Does Anoxia Induce Cell Swelling in Carp Brains? In Vivo MRI Measurements in Crucian Carp and Common Carp

Annemie Van der Linden,1 Marleen Verhoye,1 and Göran E. Nilsson2

 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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1. Schematic presentation of the water flow-through system mounted in the bore of the magnet (see also Fig. 2A) and connected to an aquarium and a set of control and monitoring devices to ensure the appropriate experimentally defined environmental parameters during the magnetic resonance imaging (MRI) experiment; 1, thermostat; 2, 100-l aquarium; 3, pump; 4, aeration; 5, bubbling with nitrogen; 6, flow rate, oxygen level, temperature and pH control; 7, water inflow; 8, Helmholtz-like radio frequency coil; 9, water outflow; 10, animal temperature control.



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Fig. 2. A: animal-holding device containing an anesthetized crucian carp. A constant irrigation of the gills is ensured by a continuous flow of water (containing 0.011% MS 222 as anesthetic) through a tube fixed in the mouth of the fish (500 ml/min). B: dedicated RF headphone antenna. C: animal-holding device with RF antenna mounted such that it allows MRI of the head. This set-up is inserted in the bore of the magnet of the magnetic resonance instrument.

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 (delta ) of 10 ms, a diffusion-gradient separation time (Delta ) 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
<IT>S</IT><SUB><IT>i</IT></SUB><IT>=</IT><IT>S</IT><SUB><IT>0</IT></SUB><IT> exp</IT>(−<IT>TE<SUB>i</SUB>/T<SUB>2</SUB></IT>)
with Si the pixel signal intensity of the SE image acquired with an echo time TEi (TE1 = 18 ms, TE2 = 34 ms, TE3 = 50 ms). This pixel-by-pixel fitting resulted in T2 maps, images in which the gray level of each pixel represents the fit-parameter T2.

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)
<IT>S</IT><SUB><IT>DWi</IT></SUB><IT>=</IT><IT>S</IT><SUB><IT>DW0</IT></SUB><IT> exp</IT>[−(<IT>b</IT><SUB><IT>ix</IT></SUB><IT>−</IT><IT>b</IT><SUB><IT>0x</IT></SUB>)<IT>ADC</IT>]
For different regions of interest (ROI), we determined the transversal relaxation (T2) and the apparent diffusion coefficient (ADC). The different ROIs were determined on the high resolution horizontal T2-weighted images (Fig. 3, A and B from 3 to 12) and copied to the corresponding ADC and T2 maps obtained at different time points. The ROIs were chosen in the brain regions indicated for both carp and crucian carp on Fig. 3: telencephalon, hypothalamus, optic lobe, vagal lobe, and cerebellum.



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Fig. 3. High-resolution MRIs obtained on anesthetized common carp (A, 1-12) and crucian carp (B, 1-12). The image resolution is 156 µm. A1 and B1: a midsagittal image through the head of the fish showing the brain and the spinal cord. A2 and B2: the same image but with superimposed, the horizontal magnetic resonance sectioning plane with consecutive slices of 1-mm thickness (from 3 to 12). A, 3-12, and B, 3-12, represent an entire set of consecutive horizontal MRIs showing the following structures: h, hypothalamus; ot, optic tectum; vl, vagal lobe; sc, spinal cord; t, telencephalon; cb, cerebellum.

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
ADC=<FR><NU>ADC<SUB>meas</SUB></NU><DE>ADC<SUB>meas</SUB>(waterphantom)</DE></FR> 2000(10<SUP>−12</SUP> m<SUP>2</SUP>/s)
with a theoretical ADC value of 2,000 10-12 m2/s for free water at T = 20°C.

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:
%ADC change=100 ∗ <FR><NU>ADC(anoxia or normoxic recovery)</NU><DE>ADC(normoxia)</DE></FR>

%T<SUB>2</SUB> change=100 ∗ <FR><NU>T<SUB>2</SUB>(anoxia or normoxic recovery)</NU><DE>T<SUB>2</SUB>(normoxia)</DE></FR>
Absolute values were compared when results were obtained from different individuals such as when comparing crucian carp exposed to normoxia and 1, 2, 5, and 24 h anoxia. Results are reported as means ± SD. One-sample Student's t-tests (2-sided; with Ho: sample mean of % values = 100) was performed on the % values while two-sample Student's t-test (2 sided) was used on absolute values. The significance level was set at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 × 10-12 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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Fig. 4. Percent change (mean ± SD) vs. normoxic value of the apparent diffusion coefficient (ADC) as monitored on-line in different regions of the brain of common carp submitted to normoxia (control situation), 2 h anoxia, and 100 min of normoxic recovery. One-sample 2-sided t-test comparison with initial normoxic situation (*P < 0.05, **P < 0.01, ***P < 0.001). Absolute (mean ± SD) normoxic ADC value for optic lobes was 397 ± 80 × 10-12 m2/s, for vagal lobes was 388 ± 52 × 10-12 m2/s, for hypothalamus was 451 ± 78 × 10-12 m2/s, for telencephalon was 429 ± 34 × 10-12 m2/s, and for cerebellum was 332 ± 84 × 10-12 m2/s.

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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Fig. 5. Percent change (mean ± SD) vs. normoxic value of the T2 as monitored on-line in different regions of the brain of common carp submitted to normoxia (control situation), 2 h anoxia, and 100 min of normoxic recovery. One-sample 2-sided t-test comparison with initial normoxic situation (*P < 0.05, **P < 0.01, ***P < 0.001). Absolute (mean ± SD) normoxic T2 value for optic lobes was 58 ± 9 ms, for vagal lobes was 57 ± 7 ms, for hypothalamus was 64 ± 13 ms, for telencephalon was 61 ± 9 ms, and for cerebellum was 48 ± 3 ms.

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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Fig. 6. Mean brain volume (mm3) (±SD) of common carp (n = 5) and crucian carp (n = 5) as measured on-line when the carp were exposed to changing oxygen concentrations: starting with normoxia, followed by 2 h anoxia and subsequent 100 min of normoxic recovery.



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Fig. 7. In vivo high-resolution MRIs obtained from the same common carp on normoxia (A), 2 h anoxia (B), and 100 min normoxic recovery after 2 h anoxia (C). The images are obtained at different levels in the brain, from hypothalamus (far left) up to the level of the optic and vagal lobes (far right) and illustrate the disappearance of the ventricles (e.g., the hyperintense structures in the optic lobe seen on the images of the 3rd and 4th columns) and the slight broadening of the brain on anoxia and normoxic recovery.

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 × 10-12 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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Fig. 8. Percent change (mean ± SD) vs. normoxic value of the ADC as monitored on-line in different regions of the brain of crucian carp submitted to normoxia (control situation), 2 h anoxia, and 100 min of normoxic recovery. One-sample 2-sided t-test comparison with initial normoxic situation (*P < 0.05, **P < 0.01, ***P < 0.001). Absolute (mean ± SD) normoxic ADC value for optic lobes was 508 ± 40 × 10-12 m2/s, for vagal lobes was 472 ± 86 × 10-12 m2/s, for hypothalamus was 553 ± 56 × 10-12 m2/s, for telencephalon was 513 ± 62 × 10-12 m2/s, and for cerebellum was 395 ± 34 × 10-12 m2/s.

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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Fig. 9. Percent change (mean ± SD) vs. normoxic value of T2 as monitored on-line in different regions of the brain of crucian carp submitted to normoxia (control situation), 2 h anoxia, and 100 min of normoxic recovery. One-sample 2-sided t-test comparison with initial normoxic situation (*P < 0.05, **P < 0.01, ***P < 0.001). Absolute (mean ± SD) normoxic T2 value for optic lobes was 44 ± 1 ms, for vagal lobes was 44 ± 3 ms, for hypothalamus was 50 ± 1 ms, for telencephalon was 50 ± 1 ms, and for cerebellum was 44 ± 1 ms.

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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Fig. 10. ADC values (mean ± SD) determined in different regions of the crucian carp brain under normoxia (control situation, n = 5), 1 h anoxia (n = 5), 2 h anoxia (n = 5), 5 h anoxia (n = 5), 24 h anoxia (n = 5), and 100 min of normoxic recovery after 24 h anoxia (n = 5). Two-sample, 2-sided t-test comparison with initial normoxic situation (*P < 0.05, **P < 0.01, ***P < 0.001).

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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Fig. 11. T2 values (mean ± SD) determined in different regions of the crucian carp brain under normoxia (control situation), 1 h anoxia, 2 h anoxia, 5 h anoxia, 24 h anoxia, and 100 min of normoxic recovery after 24 h anoxia. Two-sample, 2-sided t-test comparison with initial normoxic situation (*P < 0.05, **P < 0.01, ***P < 0.001).

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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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.


    ACKNOWLEDGMENTS

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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

0022-3077/01 $5.00 Copyright © 2001 The American Physiological Society