
Some anesthetics have cerebroprotective capacities (Bickler et
al. 1995). However, comparative experiments on nonanesthetized crucian carp using only the muscular relaxant alloferine provided similar results (data not shown). The MS 222 dose was such that the
animals could easily be kept anesthetized for up to 6 h, the duration of an entire experiment, and still recover uneventfully from
this treatment.
The ADC, reflecting the Brownian motion of water molecules in the
tissues, depends on the temperature. All brain ADC data available in
the literature have been obtained from mammals and therefore at 37°C.
Studying the impact of hypothermia on the ADC of rat brain,
Jiang et al. (1994) found that the ADC decreases with
1.5%/°C. Our measurements on carps were done at 18°C, which is
19° lower than the average temperature of the investigated mammals.
Thus our measurements should provide ADC values 28.5% lower than those
measured in, e.g., rat brain, 600-700 × 10
12
m2/s, thus between 429 and 526 × 10
12
m2/s. This is close to what we actually measured:
399 ± 74 × 10
12
m2/s for the common carp and 487 ± 60 × 10
12
m2/s for the crucian carp.
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ACKNOWLEDGMENTS |
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We thank H. Denis for management of the fish culture facility.
This work was financially supported by the Belgian National Science Foundations (Fund for Scientific Research), the University of Antwerp (University Center Antwerp), and the Research Council of Norway.
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FOOTNOTES |
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Address for reprint requests: A. Van der Linden, Bio-Imaging Lab, University Center Antwerp, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium (E-mail: avadelin{at}ruca.ua.ac.be).
Received 8 March 2000; accepted in final form 22 September 2000.
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REFERENCES |
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