Dosing‐Time–Dependent Differences in Lipopolysaccharide‐Induced Liver Injury in Rats

Dosing‐time–dependent differences in lipopolysaccharide (LPS)‐induced liver injury were examined in rats housed under a 12 h light∶dark (LD) cycle. LPS (5 mg/kg) was intravenously injected into different groups of rats at 2, 14, or 20 h after light on (HALO). Elevations in serum liver enzymes after 14 HALO were significantly greater than those after 2 HALO. These parameters were lower in rats given LPS at 20 HALO, compared to 14 HALO. The number of polymorphonuclear cells (PMN) in the liver and the amount of hepatic myeloperoxidase activity, which reflects the number of PMN in liver tissues, was significantly greater in the 14 than in the 2 HALO group. In addition, hepatic interleukin‐6 (IL‐6) production in the 14 HALO group was enhanced compared to that in the 2 HALO trial. These results suggest that LPS‐induced liver injury is greater during the early active than during the early resting period. Dosing‐time–dependent variation in the accumulation of PMN in the liver and, potentially, subsequent IL‐6 production in liver tissues might be involved in this phenomenon.     

Changes of α-Glycerophosphate Dehydrogenase Activity in Fatty Liver of Rats by Amino Acid Imbalance

An aminopeptidase was purified from an aqueous extract of mullet roe in the presence of 2-mercaptoethanol by fractionation with ammonium sulfate and column chromatography on DEAE-cellulose and Sephadex G-200. The molecular weight of the enzyme was 184,000 by gel filtration, and the enzyme appeared to consist of two homogenous subunits. The optimal pH and optimal temperature for activity were 7.4 and 45°C, respectively. Puromycin, p-chloromercuribenzoic acid, and o-phenanthroline inhibited the enzyme n on-competitively (their Ki = 1.34 μm, 0.113mm and 0.145 mm, respectively), while 2-mercaptoethylamine was competitive (Ki = 0.056 mm). The enzyme was also inhibited by l-amino acids, in particular glutamic acid. The enzyme could hydrolyze a variety of α-aminoacyl β-naphthylamides and was most active on l-alanyl-β-naphthylamide. Judging from these properties, the mullet roe aminopeptidase resembles soluble alanyl amino-peptidase [EC 3.4.11.14].     

Age‐related Changes in the Circadian and Homeostatic Regulation of Human Sleep

The reduction of electroencephalographic (EEG) slow‐wave activity (SWA) (EEG power density between 0.75–4.5 Hz) and spindle frequency activity, together with an increase in involuntary awakenings during sleep, represent the hallmarks of human sleep alterations with age. It has been assumed that this decrease in non‐rapid eye movement (NREM) sleep consolidation reflects an age‐related attenuation of the sleep homeostatic drive. To test this hypothesis, we measured sleep EEG characteristics (i.e., SWA, sleep spindles) in healthy older volunteers in response to high (sleep deprivation protocol) and low sleep pressure (nap protocol) conditions. Despite the fact that the older volunteers had impaired sleep consolidation and reduced SWA levels, their relative SWA response to both high and low sleep pressure conditions was similar to that of younger persons. Only in frontal brain regions did we find an age‐related diminished SWA response to high sleep pressure. On the other hand, we have clear evidence that the circadian regulation of sleep during the 40 h nap protocol was changed such that the circadian arousal signal in the evening was weaker in the older study participants. More sleep occurred during the wake maintenance zone, and subjective sleepiness ratings in the late afternoon and evening were higher than in younger participants. In addition, we found a diminished melatonin secretion and a reduced circadian modulation of REM sleep and spindle frequency—the latter was phase‐advanced relative to the circadian melatonin profile. Therefore, we favor the hypothesis that age‐related changes in sleep are due to weaker circadian regulation of sleep and wakefulness. Our data suggest that manipulations of the circadian timing system, rather than the sleep homeostat, may offer a potential strategy to alleviate age‐related decrements in sleep and daytime alertness levels.     

Exposure to T‐Cycles of 22 and 23 h during Lactation Modifies the Later Dissociation of Motor Activity and Temperature Circadian Rhythms in Rats

Early environmental conditions may affect the development and manifestation of circadian rhythms. This study sought to determine whether the maintenance of rats under different T‐cycles during lactation influences the subsequent degree of dissociation of the circadian rhythms of motor activity and core body temperature. Two groups of 22 day‐old Wistar rats were kept after weaning under T‐cycles of 22 h (T22) or 23 h (T23) for 70 days. Subsequently, they were kept in constant darkness (DD). Half of the animals in each group were born and reared under these experimental conditions, while the other half were reared until weaning under 24 h LD cycles (T24). Rats transferred from T24 to T22 or T23 showed two circadian components in motor activity and temperature, one entrained by light and the other free‐running. In T22, there was also desynchronization between temperature and motor activity. Rats submitted to T23 from birth showed higher stability of the 23 h component than rats transferred from T24 to T23 after weaning. However, in comparison to rats born under T24 and subsequently changed to T22, animals submitted to T22 from birth showed shorter values of the period of the non‐light‐dependent component during T22, more aftereffects when transferred to DD, and a lack of desynchronization between motor activity and temperature. The results suggest that T‐cycles in the early environment may modify overt rhythms by altering the internal coupling of the circadian pacemaker.     

Gender Dependency of Circadian Blood Pressure and Heart Rate Profiles in Spontaneously Hypertensive Rats: Effects of Beta‐Blockers

This study investigated (i) blood pressure (BP), heart rate (HR), and their relation to urinary NOx and eNOS protein expression in male and female spontaneously hypertensive rats (SHR), as well as (ii) gender‐dependent cardiovascular effects of nebivolol (NEB) in comparison to metoprolol (MET) in SHR. BP and HR were measured telemetrically after a single intraperitoneal application of NEB or MET at 07.00 and 19.00 h in male rats and at 19.00 h in proestrus female rats. The two β‐blockers varied in time of decreasing BP and HR and also in duration. In males, MET decreased BP and HR for few hours exclusively when applied at the onset of the activity phase (i.e., at 19.00 h), while after its application at 07.00 h, BP and HR were unchanged. In females, MET also caused a short‐lasting BP and HR reduction, with the effect being more pronounced than in males. In males, NEB at either dosing time decreased HR and BP to a greater extent than did MET. This effect was evident both during the activity and rest periods and persisted for at least five days. In females, NEB provoked a similar, but more pronounced, effect on BP and HR in comparison to males. These findings demonstrate that significant gender‐dependent differences in the circadian profile of BP and HR exist. BP and urinary NOx as well as eNOS expression are inversely correlated, and the cardiovascular effects of NEB and MET vary, depending on the time of application as well as gender.     

Inhibition of Calcium/Calmodulin-Dependent Protein Kinase IIα Suppresses Oxidative Stress in Cerebral Ischemic Rats Through Targeting Glucose 6-Phosphate Dehydrogenase

Ischemic stroke is a leading cause of mortality and morbidity worldwide, and oxidative stress plays a significant role in the ischemia stage and reperfusion stage. Previous studies have indicated that both calcium/calmodulin-dependent protein kinase II (CaMKII) and glucose 6-phosphate dehydrogenase (G6PD) are involved in the oxidative stress. Thus, the aim of this study was to investigate the roles of CaMKIIα, an important isoform of CaMKII, and G6PD in a rat model of middle cerebral artery occlusion (MCAO). Intracerebroventricular injection of small interfering ribonucleic acid (siRNA) for CaMKIIα was performed at 48 h pre-MCAO surgery. Immunofluorescence Staining and western blot were performed to detect the expression of p-CaMKIIα and G6PD in the cortices. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining was performed to investigate the infarct volume. In addition, neurological deficit, reactive oxygen species (ROS), ratio of reduced-to-oxidized glutathione (GSH/GSSG) and ratio of reduced-to-oxidized oxidized nicotinamide adenine dinucleotide phosphate (NADPH/NADP⁺) were assessed. The results indicated that both p-CaMKIIα and G6PD were widely located in the neurons and astrocytes, and their expression was gradually increased in the cortices after MCAO, which was accompanied by increased level of ROS and decreased levels of GSH/GSSG and NADPH/NADP⁺. However, after treatment with siRNA for CaMKIIα, p-CaMKIIα expression was decreased and G6PD expression was increased. Moreover, inhibition of CaMKIIα improved the neurological deficit, reduced the infarct volume, decreased the level of ROS and increased the levels of GSH/GSSG and NADPH/NADP⁺. The results suggested that CaMKIIα inhibition exerted neuroprotective effects through regulating G6PD expression, which provides a new target for prevention and treatment of stroke.

     

Anti-Apoptotic Effects of 3,3’,5-Triiodo-L-Thyronine in the Liver of Brain-Dead Rats

Background

Thyroid hormone treatment in brain-dead organ donors has been extensively studied and applied in the clinical setting. However, its clinical applicability remains controversial due to a varying degree of success and a lack of mechanistic understanding about the therapeutic effects of 3,3’,5-Triiodo-L-thyronine (T₃). T₃ pre-conditioning leads to anti-apoptotic and pro-mitotic effects in liver tissue following ischemia/reperfusion injury. Therefore, we aimed to study the effects of T₃ pre-conditioning in the liver of brain-dead rats.

Methods

Brain death (BD) was induced in mechanically ventilated rats by inflation of a Fogarty catheter in the epidural space. T₃ (0.1 mg/kg) or vehicle was administered intraperitoneally 2 h prior to BD induction. After 4 h of BD, serum and liver tissue were collected. RT-qPCR, routine biochemistry, and immunohistochemistry were performed.

Results

Brain-dead animals treated with T₃ had lower plasma levels of AST and ALT, reduced Bax gene expression, and less hepatic cleaved Caspase-3 activation compared to brain-dead animals treated with vehicle. Interestingly, no differences in the expression of inflammatory genes (IL-6, MCP-1, IL-1β) or the presence of pro-mitotic markers (Cyclin-D and Ki-67) were found in brain-dead animals treated with T₃ compared to vehicle-treated animals.

Conclusion

T₃ pre-conditioning leads to beneficial effects in the liver of brain-dead rats as seen by lower cellular injury and reduced apoptosis, and supports the suggested role of T₃ hormone therapy in the management of brain-dead donors.     

Circadian rhythm of activity and sleep‐wakefulness in elderly institutionalized persons

Abstract

The circadian rhythms of blood pressure (BP) and heart rate (HR) were documented in 30 patients for a 24‐hour period before and during the 24 hours that included unilateral surgery for senile cataract or retinal detachment. The patients were premedicated with diazepam. Anaesthesia was induced at a fixed time (09.00) in all patients with thiopentone, and muscle relaxation was with pancuronium. Maintenance was with enflurane in 15 patients and with fentanyl and droperidol in the rest. Though the intraoperative changes in haemodynamic parameters were dissimilar with the two types of maintenance agents, but both types had a similar effect on the circadian rhythms of blood pressure and heart rate. Whereas preoperatively the BP and HR circadian rhythms were nearly in phase, with their peaks in the late morning to early afternoon, the postoperative rhythms underwent a dissociation to a phase shift in the BP 24‐h pattern. The phase effect may be hypothetically attributed to direct pharmacological actions or to masking effects.     

Effects of Three‐Hour Restricted Food Access during the Light Period on Circadian Rhythms of Temperature,Locomotor Activity,and Heart Rate in Rats

The effects of food on biological rhythms may influence the findings of chronopharmacological studies. The present study evaluated the influence of a restricted food access during the rest (light) span of nocturnally active Wistar rats on the 24 h time organization of biological functions in terms of the circadian rhythms of temperature (T), heart rate (HR), and locomotor activity (LA) in preparation for subsequent studies aimed at evaluating the influence of timed food access on the pharmacokinetics and pharmacodynamics of medications. Ten‐wk‐old male Wistar rats were housed under controlled 12:12 h light:dark (LD) environmental conditions. Food and water were available ad libitum, excepted during a 3 wk period of restriction. Radiotelemetry transmitters were implanted to record daily rhythms in T, HR, and LA. The study lasted 7 wk and began after a 21‐d recovery span following surgery. Control baseline data were collected during the first wk (W1). The second span of 3 wk duration (W2 to W4) consisted of the restricted feeding regimen (only 3 h access to food between 11:00 and 14:00 h daily) during the L (rest span) under 12:12 h LD conditions. The third period of 3 wk duration (W5 to W7) consisted of the recovery span with ad libitium normal feeding. Weight loss in the amount of 5% of baseline was observed during W1 with stabilization of body weight thereafter during the remaining 2 wk of food restriction. The 3 h restricted food access during the L rest span induced a partial loss of circadian rhythmicity and the emergence of 12 h rhythms in T, HR, and LA. Return to ad libitum feeding conditions restored circadian rhythmicity in the manner evidenced during the baseline control span. Moreover, the MESORS and amplitudes of the T, HR, and LA 24 h patterns were significantly attenuated during food restriction (p<0.001) and then returned to initial values during recovery. These changes may be interpreted as a masking effect, since T, HR, and LA are known to directly react to food intake. The consequences of such findings on the methods used to conduct chronokinetic studies, such as the fasting of animals the day before testing, are important since they may alter the temporal structure of the organism receiving the drug and thereby compromise findings.     

Drotaverine Inhibitor of PDE4: Reverses the Streptozotocin Induced Alzheimer’s Disease in Mice

Neurochemical Research - Alzheimer’s disease (AD) is a progressive neurodegenerative disease associated with decline in memory and cognitive impairments. Phosphodiesterase IV (PDE4) protein,...     

Circadian Rhythm of the Liver of Male Rats Dosed with Phenobarbital—I. Organ Weight,Cellular Structures,Glycogen Contents and Mitotic Activity

The circadian rhythm of the liver, namely organ weight, cellular structures (by light-microscopy), glycogen content (by periodic acid-Schiff (PAS) reaction) and mitotic activity, was studied in 166 male Sprague-Dawley rats orally treated daily at 0800-0900 with 70 (study 1) or 50 (study 2) mg/kg phenobarbital (PB) for 7days. Thereafter, eight (study 1) or five (study 2) rats each were studied at 4-hr intervals at 1000, 1400, 1800, 2200, 0200, 0600 and 1000 through till the following day. The lighting schedule in the colony was 12:12, light:dark (light from 0600 to 1800). The liver weight was raised in PB-treated rats at all times of the day compared to controls and showed a distinct circadian rhythm with a peak at 1000 and a minimum at 2200 in PB-treated rats and the controls. The circadian rhythm of cellular structures was closely related to the hepatic glycogen content which was in good agreement with the controls, but at 1400 and 1800 the glycogen particles were more distinctly diminished in the enlarged centrilobular hepatocytes of PB-treated rats. The mitotic activity of hepatocytes was markedly increased in rats treated with PB but showed the same circadian rhythm as controls with a peak at 1000.     

The Function of Circadian RNA‐Binding Proteins and Their cis‐Acting Elements in Microalgae

An endogenous clock regulates the temporal expression of genes/mRNAs that are involved in the circadian output pathway. In the bioluminescent dinoflagellate Gonyaulax polyedra circadian expression of the luciferin‐binding protein (LBP) is controlled at the translational level. Thereby, a clock‐controlled RNA‐binding protein, called circadian controlled translational regulator (CCTR), interacts specifically with an UG‐repeat, which is situated in the lbp 3^′ UTR. Its binding activity correlates negatively with the amount of LBP during a circadian cycle. In the green alga Chlamydomonas reinhardtii, a clock‐controlled RNA‐binding protein (CHLAMY 1) was identified, which represents an analog of the CCTR from the phylogenetically diverse alga G. polyedra. CHLAMY 1 binds specifically to the 3^′ UTRs of several mRNAs and recognizes them all via a common cis‐acting element, composed of at least seven UG‐repeats. The binding strength of CHLAMY 1 is strongest to mRNAs, whose products are key components of nitrogen metabolism resulting in arginine biosynthesis as well as of CO₂ metabolism. Since temporal activities of processes involved in nitrogen metabolism have an opposite phase than CHLAMY 1 binding activity, the protein might repress the translation of the cognate mRNAs.     

Circadian Activity Rhythms and Phase‐Shifting of Cultured Neurons of the Rat Suprachiasmatic Nucleus

The mammalian suprachiasmatic nucleus (SCN) is the major endogenous pacemaker that coordinates various daily rhythms including locomotor activity and autonomous and endocrine responses, through a neuronal and humoral influence. In the present study we examined the behavior of dispersed individual SCN neurons obtained from 1‐ to 3‐day‐old rats cultured on multi‐microelectrode arrays (MEAs). SCN neurons were identified by immunolabeling for the neuropeptides arginine‐vasopressin (AVP) and vasoactive intestinal polypeptide (VIP). Single SCN neurons cultured at low density onto an MEA can express firing rate patterns with different circadian phases. In these cultures we observed rarely synchronized firing patterns on adjacent electrodes. This suggests that, in cultures of low cell densities, SCN neurons function as independent pacemakers. To investigate whether individual pacemakers can be influenced independently by phase‐shifting stimuli, we applied melatonin (10 pM to 100 nM) for 30 min at different circadian phases and continuously monitored the firing rate rhythms. Melatonin could elicit phase‐shifting responses in individual clock cells which had no measurable input from other neurons. In several neurons, phase‐shifts occurred with a long delay in the second or third cycle after melatonin treatment, but not in the first cycle. Phase‐shifts of isolated SCN neurons were also observed at times when the SCN showed no sensitivity to these phase‐shifting stimuli in recordings from brain slices. This finding suggests that the neuronal network plays an essential role in the control of phase‐shifts.     

NTPDase and 5′-Nucleotidase Activities in Synaptosomes of Hippocampus and Serum of Rats Subjected to Homocysteine Administration

Patients with homocystinuria, an inborn error of metabolism, present neurological dysfunction and commonly experience frequent thromboembolic complications. The nucleoside triphosphate diphosphohydrolase (NTPDase) and 5'-nucleotidase enzymes regulate the nucleotide/nucleoside ratio in the central nervous system and in the circulation and are thought to be involved in these events. Thus, the current study investigated the effect of homocysteine administration on NTPDase and 5'-nucleotidase activities, in the synaptosomal fraction of rat hippocampus, and on nucleotidase activities in rat serum. Twenty-nine-day-old Wistar rats were divided in two groups: group I (control), animals received 0.9% saline; group II (homocysteine-treated), animals received one single subcutaneous injection of homocysteine (0.6 micromol/g). Rats were killed 1 h after the injection. NTPDase and 5'-nucleotidase activities from brain and serum were significantly increased in the homocysteine-treated group. Results show that, in hippocampus, ATP and ADP hydrolysis increased by 20.5% and 20%, respectively, and AMP hydrolysis increased by 48%, when compared to controls. In serum, ATP and ADP hydrolysis increased 136% and 107%, respectively, and AMP hydrolysis increased 95%, in comparison to controls. The current data strongly indicate that in vivo homocysteine administration alters the activities of the enzymes involved in nucleotide hydrolysis, both in the central nervous system and in the serum of adult rats.     

Characteristics of Triose Phosphate／Phosphate Translocator from Wheat and Its Role in the Distribution of Assimilates

In plants, triose phosphate/phosphate translocator (TPT) is the first regulation point forpartitioning of photosynthate between source and sink. Studies on the characteristic of TPT and itsregulation on the distribution of assimilates are critical for improving the utilization rate of photosyntheticassimilates. Chloroplasts with intactness of more than 91% and high purity were isolated from wheat( Triticurn aestivurn L. cv. Jing 411) leaves. Analysis of SDS-PAGE and labeling with an irreversible specificinhibitor, [H3]2^-DIDS (4, 4‘-diisothiocyano-2, 2‘-stilbenedisulfonate, DIDS) demonstrated that wheat TPTwas a chloroplast membrane protein with a 35 kD molecular weight, which comprised about 15% of the totalmembrane proteins of chloroplasts. Western blotting analysis showed that wheat TPT is uniquelydistributed in the envelope membrane of chloroplasts, but not detected in the membranes of vacuoles andmitochondria. The silicone-oil-layer centrifugation system was employed to study the kinetic properties ofTPT. The results showed that the maximal transport activity of TPT was the highest for dihydroxyacetonephosphate (DHAP)/inorganic phosphate (Pi), then for phosphoenolpyruvate (PEP)/Pi and glucose-6-phosphate (G6P)/Pi. The Km value of TPT was the lowest for DHAP, followed by Pi, PEP and G6P,therefore the most preferred substrate of TPT is DHAP. The transport of wheat TPT to DHAP was stronglyinhibited by DIDS with a degree of 95%. Inhibition of TPT transport activity led to an obvious accumulationof starch in chloroplasts, therefore the TPT protein of wheat controls the export of TP out of chloroplastsinto cytosol. Except for the need of participating in the Calvin cycle, the ratio of TP exported out ofchloroplast to the one used for synthesizing starch was at least 93.6:6.4. The TPT protein from wheat hasmuch high transport efficiency, which plays an important role in the regulation of the distribution ofassimilates in wheat chloroplasts.     

Effects of a Seven‐Day Continuous Infusion of Ropivacaine on Circadian Rhythms in the Rat

The present study was conducted to evaluate the effect of a 7 d continuous infusion of ropivacaine on the 24 h rhythms of body temperature, heart rate, and locomotor activity. After an initial 7 d baseline, rats were randomly divided into two groups of 4 rats each to receive ropivacaine or saline via an osmotic pump for 7 consecutive days. The pumps were removed thereafter and observed during a 7 d recovery span. The studied circadian rhythms were measured by radiotelemetry throughout each of the 7 d periods. An additional group of 4 rats was studied under the same experimental conditions to assess the plasma levels of ropivacaine on days 3 and 8 following pump implantation. Our results indicate that ropivacaine does not induce loss of the circadian rhythms of body temperature, heart rate, or locomotor activity; a prominent period of 24 h was found for all variables in all animals, before, during, and after ropivacaine treatment. However, ropivacaine treatment did modify some characteristics of the rhythms; it increased the MESOR (24 h mean) of the heart rate and locomotor activity rhythms and advanced the acrophase (peak time) of the locomotor activity circadian rhythm. The present study indicates that the circadian rhythms of heart rate and locomotor activity are modified after continuous infusion of ropivacaine, which is of particular interest, given the potential cardiotoxicity of this local anesthetic agent.     

Effects of Oxidized Cooking Oil and α-Lipoic Acid on Liver Antioxidants: Enzyme Activities and Lipid Peroxidation in Rats Fed a High Fat Diet

The effects of administration of oxidized rapeseed oil and α-lipoic acid on activities of hepatic antioxidant enzymes and lipid peroxidation were studied in laboratory rats. There was an increase of the activities of superoxide dismutase, glutathione peroxidase, glutathione reductase, and glucose-6-phosphate dehydrogenase in rats fed a high fat diet to which 10% oxidized oil was added. Administration of α-lipoic acid resulted in a decrease of the activities of these enzymes. Addition of oxidized oil also resulted in increased production of oxygen radicals, evidenced by elevated malondialdehyde production. Such effect was counteracted by administration of α-lipoic acid.     

Effect of NO Synthase Inhibition on Cardiovascular Circadian Rhythms in Wild‐Type and eNOS‐Knock‐Out Mice

Cardiovascular functions (blood pressure [BP], heart rate [HR]) were radiotelemetrically studied in endothelial nitric oxide synthase (NOS) knock‐out mice (eNOS‐/‐) and their wild type C57BL/6 (WT) controls. Studies were performed with and without inhibition of the NOS with the non‐specific inhibitor N^ω‐Nitro‐L‐Arginin‐Methylester (L‐NAME). Six eNOS‐/‐and five WT mice, kept under a light:dark schedule of 12:12 h (lights on 07:00 h), were treated with L‐NAME in tap water containing different concentrations (94, 282, and 940 mg/kg) each for three days. Under control conditions, the eNOS‐/‐mice are mildly hypertensive in comparison to WT. L‐NAME increased systolic [SBP] and diastolic [DBP] blood pressures in WT mice to the levels of eNOS‐/‐mice after two days of L‐NAME application with no dose‐dependency, whereas L‐NAME had no effects on SBP and DBP in eNOS‐/‐mice. In neither mouse strain were the circadian rhythms in BP and HR affected by drug treatment. The similarity of the 24 h BP profiles in eNOS‐/‐and L‐NAME‐treated WT mice support the notion that only the enothelial NOS and not other NOS isoenzymes are of importance for hypertension in the knock‐out mouse strain.