Preclinical Rat Model Research Articles

Blood-Based Lateral-Flow Immunoassays Dipstick Test for Damaged Mitochondrial Electron Transport Chain in Pyruvate Treated Rats with Combined Blast Exposure and Hemorrhagic Shock

Blast trauma presents a unique challenge due to its complex mechanism of injury, which impacts the brain and other vital organs through overpressure waves and internal bleeding. Severe blood loss leads to an inadequate oxygen supply and insufficient fuel delivery to cells, impairing ATP production by mitochondria—essential for cell survival. While clinical symptoms of metabolic disruption are evident soon after injury, the molecular, cellular, and systemic damage persists for days to years post-injury. Current challenges in treating traumatic brain injury (TBI) stem from (1) the lack of early blood-based biomarkers for detecting metabolic failure and mitochondrial damage and (2) the limited success of mitochondrial-targeted therapeutic strategies. Objectives: To identify blood-based mitochondrial biomarkers for evaluating the severity of brain injuries and to investigate therapeutic strategies targeting mitochondria. Methods: A preclinical rat model subjected to blast exposure, with or without hemorrhagic shock (HS), followed by resuscitation was utilized. Blood samples were obtained at baseline (T0), post-injury (T60), and at the conclusion of the experiment (T180), and analyzed using a validated dipstick assay to measure mitochondrial enzyme activity. Results: Blast and HS injuries led to a significant decrease in the activity of mitochondrial enzymes, including complex I, complex IV, and the pyruvate dehydrogenase complex (PDH), compared to baseline (p < 0.05). Concurrently, blood lactate concentrations were significantly elevated (p < 0.001). An inverse correlation was observed between mitochondrial enzyme dysfunction and blood lactate levels (p < 0.05). Treatment with sodium pyruvate post-injury restored complex I, complex IV, and PDH activity to near-baseline levels, corrected hyperlactatemia, and reduced reactive oxygen species (ROS) production by mitochondria. Conclusions: Serial monitoring of blood mitochondrial enzyme activity, such as complex I, complex IV, and PDH, may serve as a valuable tool for prognostication and guiding the use of mitochondrial-targeted therapies. Additionally, mitochondrial enzyme assays in blood samples can provide insights into the global redox status, potentially paving the way for novel therapeutic interventions in TBI.

CXCR2 immunomodulatory therapy protects against microstructural white matter injury and gait abnormalities but does not mitigate deficits of cognition in a preclinical model of cerebral palsy.

Minimizing central nervous system (CNS) injury from preterm birth depends upon understanding the critical pathways that underlie essential neurodevelopmental and CNS pathophysiology. Signaling by chemokine (C-X-C motif) ligand 1 (CXCL1) through its cognate receptor, CXCR2 [(C-X-C motif) receptor 2] is essential for neurodevelopment. Increased CXCR2 signaling, however, is implicated in a variety of uterine and neuropathologies, and their role in the CNS injury associated with perinatal brain injury is poorly defined. To evaluate the long-term efficacy of CXCR2 blockade in functional repair of brain injury secondary to chorioamnionitis (CHORIO), we used an established preclinical rat model of cerebral palsy. We tested the hypothesis that transient postnatal CXCR2 antagonism with SB225002 would reduce gait deficits, hypermobility, hyperactivity, and disinhibition concomitant with repair of functional and anatomical white and gray matter injury. CHORIO was induced in pregnant Sprague Dawley rats on embryonic day 18 (E18). SB225002 (3 mg/kg) was administered intraperitoneally from postnatal day 1 (P1)-P5. Rats were aged to adulthood and tested for gait, open-field behavior and cognitive and executive function deficits using a touchscreen cognitive assessment platform. Results show that transient CXCR2 blockade attenuated microstructural white matter injury after CHORIO consistent with improved anatomical connectivity, and mitigated deficits in gait coordination, posture, balance, paw placement, and stepping (p < 0.05). Animals with CHORIO were hyperactive and hypermobile with fMRI deficits in neural circuitry central to cognition. However, CXCR2 antagonism in CHORIO animals did not normalize open-field behavior, neural activity, or cognition on a touchscreen task of discrimination learning (all p > 0.05). Studies in CXCR2 knockout mice confirmed significantly impaired cognitive performance independent of CHORIO. Taken together, transient postnatal blockade of CXCR2 ameliorates aspects of the lasting neural injury after CHORIO including normalizing gait deficits and white matter injury. However, improvement in essential functional and cognitive domains are not achieved limiting the utility of this therapeutic approach for treatment of perinatal brain injury. This study emphasizes the complex, multi-faceted role of chemokines in typical neurodevelopment, circuit formation, neural network function, and injury response.

Application of Microsponge Drug Platform to Enhance Methotrexate Administration in Rheumatoid Arthritis Therapy.

This study aimed to develop a novel nanotechnological slow-release drug delivery platform based on hyaluronic acid Microsponge (MSP) for the subcutaneous administration of methotrexate (MTX) in the treatment of rheumatoid arthritis (RA). RA is a chronic autoimmune disease characterized by joint inflammation and damage, while MTX is a common disease-modifying antirheumatic drug (DMARD), the conventional use of which is limited by adverse effects and the lack of release control. MSP were synthesized as freeze-dried powder to increase their stability and allow for a facile reconstitution prior to administration and precise MTX dosing. A highly stable and rounded-shaped micrometric MSP, characterized by an open porosity inner structure, achieved both a high MTX loading efficiency and a slow release of MTX after injection. Our drug release assays indeed demonstrated a characteristic drug release profile consisting of a very limited burst release in the first few hours, followed by a slow release of MTX sustained for over a month. By means of a preclinical rat model of RA, the administration of MTX-loaded MSP proved to nearly double the therapeutic efficacy compared to sole MTX, according to a steep reduction in arthritic score compared to control groups. The preclinical study was replicated twice to confirm this improvement in performance and the safety profile of the MSP. This study suggests that the MSP drug delivery platform holds significant potential for clinical use in improving RA therapy by enabling the sustained slow release of MTX, thereby enhancing therapeutic outcomes and minimizing side effects associated with conventional burst-release drug administration.

A dual phase biologic augmentation of rotator cuff healing in a preclinical rat model using IL-17F and low-dose lithium

BackgroundRotator cuff tendon tears are a common cause of shoulder dysfunction in adults. Yet, impaired healing continues to result in higher failures rates after surgical repair resulting in patient dysfunction and prolonged recovery. This has spurred increased investigation of biologic augmentation to improve tendon healing. This study examines the outcome of peritendinous IL-17F administration and oral low-dose lithium carbonate on rotator cuff healing following acute surgical repair in a rat model. MethodsTreatment group included an open supraspinatus peel and repair followed by a local injection of IL-17F at the bone-tendon interface and a 7-day course of oral lithium carbonate. Control rats received no additional intervention prior to surgical closure. Evaluation of healing was then preformed using MRI imaging, biomechanical testing, and histological analysis at the bone-tendon interface. ResultsEighteen rats (9 control, 9 experimental) underwent complete testing. Combined treatment of peri-tendinous IL-17F and oral low-dose lithium carbonate after rotator cuff repair improved rotator cuff healing in all outcomes when compared to controls. MRI imaging in the treatment group showed complete healing of all supraspinatus tendons across the anatomic footprint after repair. We also found significant increases in biomechanical stiffness compared to controls (p <0.01). At the histological level, treatment groups also had decreased osteoclasts (p<0.001), and increased histologic organization of fibroblasts compared to controls. These findings are consistent with an increase in biomechanical stiffness. ConclusionWe demonstrated that the combined treatment of IL-17F and oral low-dose lithium improved rotator cuff tendon healing quality following acute surgical repair in a rat model.

Lithium-containing 45S5 Bioglass-derived glass-ceramics have antioxidant activity and induce new bone formation in a rat preclinical model of type 1 diabetes mellitus

Diabetes mellitus (DM) has been associated with complications that affect the skeletal system, such as alterations in bone repair, osteoporosis, and an increased risk of fractures. In this context, the use of biomaterials able to promote osteogenic differentiation and, at the same time, limit the oxidative stress induced by DM offers a novel perspective to ensure the repair of diabetic bone tissue. Since lithium (Li) has been recently identified as a biologically active ion with osteogenic and antioxidant properties, the localized and controlled release of Li ions from bioactive glass-ceramic materials represents a promising therapeutic alternative for the treatment of bone lesions in DM. Thus, the aim of this study was to evaluate the potential osteogenic and antioxidant effects of glass-ceramic microparticles derived from a 45S5-type bioactive glass (Bioglass) containing (% by weight) 45% SiO2, 24.5% Na2O, 24.5% CaO, and 6% P2O5, in which Na2O was partially substituted by 5% of Li2O (45S5.5Li), in an experimental model of type 1 DM (DM1). The results obtained demonstrate, for the first time, that both 45S5 and 45S5.5Li glass-ceramic microparticles possess antioxidant activity and stimulate bone formationin vivoboth under physiological conditions and under experimental DM1 in rats. In this sense, they would have potential application as inorganic osteogenic agents in different strategies of bone tissue regenerative medicine.

Multiple Benefits of Empagliflozin in PCOS: Evidence from a Preclinical Rat Model.

Polycystic ovary syndrome (PCOS) is the most common complex endocrinological condition of women that is associated with infertility and metabolic disorders during the reproductive period. Recently, a great deal of research has focused on the etiopathogenesis of this disorder and the modulation of therapeutic approaches. There are still many controversies in the choice of therapy, and metformin is one of the most commonly used agents in the treatment of PCOS. Considering the link between metabolic disorders and PCOS, glycemic status is crucial in these patients, and sodium-glucose cotransporter type 2 inhibitors (SGLT2is) represent a potentially promising new therapeutic approach. These drugs have been shown to improve glucose metabolism, reduce adipose tissue, decrease oxidative stress, and protect the cardiovascular system. These data prompted us to investigate the effects of empagliflozin (EMPA) in a PCOS rat model and compare them with the effects of metformin. We confirmed that EMPA positively affects somatometric parameters, glucose and lipid metabolism, and the levels of sex hormones, as well as reduces oxidative stress and improves ovarian function and morphology. Administration of EMPA at doses of 5 mg/kg, 15 mg/kg, and 45 mg/kg during a 4-week treatment period improved, as induced by estradiol valerate and a high-fat diet, the metabolic and reproductive statuses in a PCOS rat model. The best effects, which were comparable to the effects of metformin, were achieved in groups receiving the middle and highest applied doses of EMPA. These results may prompt further clinical research on the use of EMPA in patients with PCOS.

Acupuncture improves neuroendocrine defects in a preclinical rat model of reproductive aging

AimsClinical data supports electroacupuncture (EA) as an effective treatment for female reproductive disorders especially gonadotropin abnormalities. This study aims to detect the mechanism of EA that improves the neuroendocrine defects particularly the luteinizing hormone (LH) surge failure in early reproductive aging females. Materials and methodsMiddle-aged ovariectomized rats primed with hormone were treated by EA at acupoints CV4 and SP6 and undergone LH assay. Morphological experiments detected the activation of Kiss1 cells in the anteroventral periventricular nucleus (AVPV). Using targeted liquid chromatography with tandem mass spectrometry (LC-MS/MS) and RNA-sequencing, we determined the concentrations of neurotransmitter metabolites and transcriptomics in AVPV. Key findingsEA significantly increased c-Fos and c-Fos-positive Kiss1 cells in the middle-aged AVPV as well as the total and peak LH release. Targeted LC-MS/MS and RNA-sequencing of AVPV identified differential neurotransmitters in the middle-aged females including Acetylcholine chloride, 5-Hydroxyindole-3-aceticacid, Kynurenine, Histamine, L-Histidine and L-Glycine, while EA decreased the concentration of Acetylcholine chloride. Totally 1255 differentially expressed genes modulated by EA were strongly implicated in neurotransmitter transport and KEGG pathways involved neuroactive ligand-receptor interaction, glutamatergic and gamma-aminobutyric acid-mediated synapse. Specifically, the mRNAs associated with the LH surge such as hormone receptor Pgr, adrenoceptor Adra1a, neurotransmitter transporters Slc17a6 and Slc32a1, glutamate decarboxylase Gad2 and Kiss1 were markedly altered by EA. SignificanceThese findings showed that the age-related reduction of LH surge occurred via differential neurotransmitter metabolisms and altered transcriptions in AVPV, which proposed EA-based therapy for improving responsiveness of the hypothalamus to hormone in women with advanced age.

Examining Brain Activity Responses during Rat Ultrasonic Vocalization Playback: Insights from a Novel fMRI Translational Paradigm.

Despite decades of preclinical investigation, there remains limited understanding of the etiology and biological underpinnings of anxiety disorders. Sensitivity to potential threat is characteristic of anxiety-like behavior in humans and rodents, but traditional rodent behavioral tasks aimed to assess threat responsiveness lack translational value, especially with regard to emotionally valenced stimuli. Therefore, development of novel preclinical approaches to serve as analogues to patient assessments is needed. In humans, the fearful face task is widely used to test responsiveness to socially communicated threat signals. In rats, ultrasonic vocalizations (USVs) are analogous social cues associated with positive or negative affective states that can elicit behavioral changes in the receiver. It is therefore likely that when rats hear aversive alarm call USVs (22 kHz), they evoke translatable changes in brain activity comparable with the fearful face task. We used functional magnetic resonance imaging in male and female rats to assess changes in BOLD activity induced by exposure to aversive 22 kHz alarm calls emitted in response to threatening stimuli, prosocial (55 kHz) USVs emitted in response to appetitive stimuli, or a computer-generated 22 kHz tone. Results show patterns of regional activation that are specific to each USV stimulus. Notably, limbic regions clinically relevant to psychiatric disorders (e.g., amygdala, bed nucleus of the stria terminalis) are preferentially activated by either aversive 22 kHz or appetitive 55 kHz USVs. These results support the use of USV playback as a promising translational tool to investigate affective processing under conditions of distal threat in preclinical rat models.

Air-ventilated normothermic mechanical perfusion improves susceptibility to donation after circulatory death and cold preservation-induced cholestatic liver injury through PPAR-γ/UGT1A1 axis.

End-ischemic normothermic mechanical perfusion (NMP) could provide a curative treatment to reduce cholestatic liver injury from donation after circulatory death (DCD) in donors. However, the underlying mechanism remains elusive. Our previous study demonstrated that air-ventilated NMP could improve functional recovery of DCD in a preclinical NMP rat model. Here, metabolomics analysis revealed that air-ventilated NMP alleviated DCD- and cold preservation-induced cholestatic liver injury, as shown by the elevated release of alanine aminotransferase (ALT), aspartate aminotransferase (AST), bilirubin, and γ-glutamyl transferase (GGT) in the perfusate (p < .05) and the reduction in the levels of bile acid metabolites, including ω-muricholic acid, glycohyodeoxycholic acid, glycocholic acid, and glycochenodeoxycholate (GCDC) in the perfused livers (p < .05). In addition, the expression of the key bile acid metabolism enzyme UDP-glucuronosyltransferase 1A1 (UGT1A1), which is predominantly expressed in hepatocytes, was substantially elevated in the DCD rat liver, followed by air-ventilated NMP (p < .05), and invitro, this increase was induced by decreased GCDC and hypoxia-reoxygenation in the hepatic cells HepG2 and L02 (p < .05). Knockdown of UGT1A1 in hepatic cells by siRNA aggravated hepatic injury caused by GCDC and hypoxia-reoxygenation, as indicated by the ALT and AST levels in the supernatant. Mechanistically, UGT1A1 is transcriptionally regulated by peroxisome proliferator-activator receptor-γ (PPAR-γ) under hypoxia-physoxia. Taken together, our data revealed that air-ventilated NMP could alleviate DCD- and cold preservation-induced cholestatic liver injury through PPAR-γ/UGT1A1 axis. Based on the results from this study, air-ventilated NMP confers a promising approach for predicting and alleviating cholestatic liver injury through PPAR-γ/UGT1A1 axis.

Preclinical Evaluation of Sigma 1 Receptor Antagonists as a Novel Treatment for Painful Diabetic Neuropathy.

The global prevalence of diabetes is steadily rising, with an estimated 537 million adults affected by diabetes in 2021, projected to reach 783 million by 2045. A severe consequence of diabetes is the development of painful diabetic neuropathy (PDN), afflicting approximately one in every three diabetic patients and significantly compromising their quality of life. Current pharmacotherapies for PDN provide inadequate pain relief for many patients, underscoring the need for novel treatments that are both safe and effective. The Sigma 1 Receptor (S1R) is a ligand-operated chaperone protein that resides at the mitochondria-associated membrane of the endoplasmic reticulum. The S1R has been shown to play crucial roles in regulating cellular processes implicated in pain modulation. This study explores the potential of PW507, a novel S1R antagonist, as a therapeutic candidate for PDN. PW507 exhibited promising in vitro and in vivo properties in terms of ADME, toxicity, pharmacokinetics, and safety. In preclinical rat models of Streptozotocin-induced diabetic neuropathy, PW507 demonstrated significant efficacy in alleviating mechanical allodynia and thermal hyperalgesia following both acute and chronic (2-week) administration, without inducing tolerance and visual evidence of toxicity. To the best of our knowledge, this is the first report to evaluate an S1R antagonist in STZ-induced diabetic rats following both acute and 2-week chronic administration, offering compelling preclinical evidence for the potential use of PW507 as a promising therapeutic option for PDN.

MicroPET Imaging of Riboflavin Transporter 3 Expression in Myocardial Infarction/Reperfusion Rat Models with Radiofluorinated Riboflavin.

Riboflavin transporter 3 (RFVT3) represents a potential cardioprotective biotarget in energetic metabolism reprogramming after myocardial infarction/reperfusion (MI/R). This study investigated the feasibility of noninvasive real-time quantification of RFVT3 expression after MI/R with an radiolabeled probe 18F-RFTA in a preclinical rat model of MI/R. The tracer 18F-RFTA was radio-synthesized manually and characterized on the subjects of radiolabeling yield, radiochemical purity, and stability in vivo. MI/R and sham-operated rat models were confirmed by cardiac magnetic resonance imaging (cMRI) and single-photon-emission computed tomography (SPECT) myocardial perfusion imaging (MPI) with technetium-99m sestamibi (99mTc-MIBI). Positron emission tomography (PET) imaging of MI/R and sham-operated rat models were conducted with 18F-RFTA. Ex vivo autoradiography and RFVT3 immunohistochemical (IHC) staining were conducted to verify the RFVT3 expression in infarcted and normal myocardium. 18F-RFTA injection was prepared with high radiochemical purity (>95%) and kept stable in vitro and in vivo. 18F-RFTA PET revealed significant uptake in the infarcted myocardium at 8 h after reperfusion, as confirmed by lower 99mTc-MIBI perfusion and decreased intensity of cMRI. Conversely, there were only the tiniest uptakes in the normal myocardium and blocked infarcted myocardium, which was further corroborated by ex vivo autoradiography. The RFVT3 expression was further confirmed by IHC staining in the infarcted and normal myocardium. We first demonstrate the feasibility of imaging RFVT3 in infarcted myocardium. 18F-RFTA is an encouraging PET probe for imaging cardioprotective biotarget RFVT3 in mitochondrial energetic metabolism reprogramming after myocardial infarction. Noninvasive imaging of cardioprotective biotarget RFVT3 has potential value in the diagnosis and therapy of patients with MI.

Dual‐Crosslinked Bioactive Hydrogel Scaffold for Accelerated Repair of Genital Tract Defect

AbstractReproductive health concerns like Mayer‐Rokitansky‐Küster‐Hauser (MRKH) syndrome are prevalent in today's society. MRKH syndrome is a condition that severely affects women's sexual life, fertility, and mental health and has a high prevalence of one out of 5000 female births. Vaginoplasty is the primary method to regain patients’ reproductive health. However, conventional vaginoplasty faces various challenges, including complex and non‐customized treatment procedures causing intense pains and complications. To bring new advances to vaginoplasty, a 3D‐printed hydrogel scaffold is developed to provide satisfactory mechanical support and bioactivity for accelerating defect repair after surgery. The hydrogel scaffold consisting of gelatin methacryloyl (gelMA) and carrageenan (Car) is custom 3D‐printed using an ambient temperature printing system. Furthermore, the scaffold undergoes dual‐crosslinking through chemical crosslinking of gelMA and ionic crosslinking of Car with magnesium ions (Mg2+). This dual‐crosslinking strategy substantially improves the overall mechanical properties of the scaffold and introduces bioactive Mg2+. The sustained release of Mg2+ plus the extracts from the dual‐crosslinked scaffold significantly promotes cell proliferation, migration and angiogenesis. In a preclinical rat model with penetrating genital tract defects mimicking vaginoplasty, the implantation of dual‐crosslinked scaffold repairs the penetrating wounds to near‐normal levels within one week, showing potential as an alternative for better regaining reproductive health.

Repeated birth injuries lead to long-term pelvic floor muscle dysfunction in the preclinical rat model

ObjectivesVaginal childbirth is a key risk factor for pelvic floor muscle injury and dysfunction, and subsequent pelvic floor disorders. Multiparity further exacerbates these risks. Using the rat model, validated for the studies of the human pelvic floor muscles, we have previously identified that a single simulated birth injury results in pelvic floor muscle atrophy and fibrosis. We hypothesized that multiple birth injuries would further overwhelm the muscle regenerative capacity, leading to functionally relevant pathological alterations long-term. Study DesignSprague-Dawley rats underwent simulated birth injury and were allowed to recover for 8 weeks before undergoing additional birth injury. Animals were sacrificed at acute (3- and 7-days post injury), subacute (21-, 28-, and 35-days post-injury), and long-term (8- and 12-weeks post-injury) time points post-second injury (N=3-8/time point), and the pubocaudalis portion of the rat levator ani complex was harvested to assess the impact of repeated birth injuries on muscle mechanical and histomorphological properties. The accompanying transcriptional changes were assessed by a customized NanoString panel. Uninjured animals were used as controls. Data with a parametric distribution were analyzed by a two-way analysis of variance followed by post hoc pairwise comparisons using Tukey’s or Sidak’s tests; non-parametrically distributed data were compared with Kruskal-Wallis test followed by pairwise comparisons with Dunn’s test. Data, analyzed using GraphPad Prism v8.0, San Diego, CA, are presented as mean ± SEM or median (range). ResultsFollowing the 1st simulated birth injury, active muscle force decreased acutely relative to uninjured controls (12.9±0.9 vs 25.98±2.1 g/mm2, P<0.01). At 4 weeks, muscle active force production recovered to baseline and remained unchanged at 8 weeks after birth injury (P>0.99). Similarly, precipitous decrease in active force was observed immediately after repeated birth injury (18.07±1.2 vs 25.98±2.1 g/mm2, P<0.05). In contrast to the functional recovery after a single birth injury, a long-term decrease in muscle contractile function was observed up to 12 weeks after repeated birth injuries (18.3±1.6 vs 25.98±2.1 g/mm2, P<0.05). Fiber size was smaller at the long-term time points after 2nd injury compared to the uninjured group (12 weeks vs uninjured control: 1485 (60.7-5000) vs 1989 (65.6-4702) μm2, P<0.0001). The proportion of fibers with centralized nuclei, indicating active myofiber regeneration, returned to baseline at 8 weeks post-1st birth injury, (P=0.95), but remained elevated as far as 12 weeks post-2nd injury (12 weeks vs uninjured control: 7.1±1.5 vs 0.84±0.13%, P<0.0001). In contrast to the plateauing intramuscular collagen content after 4 weeks post-1st injury, fibrotic degeneration increased progressively over 12 weeks after repeated injury (12 weeks vs uninjured control: 6. 7±1.1 vs 2.03±0.2%, P<0.001). Prolonged expression of pro-inflammatory genes accompanied by a greater immune infiltrate was observed after repeated compared to a single birth injury. ConclusionsOverall, repeated birth injuries led to a greater magnitude of pathological alterations compared to a single injury, resulting in more pronounced pelvic floor muscle degeneration and muscle dysfunction in the rat model. The above provides a putative mechanistic link between multiparity and the increased risk of pelvic floor dysfunction in women.

The Impacts of Adolescent Cannabinoid Exposure on Striatal Anxiety- and Depressive-Like Pathophysiology Are Prevented by the Antioxidant N-Acetylcysteine

BackgroundExposure to Δ9-tetrahydrocannabinol (THC) is an established risk factor for later-life neuropsychiatric vulnerability, including mood- and anxiety-related symptoms. The psychotropic effects of THC on affect and anxiogenic behavioral phenomena are known to target the striatal network, particularly the nucleus accumbens, a neural region linked to mood and anxiety disorder pathophysiology. THC may increase neuroinflammatory responses via the redox system and dysregulate inhibitory and excitatory neural balance in various brain circuits, including the striatum. Thus, interventions that can induce antioxidant effects may counteract the neurodevelopmental impacts of THC exposure. MethodsIn the current study, we used an established preclinical adolescent rat model to examine the impacts of adolescent THC exposure on various behavioral, molecular, and neuronal biomarkers associated with increased mood and anxiety disorder vulnerability. Moreover, we investigated the protective properties of the antioxidant N-acetylcysteine against THC-related pathology. ResultsWe demonstrated that adolescent THC exposure induced long-lasting anxiety- and depressive-like phenotypes concomitant with differential neuronal and molecular abnormalities in the two subregions of the nucleus accumbens, the shell and the core. In addition, we report for the first time that N-acetylcysteine can prevent THC-induced accumbal pathophysiology and associated behavioral abnormalities. ConclusionsThe preventive effects of this antioxidant intervention highlight the critical role of redox mechanisms underlying cannabinoid-induced neurodevelopmental pathology and identify a potential intervention strategy for the prevention and/or reversal of these pathophysiological sequelae.

In Vitro Human Endometrial Cells and In Vivo Rat Model Studies Suggest That Ulipristal Acetate Impacts Endometrial Compatibility for Embryo Implantation

Ulipristal acetate (UPA) and levonorgestrel are used as emergency hormonal contraceptives. Although both are highly effective in preventing pregnancy, UPA shows efficacy even when taken up to 120 h after unprotected sexual intercourse. To investigate whether the mechanism of UPA's contraceptive action involves post-fertilization effects. In vitro and in vivo studies using cultured human endometrial cells and a pre-clinical rat model. Endometrial cells treated with UPA showed changes in the expression of receptivity gene markers and a significant decrease in trophoblast spheroids attached to the cultured cells. In addition, administration of UPA to female unmated rats decreased the expression of implantation-related genes in the endometrium and inhibited the number of implantation sites in the mated group compared to the non-treated group. These results support that UPA as an emergency contraceptive might have post-fertilization effects that may affect embryo implantation.

Therapeutic Effect of Alpha Lipoic Acid in a Rat Preclinical Model of Preeclampsia: Focus on Maternal Signs, Fetal Growth and Placental Function.

Chronic hypertension is a major risk factor for preeclampsia (PE), associated with significant maternal and neonatal morbidity. We previously demonstrated that pregnant stroke-prone spontaneously hypertensive rats (SHRSP) display a spontaneous PE-like phenotype with distinct placental, fetal, and maternal features. Here, we hypothesized that supplementation with alpha lipoic acid (ALA), a potent antioxidant, during early pregnancy could ameliorate the PE phenotype in this model. To test this hypothesis, timed pregnancies were established using 10 to 12-week-old SHRSP females (n = 19-16/group), which were assigned to two treatment groups: ALA (injected intraperitoneally with 25 mg/kg body weight ALA on gestation day (GD1, GD8, and GD12) or control, receiving saline following the same protocol. Our analysis of maternal signs showed that ALA prevented the pregnancy-dependent maternal blood pressure rise (GD14 blood pressure control 169.3 ± 19.4 mmHg vs. 146.1 ± 13.4 mmHg, p = 0.0001) and ameliorated renal function, as noted by the increased creatinine clearance and improved glomerular histology in treated dams. Treatment also improved the fetal growth restriction (FGR) phenotype, leading to increased fetal weights (ALA 2.19 ± 0.5 g vs. control 1.98 ± 0.3 g, p = 0.0074) and decreased cephalization indexes, indicating a more symmetric fetal growth pattern. This was associated with improved placental efficiency, decreased oxidative stress marker expression on GD14, and serum soluble fms-like tyrosine kinase 1 (sFlt1) levels on GD20. In conclusion, ALA supplementation mitigated maternal signs and improved placental function and fetal growth in SHRSP pregnancies, emerging as a promising therapy in pregnancies at high risk for PE.

Inspiratory Neural Network Complexity Quantified by Approximate Entropy (ApEn) Analysis in SN 6-OHDA-induced Rat Model of Parkinson’s Disease

Parkinson’s disease (PD) is a multifaceted chronic and progressive neurodegenerative disease that includes a broad range of motor and non-motor symptoms (NMS). Breathing abnormalities are a common NMS in PD, and despite respiratory dysfunction being a major problem in PD as well as respiratory complications being the most common cause of death in PD patients, this NMS receives little attention. Over the last decade, a number of preclinical experimental rat models of PD generated by nigrostriatal site-targeted administration of the neurotoxin 6-hydroxydopamine (6-OHDA) have been used to evaluate various aspects of ventilatory control in PD. While all of these models have been shown to exhibit basal and chemical control of breathing abnormalities that are consistent with those in clinical reports, no studies have assessed the complex intrinsic dynamics that are associated with the central inspiratory neural network in PD. Since preclinical rat models of PD have also been shown exhibit neuroanatomical changes in brainstem regions involved in ventilatory control, it is likely that changes in inspiratory neural network dynamics, which can be assessed using nonlinear dynamical analysis methods, are also present in PD. To begin to assess this possibility, we quantified the complexity of 20 consecutive inspiratory (diaphragm EMG) bursts recorded during basal breathing in urethane-anesthetized spontaneously breathing adult female rats at 4-weeks after unilateral injection of 6-OHDA (n=18) or vehicle (VEH; n=14) directly into the substantia nigra (SN). For these analyses, we calculated approximate entropy (ApEn), which provides a statistical measure of regularity (orderliness) of a signal in the time domain, with values for the embedding dimension m and the tolerance r set to m=3 and r=0.44 SD. ApEn values were significantly higher in 6-OHDA-injected rats compared to VEH-injected rats (0.6169±0.0069 vs 0.5894±0.0087; P=0.017) albeit TI and Tpeak/TI were similar in both groups (TI: 305.8±9.5 vs 300.0±12.7 ms, P=0.713; Tpeak/TI: 51.0±1.3 vs 51.3±1.1%, P=0.904). Other significant differences in temporal features included higher breathing frequencies (93.4±2.4 vs 82.0±3.2 bpm; P=0.007), reduced TE (in ms: 334.7±12.7 vs 445.5±27.4; P=0.001), and decreased inspiratory duty cycle (in %: 47.2±1.1 vs 40.8±2.0; P=0.006) in 6-OHDA (compared to VEH)-injected rats. These observations suggest that respiratory dysfunction in PD is also accompanied by reorganization of the central inspiratory neural network that is associated with greater irregularity or less system order. Additional experiments are needed to identify the precise neural mechanisms underlying this higher system complexity. NIH NS101737; Thomas Hartman Center for Parkinson's Disease Research at Stony Brook University. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Progesterone Supplementation Improves the Maternal Syndrome of Superimposed Preeclampsia in the Dahl Salt Sensitive Rats

Introduction: Preeclampsia (PE) is characterized by new onset of hypertension after 20 weeks of gestation. It affects 5-7% of all pregnancies in the U.S., and is associated with reduced fetal weight, inflammation and hypertension (HTN). Importantly, 30% of HTN disorders in pregnancy are caused by chronic HTN that is present prior to pregnancy which increases the risk of superimposed PE (SIPE). The mechanisms responsible for the pathogenesis of PE and SIPE are unclear and currently the only treatment is early delivery of the fetus. Progesterone is important for the establishment and maintenance of pregnancy. We have previously shown that progesterone supplementation with 17-hydroxyprogesterone caproate (17-OHPC) improves inflammation, fetal weight and blood pressure in the preclinical RUPP rat model of PE, however the mechanism for this in SIPE is still unknown. Hypothesis: This study was designed to test the hypothesis that 17-OHPC reduces inflammation while improving maternal blood pressure in the preclinical pregnant Dahl Salt Sensitive (DS) rat model of SIPE. Methods: 17-OHPC (3.32mg/kg) or vehicle (Saline) was administrated intraperitoneally on gestation day (GD) 15 to normal pregnant (NP) Sprague-Dawley (SD) and pregnant Dahl Salt Sensitive (DS) rats. On GD 18, Uterine Artery Resistance Index (UARI) was measured by Vevo Doppler Ultrasound and carotid catheters were inserted. On GD 19, mean arterial blood pressure (MAP) and samples were collected. All data are expressed as mean ± standard error means (SEM). Results: MAP was 105±5 mmHg in SD+ Saline rats (n=7) and 105±4 mmHg in SD+17-OHPC rats (n=4), 137±3 mmHg in DS + Saline rats (p&lt;0.05, n=11), which improved to 125±4 mmHg in DS+17-OHPC rats (p&lt;0.05, n=7). Pup and Placenta weights were 2.1± 0.1 g, 0.6 ± 0.1 g in SD + saline rats and significantly reduced to 1.4±0.1 g, 0.5±0.1 g in DS+ Saline rats (p&lt;0.05). Neither placental weight nor pup weight was affected by 17-OHPC. UARI was 0.5 ±0.1 in SD+ Saline rats (n=6) and 0.5 ±0.1 in in SD+17-OHPC (n=4), 0.7±0.1 in DS+Saline rats (n=6, p&lt;0.05), which reduced to 0.5±0.1 in DS+17-OHPC (n=7, p&lt;0.05). TNF-alpha levels were 3.0±1.0 pg/mL in SD+ Saline rats, 11.0±1.2 in DS + Saline rats, which reduced to 4.1±1.6 in DS+17-OHPC (n=4, p&lt;0.05). Importantly, circulating and placental CD4+ T cells were 9.3±3.7 % Gate and 6.4± 1.6 % Gate in SD+ Saline rats (n=4), 38.9 ±2.9 % Gate and 33.1±4.3 % Gate in DS + Saline rats (n=6), which significantly reduced to 26.3 ± 0.1 % Gate and 3.4±1.2 % Gate in DS + 17-OHPC. Conclusion: Collectively, our findings demonstrate that 17-OHPC reduces inflammation and hypertension in the Dahl Salt Sensitive rat model of SIPE. Supported by NIH P20GM121334. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Effects of Acute and Chronic Gabapentin Treatment on Cardiovascular Function of Rats

Gabapentin (GBP), a GABA analogue, is primarily used as an anticonvulsant to treat partial seizures and neuropathic pain. While a majority of the side effects are associated with the nervous system, emerging evidence suggests a high risk for heart disease in the patients taking GBP. Given GBP can bind with high affnity to the α2δ subunits of voltage-gated Ca2+ channels (VGCCs) to block Ca2+ influx of cardiomyocytes, we hypothesize GBP suppresses cardiac function by evoking calcium signaling dysfunction. By using a preclinical rat model treated with GBP, we investigated (1) the acute cardiovascular responses to GBP (bolus i.v. injection; 50 mg/kg, 100 mg/ml x 0.1 ml in 3 min) and (2) the effects of chronic GBP treatment (i.p. 100 mg/kg/day x 7 days) on cardiovascular function and the myocardial proteome. Under isoflurane-anesthesia, blood pressure (BP), heart rate (HR), and left ventricular (LV) hemodynamics of the rats were measured using two Millar pressure transducers; one was implanted in abdominal aorta via right femoral artery, and the another was implanted in the LV chamber via right carotid artery. The LV myocardium was analyzed by proteomics, bioinformatics, and western blot to explore the molecular mechanisms underlying GBP-induced cardiac dysfunction, with the brain cortex used as a control tissue. In experiment (1), we found that i.v. GBP treatment significantly decreased BP, HR, maximal LV pressure, and maximal and minimal dP/dt, whereas it increased IRP-AdP/dt, Tau, systolic, diastolic, and cycle durations (*p &lt; 0.05 and **p &lt; 0.01 vs baseline; n = 4/group). This cardiovascular inhibition started at the injection time point and lasted at least 60 min, with a maximal effect appearing at 30 min post injection. In experiment (2), we found that chronic GBP treatment resulted in hypotension, bradycardia, and LV systolic dysfunction, with no change in plasma norepinephrine. In the myocardium of these rats, we used mass spectra to identify 109 differentially expressed proteins involved in calcium pathways, cholesterol metabolism, and galactose metabolism. Particularly, we found that calmodulin, a key protein of intracellular calcium signaling, was significantly upregulated by GBP in the heart but not in the brain, suggesting a myocardium-specific dysregulation of calcium signaling pathway, which may play a critical role in GBP-induced cardiac inhibition. Subsequent bioinformatics assays revealed a reduced Ca2+ release of sarcoplasmic reticulum (SR) and increased electrophoretic Ca2+ uptake into the matrix of mitochondria in the hearts of GBP-treated rats. In summary, we found that acute and chronic GBP treatments suppressed cardiovascular function in rats, which is attributed to abnormal calcium signaling in cardiomyocytes where GBP binds to VGCCs to reduce extracellular Ca2+ influx and cytosolic Ca2+ release from SR, resulting in blockage of excitation-contraction coupling. These data reveal a novel side effect of GBP independent of the nervous system, providing important translational evidence to suggest that GBP can evoke adverse cardiovascular events by depression of myocardial function. This project is funded by NIH R01 HL 160820. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of Serum Interleukin-22 as Novel Biomarker in Pulmonary Hypertension: A Translational Study.

Growing evidence suggests the crucial involvement of inflammation in the pathogenesis of pulmonary hypertension (PH). The current study analyzed the expression of interleukin (IL)-17a and IL-22 as potential biomarkers for PH in a preclinical rat model of PH as well as the serum levels in a PH patient collective. PH was induced by monocrotalin (60 mg/kg body weight s.c.) in 10 Sprague Dawley rats (PH) and compared to 6 sham-treated controls (CON) as well as 10 monocrotalin-induced, macitentan-treated rats (PH_MAC). Lung and cardiac tissues were subjected to histological and immunohistochemical analysis for the ILs, and their serum levels were quantified using ELISA. Serum IL levels were also measured in a PH patient cohort. IL-22 expression was significantly increased in the lungs of the PH and PH_MAC groups (p = 0.002), whereas increased IL17a expression was demonstrated only in the lungs and RV of the PH (p < 0.05) but not the PH_MAC group (p = n.s.). The PH group showed elevated serum concentrations for IL-22 (p = 0.04) and IL-17a (p = 0.008). Compared to the PH group, the PH_MAC group demonstrated a decrease in IL-22 (p = 0.021) but not IL17a (p = n.s.). In the PH patient collective (n = 92), increased serum levels of IL-22 but not IL-17a could be shown (p < 0.0001). This elevation remained significant across the different etiological groups (p < 0.05). Correlation analysis revealed multiple significant relations between IL-22 and various clinical, laboratory, functional and hemodynamic parameters. IL-22 could serve as a promising inflammatory biomarker of PH with potential value for initial diagnosis, functional classification or even prognosis estimation. Its validation in larger patients' cohorts regarding outcome and survival data, as well as the probability of promising therapeutic target structures, remains the object of further studies.