Day-old Rats Research Articles

INFLUENCE OF MATERNAL SUBHEPATIC CHOLESTASIS ON KIDNEY DEVELOPMENT IN SECOND-GENERATION OFFSPRING

Background. Maternal cholestasis of pregnancy was found to have a negative effect on the kidney structure of firstgeneration offspring. It is not known whether changes in the urinary system will occur in second-generation offspring. Objective. To establish the peculiarities of kidney development in second-generation offspring from rats with cholestasis of pregnancy. Material and methods. Second generation 15-, 45- and 90 day-old rats born from males and females developed in maternal cholestasis. Surgical, somatometric, histological, morphometric and statistical methods of research were used in the experiment. Results. Maternal cholestasis induced on the 17th day of pregnancy leads to structural changes in the kidneys of second-generation offspring. These changes include a decrease in the weight of kidneys of experimental animals, a decrease in the width of their cortical substance, a decrease in the diameter of convoluted proximal and distal tubules of nephrons and the development of structural abnormalities in the epitheliocytes of tubule sections. Hypoplastic changes in renal nephrons persist up to 90 days of postnatal development of animals. Conclusions. Maternal cholestasis induces hypoplastic abnormalities in the kidneys of second-generation rats.

Protective role of hispolon and its derivatives against apoptosis in cortical neurons induced by electromagnetic radiation from 4G mobile phone.

Electromagnetic radiation (EMR) from wireless devices, particularly mobile phones, is a potentially growing public health concern. In this study, the neuronal effects of EMR on primary cortical neurons (PCNs) from neonatal rat cerebral cortex and the protective role of hispolon (HIS) and its derivatives were investigated as a measure of cranial exposure during mobile phone use.PCNs were isolated and cultured from day-old neonatal rats, then exposed for 2 h to EMR emitted by a mobile phone operating at a frequency of 2100 MHz with 1.6 W/Kg specific absorption rate (SAR) in call-answered mode treated with HIS and its derivatives. The induction of apoptosis through modulation of pro and anti-apoptotic genes via mitochondrial pathway and the protection by the test compounds was assessed. Pyrazole derivatives decreased apoptosis by modulating the levels of pro and anti-apoptotic genes by reducing the levels of reactive oxygen species (ROS) via mitochondrial damage, which was observed in the EMR exposed PCNs. The pyrazole compounds were found to have antioxidative and anti-apoptotic properties. Thus, the neuroprotective mechanisms of the pyrazole derivatives can be investigated further, which may make them appropriate as lead compounds in developing neuroprotective formulations.

CXCL1/CXCR2 is involved in white matter injury in neonatal rats via the gut–brain axis

BackgroundThis study aimed to investigate whether CXCL1/CXCR2 mediates intestinal injury or white matter injury by delivering inflammatory mediators through the gut–brain regulation axis.MethodsNeonatal SD rats, regardless of sex, were administered 3% dextran sulfate sodium via intragastric administration at different time points to construct necrotizing enterocolitis (NEC) models. Meanwhile, hypoxia and ischemia were induced in 3 day-old SD rats to construct hypoxic–ischemic brain injury (HIBI) and NEC + HIBI models, without gender discrimination. Hematoxylin–eosin staining was used to observe pathological changes in neonatal rat intestinal and brain tissues. Western blotting detected CXCL1 and CXCR2 expression in NEC, HIBI, and NEC + HIBI rat intestinal and brain tissues.ResultsCompared with normal rats, pathological damage to periventricular white matter was observed in the NEC group. In addition to the increased mortality, the histopathological scores also indicated significant increases in brain and intestinal tissue damage in both HIBI and NEC + HIBI rats. Western blotting results suggested that CXCL1 and CXCR2 expression levels were upregulated to varying degrees in the intestinal and brain tissues of NEC, HIBI, and NEC + HIBI neonatal rats compared to that in the normal group. Compared with the HIBI group, the expression of CXCL1 and CXCR2 continued to increase in NEC + HIBI rats at different time points.ConclusionsCXCL1/CXCR2 may be involved in white matter injury in neonatal rats by delivering intestinal inflammatory mediators through the gut–brain axis.

In vitro study of the toxic and teratogenic effects of prednisolone, azathioprine and mycophenolate mofetile on embryological development of rats

This study aimed to evaluate the effects of the glucocorticoid prednisolone, the mycophenolic acid prodrug, azathioprine, and the fungi fermentation end product, mycophenolate mofetile on the embryological development of rats. Nine day-old rat embryos were cultured in rat serum containing prednisolone at varying concentrations (5–30 µg/ml) for 48 h. The test groups were cultured separately in rat serum containing 0.3–10 µg/ml azathioprine and 1–10 µg/ml mycophenolate mofetile. Embryonic development parameter effects of both drugs in combination with prednisolone (20 µg/ml) were studied using morphological methods, with special attention given to the incidence of malformations. The genotoxic effects of agents evaluated with the TUNEL test revealed that prednisolone is not a cause of developmental toxicity. The maximum safe dose of prednisolone that could be used in combination with other immunosuppressive agents was determined to be 20 µg/ml. Azathioprine was found to be toxic and teratogenic for the rat embryos beginning at a dose of 1 µg/ml. Dose-dependent toxic and teratogenic effects of mycophenolate mofetile were detected at doses lower than normal clinical ones.

Hippocampal chloride transporter KCC2 contributes to excitatory GABA dysregulation in the developmental rat model of schizophrenia

Recent studies have revealed an altered expression of NKCC1 and KCC2 in prefrontal cortex (PFC) and hippocampus of schizophrenic patients. Despite extensive considerations, the alteration of NKCC1 and KCC2 co-transporters at different stages of development has not been fully studied. Therefore, we evaluated the expression of these transporters in PFC and hippocampus at time points of four, eight, and twelve weeks in post-weaning social isolation rearing rat model. For this purpose, 23–25 days-old rats were classified into social- or isolation-reared groups. The levels of NKCC1 and KCC2 mRNA expression were evaluated at hippocampus or PFC regions at the time-points of four, eight, and twelve weeks following housing. Post-weaning isolation rearing decreased the hippocampal KCC2 mRNA expression level, but does not affect the NKCC1 mRNA expression. However, no significant difference was observed in the PFC mRNA levels of NKCC1 and KCC2 in the isolation-reared group compared to the socially-reared group during the course of modeling. Further, we assessed the therapeutic effect of selective NKCC1 inhibitor bumetanide (10 mg/kg), on improvement of prepulse inhibition (PPI) test on twelve weeks isolation-reared rats. Intraperitoneal administration of bumetanide (10 mg/kg) did not exert beneficial effects on PPI deficit. Our findings show that isolation rearing reduces hippocampal KCC2 expression level and may underlie hippocampal GABA excitatory. In addition, 10 mg/kg bumetanide is not effective in improving the reduced PPI of twelve weeks isolation-reared rats.Collectively, our findings show that hippocampal chloride transporter KCC2 contributes to excitatory GABA dysregulation in the developmental rat model of schizophrenia.

Post-acute delivery of α5-GABAA antagonist, S 44819, improves functional recovery in juvenile rats following stroke

Hypo-excitability was reported in the peri-infarct tissue following stroke, an effect counteracted by a blockage of α5-GABAA receptors in adult rodents. Our present study aims to evaluate the effect of a selective α5-GABAA receptor antagonist, S 44819, in stroke in juvenile animals. We have set up and characterized an original model of transient ischemic stroke in 28 day-old Sprague-Dawley rats (45-min occlusion of the middle cerebral artery by intraluminal suture). In this model, S 44819 (1, 3 and 10 mg/kg, b.i.d) was orally administered from day 3 to day 16 after stroke onset. Sensorimotor recovery was assessed on day 1, day 9 and day 16 after stroke onset. Results show that rats treated with S 44819 at the doses of 3 and 10 mg/kg displayed a significant improvement of the neurological deficits (neuroscore) on day 9 and day 16, when compared with animals treated with vehicle. Grip-test data analysis reveals that rats treated with S 44819 at the dose of 3 mg/kg displayed a better recovery on day 9 and day 16. These results are in agreement with those previously observed in adult rats, demonstrating that targeting α5-GABAA receptors improves neurological recovery after stroke in juvenile rats.

Optimal Valve Selection in Contemporary Transcatheter Aortic Valve Replacement for Women with Small Aortic Annuli

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 (150 nmol/0.5 µL) 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 24 h 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.

MO1016MULTIPLE-ORGAN DAMAGE FOLLOWING PERINATAL ASPHYXIA IN RAT MODEL

Abstract Background and Aims Perinatal asphyxia (PA) is associated with more than half a million mature newborn deaths yearly. It may lead to severe complications including hypoxic encephalopathy, renal- hepatic- and cardiovascular injury, as well as respiratory distress, Basic research and clinical trials mainly focus on mitigating central nervous system damage by selective head or whole body cooling, which is currently the only routinely used treatment in clinical practice. However, the extent of PA-associated multi-organ damage is not clarified yet and effective therapies are lacking. Our aim was to investigate the acute renal, hepatic and cardiac impairment following PA and to identify pathways involved in the pathomechanism. In addition, we aimed to explore long-term effects of PA on permanent organ damage and susceptibility to ischemia/-reperfusion injury in adulthood. Method Postnatal 7 day-old male Wistar rat pups (n=5-10/group) were randomly grouped as follows: (i) Baseline; (ii) Control; (iii) PA. The PA group was separated from the dam and received asphyxic gas mixture (4% O2; 20% CO2 in N2) for 15 minutes, while Control animals received normal air following separation. Serum and tissue samples were collected after 4 (T4) or 24 (T24) hours. In a second experiment 35 min bilateral renal ischemic insult was performed on control and PA rats aged 6 months (n=6-7/group). Serum and tissue samples were collected 24 (T24 IR) hours after reperfusion (Figure 1). Serum levels of electrolytes, kidney and liver functional parameters, and myocardial ischemic protein Troponin I were determined. Highly selective and sensitive tubular injury markers (Kim1, Ngal) were measured. Expressions of hypoxic (Hif1a, Hif2a) inflammatory (Il1α, Il1β, Il6, Tnfα, Mcp1, Tlr2), apoptotic (Bax, Bcl-2) and angiogenic genes (Vegf, Epo) and heat shock proteins (Hsp27, Hsp72) were investigated. Periodic-Acid Schiff stained kidney sections and Hematoxylin & Eosin stained liver sections were evaluated for structural injury. Results Blood urea nitrogen (BUN) and serum GPT were elevated at T4 following PA. Kim1, Ngal and heat shock protein expressions were increased, inflammatory and angiogenic pathways were activated in the kidney after PA. In the liver hypoxic and apoptotic pathways were activated at T24 in controls and after asphyxia, but not in the Baseline group. Vacuolisation, cytoplasmic degradation, and the onset of necrosis were observed in the liver following PA. Serum Troponin I was elevated indicating myocardial damage, moreover inflammatory cytokines and heat shock proteins increased in the heart. In adult PA rats BUN levels were elevated, suggesting a long-term detrimental effect of PA on renal function. In addition, adult PA rats were more susceptible to renal ischemic insult, confirmed by higher serum creatinine and GPT levels, as well as increased expression of tubular injury, hypoxic and inflammatory markers compared to Control rats subjected to ischemia. Conclusion Acute renal, hepatic and myocardial impairment was observed after PA. These results may justify the need for clinical follow-up and novel treatment strategies for possible multi-organ damage. The molecular pathways described here are potential targets for therapeutic intervention. In addition, birth asphyxia may increase sensitivity to renal injury in adulthood, which may be worth considering in clinical situations with potential renal impairment.

Treatment With Resveratrol Ameliorates Mitochondrial Dysfunction During the Acute Phase of Status Epilepticus in Immature Rats.

The aim of the present study was to elucidate the effect of resveratrol (natural polyphenol) on seizure activity, production of ROS, brain damage and mitochondrial function in the early phase of status epilepticus (SE), induced in immature 12 day-old rats by substances of a different mechanism of action (Li-pilocarpine, DL-homocysteic acid, 4-amino pyridine, and kainate). Seizure activity, production of superoxide anion, brain damage and mitochondrial function were assessed by EEG recordings, hydroethidium method, FluoroJadeB staining and Complex I activity measurement. A marked decrease of complex I activity associated with the acute phase of SE in immature brain was significantly attenuated by resveratrol, given i.p. in two or three doses (25 mg/kg each), 30 min before, 30 or 30 and 60 min after the induction of SE. Increased O2.– production was completely normalized, brain damage partially attenuated. Since resveratrol did not influence seizure activity itself (latency, intensity, frequency), the mechanism of protection is likely due to its antioxidative properties. The findings have a clinical relevance, suggesting that clinically available substances with antioxidant properties might provide a high benefit as an add-on therapy during the acute phase of SE, influencing also mechanisms involved in the development of epilepsy.

488 Understanding astrocyte responses to perinatal white matter injury and their role in disease pathogenesis

White matter injury (WMI) is a complication of preterm birth and a major cause of long-term neurological morbidity. The disease is characterized by reactive microgliosis and astrocytosis, defective oligodendrocyte maturation, and in severe cases, neuronal death. Reactive astrocytes are known to play important roles in the pathogenesis of many diseases, in some cases favoring repair and in others contributing to neurodegeneration. The specific nature of astrocyte reactivity after WMI remains obscure. Here we report the results of experiments aimed to define astrocyte polarization after white matter injury and its contribution to disease outcomes. WMI was induced in 2 day-old rat pups using a combination of hypoxic-ischemic and inflammatory insults. We used immunopanning to purify astrocytes from brains of injured and control rats. mRNA isolated from these cells was used for microfluidic qRT-PCR analysis of a panel of known markers of reactive astrocyte subtypes. In situ hybridization was used to define the spatial distribution of the reactive astrocytes within the rodent brain. In situ hybridization experiments demonstrate the presence of inflammatory reactive astrocytes in subcortical white matter tracts after WMI in our rodent disease model. qRT-PCR using mRNA from pre- and post-injury astrocytes reveals the temporal regulation of astrocyte reactivity over time after injury. Ongoing experiments using transgenic mice aim to modulate the astrocyte response to WMI and to quantify consequent changes in disease outcomes. We demonstrate the formation of inflammatory reactive astrocytes in a rodent model of WMI and present work in progress to characterize the astrocyte response in perinatal WMI. Should ongoing experiments demonstrate that the formation of inflammatory astrocytes is a pivotal step in WMI pathophysiology, this finding will open the door to new therapeutic strategies for the treatment of WMI.

The presence of chemosensory cues transmitted through the mother's milk increases the search and ingestion responses toward bitter and sour solutions

Olfaction is of major importance during early stages of life in altricial species. This sense allows newborns to develop different behaviors that will allow them to survive. Odors tend to be associated to contextual stimuli (such as warmth); this, in turn, enables the pups to recognize when to withdraw or approach. At the same time, olfaction modulates the acceptance of aversive flavors. The increase of approach responses toward a bitter substance during early life is enhanced by stimulation with familiar, pre-exposed odors. Newborn rats exhibit heightened grasp responses toward an artificial nipple dispensing quinine, and drink more of this bitter solution, in the presence of a pre-exposed odor (lemon or the mother's odor). The present research assessed the replicability of previous results by pre-exposing the subjects to the scent through maternal milk and using solutions with different aversive tastes. Half of the subjects (3 day-old Wistar rats) were pre-exposed to lemon odor through the maternal milk (the mother had previously ingested the lemon essence via an intragastric injection); 4 h later, all the rats were evaluated in the presence of the lemon odor with an artificial nipple containing quinine, citric acid, saline solution, or water. The results showed enhanced seeking and intake of the bitter (quinine) and sour solution (citric acid). However, this did not occur when the nipple contained water or saline solution. The evidence suggests that: During the early stages of development, familiar odors regulate the acceptance of non-palatable, otherwise rejected, flavors; and that the route of transmission of the pre-exposed odor can be through air, or through food (amniotic fluid in previous studies and, in this case, breast milk), that is, via the retronasal and orthonasal routes.

Antidiabetes of Combination of Fractionated-extracts of Andrographispaniculata and Centellaasiatica in Neonatal Streptozotocin-induced Diabetic Rats

Many medicinal plants are widely grown in South- and Southeast Asia countries. Some of them are traditionally used for treatment of diabetes mellitus such as Andrographispaniculata and Centellaasiatica. In the study, we provided fractionated-extracts of A. paniculata and C. asiatica to increase the concentration of their active compounds and eliminate unexpected substances. The aim of the study was to evaluate the antidiabetes effect of the combination of their fractionated-extracts in male neonatal streptozotocin (STZ)-induced diabetic rats. In the study, diabetes was injected intraperitoneally 90mg.kg-1 BWSTZ to two day-old rats. At the age of three months, the rats were administered with the combination of both fractionated-extracts for 14 consecutive days. We evaluated antidiabetes with parameters of blood glucose levels, morphology of pancreatic islet, β-cell density as well as immunohistochemical pancreatic insulin. The levels of MDA, SOD and GPx were also determined about oxidative stress change after treatment with the combination. In the study, the combination succeeded to lower the blood glucose level in neonatal STZ-induced diabetic rats. Inline with fact, improvement of rat pancreatic islets and β cells density, as well as moderate restoration of pancreatic insulin, were observed after treatment with the combination. The substance could decrease the level of MDA, and increase the levels of SOD and GPx. In conclusion, the combination of fractionated-extracts of A. paniculata and C. asiatica exhibited potential antidiabetic effect to its antioxidative effect in male neonatal STZ-induced diabetes rats.

P0556MULTI-ORGAN DAMAGE FOLLOWING PERINATAL ASPHYXIA IN RAT MODEL

Abstract Background and Aims Perinatal asphyxia (PA) leads to the death of more than half a million mature newborns yearly. PA is associated with several serious complications, including hypoxic encephalopathy, renal- hepatic- and cardiovascular injury, as well as respiratory distress. Basic research and clinical trials mainly focus on mitigating central nervous system damage by selective head or whole body cooling, which is currently the only routinely used treatment in clinical practice. However, the extent of PA-associated multi-organ damage is not clarified yet and effective therapies are lacking. The aim of the present study was to determine the acute renal, hepatic and cardiac impairment following PA and to identify pathways involved in the pathomechanism. In addition, we aimed to investigate long-term effects of PA on permanent organ damage and susceptibility to ischemia/-reperfusion injury in adulthood. Method Postnatal 7 day-old male Wistar rat pups (n=5-10/group) were randomly grouped as follows: (i) Baseline; or separated from the dam and placed in a treatment chamber for 15 minutes (ii) Control normal air; (iii) PA 4% O2; 20% CO2 in N2 gas mixture. Serum and tissue samples were collected after 4 (T4) and 24 (T24) hours respectively. In a second experiment 35 min bilateral renal ischemic insult was performed on control and PA rats aged 6 months (n=6-7/group). Serum and tissue samples were collected 24 (T24 IR) hours after reperfusion (Figure 1). Serum levels of electrolytes, kidney and liver functional parameters, and myocardial ischemic protein Troponin I were determined. Highly selective tubular injury markers (Kim1, Ngal) were measured. Expressions of hypoxic (Hif1a, Hif2a) inflammatory (Il1α, Il1β, Il6, Tnfα, Mcp1, Tlr2), apoptotic (Bax, Bcl-2) and angiogenic genes (Vegf, Epo) and heat shock proteins (Hsp27, Hsp72) were investigated. Periodic-Acid Schiff staining and CD45 immunostaining on kidney tissue sections, and Hematoxylin & Eosin staining on liver tissue sections were performed. Results Blood urea nitrogen (BUN) and serum GPT were elevated at T4 following PA. Kim1, Ngal and heat shock protein expressions were increased, inflammatory and angiogenic pathways were activated in the kidney after PA. CD45 staining showed increased leukocyte infiltration. In the liver hypoxic and apoptotic pathways were activated at T24 in controls and after asphyxia, but not in the Baseline group. Vacuolisation, cytoplasmic degradation, and the onset of necrosis were observed in the liver following PA. Serum Troponin I was elevated indicating myocardial damage, moreover inflammatory cytokines and heat shock proteins increased in the heart. In adult PA rats BUN levels were elevated, suggesting a long-term detrimental effect of PA on renal function. In addition, adult PA rats were more susceptible to renal ischemic insult, confirmed by higher serum creatinine and GPT levels, as well as increased expression of tubular injury, hypoxic and inflammatory markers compared to Control rats subjected to ischemia. Conclusion Acute renal, hepatic and myocardial impairment was observed after PA. These results may justify the need for clinical follow-up and novel treatment strategies for possible multi-organ damage. The molecular pathways described here are potential targets for therapeutic intervention. In addition, birth asphyxia may increase sensitivity to renal injury even in adulthood, which may be worth considering in clinical situations with potential renal impairment such as major surgeries.

Hypercaloric diet models do not develop heart failure, but the excess sucrose promotes contractility dysfunction.

Several diseases are associated with excess of adipose tissue, and obesity is considered an independent risk factor for the development of cardiac remodeling and heart failure. Dietary aspects have been studied to elucidate the mechanisms involved in these processes. Thus, the purpose was the development and characterization of an obesity experimental model from hypercaloric diets, which resulted in cardiac remodeling and predisposition to heart failure. Thirty- day-old male Wistar rats (n = 52) were randomized into four groups: control (C), high sucrose (HS), high-fat (HF) and high-fat and sucrose (HFHS) for 20 weeks. General characteristics, comorbidities, weights of the heart, left (LV) and right ventricles, atrium, and relationships with the tibia length were evaluated. The LV myocyte cross sectional area and fraction of interstitial collagen were assayed. Cardiac function was determined by hemodynamic analysis and the contractility by cardiomyocyte contractile function. Heart failure was analyzed by pulmonary congestion, right ventricular hypertrophy, and hemodynamic parameters. HF and HFHS models led to obesity by increase in adiposity index (C = 8.3 ± 0.2% vs. HF = 10.9 ± 0.5%, HFHS = 10.2 ± 0.3%). There was no change in the morphological parameters and heart failure signals. HF and HFHS caused a reduction in times to 50% relaxation without cardiomyocyte contractile damage. The HS model presented cardiomyocyte contractile dysfunction visualized by lower shortening (C: 8.34 ± 0.32% vs. HS: 6.91 ± 0.28), as well as the Ca2+ transient amplitude was also increased when compared to HFHS. In conclusion, the experimental diets based on high amounts of sugar, lard or a combination of both did not promote cardiac remodeling with predisposition to heart failure under conditions of obesity or excess sucrose. Nevertheless, excess sucrose causes cardiomyocyte contractility dysfunction associated with alterations in the myocyte sensitivity to intracellular Ca2+.

Increased Hypothalamic Projections to the Lateral Hypothalamus and Responses to Leptin in Rat Neonates From High Fat Fed Mothers.

The lateral hypothalamus (LHA) is a central hub in the regulation of food intake and metabolism, as it integrates homeostatic and hedonic circuits. During early development, maturing input to and output from the LHA might be particularly sensitive to environmental dietary changes. We examined the effects of a maternal high fat diet (HFD, 60% Kcal in fat) on the density of hypothalamic projections to the orexin (ORX-A) field of the LHA in 10 day-old (PND10) rat pups using retrograde labeling with fluorescent microspheres. We also compared responsiveness of phenotypically identified LHA neurons to leptin administration (3 mg/kg, bw) between pups from control (CD) or high fat (HFD) fed mothers on PND10 and 15-16, at the onset of independent feeding. HFD pups exhibited a higher density of LHA projections (p = 0.05) from the ventromedial hypothalamus (VMH) compared to CD pups and these originated from both SF-1 and BDNF-positive neurons in the VMH. Increased circulating leptin levels in HFD pups, particularly on PND15-16 was consistent with enhanced pSTAT3 responses to leptin in the orexin (ORX-A) field of the LHA, with some of the activated neurons expressing a GABA, but not CART phenotype. ORX-A neurons colocalizing with pERK were significantly higher in PND15-16 HFD pups compared to CD pups, and leptin-induced increase in pERK signaling was only observed in CD pups. There was no significant effect of leptin on pERK in HFD pups. These results suggest that perinatal maternal high fat feeding increases hypothalamic projections to the ORX-A field of the LHA, increases basal activation of ORX-A neurons and direct responsiveness of LHA neurons to leptin. Since these various LHA neuronal populations project quite heavily to Dopamine (DA) neurons in the ventral tegmental area, they might participate in the early dietary programming of mesocorticolimbic reward circuits and food intake.

Effect of catechin isolate from gmb4 clone green tea on oxidative stress and apoptosis in experimental cataract

The purpose of this research is to investigate the effect of catechin Isolate from GMB4 Clone Green Tea on apoptotic cell death and oxidative stress in the lens of rats with cataract. Cataract Isolate from GMB4 Clone Green Tea was induced by intraperitoneal injection of 19μmol/kg sodium selenite to ten day-old Wistar rats. The neonatal rats were randomly divided into five groups (n=5 in each group): a control group, and four cataract-induction groups, treated with either 0, 50, 100, 200mg/kg catechin Isolate from GMB4 Clone Green Tea. We performed slit-lamp bio microscopic analysis, level of GSH and GR, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay and immunohistochemistry for caspase-3. Both eyes of all rats in Group 1 did not exhibit cataract formation. In Group 2, one out of five (20%) developed grade 3 cataracts and the remaining four out of five (80%) developed grade 4 cataracts. The difference in exhibited cataract in the lens of all rats between Group 2 and any eyes of groups 3 or 4 and 5 were significant (P = 0.022, 0,001, 0,001). The grade of cataract formation was decrease in group 3, 4 and 5. The mean GSH and Caspase-3 levels lenses of group II rats were significantly (P < 0.01) lower than the levels in Group I, Group III, Group IV and Group V. Apoptotic cell death and oxidative stress in the lens that increased following cataract formation in rats was suppressed by cathechin Isolate From GMB4 Clone Green Tea.

Persistent vs transient alteration of folliculogenesis and estrous cycle after neonatal vs adult exposure to Bisphenol A

Exposure to bisphenol A (BPA), a ubiquitous endocrine-disrupting chemical (EDC), is known to produce variable effects on female puberty and ovulation. This variability of effects is possibly due to differences in dose and period of exposure. Little is known about the effects of adult exposure to environmentally relevant doses of this EDC and the differences in effect after neonatal exposure. This study sought to compare the effects of neonatal vs adult exposure to a very low dose or a high dose of BPA for 2 weeks on ovulation and folliculogenesis and to explore the hypothalamic mechanisms involved in such disruption by BPA. One-day-old and 90-day-old female rats received daily subcutaneous injections of corn oil (vehicle) or BPA (25 ng/kg/d or 5 mg/kg/d) for 15 days. Neonatal exposure to both BPA doses significantly disrupted the estrous cycle and induced a decrease in primordial follicles. Effects on estrous cyclicity and folliculogenesis persisted into adulthood, consistent with a disruption of organizational mechanisms. During adult exposure, both doses caused a reversible decrease in antral follicles and corpora lutea. A reversible disruption of the estrous cycle associated with a delay and a decrease in the amplitude of the LH surge was also observed. Alterations of the hypothalamic expression of the clock gene Per1 and the reproductive peptide phoenixin indicated a disruption of the hypothalamic control of the preovulatory LH surge by BPA.

Heat shock response in noise-induced hearing loss: effects of alanyl-glutamine dipeptide supplementation on heat shock proteins status

IntroductionThe 72kDa heat shock protein, HSP72, located intracellularly provides cochlear cytoprotective and anti-inflammatory roles in the inner ear during stressful noise challenges. The expression of intracellular HSP72 (iHSP72) can be potentiated by alanyl-glutamine dipeptide supplementation. Conversely, these proteins act as pro-inflammatory signals in the extracellular milieu (eHSP72). ObjectiveWe explore whether noise-induced hearing loss promotes both intracellular and extracellular HSP72 heat shock response alterations, and if alanyl-glutamine dipeptide supplementation could modify heat shock response and prevent hearing loss. MethodsFemale 90 day-old Wistar rats (n=32) were randomly divided into four groups: control, noise-induced hearing loss, treated with alanyl-glutamine dipeptide and noise-induced hearing loss plus alanyl-glutamine dipeptide. Auditory brainstem responses were evaluated before noise exposure (124dB SPL for 2h) and 14days after. Cochlea, nuclear cochlear complex and plasma samples were collected for the measurement of intracellular HSP72 and extracellular HSP72 by a high-sensitivity ELISA kit. ResultsWe found an increase in both iHSP72 and eHSP72 levels in the noise-induced hearing loss group, which was alleviated by alanyl-glutamine dipeptide treatment. Furthermore, H-index of HSP72 (plasma/cochlea eHSP72/iHSP72 ratio) was increased in the noise-induced hearing loss group, but prevented by alanyl-glutamine dipeptide treatment, although alanyl-glutamine dipeptide had no effect on auditory threshold. ConclusionsOur data indicates that cochlear damage induced by noise exposure is accompanied by local and systemic heat shock response markers. Also, alanyl-glutamine reduced stress markers even though it had no effect on noise-induced hearing loss. Finally, plasma levels of 72kDa heat shock proteins can be used as a biomarker of auditory stress after noise exposure.

Pairing Electrophysiology and Single Cell RNA Sequencing to Identify Mechanisms of Cellular pH/CO2 Sensitivity in Respiratory Chemoreceptor Neurons

Respiratory chemoreceptors are specialized neurons that are excited by hypercapnic acidosis (HA) and encode brain pH and/or CO2 levels to drive the appropriate changes in breathing. A subset of serotonin‐producing (5‐HT) neurons develop an apparent intrinsic chemosensitivity to HA after ~P12, but the molecular mechanisms driving their cellular pH/CO2 sensitivity remains unknown. Given that Sudden Infant Death Syndrome (SIDS) in humans has been linked to developmental 5‐HT system defects and potential alterations in ventilatory chemoreflexes, we aimed to develop a new technique to identify transcriptional differences among pH‐sensitive and ‐insensitive 5‐HT neurons in order to ultimately define the genetic determinants of cellular pH/CO2 sensitivity. Acute brainstem slices (200 uM) were prepared from 18–23 day‐old transgenic Dahl Salt Sensitive rats that express eGFP in all 5‐HT neurons (SSeGFP). Firing rates were measured via cell‐attached patch in eGFP+ 5‐HT neurons superfused with an artificial CSF (aCSF) containing inhibitors for synaptic blockade (10 mM CNQX, 50 mM D‐AP5, 20 mM Gabazine) while cycling between control (5% CO2 bal. O2; pH= 7.36; 5 min) and HA (15% CO2 bal. O2; pH= 7.10; 10 min) conditions. Once the cellular phenotype was established for pHsensitive (n=6 cells) or ‐insensitive (n=5) cells, a second electrode filled with small amounts (&lt;1ul) of intracellular solution spiked with RNAse inhibitor was used to isolate the cellular contents for subsequent single cell RNA Sequencing (scRNA‐Seq). Specificity of cellular isolation was confirmed by high levels of expression of several serotonin and neuronal genes, and a lack of glial gene expression by sc‐qRT‐PCR (n=7) or sc‐RNAseq. The scRNA‐Seq read summary indicated an average of ~45M reads, a quality score of ~33.5, and ~80% mapping rate per sample, from which &gt;10,000 known genes were quantified. However, small sample sizes prevented obtaining statistical significance for differential expression (Q &gt; 0.05). Using R software packages, unsupervised analyses (hierarchical clustering and principal component analyses) failed to clearly separate samples by phenotype, and a power analysis based on FPKM values from the 11 samples suggested sample sizes of 20–60 cells/group are required. However, a supervised analysis (pre‐defining experimental groups) using Support Vector Machine (SVM) learning coupled with Recursive Feature Elimination (i.e. creates a model with all genes and recursively reduces the number of genes to identify the fewest genes to explain the predefined phenotype) revealed a candidate molecular signature for CO2 sensitive 5‐HT neurons. Overall, we demonstrated we can obtain transcriptomic data from electrophysiologically‐characterized 5‐HT neurons at the single cell‐level, and analyses of these pilot data suggest unsupervised and supervised machine learning approaches may identify unique genetic profiles in pH‐sensitive 5‐HT neurons. These data provide the field of respiratory physiology with preliminary markers of 5‐HT neuron CO2/pH sensitivity and validate that this approach may be used to address questions pertaining to genetic determinants of a given cellular phenotype.Support or Funding InformationNIH R01 HL12358 04 &amp; Parker B. Francis FoundationThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Neonatal exposure to the experimental environment or ketamine can induce long-term learning dysfunction or overmyelination in female but not male rats.

Ketamine can induce neurotoxicity after exposures to the developing brain. To investigate whether ketamine at subanesthetic dosage or its environmental condition can cause long-term cognitive dysfunction after multiple exposures in male or female neonatal rats, postnatal day 5 (P5)-day-old Sprague-Dawley rats were randomized into three groups: ketamine group, vehicle group, and control group (no disturbance). Learning and memory abilities from P60 to P65 and immunofluorescence tests for myelin basic protein (MBP) in gray matter on P65 were conducted. The results showed that in female rats, the path length on day 1 in ketamine group and on days 1 and 2 in vehicle group was longer than that in control (P<0.05), but there was no difference between ketamine and vehicle groups (P>0.05). The mean density of MBP in the medial prefrontal cortex (mPFC) was significantly increased in vehicle and ketamine groups compared with that in control (P<0.05), and there was a significant difference between vehicle and ketamine groups (P<0.05), but MBP density was not changed in CA1 or CA3 region (P>0.05). In male rats, there were no significant differences in path length among the groups, and the density of MBP in the mPFC and hippocampus in vehicle or ketamine group was not different from that in control (P>0.05). Pearson’s correlation analysis showed that there was a positive correlation between MBP density in the mPFC and path length in adult female rats (r=0.753, P<0.01). Overall, the results suggested that neonatal female rats exposed to multiple episodes of the experimental environment can develop learning dysfunction in adulthood, which may result from overmyelination in the mPFC, but male rats were not affected. Ketamine could increase myelination in the mPFC in female rats, but it did not induce learning dysfunction in adulthood; therefore, ketamine may be a safe drug for pediatric anesthesia.