Homocarnosine levels in repeated hypothermia and hibernation

Compulsory cooling of rats (20 degrees) and susliks (20 degrees-10 degrees) leads to a considerable decrease of homocarnosine in cerebral hemispheres in midbrain and diencephalon and cerebellum. When hypothermia is repeated 4 or 7 times (in every 24 h) a more considerable decrease in the homocarnosine quantity in the rat brain takes place and it is not normalized after self-warming of the organism. After 11-12-fold hypothermia, when a certain adaptation of animals to cooling is developed, the second increase of homocarnosine content with subsequent normalization of its level in great cerebral hemispheres and in cerebellum at self-warming is observed. In dynamics of the hibernation (1, 7 and 30 days) either preservation of normothermal level or a certain increase of the homocarnosine content in all areas of the suslik brain under examination is observed.     

Dynamics of the heat exchange in rats upon getting out of the state of artificial hypothermia

With the help of thermometry and general calorimetry, changes in the heat exchange were determined in the non-hibernating mammals (rats) upon their getting out of artificial deep hypothermia (the temperature in the rectum was 22-24 degrees C) at the temperature in the calorimeter chamber 22 degrees C. An attempt was made to find out what part of the heal production during animal self-warming is used for increasing its body temperature and what part of the heat production is released to the environment by the animal. The experiments revealed a complex relationship between the body temperature, the heat loss, and the total heat production during the animal self-warming. The result was that the total heat production first increased and, after reaching the maximum, decreased gradually. The experiments showed that during two and more hours of observation the body temperature did not reach the starting level, the same was true for the total heat production, which was the sum of the heat loss and the heat production spent for warming the animal body.     

Effects of moderate hypothermia on constitutive and inducible nitric oxide synthase activities after traumatic brain injury in the rat

We investigated the effects of therapeutic hypothermia (30 degrees C) on alterations in constitutive (cNOS) and inducible (iNOS) nitric oxide synthase activities following traumatic brain injury (TBI). Male Sprague-Dawley rats were anesthetized with 0.5% halothane and underwent moderate (1.8-2.2 atm) parasagittal fluid-percussion (F-P) brain injury. In normothermic rats (37 degrees C) the enzymatic activity of cNOS was significantly increased at 5 min within the injured cerebral cortex compared with contralateral values (286.5+/-68.9% of contralateral value; mean+/-SEM). This rise in nitric oxide synthase activity was significantly reduced with pretraumatic hypothermia (138.8+/-17% of contralateral value; p < 0.05). At 3 and 7 days after normothermic TBI the enzymatic activity of cNOS was decreased significantly (30+/-8.4 and 28.6+/-20.9% of contralateral value, respectively; p < 0.05). However, immediate posttraumatic hypothermia (3 h at 30 degrees C) preserved cNOS activity at 3 and 7 days (69.5+/-23.3 and 78.6+/-7.6% of contralateral value, respectively; mean+/-SEM; p < 0.05). Posttraumatic hypothermia also significantly reduced iNOS activity at 7 days compared with normothermic rats (0.021+/-0.06 and 0.23+/-0.06 pmol/mg of protein/min, respectively; p < 0.05). The present results indicate that hypothermia (a) decreases early cNOS activation after TBI, (b) preserves cNOS activity at later periods, and (c) prevents the delayed induction of iNOS. Temperature-dependent alterations in cNOS and iNOS enzymatic activities may participate in the neuroprotective effect of hypothermia in this TBI model.     

The radioprotective effect of hypothermia on the immune and hematopoietic systems in mammals

The functional activity of the synthetic apparatus (parameter alpha) in blood lymphocytes, bone marrow hemopoietic cells, and thymus cells, as well as the total number of blood and bone marrow cells in rats after y-irradiation at a dose of 8 Gy in the conditions of normothermia and hypothermia (16-18 degrees C) with hypoxia-hypercapnia were investigated after 2 h and on days 1 and 4. The recovery processes in blood in both groups of rats after acute X-irradiation at a dose of 7 Gy for 36 days were analyzed too. Under hypothermia, on days 1-4 after acute gamma-irradiation, a decrease in the synthetic activity in remaining cells and devastation in the hemopoietic system were pronounced to a lesser degree. After X-irradiation, the restoration of synthetic activity in blood lymphocytes was shown to begin earlier and to finish faster in "hypothermic" rats as compared with the animals irradiated in the state of normothermia. The survival of "hypothermic" rats was 100% as compared with 30% in "normothermic" animals. Thus, the data show that hypothermia exerts a radioprotective effect on the cells of the immune and hemopoietic systems, thus enhancing the resistance of the organism to radiation.     

Acetylcholinesterase activity in brain slices of rats subjected to cooling

The acetylcholinesterase (AChE) activity is studied in rat slices of the cerebral cortex, corpus striatum, hypothalamus and medulla oblongata of rats during hypothermia (20 degrees C) and also 1 and 7 days after the posthypothermal period. Cooling of animals down to 20 degrees C is accompanied by an increase in the AChE activity in the brain both under incubation temperature of 20 degrees and 37 degrees C. Under prolonged hypothermia the AChE activity in the investigated brain regions, except for corpus striatum, returns to the control level. By the 7th day of posthypothermal period the AChE activity in corpus striatum, hypothalamus and medulla oblongata does not restore completely. The most substantial changes in the AChE activity both under hypothermia and posthypothermal period occur in corpus striatum, which obviously reflects its complicated functional role.     

Influence of cooling rate on activity of ionotropic glutamate receptors in brain slices at hypothermia

Hypothermia is a known approach in the treatment of neurological pathologies. Mild hypothermia enhances the therapeutic window for application of medicines, while deep hypothermia is often accompanied by complications, including problems in the recovery of brain functions. The purpose of present study was to investigate the functioning of glutamate ionotropic receptors in brain slices cooled with different rates during mild, moderate and deep hypothermia. Using a system of gradual cooling combined with electrophysiological recordings in slices, we have shown that synaptic activity mediated by the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-D-aspartate receptors in rat olfactory cortex was strongly dependent on the rate of lowering the temperature. High cooling rate caused a progressive decrease in glutamate receptor activity in brain slices during gradual cooling from mild to deep hypothermia. On the contrary, low cooling rate slightly changed the synaptic responses in deep hypothermia. The short-term potentiation may be induced in slices by electric tetanization at 16  °C in this case. Hence, low cooling rate promoted preservation of neuronal activity and plasticity in the brain tissue.     

Ethylene dibromide: effects of paternal exposure on the neurotransmitter enzymes in the developing brain of F1 progeny

The effects of ethylene dibromide (EDB) exposure to male rats on several neurotransmitter enzymes have been examined in various brain regions of the F1 progeny, from 7 to 90 days of age. The choline acetyltransferase activity was significantly increased at 21 days old, in most brain regions studied in the F1 progeny of the EDB-treated males, but not at 7, 14 or 90 days old. The acetylcholinesterase activity was altered in different brain regions of the F1 progeny of the EDB-exposed males at both 14 and 21 days old but not at 7 or 90 days old. Glutamic acid decarboxylase activity was increased in corpus striatum but decreased in frontal cortex only at 21 days of age. These neurochemical changes in the developing brain of F1 progeny of EDB-treated males at low doses may be associated with behavioral abnormalities observed early in their development.     

Post-ischemic hypothermia promotes generation of neural cells and reduces apoptosis by Bcl-2 in the striatum of neonatal rat brain

Hypothermia is a potential therapy for cerebral hypoxic ischemic injury in adults and neonates. However, the mechanism of hypothermia neuroprotection after hypoxic-ischemia (HI) on the developing rat brain remains unclear. In this research, 7-day-old rats were subjected to left carotid artery ligation followed by 8% oxygen for 2h. They were divided into hypothermia (rectal temperature, 32-33°C for 24h) and normothermia (36-37°C for 24h) groups immediately after hypoxia-ischemia. All rats were given 50mg/kg/day 5-bromodeoxyuridine (BrdU) intraperitoneally at 4-6 days and sacrificed at 1 or 2 weeks after HI. There was a significant decrease in infarct volume in the hypothermia group at 7 days after HI compared with that in the normothermia group. The numbers of nestin-labeled cells did not change greatly, but β-tubulin III (Tuj-1) immuno-positive cells increased significantly in the striatum at 1 and 2 weeks after HI in the hypothermia compared to normothermia group. Neurogenesis was assessed by double immunohistochemical/immunofluorescent labeling of BrdU with nestin, Tuj-1 or microtubule-associated protein 2 (Map-2). Newborn neural progenitors (BrdU(+)-nestin(+)) did not change dramatically, but newborn immature (BrdU(+)-Tuj-1(+)) and mature (BrdU(+)-Map-2(+)) neurons increased significantly in the hypothermia compared with normothermia group. Meanwhile, the apoptosis rate of neural precursors, immature and mature neurons, assessed by double labeling of active Casp-3 with nestin/Tuj-1/Map-2, decreased noticeably in the hypothermia compared with normothermia group. We also found that hypothermia significantly increased expression of Bcl-2, which coexisted with nestin/Tuj-1/Map-2. Inhibition of Bcl-2 expression reversed the decreased apoptosis rate of neural precursors and neurons in hypothermia animal striatum of neonatal rat brain. These results suggest that neuroprotection effects of hypothermia on injured developing rat brain may associate with enhanced generation of neuronal cells and Bcl-2-mediated reduction of apoptosis of these cells. These observations are noteworthy regarding clinical hypothermia therapy following cerebral HI injury during the perinatal period.     

Changes in the incorporation of labeled amino acids into separate protein fractions of the cerebral tissue in rats after intrauterine hypoxia

Incorporation of 14C-amino acid mixture into the cortex and cerebellum protein was studied in 7, 15, 30 days old rats after prenatal hypoxia. Prenatal hypoxic rats was shown to have alteration of the pattern of incorporation of label predecessors into brain protein. Prenatal hypoxia led to significant decrease of incorporation value at 30,000 molecular weight fractions. It is assumed that prenatal hypoxia results in selective changes of the brain protein synthesis.     

Post-intoxication hypothermia and processes of free radical lipid peroxidation in the rat brain and heart in organophosphorus cholinesterase inhibitor poisoning]

The activity of lipid peroxidation (LP) of the brain and myocardium as well as the intensity of the body hypothermia during 30 days after a single introduction of poisons at a dose < D50 were studied on the models of induced toxicosis in rats due to two organophosphorus compounds (malathion and armin). It has been established that the maximums of diene conjugates and Schiff's bases accumulation in the rat organs poisoned with malathion and the intensity of hypothermic response on days 14 and 21 after intoxication coincided. The similar elevated lipid peroxidation was associated with esophageal hypothermia on day 14 after armin administration. Therefore, because of hypothermia, the influence of organophosphorus compounds on lipids of the rat brain and heart in the post-intoxication period may include the free radical ways of their peroxidation.     

EFFECTS OF ENVIRONMENTAL COMPLEXITY ON AMINO ACID INCORPORATION INTO RAT CORTEX AND HIPPOCAMPUS IN VIVO

Abstract— Amino acid incorporation in vivo was investigated in the cortex and hippocampus of rats raised in enriched and deprived environments for various periods of time following weaning. At early times after weaning (7 days), the incorporation of l -[³H]leucine into all sub-cellular fractions of both cortex and hippocampus was higher in enriched than in deprived rats. At 16 days, incorporation into synaptosomal sub-cellular fractions was higher in enriched than in deprived hippocampus, and lower in enriched than in deprived cortex; incorporation into perikaryal fractions of both brain regions was the same in the two groups of animals. Incorporation into subcortical nuclear protein fractions was higher in enriched rats at this time. At 35 days, the only difference between enriched and deprived rats was a lower incorporation into cortical synaptosomal sub-fractions in the former. Experiments involving double labelling and electrophoresis indicate that there is no stimulation or inhibition of the synthesis of any particular protein in hippocampal nuclear and synaptosomal sub-fractions of enriched rats. Synaptosomal proteins of cortex have a greater half-life in enriched than in deprived rats; proteins of perikaryal fractions of cortex, and of all fractions of hippocampus, are turning over at the same rate in enriched and deprived animals.     

Dynamics of the heat exchange in rats upon getting out of the state of artificial deep hypothermia

With the help of thermometry and general calorimetry the changes in the heat exchange were determined in the rats upon their getting out of artificial deep hypothermia (the temperature in the rectum was 20 degrees C) at the temperature in the calorimeter chamber 20 degrees C. An attempt was made to find out what part of the heat production during an animal self-warming is used for increasing its body temperature and what part of the heat production is released to the environment by the animal. The experiments revealed a complex relationship between the body temperature, the heat loss, and the total heat production during the animal selfwarming. The total heat production first increased and, after reaching the maximum, decreased gradually. Moreover, the experiments showed that during three and more hours of observation the body temperature did not reach the initial level, the same was true for the total heat production, which in these experiments was the sum of the heat loss and the production spent for warming the animal body.     

Reversible deep whole-body hypothermia in rats

The effect of deep hypothermia on the rat's organism was investigated without the use of hypercapnic hypoxia or administration of medicine. The possibility of resuscitation and rehabilitation of rats after deep hypothermia depending on different rate of heating have been also studied. It has been observed that the cardiac activity stopped at about 3 degrees C. We supposed that the extremely intensive heating and temperature rise of an organism were both limiting factors. We have discovered that the low intensity of heating was the next factor of limitation which was connected with the duration of clinical death. These experiments showed that the intensive cooling and heating (about 0.4 degrees C per min.) are the best for resuscitation and rehabilitation after deep hypothermia.     

Changes in the electroencephalographic and gamma-aminobutyric acid transaminase and succinic semialdehyde dehydrogenase in the allergen induced rat brain

Electroencephalographic activity and gamma-Aminobutyric acid Transaminase together with Succinic semialdehyde dehydrogenase activity changes produced by sensitization with Prosopis juliflora pollen allergen were studied in the cerebral cortex and hypothalamus of the rat brain. Electrical activity of EEG recording begins to appear on 3rd day after sensitization with maximum increase in activity was found on day 9 and decreased after that. A sudden increase in electrical activity was produced in 9th day sensitized rat with 10 min after giving challenging dose intravenously. The measurement of enzymatic activity of GABA-T and SSA-DH showed decrease and increase in 3, 9, 15 and 30 days sensitized rat hypothalamus and cerebral cortex whole homogenate and mitochondrial fractions. A maximum changes in enzymatic activity was found in 9th day sensitized rat with significant alterations after giving sudden stress as challenging dose. These changes in EEG activity and GABA-ergic neurotransmitter in allergenic rats showed the immunoregulatory role of nervous system mediated via GABA shunt.     

A comparative analysis of the functional stability (based on electrophysiological criteria) of slices of the suslik and guinea pig brains maintained under deep hypothermia]

The effect of prolonged deep cooling has been investigated in hippocampal and septal slices from the brain of hibernating and active ground squirrels, as well as of the guinea pigs. The slices were kept at low temperatures (2-4 degrees C) for various periods of time (from several hours to 6 days) and were periodically tested in a warm (31 degrees C) incubation medium. Hippocampal field potentials (mainly of the field CA1) and spontaneous activity of single neurones of the medial septum were recorded. Significant differences were observed in the recovery of functional activity of the slices after their preparation as well as after cooling between experimental groups of animals. Slices from hibernating ground squirrels retained their activity for 7-9 days, those from active ones--for 6-7 days, whereas slices from guinea pigs did not recover their functional activity after cooling for more than 1-2 days.     

The cytokinins of cultured carrot cells

Summary Cytokinin-autotrophic strains of carrot callus contained active substances with Chromatographic mobilities on Sephadex LH-20 corresponding to 6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine (Zeatin, Z), 6-(3-methylbut-2-enylamino)purine (iP) and the ribosides (9R)Z and (9R)iP. The apparent major activity was found in a fraction, with an elution volume of 242–291 ml. Hydrolysis of this fraction with HCl and -Glucosidase gave rise to Z, indicating that the major active compound is a polar conjugate of Zeatin.In all experiments the extracts were tested immediately after preparation; deep freeze storage, led to a considerable loss of activity in polar fractions. while the free base cytokinins and their ribosides showed increased activity levels.Analogous results were obtained by means of paper chromatography.     

Niaspan Attenuates the Adverse Effects of Bone Marrow Stromal Cell Treatment of Stroke in Type One Diabetic Rats

Aims

Our previous studies have found that bone-marrow-stromal cells (BMSC) therapy improves functional recovery after stroke in non-diabetic rats while increases brain hemorrhage and induces arteriosclerosis-like changes in type-one-diabetic (T1DM) rats. Niaspan treatment of stroke increases vascular stabilization, decreases brain hemorrhage and blood-brain-barrier (BBB) leakage in T1DM rats. We therefore tested the hypothesis that combination therapy of BMSC with Niaspan attenuates the side effects of BMSC monotherapy in T1DM rats.

Methods

T1DM-rats induced by streptozotocin were subjected to 2 hours of middle-cerebral-artery occlusion (MCAo) and treated with: 1) PBS; 2) BMSC (5×10⁶); 3) Niaspan (40 mg/kg) daily for 14 days; 4) BMSC (5×10⁶) +Niaspan (40 mg/kg, daily for 14 days) combination starting at 24 hours after MCAo. All rats were monitored for 14 days.

Results

Combination BMSC+Niaspan treatment of T1DM-MCAo rats did not increase brain hemorrhage, and significantly decreased BBB leakage and vascular arteriosclerosis-like changes as well as decreased Angiogenin, matrix metalloproteinase 9 (MMP9) and ED1 expression in ischemic brain and internal-carotid-artery compared to non-treatment control and BMSC monotherapy animals.

Conclusions

Combination therapy using BMSC with Niaspan decreases BBB leakage and cerebral arteriosclerosis-like changes. These beneficial effects may be attributed to the decreased expression of Angiogenin, MMP9 and ED1.     

Protein metabolism by rat brain synaptosomes during learning

The synthesis and degradation of rat brain synaptosomal proteins were studied in three groups of animals: trained the behaviour pattern in the maze, "pseudo-trained" and control. These processes were assessed from protein specific radioactivity after 1, 3 days and after 1, 3, 6 and 9 weeks following intraventricular injection of 14C-lysine. The experiments showed three fractions differing in overall values of half-life (T50). An increase in specific radioactivity of brain proteins of trained animals was revealed as compared to that in "pseudo-trained" and control rats. T50 was recorded to rise for slow-metabolizing fractions of synaptosomal proteins of trained rats. Participation of synaptosomal proteins in the mechanisms of long-term memory is discussed.     

In vitro study of the aromatic hydroxylation of 1-methyltetrahydro-beta-carboline (methtryptoline) in rat

The incubation of 1-methyltetrahydro-beta-carboline (1-MeTHBC) with hepatocytes isolated from 3-methylcholanthrene-treated rats led to formation of the 5-, 6- and 7-hydroxylated products. The hydroxylating activity was associated with the microsomal fraction as indicated by testing different subcellular fractions. The highest activity for hydroxylating 1-MeTHBC was found in liver which was about ten times as active as lung. Only a trace amount of hydroxylating activity was present in brain and kidney tissue. Analysis using chiral gas chromatography revealed an unequal abundance of enantiomers in all three products. The formation of the 5-, 6- and 7-hydroxylated products was confirmed in vivo by analysis of 24 h urine samples after intraperitoneal administration of 1-MeTHBC to 3-methylcholanthrene-treated rats.     

Localization of vicilin peptidohydrolase in the cotyledons of mung bean seedlings by immunofluorescence microscopy

Vicilin peptidohydrolase, the protease that hydrolyzes the reserve proteins in the cotyledons of mung bean (Vigna radiata) seedlings, has been localized intracellularly by immunofluorescence microscopy using monospecific antibodies against the enzyme and rhodamine-coupled goat-anti-rabbit immunoglobulin G's. The enzyme can first be visualized after 3 days of seedling growth and is associated with small foci within the cytoplasm of the storage parenchyma cells farthest from the vascular bundles. On the 4th day of growth, the protease is also present in the numerous large protein bodies within these cells. Vicilin peptidohydrolase is known to be synthesized de novo starting on the 3rd day of growth. Our observations are therefore consistent with the interpretation that the enzyme is synthesized in the cytoplasm and subsequently transported to the protein bodies.