Day-old Rats Research Articles

Expression of Neurotrophic Factors and Their Receptors in Rat Posterior Taste Bud Cells

Taste is an important sense in survival and growth of animals. The growth and maintenance of taste buds, the receptor organs of taste sense, are under the regulation of various neurotrophic factors. But the distribution aspect of neurotrophic factors and their receptors in distinct taste cell types are not clearly known. The present research was designed to characterize mRNA expression pattern of neurotrophic factors and their receptors in distinct type of taste cells. In male 45-60 day-old Sprague-Dawley rats, epithelial tissues with and without circumvallate and folliate papillaes were dissected and homogenized, and mRNA expressions for neurotrophic factors and their receptors were determined by RT-PCR. The mRNA expressions of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), receptor tyrosine kinase B (TrkB), exclusion of nerve growth factor (NGF), neurotrophin-4/5 (NT4/5), receptor tyrosine kinase A (TrkA), receptor tyrosine kinase C (TrkC), and p75NGFR were observed in some population of taste cell. In support of this result and to characterize which types of taste cells express NT3, BDNF, or TrkB, we examined mRNA expressions of NT3, BDNF, or TrkB in the PLCβ2 (a marker of Type II cell)- and/or SNAP25 (a marker of Type III cell)-positive taste cells by a single taste cell RT-PCR and found that the ratio of positively stained cell numbers were 17.4, 6.5, 84.1, 70.3, and 1.4 % for PLCβ2, SNAP25, NT3, BDNF, and TrkB, respectively. In addition, all of PLCβ2- and SNAP25-positive taste cells expressed NT3 mRNA, except for one taste bud cell. The ratios of NT3 mRNA expressions were 100% and 91.7% in the SNAP25- and PLCβ2-positive taste cells, respectively. However, two TrkB-positive taste cells co-expressed neither PLCβ2 nor SNAP 25. The results suggest that the most of type II or type III cells express BDNF and NT3 mRNA, but the expression is shown to be less in type I taste cells.

Acute intrastriatal injection of quinolinic acid provokes long-lasting misregulation of the cytoskeleton in the striatum, cerebral cortex and hippocampus of young rats

Quinolinic acid (QUIN) is a neuroactive metabolite of the kinurenine pathway, considered to be involved in aging and some neurodegenerative disorders, including Huntington׳s disease. In the present work we have studied the long-lasting effect of acute intrastriatal injection of QUIN (150nmol/0.5µL) in 30 day-old rats on the phosphorylating system associated with the astrocytic and neuronal intermediate filament (IF) proteins: glial fibrillary acidic protein (GFAP), and neurofilament (NF) subunits (NFL, NFM and NFH) respectively, until 21 days after injection. The acute administration of QUIN altered the homeostasis of IF phosphorylation in a selective manner, progressing from striatum to cerebral cortex and hippocampus. Twenty four hours after QUIN injection, the IFs were hyperphosphorylated in the striatum. This effect progressed to cerebral cortex causing hypophosphorylation at day 14 and appeared in the hippocampus as hyperphosphorylation at day 21 after QUIN infusion. PKA and PKCaMII have been activated in striatum and hippocampus, since Ser55 and Ser57 in NFL head domain were hyperphosphorylated. However, MAPKs (Erk1/2, JNK and p38MAPK) were hyperphosphorylated/activated only in the hippocampus, suggesting different signaling mechanisms in these two brain structures during the first weeks after QUIN infusion. Also, protein phosphatase 1 (PP1) and 2B (PP2B)-mediated hypophosphorylation of the IF proteins in the cerebral cortex 14 after QUIN injection reinforce the selective signaling mechanisms in different brain structures. Increased GFAP immunocontent in the striatum and cerebral cortex 24h and 14 days after QUIN injection respectively, suggests reactive astrocytes in these brain regions. We propose that disruption of cytoskeletal homeostasis in neural cells takes part of the long-lasting molecular mechanisms of QUIN toxicity in adolescent rats, showing selective and progressive misregulation of the signaling mechanisms targeting the IF proteins in the striatum, cerebral cortex and hippocampus with important implications for brain function.

Blood brain barrier is impermeable to solutes and permeable to water after experimental pediatric cardiac arrest

Pediatric asphyxial cardiac arrest (CA) results in unfavorable neurological outcome in most survivors. Development of neuroprotective therapies is contingent upon understanding the permeability of intravenously delivered medications through the blood brain barrier (BBB). In a model of pediatric CA we sought to characterize BBB permeability to small and large molecular weight substances. Additionally, we measured the percent brain water after CA. Asphyxia of 9min was induced in 16–18 day-old rats. The rats were resuscitated and the BBB permeability to small (sodium fluorescein and gadoteridol) and large (immunoglobulin G, IgG) molecules was assessed at 1, 4, and 24h after asphyxial CA or sham surgery. Percent brain water was measured post-CA and in shams using wet-to-dry brain weight. Fluorescence, gadoteridol uptake, or IgG staining at 1, 4h and over the entire 24h post-CA did not differ from shams, suggesting absence of BBB permeability to these solutes. Cerebral water content was increased at 3h post-CA vs. sham. In conclusion, after 9min of asphyxial CA there is no BBB permeability over 24h to conventional small or large molecule tracers despite the fact that cerebral water content is increased early post-CA indicating the development of brain edema. Evaluation of novel therapies targeting neuronal death after pediatric CA should include their capacity to cross the BBB.

Dopamine preferentially inhibits NMDA receptor-mediated EPSCs by acting on presynaptic D1 receptors in nucleus accumbens during postnatal development.

Nucleus accumbens (nAcb), a major site of action of drugs of abuse and dopamine (DA) signalling in MSNs (medium spiny neurons), is critically involved in mediating behavioural responses of drug addiction. Most studies have evaluated the effects of DA on MSN firing properties but thus far, the effects of DA on a cellular circuit involving glutamatergic afferents to the nAcb have remained rather elusive. In this study we attempted to characterize the effects of dopamine (DA) on evoked glutamatergic excitatory postsynaptic currents (EPSCs) in nAcb medium spiny (MS) neurons in 1 to 21 day-old rat pups. The EPSCs evoked by local nAcb stimuli displayed both AMPA/KA and NMDA receptor-mediated components. The addition of DA to the superfusing medium produced a marked decrease of both components of the EPSCs that did not change during the postnatal period studied. Pharmacologically isolated AMPA/KA receptor-mediated response was inhibited on average by 40% whereas the isolated NMDA receptor-mediated EPSC was decreased by 90%. The effect of DA on evoked EPSCs were mimicked by the D1-like receptor agonist SKF 38393 and antagonized by the D1-like receptor antagonist SCH 23390 whereas D2-like receptor agonist or antagonist respectively failed to mimic or to block the action of DA. DA did not change the membrane input conductance of MS neurons or the characteristics of EPSCs produced by the local administration of glutamate in the presence of tetrodotoxin. In contrast, DA altered the paired-pulse ratio of evoked EPSCs. The present results show that the activation D1-like dopaminergic receptors modulate glutamatergic neurotransmission by preferentially inhibiting NMDA receptor-mediated EPSC through presynaptic mechanisms.

Adenosine receptor blockade impairs the ability of rat pups to autoresuscitate from primary apnea during repeated exposure to hypoxia (LB790)

Autoresuscitation (AR) failure from hypoxic‐induced apnea by gasping has been suggested to play a role in sudden infant death. Little is known, however, about the factors that influence gasping and its ability to effect autoresuscitation in the newborn. The present experiments were carried out on 18 5‐6 day‐old rat pups to investigate the role of adenosine acting via A1 receptors in mediating AR from hypoxic‐induced apnea by gasping. DPCPX (20 mg/kg; a selective adenosine A1‐receptor antagonist) attenuated the heart rate response to hypoxia and decreased the number of hypoxic episodes tolerated before AR failure (DPCPX 10±2 (n=9) versus vehicle 17±5 (n=9); p<0.001). Following vehicle, death was preceded most often by AV dissociation whereas following DPCPX death was preceded most often by an isoelectric electrocardiogram. Thus, our data provide evidence that adenosine acting via A1 receptors plays an important role in promoting AR from hypoxic‐induced apnea by gasping.Grant Funding Source: CIHR

Resveratrol potentiates effects of simvastatin on inhibition of rat ovarian theca-interstitial cells steroidogenesis

BackgroundPolycystic ovary syndrome (PCOS) is characterized by ovarian enlargement, hyperplastic theca compartment and increased androgen production due to, at least in part, excessive expression of several key genes involved in steroidogenesis. Previously, our group has demonstrated that simvastatin, competitive inhibitor of 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase), a rate-limiting step of the mevalonate pathway, reduces rat-theca interstitial cell steroidogenesis by inhibiting Cyp17a1 gene expression, the key enzyme of the androgen biosynthesis pathway. Recently, we demonstrated that resveratrol, a bioflavonoid abundant in red grapes, decreases rat theca-interstitial cell steroidogenesis and this suppressive effect is mediated through mechanisms independent of the mevalonate pathway. The present study evaluated the effect of combining simvastatin and resveratrol treatments on rat theca-interstitial cell steroidogenesis.MethodsRat theca-interstitial cells isolated from 30 day-old female rats were cultured for up to 48 h with or without simvastatin (1 μM) and/or resveratrol (3-10 μM). Steroidogenic enzymes gene expression was evaluated by quantitative real time PCR and steroid levels were measured by liquid chromatography-mass spectrometry. Comparisons between groups were performed using ANOVA and Tukey test.ResultsResveratrol potentiated inhibitory effects of simvastatin on androstenedione and androsterone production in theca-interstitial cells. This suppressive effect correlated with profound inhibition in Cyp17a1 mRNA expression in the presence of a combination of resveratrol and simvastatin.ConclusionsThe present findings indicate that resveratrol potentiates the simvastatin-induced inhibitory effect on theca-interstitial cell androgen production, raising the possibility of development of novel treatments of PCOS.

Effects of FK506 on long-term potentiation observed by optical imaging in organotypic hippocampal slice culture

Electrophysiological and biochemical assays were used to observe long-term changes in synaptic efficacy following electrical and/or pharmacological manipulation of synaptic function. We investigated the effects of FK506 on synaptic plasticity in organotypic hippocampal slice culture (OHSC) using optical imaging system. Hippocampal slices were prepared from 6-7 day-old rats using a tissue chopper and placed on a membrane insert. 18 hr after kainic acid (KA) treatment, significant delayed neuronal death which was quantified by propidium iodide, NeuN, and TUNEL staining was detected in CA3 area at 24 hr after KA treatment. The neuronal death was prevented significantly at 24 hr after 0.1 μM FK506 treatment. In the electrophysiology study, optical signals were observed by long-term potentiation (LTP) followed by the stimulation of the Schaffer collateral pathway. The improvement in amount of LTP was found in the FK506-treated group. These results suggest that FK506 may have a beneficial role in recovery of synaptic efficacy following KA-induced neuronal injury.

Effects of Obesity and 7,12-Dimethylbenz(A) Anthracene (DMBA) Treatment on Liver Cytochrome P4501A1 and 1B1 Expression in Ovariectomized Obese Zucker Rats

Obesity is a risk factor for postmenopausal breast cancer development. We have shown that obesity increases DMBA-induced mammary tumor development in intact and ovariectomized Zucker rats. Several data suggest that DMBA requires metabolic activation to exert its carcinogenic effects. The objective of this study was to investigate the effect of obesity on hepatic expression of cytochrome P450 CYP1A1 and CYP1B1 following DMBA treatment in obese and lean ovariectomized Zucker rats. Forty day-old, ovariectomized obese (n=20) and lean (n=20) Zucker rats were placed on AIN-93 G diet and 10 days later were orally gavaged with either with sesame oil (control) or with 65 mg/kg DMBA in sesame oil. All rats were sacrificed 24 hours post-DMBA treatment. Liver microsomes were prepared, and CYP1A1 and CYP1B1 expression was measured by Western blotting using goat anti-mouse CYP1A1 and CYP1B1 antibodies. DMBA treatment significantly (p<0.001) increased expression of CYP1A1 in lean and obese ovariectomized rats compared to the control group. CYP1B1 expression was not affected by obesity or DMBA. These data suggest that DMBA can increase the expression of enzymes that are responsible for DMBA metabolism in the ovariectomized Zucker rat model.

Differences in age modulates neutrophils function

The sexual maturation in all mammals is the period in which the quiescent gonads are activated by gonadotropins from anterior pituitary, increasing the secretion of sexual hormones. Sexual maturation it is also related with the development of several other body features such as body mass and maturation of the circulatory, skeletal and hematopoietic systems. The aim of the present study was to evaluate the function of neutrophils submitted to in vivo lower and higher concentration of testosterone (sexually immature: 60 days and sexually mature: 90 days). Using different approaches we evaluated cell viability and function and gene expression in rat neutrophils from 60 and 90 days-old animals. Neutrophils from 90 days-old rats showed a decrease in phagocytic and fungicidal capacity, without change in cellular viability. Additionally, we verified that sexual maturation induced increase in production of reactive oxygen and nitrogen species (RONS) and also in TNF-α, IL-6 and IL-10 cytokines. In conclusion, our data suggest that increase in testosterone levels induced significant alteration in neutrophil function, impairing phagocytic capacity.

Limitations of Mild, Moderate, and Profound Hypothermia in Protecting Developing Hippocampal Neurons After Simulated Ischemia.

Mild hypothermia (33°C-34°C) after cerebral ischemia in intact animals or ischemia-like conditions in vitro reduces neuron death. However, it is now clear that more profound hypothermia or delayed hypothermia may not provide significant protection. To further define the limitations of hypothermia after cerebral ischemia, we used hippocampal slice cultures to examine the effects of various degrees, durations, and delays of hypothermia on neuron death after an ischemia-like insult. Organotypic cultures of the hippocampus from 7- to 8 day-old rat pups were cooled to 32°C, 23°C, 17°C, or 4°C immediately or after a 2-4 hour delay from an injurious insult of oxygen and glucose deprivation (OGD). Cell death in CA1, CA3 and dentate regions of the cultures was assessed 24 hours later with SYTOX® or propidium iodide, both of which are fluorescent markers labeling damaged cells. OGD caused extensive cell death in CA1, CA3, and dentate regions of the hippocampal cultures. Hypothermia (32°C, 23°C and 17°C) for 4-6 hours immediately after OGD was protective at 24 hours, but when hypothermia was applied for longer periods or delayed after OGD, no protection or increased death was seen. Ultra-profound hypothermia (4°C) increased cell death in all cell areas of the hippocampus even when after a milder insult of only hypoxia. In an in vitro model of recovery after an ischemia-like insult, mild to profound hypothermia is protective only when applied without delay and for limited periods of time (6-8 hours). Longer durations of hypothermia, or delayed application of the hypothermia can increase neuron death. These findings may have implications for clinical uses of therapeutic hypothermia after hypoxic or ischemic insults, and suggest that further work is needed to elucidate the limitations of hypothermia as a protective treatment after ischemic stress.

Acute effects of ethanol on GABAA and glycine currents in the lateral habenula neurons of young rats.

Compelling evidence has shown a pivotal role of dopaminergic function in drug addiction. Recently, the lateral habenula (LHb) has attracted a great deal of attention as another target for abused drugs in the brain because its role in regulating dopaminergic system, among others. GABA and glycine are major inhibitory neurotransmitters. Their corresponding receptors are key targets of ethanol. The properties of these receptors in LHb neurons and their responses to ethanol in particular however, remain unknown. Using the patch clamp techniques, we examined the effects of ethanol on the chloride currents elicited by GABA and glycine in LHb neurons acutely dissociated from 10-20 day-old Sprague–Dawley rats. We show that GABA concentration-dependently elicited a bicuculline sensitive inward current in 96% (130/140) of the neurons tested. Ethanol (43.2 mM) suppressed current elicited by a wide range of concentrations (1-300 μM) of GABA in 74% (35/47) cells tested. Ethanol suppression is dependent on its concentrations but not on membrane potentials of the neurons. Moreover, glycine concentration-dependently elicited an inward current in 94% (112/120) of the neurons tested. Both strychnine and picrotoxin concentration dependently suppressed glycine current with IC50 of 220 nM and 813 μM, respectively. Ethanol (43.2 mM) potentiated current elicited by unsaturated but not saturated concentrations of glycine. Thus, the LHb neurons of young rats contain both functional GABAA and glycine receptors which are sensitive to ethanol at pharmacologically relevant concentrations. These effects of ethanol might be important in the control of the activity and output of LHb neurons.

The influence of sustained dual-factor presentation on the expansion and differentiation of neural progenitors in affinity-binding alginate scaffolds

Biomaterials capable of controlling the release of multiple growth factors (GFs) could potentially promote the integration of co-transplanted neural progenitor cells (NPCs) and stimulate the plasticity and regenerability of the lesioned spinal cord. As a first step towards the employment of such a vehicle for cell therapy, this study examined the capability of an alginate-sulphate/alginate scaffold, able to capture and rigorously control the release of GFs, to promote the expansion and lineage differentiation of NPCs in vitro. Epidermal growth factor (EGF) and fibroblast growth factor-2 (bFGF) were affinity-bound to alginate-sulphate (200 ng/scaffold) and the bioconjugates were mixed with partially calcium-crosslinked alginate. NPCs isolated from 18 day-old rat embryo brains and seeded into the scaffold during preparation were found to proliferate and differentiate within the vehicle. A continuous release of both bFGF and EGF was noted for a period of 21 days. The concentrations of released GFs were sufficient to promote extensive NPC proliferation at initial cultivation times; the number of neurospheres in the scaffold was twice the number found in the 2D cultures supplemented with 20 ng/ml each factor every 3 days. Between days 10-14, when the GF concentrations had substantially declined, extensive cell migration from the neurospheres as well as lineage differentiation were noted in the scaffold; immunocytochemical analyses confirmed the presence of neurons, astrocytes and oligodendrocytes.The scaffold has a potential to serve as cell delivery vehicle, with proven capability to promote cell retention and expansion, while enabling NPC lineage differentiation in situ.

Flujo mesentérico en un modelo experimental por isquemia-reperfusión en ratas

IntroductionMaintained acute occlusion followed by reperfusion of the superior mesenteric artery (SMA) in a few hours can trigger irreversible bowel damage. The aim of the study was to determine the changes in mesenteric flow measured by colour Doppler Ultrasound and correlating with histological lesions in an experimental model of ischaemia-reperfusion. Method and materialThree groups of Sprague-Dawley 17 day-old rats were studied (control, ischemia and reperfusion). The model used was ischaemia-reperfusion over the SMA. Intra-abdominal ultrasound was then performed. The parameters recorded were: Maximum systolic velocity (MSV), pulsatility index (PI), resistance (RI) and systole-diastole (S/D). The histological variables were: intestinal lesion (Wallace/Keenan-Chiu scale), morphometrics (mean villus height [MVH]), and goblet cells. The Spearman (rs) correlation was used. ResultsThe MSV in the reperfusion group was 74.3cm/s, the PI 7.33 and S/D 25.75 in the SMA, which were higher than the controls (41.35cm/s; 3.12 and 12.45, respectively). A direct association (P<.01) was found between MSV, PI and S/D regarding: Wallace/Kennan scoring system (rs = 0.655; rs = 0.593; rs = 0.63) and the Chiu (rs = 0.569; rs = 0.522; rs = 0.47). While the correlation was the reverse (P<.01) when associated with the MVH (rs = –0,495; rs = –0,452; rs = –0,459) and goblet cells of the colon (rs = –0,525; rs = –0,45; rs = –0,518). ConclusionsThe reperfusion phase increased mesenteric flow expressed by the MSV and PI and could significantly predict the potential bowel damage at macroscopic and microscopic level.

Repeated exposure to propofol potentiates neuroapoptosis and long-term behavioral deficits in neonatal rats

Previous studies have shown that exposure of the immature brain to drugs that block NMDA glutamate receptors or drugs that potentiate GABAA receptors can trigger widespread neuroapoptosis. Almost all currently used general anesthetics have either NMDA receptor blocking or GABAA receptor enhancing properties. Propofol, a new intravenous anesthetic, is widely used in pediatric anesthesia and intensive care practice whose neurotoxicity on brain development remains unknown. We investigated the effects of neonatal propofol anesthesia on neuroapoptosis and long-term spatial learning/memory functions. Propofol was administered to 7 day-old rats either as a single dose or in 7 doses at concentrations sufficient to maintain a surgical plane of anesthesia. Immunohistochemical studies revealed a significant increase in the levels of caspase-3 in the hippocampal CA1 region after propofol administration. At postnatal day 34, light microscopic observations revealed a significant reduction in neuronal density and apparent morphological changes in the pyramidal cells of rats that had received 7 doses of propofol. These rats showed a longer escape latency/path length, less time spent in the target quadrant and fewer original platform crossings in the Morris Water Maze test. This treatment also produced a remarkable reduction in the levels of excitatory neurotransmitters in the cortex and the hippocampus as measured by high performance liquid chromatography. Repeated exposure to propofol induced exposure-time dependent neuroapoptosis and long-term neurocognitive deficits in neonatal rats. The neurocognitive deficits may be attributed to neuronal loss and a reduction of excitatory neurotransmitter release in the cortex and hippocampus.

Mannitol Protects Hair Cells Against Tumor Necrosis Factor α–Induced Loss

Mannitol has otoprotective effects against tumor necrosis factor (TNF) α-induced auditory hair cell (HC) loss. Mannitol has been demonstrated to possess cytoprotective effects in several organ systems. Its protective effect on postischemic hearing loss has also been shown. Mannitol's otoprotective mechanism and site of action are at present unknown. Organ of Corti (OC) explants were dissected from 3 day-old rat pups. The safety (nonototoxicity) of mannitol was assessed at 4 different concentrations (1-100 mM). Three experimental arms were designed including: a control group, TNFα group, and TNFα + mannitol group. Cell viability was determined by counts of fluorescein isothiocyanate (FITC) phalloidin stained HC. Immunofluorescence assay of phospho-c-Jun and the proapoptotic mediators, cleaved caspase-3, apoptosis inducing factor (AIF), and endonuclease G (Endo G) were performed. Analysis of HC density confirmed the safety of mannitol at concentration ranges of 1 to 100 mM. The ototoxic effect of TNFα was demonstrated (p < 0.05). The otoprotective effect of 100 mM mannitol in TNFα-challenged OC explants was also demonstrated (p < 0.001). Mannitol treatment reduced the high levels of phospho-c-Jun observed in the TNFα-challenged group. AIF cluster formation and EndoG translocation into the nuclei of HCs were also reduced by mannitol treatment. Mannitol significantly reduces the ototoxic effects of TNFα against auditory HC's potentially by inhibiting c-Jun N terminal kinase (JNK) activation pathway and AIF, EndoG nuclear translocation. This local otoprotective effect may have therapeutic implications in inner ear surgery, for example, cochlear implants, protection of residual hearing, as well as implications for postischemic inner ear insults.

Effect of retinoic acid on the nitrergic innervation of meibomian glands in rats

The purpose of this study was to investigate the effect of prenatal administration of retinoic acid (RA) on the development of nicotinamide adenine dinucleotide diaphorase (NADPH-d) positive structures in the rat Meibomian glands. One mg/kg of RA was applied to pregnant Wistar rats intraperitonaelly during the gestational period in each of the 12th–14th embryonic days (totally 3 mg/kg). Sections of the central upper eyelids were investigated in rat pups on the 14th postnatal day. They were processed histochemically for NADPH-d, to study the presence and distribution of nitric oxide synthase (NOS) positive nerve structures. NADPH-d staining of Meibomian glands was compared in two groups of rat pups. In the control group, eyelids of 14 day-old rats were studied with no experimental intervention. The second group consisted of rat pups which were prenatally administered the excess of RA. Histochemical analysis of control eyelids revealed numerous NADPH-d well-stained acini of Meibomian glands arranged tightly into groups. Intensively stained vessels and NADPH-d/NOS-positive nerve fibers bordered acini of Meibomian glands. These structures were present in the submucosal layer as well. The analysis of RA group showed less numerous, shrunken acini of Meibomian glands that were seen not only smaller in size, but also in density of their staining and the amount of nitrergic nerve fibers around acini were considerably lowered. In the submucosa differences were noticed compared to the control group, there were numerous NADPH-d stained vessels accompanied by NADPH-d/NOS-positive nerve fibers. The excess of RA during the prenatal period may influence on the development and morphology of NADPH-d positive structures of rat's Meibomian glands.

Disrupted cytoskeletal homeostasis, astrogliosis and apoptotic cell death in the cerebellum of preweaning rats injected with diphenyl ditelluride

In the present report 15 day-old rats were injected with 0.3μmol of diphenyl ditelluride (PhTe)2/kg body weight and parameters of neurodegeneration were analyzed in slices from cerebellum 3 and 6 days afterwards. The earlier responses, at day 3 after injection, included hyperphosphorylation of intermediate filament (IF) proteins from astrocyte (glial fibrillary acidic protein – GFAP – and vimentin) and neuron (low-, medium- and high molecular weight neurofilament subunits: NF-L, NF-M and NF-H); increased mitogen-activated protein kinase (MAPK) (Erk and p38MAPK) and cAMP-dependent protein kinase (PKA) activities. Also, reactive astrogliosis takes part of the early responses to the insult with (PhTe)2, evidenced by upregulated GFAP in Western blot, PCR and immunofluorescence analysis. Six days after (PhTe)2 injection we found persistent astrogliosis, increased propidium iodide (PI) positive cells in NeuN positive population evidenced by flow cytometry and reduced immunofluorescence for NeuN, suggesting that the in vivo exposure to (PhTe)2 progressed to neuronal death. Moreover, activated caspase 3 suggested apoptotic neuronal death. Neurodegeneration was related with decreased [3H]glutamate uptake and decreased Akt immunoreactivity, however phospho-GSK-3-β (Ser9) was not altered in (PhTe)2 injected rat. Therefore, the present results show that the earlier cerebellar responses to (PhTe)2 include disruption of cytoskeletal homeostasis that could be related with MAPK and PKA activation and reactive astrogliosis. Akt inhibition observed at this time could also play a role in the neuronal death evidenced afterwards. The later events of the neurodegenerative process are characterized by persistent astrogliosis and activation of apoptotic neuronal death through caspase 3 mediated mechanisms, which could be related with glutamate excitotoxicity. The progression of these responses are therefore likely to be critical for the outcome of the neurodegeneration provoked by (PhTe)2 in rat cerebellum.

Influence of electromagnetic fields on reproductive system of male rats

Purpose: Reports of declining male fertility have renewed interest in the role of environmental and occupational exposures in the etiology of human infertility. The aim of the present work was to investigate the effect of 10 GHz exposure on the male Wistar rat's reproductive system and to find out the possible causative factors.Materials and methods: The study was divided into sham-exposed and exposed groups. Seventy day-old rats were exposed to 10 GHz microwave radiation for 2 h per day for 45 days at power density 0.21 mW/cm2 and specific absorption rate (SAR) of 0.014 W/kg. After the end of the experiment, blood samples were collected for the estimation of in vivo chromosomal aberration damage and micronucleus test. Spermatozoa were taken out for estimation of Caspase-3, comet assay, testosterone and electron microscopy and compared with sham-exposed.Results: The study of scanning electron microscopic revealed shrinkage of the lumen of the seminiferous tubules. Apoptotic bodies were found in exposed group. A flow cytometry examination showed formation of micronuclei body in lymphocytes of exposed group. Comet assay confirmed DNA (deoxyribonucleic acid) strand break. Testosterone level was found significantly decreased with the shrinkage of testicular size.Conclusions: 10 GHz field has an injurious effect on fertility potential of male-exposed animals.

Periventricular leukomalacia long-term prognosis may be improved by treatment with UDP-glucose, GDNF, and memantine in neonatal rats

The therapeutic effects of UDP-glucose (UDPG), an endogenous agonist of GPR17 that may promote the self-repair of white matter, glial cell line-derived neurotrophic factor (GDNF), a neurotrophic factor correlated with the growth and survival of nerve cells, and memantine, an antagonist of NMDA receptors, were evaluated for functional improvement of neonatal rats with experimental periventricular leukomalacia (PVL). Five day-old neonatal rat pups were subjected to an ischemia-induced model of PVL. The pups were then randomly divided into sham, PVL, PVL plus UDPG, PVL plus GDNF, and PVL plus memantine groups. All pups were weighed and the age at first eye opening recorded. Pathological changes and myelin sheath formation in the white matter were assessed under both light and electron microscopy on day 7 and 21 after induction of PVL. Values of escape latency (EL) and swimming distance (SD) in Morris water maze test, and the modified inclined plane scores in Rivlin inclined plane test were recorded for rats on day 26. Pups in the PVL group were found to be significantly lower in weight (p<0.05), delayed in age at first eye opening (p<0.01), and impaired in their inclined plane (p<0.01) and Morris water maze (p<0.01) performance compared with those in the sham, UDPG, GDNF and memantine groups. Histopathological grading of the white matter classified all pups in the PVL group with significantly more severe injury (p<0.01), and the number and thickness of their myelin sheaths were significantly less (p<0.01), compared to the UDPG, GDNF, memantine, or sham groups. These results indicate that treatment with UDPG, GDNF, and memantine may significantly improve long-term prognosis in neonatal rats with cerebral white matter injury, characteristic of PVL.

Fructose Rich Diet-Induced High Plasminogen Activator Inhibitor-1 (PAI-1) Production in the Adult Female Rat: Protective Effect of Progesterone

The effect of progesterone (P4) on fructose rich diet (FRD) intake-induced metabolic, endocrine and parametrial adipose tissue (PMAT) dysfunctions was studied in the adult female rat. Sixty day-old rats were i.m. treated with oil alone (control, CT) or containing P4 (12 mg/kg). Rats ate Purina chow-diet ad libitum throughout the entire experiment and, between 100 and 120 days of age drank ad libitum tap water alone (normal diet; CT-ND and P4-ND) or containing fructose (10% w/v; CT-FRD and P4-FRD). At age 120 days, animals were subjected to a glucose tolerance test or decapitated. Plasma concentrations of various biomarkers and PMAT gene abundance were monitored. P4-ND (vs. CT-ND) rats showed elevated circulating levels of lipids. CT-FRD rats displayed high (vs. CT-ND) plasma concentrations of lipids, leptin, adiponectin and plasminogen activator inhibitor-1 (PAI-1). Lipidemia and adiponectinemia were high (vs. P4-ND) in P4-FRD rats. Although P4 failed to prevent FRD-induced hyperleptinemia, it was fully protective on FRD-enhanced plasma PAI-1 levels. PMAT leptin and adiponectin mRNAs were high in CT-FRD and P4-FRD rats. While FRD enhanced PMAT PAI-1 mRNA abundance in CT rats, this effect was absent in P4 rats. Our study supports that a preceding P4-enriched milieu prevented the enhanced prothrombotic risk induced by FRD-elicited high PAI-1 production.