Selective vulnerability of the hippocampus to the cytotoxic edema; magnetic resonance imaging and fluorescence microscopy studies in the rats.

OBJECTIVES: Changes in the hippocampus induced by water intoxication were studied using fluorescence microscopy (FM) and magnetic resonance imaging (MRI). METHODS: In three animals (rats), intracellular/extracellular distribution of Evans blue (EB) in cerebral cortex and hippocampus of both hemispheres was revealed by injection of EB into the internal carotid artery (ICA) in hyperhydrated rats (water intoxication, WI). A total of 8 experimental rats were used for the MRI study. The animals were scanned before WI, then the experimental brain edema was induced by WI and MR scanning was performed at day 1 and day 8 after WI. Besides standard T2-weighted imaging an apparent diffusion coefficient (ADC) and transverse relaxation time (T2) were evaluated. RESULTS: Hyperhydration brought about the largest intracellular deposits of EB in CA3 hippocampal region, followed by the cerebral cortex and CA1 hippocampal region with the lowest amount of intracellular EB in the dentate gyrus. A higher apparent diffusion coefficient (corresponding to a vasogenic edema) was found the first day after hyperhydration in the cortex and in the CA1 and CA3 regions with no changes in dentate gyrus. CONCLUSION: Both FM and MRI confirmed a selectively higher vulnerability to hyperhydration and hyponatremia (achieved by water intoxication) of the hippocampal cells compared to dentate gyrus cells.

Intracranial pressure and mean arterial pressure monitoring in freely moving rats via telemetry; pilot study.

OBJECTIVES: Accurate values of the intracranial pressure (ICP) and mean arterial pressure (MAP) are the prerequisite for calculating cerebral perfusion pressure (CPP). Increased ICP values decrease CPP. The origin of ICP increase in the clinical cases after brain ischemia and diffuse brain injury is the cellular brain edema (CE). Short-term monitoring of ICP and MAP is possible only in the unconscious patients, in experiments with rats it used to be possible only in general anesthesia. Long-term monitoring of ICP or MAP in the clinical practice is not possible. We therefore introduce an experimental model with telemetric monitoring. METHODS: ICP (subdurally) and MAP (intracarotically) were monitored in freely moving rats for 72 hours by DSI™ (Data Sciences International) telemetry system. The control group consisted of 8 rats, the experimental group had 8 animals with CE-induced by water intoxication. RESULTS: The mean MAP, ICP and CPP values were significantly higher in the experimental group. Average values of MAP were 19.9 mmHg (18%), ICP 5.3 mmHg (55%), CPP 14.5 mmHg (15% higher). CONCLUSION: The results of the pilot study verified possibilities of long-term telemetric monitoring of the mean arterial and intracranial pressures for the determination of current cerebral perfusion pressure in freely moving rats under physiological conditions and with increased intracranial pressure due to the induced cerebral edema. Detailed analysis of the course of the curves in the experimental group revealed episodes of short-term CPP reduction below the optimum value of 70 mmHg. Interpretation of these episodes requires simultaneous monitoring of rat behavior.

Cytotoxic brain edema induced by water intoxication and vasogenic brain edema induced by osmotic BBB disruption lead to distinct pattern of ICP elevation during telemetric monitoring in freely moving rats.

OBJECTIVES: A novel method of long-term telemetric monitoring of mean arterial pressure (MAP) and intracranial pressure (ICP) for the determination of current cerebral perfusion pressure (CPP) and the time course of ICP in freely moving rats under physiological conditions and with increased ICP due to the induced cerebral edema were studied. METHODS: The brain edema, that caused volume enlargement and ICP elevation was achieved in entirely experimental conditions without any parallel pathological process. Vasogenic/extracellular edema was induced by osmotic blood-brain barrier disruption (BBBd) and for induction of cytotoxic/intracellular edema the water intoxication model (WI) was used. RESULTS: The results showed significantly elevated values of ICP both in conditions of osmotic blood-brain barrier disruption (BBBd model) and cytotoxic/intracellular edema (WI model) compared to intact rats. The average values of ICP were significantly higher in WI model compared to osmotic BBBd model. Distinct pattern of elevated ICP, related to the selected way of experimental brain edema induction, was found. In the experimental model of osmotic BBB disruption, the elevation of ICP started earlier but was of very short duration. In WI model the elevation of ICP was present during the whole period of monitoring. CONCLUSION: Our results indicate that purely experimental models of brain edema (WI, BBBd) without any parallel pathological process can compromise the basic brain homeostatic activity.

Locomotion in young rats with induced brain cellular edema - effects of recombinant human erythropoietin.

OBJECTIVES: Effect of recombinant human erythropoietin (rhEPO) on spontaneous motor activity was tested in young rats after intraperitoneal (i.p.) administration of rhEPO, followed by induction of cellular brain edema (CE). Induced changes in the spontaneous horizontal locomotor activity was studied by open field test (OFT). METHODS: CE was induced by water intoxication (WI) using standard method of fractional hyperhydration accompanied with desmopressin administration. Using the accepted method of OFT average time spent in locomotion (s) was determined. 48 young rats at the age of 25, and 35 days were divided into three groups - controls, rats after WI (OFT followed after 44 hours), and rats administered with rhEPO prior to application WI (OFT after 48 hours). RESULTS: In 35-day-old rats rhEPO administration increased the spontaneous locomotor activity, previously decreased by cellular edema. In 25-day-old rats, rhEPO administration prior to the induced CE, decreased spontaneous locomotor activity. CONCLUSION: Presented results demonstrate the neuroprotective capacity of rhEPO, manifested by elimination of the suppressive influence of CE on the locomotion in 35-day-old rats. In 25-day-old rats the neuroprotective effect was not present. These results confirmed that the 10 day interval in the development may represent a different stage of brain maturation in the relation to the neuroprotective effect of rhEPO.

Cellular brain edema induced by water intoxication in rat experimental model.

OBJECTIVES: This paper presents our own rat model of the cellular brain edema, induced by water intoxication (WI). The basic principle of the model is an osmotic imbalance in the cell membrane followed by an intracellular flow of sodium and simultaneous accumulation of water leading to the subsequent increase of BBB permeability. METHODS: The usefulness of the model was tested in precisely specified conditions whose results were clearly expressed. The procedure determined both how WI induces cellular edema as well as the disturbances caused by cellular edema. RESULTS: The evidence of existing cellular edema with increased BBB permeability was proved by intracellular accumulation of intravital dye with a large molecular size; increased brain-water content was confirmed by using the dry/wet weight method and by the decrease in CT density; the elevated intracranial pressure (ICP) due to the expanding volume was determined by continuous monitoring the ICP; the structural lesions were proved by identification of the myelin disintegration; and the impaired nervous functions was demonstrated by the of open field test method. CONCLUSION: Our experimental model can help the future studies of pathophysiology of cellular brain edema and is suitable for testing neuroprotective agents.

An experimental model of the "dual diagnosis": Effect of cytotoxic brain edema plus peripheral neuropathy on the spontaneous locomotor activity of rats.

OBJECTIVE: The aim of the study was to find how a simultaneous impairment of the CNS (cellular brain edema induced by water intoxication) and PNS (blockade of the right forelimb brachial plexus by local anesthewtic - Marcaine) affects spontaneous locomotor activity of adult rats. METHODS: Rats were divided into groups of animals without water intoxication (without WI) - A,B,C, and those that were water intoxicated (induction of brain edema - after WI) - D,E,F. Both groups were further divided into intact ones (A,D), animals with PNS lesion (Marcaine) (B,E) and sham-operated animals (C,F). Locomotor activity (LA) of the rats was tested by the open field test. RESULTS: LA of rats with both CNS and PNS impairment (WI + Marcaine) was significantly suppressed compared to the activity of control rats. Comparison of LA of rats with a single lesion - PNS impairment only (Marcaine only), CNS lesion only (WI) to those animals with both lesions (WI + Marcaine) revealed even larger decrease of LA of rats with combined lesions, which represents a model of the dual diagnosis. Also the pattern of behaviour of rats in both sham operated groups was different, which apparently depended on water intoxication. CONCLUSION: The presented results show that the LA of rats with combined lesions is significantly lower compared to the activity of rats with a single lesion in the CNS or PNS. Results also indicate that the already induced endoneurial edema prevents subsequent accumulation of water applied to the intimate vicinity of the peripheral nervous structures.

Study of locomotion, rearing and grooming activity after single and/or concomitant lesions of central and peripheral nervous system in rats.

OBJECTIVE: Locomotion, rearing and grooming represent different forms of behaviour and motor activity in rats. In this study, changes in these activities were analysed in relation to impaired function of the nervous system by single and/or concomitant lesions representing an experimental model of the dual diagnosis. METHODS: 32 rats were divided into 4 groups of 8 rats: intact rats, rats with single lesion of peripheral nervous system (PNS) - Marcaine neuropathy, rats with single CNS lesion - cellular brain edema induced by water intoxication, and the concomitant lesions (combination of CNS and PNS lesion in one rat). Water intoxication was performed in a standard way by fractionated hyperhydration. The average time spent by locomotion, rearing and grooming was registered and analyzed using an open field test. RESULTS: All activities of the rats after water intoxication became inhibited due to the generally suppressive effect of brain edema. Lesion of PNS reduced activity in locomotion only, because for rearing and grooming activities, the function of the forelimb is not dominant. Combination of lesions (dual diagnosis) reduced locomotion and rearing activity more than single lesions, and enhances the stressogenic effect, which was manifested by a long periods of grooming. CONCLUSION: Results of our study confirmed the physiological and pathophysiological differences in the movement stereotype between locomotion, rearing and grooming caused by the characteristics and algorithms of the movements, which are inborn to rats - the dominant role of the forelimbs in locomotion, the dominant exploratory activity in rearing, and the precise syntactic movement pattern in grooming.

Neuronal excitability changes depend on the time course of cellular edema induced by water intoxication in young rats.

OBJECTIVES: The aim of the study was to determine whether the functional state of neurons is affected by the duration of the induced cellular edema and by the age of animals tested. The cellular edema was induced by water intoxication and neuronal functions were tested by the standard method of electrical stimulation of neurons of the cerebral cortex. METHODS: water intoxication was induced by standard method of fractionated hyperhydration. Excitability of cortical neurons was tested by cortical stimulation with the intensity required to induce cortical afterdischarge (AD). Animals were divided into three experimental groups (B, C, D) and three control groups (AB, AC, AD). Experimental groups differed in age of water intoxication (12 or 25 days) and age of excitability testing (25 or 35 days). Changes in the duration of AD (seconds) were statistically evaluated. RESULTS: Duration of cortical afterdischarges (AD) in the control groups was at the level literature data. In all experimental groups (B, C, D), excitability of cortical neurons was markedly inhibited. AD was possible to induce only in some of the animals and its average duration was significantly shorter than in control groups. CONCLUSION: This inhibitory effect can be explained by persistent impairment of astrocyte-to-neuron communication, which plays a key role in the process of formation of structural and functional changes during cellular edema. Some of the functional manifestations of the developing edema are influenced by the age of experimental animals. At least some events of this process are not influenced by the age of experimental animals.

Biochemical manifestations of the nervous tissue degradation after the blood-brain barrier opening or water intoxication in rats.

OBJECTIVES: The aim of the study was to determine changes of biomarkers of nervous tissue degradation in experimental model of osmotic blood-brain barrier opening or water intoxication and to find whether they correspond to changes in well defined clinical entities. METHODS: In the cerebro-spinal fluid taken via the suboccipital puncture, myelin basic protein (MBP ng/ml), neuron-specific enolase (NSE ng/ml) and TAU-protein (Tau pg/ml) were determined by ELISA in 19 controls and 29 experimental rats several hours or one week after the experimental intervention. RESULTS: Significant difference between the control and experimental groups was revealed only for the concentration of myelin basic protein. After the BBB opening, its level dramatically increased within hours and dropped back to control values within one week. Water intoxication induced only dilutional hypoproteinorachia. No significant changes were found in NSE and levels of TAU-protein were not detectable. CONCLUSION: 1. Increased permeability of cytoplasmic membranes induced by water intoxication does not alter any of monitored CSF biomarkers. 2. Osmotic opening of the BBB in vivo experiment without the presence of other pathological conditions leads to a damage of myelin, without impairment of neurons or their axons.

Neuronal excitability after water intoxication in young rats.

OBJECTIVES: Our previous experiments with animal models revealed that water intoxication induces brain oedema and opens plasma membranes. Present study is aimed to determine whether the standard method of hyperhydration can influence cerebral microenvironment also in young rats. Neuronal functions were tested by standard electrical cortical stimulation. METHODS: Hyperhydration was induced by administration of distilled water (DW) intraperitoneally. Three groups of young rats were used: 12, 25, and 35-day-old. Cortical excitability was tested 19 to 20 hours after DW administration by electrical stimulation of the sensorimotor cortex with intensity necessary to elicit cortical afterdischarges (AD). Water content in the brain was estimated by dry/wet ratio and value of natremia by standard biochemical examination. Control animals of the same age groups were tested in the same way, only they did not receive DW. RESULTS: Brain water content in hyperhydrated animals was smaller than in controls in all studied age groups. Natremia was the same (normal) in both the hyperhydrated and control animals aged 25 days. Excitability of cortical neurons in young hyperhydrated animals was significantly inhibited in comparison to the same age groups of controls. CONCLUSION: Hyperhydration induced in young rats (12, 25, 35-day-old) had different effects than in adults. Absence of hyponatremia, lower water content in the brain and significant inhibition of cortical excitability can be explained on the basis of ontogenetically dependent aquaporine expression (AQP 4) and different activity of ionic membrane transporters.

CT density decrease in water intoxication rat model of brain oedema.

OBJECTIVES: The aim of this study was to determine whether water intoxication affects the radiodensity of brain tissue in CT scan examination in the rat model of brain oedema. METHODS: A standard CT scan of the brain was obtained in a group of rats, first at control conditions (controls - CG) and then after hyperhydration (oedema model-EG) in the region of interest (ROI) corresponding to the area of coronary sections with pixel size 0.125 mm in position A (bregma +2.43 mm), position B (bregma -2.92 mm), position C (bregma -12.73 mm). Densitometrically determined mean values (MV), expressed in Hounsfield units (HU) were processed by standard statistical methods. RESULTS: The average MV density was 120.49±6.79 HU for the control measurement and 88.01±4.72 HU after the hyperhydration, which represents decrease in the density by 32.48 HU (p<0.001). In the control measurement the average value of HU for the position A was 121.98, for position B 112.4 and for position C 127.08. In conditions of hyperhydration, the average MV density in position A was 89.95 HU in position B 84.67 HU and in position C 89.43 HU. The differences between the CG and EG were in all positions A, B, C statistically significant (p<0.001). In the control measurement, the differences between position A×B (p<0.05) and B×C (p<0.001) were statistically significant. After hyperhydration no significant difference between the position A, B, C was found. CONCLUSION: water intoxication caused by hyperhydration in rats can induce diffuse brain oedema, which is reflected in the CT examination by the decrease of brain tissue density, expressed in HU. The value of the measured density depends on the location and size of the measured brain area.

Effect of methylprednisolone on the axonal impairment accompanying cellular brain oedema induced by water intoxication in rats.

OBJECTIVES: Our previous experiments proved that methylprednisolone (MP) can significantly reduce axonal impairment accompanying extracellular oedema induced by the osmotic challenge (load) on the blood-brain barrier (BBB). The aim of the present work was to identify whether MP can affect myelin impairment accompanying intracellular oedema induced by water intoxication. METHODS: For induction of cellular brain oedema, the standard model of water intoxication was chosen. Animals received distilled water in amount corresponding to 15% of the animal's body weight. The volume was divided into three parts and administered intraperitoneally in 8 hours interval. Axonal changes were recognized as signs of myelin disintegration (oedematous distensions, axonal swelling, vesicles, varicosities) at histological sections stained with Black Gold and classified into four grades of myelin degradation. Hippocampal CA1 and CA3 areas and the dentate gyrus were selected for the study. Methylprednisolone was administered either intraperitoneally or intracarotically. Its effect was studied in two different time intervals: in the acute group (30 minutes after hyperhydration and MP application) and in chronic one (1 week after hyperhydration and MP application). RESULTS: In both the acute and chronic groups, cellular oedema induced by water intoxication brought about apparent damage of myelin (compared to control animals p<0.0001). Intracarotic injection of MP was not able to influence myelin integrity changes either in the acute or in chronic group. However, intraperitoneal administration of MP increased the level of myelin deterioration in the acute group (p 0.05), but improved myelin changes in the chronic group (p<0.005). CONCLUSION: The effect of MP on axonal impairment during cellular brain oedema induced by water intoxication differs from that during the extracellular osmotic oedema. In the extracellular oedema, cellular metabolism is not significantly affected and myelin changes can be influenced by the neuroprotective effect of MP. The primary cause of cellular oedema is a disorder of cellular metabolism and myelin impairment is one of the structural consequences of such disorder. That is why the myelin changes are not affected by MP administration in a consistent and specific manner.

Methylprednisolone reduces axonal impairment in the experimental model of brain oedema.

OBJECTIVES: In our earlier paper we demonstrated that opening of the blood-brain barrier with an osmotic insult induces brain oedema which represents a factor triggering axonal impairment accompanied with myelin disintegration. The aim of the present study was to find whether methylprednisolone can reduce such axonal impairment in our model of brain oedema. METHODS: Brain oedema was induced by osmotic blood-brain barrier opening with 20% mannitol applied selectively into the internal carotid. Axonal changes were recognized as signs of myelin disintegration (oedematous vesicles, varicosity, myelin fragmentation) at histological sections stained with Black Gold in hippocampal areas CA1 and CA3 and in the dentate gyrus at time intervals of 30 minutes (acute group) or one week (chronic group) after the blood-brain barrier opening. At the same time intervals methylprednisolone was applied in two different ways - into peritoneal cavity or into the right carotid artery. RESULTS: Impairment of the axonal integrity (changes of the myelin sheet integrity) was identified in all areas studied in both experimental groups. Whereas in the control group axons were of the uniform diameter, in the experimental groups various forms of myelin disintegration were observed. Methylprednisolone reduced the degree of myelin disintegration in both time intervals with the highest effect in the acute group with the intracarotic administration. CONCLUSION: Methylprednisolone can effectively reduce myelin changes accompanying brain oedema induced by blood-brain barrier opening with an osmotic insult.

Time-dependent axonal impairment in experimental model of brain oedema.

OBJECTIVES: Clinically very serious condition of ischaemia and brain injury which are often associated with brain oedema is frequently accompanied by the impairment of the structural integrity of axons. We wondered whether the brain oedema (without ischemia brain injury) can induce structural axonal impairment. METHODS: Brain oedema was induced by osmotic blood-brain barrier opening with 20% mannitol applied selectively into the internal carotid. Axonal changes were recognized as signs of myelin disintegration (oedematous vesicles, varicosity, myelin fragmentation) at histological sections stained with Black Gold in hippocampal areas CA1 and CA3 and in the dentate gyrus and cerebral cortex at time intervals of one hour, one day, three days and one week after the oedema induction. RESULTS: Impairment of the structural integrity was identified in myelin sheets in all areas studied in all experimental groups. Whereas in the control group axon were of the uniform diameter, in the experimental groups various forms of myelin disintegration were observed. The progression of myelin damage depended on the time elapsed after the oedema induction. CONCLUSION: Opening the blood-brain barrier with an osmotic insult induces brain oedema which represents a factor triggering axonal impairment accompanied with myelin changes. The development of axonal changes initiated by brain oedema only (without ischemia brain injury) is a novel observation.

Cytogenetic analyses in 81 patients with brain gliomas: correlation with clinical outcome and morphological data.

Specific gene mutations, loss of heterozygosity, deletions and/or amplifications of entire chromosomal regions and gene silencing have been described in gliomas. 82 samples from 81 patients were investigated to detect the deletion of TP53, RB1, CDKN2A genes, deletion of 1p36 and 19q13.3 region, amplification of EGFR gene, trisomy of chromosome 7 and monosomy of chromosome 10 in glial cells. Dual-colour interphase fluorescence in situ hybridization (I-FISH) with locus-specific and/or chromosome enumeration DNA probes were used for cytogenetic analyses. In the study, molecular cytogenetic analyses were successfully performed in 74 patients (91.3%) and were uninformative in 7 only (8.7%). The cytogenetic analyses were correlated with morphological data and clinical outcome. I-FISH was the essential part of diagnostics. In comparison with the clinical data, the patients' age seems to be a factor more important for the overall survival, rather than cytogenetic findings in glial tumours. The combined deletion of 1p36 and 19q13.3 chromosomal regions predicts longer overall survival for patients with oligodendroglial tumours.