Juvenile Wistar Rats Research Articles

Azoospermia and multi-organ damage in juvenile rats exposed to α-Terpineol from weaning to sexual maturity

This study aimed to evaluate the repeated oral administration of α-terpineol in juvenile Wistar rats over a 70-day period. The objective was to assess the potential systemic and reproductive toxicity of α-terpineol when administered by gavage at doses of 75, 150, and 300 mg/kg/day to juvenile Wistar rats for 70 days from postnatal day 24. The control group received corn oil for 70 days. During the study, various parameters were evaluated, including clinical signs, body weight, food intake, neurobehavioral observations, haematology, serum biochemistry, organ weights, steroidogenic gene expression, and histopathological examination. No toxicity-related changes were observed in body weight, food intake, neurobehavioral observations, or steroidogenic gene expression. However, sperm evaluation revealed a complete absence of sperm and delayed sexual maturation. Total cholesterol was significantly elevated in both sexes, and serum testosterone was reduced at the 150 and 300 mg/kg doses. Microscopic examination showed severe pathological changes in the testes, epididymis, liver, and kidneys of both males and females. After the 14-day recovery period, total cholesterol levels returned to the normal range, but no recovery was observed in the other organs. The no-observed-adverse-effect level was 75 mg/kg/day for male rats based on the histopathological findings in the testes, liver, and kidneys, and for female rats based on the kidney and liver histopathology.

Moderate aerobic training counterbalances the deleterious effect of undernutrition on oxidative balance and mitochondrial markers

The state of Maternal Protein Malnutrition (MPM) is associated with several deleterious effects, including inflammatory processes and dysregulation in oxidative balance, which can promote neurodegeneration. On the other hand, it is known that aerobic exercise can promote systemic health benefits, combating numerous chronic diseases. Therefore, we evaluate the effect of aerobic exercise training (AET) on indicators of mitochondrial bioenergetics, oxidative balance, endoplasmic reticulum stress, and neurotrophic factor in the prefrontal cortex of malnourished juvenile Wistar rats. Pregnant Wistar rats were fed with a diet containing 17% or 8% casein during pregnancy and lactation. At 30 days of life, male offspring were divided into 4 groups: Low-Protein Control (LS), Low-Protein Trained (LT), Normoprotein Control (NS), and Normoprotein Trained (NT). The trained groups performed an AET for 4 weeks, 5 days a week, 1 h a day per session. At 60 days of life, the animals were sacrificed and the skeletal muscle, and prefrontal cortex (PFC) were removed to evaluate the oxidative metabolism markers and gene expression of ATF-6, GRP78, PERK and BDNF. Our results showed that MPM impairs oxidative metabolism associated with higher oxidative and reticulum stress. However, AET restored the levels of indicators of mitochondrial bioenergetics, in addition to promoting resilience to cellular stress. AET at moderate intensity for 4 weeks in young Wistar rats can act as a non-pharmacological intervention in fighting against the deleterious effects of a protein-restricted maternal diet.

Increased Depressive-like, Anxiety-like, and Perseverative-like Behavior in Binge Eating Model in Juvenile Rats.

The aim of the present study was to evaluate depressive-like, anxiety-like, and perseverative-like behaviors in a binge eating model. Juvenile Wistar rats, using the binge eating model, were compared to caloric restriction, induced stress, and control groups. Rats of the induced stress group presented binge-like behaviors in standard food intake in the second cycle of the experiment when compared to the caloric restriction group and the binge eating model group. Depressive-like behavior was observed in the binge eating model group with longer immobility time (p < 0.001) and less swim time (p < 0.001) in comparison to the control group. Anxiety-like behavior was observed by shorter duration of burying latency in the binge eating model group when compared to the induced stress group (p = 0.04) and a longer duration of burying time when compared to the control group (p = 0.02). We observed perseverative-like behavior by the binge model group, who made more entries to the new arm (p = 0.0004) and spent a longer time in the new arm when compared to the control group (p = 0.0001). Our results show differences in behaviors between the groups of rats studied. These results suggest that calorie restriction-refeeding, along with stress, may lead to depressive-like, anxiety-like, and perseverative-like behavioral changes in male Wistar rats.

Adolescent food insecurity in female rodents and susceptibility to diet-induced obesity

Food insecurity is defined as having limited or uncertain access to nutritious foods, and adolescent food insecurity is associated with obesity and disordered eating behaviors in humans. We developed a rodent model of adolescent food insecurity to determine whether adolescent food insecurity per se promotes increased susceptibility to diet-induced obesity and altered eating behaviors during adulthood. Female juvenile Wistar rats were singly housed and assigned to three experimental diets: food-secure with standard chow (CHOW), food-secure with a high-fat/sugar Western diet (WD), and food-insecure with WD (WD-FI). Food-secure rats (CHOW and WD) received meals at fixed feeding times (9:00, 13:00, and 16:00). WD-FI rats received meals at unpredictable intervals of the above-mentioned feeding times but had isocaloric amounts of food to WD. We investigated the impact of adolescent food insecurity on motivation for sucrose (Progressive Ratio), approach-avoidance behavior for palatable high-fat food (Approach-Avoidance task), and susceptibility to weight gain and hyperphagia when given an obesogenic choice diet. Secondary outcomes were the effects of food insecurity during development on anxiety-like behaviors (Open Field and Elevated Plus Maze) and learning and memory function (Novel Location Recognition task). Rodents with adolescent food insecurity showed a greater trend of weight gain and significantly increased fat mass and liver fat accumulation on an obesogenic diet in adulthood, despite no increases in motivation for sucrose or high-fat food. These data suggest that adolescent unpredictable food access increases susceptibility to diet-induced fat gain without impacting food motivation or food intake in female rodents. These findings are among a small group of recent studies modeling food insecurity in rodents and suggest that adolescent food insecurity in females may have long-term implications for metabolic physiology later in life.

Probiotic Bacteria from Human Milk Can Alleviate Oral Bovine Casein Sensitization in Juvenile Wistar Rats.

This study aims to see if probiotic bacteria from human milk could ameliorate oral cow's milk sensitization. The probiotic potential of the SL42 strain isolated from the milk of a healthy young mother was first determined. Rats were then randomly gavaged with cow's milk casein without an adjuvant or assigned to the control group. Each group was further subdivided into three groups, with each receiving only Limosilactobacillus reuteri DSM 17938, SL42, or a phosphate-buffered saline solution. Body weight, temperature, eosinophils, serum milk casein-specific IgE (CAS-IgE), histamine, and serum S100A8/A9 and inflammatory cytokine concentrations were measured. The animals were sacrificed after 59 days; histological sections were prepared, and the spleen or thymus weights, as well as the diversity of the gut microbiota, were measured. On days 1 and 59, SL42 abridged systemic allergic responses to casein by dropping histamine levels (25.7%), CAS-specific IgE levels (53.6%), eosinophil numbers (17%), S100A8/9 (18.7%), and cytokine concentrations (25.4-48.5%). Analyses of histological sections of the jejunum confirmed the protective effect of probiotic bacteria in the CAS-challenged groups. Lactic acid bacteria and Clostridia species were also increased in all probiotic-treated groups. These findings suggest that probiotics derived from human milk could be used to alleviate cow's milk casein allergy.

Calcium Supplementation Ameliorates Cerebellar Oxidative Stress in Lactational Aluminum-induced Neurotoxicity in Rats.

The neurotoxic effects of aluminum exposure during the critical period of neurodevelopment have been well documented. This study investigated the known protective effects of calcium supplementation on the cerebellum of juvenile Wistar rats following aluminum-induced neurotoxicity during lactation. Four groups of juvenile rats were exposed via lactation to distilled water (control group), aluminum (40 mg/kg/d), calcium supplement (50 mg/kg/d), and a combination of both aluminum and calcium from postnatal day 4 to day 28. The cerebella of the animals were excised to access the levels of antioxidant enzymes (superoxide dismutase [SOD], glutathione peroxidase [GPx]), lipid peroxidation (malondialdehyde), histomorphological alterations (hematoxylin and eosin staining), Nissl profile (cresyl fast violet staining), and glial activation (glial fibrillary acidic protein immunohistochemistry). Lactational aluminum significantly decreased the activities of superoxide dismutase and glutathione peroxidase while exacerbating lipid peroxidation and reactive astrocyte in cerebellar lysates. Lactational calcium supplementation normalized the activities of SOD and GPx, thereby preventing excessive lipid peroxidation and glial activation. Despite no apparent changes in the general histology of the cerebellum, aluminum-induced chromatolysis changes in the Purkinje cell layer, which was counteracted by the antioxidant propensities of calcium supplementation. These findings support that calcium supplementation significantly protects the cerebellum against aluminum-induced oxidative stress, chromatolysis, and neuroinflammation.

Undernutrition during development modulates endoplasmic reticulum stress genes in the hippocampus of juvenile rats: Involvement of oxidative stress

To evaluate whether exercise training mitigates the deleterious effects of undernutrition during the developmental period in juvenile Wistar rats. Pregnant Wistar rats were fed with a diet containing 17 % or 8 % casein during pregnancy and lactation. At 30 days of life, male offspring were divided into 4 groups: Low-Protein non-trained (LS), Low-Protein Trained (LT), Normoprotein non-trained (NS), and Normoprotein Trained (NT). Trained rats performed aerobic exercise training (AET) for 4 weeks, 5 days a week, 1 h a day. 24 h from the last day of training, the animals were sacrificed. The tissues were removed to analyze indicators of mitochondrial metabolism, oxidative stress, and gene expression of GRP78, PERK, ATF6 ER stress markers, and BDNF. The results showed that undernutrition during development promotes deleterious effects on mitochondrial oxidative metabolism and induces reticulum stress in the hippocampus of juvenile rats. On the other hand, AET improves mitochondrial function and increases enzymatic and non-enzymatic antioxidant capacity, as well as declines ER stress. AET at moderate intensity for 4 weeks in male juvenile Wistar rats acts as a lifestyle intervention opposing the negative effects induced by a protein-restricted maternal diet.

Structural Changes in the Medial Prefrontal Cortex and Anterior Cingulate Cortex of Dehydroepiandrosterone-Induced Wistar Rat Model of Polycystic Ovarian Syndrome.

Polycystic ovary syndrome (PCOS) is a complex endocrine disorder in women that is associated with an increased risk of infertility. This study aims to evaluate the neurobehavioral and neurochemical changes along with the associated changes in the medial prefrontal cortex (mPFC) and anterior cingulate cortex (ACC) of the dehydroepiandrosterone (DHEA)-induced PCOS model rats. A total of 12 female juvenile Wistar rats (30 to 50 g) about 22 to 44 days old were divided into 2 groups. The control group received sesame oil while the PCOS group received sesame oil plus DHEA. All treatment was done via daily subcutaneous injection for 21 days. Subcutaneous DHEA-induced PCOS significantly depleted the line crossing and rearing frequency in the open field, along with the percentage of the time in the white box, line crossing, rearing, and peeping frequency in the black and white box, and the percentage of alternation in the Y-maze. PCOS significantly increased the immobility time, freezing period, and the percentage of time in the dark area in the forced swim test, open field test, and black and white box, respectively. The level of luteinizing hormone, follicle-stimulating hormone, malondialdehyde (MDA), reactive oxygen species (ROS), and interleukin-6 (IL-6) increased significantly, while norepinephrine depleted significantly with an obvious decrease in the brain-derived neurotrophic factor level in the PCOS model rats. PCOS rats exhibited cystic follicles in the ovaries and necrotic or degenerative like features in the hippocampal pyramidal cells. DHEA-induced PCOS results in anxiety and depressive behavior with structural alteration in rats, possibly through the elevation of MDA, ROS, and IL-6 levels, which also attributes to impaired emotional and executive functions in the mPFC and ACC.

Proanthocyanidin from Vitis vinifera attenuates memory impairment due to convulsive status epilepticus

This study investigated the effects of proanthocyanidin-rich fraction (PRF) of Vitis vinifera seed extract on the markers of hippocampal-dependent memory in convulsive status epilepticus (CSE) rat model.One hundred juvenile Wistar rats were randomized into 6 groups. Group 1 (n = 10) received propylene glycol (PG 0.1 ml/100 g) intraperitoneally (i.p), while convulsion was induced in groups 2–6 (n = 18 each) using lithium (127 mg/kg i.p) and pilocarpine hydrochloride (40 mg/kg i.p). The established CSE rats in groups 2–6 received a daily treatment of PG (0.1 ml i.p), PRF (30 mg/kg i.p), PRF (20 mg/kg BW i.p), PRF (10 mg/kg BW i.p) or diazepam (5 mg/kg BW i.p) for seven days. Thereafter, they were kept untreated but with access to feed and water for 21 days. The control and CSE-treated rats were subjected to behavioral tests, while the biochemical and histomorphological evaluations of the hippocampus were done after the sacrifice. The results were presented as mean ± SEM in graphs or tables. The level of significance was considered when p < 0.05.There was significant decrease in the hippocampal-dependent memory, hippocampal weight and an increased malondialdehyde concentration following CSE. The activities of acetylcholinesterase decreased significantly in the PRF-treated CSE rats. The hippocampal glial cells and granule count increased significantly following CSE, with various neurodegenerative features in the CA1 of the hippocampus. These derangements were attenuated significantly following PRF treatment.Memory impairment following CSE may be attenuated with the administration of PRF from V. vinifera seed in rats.

The role of intermittent PTH administration in conjunction with allogenic stem cell treatment to stimulate fracture healing.

AimsA growing number of fractures progress to delayed or nonunion, causing significant morbidity and socioeconomic impact. Localized delivery of stem cells and subcutaneous parathyroid hormone (PTH) has been shown individually to accelerate bony regeneration. This study aimed to combine the therapies with the aim of upregulating fracture healing.MethodsA 1.5 mm femoral osteotomy (delayed union model) was created in 48 female juvenile Wistar rats, aged six to nine months, and stabilized using an external fixator. At day 0, animals were treated with intrafracture injections of 1 × 106 cells/kg bone marrow mesenchymal stem cells (MSCs) suspended in fibrin, daily subcutaneous injections of high (100 μg/kg) or low (25 μg/kg) dose PTH 1-34, or a combination of PTH and MSCs. A group with an empty gap served as a control. Five weeks post-surgery, the femur was excised for radiological, histomorphometric, micro-CT, and mechanical analysis.ResultsCombination therapy treatment led to increased callus formation compared to controls. In the high-dose combination group there was significantly greater mineralized tissue volume and trabecular parameters compared to controls (p = 0.039). This translated to significantly improved stiffness (and ultimate load to failure (p = 0.049). The high-dose combination therapy group had the most significant improvement in mean modified Radiographic Union Score for Tibia fractures (RUST) compared to controls (13.8 (SD 1.3) vs 5.8 (SD 0.5)). All groups demonstrated significant increases in the radiological scores – RUST and Allen score – histologically compared to controls.ConclusionWe demonstrate the beneficial effect of localized MSC injections on fracture healing combined with low- or high-dose teriparatide, with efficacy dependent on PTH dose.Cite this article: Bone Joint Res 2021;10(10):659–667.

Postnatal exposure to fluoxetine led to cognitive-emotional alterations and decreased parvalbumin positive neurons in the hippocampus of juvenile Wistar rats.

The exposure to selective serotonin reuptake inhibitors (SSRIs) during development results in behavioural impairment in adulthood in humans and animal models. Indeed, serotonergic overexpression in early life leads to structural and functional changes in brain circuits that control cognition and emotion. However, the effects of developmental exposure to these substances on the behaviour of adolescent rats are conflicting and remain poorly characterised. We performed a behavioural screening to investigate the effects of postnatal exposure to fluoxetine on memory and behaviours related to anxiety, anhedonia, and depression, as well we evaluate the parvalbumin expression in hippocampus of juvenile (~PND45) female and male rats. Fluoxetine (daily 20 mg/kg s.c. injections from PND7-PND21)- or vehicle-treated adolescent rats went through several behavioural tasks (from PND 38 to PND52) and were subject to transcardial perfusion and brain removal for immunohistochemical analysis (PND53). We found that postnatal exposure to fluoxetine increased anxiety- and depression-like behaviours in the open field and sucrose preference and forced swimming tests, respectively. In addition, this treatment induced working memory and short-term (but not long-term) recognition memory impairments, and reduced parvalbumin-positive interneurons in the hippocampus. In addition, the results revealed developmental sex-dependent effects of fluoxetine postnatal treatment on adolescent rats' behaviour. These outcomes indicate that affective disorders and mnemonic alterations caused by SSRIs perinatal exposure can be present at adolescence.

Effects of Spondias mombin leaf extracts on testicular histology and semen characteristics of prepubertal (juvenile) Wistar rats

Introduction: Spondias Mombin is an Anacardiaceae reported in folklore to be a useful therapeutic agent for some reproduction-related issues. This work seeks to explore, preliminarily, the fertility potentials of Spondias Mombin extracts on juvenile Wistar rats. Methods: One hundred pre-pubertal male Wistar rats divided into four treatment groups were studied for testicular histology, semen evaluation and haematocrit following oral administration of 800mg/kg Body weight (BW) of ethyl acetate (A), isopropanol (B) and methanol (C) Spondias mombin leaf ex-tracts and propylene glycol (D) for 35 days. Five rats were sacrificed from each group every seven days of extract administration for the aforementioned evaluations using standard methods. Results: Body weights increased markedly from week 1 to week 3 in all the groups and the weight gain continued with the Isopropanol (ISP) group till week 5 (48.00±10.36g) in contrast to fluctuations ob-served in other groups. All extract-treated groups had peak Packed Cell Volume (PCV) at week 2 with highest (54.00±3.16) seen in methanol (METH) group. All the groups experienced transition from azoospermia at week 1 to sexual maturity by week 5 as evidenced by progressive increase in the spermatozoa parameters evaluated. At week two, the EA group was the only group with motility score (67.00±5.38%) suggestive of puberty attainment. Normal histology was observed in testes and epididymides in all groups after 5 weeks of extract administration. Significance: Ethyl acetate leaf extract of Spondias mombin appeared to fast track attainment of puberty in Wistar rat at 800mg/kg BW. The plant’s leaf methanol extract at the same concentration appeared to boost PCV and promote fertility in Wistar rats significantly.

A Study of 4G Radiofrequency Radiation effects on Juvenile Wistar Rats Cerebellum and Potential Attenuative Properties of Fish Oil Omega-3 Fat

Introduction: Technological devices have become a very important part of our everyday life. These electronic devices create an artificial electromagnetic field (EMF) and emit radiofrequency radiations (RFR) which have been reported to have deleterious effects on various tissues in living organisms. Fish oil is a rich source of the n-3 (also known as omega-3) fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The cerebellum plays an important role in motor control and the coordination, precision and timing of movements, as well as in motor learning. This study investigated the effect of radiofrequency radiation on the structures of the cerebellum of adolescent Wistar rats, its effect on pre-pubertal development and the potential attenuative properties of fish oil omega-3 fat.&#x0D; Materials and Methods: Forty (n=40) adolescent Wistar rats of approximately 35 days old were divided into five groups labelled A-E. A 4G RFR-emitting WI-FI device served as the RFR source. The 4G radiofrequency transmitter was installed at a distance of 0.5 meters from the base of the cage housing the experimental animals in each group. The fish oil was administered orally to the animals. Rats were sacrificed after 28 days of treatment and their brains harvested. The cerebellar tissues were excised and processed for histological, immunohistochemical and biochemical observations. Neurobehavioural studies including Barnes Maze and beam walk were carried out before sacrifice.&#x0D; Results: 4G radiofrequency radiation caused distortions in the morphology and spatial arrangement of Purkinje cells in the cerebellar tissues. It also induced neuroinflammation as marked by enhanced astrocyte reactions. Also, behavioural aberrations including negative changes in key memory parameters such as increased latency and error poke in Barnes maze assays, and increased movement parameter errors were attributable to FRR effects. Also, aberrations in neurotransmitters activities levels and vital neurochemistry enzymes were observed. The administered Omega-3 fat had an attenuative effect on the affected cortical structures, neurochemistry and selected neurobehavioural parameters.

Роль тиреоїдних гормонів у формуванні просторової пам’яті щурів у ранньому онтогенезі

Changes in behavioral and mnestic activity, as well as their neurochemical support in thyroid dysfunction were determined in juvenile Wistar rats. Behavioral activity was studied in an elevated cruciform labyrinth, the study of spatial memory was performed by the development of protective avoidance reaction in the Morris water labyrinth, and by the production of food reactions in the 8-beam labyrinth. The content of free amino acids of the neurotransmitter spectrum and serotonin was determined by thin layer chromatography followed by spectrophotometry. It was found that thyroid dysfunction in early ontogenesis was accompanied by significant impairments of emotional and cognitive activity depending on the thyroid status of rats; differences in the mechanisms of the formation of spatial memory with negative and positive reinforcement were also found. At the same time, the anxiolytic type of behavior and formation of spatial memory in juvenile animals with experimental hyperthyroidism are possibly provided by an increase in GABA content in the neocortex by 40% and a decrease in serotonin level in the hippocampus by 32%. Experimental hypothyroidism caused an anxiogenic effect and cognitive impairment, which were accompanied by an excessive increase by 51% in the neocortical serotonin content.

Acute Alcohol Tissue Damage: Protective Properties of Betaine

Teenage binge drinking is a major health issue; however, there is a paucity of data on liver injury. Herein, we investigated how acute ethanol affects juvenile hepatic cells through changes in oxidative stress, apoptosis, and liver function, as well as the ability of betaine, which can replen-ish the antioxidant glutathione and mitigate oxidative injury. Juvenile male Wistar rats were given either water or betaine (2% w/v) for 6 days and treated with either saline 0.15 mol/L NaCl or ethanol (75 mmol/kg bodyweight). After 24 h, liver enzymes, oxidative damage, apoptosis, and parameters of antioxidant enzyme activity were examined. Acute ethanol increased hepatic enzymes (99%, P &lt; 0.05). Total protein and albumin levels were reduced by 14 and 18% (P &lt; 0.001), respectively, which was prevented by betaine treatment. Cytosolic cytochrome c increased by 59% (P &lt; 0.05), corresponding to a decrease in mitochondrial cytochrome c content, which was ameliorated with betaine. Cytosolic glutathione peroxidase was reduced with alcohol (P &lt; 0.05) and was prevented with betaine. Subtle changes were observed in catalase, superoxide dismutase, glutathione reductase, and complex I activity after ethanol treatment. In summary, whilst juvenile animals appear to have higher basal levels of antioxidant enzymes, betaine conferred some protection against alcohol-induced oxidative stress.

Similar hepatoprotective effectiveness of Diphenyl diselenide and Ebselen against cisplatin-induced disruption of metabolic homeostasis and redox balance in juvenile rats

Cisplatin is an antineoplastic drug well recognized for its success in the battle against several types of cancer in adult, juvenile, and child populations. Meanwhile, this drug is also famous due to its serious side effects, such as hepatotoxicity. This study evaluated the hepatoprotective effectiveness of Diphenyl Diselenide (PhSe)2 and Ebselen in a model of cisplatin-induced toxicity in juvenile rats. Juvenile Wistar rats received a single intraperitoneal (i.p) injection of cisplatin (6 mg/kg) or saline solution, at postnatal day (PND) 21. Ebselen (11 mg/kg) or (PhSe)2 (12 mg/kg) was intragastrically (i.g) administered in rats from PND 21 to PND 25. At PND 26, the blood and liver were collected for the biochemistry assays. A single administration of cisplatin was enough to alter the makers of hepatic function (an increase of AST activity) and the blood lipid profile (an increase of cholesterol and triglycerides, TG). The cisplatin-induced metabolic disruption was demonstrated by the increase of hepatic glycogen and TG contents and hexokinase, glucose-6-phosphatase, and tyrosine aminotransferase activities; a decrease of citrate synthase activity and the levels of GLUT-2. Cisplatin-induced hepatic oxidative stress was characterized by an increase in reactive oxygen species, TBARS, protein carbonyl, and Nox levels as well as the decrease in NPSH levels. Ebselen and (PhSe)2 were effective against all alterations caused by this chemotherapy medication. The present findings highlight the (PhSe)2 and Ebselen similar hepatoprotective effectiveness against cisplatin-induced disruption of metabolic homeostasis and redox balance in juvenile rats.

Early-life stress affects behavioral and neurochemical parameters differently in male and female juvenile Wistar rats.

Neonatal handling is an early life stressor that leads to behavioral and neurochemical changes in adult rats in a sex-specific manner and possibly affects earlier stages of development. Here, we investigated the effects of neonatal handling (days 1-10 after birth) on juvenile rats focusing on biochemical parameters and olfactory memory after weaning. Male neonatal handled rats performed more crossings on the hole-board task, increased Na+ /K+ -ATPase activity in the olfactory bulb, and decreased acetylcholinesterase activity in the hippocampus versus non-handled males. Female neonatal handled animals increased the number of rearing and nose-pokes on the hole-board task, decreased glutathione peroxidase activity, and total thiol content in the hippocampus versus non-handled females. This study reinforces that early life stress affects behavioral and neurochemical parameters in a sex-specific manner even before the puberty onset.

SUN-608 Juvenile Toxicity Study of Livoletide: A Peptide Analogue of Unacylated Ghrelin for the Treatment of Hyperphagia in Prader-Willi Syndrome

Background: Prader-Willi syndrome (PWS) is a rare endocrine disorder characterized by hyperphagia and abnormal food-related behaviors that contribute to severe morbidity, early mortality, and significant burdens for patients and caregivers. Dysregulation of acylated ghrelin (AG) and unacylated ghrelin (UAG) in people with PWS and hyperphagia has led to the hypothesis that replacing the relative deficit of UAG observed in this population will reduce hyperphagia. Livoletide is a cyclic, 8 amino acid analogue of UAG being developed as a potential treatment for hyperphagia and food-related behaviors associated with PWS. In a previous Phase 2a study (n=47) in people with PWS, livoletide improved food-related behaviors and hyperphagia scores relative to placebo. An extensive nonclinical safety program to support clinical development of livoletide has been conducted. Here we report the results of study of livoletide in juvenile rats. Methods: Male and female juvenile Wistar rats were administered livoletide (10, 25, or 75 mg/kg/day, subcutaneous) once daily from the age of weaning (postnatal day 21) until 12 weeks of age (n=32/sex/group). Subgroups were sacrificed at the end of the dosing period or after a recovery period. Toxicity was assessed based on local tolerance, clinical observations, body weights, food consumption, tibia length, bone densitometry, behavioral tests, IGF1 (insulin-like growth factor-1), LH (luteinizing hormone), and other endpoints. Macroscopic (necropsy), microscopic/histopathological, and caesarean examinations were also performed. Toxicokinetic (TK) evaluations were performed at various time-points after dosing to assess exposure. Results: Livoletide was not associated with treatment-related clinical signs or any relevant changes in hematological, coagulation, clinical chemistry parameters or urine analysis. Body weight and food consumption were unaffected. Livoletide did not affect systemic concentrations of IGF-1 or LH. No adverse effects of livoletide on bone growth, mating performance, or fertility were observed. Pups delivered by caesarian section did not exhibit abnormalities. Results were consistent with a lack of effect of livoletide on growth hormone signaling. Histological changes were limited to minimal local reversible reactions at injection sites that were non-adverse and observed at low incidence and severity. Conclusions: Livoletide was well-tolerated and not associated with evidence of overt systemic toxicity. The no observed adverse effect level (NOAEL) of 75 mg/kg/day was associated with Cmax and AUC0-24h values of 72.6/83.2 μg/mL and 169/144 μg.h/mL (males/females), respectively. These exposures are >100-fold above the anticipated clinical exposures in the Phase 2b/3 ZEPHYR study, which is enrolling people ages 4 to 65 with PWS.

Deficit of Long-Term Potentiation Induction, but Not Maintenance, in the Juvenile Hippocampus after Neonatal Proinflammatory Stress

CA3-CA1 long-term potentiation (LTP) in the hippocampal slices from juvenile Wistar rats was studied to reveal factors potentially contributing to different sensitivity to neonatal proinflammatory stress (NPS). NPS was induced by intra­peritoneal injections of bacterial lipopolysaccharide (LPS) to neonatal rats (two injections of LPS, or saline in the control group, consecutively on postnatal days 3 and 5 [PND3 and PND5]). In females, a significant effect of NPS on hippocampus development was associated with modifications of long-term synaptic plasticity, the synapses becoming more resistant to LTP induction. LTP deficit in the slices of the NPS group was not associated with a decrease in LTP maintenance, since late LTP generally corresponded to early LTP magnitude, similar in all groups. Moreover, partial correlation revealed significantly higher residual LTP 1 h after high-frequency stimulation in the NPS groups compared to the corresponding value of early LTP in the control groups, suggesting improved consolidation. Both effects were evident in NPS females. A number of males responded to NPS similarly to females, while others were relatively resistant to NPS exposure, a significant increase in variability of LTP magnitude being revealed in NPS males compared to respective females and the control groups. We suggest that postnatal development of long-term plasticity after NPS is similar in animals of both sexes; however, additional specific factor(s) may promote a relative resistance of the male brain.

Selective Peripheral Chemoreceptor Hyperreflexia Evoked after Sympathetic Stimulation

Previous studies have demonstrated that the integrity of the carotid bodies is essential for the development and maintenance of hypertension. In this condition, both increased tonicity and hyperreflexia are displayed by chemosensitive neurons in the petrosal ganglia of spontaneous hypertensive rats (SHR). However, what drives carotid body hyper‐excitability is not known. We hypothesised this was triggered by the sympathetic nerves innervating the carotid body, which originate from the superior cervical ganglion (SCG). The aim of our study was test if activation of the SCG could modulate carotid body activity. Experiments were carried out in 6 juvenile Wistar rats (60–80g) using the decerebrated in situ working heart‐brainstem preparation (WHBP). Simultaneous recordings of phrenic (PN) and thoracic sympathetic nerves (tSNA) were obtained using glass suction electrodes. Heart rate (HR) was derived from the electrocardiogram (ECG). Chemoreflex was activated using NaCN (50μL, 0.04%, i.a.) and the SCG was stimulated electrically (ES; 30Hz, 2 ms, 10V) using a Grass SD9 stimulator via a bipolar microelectrode. The chemoreflex was evoked before and immediately after ES of the SCG. Changes in sympathetic activity, perfusion pressure, phrenic and heart rate were analysed quantitatively. Data are displayed as mean ± SEM with a level of significance set at P&lt;0.05; differences were tested using paired student‐t test.The only component of the chemoreflex augmented by SCG stimulation was the evoked increase in sympathetic activity. In Wistars, the control response of 29.3 ± 3.2% from baseline was augmented to 46.04 ± 3.58% after ES (P=0.006). Within 10 min of the stimulation, the sympatho hyperreflexia had returned to control (20.24 ± 5.96%). SHR also presented sympatho hyperreflexia (43.0 ± 6.3 vs 62.9 ± 9.2 %, P= 0.015) but the change of sensitization was of similar magnitude between rat strains. The SCG induced carotid body hyperreflexia was prevented by application of lignocaine into the SCG. We conclude that stimulation of the efferent sympathetic nerves innervating the carotid body can increase the gain of the chemoreflex symathoexcitatory response. We are currently assessing whether chemoreflex hyperreflexia observed in neurogenic hypertension is mediated by sympathetic nerves innervating the carotid body.Support or Funding InformationHealth Research Council of New Zealand