Positive impact of levetiracetam on emotional learning and memory in naive mice

AimsThe effect of an antiepileptic drug on cognitive function is of primary importance with respect to the patient's quality of life. Levetiracetam (LEV) is a novel antiepileptic drug used to treat epilepsy, but its effects on spatial and emotional learning and memory are not yet well understood. The goal of our study was to establish the effects of LEV (17 and 54 mg/kg, intraperitoneally (IP)) on spatial memory retrieval in the Morris water maze test and on acquisition and memory formation in the passive avoidance (PA) test in naive mice.Main methodsThe subjects were adult male BALB/c mice. Spatial learning and memory was established with the Morris water maze (MWM) test. The ‘time spent in escape platforms quadrant’ and the ‘distance to platform’ analyses were measured using a video tracking system to determine spatial memory function. Emotional learning and memory were determined with a one-trial, step-through passive avoidance test.Key findingsIn the MWM test, LEV (17 and 54 mg/kg) neither affected the time spent in the target quadrant nor altered the distance to platform. Moreover, LEV had no effect on swim speed. In the PA task, LEV (17 and 54 mg/kg) significantly prolonged retention latency.SignificanceOur results indicate that LEV did not alter spatial memory retrieval in the MWM test, but it did show some ameliorating effects on acquisition and memory formation in the PA test in naive mice.     

Medial-frontal cortex hypometabolism in chronic phencyclidine exposed rats assessed by high resolution magic angle spin 11.7 T proton magnetic resonance spectroscopy

BackgroundProton magnetic resonance spectroscopy (¹H-MRS) clinical studies of patients with schizophrenia document prefrontal N-acetylaspartate (NAA) reductions, suggesting an effect of the disease or of antipsychotic medications. We studied in the rat the effect of prolonged exposure to a low-dose of the NMDA glutamate receptor antagonist phencyclidine (PCP) on levels of NAA, glutamate and glutamine in several brain regions where metabolite reductions have been reported in chronically medicated patients with schizophrenia.MethodsTwo groups of ten rats each were treated with PCP (2.58 mg/kg/day) or vehicle and were sacrificed after 1 month treatment. Concentrations of neurochemicals were determined with high resolution magic angle (HR-MAS) ¹H-MRS at 11.7 T in ex vivo punch biopsies from the medial frontal and cingulate cortex, striatum, nucleus accumbens, amygdala and ventral hippocampus.ResultsPCP treatment reduced NAA, glutamate, glycine, aspartate, creatine, lactate and GABA in medial frontal cortex. In the nucleus accumbens, PCP reduced levels of NAA, aspartate and glycine; similarly aspartate and glycine were reduced in the striatum. Finally the amygdala and hippocampus had elevations in glutamine and choline, respectively.ConclusionsLow-dose PCP in rats models prefrontal NAA and glutamate reductions documented in chronically-ill schizophrenia patients. Chronic glutamate NMDA receptor blockade in rats replicates an endophenotype in schizophrenia and may contribute to the prefrontal hypometabolic state in schizophrenia.     

Genistein attenuates D-galactose-induced oxidative damage through decreased reactive oxygen species and NF-κB binding activity in neuronal PC12 cells

AimsWe investigated the mechanism of D-galactose (DG)-induced oxidative damage and the neuroprotective action of genistein in PC12 cells.Main methodsPC12 cells were treated with 40 mM DG dissolved in medium containing 85% RPMI1640, 10% HBS and 5% FBS with or without genistein. We measured the protein expression of β-amyloid (Aβ), advanced glycation end products (AGEs), IκB-α and manganese-superoxide dismutase (MnSOD) by western blotting, intracellular reactive oxygen species (ROS) by 2, 7-dichlorofluorescin-diacetate, and the binding activity of nuclear factor kappa B (NF-κB) by electrophortic mobility shift assay.Key findingsDG (40 mM) completely retarded cell growth after incubation for 72 h, and this effect was not due to osmotic changes, as 40 mM mannitol had no effect. Mechanistically, we found that DG increased intracellular ROS starting at 4 h and increased Aβ and AGEs at 24 h. DG treatment for 24 h also increased the binding activity of NF-κB but strongly decreased the expression of IκB-α protein. Furthermore, DG treatment for 48 h increased MnSOD protein expression. All these effects of DG were effectively inhibited by genistein (0.5–10 μM).SignificanceThe present study indicates that the protection of genistein against DG-induced oxidative stress in PC12 cells, and the effect is likely mediated by decreased intracellular ROS and binding activity of NF-κB.     

Effect of different propranolol doses on skeletal structural and mechanic efficiency in an animal model of growth retardation

ObjectiveTo assess in a growth retardation (GR) model the impact of different propranolol (P) doses on anthropomorphometric and biomechanical variables of the appendicular skeleton.Materials and methodsTwenty-one day-old male Wistar rats were divided into the following groups: control (C), C + P3.5 (CP3.5); C + P7 (CP7); C + P10.5 (CP10.5); C + P14 (CP14); ED, ED + P3.5 (EDP3.5); ED + P7 (EDP7); ED + P10.5 (EDP10.5), and ED + P14 (EDP14). Control animals with/without P were fed a rodent diet ad libitum. GR rats with/without P were given 80% of the same diet per 100 g body weight for 4 weeks (T4). Propranolol 3.5, 7, 10.5, and 14 mg/kg/day was intraperitoneally injected 5 days/week for 4 weeks to the CP3.5 and EDP3.5; CP7 and EDP7; CP10.5 and EDP10.5, and CP14 and EDP14 groups respectively.ResultsAt T4, energy restriction had negative effects upon overall growth, femur, and its mechanical competence. Propranolol improved bone rigidity in GR animals at doses of 7 and 10.5 mg/kg/day, with a maximum response at 7 mg/kg/day.ConclusionsPropranolol 7 mg/kg/day would be the most effective dose for modeling incorporation of bone, as shown by the increased skeletal structural and mechanic efficiency in this animal model of growth retardation. Such effect may result from maintenance of mechanosensor viability, changes in its sensitivity, the biomechanical reference point and/or effector response in GR rats.     

Effects of different physical training protocols on ventricular oxidative stress parameters in infarction-induced rats

AimPhysical exercise is important in the prevention and treatment of cardiovascular diseases. Nevertheless, controversy remains around type and intensity of effort required for significant biochemical protective changes. This study investigates two exercise protocols on ventricular oxidative parameters in rats post-infarction.Main methodsThirty-six 2-month-old male Wistar rats were divided in two groups (n = 18): Sham and acute myocardial infarction (AMI) conducted by blocking the coronary artery. Thirty days after AMI, animals were divided in 6 subgroups (n = 6): sham, sham + continuous training (60 min), sham + interval training, AMI, AMI + continuous training, and AMI + interval training. Training was conducted in water (30–32 °C) 5 times a week for 6 weeks. Animals were sacrificed 48 h after the last exercise routine. Left ventricles were used for oxidative stress analyses (antioxidant enzyme activity and level, oxidative damage) and HIF1α and cit c oxidase expression.Key findingsAfter AMI, both exercise models decreased superoxide levels significantly. Training routines did not alter SOD expression and activity, though CAT expression increased with continuous training and GPX level diminished in both training groups, which coincided with the increase in GPX activity. Lipid damage decreased only in the continuous training group, while protein damage decreased only in the interval training group. Cytochrome C increased in both groups, while HIF-1 α dropped significantly after both exercise protocols.SignificanceSignificant improvement occurred in myocardium redox status in rats challenged with AMI after different training routines. However, continuous training seems to be more efficient in improving the parameters analyzed.     

Role of the central amygdala GABA-A receptors in morphine state-dependent memory

AimsIn the present experiments, the effects of bilateral microinjections of the GABA-A receptor agonist and/or antagonist into the central amygdala (CeA) on morphine state-dependent memory were examined.Main methodsIn order to assess memory retrieval, a step-through passive avoidance task was used in adult male Wistar rats.Key findingsSubcutaneous (s.c.) administration of morphine (5 and 7.5 mg/kg) immediately after training (post-training) decreased the memory retrieval. Pre-test administration of the opioid (7.5 mg/kg) also induced amnesia. The response induced by post-training morphine (7.5 mg/kg) was significantly reversed by pre-test administration of the drug (5 and 7.5 mg/kg), indicating morphine state-dependent memory. Pre-test intra-CeA microinjection of muscimol, a GABA-A receptor agonist (0.01, 0.02 and 0.03 µg/rat) reduced morphine state-dependent memory. However, the same doses of muscimol by itself had no effect on memory retrieval. Furthermore, pre-test intra-CeA microinjection of bicuculline, a GABA-A receptor antagonist by itself did not alter memory retrieval. The antagonist also did not change post-training morphine (7.5 mg/kg)-induced amnesia, but in combination with a lower dose of morphine (0.5 mg/kg), improved memory performance. Moreover, muscimol's ability to interfere with morphine state-dependent memory was reversed by co-injection of bicuculline.SignificanceThe results suggest that GABA-A receptor mechanism of the CeA may influence morphine state-dependent memory.     

μ1- and 5-HT1-dependent mechanisms in the anterior pretectal nucleus mediate the antinociceptive effects of retrosplenial cortex stimulation in rats

AimThis study examines if injection of cobalt chloride (CoCl₂) or antagonists of muscarinic cholinergic (atropine), μ₁-opioid (naloxonazine) or 5-HT₁ serotonergic (methiothepin) receptors into the dorsal or ventral portions of the anterior pretectal nucleus (APtN) alters the antinociceptive effects of stimulating the retrosplenial cortex (RSC) in rats.Main methodChanges in the nociceptive threshold were evaluated using the tail flick or incision pain tests in rats that were electrically stimulated at the RSC after the injection of saline, CoCl₂ (1 mM, 0.10 μL) or antagonists into the dorsal or ventral APtN.Key findingsThe injection of CoCl₂, naloxonazine (5 μg/0.10 μL) or methiothepin (3 μg/0.10 μL) into the dorsal APtN reduced the stimulation-produced antinociception from the RSC in the rat tail flick test. Reduction of incision pain was observed following stimulation of the RSC after the injection of the same substances into the ventral APtN. The injection of atropine (10 ng/0.10 μL) or ketanserine (5 μg/0.10 μL) into the dorsal or ventral APtN was ineffective against the antinociception resulting from RSC stimulation.Significanceμ₁-opioid- and 5-HT₁-expressing neurons and cell processes in dorsal and ventral APtN are both implicated in the mediation of stimulation-produced antinociception from the RSC in the rat tail flick and incision pain tests, respectively.     

Baicalin upregulates the genetic expression of antioxidant enzymes in Type-2 diabetic Goto-Kakizaki rats

AimThe primary purpose of this study was to characterize and investigate the antioxidant and anti-diabetic activities of the flavonoid baicalin in type 2 diabetic Goto-Kakizaki rats.Main methodsFour groups of Goto-Kakizaki rats (n = 6) were subjected to the following oral treatments for 30 days: (1) metformin — 500 mg/kg (2) baicalin — 120 mg/kg (3) metformin 500 mg/kg and baicalin — 120 mg/kg (4) vehicle treated diabetic controls receiving distilled water. The plasma glucose, triglyceride, total cholesterol, lipid peroxide and protein carbonyl contents were measured on a weekly basis. Following the completion of the treatment, the rats were sacrificed and their blood, heart, pancreatic and hepatic tissues were collected for analysis. The antioxidant enzyme activities as well as their expression were quantified using Western Blot, microarray and RT-PCR.Key findingsThe respective analyses showed that the baicalin- and the metformin and baicalin-treated groups had statistically significant increases (p < 0.05) in the activity and expression of the antioxidant enzymes (superoxide dismutase, catalase and glutathione peroxidase) compared with vehicle- and metformin-treated groups. Further complementing the antioxidant enzyme activity increases, the oxidative stress markers of plasma lipid peroxide and protein carbonyl contents were reduced in these groups as well. These treatment groups also had reduced plasma total cholesterol and triglyceride levels compared with vehicle-treated and metformin-treated groups (p < 0.05).SignificanceBaicalin was an efficient antioxidant in reducing hyperglycemia-induced oxidative stress through the increased expression of antioxidant enzyme activities. It was also an efficient anti-hypertriglyceridemic as well as anti-hypercholesterolemic agent compared with metformin.     

17-β estradiol in the acetylcholinesterase activity and lipid peroxidation in the brain and blood of ovariectomized adult and middle-aged rats

AimsTo investigate the 17-β estradiol in the acetylcholinesterase activity and lipid peroxidation in the brain and blood of ovariectomized rats of different ages.Main methodsAnimals were randomly assigned into three experimental groups of each age (n = 6). Control groups consisted of adult (sham-A) and middle-aged (sham-MA) female rats, ovariectomized adult (OVX-A) and middle-aged (OVX-MA) rats without estrogen therapy reposition, and ovariectomized adult (OVX + E2-A) and middle-aged (OVX + E2-MA) rats treated with 17-β estradiol for 30 days. After this period, AChE activity and lipid peroxidation were measured in the brain and blood.Key findingsThe AChE activity increased (p < 0.05) in striatum (ST) in OVX-A, OVX + E2-A and OVX-MA, and hippocampus (HP) in OVX-MA. The enzyme activity decreased (p < 0.05) in ST of OVX + E2-MA, and cerebral cortex (CC) in OVX + E2-A, OVX-MA and OVX + E2-MA. Blood AChE activity increased (p < 0.05) in OVX + E2-A and decreased (p < 0.05) in OVX-MA. Lymphocyte AChE activity increased (p < 0.05) in OVX-A and OVX + E2-A and decreased (p < 0.05) in OVX-MA. Lipid peroxidation increased (p < 0.05) in ST of OVX-A, CC of OVX-A and OVX-MA, HP of OVX-A, and cerebellum (CE) of OVX-A, OVX-MA, and OVX + E2-MA. Lipid peroxidation decreased (p < 0.05) in ST, CC and CE of OVX + E2-A, and ST and HP of OVX + E2-MA. Similar values of lipid peroxidation to control groups were found in ST and HP of OVX-MA, HP of OVX + E2-A and CC of OVX + E2-MA.Significance17-β estradiol is able to modulate the AChE activity and non-neuronal cholinergic response as well as to reduce lipid peroxidation. Its response is dependent on the age and brain structure analyzed.     

Different roles of zinc plus arachidonic acid on insulin sensitivity between high fructose- and high fat-fed rats

AimsThis study was to determine the effects of zinc plus arachidonic acid (ZA) treatment on the insulin action in the specific ZA target organs using hyperinsulinemic euglycemic clamp method.Main methods18 Sprague–Dawley rats weighing ~ 130 g were divided into 3 groups of 6 rats and treated them with 1) normal rat chow, 2) high fructose (60.0%) diet only, or 3) the same fructose diet plus drinking water containing 10 mg zinc plus 50 mg arachidonic acid (AA)/L. In a separate study, male Wistar rats weighing ~ 250 g were fed normal rat chow (n = 4) or high fat (66.5%) diet with drinking water containing zero (n = 9) or 10 mg AA plus 20 mg zinc /L (n = 9). After 4 week treatment, insulin action was assessed using the hyperinsulinemic eguglycemic clamp technique.Key findingsHigh fructose feeding impaired suppression of hepatic glucose output by insulin compared to controls during the clamp procedure (4.39 vs. 2.35 mg/kg/min; p < 0.05). However, ZA treatment in high fructose-fed rats showed a significant improvement of hepatic insulin sensitivity compared to non-treatment controls (4.39 vs. 2.18 mg/kg/min; p < 0.05). Glucose infusion rates in Wistar rats maintained on a high fat diet (HFD) were significantly lower compared to control rats (22.8 ± 1.3 vs. 31.9 ± 1.4 mg/kg/min; p < 0.05). ZA treatment significantly improved (~ 43%) peripheral tissue insulin sensitivity in HFD fed animals (26.7 ± 1.3 [n = 9] vs. 22.8 ± 1.3 mg/kg/min; p < 0.05).SignificanceThese data demonstrate that ZA treatment is effective in improving glucose utilization in hyperglycemic rats receiving either a high-fructose or a high-fat diet.     

Intraperitoneal kisspeptin-10 administration induces dose-dependent degenerative changes in maturing rat testes

AimsKisspeptin, a peptide secreted by hypothalamic neurons, is a critical regulator of reproduction and puberty but its role in the regulation of gonadal maturation in sexually immature males is elusive. The present study investigated the effects of 12 days of pulsatile kisspeptin administration on gonadotropins and testosterone release and maturation of immature male gonads.Main methodsKisspeptin-10 was administered intraperitoneally at different dosage concentrations (1 μg, 1 ng, and 10 pg) to 5 weeks old prepubertal male rats, twice daily for 12 days. Plasma LH, FSH and testosterone concentrations were measured through competitive-binding radioimmunoassay. Spermatogenesis was studied mainly at stage VII of the spermatogenic cycle through light and electron microscopy.Key findingsAt the end of the treatments plasma LH and testosterone concentrations were reduced significantly at 1 ng and 1 μg kisspeptin doses (P < 0.05; P < 0.01). Type A spermatogonia, preleptotene spermatocytes, pachytene spermatocytes, step 7 spermatids, elongated spermatids and daily sperm production decreased significantly (P < 0.05). Sertoli cell efficiency and total support capacity of Sertoli cells were reduced at all doses (P < 0.05). Meiotic index decreased (P < 0.05) at 1 μg dose only, whereas coefficient of mitosis increased at 1 ng and 1 μg (P < 0.01) kisspeptin doses. Histologically, degeneration of seminiferous tubules was evident showing tubular necrosis, multinucleated giant cell formation, intratubular vacuolization, widened lumen and deshaped germ cells. Marked ultrastructural changes characterized by thin basal laminae, enlarged intratubular spaces, abnormal acrosome and disrupted germ cells were noticeable.SignificanceIn conclusion long-term kisspeptin-10 administration negatively regulates gonadal maturation in prepubertal testes.     

Evaluation of vitamin E in the treatment of erectile dysfunction in aged rats

AimsThe present study aims to elucidate the role of oxidative stress in erectile dysfunction associated with aging and to investigate the effect of treatment with vitamin E in this respect.Main methodsRats were divided into four groups: young (3-month-old), aged rats (18-month-old), aged rats given 80 IU of vitamin E/rat/day for 21-days, aged rats given 5 mg/kg of sildenafil/day for 21-days. Intracavernosal pressure/mean arterial pressure (ICP/MAP), nitric oxide production, TBARS, GSH levels and SOD activity in corpus cavernosum were measured.Key findingsSignificant decrease in ICP/MAP was observed in aged rats at both low and high frequency of stimulation. Significant increase in ICP/MAP was observed in aged rats treated with vitamin E over the range of 0.8 to 5 Hz but young control values were not restored. Percentage potentiation of ICP/MAP than aged group at 0.8 Hz was 326 ± 41.3% and 897 ± 72.2% for vitamin E and sildenafil respectively. Decreased levels of NO₂/NO₃ and SOD activity in the penile tissue observed with aging were elevated back to control by either vitamin E or sildenafil. Penile concentration of TBARS was 20.86 ± 0.83 for aged rats vs. 11.39 ± 0.79 nmol/g tissue for young controls. Both vitamin E and sildenafil reduced penile TBARS in aged rats.SignificanceThis study proves that antioxidant therapy with vitamin E ameliorates the age-associated erectile dysfunction. Sildenafil may exert some antioxidant properties which add to the advantages of its long-term use. The effect of combinations of low-dose sildenafil and vitamin E on age-associated erectile dysfunction merits to be studied.     

NADPH-d and Fos reactivity in the rat spinal cord following experimental spinal cord injury and embryonic neural stem cell transplantation

AimsThe aim of this study is to determine the role of nitric oxide (NO) in neuropathic pain and the effect of embryonic neural stem cell (ENSC) transplantation on NO content in rat spinal cord neurons following spinal cord injury (SCI).Main methodsNinety adult male Sprague–Dawley rats were divided into 3 groups (n = 30, each): control (laminectomy), SCI (hemisection at T12–T13 segments) and SCI + ENSC. Each group was further divided into sub-groups (n = 5 each) based on the treatment substance (L-NAME, 75 mg/kg/i.p.; l-arginine, 225 mg/kg/i.p.; physiological saline, SF) and duration (2 h for acute and 28 days for chronic groups). Pain was assessed by tail flick and Randall–Selitto tests. Fos immunohistochemistry and NADPH-d histochemistry were performed in segments 2 cm rostral and caudal to SCI.Key findingsTail-flick latency time increased in both acute and chronic L-NAME groups and increased in acute and decreased in chronic l-arginine groups. The number of Fos (+) neurons decreased in acute and chronic L-NAME and decreased in acute l-arginine groups. Following ENSC, Fos (+) neurons did not change in acute L-NAME but decreased in the chronic L-NAME groups, and decreased in both acute and chronic l-arginine groups. NADPH-d (+) neurons decreased in acute L-NAME and increased in l-arginine groups with and without ENSC transplantation.SignificanceThis study confirms the role of NO in neuropathic pain and shows an improvement following ENSC transplantation in the acute phase, observed as a decrease in Fos(+) and NADPH-d (+) neurons in spinal cord segments rostral and caudal to injury.     

Mesenchymal stromal cells prolong the lifespan in a rat model of amyotrophic lateral sclerosis

Background aimsAmyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder characterized by the loss of brain and spinal cord motor neurons (MN). The intraspinal and systemic grafting of mesenchymal stromal cells (MSC) was used to treat symptomatic transgenic rats overexpressing human superoxide dismutase 1 (SOD1) in order to alleviate the disease course and prolong the animals’ lifespan.MethodsAt the age of 16 weeks (disease onset) the rats received two grafts of MSC expressing green fluorescent protein (GFP⁺ MSC) on the same day, intraspinally (10⁵ cells) and intravenously (2 × 10⁶ cells). Sham-treated animals were injected with phosphate-buffered saline (PBS). Motor activity, grip strength and body weight were tested, followed by immunohistochemical analysis.ResultsThe combined grafting of MSC into symptomatic rats had a significant effect on motor activity and grip strength starting 4 weeks after transplantation. The lifespan of animals in the treated group was 190 ± 3.33 days compared with 179 ± 3.6 days in the control group of animals. Treated rats had a larger number of MN at the thoracic and lumbar levels; these MN were of larger size, and the intensity of terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL) staining in the somas of apoptotic MN at the thoracic level was much lower than in sham-treated animals. Transplanted GFP⁺ MSC survived in the spinal cord until the end stage of the disease and migrated both rostrally and caudally from the injection site.ConclusionsIntraspinal and intravenous transplantation of MSC has a beneficial and possibly synergistic effect on the lifespan of ALS animals.     

Involvement of dorsal hippocampal muscarinic cholinergic receptors on muscimol state-dependent memory of passive avoidance in mice

AimsIn the present study, the effects of bilateral intra-dorsal hippocampal (intra-CA1) injections of cholinergic agents on muscimol state-dependent memory were examined in mice.Main methodsA single-trial step-down passive avoidance task was used for the assessment of memory retention in adult male NMRI mice.Key findingsPre-training intra-CA1 administration of a GABA-A receptor agonist, muscimol (0.05 and 0.1 μg/mouse) dose dependently induced impairment of memory retention. Pre-test injection of muscimol (0.05 and 0.1 μg/mouse, intra-CA1) induced state-dependent retrieval of the memory acquired under pre-training muscimol (0.1 μg/mouse, intra-CA1) influence. Pre-test intra-CA1 injection of an acetylcholinesterase inhibitor, physostigmine (0.5 and 1 μg/mouse, intra-CA1) reversed the memory impairment induced by pre-training administration of muscimol (0.1 μg/mouse, intra-CA1). Moreover, pre-test administration of physostigmine (0.5 and 1 μg/mouse, intra-CA1) with an ineffective dose of muscimol (0.025 μg/mouse, intra-CA1) significantly restored the retrieval and induced muscimol state-dependent memory. Pre-test intra-CA1 administration of physostigmine (0.25, 0.5 and 1 μg/mouse) by itself cannot affect memory retention. Pre-test intra-CA1 injection of the muscarinic receptor antagonist, atropine (1 and 2 μg/mouse) 5 min before the administration of muscimol (0.1 μg/mouse, intra-CA1) dose dependently inhibited muscimol state-dependent memory. Pre-test intra-CA1 administration of atropine (0.5, 1 and 2 μg/mouse) by itself cannot affect memory retention.SignificanceThe results suggest that muscarinic cholinergic mechanism of the CA1 may influence muscimol state-dependent memory.     

Coexistence of insulin resistance and increased glucose tolerance in pregnant rats: a physiological mechanism for glucose maintenance

AimThe contribution of insulin resistance (IR) and glucose tolerance to the maintenance of blood glucose levels in non diabetic pregnant Wistar rats (PWR) was investigated.Main methodsPWR were submitted to conventional insulin tolerance test (ITT) and glucose tolerance test (GTT) using blood sample collected 0, 10 and 60 min after intraperitoneal insulin (1 U/kg) or oral (gavage) glucose (1 g/kg) administration. Moreover, ITT, GTT and the kinetics of glucose concentration changes in the fed and fasted states were evaluated with a real-time continuous glucose monitoring system (RT-CGMS) technique. Furthermore, the contribution of the liver glucose production was investigated.Key findingsConventional ITT and GTT at 0, 7, 14 and 20 days of pregnancy revealed increased IR and glucose tolerance after 20 days of pregnancy. Thus, this period of pregnancy was used to investigate the kinetics of glucose changes with the RT-CGMS technique. PWR (day 20) exhibited a lower (p < 0.05) glucose concentration in the fed state. In addition, we observed IR and increased glucose tolerance in the fed state (PWR-day 20 vs. day 0). Furthermore, our data from glycogenolysis and gluconeogenesis suggested that the liver glucose production did not contribute to these changes in insulin sensitivity and/or glucose tolerance during late pregnancy.SignificanceIn contrast to the general view that IR is a pathological process associated with gestational diabetes, a certain degree of IR may represent an important physiological mechanism for blood glucose maintenance during fasting.     

Indoxyl sulfate upregulates renal expression of MCP-1 via production of ROS and activation of NF-κB, p53, ERK, and JNK in proximal tubular cells

AimsMonocyte chemotactic protein-1 (MCP-1) plays an important role in recruiting monocytes/macrophages to injured tubulointerstitial tissue. The present study examined whether indoxyl sulfate, a uremic toxin, regulates renal expression of MCP-1.Main methodsThe effect of indoxyl sulfate on the expression of MCP-1 was determined using human proximal tubular cells (HK-2 cells) and following animals: (1) Dahl salt-resistant normotensive rats (DN), (2) Dahl salt-resistant normotensive indoxyl sulfate-administered rats (DN + IS), (3) Dahl salt-sensitive hypertensive rats (DH), and (4) Dahl salt-sensitive hypertensive indoxyl sulfate-administered rats (DH + IS).Key findingsDN + IS, DH, and DH + IS rats showed significantly increased mRNA expression of MCP-1 in the kidneys compared with DN rats. DH + IS rats tended to show increased mRNA expression of MCP-1 in the kidneys compared with DH rats. Immunohistochemistry demonstrated the stimulatory effects of indoxyl sulfate on MCP-1 expression and monocyte/macrophage infiltration in the kidneys. Indoxyl sulfate upregulated mRNA and protein expression of MCP-1 in HK-2 cells. Indoxyl sulfate induced activation of ERK, p38, and JNK as well as of NF-κB and p53 in HK-2 cells. An antioxidant, and inhibitors of NF-κB, p53, ERK pathway (MEK1/2), and JNK suppressed indoxyl sulfate-induced mRNA expression of MCP-1 in HK-2 cells.SignificanceIndoxyl sulfate upregulates renal expression of MCP-1 through production of reactive oxygen species (ROS), and activation of NF-κB, p53, ERK, and JNK in proximal tubular cells. Thus, accumulation of indoxyl sulfate in chronic kidney disease might be involved in the pathogenesis of tubulointerstitial injury through induction of MCP-1 in the kidneys.     

Cryopreserved mobilized autologous blood progenitors stored for more than 2 years successfully support blood count recovery after high-dose chemotherapy

Background aimsThe ability of hematopoietic progenitor cells–apheresis (HPC-A) that have been stored for many years after cryopreservation to reconstitute hematopoiesis following high-dose chemo/radiotherapy has not been well-documented.MethodsIn this retrospective study, eight Canadian centers contributed data from 53 autologous stem cell transplants (ASCT) performed using HPC-A that had undergone long-term storage (>2 years, range 2–7 years) and 120 ASCT using HPC-A stored for <6 months (short-term storage).ResultsThe doses of nucleated and CD34⁺ cells per kilogram recipient weight were similar between the short- (mean ± SD, 4.7 ± 4.9 × 10⁸ and 6.8 ± 4.3 × 10⁶, respectively) and long- (4.0 ± 4.9 × 10⁸ and 6.1 ± 3.4 × 10⁶, respectively) term storage groups. The median days to neutrophils (absolute neutrophil count; ANC) >0.5 × 10⁹/L (median 11 days for both short- and long-term storage) and platelets >20 × 10⁹/L (median 12 and 11 for short- and long-term storage, respectively) post-ASCT were not significantly different between the two groups. When ASCT performed with <5 × 10⁶/kg CD34⁺ cells was compared there was also no difference in ANC or platelet recovery (median 12 days for both after short-term storage, and 12 and 11 days, respectively, after long-term storage). Fourteen HPC-A products stored for >5 years also showed similar count recoveries as the entire long-term storage group (median 11 days for both ANC and platelets).ConclusionsCryopreserved HPC-A can be stored for at least 5 years with no apparent loss in their ability to support hematopoietic reconstitution after high-dose chemotherapy.