Cerebral perfusion pressure and behavior monitoring in freely moving rats.

Cerebral perfusion pressure (CPP) is the net pressure gradient that drives oxygen delivery to cerebral tissue. It is the difference between the mean arterial pressure (MAP) and the intracranial pressure (ICP). As CPP is a calculated value, MAP and ICP must be measured simultaneously. In research models, anesthetized and acute monitoring is incapable of providing a realistic picture of the relationship between ICP and MAP under physiological and/or pathophysiological conditions. For long-term monitoring of both pressures, the principle of telemetry can be used. The aim of this study was to map changes in CPP and spontaneous behavior using continuous pressure monitoring and video recording for 7 days under physiological conditions (group C - 8 intact rats) and under altered brain microenvironment induced by brain edema (group WI - 8 rats after water intoxication) and neuroprotection with methylprednisolone - MP (group WI+MP - 8 rats with MP 100 mg/kg b.w. applicated intraperitoneally during WI). The mean CPP values in all three groups were in the range of 40-60 mm Hg. For each group of rats, the percentage of time that the rats spent during the 7 days in movement pattern A (standard movement stereotype) or B (atypical movement) was defined. Even at very low CPP values, the standard movement stereotype (A) clearly dominated over the atypical movement (B) in all rats. There was no significant difference between control and experimental groups. Chronic CPP values with correlated behavioral type may possibly answer the question of whether there is a specific, universal, optimal CPP at all.

Determination of brain water content by dry/wet weight measurement for the detection of experimental brain edema.

Brain edema is a fatal pathological state in which brain volume increases as a result of abnormal accumulation of fluid within the brain parenchyma. A key attribute of experimentally induced brain edema - increased brain water content (BWC) - needs to be verified. Various methods are used for this purpose: specific gravimetric technique, electron microscopic examination, magnetic resonance imaging (MRI) and dry/wet weight measurement. In this study, the cohort of 40 rats was divided into one control group (CG) and four experimental groups with 8 rats in each group. The procedure for determining BWC using dry/wet weight measurement was initiated 24 h after the completion of edema induction by the water intoxication method (WI group); after the intraperitoneal administration of Methylprednisolone (MP) together with distilled water during edema induction (WI+MP group); 30 min after osmotic blood brain barrier disruption (BBBd group); after injection of MP via the internal carotid artery immediately after BBBd (BBBd + MP group). While induction of brain edema (WI, BBBd) resulted in significantly higher BWC, there was no increase in BWC in the MP groups (WI+MP, BBBd+MP), suggesting a neuroprotective effect of MP in the development of brain edema.

Effect of methylprednisolone on experimental brain edema in magnetic resonance imaging.

Magnetic resonance imaging has been used for evaluating of a brain edema in experimental animals to assess cytotoxic and vasogenic edema by the apparent diffusion coefficient (ADC) and T2 imaging. This paper brings information about the effectiveness of methylprednisolone (MP) on experimental brain edema. A total of 24 rats were divided into three groups of 8 animals each. Rats with cytotoxic/intracellular brain edema induced by water intoxication were assigned to the group WI. These rats also served as the additional control group CG when measured before the induction of edema. A third group (WIMP) was intraperitoneally administered with methylprednisolone 100 mg/kg during water intoxication treatment. The group WI+MP was injected with methylprednisolone 50 mg/kg into the carotid artery within two hours after the water intoxication treatment. We evaluated the results in four groups. Two control groups (CG, WI) and two experimental groups (WIMP, WI+MP). Rats were subjected to MR scanning 24 h after edema induction. We observed significantly increased ADC values in group WI in both evaluated areas - cortex and hippocampus, which proved the occurrence of experimental vasogenic edema, while ADC values in groups WIMP and WI+MP were not increased, indicating that the experimental edema was not developed and thus confirming the protective effect of MP.

Forelimb Movement Disorder in Rat Experimental Model.

Study of motor activity is an important part of the experimental models of neural disorders of rats. It is used to study effects of the CNS impairment, however studies on the peripheral nervous system lesions are much less frequent. The aim of the study was to extend the spectrum of experimental models of anterior limb movement disorders in rats by blockade of the right anterior limb brachial plexus with the local anesthetic Marcaine (Ma), or with aqua for injection administered into the same location (Aq) (with control intact group C). Two other groups with anterior limb movement disorders underwent induction of cellular brain edema by water intoxication (MaWI and AqWI). Results showed a lower spontaneous motor activity of animals in all experimental groups versus controls, and lower spontaneous motor activity of animals in the MaWI group compared to other experimental groups in all categories. There was no difference in spontaneous activity between the groups Ma, Aq and AqWI. Our study indicates that alterations of spontaneous motor activity may result from the impaired forelimb motor activity induced by the anesthetic effect of Marcaine, by the volumetric effect of water, as a result of induced brain edema, or due to combination of these individual effects.

Loss of body weight is accompanying cellular brain edema induced by water intoxication in the rat.

Induction of cellular cerebral edema (CE) was achieved by a standard method of water intoxication which consisted of fractionated intraperitoneal administration of distilled water (DW) together with the injection of desmopressin (DP). Using metabolic cage, fluid and food balance was studied in two groups of eight animals: group C - control; group CE - cellular edema induced by water intoxication. For each rat the intake (food pellets and water) and excretion (solid excrements and urine) were recorded for 48 h together with the initial and final body weight. CE animals consumed significantly less food, drank less water and eliminated the smallest amount of excrements. The induction of cellular cerebral edema was accompanied with a significant loss of body weight (representing on average 13 % of the initial values) mainly due to a reduction of food intake. This phenomenon has not yet been reported.

Methylprednisolone modulates intracranial pressure in the brain cellular edema induced by water intoxication.

Continuous monitoring of the intracranial pressure (ICP) detects impending intracranial hypertension resulting from the impaired intracranial volume homeostasis, when expanding volume generates pressure increase. In this study, cellular brain edema (CE) was induced in rats by water intoxication (WI). Methylprednisolone (MP) was administered intraperitoneally (i.p.) before the start of CE induction, during the induction and after the induction. ICP was monitored for 60 min within 20 h after the completion of the CE induction by fibreoptic pressure transmitter. In rats with induced CE, ICP was increased (Mean+/-SEM: 14.25+/-2.12) as well as in rats with MP administration before the start of CE induction (10.55+/-1.27). In control rats without CE induction (4.62+/-0.24) as well as in rats with MP applied during CE induction (5.52+/-1.32) and in rats with MP applied after the end of CE induction (6.23+/-0.73) ICP was normal. In the last two groups of rats, though the CE was induced, intracranial volume homeostasis was not impaired, intracranial volume as well as ICP were not increased. It is possible to conclude that methylprednisolone significantly influenced intracranial homeostasis and thus also the ICP values in the model of cellular brain edema.

Subconvulsive dose of kainic acid transiently increases the locomotor activity of adult Wistar rats.

Kainic acid (KA) is a potent neurotoxic substance valuable in research of temporal lobe epilepsy. We tested how subconvulsive dose of KA influences spontaneous behavior of adult Wistar rats. Animals were treated with 5 mg/kg of KA and tested in Laboras open field test for one hour in order to evaluate various behavioral parameters. Week after the KA treatment animals were tested again in Laboras open field test. Finally, rat's brains were sliced and stained with Fluoro-Jade B to detect possible neuronal degeneration. Treatment with KA increased the time spent by locomotion (p<0.01), exploratory rearing (p<0.05) and animals traveled longer distance (p<0.01). These parameters tended to increase thirty minutes after KA administration. Week after the treatment we did not found differences in any measured behavioral parameter. Histology in terms of Fluoro-Jade B staining did not reveal any obvious neuronal damage in hippocampus. These results demonstrate that subconvulsive KA dose changes the behavioral parameters only transiently. Clarification of timing of the KA induced changes may contribute to understand mutual relationship between non-convulsive seizures and behavioral/cognitive consequences.

Low dose domoic acid influences spontaneous behavior in adult rats.

Domoic acid (DA) is a potent marine neurotoxine present in seafood. Intoxication by DA causes gastrointestinal symptoms like vomiting and diarrhoea and also the so-called amnesic shellfish poisoning (inflicting memory impairment and seizures). Since exposure to non-convulsive doses is relevant to the human health, we investigated the effect of low dose DA administration in adult Wistar rats. Rats were administered with DA at the dose 1.0 mg/kg and their behavior was monitored for one hour in three sessions. The first session started immediately after DA administration. The second and third session started one and two weeks later. After the third session, the histochemical analysis of the hippocampi of the animals was conducted (Fluoro-Jade B, bis-benzimide). DA increased time spent by locomotion and distance travelled in the second half of the first session and this effect was pronounced during the second and third session. Exploratory rearing was decreased by DA administration in the first half of the first session. DA influenced the grooming in biphasic manner (decrease followed by an increase of time spent by grooming). This biphasic trend was observed even two weeks after the DA administration. Histochemistry of DA treated rats did not confirm the presence of apoptotic bodies, Fluoro-Jade B positive cells were not found neither in CA1 nor CA3 area of the hippocampi. Our study revealed that a low dose of DA affect short and long-term the spontaneous behavior of rats without inducing neuronal damage.

Influence of low-dose neonatal domoic acid on the spontaneous behavior of rats in early adulthood.

Consumption of seafood containing toxin domoic acid (DA) causes an alteration of glutamatergic signaling pathways and could lead to various signs of neurotoxicity in animals and humans. Neonatal treatment with domoic acid was suggested as valuable model of schizophrenia and epilepsy. We tested how repeated early postnatal DA administration influences the spontaneous behavior of rats in adulthood. Rats were injected with 30 microg DA/kg from postnatal day (PND) 10 until PND 14. Their behavior was observed in the open field test for one hour (Laboras, Metris) at PND 35, PND 42 and PND 112. We did not find any difference between DA treated rats and animals injected with equivalent volume of saline in both test sessions at PND 35 and PND 42. DA rats at PND 112 exhibited significantly higher vertical and horizontal exploratory activity (tested parameters: locomotion, distance travelled, average speed reached during test, grooming and rearing) between the 30th-40th min of the test session and habituated over 10 min later. We conclude that at least in the given experimental design, neonatal DA treatment results in alteration of the spontaneous behavior of rats in adulthood.

Fast and delayed locomotor response to acute high-dose nicotine administration in adult male rats.

The aim of the present study was to compare the immediate and delayed locomotor response to high-dose nicotine (NIC) administration in rats. The vertical and horizontal activity of behavior in adult male rats exposed to 1 mg/kg NIC or saline (SAL) were tested in a Laboras apparatus for one hour after drug application. Animals were then returned to their cages and housed for another seven days. After this period all animals were placed in Laboras again and their behavioral pattern was retested for another period of one hour (delayed response). Horizontal activity: immediately after nicotine administration animal were less mobile (first 2-minutes interval), when compared with controls. The immobilization effect of nicotine disappeared within 4 minutes and during whole first 10-minutes interval time spent by locomotion did not differ from controls. Locomotion activity of animals treated with nicotine increased robustly in following 10 minutes and remained significantly higher in 2nd, 3rd and 5th 10-minutes interval. Vertical activity: Rearing frequency was significantly lowered by NIC administration in first two minutes of the experiment and the same was found when the duration of rearing was analyzed. Lower rearing intensity of NIC treated animals disappeared in 4 minutes and was finally higher during whole test session as compared with controls. When duration of rearing was analyzed it was significantly longer in NIC treated animals. In majority of observed behavioral aspects there were no differences between NIC treated rats and controls seven days after NIC or SAL treatment. Our results reflect effect of NIC and we conclude that NIC significantly influences behavior of experimental animals.

Influence of allopurinol on evoked cortical afterdischarges during early ontogenesis.

The aim of our study was to test the hypothesis, whether repeated allopurinol pre-treatment (in dose of 135 mg/kg s.c.) can influence changes of brain excitability caused by long-term hypoxia exposition in young immature rats. Rat pups were exposed together with their mother in to an intermittent hypobaric hypoxia (simulated altitude of 7 000 m) since the day of birth till the 11th day (youngest experimental group) or 17th day for 8 hours a day. Allopurinol was administered daily immediately before each hypoxia exposition. The duration of evoked afterdischarges (ADs) and the shape of evoked graphoelements were evaluated in 12, 18, 25 and 35-day-old freely moving male pups. Hypobaric hypoxia prolonged the duration of ADs in 12, 18 and 25-day-old rats. The ADs were prolonged in 35-day-old rats only after the first stimulation. Allopurinol shorted the duration of ADs only in 12-day-old pups. In older experimental group the effect of allopurinol treatment was less pronounced.

Nicotine and kainic acid effects on cortical epileptic afterdischarges in immature rats.

Aim of the study was to test the effect of nicotine (NIC) and kainic acid (KA) co-treatment in immature rats. Male Wistar albino rats (two different age groups) were chosen for the study. Experiments started on postnatal day (PD) 8 or 21 and animals were treated twice a day for three days with nicotine, fourth day KA was administered. Animals at PD12 (PD25 respectively) were examined electrophysiologically for cortical epileptic afterdischarges (ADs). First cortical ADs in PD12 animals were longer, when compared to PD25 rats (group treated with both substances). Nor NIC or KA treatment affected the length of discharges in PD12 rats. Older experimental group exhibited the shortening of the first ADs (group treated with NIC and KA, compared with groups exposed to single treatment). Few changes were found in KA treated group - 2(nd) and 4(th) ADs were shorter when compared with first ADs. These results demonstrate that NIC treatment played minor role in seizure susceptibility of PD12 rats, sensitivity to NIC differs during ontogenesis and subconvulsive dose of KA influenced the length of discharges only in PD25 animals.

Nicotine influences the motor performance of immature rats in two different sensorimotor tasks.

The aim of this study was to assess the effect of nicotine on motor performance of immature (12-day-old) rats. We used two sensorimotor tasks (surface righting response and negative geotaxis test) to evaluate the influence of nicotine on animal's motor activity in course of 24 hours. Animals were treated intraperitoneally with two different nicotine doses (0.5 mg/kg and 1.0 mg/kg) and tested in four sessions (1 minute, 10 minutes, 1 hour and 24 hours after the injection). We concluded that nicotine significantly influences the motor behaviour in 12-day-old rats and this effect is dose dependent.

Is learning ability and spatial memory in rats influenced by single dose of nicotine?

A lot of studies have been concentrated on an effect of a short or a long-term administration of nicotine in humans or in animals. The negative effects on the human organism have been known for a long time, but these health problems are known especially from observing smokers. The number of tasks in human and in animals with accent on positive effect of nicotine has increased especially with regard to improvement of cognitive functions. The aim of this study was to investigate, how much a single dose of nicotine can influence the learning ability in rats. Male Wistar albino rats, 25-day-old, were studied. Two groups of animals received an intraperitoneal (i.p.) injection of nicotine in two different doses (0.75 mg/kg and 1.00 mg/kg b.w.). The third group received a single i.p. injection of saline in the equal volume (the control group). After the drug application the escape latency and the path length were measured and assessed in two periods of sessions in the Morris water maze. In our study no explicit changes of learning ability after a single nicotine injection was confirmed. Only at the third day of the task the trajectory was shorter (p<0.05) but this result appears too isolated. So we cannot exclude that such improvement was caused by other factors than by the nicotine administration.

Effect of the single-dose of nicotine-administration on the brain bioelectrical activity and on behaviour in immature 12-day-old rats.

A variety of current studies is concentrated on the effect of short-term or long-term administration of nicotine in humans and in animals. The aim of this study was to investigate the effect of nicotine after a single administration in two different doses on the brain bioelectrical activity and on behaviour and motor activity in young, immature rats. Male Wistar albino rats, 12-day-old, were used in the experiment. Two groups were administrated by one intraperitoneal (i.p.) injection of nicotine in two various doses. The last (the third) group, which was given one i.p. saline injection, served as a control group. The group with lower dose of nicotine (0.75 mg/kg body weight) showed only mild alteration of the electrocorticogram (ECoG), and no behavioural or motor changes. In the second group (with higher dose of nicotine--1.00 mg/kg), epileptiform discharges manifested in about 50% of animals. Those animals showed also changes in motor activity (tremor of hindlimbs), but only slightly expressed within the time when epileptiform changes occurred in the ECoG. Routine behaviour and locomotion was observed only in a part of animals. In the third group (control group) no changes in bioelectrical activity, in behaviour or in motor activity were observed. We conclude that even a single dose of nicotine can evoke alteration in the ECoG, in behaviour and in motor activity of immature rats. On the other hand, the quantity, quality and length of ECoG abnormalities as well as parameters of behaviour were closely related to the dose of nicotine.

Magnesium and posthypoxic changes of nitrergic population in rat hippocampus.

We used NADPH-diaphorase staining to study effects of magnesium pre-treatment during long-lasting hypoxia on the brain structure of rats. NADPH-diaphorase is an enzyme co-localized in neurons with NO-synthase that is responsible for NO synthesis. NO participates in hypoxic-ischaemic injury of the brain. Hypoxia was induced in consecutive days from the 2nd till the 11th day of postnatal life in a hypobaric chamber (for 8 hours per day). Magnesium was administered before each hypoxia exposition. At the age of 12 days, the animals were transcardially perfused with 4% buffered neutral paraformaldehyde under the deep thiopental anaesthesia. Cryostat sections were stained to identify NADPH-diaphorase positive neurons that were then quantified in five hippocampal regions. In comparison to the control animals, intermittent hypoxia brought about higher density of NADPH-diaphorase positive neurons in all studied areas of the hippocampal structure: in CA1 and CA3 areas of the hippocampus and in hilus, in the dorsal and ventral blades of the dentate gyrus. Magnesium pre-treatment during hypoxia reduced number of NADPH-diaphorase positive neurons in all studied areas.

Changes of hippocampal neurons after perinatal exposure to ethanol.

The effect of ethanol on the structural development of the central nervous system was studied in offspring of Wistar rats, drinking 20 % ethanol during pregnancy and till the 28th day of their postnatal life. The structural changes in the hippocampus and dentate gyrus were analyzed at the age of 18, 35 and 90 days. A lower width of pyramidal and granular cell layers, cell extinction and fragmentation of numerous nuclei were found in all experimental animals compared to control animals. The extent of neural cell loss was similar in all monitored areas and in all age groups. At the age of 18 and 35 days, the degenerating cells were observed in the CA1 and CA3 area of the hippocampus and in the ventral and dorsal blade of the dentate gyrus. Numerous glial cells replaced the neuronal population of this region. Some degenerating cells with fragmented nuclei were observed at the age of 90 days. Our experiments confirmed the vulnerability of the developing central nervous system by ethanol intake during the perinatal period and revealed a long-lasting degeneration process in the hippocampus and dentate gyrus.

Alcohol abuse in mothers during gravidity and breastfeeding brings changes of hippocampal neurons in their offspring.

Neurotoxic effect of ethanol on the CNS of laboratory rats in the prenatal and postnatal period was studied. Another aim of the experiment was to analyse structure of the hippocampus after the prenatal and postnatal exposure to alcohol and to identify the most vulnerable hippocampal regions. Pregnant Wistar rats of our own breed received 20% alcohol p.o. ad libitum every day since the conception to the 18th day of postnatal life of their offspring. Since the birth (the day 1) till the age of 18 days offspring were kept together with their mother and were exposed to postnatal alcohol effect (alcohol in the breast milk). At the age of 18 days animals were perfused under deep thiopental anaesthesia with buffered solution of paraformaldehyde. Serial sections were stained with Fluoro-Jade B and DNA specific dye bis-benzimide (Hoechst No 33258). Brains of young rats aged 18 days were analysed under the light microscope Olympus Provis AX-70 with epifluorescence. In CA1 and CA3 areas and in Gyrus dentatus of the hippocampus, groups of degenerating cells were observed. In all offspring some cells with fine granulated karyons were identified, which were accompanied with high numbers of glial cells. Our results demonstrate the neurotoxic effects of alcohol and the high vulnerability of the developing CNS. The identification of cells with segmented karyons indicates the role of apoptotic mechanism in the cell death.

Effect of magnesium pre-treatment on the hippocampal NOS activity during long-lasting intermittent hypoxia.

Influence of magnesium pre-treatment during repetitive hypoxia was studied in the hippocampus of rats by histochemical analysis (NADPH-diaphorase staining). NADPH-diaphorase occurs concurrently with NO-synthase that is responsible for NO synthesis. Rat pups were kept together with their mother for 8 hours a day in a hypobaric chamber at a simulated altitude of 7,000 m since the day of birth till the 17th day. The first group of animals was exposed to the repeated hypoxia; the second group under the same conditions was pre-treated by magnesium before the exposition to the hypoxia. Both groups were compared with intact control animals and intact animals treated with magnesium. The experimental and control animals were the transaortically perfused with 4% buffered neutral formaldehyde under thiopental anaesthesia at the age of 35 days. Brains were processed for NADPH-d staining. We estimated the density of NADPH-d positive neurons in CA1 and CA3 areas of the hippocampus and in the dentate gyrus. Intermittent hypoxia brings about higher numbers of NADPH-diaphorase positive neurons of CA1 and CA3 of the hippocampus and of the dorsal blade of dentate gyrus, in the comparison with either group of control animals. In the hilus and ventral blade of the dentate gyrus, on the contrary, the number of NADPH-d positive neurons was smaller. Magnesium pre-treatment during hypoxia decreased number of nitrergic neurons in all areas of the hippocampus except CA1 area, where the effect of magnesium was not significant. These results demonstrate that magnesium can probably have a neuroprotective effect.

Partial effect of nimodipine on evoked epileptic seizures in young rats.

The present work studied the effect of calcium channel blocker (nimodipine) on epileptic seizures elicited by electrical stimulation of somatosensory cortex in young rats exposed to hypoxia. Rats were exposed to different patterns of short-term hypoxia (hypobaric or normobaric) and reactions of cortical neurones were registered. In the youngest animals (12-day-old), the effect was minimal, in older rats prolongation or shortening of evoked epileptic seizures were registered after two types of hypoxia. The decrease of the duration of evoked epileptic seizures in control 12-day-old rats and any effect in older animals (in 25- and 35-day-old) after nimodipine administration was observed. In older rats (25- and 35-day-old) exposed to hypobaric hypoxia after pre-treatment with nimodipine (L-type calcium channel antagonist) the duration of epileptic seizures after repeated stimulation was increased.