Serotonin effect on deiodinating activity in the rat

Serotonin (5-HT) and thyroid hormones are part of a complex system modulating eating behaviour and energy expenditure. 5-Deiodinase (5-D) converts the relatively inactive thyroxine (T4) to triiodothyronine (T3), and its activity is an indirect measure of T3 production in peripheral tissues, particularly in the brain, intrascapular brown adipose tissue (IBAT), heart, liver, and kidney. We evaluated the effect of 5-HT on 5'-D activity during basal conditions and after short (30 min) cold exposure (thyroid stimulating hormone stimulation test, TST). 5'-D activity was assessed in the liver, heart, brain, kidney, and IBAT. TST increases 5'-D activity in the brain, heart, and IBAT and decreases it in kidney, leaving it unchanged in the liver. 5-HT alone did not modify 5'-D activity in the organs under study but decreased it in the IBAT, heart, and brain when injected before the TST was administered. Our results confirm the important role of 5-HT in thermoregulation, given its peripheral site of action, in modulating heat production controlling intracellular T3 production. These effects are more evident when heat production is upregulated during cold exposure in organs containing type II 5'-D, such as the brain, heart, and IBAT, which are able to modify their function during conditions that alter energy balance. In conclusion, 5-HT may also act peripherally directly on the thyroid and organs containing type II 5'-D, thus controlling energy expenditure through heat production.     

Upregulation of neuronal nitric oxide synthase, edema and cell injury following heat stress are reduced by pretreatment with EGB-761 in the rat

Rats exposed to 4 h heat stress (HS) at 38°C exhibited marked upregulation of neuronal nitric oxide synthase (nNOS) in the brain regions exhibiting blood–brain barrier (BBB) breakdown, brain edema and cell damage. Pretreatment with an anti-oxidant compound EGB-761 (an extract of Gingko biloba) administered 50 mg/kg, per os for 5 days, significantly attenuated nNOS expression, BBB disruption, brain edema and cell injury. These results suggest that EGB-761 is neuroprotective in heat stress and this effect of the compound is related with the inhibition of NOS expression, not reported earlier.     

A new antioxidant compound H-290/51 attenuates heat shock protein (HSP 72 kD) response, edema and cell injury following acute heat exposure. An experimental study using light and electron microscopy in the rat

Rats exposed to 4 h heat stress at 38°C exhibited upregulation of heat shock protein (HSP 72 kD) expression in several brain regions associated with brain edema and cell injury. Pretreatment with a new anti-oxidant compound H-290/51 (50 mg/kg, per os, 30 min before stress) significantly attenuated HSP expression, brain edema and cell injury. These results suggest that oxidative stress associated with brain edema plays important roles in HSP expression, not reported earlier.     

Triiodothyronine deiodinating activity in brown adipose tissue after short cold stimulation test in trained and untrained rats

Interscapular brown adipose tissue (IBAT) activity is controlled by sympathetic nervous system, and factors that influence thermogenesis appear to be centrally connected to the sympathetic outflow to IBAT. Cold exposure produces a rise in BAT temperature, which is associated with an increased thyroid activity, elevated serum levels of 3,5,3'-triiodothyronine (T3), and an increased rate of T3 production. This study evaluated the effect of swimming training on 5'-triiodothyronine deiodinase (5'-D) activity in IBAT under normal environmental conditions and after short (30 min) cold exposure (TST stimulation test). 5'-D activity is lower in trained rats at basal condition, and TST increases 5'-D in IBAT of both untrained and trained rats. However, this increase is lower in trained rats. Training reduces the deiodinating activity in normal environmental conditions as well as after short cold exposure. Probably, other compensatory mechanisms of heat production are active in trained rodents.     

Effect of heat exposure on the activity of monoamine oxidase in the rat brain and interscapular brown adipose tissue

The exposure of Wistar male rats (200±20 g) to high ambient temperature (38°C) for 20 and 60 min induced an equal decrease in hypothalamic, brain stem and hippocampal monoamine oxidase activity when compared to controls. The interscapular brown adipose tissue monoamine oxidase activity, as well as oxygen consumption and rectal temperature were increased only after a 60 min heat exposure. The adrenal function, assessed by dopamine-beta-hydroxylase activity and cholesterol concentration, was enhanced both after 20 and 60 min. In conclusion, heat induced the increase in adrenal function and interscapular brown adipose tissue monoamine oxidase activity, but the decrease in that of the brain.     

WST-1-based cell cytotoxicity assay as a substitute for MTT-based assay for rapid detection of toxigenic Bacillus species using CHO cell line

Bacillus cereus continues to be one of the important foodborne pathogens due to its ability to produce various heat-labile and -stable toxins. Several methods have been developed to assess the pathogenicity of the B. cereus strains; however, most of these take more than 2–3 days to provide confirmatory results. In this study we standardized a one-step cytotoxicity assay using WST-1 (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene disulfonate) and compared with the traditional MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)-based assay for rapid detection of cytotoxic Bacillus spp. using Chinese hamster ovary (CHO) cell line. Crude toxin preparations from 50 isolates of Bacillus spp. were exposed to CHO cell line for 1 h or 24 h and the cytotoxicity was determined by using WST-1 and MTT-based methods. Most B. cereus strains and some strains of other Bacillus species from our collection or from food sources showed comparably high cytotoxicity using either of the methods (P = 0.81); however, WST-1 assay provided results in only 3 h while MTT assay in 44–52 h. A positive correlation (R² = 0.93) between WST-1 and MTT assays strongly suggests that the WST-1-based cytotoxicity assay could be used as an alternative method to MTT assay for rapid (3 h) confirmation of toxigenic Bacillus species in foods prior to their retail distribution or consumption.     

Capsaicin-evoked bradycardia in anesthetized guinea pigs is mediated by endogenous tachykinins

The present study was done to characterize the effects of endogenous tachykinins on heart rate in urethane-anesthetized guinea pigs. Intravenous injection of capsaicin (32 nmol/kg) was used to evoke release of tachykinins and calcitonin gene-related peptide (CGRP) from cardiac sensory nerve fibers. Such injections caused a brief decrease in heart rate (− 37 ± 7 beats/min, n = 6) that was followed by a more prolonged increase (+ 44 ± 10 beats/min). Blood pressure was lowered by − 11 ± 2 mmHg. Bilateral vagotomy did not affect the chronotropic or depressor responses to capsaicin, but atropine (1 µmol/kg) nearly abolished the bradycardic response (− 8 ± 3 beats/min, n = 7). Combined blockade of NK₂ and NK₃ receptors, with SR48968 and SR14801 respectively, also caused a significant reduction of capsaicin-evoked bradycardia (− 14 ± 3 beats/min, n = 4) but did not affect bradycardia evoked by vagal nerve stimulation. Blockade of CGRP receptors eliminated capsaicin-evoked tachycardia and prolonged the capsaicin-evoked bradycardia. These findings suggest that capsaicin-evoked bradycardia in the anesthetized guinea pig is mediated by tachykinins that stimulate cardiac cholinergic neurons. This effect appears to be truncated by the positive chronotropic action of CGRP that is also released from cardiac afferents by capsaicin.     

Glutathion content, rate of apoptosis, and brown adipose tissue mass in rats exposed to different ambient temperatures

1. Amount of glutathione (GSH) in the interscapular brown adipose tissue (IBAT) of cold-adapted (5±1 °C), and 1, 3 and 7 days re-adapted (22±1 °C) rats were examined.

2. The higher amount of GSH was found in the IBAT of cold-adapted rats compared to control animals (22±1 °C).

3. After 1 and 7 days of re-adaptation, the level of GSH was still significantly higher compared to control group.

4. On the third day of re-adaptation, the quantity of GSH in the tissue was significantly lower compared to the cold-adapted group.

5. In cold-adapted animals, intensity of apoptosis in IBAT was lower, whereas in re-adapted animals (3rd and 7th day) it was higher than in control group.

6. This study indicates that GSH depletion is associated with an enhanced apoptosis in IBAT during re-adaptation.

The effect of adaptation to cold and re-adaptation to room temperature on the level of glutathione in rat tissues

1. Glutathione (GSH) was assayed in plasma, liver and IBAT in control (22±1°C) and cold adapted rats (45 days in 5±1°C), and in rats cold adapted and brought back to room temperature after 1, 3, 7 and 15 days.

2. Adaptation to the cold led to reduced GSH in the liver and plasma while the level in intrascapular brown adipose tissue (IBAT) was increased in comparison to controls.

3. On the first day of re-adaptation, plasma GSH was similar to the control level, as were hepatic levels on the first and fifteenth day, but GSH in IBAT remained higher even after fifteen days.

C-fos expression in rat brain during heat stress

Rats, under urethane anesthesia, 0, 20, 40 or 80 min after the start of heat stress (42°C) were sacrificed for determination of c-fos expression in different brain regions. In situ hybridization and immunocytochemistry methods were used, respectively, for determination of c-fos mRNA and protein, respectively. In general, either colon temperature (T_CO), mean arterial pressure (MAP), local cerebral blood flow (CBF) or c-fos expression in different brain regions (including the preoptic area, supraoptic nuclei, paraventricular nuclei, thalamus, amygdala, nucleus tract solitarii, area postrema and ventrolateral medulla) increased at 20–40 min after the start of heat exposure. However, the heatstroke, which appears as profound decreases in both MAP and local CBF and increases in T_CO, was produced 80 min after heat stress. The c-fos expression was heavily induced in all these brain regions after the onset of heatstroke. The data suggest that c-fos expression in rat brain during heatstroke is associated with hyperthermia, arterial hypotension or cerebral ischemia.     

Copper-Zinc Containing and Manganese Containing Superoxide Dismutase in the Ground Squirrel/Citellus Citellus/—THE Effect of Hibernation

The distribution of Copper-Zinc containing and Manganese-containing superoxide dismutase in the liver, kidney, interscapular brown adipose tissue (IBAT) and brain of the ground squirrel, as well as the effect of hibernation, was studied. Activity of both forms of SOD was highest in the liver and lowest in the brain. Activity of the Mn SOD in relation to total SOD was higher in the liver and kidney of the ground squirrel as compared with results reported for other rodents. The highest activity of Mn SOD in relation to total SOD was found in the IBAT and brain (36% and 49%, respectively). Total SOD activity per mg proteins and per g wet mass in IBAT and brain of hibernating animals was increased: for IBAT, p < 0,05 and p < 0,025, respectively; for brain, p < 0,01 and p < 0,025, respectively. Protein content in hibernating ground squirrel was not significantly changed. In the hibernating ground squirrel CuZn SOD activity in IBAT and brain was higher as compared with the active animal (p < 0,025 and p < 0,005, respectively). In the liver and kidney CuZn SOD was not significantly changed during the hibernation. In the liver and brain of the hibernating animals a lower Mn SOD activity was found (p < 0,005 and p < 0,05, respectively).     

Augmented cocaine conditioned place preference in rats pretreated with systemic ghrelin

The physiological mechanism through which food restriction (FR) enhances the biobehavioral actions of psychostimulants is unknown but may involve the gut peptide ghrelin. Plasma levels of ghrelin are increased by FR and reduced by eating. Moreover, systemically administered ghrelin crosses into the brain and is known to augment the locomotor-stimulating effects of cocaine [COC: Wellman et al., 2005]. This study sought to determine whether pretreatment with ghrelin (5 nmol) would enhance the rewarding properties of COC (0.0, 0.312, 0.625, or 1.25 mg/kg i.p.) as measured by conditioned place preference (CPP). Adult male Sprague–Dawley rats were given free access to both sides of a CPP chamber to determine initial side preference. The rats were then confined for 30 min to either their preferred side or non-preferred side on 8 consecutive days. When rats were confined to the least preferred side, each was injected with 0.5 ml (i.p.) of either ghrelin (5 nmol) or saline 1 h before the conditioning trial and then injected (i.p.) with one of the COC doses immediately prior to the conditioning trial. On alternate days, rats were injected with vehicle one hour before and again immediately before the conditioning trial. Place preference scores were computed as the differences in time (min) spent on the least preferred side of the chamber for the pre-test and the postconditioning test, covaried by the initial degree of preference (% time spent on the black side during the pre-test). These analyses indicated a significant interaction between ghrelin pretreatment and COC dose on changes in preference scores. Significantly higher place preference scores were noted for rats treated with either 0.312 or 0.625 mg/kg COC doses, but only when these COC doses were preceded by administration of 5 nmol ghrelin. In contrast, saline pretreated rats exhibited significant CPP at the 1.25 mg/kg COC dose, but the ghrelin pretreated group did not. These results provide partial support for the contention that ghrelin pretreatment can augment the rewarding effects of sub-threshold doses of COC in a CPP procedure. Moreover, these findings are consistent with the view that ghrelin may play a role in the capacity of FR to augment psychostimulant action.     

Phosphoinositide hydrolysis increase by angiotensin-(1--7) in neonatal rat brain

Angiotensin (Ang)-(1–7) is an endogenous peptide hormone of the renin–angiotensin system which exerts diverse biological actions, some of them counterregulate Ang II effects. In the present study potential effect of Ang-(1–7) on phosphoinositide (PI) turnover was evaluated in neonatal rat brain. Cerebral cortex prisms of seven-day-old rats were preloaded with [³H]myoinositol, incubated with additions during 30 min and later [³H]inositol-phosphates (IPs) accumulation quantified. It was observed that PI hydrolysis enhanced 30% to 60% in the presence of 0.01 nM to 100 nM Ang-(1–7). Neither 10 nM [D-Ala⁷]Ang-(1–7), an Ang-(1–7) specific antagonist, nor 10 nM losartan, an angiotensin II type 1 (AT₁) receptor antagonist, blocked the effect of 0.1 nM Ang-(1–7) on PI metabolism. The effect of 0.1 nM Ang-(1–7) on PI hydrolysis was not reduced but it was even significantly increased in the simultaneous presence of [D-Ala⁷]Ang-(1–7) or losartan. PI turnover enhancement achieved with 0.1 nM Ang-(1–7) decreased roughly 30% in the presence of 10 nM PD 123319, an angiotensin II type 2 (AT₂) receptor antagonist. The antagonists alone also enhanced PI turnover. Present findings showing an increase in PI turnover by Ang-(1–7) represent a novel action for this peptide and suggest that it exerts a function in this signaling system in neonatal rat brain, an effect involving, at least partially, angiotensin AT₂ receptors.     

Chronotoxicity of Nedaplatin in Rats

Chronotoxicologic profiles of nedaplatin, a platinum compound, were evaluated in rats maintained under a 12 light/12 dark cycle with light from 07:00 h to 19:00 h. Nedaplatin (5 mg/kg) was injected intravenously, once a week for 5 weeks at 08:00 h or 20:00 h. The suppression of body weight gain and reduction of creatinine clearance were significantly greater with the 20:00 h than 08:00 h treatment. Accumulation of nedaplatin in the renal cortex and bone marrow were also greater with 20:00 h treatment. There were significant relationships between the nedaplatin content in the kidney and bone marrow and degree of injury to each. These results suggest that the nedaplatin-induced toxicity depends on its dosing-time, and it is greater with treatment at 20:00 h, during the active phase. The dosing-time dependency in the accumulation of nedaplatin in the tissue of the organs might be involved in this chronotoxicologic phenomenon.     

Role of inferior olive and thoracic IML neurons in nonshivering thermogenesis in rats

Removal of the midbrain tonic inhibitory mechanism on nonshivering thermogenesis (NST) results in increased temperatures of the interscapular brown adipose tissue (IBAT) and rectum (T(IBAT) and T(rec), respectively) via an enhanced central sympathetic output. Because it is unlikely that neurons (primary) of the midbrain inhibitory mechanism tonically inhibit the IBAT monosynaptically, there must be secondary or tertiary neurons posterior to the midbrain. Such neurons, therefore, may increase their activity during enhanced NST after removal of the midbrain tonic inhibition. The aim of the present experiments was to localize these secondary or tertiary neurons and establish descending neuronal pathway(s) that may project to the major NST effector IBAT. T(IBAT) and T(rec) increases induced by removal of the tonic inhibition by midbrain procaine microinjections were accompanied with appearance of c-Fos-positive neurons in the inferior olive (IO) and the intermediolateral (IML) cell column of the thoracic spinal cord. Electrical stimulation of and L-glutamate microinjections into the IO increased T(IBAT) and T(rec). Midbrain procaine-induced T(IBAT) and T(rec) increases were blocked by electrolytic IO lesions. These results suggest that central thermal signals produced from the lower midbrain are transmitted to IBAT through the IO and IML and that the IO has a role in the central sympathetic functions.     

Ascorbate and H2O2 induced oxidative DNA damage in Jurkat cells

The effect of vitamin C (ascorbate) on oxidative DNA damage was examined by first incubating cells with dehydroascorbate, which boosts the intracellular concentration of ascorbate, and then exposing cells to H₂O₂. Oxidative DNA damage was estimated by the analysis of 5-hydroxy-2′-deoxycytidine (oh⁵dCyd) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (oxo⁸dGuo). The presence of a high concentration of ascorbate (30 mM), compared to the absence of ascorbate in cells, when exposed to H₂O₂ (200 μM), resulted in a remarkable sensitization of oh⁵dCyd from 2.7 ± 0.6 to 40.8 ± 6.1 lesions /10⁶ dCyd (15-fold). In contrast, the level of oxo⁸dGuo increased from 8.4 ± 0.4 to 12.1 ± 0.5 lesions/10⁶ dGuo (50%). The formation of oh⁵dCyd was also observed at lower concentrations of intracellular ascorbate and exogenous H₂O₂. Additional studies showed that replacement of H₂O₂ with tert-butyl hydroperoxide completely abolished damage, and that preincubation with iron and desferroxamine increased and decreased this damage, respectively. The latter studies suggest that a Fenton reaction is involved in the mechanism of damage. In conclusion, we report a novel model system in which ascorbate sensitizes H₂O₂-induced oxidative DNA damage in cells, leading to elevated levels of oh⁵dCyd and oxo⁸dGuo, with a strong bias toward the formation of oh⁵dCyd.     

Gene-specific oxidative lesions in aged rat brain detected by polymerase chain reaction inhibition assay

An exposure of isolated rat brain genomic DNA to oxidative stress in the form of iron salts (Fe²⁺) and ascorbate results in gene-specific DNA lesions detectable by a quantitative polymerase chain reaction (PCR) based assay in which PCR amplification efficiency of the affected genes (e.g. β-actin and p53) is grossly impaired. Such oxidative DNA lesions are prevented by hydroxyl radical scavengers like mannitol (20 mM) and sodium benzoate (20 mM) or by the antioxidant enzyme catalase (50 μg/ml) present in the incubation mixture during exposure to Fe²⁺ and ascorbate. When brain DNA isolated from young (4-6 months of age) and aged (20-24 months of age) rats are analyzed similarly by the PCR based method, the amplification levels of β-actin and p53 genes are noticeably decreased in the case of aged rat indicating an accumulation of gene-specific DNA lesions during brain aging.     

Developmental methylation of the regulatory region of HoxB5 gene in mouse correlates with its tissue-specific expression

We have studied the dynamics of de novo CpG methylation in the regulatory region of one of the homeobox gene HoxB5 during mouse development by sodium bisulfite sequencing. Methylation pattern was examined at embryonic day 18.5 and adult in kidney and spleen while in the liver the same exercise has been done in 11.5 dpc, 18.5 dpc, 5 dpp and in adult. In the liver at 11.5 dpc, all the 47 contiguous sites (including a CpG island from 2035 to 2330 bp) at 5' regulatory region of HoxB5 were unmethylated. Random methylation commences from 18.5 dpc and continues in 5 dpp and in the adult. In the kidney at 18.5 dpc, 26 CpGs were examined (excluding the CpG island region) and all of them were unmethylated but the fetal spleen had at least a few sites considerably methylated. In the adult there was a low level methylation in the kidney, on the other hand, in the spleen, all the CpGs were methylated except a few sites and certain sites were totally methylated. Thus in the adult, the level of methylation was much higher than in the fetal stage. On the other hand semi-quantitative RT-PCR revealed that the extent of expression of HoxB5 was higher in embryonic stages than in the adult. Thus HoxB5 is a good paradigm to support that the developmental methylation of HoxB5 and its expression pattern show an inverse correlation.     

Disturbance of circadian rhythms in analgosedated intensive care unit patients with and without craniocerebral injury

Melatonin, cortisol, heart rate, blood pressure, spontaneous motor activity, and body temperature follow stable circadian rhythms in healthy individuals. These circadian rhythms may be influenced or impaired by the loss of external zeitgebers during analgosedation, critical illness, continuous therapeutic intervention in the intensive care unit (ICU), and cerebral injury. This prospective, observational, clinical study examined 24 critically ill analgo-sedated patients, 13 patients following surgery, trauma, or acute respiratory distress (ICU), and 11 patients with acute severe brain injury following trauma or cerebral hemorrhage (CCI). Blood samples for the determination of melatonin and cortisol were obtained from each patient at 2 h intervals for 24 h beginning at 18:00 h on day 1 and ending 16:00 h on day 2. Blood pressure, heart rate, body temperature, and spontaneous motor activity were monitored continuously. Level of sedation was assessed using the Ramsey Sedation Scale. The severity of illness was assessed using the APACHE-II-score. The time series data were analyzed by rhythm analysis with the Chronos-Fit program, using partial Fourier series with up to six harmonics. The 24 h profiles of all parameters from both groups of patients were greatly disturbed/abolished compared to the well-known rhythmic 24 h patterns in healthy controls. These rhythm disturbances were more pronounced in patients with brain injury. The results of this study provide evidence for a pronounced disturbance of the physiological temporal organization in ICU patients. The relative contribution of analgosedation and/or brain injury, however, is a point of future investigation.