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Neonatal asphyxia and hyperthermia and cognitive deficits in adult rats: Role of iron
ARTICLE IN PRESS Journal of Thermal Biology 34 (2009) 391–400
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Neonatal asphyxia and hyperthermia and cognitive deficits in adult rats: Role of iron Justyna Rogalska , Micha" Caputa, Katarzyna Pia˛tkowska, Anna Nowakowska Poland Department of Animal Physiology, Institute of General and Molecular Biology, N. Copernicus University, ul. Gagarina 9, 87-100 Torun,
a r t i c l e in fo
abstract
Article history: Received 5 June 2009 Accepted 25 August 2009
We compared effects of a critical neonatal anoxia, applied in Wistar rats at body temperatures of 33, 37 and 39 1C, on memory performance in adulthood. Because hyperthermic–anoxic neonates suffer from hyperferremia an additional group of rats, exposed to anoxia at 39 1C, was injected with deferoxamine, a chelator of iron. At the age of 4 and 12 months all rats were examined in hole board, typical maze and Morris maze. The memory was disturbed by neonatal anoxia at 39 1C. The disturbances were prevented by both the naturally reduced body temperature and by deferoxamine. In conclusion, neonatal hyperthermia induces iron-mediated, extremely delayed postanoxic cognitive disturbances in adulthood. & 2009 Elsevier Ltd. All rights reserved.
Keywords: Rats Neonatal asphyxia Hyperthermia Iron Deferoxamine Behaviour Memory disturbances
1. Introduction Experimental birth asphyxia results in both immediate (Tan et al., 1999; Skoff et al., 2001; Buonocore et al., 2002) and delayed (Dell’Anna et al., 1991; Nyakas et al., 1996; Perlman, 2001; Mikati et al., 2005) damage to the brain. The mechanism of hypoxicischaemic neuronal cell death in animals and humans after hypoxia-ischaemia includes neuronal necrosis and apoptosis (Perlman, 2006). Accumulation of free radicals within the mitochondria triggers c-aspartate proteases (caspases) pathway leading to apoptosis, thus amplifying initial damage caused by necrosis (Santore et al., 2002). The most extreme delays in the damage are connected with oxidative stress, which is catalysed by iron deposited in the asphyxiated brain (Kondo et al., 1995; Buonocore et al., 1998). Also iron supplementation in the critical postnatal period leads to oxidative stress in adult rats (Dal-Pizzol et al., 2001). Neonatal anoxia/ischaemia is also known as a common cause of delayed learning disturbances in juvenile and adult rodents (Dell’Anna et al., 1991; Buwalda et al., 1995; Nyakas et al., 1996; Mikati et al., 2005). Because the insult affects the hippocampal area (Papazisis et al., 2008), in particular, failure of spatial memory is the main learning disturbance (Dell’Anna et al., 1991; Nyakas et al., 1996; Mikati et al., 2005).
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E-mail address: [email protected] (J. Rogalska). 0306-4565/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2009.08.003
Previously we have demonstrated that neonatal asphyxia, in rats incubated at an elevated ambient temperature to keep their rectal temperature at 39 1C, induced plasma hyperferremia (Caputa et al., 2001) followed by iron accumulation in the frontal cortex, the hippocampus and the corpus striatum (Rogalska et al., 2006b). These disturbances were prevented when the asphyxiated rats were allowed to maintain their body temperature at physiological neonatal level of 33 1C. The hyperthermic neonatal exposure to anoxia causes lifelong emotional disturbances, such as stress-induced hyperactivity in juvenile rats (Rogalska et al., 2004) and reduced alertness to external stimuli signaling potential dangers in adult (Caputa et al., 2005) and old (Rogalska et al., 2006a) rats. These abnormalities were prevented by both the neonatal-age-specific physiological reduction in body temperature and postanoxic injection of deferoxamine (DF), a chelator of iron (Rogalska et al., 2004, 2006a; Caputa et al., 2005). It must be stressed that deferoxamine protects the rat brain against the above-mentioned iron accumulation (Rogalska et al., 2006b). There are, however, some controversies concerning neuroprotective effects of post-hypoxic-ischaemic reductions in body temperature. The reduction in body temperature delays but does not prevent cerebral injury in rats exposed to the insult in their infancy (Trescher et al., 1997). Surprisingly, some hippocampal neurons, previously salvaged due to body cooling, are extremely vulnerable to secondary ischaemic attacks (De Bow and Colbourne, 2003). Deferoxamine decreases hypoxic-ischaemic and reperfusionassociated brain injury in animals by blocking the formation of reactive oxygen species via inhibition of Fenton reaction.
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Moreover, the drug exerts its effect in neonatal rats by activation of the hypoxia-inducible transcription factor-1a (HIF-1a), a master switch of the brain’s response to hypoxia (Hamrick et al., 2005). There are, however, to the best of our knowledge, no published studies that have evaluated the effects of DF, under experimental anoxic conditions, on the memory processes in adult rats. Hypothermia applied during and after hypoxia-ischaemia seems to be the most attractive protective strategy because of multiple effects at different levels within pathways that contribute to brain injury after the insult (Perlman, 2006). Recent clinical trials confirmed that selective head cooling may prevent encephalopathy in term infants at highest risk for perinatal hypoxic-ischaemic brain injury (Gluckman et al., 2005). The efficacy and safety of whole-body cooling initiated within 6 h of age to a depth of 33.5 1C for 72 h duration has also been recently demonstrated (Shankaran et al., 2008). Recent clinical trials showed that relatively high temperatures applied in incubators during usual care after hypoxia-ischaemia is associated with increased risk of adverse outcomes (Laptook et al., 2008). Infants with pyrexia of 38 1C or more had an elevated rate of unfavorable outcome (Wyatt et al., 2007). Accordingly, the aim of the present study was to investigate long-term effects of neonatal anoxia, hyperthermia and pharmacological suppressing of hyperferremia with deferoxamine on development of spatial memory in rats. A battery of appropriate behavioural tests, such as hole board, Morris water maze and typical maze test, were performed at 4 and 12 months of age following the neonatal insult. We also applied the light-dark discrimination learning test (Li et al., 1999) to check both the acquisition and the latency of retention of the one-trial passive avoidance memory.
2. Materials and methods All experiments described in the present paper were performed according to the rules of the State Committee on the Use and Care of Laboratory Animals (permission of the Local Committee No. 6/2003). 2.1. Animals A total of 128 newborn Wistar rats of 2 days of age, of both sexes, weighting 7–8 g were used. The newborns were taken from their mothers (housed in acrylic cages lined with wood shavings, at a room temperature of 20–22 1C) and were exposed to a critical anoxia at various body temperatures. The experimental animals were divided equally among groups described in the Section 2.2. Prior to weaning, pups were housed with their own dams. The weanlings were sorted according to sexes and all males (79 animals) were used for the behavioural experiments. The males were housed in plastic cages at light:dark conditions of 12 h:12 h and were fed with commercial chow pellets and provided with water ad libitum. When they reached the age of 4 months and then at the age of 12 months they were subjected to the behavioural tests described in the Section 2.3. 2.2. Exposure to anoxia The procedures used to elicit anoxia, to manipulate neonatal body temperature, and pharmacological treatment of the rats were described in details in our previous work (Rogalska and Caputa, 2005). Briefly, prior to the manipulations, miniature thermocouples (0.1 mm in diameter) were inserted 5 mm into the rectum of the rat pups. Then the pups, divided into the following
seven groups, were individually transferred to 200 ml plethysmographic chambers, kept at different temperatures to stabilize body temperature at three different levels: (i) 33 1C (which is the normal body temperature of newborn rats (Rogalska and Caputa, 2005))—single anoxic and control groups, (ii) 37 1C (which is the body temperature of healthy adults)—single anoxic and control groups, and (iii) 39 1C (which is body temperature typical of febrile adults)—two anoxic and a single control group. To elicit anoxia, the plethysmographic chambers were flushed with 100% nitrogen. Rats of the extra group exposed to anoxia at the highest body temperature of 39 1C, were injected with DF (deferoxamine mesylate, Sigma-Aldrich, Chemie, GmbH, Steinheim, Germany), a chelator of iron, to match the nontreated anoxic animals. The drug was injected subcutaneously (100 mg/kg s.c.) twice: immediately after anoxia and 24 h later. The anoxia was continued for 25 min (Buwalda et al., 1995; Nyakas et al., 1996) or it was terminated earlier in hyperthermic groups (to prevent lethal asphyxia) when a critical accelerated and shallow gasping phase (identified barometrically in the plethysmographic chambers) occurred (Caputa et al., 2001; Rogalska and Caputa, 2005). It must be stressed that this procedure allows keeping mortality rate at 0%. After anoxia the animals were exposed to atmospheric air at unchanged temperature for 120 min. Control rats, were exposed to atmospheric air throughout the same period (145–130 min) in the respective thermal conditions. 2.3. Behavioural tests The experiments started when the rats were 4 months old and were repeated (except for the light–dark discrimination test, which could not be repeated for technical reasons described in the next section) at the age of 12 months. Each behavioural test was performed in a sound-isolated room with lighting conditions and environmental cues held constant throughout testing. The experimenter stayed in the room adjacent to the one in which the experiments were performed. Before testing of the next animal, the floor and walls of the apparatus were carefully cleaned to remove the smell traces left by previous rats. To avoid influences of circadian rhythms on performance of the experimental animals each behavioural test was started at 9 a.m. The behaviour of rats during the tests was videotaped and then analysed with the EthoVision 2.3 software (Noldus, Wageningen, Netherlands). 2.3.1. Light–dark discrimination test At 4 months of age the rats were subjected to the passive avoidance task (light–dark discrimination test) described by Li et al. (1999). A two-compartment step-through apparatus was used. The white-painted and illuminated compartment was separated from the black-painted and nonilluminated compartment (dimensions: 0.24 0.24 0.40 m3 each) by a 0.24 0.40 m2 sliding door (all made of plywood). The dark compartment was supplied with a metal grid floor for the delivery of electric foot shocks. During the first session all rats were subjected to a single pretraining trial. They were placed in the illuminated compartment for 4 min. On the following day, in the acquisition trial, the rat was placed in the illuminated compartment, and the time that elapsed until it entered the dark compartment (acquisition latency) was recorded. As soon as the rat entered the dark compartment, the door was closed and an electric shock (50 Hz, 100 V for 1 s) was applied twice (at 2 s interval) to the grid by an adjustable shock generator (home-made). Then the rat was placed in its home cage. A retention trial was carried out by placing the
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rat in the illuminated compartment again 24 h after the acquisition trial. The time (retention latency) of entering the dark compartment, and the time spent in the illuminated compartment in the retention trial over a 3-min period (excluding any excursions into the dark compartment) were recorded. If the rat did not enter the dark compartment within 180 s, the retention test was interrupted, and the maximum score of 180 s was assigned. Because the rats used in this investigation were 2 months later implanted with transmitters for telemetric recording of body temperature and motor activity (to be published in a separate paper) we had to cancel their second testing at the age of 12 months (there was a risk of damage to the transmitters by the electric shocks). 2.3.2. Hole board spatial learning test The equipment described by Buwalda et al. (1995) was applied to study water-motivated spatial learning behaviour. The hole board (dimensions: 0.7 0.7 0.45 m3) is a test apparatus, in which animals have to memorize a random pattern of 4 holes filled with water, accessible to them, out of 20 holes (35 mm diameter, 40 mm depth) distributed equidistantly in a floor plate. Each experiment started 24 h after water deprivation (we decided to replace hunger-motivated learning, which is commonly used in this test, with thirst motivated learning, because the latter seems to be much stronger as a drive). Before testing the rats were placed in a start-box (0.2 0.7 0.45 m3) adjacent to the hole board arena. After 10 s the guillotine door was opened and animals were allowed to visit the arena. During the first 2 days the rats were placed four times daily for 5 min periods in the hole board with water available in all holes in order to get used to visiting the holes for reward and so the animals had an opportunity to rehydrate. The spatial learning started on the third day. Four holes arranged in a fixed, randomly chosen pattern were supplied with accessible water hidden 30 mm under the floor level. Remaining 16 holes also contained water but it was inaccessible (covered with a wire mesh). A visit to a hole was scored when the nose of the rat was placed in it. Rats were removed 3 min after the start of each trial. The testing lasted 5 days. The reference memory ratio (RMR) was defined as RMR ¼
x yþz
where x is a number of visits and revisits to the baited set of holes, y is four minus the number of visits to baited holes and z is total number of visits and revisits to all holes. The overall range of the RMR quotient extends from 0 (no baited holes visited) to 1 (only the four baited holes visited). 2.3.3. Typical maze The typical maze (dimensions: 0.7 0.7 m2) was surrounded by walls 0.45 m high. Again, we used water-deprived rats, which were given a cup of water (2 ml) as a reinforcement. The test was performed three times every 5 days and the animals were tested twice a day at 4-h intervals between the tests, six times altogether. The water deprivation was planned as follows: 36 h prior to the first session of the day and then for another 4 h until completion of the second test of the day. The animals were placed at the starting point of the maze and had to reach the water cup without any other reinforcement. The latency to reach the reward was recorded and analysed. 2.3.4. Morris water maze A circular swimming pool (1.40 m in diameter and 0.48 m in height) was filled with water ( 25 1C) to a depth of 0.30 m. Water
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was rendered opaque by addiction of a small quantity of powdered milk and some beet juice was added to contrast white fur of rats. Four points around the circumference of the pool were arbitrarily designated: North, South, East, and West, which allowed to divide the pool area into 4 quadrants (NW, SW, etc.). A circular goal platform (90 mm in diameter) laid 20 mm under the surface of the water, in the middle of the SW quadrant for all trials. The rats were given four consecutive trials a day (with 20 s intervals) for 5 days, according to the procedure described by Morris (1984). The animals were dropped into water from different quadrants on each trial (always facing the wall of the pool), and had to learn to navigate to the invisible platform using the spatial cues available in the room. The sequence of starting points was changed during consecutive days of the test. Rats that failed to reach the platform within 120 s were gently guided to it and allowed to climb on. The latency of reaching the platform in all trials was recorded. After the fourth trial of the last session, the rats were submitted to a probe test for spatial bias. The platform was removed and the rat, starting from the opposite quadrant, was allowed a 2-min search for the platform. The path was recorded on videotape and a spatial bias regarded as a number of crossings the SW quadrant of the pool (where the platform was previously hidden) was measured. 2.4. Data analysis All results were tested for normality with Kolmogorov– Smirnov test. All variables of rat’s behaviour in the passive avoidance test were analysed with two-way ANOVA in a 3 2 factorial design, where temperature (33, 37, and 39 1C) and experimental conditions (control and anoxia) were the factors. A three-way analysis of variance (temperature experimental conditions trial) with repeated measures on the third factor was applied to analyse the results of rat’s behaviour in hole board test (3 2 5), water maze (3 2 5) and typical maze (3 2 6). The effect of treatment with DF in hyperthermic groups in light– dark discrimination test was analysed by one-way ANOVA and the effect of DF in the hole board test, water maze and typical maze—by two-way ANOVA (experimental conditions trial) with repeated measures on the last factor. Each analysis was followed by multiple comparison using Newman–Keuls post hoc test. The significance level was set at p o0.05 for all statistical tests. Paired Student’s t-test was used to analyse the progress in learning in particular groups of rats. Data analyses were performed with the use of Statistica (StatSoft, Cracow, Poland).
3. Results 3.1. Light–dark discrimination test There was a significant effect of neonatal body temperature on retention latency (Fig. 1A; F2,56 =6.79; po0.01), but no effect of neonatal anoxia (F1,56 = 0.64; n.s.), and no interactions between both factors (F2,56 = 0.08; n.s.). The latency of both control and anoxic rats maintained at physiological body temperature of 33 1C tended to be longer than that of the rats exposed to neonatal anoxia at body temperatures of 37 and 39 1C. Postanoxic DF treatment of hyperthermic newborns did not influence the latency (F2,22 =2.26; n.s.) (Fig. 1A, right side). However, the latency tended to be longer in DF-treated animals than that in their hyperthermic (39 1C) counterparts exposed to neonatal anoxia. The time spent in the light compartment in the retention trial (Fig. 1B) was significantly influenced by temperature (F2,56 = 9.58;
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Fig. 1. Effects of neonatal anoxia, neonatal body temperature and chelation of iron with deferoxamine (DF) upon passive avoidance memory: retention latency (A) and time spent in the light compartment (B) in the retention trial over 3-min period (excluding any excursions into the dark compartment) in young adult (4 months old) rats which were exposed 24 earlier to electric foot shock in the dark compartment. The data are presented as mean values+ S.E.M., *p o0.05 for two-way ANOVA or #p o0.05 for oneway ANOVA. Numbers inside or under columns indicate numbers of animals in individual groups.
p o0.001). However, there were no effects of neonatal anoxia (F1,56 = 1.83; n.s.), and no interactions between both factors (F2,56 = 0.53; n.s.). Post hoc analysis revealed that rats exposed to neonatal anoxia at body temperatures of 37 and 39 1C spent less time in the light compartment than both control and anoxic rats kept postpartum at body temperature of 33 1C (p o0.05). There was also a significant effect of experimental conditions (F2,22 = 3.69; p o0.05) in three groups of rats exposed to neonatal hyperthermia (Fig. 1B, right side). The time spent in the light compartment was significantly longer in DF-treated rats than that in the rats exposed to neonatal anoxia at the same body temperature (p o0.05).
3.2. Hole board test Both neonatal body temperature and neonatal anoxia clearly influenced the learning of 4 months old rats (Fig. 2A). Reference memory ratio (RMR) decreased with increasing neonatal body temperature (F2,42 = 34.44; p o0.001) and was further reduced in rats exposed to neonatal anoxia (F1,42 =11.86; p o0.01) but there was no significant interaction between those factors (F2,42 = 0.15; n.s.). The increase of reference memory ratio during 5 consecutive testing days was observed in all experimental groups (F4,168 =63.97; p o0.001). Interactions of temperature and trial as well as anoxia and trial were also significant (F8,168 = 3.74; po0.001; F4,168 = 2.46; p o0.05). Post hoc test revealed that the performance of the animals exposed to neonatal anoxia at 39 1C was significantly lower than that of the asphyxiated rats kept postpartum at body temperature of 33 1C on days 3 (po0.05), 4 (po0.01) and 5 (p o0.05) (Fig. 2A). There were also significant differences of the performance between rats exposed to neonatal anoxia at 37 and 33 1C on days 3 (po0.05), 4 (po0.05) and 5 (po0.001). All groups started from the same RMR level, however, the progress of learning was clearly disturbed by neonatal exposure to hyperthermia and anoxia. There were significant differences between the three hyperthermic groups (Fig. 2A, right side; F2,19 = 5.01; p o0.05). There were also significant effects of the trial (F4,76 = 21.89; po 0.001), but there were no significant interactions between both factors (F8,76 =1.27; n.s.). Post hoc analysis demonstrated that from the 3rd day of the experiment on, DF-treated anoxic rats showed significant improvement in memory performance in comparison to their untreated counterparts at the same neonatal body temperature (day 3: po0.05; day 4: p o0.01; day 5: p o0.05). As far as the performance in the hole board in 12 months old rats is concerned (Fig. 2B) the analysis shows a significant main
effect of all three experimental factors: temperature (F2,34 = 7.75; po0.01), anoxia (F1,34 =8.75; po0.01) and trial (F4,136 = 16.75; po0.001). Interaction of temperature and anoxia (F2,34 =6.77; po0.01) was also significant. The efficacy of learning was better in control and anoxic rats allowed to maintain neonatal body temperature at 33 1C than that in rats exposed to neonatal anoxia at body temperatures of 37 and 39 1C. Further analysis with post hoc test revealed that the value of RMR on day 5 was lower in animals exposed to neonatal anoxia at 37 and 39 1C than that in anoxic rats kept postpartum at body temperature of 33 1C (p o0.01). The difference in RMR between the first and last day of the experiment in animals kept neonatally at physiological body temperature, both in control and anoxic groups, was significant (p o0.05, Student’s t-test), whereas in anoxic rats exposed to neonatal hyperthermia (body temperature of 37 and 39 1C) there was no progress in the performance in consecutive days of the experiment. There was a significant effect of the experimental conditions (F2,14 = 6.57; p o0.01) and trial (F4,56 =7.82; p o0.001) in three groups of rats exposed to neonatal body temperature of 39 1C (Fig. 2B, right side). Chelation of iron with DF prevented the impairment of learning.
3.3. Typical maze test Latency of reaching the reward in typical maze in 4 months old rats (Fig. 3A) was significantly influenced by neonatal anoxia (F1,41 = 5.82; po0.05) and trial (F5,205 = 38.68; p o0.001), and there was a significant interaction between these factors (F5,205 = 2.76; po0.05). On the other hand, the latency was not significantly different when analysed for temperature (F2,41 = 0.85; n.s.). Further analysis evidenced that on the first day of the experiment the latency in animals exposed to neonatal anoxia at 37 1C was significantly longer than that in anoxic rats allowed to maintain neonatal body temperature at 33 1C (p o0.05) and (surprisingly) than that in rats at the highest neonatal body temperature of 39 1C (p o0.001). DF treatment (Fig. 3A, right side) had no effect on the latency (F2,24 =0.06; n.s.). Two-way ANOVA shows only the main significant effect of trial (F5,120 = 16.69; po0.001), but no interaction between the factors (F10,120 = 0.80; n.s.). All groups showed a progressive decrease of latency in the subsequent trials. However, the performance was the best in control and anoxic rats maintained at physiological body temperature of 33 1C and in hyperthermic rats treated postanoxia with deferoxamine as well.
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Fig. 2. Effects of neonatal anoxia, neonatal body temperature and chelation of iron with deferoxamine (DF) upon reference memory ratio (RMR) in the hole board learning during 5 consecutive testing days (indicated by numbers below columns) in rats at the age of 4 months (panel A) and 12 months (panel B). The data are presented as mean values+ S.E.M. Number of animals in each group is indicated inside the column showing results of the initial testing day. Statistical differences are described in the text.
The disturbances due to neonatal exposure to anoxia and hyperthermia persisted in the same rats tested at the age of 12 months (Fig. 3B). In general, rats exposed to neonatal anoxia at 37 and 39 1C in all trials required more time to reach the water cup than rats kept postpartum at body temperature of 33 1C both in control and anoxic groups. There were significant main effects of neonatal body temperature (F2,40 = 4.99; p o0.01), anoxia (F1,40 = 5.15; po0.05) and interaction between these factors (F2,40 = 4.28; p o0.05). A significant effect of trial (F5,2 =17.0; p o0.001) indicates that time to reach the water cup shortened during subsequent sessions. Clear-cut differences between groups are seen already on the first day of the test. The time to reach the reward was significantly shorter in rats allowed to maintain their neonatal body temperature at 33 1C, both in control and anoxic groups, than that in animals exposed to neonatal anoxia at 37 1C (po0.05). During the consecutive days the performance tended to be superior in rats allowed to maintain the reduced neonatal body temperature. There were significant differences between the three hyperthermic groups (F2,12 = 3.98; p o0.05) and trials (F5,85 =8.95; p o0.001), and no significant interactions between both factors (F10,85 = 1.08; n.s.) (Fig. 3B, right side). DF-treated rats tended to perform better than the remaining animals. 3.4. Morris water maze test Neonatal anoxia combined with hyperthermia affected also the performance of the rats in the water maze (Figs. 4 and 5). The latency
to find the hidden platform in 4 months old rats (Fig. 4A) increased with increasing neonatal body temperature (F2,59 =6.36; po0.01). However, it was not significantly different when analysed for treatment (F1,59 =0.45; n.s.). Rats exposed to anoxia under hyperthermic conditions (39 1C) performed worse than did anoxic rats kept postpartum at body temperature of 33 1C (po0.001). DF-treated rats found the platform in a significantly shorter time (po0.05; Fig. 4A, right side) than their untreated anoxic counterparts at the same neonatal body temperature. Eight months later there were no clear differences in the navigation learning and the rats apparently remembered their earlier training (Fig. 4B). The performance was not influenced by neonatal temperature (F2,53 = 2.58; n.s.) and anoxia (F1,53 =0.004; n.s.). There were no significant differences between the three hyperthermic groups (right side of Fig. 4B; F2,26 =1.41; n.s.). There was, however, a significant effect of trial (F4,104 = 3.83; p o0.01), but no interaction between both factors (F8,104 = 0.91; n.s.). The latencies in DF-treated rats in each trial were shorter that those in their nontreated counterparts. As far as the spatial bias performance in 4 months old rats is concerned (Fig. 5A), it worsened with increasing neonatal body temperature (F2,59 =5.09; po 0.01) but was not significantly reduced in rats exposed to neonatal anoxia (F1,59 = 0.30; n.s.) and there was no interaction between these factors (F2,59 = 0.74; n.s.). The analysis of the spatial bias performance in the same rats at the age of 12 months (Fig. 5B) showed a significant main effect of both experimental factors: temperature (F2,53 = 3.74; p o0.05) and anoxia (F1,53 = 7.68; po0.01) but no interaction between them
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Fig. 3. Effects of neonatal anoxia, neonatal body temperature and chelation of iron with deferoxamine (DF) on spatial learning performance (expressed as latency of reaching the reward) in rats at the age of 4 months (panel A) and 12 months (panel B) placed in the typical maze during 6 consecutive trials (indicated by numbers below columns). The data are presented as mean values+ S.E.M. Number of animals in each group is indicated inside the column showing results of the initial testing day. Statistical differences are described in the text.
(F2,53 = 1.15; n.s.). Post hoc test revealed cognitive impairment in animals exposed to neonatal anoxia at 39 1C compared to those kept postpartum at body temperature of 33 1C, both in control and anoxic groups (p o0.05). A comparison of the spatial bias performance in three groups of rats exposed to neonatal body temperature of 39 1C showed significant main effects of treatment, both in 4 (F2,30 = 3.81; p o0.05; Fig. 5A) and 12 months old animals (F2,26 = 4.34; p o0.05; Fig. 5B). Post hoc analysis demonstrated that the performance in DF-treated rats was superior to that in their counterparts at the same neonatal body temperature, both in untreated control and anoxic groups (p o0.05) (Fig. 5A and B, right side).
4. Discussion There are some controversies concerning appropriate experimental models of hypoxic-ischaemic injury of the brain as well as various models of neuroprotective reductions in body temperature. One of the controversies concerns an adequate animal model of birth asphyxia. The majority of authors argue that 7-day-old rats are the most suitable (Trescher et al., 1997; Palmer et al., 1999; Mishima et al., 2004; Papazisis et al., 2008; van der Kooij et al., 2009) because they have brain maturation that corresponds to the term babies. On the other hand, the preterm infants are particularly susceptible to perinatal pathology (Sullivan, 1988;
Buonocore et al., 1998, 2002; Perlman, 2001; Rao and Georgieff, 2002). Therefore, in our experimental approach we keep on using 2-day-old rats as a model of the preterm birth asphyxia. This experimental approach is unique in terms of a pattern of body temperature manipulation (we use clamping of body temperature at various levels both during anoxia as well as over 120 min postanoxia). The present investigation has confirmed previous experimental data (Dell’Anna et al., 1991; Nyakas et al., 1996) that neonatal anoxia induces disturbances in spatial memory performance in adult rats. Moreover, we were able to show that such impairment is always connected with a significantly elevated body temperature and that normal body temperature of newborn rats, which is as low as 33 1C, protects them against the postanoxic disturbances. It must be stressed that all newborns of this group tolerated, highly repeatedly, the full-time (25 min) exposure to anoxia, while rats forced to maintain their body temperature at 39 1C tolerated anoxia for no longer than 10 min, which confirms the results of our previous paper (Rogalska and Caputa, 2005). In the present investigation postanoxic and posthyperthermic disturbances in spatial memory performance persisted in adult rats at the age of 4 and 12 months. Such a delayed postanoxic impairment is possibly induced by oxidative stress catalysed by iron, since postanoxic treatment of the hyperthermic newborns with deferoxamine, a chelator of iron, prevented the memory deficits.
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Fig. 4. Effects of neonatal anoxia, neonatal body temperature and chelation of iron with deferoxamine (DF) on spatial learning performance (expressed as latency of reaching the hidden platform) of rats at the age of 4 months (A), and 12 months (B) in water maze during 5 consecutive days (indicated by numbers below columns) of the experiment. The data are presented as mean values+ S.E.M. Statistical differences are indicated in the text.
Fig. 5. Effects of neonatal anoxia, neonatal body temperature and chelation of iron with deferoxamine (DF) upon memory retention of rats at the age of 4 months (A), and 12 months (B) in water maze during spatial bias task. The data are presented as mean values+ S.E.M., *p o0.05, **p o 0.01 for two-way ANOVA or #p o 0.05 for one-way ANOVA. Numbers inside columns indicate numbers of animals in individual groups.
In our previous papers we have shown that neonatal exposure of rats to anoxia under hyperthermic conditions leads to plasma hyperferremia (Caputa et al., 2001), which is followed by iron accumulation in the brain (Rogalska et al., 2006b). Asphyxiated preterm infants also suffer from free iron release in erythrocytes (Buonocore et al., 1998) enhancing the risk of oxidative injury (Sullivan, 1988; Buonocore et al., 2002). Increased body iron stores also contribute to the stroke progression in adult patients with a cerebral infarction (Davalos et al., 2000). Iron is necessary as a
cofactor of myelinization, yet the excessive cerebral iron deposition in asphyxiated neonatal rats (Palmer et al., 1999; Rogalska et al., 2006b) cannot support myelinization because the insult leads to reduced cerebral concentration of myelin basic protein and proteolipid protein and results in death of oligodendroglial precursors in the brain of neonatal mice (Skoff et al., 2001). Altogether, the accumulation of iron in the brain of asphyxiated newborns is unequivocally harmful. This conclusion is strongly supported by Dal-Pizzol’s et al. (2001) results showing that rats
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treated orally with high doses of iron in neonatal period suffer from oxidative damage to the brain even at the age of 3 months. There are several studies showing neuroprotective effect of DF due to iron chelation. Administration of DF resulted in a reduction in the number of damaged neurons in the CA1 region of hippocampus and tended to decrease damage to the region of CA3 and DG (Papazisis et al., 2008). Alterations in gene expression following DF treatment have also been described. One of them is an increase in expression of HIF1 alpha, which is suggested to promote cell survival in hypoxic tissues (Hamrick et al., 2005). Recently, van der Kooij and colleagues (2009) have found that deferoxamine did not prevent gray or white matter damage in 7 days old rats, which underwent right common carotid artery occlusion followed by a 90-min exposure to 8% oxygen. However, in the present paper we were able to show that deferoxamine normalizes cognitive processes in adult rats exposed to neonatal anoxia. Therefore, it is possible that DF plays a more significant role in the immature brain since iron accumulation is most pronounced in the early development. Damage to the rabbit brain following fetal hypoxia was clearly accelerated during initial 8 min of reoxygenation, which was associated with enhanced free radical production (Tan et al., 1999). This neurotoxic process is prevented by a moderate decrease in brain temperature (Globus et al., 1995; Nyakas et al., 1996; Colbourne et al., 1997). Therefore, reduced body temperature, applied in this investigation both during anoxia and immediately postanoxia, is likely to provide maximal neuroprotection, both immediate and extremely delayed (the latter being a consequence of the prevented cerebral iron accumulation). Postanoxic pathology manifests itself as both histological and behavioural abnormalities. In rats subjected to neonatal hypoxiaischaemia injury of the brain is observed from second week on, and progresses up to fifth month of their life (Mishima et al., 2004).The authors conclude, however, that quantitative histological analysis of the injury is less sensitive method of the evaluation than behavioural analysis. Postanoxic damage to the brain is connected not only with neuronal death but also with dendritic athrophy (Titus et al., 2007), which is indetectable histologically. Moreover, the cognitive and behavioural problems occur in premature infants in connection with the presence or absence of neuroimaging abnormalities. At adolescence they display specific deficits in everyday memory when compared with full-term controls (Briscoe et al., 2001). This is why we have decided to focus on behavioural effects of neonatal asphyxia in rats. All spatial memory tests applied in this investigation showed conspicuous cognitive disturbances in rats exposed to neonatal anoxia under hyperthermic conditions. Nevertheless, there were some subtle differences among the tests. The light–dark discrimination test revealed that the both groups of neonatal rats kept at their natural body temperature of 33 1C as well as rats exposed to neonatal anoxia under hyperthermic conditions and then injected with deferoxamine spent significantly more time in the light compartment (they previously had been subjected to the electric shock in the dark compartment) than the animals suffering from neonatal anoxia under hyperthermic conditions and untreated. In the hole board test older (12 months old) rats tended to perform worse than they did 8 months earlier. In contrast, the performances in both the typical maze and the water maze tended to improve in the older animals. This leads to a conclusion that of these three tests the experience played a role in the latter two but not in the former. Obviously, the older animals must have remembered the way to reach the goal in the typical maze and the water maze. Therefore, both tasks can be regarded as less challenging than the hole board task.
Actually, hole board task showed persistent, clearly harmful main effect of neonatal anoxia and neonatal body temperature, and a highly significant interaction between these factors. The remaining tests confirmed the main effect of neonatal body temperature (light–dark discrimination test and water maze in rats at the age of 4 months) or the main effect of neonatal anoxia (typical maze in rats 4 months old) or the main effect of both of them (typical maze and spatial bias in water maze at the age of 12 months). In each of the tests post hoc analysis evidenced clear-cut disturbances of learning in rats exposed to anoxia at body temperature of 39 1C, in comparison with the performance of animals exposed to anoxia at physiological body temperature of 33 1C. The combined harmful effect of neonatal hyperthermia and anoxia is not surprising. On the other hand, the selective effect of such a moderate and transient hyperthermia is an unexpected phenomenon. Prominent harmful effects of elevated temperature in animals subjected to hypoxia/ischaemia were observed when temperature elevations occurred immediately after the insult and were continued for long periods (Reglodi et al., 2000). This suggests that the neonatal brain is extremely susceptible to hyperthermia. There is a strong clinical support for such a conclusion. Namely, a prospective cohort study of 4915 low-risk women at term showed the association of maternal fever with neonatal encephalopathy, which was independent of other known intrapartum risk factors (Impey et al., 2001). In the previous papers (Rogalska et al., 2004, 2006a; Caputa et al., 2005) we were able to show that both the reduced body temperature and chelation of iron prevents postanoxic disturbances in stress responses of rats from their youth up to senescence. The present investigation implies the same conclusion concerning spatial memory disturbances. This strengthens an argument that asphyxiated human newborns should be moderately cooled to prevent not only early neurological deterioration, which has been shown in 12-months follow-up (Gunn et al., 1998; Compagnoni et al., 2002) but also a variety of much more delayed disturbances such as attention deficit-hyperactivity disorder (ADHD) in childhood and cognitive failure in adulthood. The pathological effect of the postasphyxic iron-mediated oxidative stress, indirectly shown in the present investigation, is also postulated in recent neonatological reviews. There is some cause for concern about infants who receive iron supplements or blood transfusions (Sullivan, 1988; Rao and Georgieff, 2002) as well as those who are resuscitated with 100% oxygen (Vento et al., 2003). Unfortunately, in contrast to the firmly established knowledge of both preventive (Rogalska and Caputa, 2005; Caputa, 2006) and defensive (Rogalska and Caputa, 2005; Caputa, 2006) reduction in body temperature of asphyxiated newborn rats, such data concerning human newborns are extremely scarce. For the last decades asphyxiated preterm infants are being forced to stabilize their body temperature at 37 1C by mean of incubators. Recording natural thermal responses of asphyxiated human newborns has not been continued for 50 years now! The results of one of those early investigations are similar to those obtained recently in experiments on animal models. Namely, body temperature of moderately asphyxiated babies was 1.5 1C lower than that of their healthy counterparts maintained under the same external conditions (Burnard and Cross, 1958). We conclude that there is an urgent need for revitalization of such studies. Because the neonatal community is conservative with respect to acceptation of such a moderate body or head cooling as a new ‘‘standard of care’’ for asphyxiated babies (Kirpalani et al., 2007) the researchers should do their best to convince the community to this procedure. It is generally acknowledged that there is no information regarding the optimal management of infants with hypoxicischaemic encephalopathy (Laptook et al., 2008). The range of ambient temperatures at which babies start sweating to lose heat
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is surprisingly wide (33–38 1C) (Laptook et al., 2008). Infants exposed to maternal fever have a greater likelihood of a low 1 min Apgar score, poor respiratory effort, and the need for bag-mask ventilation in the delivery room. A case-control study design demonstrated that intrapartum fever, which was unlikely to be infectious in origin, was associated with a 3.4-fold risk of unexplained early-onset seizures in term infants (Lieberman et al., 2000). Important associations between reduced or elevated body temperature at birth and subsequent morbidity and mortality (Laptook et al., 2008) suggest that change in temperature at birth causes brain injury (hyperthermia) or a neuroprotection (anapyrexia).
Acknowledgements This work was supported by the grant of 4.P05A.059.16 from the Polish State Committee for Scientific Research.
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Journal of Thermal Biology journal homepage: www.elsevier.com/locate/jtherbio
Neonatal asphyxia and hyperthermia and cognitive deficits in adult rats: Role of iron Justyna Rogalska , Micha" Caputa, Katarzyna Pia˛tkowska, Anna Nowakowska Poland Department of Animal Physiology, Institute of General and Molecular Biology, N. Copernicus University, ul. Gagarina 9, 87-100 Torun,
a r t i c l e in fo
abstract
Article history: Received 5 June 2009 Accepted 25 August 2009
We compared effects of a critical neonatal anoxia, applied in Wistar rats at body temperatures of 33, 37 and 39 1C, on memory performance in adulthood. Because hyperthermic–anoxic neonates suffer from hyperferremia an additional group of rats, exposed to anoxia at 39 1C, was injected with deferoxamine, a chelator of iron. At the age of 4 and 12 months all rats were examined in hole board, typical maze and Morris maze. The memory was disturbed by neonatal anoxia at 39 1C. The disturbances were prevented by both the naturally reduced body temperature and by deferoxamine. In conclusion, neonatal hyperthermia induces iron-mediated, extremely delayed postanoxic cognitive disturbances in adulthood. & 2009 Elsevier Ltd. All rights reserved.
Keywords: Rats Neonatal asphyxia Hyperthermia Iron Deferoxamine Behaviour Memory disturbances
1. Introduction Experimental birth asphyxia results in both immediate (Tan et al., 1999; Skoff et al., 2001; Buonocore et al., 2002) and delayed (Dell’Anna et al., 1991; Nyakas et al., 1996; Perlman, 2001; Mikati et al., 2005) damage to the brain. The mechanism of hypoxicischaemic neuronal cell death in animals and humans after hypoxia-ischaemia includes neuronal necrosis and apoptosis (Perlman, 2006). Accumulation of free radicals within the mitochondria triggers c-aspartate proteases (caspases) pathway leading to apoptosis, thus amplifying initial damage caused by necrosis (Santore et al., 2002). The most extreme delays in the damage are connected with oxidative stress, which is catalysed by iron deposited in the asphyxiated brain (Kondo et al., 1995; Buonocore et al., 1998). Also iron supplementation in the critical postnatal period leads to oxidative stress in adult rats (Dal-Pizzol et al., 2001). Neonatal anoxia/ischaemia is also known as a common cause of delayed learning disturbances in juvenile and adult rodents (Dell’Anna et al., 1991; Buwalda et al., 1995; Nyakas et al., 1996; Mikati et al., 2005). Because the insult affects the hippocampal area (Papazisis et al., 2008), in particular, failure of spatial memory is the main learning disturbance (Dell’Anna et al., 1991; Nyakas et al., 1996; Mikati et al., 2005).
Corresponding author. Tel.: + 48 103356 6112631; fax: + 48 103356 6114772.
E-mail address: [email protected] (J. Rogalska). 0306-4565/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.jtherbio.2009.08.003
Previously we have demonstrated that neonatal asphyxia, in rats incubated at an elevated ambient temperature to keep their rectal temperature at 39 1C, induced plasma hyperferremia (Caputa et al., 2001) followed by iron accumulation in the frontal cortex, the hippocampus and the corpus striatum (Rogalska et al., 2006b). These disturbances were prevented when the asphyxiated rats were allowed to maintain their body temperature at physiological neonatal level of 33 1C. The hyperthermic neonatal exposure to anoxia causes lifelong emotional disturbances, such as stress-induced hyperactivity in juvenile rats (Rogalska et al., 2004) and reduced alertness to external stimuli signaling potential dangers in adult (Caputa et al., 2005) and old (Rogalska et al., 2006a) rats. These abnormalities were prevented by both the neonatal-age-specific physiological reduction in body temperature and postanoxic injection of deferoxamine (DF), a chelator of iron (Rogalska et al., 2004, 2006a; Caputa et al., 2005). It must be stressed that deferoxamine protects the rat brain against the above-mentioned iron accumulation (Rogalska et al., 2006b). There are, however, some controversies concerning neuroprotective effects of post-hypoxic-ischaemic reductions in body temperature. The reduction in body temperature delays but does not prevent cerebral injury in rats exposed to the insult in their infancy (Trescher et al., 1997). Surprisingly, some hippocampal neurons, previously salvaged due to body cooling, are extremely vulnerable to secondary ischaemic attacks (De Bow and Colbourne, 2003). Deferoxamine decreases hypoxic-ischaemic and reperfusionassociated brain injury in animals by blocking the formation of reactive oxygen species via inhibition of Fenton reaction.
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Moreover, the drug exerts its effect in neonatal rats by activation of the hypoxia-inducible transcription factor-1a (HIF-1a), a master switch of the brain’s response to hypoxia (Hamrick et al., 2005). There are, however, to the best of our knowledge, no published studies that have evaluated the effects of DF, under experimental anoxic conditions, on the memory processes in adult rats. Hypothermia applied during and after hypoxia-ischaemia seems to be the most attractive protective strategy because of multiple effects at different levels within pathways that contribute to brain injury after the insult (Perlman, 2006). Recent clinical trials confirmed that selective head cooling may prevent encephalopathy in term infants at highest risk for perinatal hypoxic-ischaemic brain injury (Gluckman et al., 2005). The efficacy and safety of whole-body cooling initiated within 6 h of age to a depth of 33.5 1C for 72 h duration has also been recently demonstrated (Shankaran et al., 2008). Recent clinical trials showed that relatively high temperatures applied in incubators during usual care after hypoxia-ischaemia is associated with increased risk of adverse outcomes (Laptook et al., 2008). Infants with pyrexia of 38 1C or more had an elevated rate of unfavorable outcome (Wyatt et al., 2007). Accordingly, the aim of the present study was to investigate long-term effects of neonatal anoxia, hyperthermia and pharmacological suppressing of hyperferremia with deferoxamine on development of spatial memory in rats. A battery of appropriate behavioural tests, such as hole board, Morris water maze and typical maze test, were performed at 4 and 12 months of age following the neonatal insult. We also applied the light-dark discrimination learning test (Li et al., 1999) to check both the acquisition and the latency of retention of the one-trial passive avoidance memory.
2. Materials and methods All experiments described in the present paper were performed according to the rules of the State Committee on the Use and Care of Laboratory Animals (permission of the Local Committee No. 6/2003). 2.1. Animals A total of 128 newborn Wistar rats of 2 days of age, of both sexes, weighting 7–8 g were used. The newborns were taken from their mothers (housed in acrylic cages lined with wood shavings, at a room temperature of 20–22 1C) and were exposed to a critical anoxia at various body temperatures. The experimental animals were divided equally among groups described in the Section 2.2. Prior to weaning, pups were housed with their own dams. The weanlings were sorted according to sexes and all males (79 animals) were used for the behavioural experiments. The males were housed in plastic cages at light:dark conditions of 12 h:12 h and were fed with commercial chow pellets and provided with water ad libitum. When they reached the age of 4 months and then at the age of 12 months they were subjected to the behavioural tests described in the Section 2.3. 2.2. Exposure to anoxia The procedures used to elicit anoxia, to manipulate neonatal body temperature, and pharmacological treatment of the rats were described in details in our previous work (Rogalska and Caputa, 2005). Briefly, prior to the manipulations, miniature thermocouples (0.1 mm in diameter) were inserted 5 mm into the rectum of the rat pups. Then the pups, divided into the following
seven groups, were individually transferred to 200 ml plethysmographic chambers, kept at different temperatures to stabilize body temperature at three different levels: (i) 33 1C (which is the normal body temperature of newborn rats (Rogalska and Caputa, 2005))—single anoxic and control groups, (ii) 37 1C (which is the body temperature of healthy adults)—single anoxic and control groups, and (iii) 39 1C (which is body temperature typical of febrile adults)—two anoxic and a single control group. To elicit anoxia, the plethysmographic chambers were flushed with 100% nitrogen. Rats of the extra group exposed to anoxia at the highest body temperature of 39 1C, were injected with DF (deferoxamine mesylate, Sigma-Aldrich, Chemie, GmbH, Steinheim, Germany), a chelator of iron, to match the nontreated anoxic animals. The drug was injected subcutaneously (100 mg/kg s.c.) twice: immediately after anoxia and 24 h later. The anoxia was continued for 25 min (Buwalda et al., 1995; Nyakas et al., 1996) or it was terminated earlier in hyperthermic groups (to prevent lethal asphyxia) when a critical accelerated and shallow gasping phase (identified barometrically in the plethysmographic chambers) occurred (Caputa et al., 2001; Rogalska and Caputa, 2005). It must be stressed that this procedure allows keeping mortality rate at 0%. After anoxia the animals were exposed to atmospheric air at unchanged temperature for 120 min. Control rats, were exposed to atmospheric air throughout the same period (145–130 min) in the respective thermal conditions. 2.3. Behavioural tests The experiments started when the rats were 4 months old and were repeated (except for the light–dark discrimination test, which could not be repeated for technical reasons described in the next section) at the age of 12 months. Each behavioural test was performed in a sound-isolated room with lighting conditions and environmental cues held constant throughout testing. The experimenter stayed in the room adjacent to the one in which the experiments were performed. Before testing of the next animal, the floor and walls of the apparatus were carefully cleaned to remove the smell traces left by previous rats. To avoid influences of circadian rhythms on performance of the experimental animals each behavioural test was started at 9 a.m. The behaviour of rats during the tests was videotaped and then analysed with the EthoVision 2.3 software (Noldus, Wageningen, Netherlands). 2.3.1. Light–dark discrimination test At 4 months of age the rats were subjected to the passive avoidance task (light–dark discrimination test) described by Li et al. (1999). A two-compartment step-through apparatus was used. The white-painted and illuminated compartment was separated from the black-painted and nonilluminated compartment (dimensions: 0.24 0.24 0.40 m3 each) by a 0.24 0.40 m2 sliding door (all made of plywood). The dark compartment was supplied with a metal grid floor for the delivery of electric foot shocks. During the first session all rats were subjected to a single pretraining trial. They were placed in the illuminated compartment for 4 min. On the following day, in the acquisition trial, the rat was placed in the illuminated compartment, and the time that elapsed until it entered the dark compartment (acquisition latency) was recorded. As soon as the rat entered the dark compartment, the door was closed and an electric shock (50 Hz, 100 V for 1 s) was applied twice (at 2 s interval) to the grid by an adjustable shock generator (home-made). Then the rat was placed in its home cage. A retention trial was carried out by placing the
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rat in the illuminated compartment again 24 h after the acquisition trial. The time (retention latency) of entering the dark compartment, and the time spent in the illuminated compartment in the retention trial over a 3-min period (excluding any excursions into the dark compartment) were recorded. If the rat did not enter the dark compartment within 180 s, the retention test was interrupted, and the maximum score of 180 s was assigned. Because the rats used in this investigation were 2 months later implanted with transmitters for telemetric recording of body temperature and motor activity (to be published in a separate paper) we had to cancel their second testing at the age of 12 months (there was a risk of damage to the transmitters by the electric shocks). 2.3.2. Hole board spatial learning test The equipment described by Buwalda et al. (1995) was applied to study water-motivated spatial learning behaviour. The hole board (dimensions: 0.7 0.7 0.45 m3) is a test apparatus, in which animals have to memorize a random pattern of 4 holes filled with water, accessible to them, out of 20 holes (35 mm diameter, 40 mm depth) distributed equidistantly in a floor plate. Each experiment started 24 h after water deprivation (we decided to replace hunger-motivated learning, which is commonly used in this test, with thirst motivated learning, because the latter seems to be much stronger as a drive). Before testing the rats were placed in a start-box (0.2 0.7 0.45 m3) adjacent to the hole board arena. After 10 s the guillotine door was opened and animals were allowed to visit the arena. During the first 2 days the rats were placed four times daily for 5 min periods in the hole board with water available in all holes in order to get used to visiting the holes for reward and so the animals had an opportunity to rehydrate. The spatial learning started on the third day. Four holes arranged in a fixed, randomly chosen pattern were supplied with accessible water hidden 30 mm under the floor level. Remaining 16 holes also contained water but it was inaccessible (covered with a wire mesh). A visit to a hole was scored when the nose of the rat was placed in it. Rats were removed 3 min after the start of each trial. The testing lasted 5 days. The reference memory ratio (RMR) was defined as RMR ¼
x yþz
where x is a number of visits and revisits to the baited set of holes, y is four minus the number of visits to baited holes and z is total number of visits and revisits to all holes. The overall range of the RMR quotient extends from 0 (no baited holes visited) to 1 (only the four baited holes visited). 2.3.3. Typical maze The typical maze (dimensions: 0.7 0.7 m2) was surrounded by walls 0.45 m high. Again, we used water-deprived rats, which were given a cup of water (2 ml) as a reinforcement. The test was performed three times every 5 days and the animals were tested twice a day at 4-h intervals between the tests, six times altogether. The water deprivation was planned as follows: 36 h prior to the first session of the day and then for another 4 h until completion of the second test of the day. The animals were placed at the starting point of the maze and had to reach the water cup without any other reinforcement. The latency to reach the reward was recorded and analysed. 2.3.4. Morris water maze A circular swimming pool (1.40 m in diameter and 0.48 m in height) was filled with water ( 25 1C) to a depth of 0.30 m. Water
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was rendered opaque by addiction of a small quantity of powdered milk and some beet juice was added to contrast white fur of rats. Four points around the circumference of the pool were arbitrarily designated: North, South, East, and West, which allowed to divide the pool area into 4 quadrants (NW, SW, etc.). A circular goal platform (90 mm in diameter) laid 20 mm under the surface of the water, in the middle of the SW quadrant for all trials. The rats were given four consecutive trials a day (with 20 s intervals) for 5 days, according to the procedure described by Morris (1984). The animals were dropped into water from different quadrants on each trial (always facing the wall of the pool), and had to learn to navigate to the invisible platform using the spatial cues available in the room. The sequence of starting points was changed during consecutive days of the test. Rats that failed to reach the platform within 120 s were gently guided to it and allowed to climb on. The latency of reaching the platform in all trials was recorded. After the fourth trial of the last session, the rats were submitted to a probe test for spatial bias. The platform was removed and the rat, starting from the opposite quadrant, was allowed a 2-min search for the platform. The path was recorded on videotape and a spatial bias regarded as a number of crossings the SW quadrant of the pool (where the platform was previously hidden) was measured. 2.4. Data analysis All results were tested for normality with Kolmogorov– Smirnov test. All variables of rat’s behaviour in the passive avoidance test were analysed with two-way ANOVA in a 3 2 factorial design, where temperature (33, 37, and 39 1C) and experimental conditions (control and anoxia) were the factors. A three-way analysis of variance (temperature experimental conditions trial) with repeated measures on the third factor was applied to analyse the results of rat’s behaviour in hole board test (3 2 5), water maze (3 2 5) and typical maze (3 2 6). The effect of treatment with DF in hyperthermic groups in light– dark discrimination test was analysed by one-way ANOVA and the effect of DF in the hole board test, water maze and typical maze—by two-way ANOVA (experimental conditions trial) with repeated measures on the last factor. Each analysis was followed by multiple comparison using Newman–Keuls post hoc test. The significance level was set at p o0.05 for all statistical tests. Paired Student’s t-test was used to analyse the progress in learning in particular groups of rats. Data analyses were performed with the use of Statistica (StatSoft, Cracow, Poland).
3. Results 3.1. Light–dark discrimination test There was a significant effect of neonatal body temperature on retention latency (Fig. 1A; F2,56 =6.79; po0.01), but no effect of neonatal anoxia (F1,56 = 0.64; n.s.), and no interactions between both factors (F2,56 = 0.08; n.s.). The latency of both control and anoxic rats maintained at physiological body temperature of 33 1C tended to be longer than that of the rats exposed to neonatal anoxia at body temperatures of 37 and 39 1C. Postanoxic DF treatment of hyperthermic newborns did not influence the latency (F2,22 =2.26; n.s.) (Fig. 1A, right side). However, the latency tended to be longer in DF-treated animals than that in their hyperthermic (39 1C) counterparts exposed to neonatal anoxia. The time spent in the light compartment in the retention trial (Fig. 1B) was significantly influenced by temperature (F2,56 = 9.58;
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Fig. 1. Effects of neonatal anoxia, neonatal body temperature and chelation of iron with deferoxamine (DF) upon passive avoidance memory: retention latency (A) and time spent in the light compartment (B) in the retention trial over 3-min period (excluding any excursions into the dark compartment) in young adult (4 months old) rats which were exposed 24 earlier to electric foot shock in the dark compartment. The data are presented as mean values+ S.E.M., *p o0.05 for two-way ANOVA or #p o0.05 for oneway ANOVA. Numbers inside or under columns indicate numbers of animals in individual groups.
p o0.001). However, there were no effects of neonatal anoxia (F1,56 = 1.83; n.s.), and no interactions between both factors (F2,56 = 0.53; n.s.). Post hoc analysis revealed that rats exposed to neonatal anoxia at body temperatures of 37 and 39 1C spent less time in the light compartment than both control and anoxic rats kept postpartum at body temperature of 33 1C (p o0.05). There was also a significant effect of experimental conditions (F2,22 = 3.69; p o0.05) in three groups of rats exposed to neonatal hyperthermia (Fig. 1B, right side). The time spent in the light compartment was significantly longer in DF-treated rats than that in the rats exposed to neonatal anoxia at the same body temperature (p o0.05).
3.2. Hole board test Both neonatal body temperature and neonatal anoxia clearly influenced the learning of 4 months old rats (Fig. 2A). Reference memory ratio (RMR) decreased with increasing neonatal body temperature (F2,42 = 34.44; p o0.001) and was further reduced in rats exposed to neonatal anoxia (F1,42 =11.86; p o0.01) but there was no significant interaction between those factors (F2,42 = 0.15; n.s.). The increase of reference memory ratio during 5 consecutive testing days was observed in all experimental groups (F4,168 =63.97; p o0.001). Interactions of temperature and trial as well as anoxia and trial were also significant (F8,168 = 3.74; po0.001; F4,168 = 2.46; p o0.05). Post hoc test revealed that the performance of the animals exposed to neonatal anoxia at 39 1C was significantly lower than that of the asphyxiated rats kept postpartum at body temperature of 33 1C on days 3 (po0.05), 4 (po0.01) and 5 (p o0.05) (Fig. 2A). There were also significant differences of the performance between rats exposed to neonatal anoxia at 37 and 33 1C on days 3 (po0.05), 4 (po0.05) and 5 (po0.001). All groups started from the same RMR level, however, the progress of learning was clearly disturbed by neonatal exposure to hyperthermia and anoxia. There were significant differences between the three hyperthermic groups (Fig. 2A, right side; F2,19 = 5.01; p o0.05). There were also significant effects of the trial (F4,76 = 21.89; po 0.001), but there were no significant interactions between both factors (F8,76 =1.27; n.s.). Post hoc analysis demonstrated that from the 3rd day of the experiment on, DF-treated anoxic rats showed significant improvement in memory performance in comparison to their untreated counterparts at the same neonatal body temperature (day 3: po0.05; day 4: p o0.01; day 5: p o0.05). As far as the performance in the hole board in 12 months old rats is concerned (Fig. 2B) the analysis shows a significant main
effect of all three experimental factors: temperature (F2,34 = 7.75; po0.01), anoxia (F1,34 =8.75; po0.01) and trial (F4,136 = 16.75; po0.001). Interaction of temperature and anoxia (F2,34 =6.77; po0.01) was also significant. The efficacy of learning was better in control and anoxic rats allowed to maintain neonatal body temperature at 33 1C than that in rats exposed to neonatal anoxia at body temperatures of 37 and 39 1C. Further analysis with post hoc test revealed that the value of RMR on day 5 was lower in animals exposed to neonatal anoxia at 37 and 39 1C than that in anoxic rats kept postpartum at body temperature of 33 1C (p o0.01). The difference in RMR between the first and last day of the experiment in animals kept neonatally at physiological body temperature, both in control and anoxic groups, was significant (p o0.05, Student’s t-test), whereas in anoxic rats exposed to neonatal hyperthermia (body temperature of 37 and 39 1C) there was no progress in the performance in consecutive days of the experiment. There was a significant effect of the experimental conditions (F2,14 = 6.57; p o0.01) and trial (F4,56 =7.82; p o0.001) in three groups of rats exposed to neonatal body temperature of 39 1C (Fig. 2B, right side). Chelation of iron with DF prevented the impairment of learning.
3.3. Typical maze test Latency of reaching the reward in typical maze in 4 months old rats (Fig. 3A) was significantly influenced by neonatal anoxia (F1,41 = 5.82; po0.05) and trial (F5,205 = 38.68; p o0.001), and there was a significant interaction between these factors (F5,205 = 2.76; po0.05). On the other hand, the latency was not significantly different when analysed for temperature (F2,41 = 0.85; n.s.). Further analysis evidenced that on the first day of the experiment the latency in animals exposed to neonatal anoxia at 37 1C was significantly longer than that in anoxic rats allowed to maintain neonatal body temperature at 33 1C (p o0.05) and (surprisingly) than that in rats at the highest neonatal body temperature of 39 1C (p o0.001). DF treatment (Fig. 3A, right side) had no effect on the latency (F2,24 =0.06; n.s.). Two-way ANOVA shows only the main significant effect of trial (F5,120 = 16.69; po0.001), but no interaction between the factors (F10,120 = 0.80; n.s.). All groups showed a progressive decrease of latency in the subsequent trials. However, the performance was the best in control and anoxic rats maintained at physiological body temperature of 33 1C and in hyperthermic rats treated postanoxia with deferoxamine as well.
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Fig. 2. Effects of neonatal anoxia, neonatal body temperature and chelation of iron with deferoxamine (DF) upon reference memory ratio (RMR) in the hole board learning during 5 consecutive testing days (indicated by numbers below columns) in rats at the age of 4 months (panel A) and 12 months (panel B). The data are presented as mean values+ S.E.M. Number of animals in each group is indicated inside the column showing results of the initial testing day. Statistical differences are described in the text.
The disturbances due to neonatal exposure to anoxia and hyperthermia persisted in the same rats tested at the age of 12 months (Fig. 3B). In general, rats exposed to neonatal anoxia at 37 and 39 1C in all trials required more time to reach the water cup than rats kept postpartum at body temperature of 33 1C both in control and anoxic groups. There were significant main effects of neonatal body temperature (F2,40 = 4.99; p o0.01), anoxia (F1,40 = 5.15; po0.05) and interaction between these factors (F2,40 = 4.28; p o0.05). A significant effect of trial (F5,2 =17.0; p o0.001) indicates that time to reach the water cup shortened during subsequent sessions. Clear-cut differences between groups are seen already on the first day of the test. The time to reach the reward was significantly shorter in rats allowed to maintain their neonatal body temperature at 33 1C, both in control and anoxic groups, than that in animals exposed to neonatal anoxia at 37 1C (po0.05). During the consecutive days the performance tended to be superior in rats allowed to maintain the reduced neonatal body temperature. There were significant differences between the three hyperthermic groups (F2,12 = 3.98; p o0.05) and trials (F5,85 =8.95; p o0.001), and no significant interactions between both factors (F10,85 = 1.08; n.s.) (Fig. 3B, right side). DF-treated rats tended to perform better than the remaining animals. 3.4. Morris water maze test Neonatal anoxia combined with hyperthermia affected also the performance of the rats in the water maze (Figs. 4 and 5). The latency
to find the hidden platform in 4 months old rats (Fig. 4A) increased with increasing neonatal body temperature (F2,59 =6.36; po0.01). However, it was not significantly different when analysed for treatment (F1,59 =0.45; n.s.). Rats exposed to anoxia under hyperthermic conditions (39 1C) performed worse than did anoxic rats kept postpartum at body temperature of 33 1C (po0.001). DF-treated rats found the platform in a significantly shorter time (po0.05; Fig. 4A, right side) than their untreated anoxic counterparts at the same neonatal body temperature. Eight months later there were no clear differences in the navigation learning and the rats apparently remembered their earlier training (Fig. 4B). The performance was not influenced by neonatal temperature (F2,53 = 2.58; n.s.) and anoxia (F1,53 =0.004; n.s.). There were no significant differences between the three hyperthermic groups (right side of Fig. 4B; F2,26 =1.41; n.s.). There was, however, a significant effect of trial (F4,104 = 3.83; p o0.01), but no interaction between both factors (F8,104 = 0.91; n.s.). The latencies in DF-treated rats in each trial were shorter that those in their nontreated counterparts. As far as the spatial bias performance in 4 months old rats is concerned (Fig. 5A), it worsened with increasing neonatal body temperature (F2,59 =5.09; po 0.01) but was not significantly reduced in rats exposed to neonatal anoxia (F1,59 = 0.30; n.s.) and there was no interaction between these factors (F2,59 = 0.74; n.s.). The analysis of the spatial bias performance in the same rats at the age of 12 months (Fig. 5B) showed a significant main effect of both experimental factors: temperature (F2,53 = 3.74; p o0.05) and anoxia (F1,53 = 7.68; po0.01) but no interaction between them
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Fig. 3. Effects of neonatal anoxia, neonatal body temperature and chelation of iron with deferoxamine (DF) on spatial learning performance (expressed as latency of reaching the reward) in rats at the age of 4 months (panel A) and 12 months (panel B) placed in the typical maze during 6 consecutive trials (indicated by numbers below columns). The data are presented as mean values+ S.E.M. Number of animals in each group is indicated inside the column showing results of the initial testing day. Statistical differences are described in the text.
(F2,53 = 1.15; n.s.). Post hoc test revealed cognitive impairment in animals exposed to neonatal anoxia at 39 1C compared to those kept postpartum at body temperature of 33 1C, both in control and anoxic groups (p o0.05). A comparison of the spatial bias performance in three groups of rats exposed to neonatal body temperature of 39 1C showed significant main effects of treatment, both in 4 (F2,30 = 3.81; p o0.05; Fig. 5A) and 12 months old animals (F2,26 = 4.34; p o0.05; Fig. 5B). Post hoc analysis demonstrated that the performance in DF-treated rats was superior to that in their counterparts at the same neonatal body temperature, both in untreated control and anoxic groups (p o0.05) (Fig. 5A and B, right side).
4. Discussion There are some controversies concerning appropriate experimental models of hypoxic-ischaemic injury of the brain as well as various models of neuroprotective reductions in body temperature. One of the controversies concerns an adequate animal model of birth asphyxia. The majority of authors argue that 7-day-old rats are the most suitable (Trescher et al., 1997; Palmer et al., 1999; Mishima et al., 2004; Papazisis et al., 2008; van der Kooij et al., 2009) because they have brain maturation that corresponds to the term babies. On the other hand, the preterm infants are particularly susceptible to perinatal pathology (Sullivan, 1988;
Buonocore et al., 1998, 2002; Perlman, 2001; Rao and Georgieff, 2002). Therefore, in our experimental approach we keep on using 2-day-old rats as a model of the preterm birth asphyxia. This experimental approach is unique in terms of a pattern of body temperature manipulation (we use clamping of body temperature at various levels both during anoxia as well as over 120 min postanoxia). The present investigation has confirmed previous experimental data (Dell’Anna et al., 1991; Nyakas et al., 1996) that neonatal anoxia induces disturbances in spatial memory performance in adult rats. Moreover, we were able to show that such impairment is always connected with a significantly elevated body temperature and that normal body temperature of newborn rats, which is as low as 33 1C, protects them against the postanoxic disturbances. It must be stressed that all newborns of this group tolerated, highly repeatedly, the full-time (25 min) exposure to anoxia, while rats forced to maintain their body temperature at 39 1C tolerated anoxia for no longer than 10 min, which confirms the results of our previous paper (Rogalska and Caputa, 2005). In the present investigation postanoxic and posthyperthermic disturbances in spatial memory performance persisted in adult rats at the age of 4 and 12 months. Such a delayed postanoxic impairment is possibly induced by oxidative stress catalysed by iron, since postanoxic treatment of the hyperthermic newborns with deferoxamine, a chelator of iron, prevented the memory deficits.
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Fig. 4. Effects of neonatal anoxia, neonatal body temperature and chelation of iron with deferoxamine (DF) on spatial learning performance (expressed as latency of reaching the hidden platform) of rats at the age of 4 months (A), and 12 months (B) in water maze during 5 consecutive days (indicated by numbers below columns) of the experiment. The data are presented as mean values+ S.E.M. Statistical differences are indicated in the text.
Fig. 5. Effects of neonatal anoxia, neonatal body temperature and chelation of iron with deferoxamine (DF) upon memory retention of rats at the age of 4 months (A), and 12 months (B) in water maze during spatial bias task. The data are presented as mean values+ S.E.M., *p o0.05, **p o 0.01 for two-way ANOVA or #p o 0.05 for one-way ANOVA. Numbers inside columns indicate numbers of animals in individual groups.
In our previous papers we have shown that neonatal exposure of rats to anoxia under hyperthermic conditions leads to plasma hyperferremia (Caputa et al., 2001), which is followed by iron accumulation in the brain (Rogalska et al., 2006b). Asphyxiated preterm infants also suffer from free iron release in erythrocytes (Buonocore et al., 1998) enhancing the risk of oxidative injury (Sullivan, 1988; Buonocore et al., 2002). Increased body iron stores also contribute to the stroke progression in adult patients with a cerebral infarction (Davalos et al., 2000). Iron is necessary as a
cofactor of myelinization, yet the excessive cerebral iron deposition in asphyxiated neonatal rats (Palmer et al., 1999; Rogalska et al., 2006b) cannot support myelinization because the insult leads to reduced cerebral concentration of myelin basic protein and proteolipid protein and results in death of oligodendroglial precursors in the brain of neonatal mice (Skoff et al., 2001). Altogether, the accumulation of iron in the brain of asphyxiated newborns is unequivocally harmful. This conclusion is strongly supported by Dal-Pizzol’s et al. (2001) results showing that rats
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treated orally with high doses of iron in neonatal period suffer from oxidative damage to the brain even at the age of 3 months. There are several studies showing neuroprotective effect of DF due to iron chelation. Administration of DF resulted in a reduction in the number of damaged neurons in the CA1 region of hippocampus and tended to decrease damage to the region of CA3 and DG (Papazisis et al., 2008). Alterations in gene expression following DF treatment have also been described. One of them is an increase in expression of HIF1 alpha, which is suggested to promote cell survival in hypoxic tissues (Hamrick et al., 2005). Recently, van der Kooij and colleagues (2009) have found that deferoxamine did not prevent gray or white matter damage in 7 days old rats, which underwent right common carotid artery occlusion followed by a 90-min exposure to 8% oxygen. However, in the present paper we were able to show that deferoxamine normalizes cognitive processes in adult rats exposed to neonatal anoxia. Therefore, it is possible that DF plays a more significant role in the immature brain since iron accumulation is most pronounced in the early development. Damage to the rabbit brain following fetal hypoxia was clearly accelerated during initial 8 min of reoxygenation, which was associated with enhanced free radical production (Tan et al., 1999). This neurotoxic process is prevented by a moderate decrease in brain temperature (Globus et al., 1995; Nyakas et al., 1996; Colbourne et al., 1997). Therefore, reduced body temperature, applied in this investigation both during anoxia and immediately postanoxia, is likely to provide maximal neuroprotection, both immediate and extremely delayed (the latter being a consequence of the prevented cerebral iron accumulation). Postanoxic pathology manifests itself as both histological and behavioural abnormalities. In rats subjected to neonatal hypoxiaischaemia injury of the brain is observed from second week on, and progresses up to fifth month of their life (Mishima et al., 2004).The authors conclude, however, that quantitative histological analysis of the injury is less sensitive method of the evaluation than behavioural analysis. Postanoxic damage to the brain is connected not only with neuronal death but also with dendritic athrophy (Titus et al., 2007), which is indetectable histologically. Moreover, the cognitive and behavioural problems occur in premature infants in connection with the presence or absence of neuroimaging abnormalities. At adolescence they display specific deficits in everyday memory when compared with full-term controls (Briscoe et al., 2001). This is why we have decided to focus on behavioural effects of neonatal asphyxia in rats. All spatial memory tests applied in this investigation showed conspicuous cognitive disturbances in rats exposed to neonatal anoxia under hyperthermic conditions. Nevertheless, there were some subtle differences among the tests. The light–dark discrimination test revealed that the both groups of neonatal rats kept at their natural body temperature of 33 1C as well as rats exposed to neonatal anoxia under hyperthermic conditions and then injected with deferoxamine spent significantly more time in the light compartment (they previously had been subjected to the electric shock in the dark compartment) than the animals suffering from neonatal anoxia under hyperthermic conditions and untreated. In the hole board test older (12 months old) rats tended to perform worse than they did 8 months earlier. In contrast, the performances in both the typical maze and the water maze tended to improve in the older animals. This leads to a conclusion that of these three tests the experience played a role in the latter two but not in the former. Obviously, the older animals must have remembered the way to reach the goal in the typical maze and the water maze. Therefore, both tasks can be regarded as less challenging than the hole board task.
Actually, hole board task showed persistent, clearly harmful main effect of neonatal anoxia and neonatal body temperature, and a highly significant interaction between these factors. The remaining tests confirmed the main effect of neonatal body temperature (light–dark discrimination test and water maze in rats at the age of 4 months) or the main effect of neonatal anoxia (typical maze in rats 4 months old) or the main effect of both of them (typical maze and spatial bias in water maze at the age of 12 months). In each of the tests post hoc analysis evidenced clear-cut disturbances of learning in rats exposed to anoxia at body temperature of 39 1C, in comparison with the performance of animals exposed to anoxia at physiological body temperature of 33 1C. The combined harmful effect of neonatal hyperthermia and anoxia is not surprising. On the other hand, the selective effect of such a moderate and transient hyperthermia is an unexpected phenomenon. Prominent harmful effects of elevated temperature in animals subjected to hypoxia/ischaemia were observed when temperature elevations occurred immediately after the insult and were continued for long periods (Reglodi et al., 2000). This suggests that the neonatal brain is extremely susceptible to hyperthermia. There is a strong clinical support for such a conclusion. Namely, a prospective cohort study of 4915 low-risk women at term showed the association of maternal fever with neonatal encephalopathy, which was independent of other known intrapartum risk factors (Impey et al., 2001). In the previous papers (Rogalska et al., 2004, 2006a; Caputa et al., 2005) we were able to show that both the reduced body temperature and chelation of iron prevents postanoxic disturbances in stress responses of rats from their youth up to senescence. The present investigation implies the same conclusion concerning spatial memory disturbances. This strengthens an argument that asphyxiated human newborns should be moderately cooled to prevent not only early neurological deterioration, which has been shown in 12-months follow-up (Gunn et al., 1998; Compagnoni et al., 2002) but also a variety of much more delayed disturbances such as attention deficit-hyperactivity disorder (ADHD) in childhood and cognitive failure in adulthood. The pathological effect of the postasphyxic iron-mediated oxidative stress, indirectly shown in the present investigation, is also postulated in recent neonatological reviews. There is some cause for concern about infants who receive iron supplements or blood transfusions (Sullivan, 1988; Rao and Georgieff, 2002) as well as those who are resuscitated with 100% oxygen (Vento et al., 2003). Unfortunately, in contrast to the firmly established knowledge of both preventive (Rogalska and Caputa, 2005; Caputa, 2006) and defensive (Rogalska and Caputa, 2005; Caputa, 2006) reduction in body temperature of asphyxiated newborn rats, such data concerning human newborns are extremely scarce. For the last decades asphyxiated preterm infants are being forced to stabilize their body temperature at 37 1C by mean of incubators. Recording natural thermal responses of asphyxiated human newborns has not been continued for 50 years now! The results of one of those early investigations are similar to those obtained recently in experiments on animal models. Namely, body temperature of moderately asphyxiated babies was 1.5 1C lower than that of their healthy counterparts maintained under the same external conditions (Burnard and Cross, 1958). We conclude that there is an urgent need for revitalization of such studies. Because the neonatal community is conservative with respect to acceptation of such a moderate body or head cooling as a new ‘‘standard of care’’ for asphyxiated babies (Kirpalani et al., 2007) the researchers should do their best to convince the community to this procedure. It is generally acknowledged that there is no information regarding the optimal management of infants with hypoxicischaemic encephalopathy (Laptook et al., 2008). The range of ambient temperatures at which babies start sweating to lose heat
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is surprisingly wide (33–38 1C) (Laptook et al., 2008). Infants exposed to maternal fever have a greater likelihood of a low 1 min Apgar score, poor respiratory effort, and the need for bag-mask ventilation in the delivery room. A case-control study design demonstrated that intrapartum fever, which was unlikely to be infectious in origin, was associated with a 3.4-fold risk of unexplained early-onset seizures in term infants (Lieberman et al., 2000). Important associations between reduced or elevated body temperature at birth and subsequent morbidity and mortality (Laptook et al., 2008) suggest that change in temperature at birth causes brain injury (hyperthermia) or a neuroprotection (anapyrexia).
Acknowledgements This work was supported by the grant of 4.P05A.059.16 from the Polish State Committee for Scientific Research.
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