Female Rats Research Articles

8245 Estradiol Interacts with the Val66Met Single Nucleotide Polymorphism in the Brain-Derived Neurotrophic Factor Gene to Alter Endometrial Thickness in a Rat Model

Abstract Disclosure: S. Moosa: None. E. Kohlmeir: None. S. Kujawa: None. D. Korol: None. A. Prakapenka: None. Brain derived neurotrophic factor (BDNF) is most known for its involvement in cell development, survival, and plasticity in the nervous system. BDNF and its cognate receptors can also be found in non-neural tissue including the ovary and uterus in humans and in animal models, suggesting that BDNF signaling may regulate reproductive functions. Indeed, disruptions in BDNF signaling are associated with health conditions related to the reproductive system, such as endometriosis. Studies show that treatment with estradiol increases expression of uterine BDNF and its cognate receptors, suggesting that estradiol can modulate BDNF signaling and its effects in the reproductive system. A common human functional single nucleotide polymorphism (SNP) in the BDNF gene, Val66Met (rs6265), is thought to decrease activity-dependent release of BDNF, altering BDNF signaling. Using a CRISPR/cas9 rat model of the human Val66Met BDNF SNP, we investigated the interacting role of estradiol and this variation in the BDNF gene on female uterine structure. Middle-aged (11-14-months-old) female rats were either homozygous for the Val allele (Val/Val; control) or the Met allele (Met/Met; BDNF SNP). All rats were ovariectomized and after three weeks randomly assigned to receive subcutaneous treatment of vehicle (sesame oil) or estradiol (17beta-estradiol benzoate) for two consecutive days. Twenty-four hours after the second injection, rats were euthanized and tissue collected, weighed, and placed in fixative. Uterine tissue weights differed significantly by treatment and by genotype such that uterine horns were heavier in estradiol-treated compared to vehicle-treated rats within each genotype and uterine horns were heavier in Met/Met compared to Val/Val rats within each treatment condition. To determine which uterine layer contributed to the weight differences, uterine horns were sectioned and stained with hematoxylin and eosin. Thickness of myometrium, endometrium, and lumen were individually measured. Although there were no treatment and no genotype effects on myometrial thickness, there were significant treatment and genotype effects on endometrial thickness. Of note, the endometrium of estradiol-treated Met/Met rats was thicker compared to vehicle-treated Met/Met rats and compared to estradiol-treated Val/Val rats, suggesting that estradiol and the BDNF gene variation interact to modulate endometrial size. Lumen circumference did not differ between genotypes but did increase with estradiol compared to vehicle treatment within each genotype. Altogether, findings suggest that BDNF genotype affects uterine structure, notably the endometrium. Differences with BDNF gene variation emerged with estradiol exposure, implicating the hormone’s role in modulating BDNF signaling and warranting future research on the interactive effects of estradiol and BDNF genotype on the endometrium. Presentation: 6/2/2024

Basolateral amygdala inputs to the nucleus accumbens shell modulate the consolidation of cued-response and inhibitory avoidance learning

The basolateral amygdala (BLA) modulates different types of memory consolidation via distinct projections to downstream brain regions in multiple memory systems. Prior studies indicate that the BLA projects to the nucleus accumbens shell (NAshell) and that these regions interact to influence some types of behavior. Moreover, previous pharmacological work suggests the BLA and NAshell interact to influence memory. However, the precise role of the BLA-NAshell pathway has never been directly investigated in the consolidation of different types of memory including cued-response, spatial, or inhibitory avoidance (IA) learning. To address this, male and female Sprague-Dawley rats received optogenetic manipulations of the BLA or BLA-NAshell pathway immediately following training in different learning tasks. An initial experiment found that optogenetically inhibiting the BLA itself immediately after training impaired cued-response retention in a Barnes maze task in males and females, confirming earlier pharmacological work in males alone. Subsequent experiments found that BLA-NAshell pathway inhibition impaired retention of cued-response and IA learning but had no effect on retention of spatial learning. However, the present work did not observe any effects of pathway stimulation immediately after cued-response or IA learning. Together, the present findings suggest the BLA modulates the consolidation of cued-response and IA, but not spatial, memory consolidation via NAshell projections.

7877 Adipose Depot Differences In Regenerative Properties Of Exosomes Derived From Human Adipose Stem Cells

Abstract Disclosure: M.A. Krause-Hauch: None. R. Patel: None. B. Osborne: None. P. Albear: None. N.A. Patel: None. Many patients struggle with chronic recalcitrant wounds that are the result of delayed healing. Underlying inflammation is one major risk factor for impaired dermal wound healing that may lead to pathologies such as infection and scarring. Adult human adipose stem cells (hASCs) have shown tremendous potential in regenerative medicine. The regenerative potential of hASCs is dependent on secreted bioactive material which is packaged and released in extracellular exosomes. We previously showed that hASC exosomes from the subcutaneous depot (sc) promoted repair in human dermal fibroblasts using in vitro assays. In this study, we evaluated exosomes derived from the omental depot (om) of hASC and compared its efficacy with sc-hASC. Our results show differences in levels of the long noncoding RNA (lncRNA) cargo between om-hASCexo and sc-hASCexo. Next, we evaluated their repair potential in a wound model in vivo. Male and female F344 rats were dorsally wounded via punch biopsy and silicone scaffolding was attached to prevent contraction. The wounds were then either not treated (control) or treated with 50μg om-hASCexo or sc-hASCexo every alternate day when each wound size was measured for 1 week to monitor healing progress. After 7 days, wounds treated with either om-hASCexo and sc-hASCexo closed significantly faster than untreated control wounds. While no difference was observed in closure percentage after 7 days for wounds treated with either om-hASCexo and sc-hASCexo, sc-hASCexo appeared to act faster earlier (1-2 days post wound) in the wound healing process while om-hASCexo acts faster during later stages (2-4 days post wound) of wound healing. The wound tissue was collected for biochemical and histological analysis. qPCR analysis on day 7 revealed that both om-hASCexo and sc-hASCexo treatment resulted in increased levels of Collagen 1 & 3 and MMP9, while levels of TGF-β decreased with treatment. These results indicate that om-hASCexo and sc-hASCexo enhanced the wound healing process while decreasing inflammation of the wound. To determine the differences between om-hASCexo and sc-hASCexo treatments on wound healing pathways, RNAseq was performed. Results show specificity in pathway activation and gene expression between the om-hASCexo and sc-hASCexo treatments. In conclusion, we have demonstrated that the regenerative mechanisms differ between the hASC depots from which exosomes are secreted. At a broader level, this finding may shed light on the body’s organ health and repair processes in normal or disease states. Presentation: 6/3/2024

7561 Maternal Sucrose Consumption During Gestation Alone Does Not Affect Glucocorticoids and Aldosterone in the Fetal Brain and Blood of Rats

Abstract Disclosure: M.M. Jung: None. D.R. Seib: None. M. Idrissi: None. H.W. Chen: None. K.K. Soma: None. Sucrose (table sugar) is a common added sugar in foods and beverages. Sucrose intake is high worldwide, yet little is known about how sucrose affects the brain and hormones. We designed an isocaloric, macro/micro-nutrient matched control diet (1% kcal sucrose) and high-sucrose diet (26% kcal sucrose) paradigm where the two diets only differ in kcal from sucrose. In this paradigm, maternal high-sucrose diet intake alters glucocorticoids (GCs) which are steroid hormones that regulate numerous physiological processes including the stress response. 16 wk of maternal sucrose intake (10 wk before mating, 3 wk during gestation, and 3 wk during lactation) increases blood and brain corticosterone levels in adult female offspring. Moreover, 13 wk of maternal sucrose intake (10 wk before mating and 3 wk during gestation) increases 11-deoxycorticosterone (DOC, corticosterone precursor) and 11-dehydrocorticosterone (DHC, corticosterone metabolite) in maternal serum, and increases aldosterone in fetal blood and brain at embryonic day 19.5 (E19.5). These results suggest that long-term maternal sucrose intake is a stressor that alters GC and aldosterone signalling. It is unclear whether a maternal high-sucrose diet only during gestation alters fetal steroid levels, or whether long-term sucrose consumption before mating is necessary. Here, we investigate the effects of maternal sucrose intake during gestation only on GCs and aldosterone in the fetal blood and brain. We hypothesize that maternal sucrose intake only during gestation will alter GC and aldosterone signaling in the fetus. We predict that maternal sucrose consumption during gestation alone will increase GC and aldosterone levels in the fetal blood and brain. Female Long-Evans rats were fed either a high-sucrose diet or a control diet starting E0.5 (day of mating confirmation) (n=15/diet). On E19.5, we collected fetal brain and blood and microdissected the regions of the fetal brain that are sensitive to GCs and aldosterone: nucleus accumbens, amygdala, hypothalamus, ventral hippocampus, and ventral tegmental area. We measured a panel of 15 steroids including GCs and aldosterone in the fetal brain (0.8-1.6 mg/region) and blood (5 µL) using liquid chromatography-tandem mass spectrometry, the gold standard for steroid quantification. Maternal high-sucrose diet intake during gestation alone did not alter maternal food intake, maternal body mass, or litter size, but increased the percentage of male offspring per litter. Maternal sucrose intake did not alter DOC, corticosterone, or DHC levels in the fetal blood and brain regions. Aldosterone was not detected in the fetal blood and brain. These results suggest that maternal sucrose intake during gestation alone does not alter GC and aldosterone levels in the developing fetus and that a longer duration of maternal sucrose intake is needed to alter offspring endocrine physiology. Presentation: 6/1/2024

7849 The EDC Mixture “NeuroMix” Has Developmental, Anxiolytic, And Obesogenic Effects In Rats

Abstract Disclosure: E. Morales-Grahl: None. L. M. Thompson: None. E.N. Hilz: None. A.C. Gore: None. Humans are exposed to a variety of endocrine disrupting chemicals (EDCs) on a daily basis through drinking water, food, and contact with everyday objects. Safety guidelines aim to keep exposure to individual EDCs below certain levels but don’t take into account the potentially unpredictable effects of simultaneous exposure to multiple EDCs. Furthermore, EDCs may exhibit non-monotonic dose responses. The present study assessed the effect of an EDC mixture at three different doses on two sets of behaviors implicated in EDC research: anxiety, and high fat and sugar consumption. Beginning in early pregnancy and until pups were weaned at day 21, Sprague Dawley rats were fed daily with NeuroMix, our lab’s EDC mixture containing 9 common industrial chemicals, at three doses (0.1x, 1x, 10x). The base dose (1x) contained EDCs below the no observed adverse effect level in humans (NOAEL). In adulthood, the female and male offspring were then subjected to an open field and light/dark test, followed by a high fat and high sucrose preference test. 0.1x NeuroMix exposure advanced the timing of vaginal opening in female offspring, while all doses increased anogenital distance in males, marking differences in sexual development. 1x NeuroMix exposure in male rats increased their time in the open field corners and their time immobile in the corners, indicating higher anxiety. No difference was seen in sucrose preference. Female rats exposed to 1x NeuroMix showed a higher preference for a high-fat diet, and gained more weight compared to the controls while on this diet. These findings demonstrate that a mixture of human-relevant chemicals, when administered perinatally to rats, have developmental, anxiogenic and obesogenic effects in a sexually dimorphic manner. We also show a non-monotonic response to our chemical mixture, with the 1x dose showing greater effects than the 10x dose. Overall, we provide evidence that even at levels considered safe for individual EDCs, there are adverse developmental and behavioral effects of EDCs when combined. Grant support: R35 ES035024, F32 ES034257. Presentation: 6/1/2024

Subchronic Toxicity of Ethanol Extract of Mangosteen Rind (Garcinia Mangostana L.) on Kidney Function in Wistar Rats

Mangosteen (Garcinia mangostana L.) is a fruit that possesses antioxidant, antibacterial, antihistamine, anti-inflammatory, and antidiabetic properties. Before this, an acute toxicity test was conducted on the ethanol extract of mangosteen rind. However, further testing is required, specifically the subchronic toxicity test, to identify any hazardous effects that may not have been found in the previous study. The objective of this study is to evaluate the amount of toxicity based on the mortality rate, alterations in the relative weight of the kidneys, and changes in renal function as shown by levels of urea and creatinine. This study is an experimental investigation that employs the Post-test Only Control Group Design methodology, with a sample of 40 rats. The rats were categorized into two groups: the control group and the treatment group. The treatment group was divided into three subgroups, each receiving a dosage of 250 mg/kg body weight. The doses were 500 milligrams per kilogram and 1000 milligrams per kilogram of body weight, respectively. The control group received simply water and feed. The duration of the treatment was 28 days. Observations were conducted over 28 days, which involved monitoring animal mortality. On the 29th day, a surgical procedure was carried out to assess the comparative weight of the kidneys and collect blood samples for analyzing the levels of urea and creatinine. The findings demonstrated no mortality in the test subjects, although there was an elevation in the relative mass of the kidneys and an increase in urea concentrations. The increase in urea levels was observed in female rats using the Kruskal–Wallis test (P = 0.019). The study was further conducted using the post-hoc Mann–Whitney test. Control female rats exhibited notable disparities when administered dosages of 250 mg/kgBW and 500 mg/kgBW. The repeated administration of an ethanolic extract derived from the peel of the mangosteen fruit had a detrimental impact on the functioning of the kidneys, as evidenced by a rise in urea concentrations. Keywords: mangosteen, toxicity test, subchronic, kidney

Herbs-Partitioned Moxibustion on the Navel in a Rat Model of Primary Dysmenorrhea with Cold Coagulation and Blood Stasis.

Primary dysmenorrhea (PDM) refers to the occurrence of spasmodic colicky pain in the lower abdomen without any obvious pelvic pathology, often accompanied by other systemic symptoms, which significantly reduces the quality of life of women with PDM, 45%-95% of menstrual women are affected by it. Despite the high incidence of PDM, it is often not well treated and is ignored by relevant researchers and women themselves. Herbs-partitioned moxibustion (HPM) is a characteristic external therapy in traditional Chinese medicine (TCM), which is widely used to treat diseases in China; it treats diseases by applying a Chinese herbal formula on the patient's acupoints and then igniting moxa sticks on the herbal formula for moxibustion therapy. The primary effectiveness of the herbs utilized in this study is meridian activation and warmth, which encourages qi circulation to relieve pain. Moxibustion's heat stimulation could hasten the infusion of the herbs into the human body. Consequently, HPM, which combines acupoint, herbal, and heat stimulations, is appropriate for treating gynecological disorders, particularly functional diseases such as PDM. This paper provides a detailed description of the procedures and precautions for HPM at the Shenque (CV8) acupoint in female experimental rats, which 0provides an experimental basis for better promoting the application and development of HPM on the navel in PDM.

Nanoscale organization of cardiac calcium channels is dependent on thyroid hormone status.

Thyroid hormone dysfunction is frequently observed in patients with chronic illnesses including heart failure which increases risk of adverse events. This study examined effects of thyroid hormones (TH) on cardiac T-tubule (TT) integrity, Ca2+ sparks, and nanoscale organization of ion channels in excitation-contraction (EC)-coupling, including L-type calcium channel (Cav1.2), ryanodine receptor-type 2 (RyR2), and junctophilin-2 (Jph2). TH deficiency was established in adult female rats by propyl-thiouracil (PTU) ingestion for 8 weeks; followed by randomization to continued PTU without or with oral triiodo-L-thyronine (T3; 10 ug/kg/d) for two additional weeks (PTU+T3). Confocal microscopy of isolated cardiomyocytes (CM) showed significant misalignment of TTs, and increased Ca2+ sparks in thyroid-deficient CMs. Density-Based Spatial Clustering of Applications with Noise (DBSCAN) analysis of STochastic Optical Reconstruction Microscopy (STORM) images showed decreased (p<0.0001) RyR2 cluster number per cell area in PTU CMs compared to euthyroid (EU) control myocytes, and this was normalized by T3-treatment. Cav1.2 channels and Jph2 localized within 210 nm radius of the RyR2 clusters were significantly reduced in PTU myocytes, and these values were increased with T3 treatment. A significant percentage of the RyR2 clusters in the PTU myocytes had neither Cav1.2 or Jph2, suggesting fewer functional clusters in EC-coupling. Nearest neighbor distances between RyR2 clusters were greater (p<0.001) in PTU cells compared to EU and T3-treated CMs that corresponds to disarray of TTs at the sarcomere z-discs. These results support a regulatory role of T3 in the nanoscale organization of RyR2 clusters and co-localization of Cav1.2 and Jph2 in optimizing EC-coupling.

Evaluation of the effect of carvedilol orodispersible tablets on ischemia-reperfusion injury and flap viability in rats: An in vivo study.

Flap surgery is an integral part of plastic surgery, and ischemia-reperfusion (I/R) injury significantly affects the viability of the flap. Carvedilol (CRV), a nonselective beta-blocker with alpha-1 blocking and antioxidant properties, and known for its potential in reducing I/R damage, was chosen as the active substance for our study. The aim of this study was to investigate the vasodilator and antioxidant effects of CRV on rat inferior epigastric artery skin flap using orally disintegrating tablets (ODTs). The optimized ODT formulation was subjected to in vivo experiments using Sprague-Dawley female rats (n = 24) divided into three groups: Group I (control, I/R), Group II (treatment, I/R + CRV), and Group III (treatment, I/R), I/R + CRV ODT). Reperfusion was then observed following the release of the microclamp from the pedicle, and the flap was then re-adapted to its original position. Control rats were given oral isotonic solution via gavage and were subjected to 8 h of ischemia and 12 h of reperfusion. Group II was given 2 mg/kg CRV oral tablets for 7 days before and after surgery. Group III was given 2 mg/kg/day CRV ODT for the same period. Biopsies were taken from the flap and histopathological and biochemical analyses including superoxide dismutase, glutathionenitric oxide, malondialdehyde, paraoxonase 1, total oxidant, and total antioxidant capacities were performed. This study demonstrates that CRV ODTs significantly increased flap viability by approximately 25% compared to the control group, highlighting their promising therapeutic potential.

Effects of Tryptophan and Physical Exercise on the Modulation of Mechanical Hypersensitivity in a Fibromyalgia-like Model in Female Rats.

Though the mechanisms are not fully understood, tryptophan (Trp) and physical exercise seem to regulate mechanical hypersensitivity in fibromyalgia. Here, we tested the impact of Trp supplementation and continuous low-intensity aerobic exercise on the modulation of mechanical hypersensitivity in a fibromyalgia-like model induced by acid saline in female rats. Twelve-month-old female Wistar rats were randomly divided into groups: [control (n = 6); acid saline (n = 6); acid saline + exercise (n = 6); acid saline + Trp (n = 6); and acid saline + exercise + Trp (n = 6)]. Hypersensitivity was caused using two intramuscular jabs of acid saline (20 μL; pH 4.0; right gastrocnemius), 3 days apart. The tryptophan-supplemented diet contained 7.6 g/hg of Trp. The three-week exercise consisted of progressive (30-45 min) treadmill running at 50 to 60% intensity, five times (Monday to Friday) per week. We found that acid saline induced contralateral mechanical hypersensitivity without changing the levels of Trp, serotonin (5-HT), and kynurenine (KYN) in the brain. Hypersensitivity was reduced by exercise (~150%), Trp (~67%), and its combination (~160%). The Trp supplementation increased the levels of Trp and KYN in the brain, and the activity of indoleamine 2,3-dioxygenase (IDO), and decreased the ratio 5-HT:KYN. Exercise did not impact the assessed metabolites. Combining the treatments reduced neither hypersensitivity nor the levels of serotonin and Trp in the brain. In conclusion, mechanical hypersensitivity induced by acid saline in a fibromyalgia-like model in female rats is modulated by Trp supplementation, which increases IDO activity and leads to improved Trp metabolism via the KYN pathway. In contrast, physical exercise does not affect mechanical hypersensitivity through brain Trp metabolism via either the KYN or serotonin pathways. Because this is a short study, generalizing its findings warrants caution.

Pharmacokinetics and related gender difference studies of four active components of Codonopsis Pilosula by LC-MS/MS determination

Ethnopharmacological relevanceCodonopsis pilosula (C. pilosula), commonly known as Dangshen in Chinese, had been used to regulate the immune, digestive, and circulatory systems of human. The reported pharmacokinetic studies on C. pilosula are mainly limited to in vivo profile studies of a single component. It has not been detected simultaneously the in vivo pharmacokinetic profiles of multiple active components as well as related gender difference after oral dosing of the extraction of C. pilosula. Aim of the studyThis study aims to reveal the pharmacokinetic characteristics of the four main active components of C. pilosula after oral dosing of its extraction in rats, and to explain the gender differences in absorption and metabolism. Materials and methodsThe plasma pharmacokinetic characteristics of four main active components of C. pilosula was explored using the established LC-MS/MS method after oral dosing of the extraction of C. pilosula in male and female rats. In vitro intestinal pouch permeability and liver microsome metabolic stability were also observed to classify the possible mechanism of gender difference existed in the pharmacokinetic profiles of the four active components in rats. ResultsFour effective components were absorbed quickly in rats after oral administration of alcoholic extract of C. pilosula (1.36 g/mL, equivalent to 2 g/mL as crude drug), and their exposure order was as follows: Atractylenolide III > Lobetyolin > Tangshenoside I > Syringin. The exposure (AUC) and peak concentration (Cmax) of Atractylenolide III in female rats were much higher than those in male rats, indicating a significant gender difference in pharmacokinetics of Atractylenolide III between female and male animals. With the help of the rat model of intestinal sac in vitro, it was found that Lobetyolin was a hypertonic compound, and both Tangshenoside I and Syringin were compounds with medium permeabiltiy. Notably, the Papp of Atractylenolide III was 3.3 × 10−6 cm/s in male rat intestinal sac assay, while that was 10 × 10−6 cm/s in female rat intestinal sac model, showing a significant gender difference in intestinal permeability (P < 0.05). After the addition of NADPH, the four compounds were reduced in a time-dependent manner, suggesting that CYP450s could catalyze their metabolism. After incubation, the remaining content of Atractylenolide III in the liver microsomes of male and female rats was 27% and 57%, respectively, suggesting slower metabolic rate of in female rat liver microsomes. ConclusionA simple, efficient and reliable LC-MS/MS method for the simultaneous determination of four active index components of C. pilosula, Lobetyolin, Tangshenoside I, Atractylenolide III and Syringin, in rat plasma was established and verified. This method was successfully applied in the pharmacokinetic study after single oral administration of the alcoholic extract of C. pilosula in rats. Gender difference was observed in the pharmacokinetic profile of Atractylenolide III in rats. Intestinal absorption and liver metabolism might be two key factors that resulted in the gender difference in exposure and pharmacokinetics of Atractylenolide III in rats. This study provides supportive data for clinical rational application of C. pilosula in individualized medication therapy.

Molecular mechanisms underlying the effects of hypo- and hyper-prolactinemia on spermatogenesis and fertility in male rats.

Hypo- and hyper-prolactinemia have deleterious effects on male reproduction, yet there is a dearth of information regarding the underlying mechanisms. The aim of this study was to delineate the molecular mechanisms by which hypo- and hyper-prolactinemia affects spermatogenesis and fertility in male rats. In vivo male rat models for hypo- and hyper-prolactinemia were established using dopamine receptor agonist, Bromocriptine (Brm), and antagonist, Fluphenazine (Flu), respectively. Effects on fertility and spermatogenesis were assessed by studying pre- and post-implantation loss, litter size, sperm parameters, hormonal profile, testicular histology, testicular cell population, and testicular transcriptome in rats. Treatment with Brm and Flu for 60 days led to subfertility, which was indicated by an increase in pre- and post-implantation loss and decrease in litter size, when mated with control female rats. Decreased sperm count was observed after both treatments, whereas reduced sperm motility was noted in Flu group. Serum FSH was unaffected, and LH was decreased by Flu treatment. Testosterone was decreased in both the groups, whereas estradiol was decreased in the Flu group. An arrest in spermatogenic cycle beyond round spermatids was observed in the Flu group. Additionally, testicular apoptosis in germ cells, mostly spermatocytes of Stage IX-XIV was noted in both the groups. Further, testicular RNA-Seq analysis revealed a total of 1539 and 824 differentially expressed genes/DEGs in Brm and Flu, respectively (Sequence Read Archive/SRA Database accession number: PRJNA1150513). Gene ontology and pathway analysis of DEGs highlighted enrichment of steroid metabolic pathway and ribosomal biogenesis pathway. Hub genes identified from the DEGs were validated by qPCR and the results showed that Uba52, Rps27a, Rpl23, Rps5, Rps16 were significantly down-regulated by Brm, whereas Rps27a, Rps29, Rps15, Rps27, Faul1 were significantly down-regulated by Flu. Hypo- and hyper-prolactinemia leads to subfertility and decreased sperm parameters possibly through an effect on steroid metabolism and ribosomal biogenesis pathway. Therefore, maintaining prolactin levels in physiological range is crucial.

Strontium-containing mineralized phospholipid coatings improve osseointegration in osteoporotic rats.

Surface treatments play an important role in enhancing the osseointegration of Titanium (Ti) and its alloys. This study introduces a method employing biomimetic hydroxyapatite (Hap) deposition guided by molecularly organized phospholipids, affixed to the metal implant surface. Using the Langmuir-Blodgett technique, phospholipids were deposited onto Ti-screws by using CaCl2 or CaCl2/SrCl2 aqueous solution in the subphase of a Langmuir trough in the target proportion (i.e. 10 and 90 mol% of Sr2+ in relation of Ca2+) followed by immersion in phosphate buffer and in supersaturated simulated body fluid. Coating composition and morphology were evaluated using infrared spectroscopy and scanning electron microscopy, respectively, while contact angle measurements assessed coating wettability and surface energy. Randomized screws were then implanted into the tibias of healthy and osteoporotic female rats (G1: Control-Machined, G2: Hap, G3: HapSr10, G4: HapSr90). Osseointegration, assessed 60 days post-implantation, included reverse torque, fluorochrome area, bone tissue-screw contact area, and linear extent of bone-screw contact. Results, grouped by surface treatment (Machined, Hap, HapSr10, HapSr90), revealed that the deposition of Hap, HapSr10, and HapSr90 resulted in thin and rough coatings composed of hydroxyapatite (Hap) on the screw surface with nanoscale pores. The coatings resulted in increased wettability and surface energy of Ti surfaces. The minerals are chemically similar to natural bone apatite as revealed by FTIR analysis. In vivo analyses indicated higher torque values for strontium-containing surfaces in the osteoporotic group (p = 0.02) and, in the control group superior torque for screw removal on the Hap surface (p = 0.023). Hydroxyapatite-treated surfaces enhance morphology, composition, and reactivity, promoting screw osseointegration in healthy and osteoporotic female rats. The incorporation of strontium into the mineral phase has been proposed to not only stimulate osteoblast activity but also reduce osteoclastic resorption, which may explain the improved outcomes observed here in experimental osteoporotic conditions.

Chronic inflammatory pain reduces fentanyl intake during early acquisition of fentanyl self-administration, but does not change motivation to take fentanyl in male and female rats

The co-occurrence of chronic pain and opioid misuse has led to numerous preclinical investigations of pain-opioid interactions to examine how pain manipulations alter the reinforcing properties of opioids. However, preclinical investigations of chronic pain effects on opioid drug self-administration have produced inconsistent results. Our previous work demonstrated that established fentanyl self-administration is resistant to change by induction of chronic inflammatory pain (Complete Freund's Adjuvant; CFA) in male and female rats, while other laboratories have shown that CFA increased fentanyl self-administration in male but not female rats when pain induction precedes self-administration, which may be a critical factor in determining the effects of chronic pain on self-administration. The present study was designed similarly to Higginbotham et al. (2022) to test the effects of CFA on fentanyl self-administration in rats that underwent pain prior to acquisition of fentanyl self-administration. Male and female rats treated with hindpaw CFA or saline were trained to intravenously self-administer (IVSA) fentanyl for 3 weeks under limited access to fentanyl (2 h per day) conditions. After 3 weeks of fentanyl IVSA acquisition, we tested motivation to take fentanyl using progressive ratio testing and dose-response testing. CFA male and female rats self-administered less fentanyl than saline-treated controls during week 1 of acquisition, but not during weeks 2–3 of acquisition. Intra-session analysis of week 1 data demonstrated that chronic inflammatory pain suppressed fentanyl intake towards the end of week 1 and at the end of each operant session. We also report no effects of chronic inflammatory pain on motivation to take fentanyl. We discuss potential methodological explanations for differences between these results and prior reports. Our findings demonstrate that CFA temporarily suppresses fentanyl IVSA in animals without changing motivation to take fentanyl or promoting escalation of opioid use, suggesting that chronic inflammatory pain is unlikely to promote long-term risk of opioid misuse.

Effect of dimephosphone in a course administration on the mechanical activity of the intestine of rats with an autism model

BACKGROUND: Gastrointestinal tract dysfunctions are a very common problem in patients with autism spectrum disorders, which aggravates the underlying course of the disease, and therefore there is a need to find safe drugs to correct gastrointestinal dysfunctions. AIM: Evaluation of intestinal contractile activity in rats with an autism model after a course of intragastric administration of dimephosphone. MATERIAL AND METHODS: The mechanical activity of the duodenum and ileum of rats with the valproate model of autism after the administration of dimephosphone was studied in vitro. To model autism, female rats were given a single subcutaneous injection of sodium valproate at a dose of 500 mg/kg in the withers area on the 13th day of pregnancy. The offspring of these rats were divided into two groups. The experimental group of rats (n=12) was given dimephosphone at a dose of 50 mg/kg for 30 days, starting from the age of 2 months, control rats (n=12) were given physiological saline in an equal volume. Another group of rats was intact (n=9), which were born from female rats not exposed to valproic acid. The effect of carbachol (10–8–10–5 M), ATP (10–7–10–4 M), 2-methylthio-ATP (10–7–10–5 M) and electrical stimulation (1–5 Hz) on the mechanical activity of isolated intestinal smooth muscle preparations was assessed. Statistical processing was performed in IBM SPSS Statistics 26.0 using one-way analysis of variance. RESULTS: It was found that in rats with autism modeling, carbachol-induced intestinal contractions and ATP-induced intestinal relaxation increased. A course of intragastric administration of dimephosphone for 30 days normalized these changes, which became indistinguishable from the corresponding indices in intact animals. Thus, ATP at a concentration of 10–4 caused relaxation of the smooth muscles of the duodenum of rats with autism modeling after a course of dimephosphone administration by 77.1±14.7%, which significantly differed from the corresponding indices in control animals — 34.4±9.4% (p 0.05) and did not differ from the indices in intact rats. Similar changes occurred in the mechanical activity of the ileum. Intestinal relaxation in rats caused by 2-methylthio-ATP did not change significantly either in the control or in the group of animals receiving dimephosphone. CONCLUSION: Dimephosphone, when administered intragastrically, normalizes the disturbances in the mechanical activity of the intestines of rats with an autism model.

LEAP2, a ghrelin receptor inverse agonist, and its effect on alcohol-related responses in rodents

The underlying neurobiology of alcohol use disorder (AUD) is complex and needs further unraveling, with one of the key mechanisms being the gut-brain peptide ghrelin and its receptor (GHSR). However, additional substrates of the ghrelin pathway, such as liver-expressed antimicrobial peptide 2 (LEAP2), an endogenous GHSR inverse agonist, may contribute to this neurobiological framework. While LEAP2 modulates feeding and reward through central mechanisms, its effects on alcohol responses are unknown. The aim of the present study was therefore to identify the impact of central LEAP2 on the ability of alcohol to activate the mesolimbic dopamine system and to define its ability to control alcohol intake. These experiments revealed that central LEAP2 (i.e. into the third ventricle) prevented the ability of alcohol to cause locomotor stimulation in male mice, suppressed the memory of alcohol reward and attenuated the dopamine release in the nucleus accumbens caused by alcohol. Moreover, central LEAP2 reduced alcohol consumption in both male and female rats exposed to alcohol for 6 weeks before treatment. However, the serum levels of LEAP2 were similar between high- and low- alcohol-consuming (male) rats. Furthermore, central LEAP2 lowered the food intake in the alcohol-consuming male rats and reduced the body weight in the females. Collectively, the present study revealed that central LEAP2 mitigates alcohol-related responses in rodents, contributing to our understanding of the ghrelin pathway’s role in alcohol effects.

Neonicotinoid pesticides: evidence of developmental neurotoxicity from regulatory rodent studies.

Neonicotinoids are the most widely used class of insecticides in the United States (U.S.). and the world. Consistent with their high use and persistence, neonicotinoids are often found contaminating drinking water and food. They are also detected in human urine, breast milk, amniotic and cerebrospinal fluids, as well as the brains of treated rodents. Neonicotinoids were once thought to pose little neurotoxic risk to humans, but a growing body of research challenges that assumption. In this study we provide the first comprehensive assessment of unpublished rodent developmental neurotoxicity (DNT) studies on five neonicotinoids that were submitted to the U.S. Environmental Protection Agency (EPA) by neonicotinoid manufacturers. Groups of female rats were administered three different doses of a neonicotinoid during pregnancy and lactation, and their offspring subjected to various neurological tests and brain measurements. We identified nicotine-like effects such as reduced brain size, indicative of neuronal cell loss. Statistically significant shrinkage of brain tissue was observed in high-dose offspring for five neonicotinoids: acetamiprid, clothianidin, imidacloprid, thiacloprid, and thiamethoxam. Two brain regions reduced in the rodent studies-the corpus callosum and caudate-putamen-tend to be smaller in people diagnosed with attention-deficit hyperactivity disorder (ADHD), and in children of mothers who smoked during pregnancy, suggesting a possible link between perinatal neonicotinoid exposure and ADHD. A decreased auditory startle reflex was reported for acetamiprid at all doses and was statistically significant in the mid- and high-dose offspring, and for clothianidin in juvenile high-dose females. No mid- or low-dose brain morphometric data were submitted for acetamiprid, imidacloprid, or thiacloprid. Thiamethoxam mid- and low-dose brain morphometric data were provided to EPA upon request. Only partial mid-dose brain morphometry data were submitted for clothianidin, but no low-dose data. Yet despite this lack of data, EPA concluded that only the high-dose brain morphometric effects were treatment-related-setting the mid-dose as the study's No Observed Adverse Effect Level (NOAEL) or failing to find a definitive NOAEL for acetamiprid, clothianidin, imidacloprid, thiacloprid and thiamethoxam. We found numerous deficiencies in EPA's regulatory oversight and data analyses. EPA dismissed statistically significant adverse effects, accepted substandard DNT studies despite lack of valid positive control data, and allowed neonicotinoid registrants to unduly influence agency decision-making. We conclude that perinatal exposure to neonicotinoids and their metabolites induces adverse, nicotine-like neurotoxic effects in rodent bioassays, and that the exposure limits set by EPA for human exposure are either not protective or not supported by available neurotoxicity data. We propose regulatory changes to empower EPA to better protect public health from developmental neurotoxins like neonicotinoids.

Sex differences in fear expression and persistence in an animal model of Post-Traumatic Stress Disorder

Post-Traumatic Stress Disorder (PTSD) is a complex psychiatric condition arising from traumatic experiences, marked by abnormal fear memories. Despite women are twice as likely as men to develop PTSD, the biological mechanisms underlying this disparity remain inadequately explored, particularly in preclinical studies involving female subjects.Previous research shows that female rats exhibit active fear responses, while males display passive behaviors. Additionally, sex differences in ultrasonic vocalizations (USVs) during fear conditioning have been observed, indicating varying emotional responses.Here, we validated a traumatic stress model consisting of footshock exposure paired with social isolation − originally developed in male rats − on females for the first time, focusing on sex differences in fear memory expression, retention and extinction. Our findings reveal that only during trauma exposure, males predominantly exhibited passive responses, whereas females demonstrated more active responses, despite both sexes emitting similar numbers of alarm USVs. Females also showed lower levels of freezing and USV emissions throughout extinction sessions and displayed a higher extinction rate compared to males. Notably, only males displayed a conditioned fear response when triggered by a single mild stressor.These findings highlight sex differences in trauma responses and fear memory processes. The study emphasizes the importance of incorporating 22-kHz USV evaluations along with other behavioral metrics for a comprehensive understanding of fear memory. This research contributes to the existing literature on traumatic stress models as well as underscores the necessity of including female subjects in preclinical studies to better inform treatment and prevention strategies tailored to both sexes.

Endothelin receptor B is required for the blood pressure-lowering effect of G protein-coupled estrogen receptor 1 in ovariectomized rats.

Activation of G protein-coupled estrogen receptor 1 (GPER1) elicits antihypertensive actions in different animal models. The endothelin-1 signaling system plays a fundamental role in blood pressure regulation. Lack of functional endothelin receptor B (ETB) evokes hypertension and salt sensitivity. GPER1 and ETB interact to promote urinary sodium excretion in female rats. We hypothesized that activation of GPER1 protects against hypertension and salt sensitivity induced by ETB antagonism in female rats. Female Sprague-Dawley rats were implanted with radiotelemetry. Animals were then subjected to ovariectomy and simultaneously implanted with minipumps to deliver either the GPER1 agonist G1 or its corresponding vehicle. Two weeks post surgery, we initiated treatment of rats with the ETB antagonist A-192621. Animals were maintained on a normal-salt diet and then challenged with a high-salt diet for an additional 5 days. Assessment of mean arterial blood pressure revealed an increase in vehicle-treated, but not G1-treated, rats in response to ovariectomy. A-192621 increased blood pressure in normal-salt diet-fed vehicle- and G1-treated rats. G1 improved the circadian blood pressure rhythms that were disrupted in A-192621-treated ovariectomized rats. Thus, although systemic GPER1 activation did not protect ovariectomized rats from hypertension and salt sensitivity induced by ETB antagonism, it maintained circadian blood pressure rhythms. Functional ETB is required to elicit the antihypertensive actions of GPER1. Additional studies are needed to improve our understanding of the interaction between G protein-coupled receptors in regulating circadian blood pressure rhythm.NEW & NOTEWORTHY Systemic G protein-coupled estrogen receptor 1 (GPER1) activation in rats prevents the increase in blood pressure evoked by ovariectomy. Blockade of endothelin receptor B negates the blood pressure-lowering impact of GPER1 in ovariectomized rats. Endothelin receptor B plays an important role in mediating the blood pressure-lowering action of GPER1 activation in female rats.

How malocclusion interferes with tissue inhibitor of metalloproteinase-1 expression and morphology of the articular cartilage of the mandible in female rats

ObjectiveThe purpose of this study was to investigate morphological alterations and tissue inhibitor of metalloproteinase-1 expression in the articular cartilage of the mandible under conditions of experimentally induced malocclusion. DesignTwenty-four 8-week-old female Wistar rats were used and divided into control and experimental groups with two different treatment periods (2 and 4 weeks). Sagittal malocclusions were orthodontically created, causing mesial movement of the first molars and distalization of the third molars unilaterally and on opposite sides of the arches. Sagittal sections of the articular cartilage of the mandible were subjected to hematoxylin and eosin and immunohistochemistry for tissue inhibitor of metalloproteinase-1. Chi-square and MannWhitney U tests were applied. ResultsAnimals treated for 2 and 4 weeks showed morphological alterations in articular cartilage of the mandible. The main findings were thickening of the posterior third, layer derangement, osteoclast activity and osteophyte formation. Among the cellular aspects, the presence of chondrocytes with condensed nuclei and cytoplasm reduction were observed. The enzyme in control animals was observed only in the mature layer. Treated animals showed immunopositive cells in the proliferative and mature layers, and in the 2-week treated group, the posterior third of the cartilage had more immunolabeled cells than control (P=0.0291). ConclusionsThe occlusal disorder caused morphological changes in articular cartilage of the mandible, probably due to the attempt to adapt to the new condition. Tissue inhibitor of metalloproteinase-1 expression may play a role as an initial modulator in the biological events observed in articular cartilage of the mandible.