Preclinical Animal Models Research Articles

Thymidine Phosphorylase Imaging Probe for Differential Diagnosis of Metabolic dysfunction-associated Steatohepatitis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) comprises simple steatosis (SS), which has a low risk of mortality, and metabolic dysfunction-associated steatohepatitis (MASH), which can progress to liver cirrhosis and hepatocellular carcinoma. Because differentiation between MASH and SS is the most important issue in the diagnosis of MASLD, the establishment of noninvasive diagnostic methods is urgently needed. In this study, we evaluated the potential of [123I]IIMU, a thymidine phosphorylase (TYMP) targeted SPECT imaging probe, for differential diagnosis of MASLD in a preclinical animal model. SS and MASH mice were prepared by feeding db/db mice with a standard diet and a methionine/choline-deficient diet, respectively. Control mice were prepared by feeding m/m mice with a standard diet. TYMP expression in the liver was evaluated by RT-PCR, western blotting, and immunohistochemistry. The biodistribution of [125I]IIMU in the three model mice was evaluated at 30min post-injection. SPECT/CT imaging studies of the three model mice were performed 30min after injection of [123I]IIMU. Hepatic TYMP expression level was the highest in the SS mice and the lowest in the MASH mice at both mRNA and protein levels. The immunohistochemistry experiment showed a patchy distribution of TYMP only in the liver of MASH mice. In the biodistribution study, the hepatic accumulation of [125I]IIMU was the highest in the SS mice and the lowest in the MASH mice. The SPECT/CT imaging study showed similar results to the biodistribution experiment. Hepatic TYMP expression level may serve as a promising imaging biomarker for differential diagnosis of SS and MASH. SPECT imaging using [123I]IIMU potentially provides a novel noninvasive diagnostic method to differentiate MASH and SS.

Pregnancy in obese women and mechanisms of increased cardiovascular risk in offspring.

Pregnancy complicated by maternal obesity contributes to an increased cardiovascular risk in offspring, which is increasingly concerning as the rates of obesity and cardiovascular disease are higher than ever before and still growing. There has been much research in humans and preclinical animal models to understand the impact of maternal obesity on offspring health. This review summarizes what is known about the offspring cardiovascular phenotype, describing a mechanistic role for oxidative stress, metabolic inflexibility, and mitochondrial dysfunction in mediating these impairments. It also discusses the impact of secondary postnatal insults, which may reveal latent cardiovascular deficits that originated in utero. Finally, current interventional efforts and gaps of knowledge to limit the developmental origins of cardiovascular dysfunction in offspring of obese pregnancy are highlighted.

Deep functional measurements of Fragile X syndrome human neurons reveal multiparametric electrophysiological disease phenotype

Fragile X syndrome (FXS) is a neurodevelopmental disorder caused by hypermethylation of expanded CGG repeats (>200) in the FMR1 gene leading to gene silencing and loss of Fragile X Messenger Ribonucleoprotein (FMRP) expression. FMRP plays important roles in neuronal function, and loss of FMRP in mouse and human FXS cell models leads to aberrant synaptic signaling and hyperexcitability. Multiple drug candidates have advanced into clinical trials for FXS, but no efficacious treatment has been identified to date, possibly as a consequence of poor translation from pre-clinical animal models to human. Here, we use a high resolution all-optical electrophysiology platform applied to multiple FXS patient-derived and CRISPR/Cas9-generated isogenic neuronal cell lines to develop a multi-parametric FXS disease phenotype. This neurophysiological phenotype was optimized and validated into a high throughput assay based on the amount of FMRP re-expression and the number of healthy neurons in a mosaic network necessary for functional rescue. The resulting highly sensitive and multiparameter functional assay can now be applied as a discovery platform to explore new therapeutic approaches for the treatment of FXS.

Towards an emerging role for anticoagulants in cancer therapy: a systematic review and meta-analysis

BackgroundAnticoagulants, renowned for their role in preventing blood clot formation, have captivated researchers’ attention for the exploitation of their potential to inhibit cancer in pre-clinical models.ObjectivesTo undertake a systematic review and meta-analysis of the effects of anticoagulants in murine cancer research models. Further, to present a reference tool for anticoagulant therapeutic modalities relating to future animal pre-clinical models of cancer and their translation into the clinic.MethodsFour databases were utilized including Medline (Ovid), Embase (Ovid), Web of science, and Scopus databases. We included studies relating to any cancer conducted in murine models that assessed the effect of traditional anticoagulants (heparin and its derivatives and warfarin) and newer oral anticoagulants on cancer.ResultsA total of 6,158 articles were identified in an initial multi-database search. A total of 157 records were finally included for data extraction. Studies on heparin species and warfarin demonstrated statistically significant results in favour of tumour growth and metastasis inhibition.ConclusionOur findings constitute a valuable reference guide for the application of anticoagulants in cancer research and explore the promising utilization of non-anticoagulants heparin in preclinical cancer research.Systematic Review RegistrationPROSPERO [CRD42024555603].

Biochanin A Attenuates Psoriasiform Inflammation by Regulating Nrf2/HO-1 Pathway Activation and Attenuating Inflammatory Signalling.

Psoriasis is a long-term inflammatory skin condition marked by an overabundance of keratinocytes and the release of pro-inflammatory cytokines in the outer layer of skin. For the comprehensive management of intermediate to advanced psoriasis, innovative biological treatments have been developed. Products for the superficial therapy of mild to moderate psoriasis are still necessary, though. Trifolium pratense contains the isoflavone biochanin A (BCA), which exhibits antiviral, antioxidant, anti-carcinogenic, and anti-inflammatory properties, and helps protect the integrity and function of the endothelium. Although investigations have not shown that BCA is effective in treating psoriasis, it has been shown to slow down the breakdown of the skin barrier by regulating keratinocyte growth. We sought to clarify the basic mechanisms behind BCA's impact on psoriasis in vitro and in vivo using experimental research via regulating Nrf2/HO-1 signaling pathway. By subjecting human primary keratinocytes to psoriasis-related cytokines, psoriasis-like keratinocytes were produced. The CCK8 test was used in this investigation to assess cell viability. BCA reduced keratinocyte growth and inflammatory cascade stimulation produced by TNF-α and IL-6, according to in vitro investigations conducted on HaCaT cells. The in vivo findings showed that six days of BCA therapy significantly decreased the skin, hematological indicators, levels of NO, TBARS, histopathological, and pro-inflammatory factors of COX-2, iNOS, NF-κB pathway. It additionally influenced the protein content of pro-inflammatory cytokines such as IL-17, IL-23, IL-1β in the epidermis along with IL-6, TNF-α among the epidermis and serum. In addition, in contrast to the IMQ group, BCA improved the skin's level of Nrf2/HO-1 protein, anti-inflammatory cytokine IL-10, and antioxidant indicators like SOD, CAT, GST, GSH, GR, and Vit-C. Ultimately, our research shows that BCA was effective in treating psoriasis in pre-clinical animal models by activating the Nrf2/HO-1 pathway, leading to an increase in antioxidant and anti-inflammatory markers.

First, do no harm: the role of preclinical animal models in predicting adverse events in gene therapy clinical trials for Duchenne muscular dystrophy and X-Linked myotubular myopathy

The occurrence of severe adverse events (SAEs) in patients with Duchenne muscular dystrophy (DMD), X-linked myotubular myopathy (XLMTM), and other neuromuscular diseases treated with adeno-associated virus (AAV) constructs has prompted studies to improve the safety and efficacy of gene therapy. Physicians have weighed the medical tenet of “first, do no harm” against the perspective of patients with progressive life-threatening conditions who may accept greater risk. Regarding SAE pathogenesis, discussion has focused on total AAV exposure and patient mutations more likely to induce immunity, while stressing the limitations of animal models in predicting adverse events. Therapeutic strategies for reducing side effects have employed more myotropic AAV serotypes and efficient transgenes. Other recommendations include excluding certain DMD gene mutations associated with SAEs and substituting less immunogenic transgenes such as utrophin (DMD) and myotubularin-related protein (XLMTM). For the sake of preclinical studies, emphasis has been placed on outbred rodents and larger animals that better predict immunity. Here, the absence of side effects in canine DMD and XLMTM models might be explained partly by phenotypic differences between affected humans and dogs. Specifically, dystrophin- and myotubularin-deficient dogs exhibit milder lesions, including less muscle fat deposition and the absence of hepatopathy, respectively, which could lead to reduced immune responses to AAV constructs. To better predict future problems, thought should be given to tracking early subclinical markers of the innate immune response, especially complement activation. Regardless of steps taken to improve the predictive value of animal models for SAEs, some questions will only be answered through human clinical trials after carefully considering the risk-benefit ratio.

Biomechanical evaluation of the porcine carpus as a potential preclinical animal model for the human carpus

Advancing successful treatments for carpal instabilities of the wrist are hindered due, in part, to limited preclinical animal models. The purpose of this study was to evaluate the forelimb of the Yucatan minipig (YP) as a preclinical animal model for the human wrist by quantifying carpal biomechanics in vitro in the intact and after two ligament transection conditions. Porcine wrist biomechanics (n = 12, 5 M, 7F) were determined in 28 range of motion (ROM) directions, in pronation-supination, and in volar-dorsal translation using a six-axis robotic musculoskeletal simulator. Testing was implemented in three conditions – intact, and after sequential transection of radial intermediate ligament (RIL) and dorsal intercarpal ligament (DIC). Mixed models were employed to examine differences in direction and conditions among male and female specimens. The intact ROM envelope was elliptical in shape and oriented toward ulnar flexion with the largest ROM about 15° from the flexion–extension axis. Transection of RIL and DIC did not alter the ROM envelope orientation, however, subtle increases in ROM were observed in extension and radial deviation following transection of both RIL and DIC. Pronation in neutral was greater than supination in all three test conditions. Volar translation increased subtly in the RIL + DIC condition. This novel study investigated the multidirectional biomechanics of the YP forelimb. ROM in the general directions of extension, radial and ulnar deviation were less than in humans, while flexion was substantially larger. These specific ligament transections had minor effects on the biomechanics of the YP forelimb.

The Goat as a Model for Temporomandibular Joint Disc Replacement: Techniques for Scaffold Fixation

A state-of-the-art scaffold capable of efficiently reconstructing the temporomandibular joint (TMJ) disc after discectomy remains elusive. The major challenge has been to identify a degradable scaffold that remodels into a TMJ disc-like tissue, and prevents increased joint pathology, among other significant complications. Tissue engineering research provides a foundation for promising approaches toward the creation of successful implants/scaffolds aiming to restore the TMJ disc. In light of improving the quality of life of patients who undergo TMJ disc removal, it is critical to establish a preclinical animal model to evaluate the properties of promising scaffolds implanted post-discectomy and to determine the most efficient implantation procedures to ensure a more reliable in-depth evaluation of the biomaterial replacing the articular disc. The present study evaluated the outcomes of two protocols for the implantation of an acellular scaffold composed of an extracellular matrix (ECM) derived from the small intestinal submucosa (SIS) of the pig, as a regenerative template for the TMJ disc in a goat model. The outcomes suggest that leaving one-half of the disc medially will allow anchoring the device to the medial aspect of the joint while avoiding lateral displacement of the ECM scaffold. The goat model is ideal to assess the longevity of tissue-engineered solutions for the TMJ disc as the goat chews for 12-16 hours a day. This study provides an important reference for selecting a suitable scaffold implantation procedure and the goat model for the development of new strategies to assess TMJ disc regeneration.

Supplemental oxygen for pulmonary embolism (SO-PE): study protocol for a mechanistic, randomised, blinded, cross-over study

BackgroundAcute pulmonary embolism (PE) mortality is linked to abrupt rises in pulmonary artery (PA) pressure due to mechanical obstruction and pulmonary vasoconstriction, leading to right ventricular (RV) dilation, increased RV...

Selecting preclinical animal models in hepatology research: A call for uniform guidelines

Selecting preclinical animal models in hepatology research: A call for uniform guidelines

Matrikine stimulation of equine synovial fibroblasts and chondrocytes results in an in vitro osteoarthritis phenotype.

Osteoarthritis (OA) is a debilitating disease that impacts millions of individuals and has limited therapeutic options. A significant hindrance to therapeutic discovery is the lack of in vitro OA models that translate reliably to in vivo preclinical animal models. An alternative to traditional inflammatory cytokine models is the matrikine stimulation model, in which fragments of matrix proteins naturally found in OA tissues and synovial fluid, are used to stimulate cells of the joint. The objective of this study was to determine if matrikine stimulation of equine synovial fibroblasts and chondrocytes with fibronectin fragments (FN7-10) would result in an OA phenotype. We hypothesized that FN7-10 stimulation of equine articular cells would result in an OA phenotype with gene and protein expression changes similar to those previously described for human chondrocytes stimulated with FN7-10. Synovial fibroblasts and chondrocytes isolated from four horses were stimulated in monolayer culture for 6 or 18 h with 1 µM purified recombinant 42 kD FN7-10 in serum-free media. At the conclusion of stimulation, RNA was collected for targeted gene expression analysis and media for targeted protein production analysis. Consistent with our hypothesis, FN7-10 stimulation resulted in significant alterations to many important genes that are involved in OA pathogenesis including increased expression of IL-1β, IL-4, IL-6, CCL2/MCP-1, CCL5/RANTES, CXCL6/GCP-2, MMP-1, MMP-3, and MMP13. The results of this study suggest that the equine matrikine stimulation model of OA may prove useful for in vitro experiments leading up to preclinical trials.

Virus-like Particles-Based Vaccine to Combat Neurodegenerative Diseases.

Neurodegenerative diseases are regarded as gradual, incurable conditions with an insidious onset. Alzheimer's disease (AD) and Parkinson's disease (PD) are two of the most prevalent neurodegenerative diseases reported globally. Developing effective treatment strategies for neurodegenerative diseases has remained a primary objective and a huge challenge for researchers. The therapeutic medications that are now approved for the treatment of neurodegenerative diseases merely treat the symptoms; the underlying pathology is not addressed. Therefore, the emergence of novel disease-modifying therapeutic modalities such as immunotherapy has opened a new path in developing effective treatments for neurogenerative diseases. Compared to other types of subunit active vaccines, virus-like particles (VLPs) are considerably more immunogenic as they present dense and repetitive viral antigen epitopes on their surface, which can trigger both humoral and cell-mediated immune responses. They are also a much safer option than the traditional inactivated and live-attenuated vaccines since they are devoid of viral genomes and are, therefore, non-pathogenic and non-infectious. Researchers have turned their attention to VLPs as an active immunotherapy candidate for AD due to the lessons learned from the AN1792 trial. Studies have shown that they effectively induce anti-Aβ, anti-tau, and anti-α-Synuclein antibodies while avoiding T-cell-related immune reactions in animal models of AD and PD. This review compiles the findings of preclinical animal model studies and clinical investigations on VLP-based vaccines for neurogenerative diseases thus far. The technical limitations and potential difficulties associated with the future application of VLP-based vaccines in patients with neurodegenerative diseases have also been covered.

Responsive Magnetic Particle Imaging Tracer: Overcoming "Always-On" Limitation, Eliminating Interference, and Ensuring Safety in Adaptive Therapy.

Magnetic particle imaging (MPI) has emerged as a novel technology utilizing superparamagnetic nanoparticles as tracers, essential for disease diagnosis and treatment guidance in preclinical animal models. Unlike other modalities, MPI provides high sensitivity, deep tissue penetration, and no signal attenuation. However, existing MPI tracers suffer from "always-on" signals, which complicate organ-specific imaging and hinder accuracy. To overcome these challenges, we have developed a responsive MPI tracer using pH-responsive PdFe alloy particles coated with a gatekeeper polymer. This tracer exhibits pH-sensitive Fe release and modulation of the MPI signal, enabling selective imaging with a higher signal-to-noise ratio and intratumoral pH quantification. Notably, this responsive tracer facilitates subtraction-enhanced MPI imaging, effectively eliminating interference from liver uptake and expanding the scope of abdominal imaging. Additionally, the tracer employs a dual-function mechanism for adaptive cancer therapy, combining pH-switchable enzyme-like catalysis with dual-key co-activation of ROS generation, and Pd skeleton that scavenges free radicals to minimize Fe-related toxicity. This advancement promises to significantly expand MPI's applicability in diagnostics and therapeutic monitoring, marking a leap forward in imaging technology.

Endocannabinoid concentrations in major depression: effects of childhood maltreatment and relation to hippocampal volume

Evidence from preclinical animal models suggests that the stress-buffering function of the endocannabinoid (eCB) system may help protect against stress-related reductions in hippocampal volume, as is documented in major depressive disorder (MDD). However, stress exposure may also lead to dysregulation of this system. Thus, pathways from marked stress histories, such as childhood maltreatment (CM), to smaller hippocampal volumes and MDD in humans may depend on dysregulated versus intact eCB functioning. We examined whether the relation between MDD and peripheral eCB concentrations would vary as a function of CM history. Further, we examined whether eCBs moderate the relation of CM/MDD and hippocampal volume. Ninety-one adults with MDD and 62 healthy comparison participants (HCs) were recruited for a study from the Canadian Biomarker Integration Network in Depression program (CAN-BIND-04). The eCBs, anandamide (AEA) and 2-arachidonylglycerol (2-AG), were assessed from blood plasma. Severe CM history was assessed retrospectively via contextual interview. MDD was associated with eCBs, though not all associations were moderated by CM or in the direction expected. Specifically, MDD was associated with higher AEA compared to HCs regardless of CM history, a difference that could be attributed to psychotropic medications. MDD was also associated with higher 2-AG, but only for participants with CM. Consistent with hypotheses, we found lower left hippocampal volume in participants with versus without CM, but only for those with lower AEA, and not moderate or high AEA. Our study presents the first evidence in humans implicating eCBs in stress-related mechanisms involving reduced hippocampal volume in MDD.

Bimagrumab: an investigational human monoclonal antibody against activin type II receptors for treating obesity.

Bimagrumab is a human monoclonal antibody that prevents activin type II receptors (ActRII) from functioning. This antibody has a higher affinity for muscle activin-2 receptors than natural ligands such as activin and myostatin, which act as negative muscle growth regulators. Blocking the activin receptor with bimagrumab could be a new pharmaceutical approach for managing patients with obesity and type 2 diabetes mellitus (T2DM). Bimagrumab has anabolic effects on skeletal muscle mass by preventing myostatin binding and other negative muscle growth regulators. Preclinical animal models have also shown that ActRII blockade promotes actions beyond skeletal muscle, including effects on brown adipose tissue (BAT) differentiation and activity. In a phase 2 randomized clinical trial, ActRII blockade with bimagrumab led to significant loss of total body fat mass (FM), lean mass (LM) gain, and metabolic improvements over 48weeks in overweight or obese patients with type 2 diabetes. The trial involved [number of participants], and the results showed [specific findings]. Currently, Bimagrumab is being evaluated for its potential to treat muscle wasting, functional loss in hip fractures and sarcopenia, as well as obesity. However, it is essential to note that Bimagrumab also blocks the effects of other ActRII ligands, which play a role in the neurohormonal axes, pituitary, gonads, and adrenal glands. These observations suggest that bimagrumab might represent a new approach for treating patients with obesity and related metabolic disturbances.

Toxicology profile of a novel GLP-1 receptor biased agonist-SAL0112 in nonhuman primates

Oral small-molecule GLP-1 receptor biased agonists exhibit promising treatment efficacy of type 2 diabetes and obesity. SAL0112 is a novel compound that has demonstrated remarkable efficacy in preclinical animal models. Herein, both in vitro and in vivo preclinical toxicity investigations were conducted to explore the safety profile of SAL0112. The HTRF assay and TR-FRET assay were utilized for cAMP detection. Patch clamp assay was employed for hERG potassium ion channel determination. Cynomolgus monkeys were used in a cardiovascular safety pharmacology study and a 13-week repeated dose toxicity study. The telemetry system was employed to detect cardiovascular indicators such as ECG, HR, and BP. During the repeated dose toxicity study, body weight, food intake, hematology, coagulation function test, serum biochemistry tests, and urine analysis were measured. Macroscopic and microscopic observations were conducted at the end of the study. TK studies were conducted on Day 1 and Day 91. SAL0112 exhibited a high degree of potency in activating the monkey GLP-1 receptor whereas had no effect on the rodent GLP-1 receptor. In contrast to Danuglipron, which demonstrated high potency on hERG with an IC50 value of 6.9 μM, the IC50 of SAL0112 on hERG was greater than 100 μM. Compared to the Vehicle Control group, no significant changes in cardiovascular indicators were observed in the cardiovascular safety pharmacology study after a single dose of SAL0112 up to 250 mg/kg (P > 0.05). A repeated dose toxicity study revealed moderate anorexigenic effects and a reduction in body weight, effects that were found to be reversible and not associated with any pathological changes. The NOAEL of SAL0112 is 150 mg/kg, providing an approximate safety margin of threefold. SAL0112 demonstrated a favorable safety profile in cynomolgus monkeys, with a substantial therapeutic window that supports the progression of this compound into clinical studies.

Cellular and Molecular Mechanisms of Hypertrophy of Ligamentum Flavum.

Hypertrophy of the ligamentum flavum (HLF) is a common contributor to lumbar spinal stenosis (LSS). Fibrosis is a core pathological factor of HLF resulting in degenerative LSS and associated low back pain. Although progress has been made in HLF research, the specific molecular mechanisms that promote HLF remain to be defined. The molecular factors involved in the onset of HLF include increases in inflammatory cytokines such as transforming growth factor (TGF)-β, matrix metalloproteinases, and pro-fibrotic growth factors. In this review, we discuss the current understanding of the mechanisms involved in HLF with a particular emphasis on aging and mechanical stress. We also discuss in detail how several pathomechanisms such as fibrosis, proliferation and apoptosis, macrophage infiltration, and autophagy, in addition to several molecular pathways involving TGF-β1, mitogen-activated protein kinase (MAPKs), and nuclear factor-κB (NF-κB) signaling, PI3K/AKT signaling, Wnt signaling, micro-RNAs, extracellular matrix proteins, reactive oxygen species (ROS), etc. are involved in fibrosis leading to HLF. We also present a summary of the current advancements in preclinical animal models for HLF research. In addition, we update the current and potential therapeutic targets/agents against HLF. An improved understanding of the molecular processes behind HLF and a novel animal model are key to developing effective LSS prevention and treatment strategies.

Regulation of β-Adrenergic Receptors in the Heart: A Review on Emerging Therapeutic Strategies for Heart Failure.

The prolonged overstimulation of β-adrenergic receptors (β-ARs), a member of the G protein-coupled receptor (GPCR) family, causes abnormalities in the density and functionality of the receptor and contributes to cardiac dysfunctions, leading to the development and progression of heart diseases, especially heart failure (HF). Despite recent advancements in HF therapy, mortality and morbidity rates continue to be high. Treatment with β-AR antagonists (β-blockers) has improved clinical outcomes and reduced overall hospitalization and mortality rates. However, several barriers in the management of HF remain, providing opportunities to develop new strategies that focus on the functions and signal transduction of β-ARs involved in the pathogenesis of HF. As β-AR can signal through multiple pathways influenced by different receptor subtypes, expression levels, and signaling components such as G proteins, G protein-coupled receptor kinases (GRKs), β-arrestins, and downstream effectors, it presents a complex mechanism that could be targeted in HF management. In this narrative review, we focus on the regulation of β-ARs at the receptor, G protein, and effector loci, as well as their signal transductions in the physiology and pathophysiology of the heart. The discovery of potential ligands for β-AR that activate cardioprotective pathways while limiting off-target signaling is promising for the treatment of HF. However, applying findings from preclinical animal models to human patients faces several challenges, including species differences, the genetic variability of β-ARs, and the complexity and heterogeneity of humans. In this review, we also summarize recent updates and future research on the regulation of β-ARs in the molecular basis of HF and highlight potential therapeutic strategies for HF.

Tissue-Engineered Three-Dimensional Platforms for Disease Modeling and Therapeutic Development.

Three-dimensional (3D) tissue-engineered models are under investigation to recapitulate tissue architecture and functionality, thereby overcoming limitations of traditional two-dimensional cultures and preclinical animal models. This review highlights recent developments in 3D platforms designed to model diseases in vitro that affect numerous tissues and organs, including cardiovascular, gastrointestinal, bone marrow, neural, reproductive, and pulmonary systems. We discuss current technologies for engineered tissue models, highlighting the advantages, limitations, and important considerations for modeling tissues and diseases. Lastly, we discuss future advancements necessary to enhance the reliability of 3D models of tissue development and disease.

7288 A Preclinical Mouse Model of Gender-Affirming Hormone Therapy: Metabolic and Behavioral Outcomes

Abstract Disclosure: A. Yasrebi: None. K. Otersen: None. O. Groh: None. E. Guthman: None. I. Shmarakov: None. S. Campbell: None. T.A. Roepke: None. The long-term metabolic effects of gender-affirming hormone therapy (GAHT) is largely unknown, in part, due to the lack of properly designed preclinical animal models. Here we propose a rodent study design, beyond the classical endocrine model of gonadectomy and steroid treatment, to accurately reflect the reality of the trans experience. Specifically, trans women do not all seek or undergo orchidectomy (ORX) instead take estrogen supplementation along with an androgen blocker such as finasteride. In this study, 40 WT C57 male mice were divided into 4 groups of 10 mice - 1) intact with oil vehicle, 2) intact with estradiol benzoate (EB, 150 μg/kg/d) and finasteride (F, 0.25 mg/kg/d), 3) ORX with oil; and ORX with EB (150 μg/kg/d). Each mouse was dosed daily for 8 weeks while on a standard chow diet. Mice were pair housed and food intake and body weight were collected weekly prior to behavioral (open field, elevated plus, Y-maze) and metabolic (body composition, metabolic rates, activity, tolerance testing) phenotyping. Intact mice treated with EB+F exhibited less avoidance behaviors, potentially due to differences in movement, with no differences between treatments in the Y-maze. Cumulatively, Intact:EB+F gained less weight than Intact:oil (3.4 ± 0.4 g vs 5.3 ± 0.4 g), while ORX:oil mice gained less than ORX:EB (2.2 ± 0.5 g vs 5.5 ± 0.7 g). These interactive effects were due to differences in the gain of fat or lean mass between ORX and intact and lower cumulative food intake in ORX:oil mice (136 ± 3.8 g vs Intact:oil: 159 ± 4.9 g; Intact:EB+F: 167 ± 6.1 g; ORX:EB: 171 ± 3.4 g). Metabolic assessments found that Intact:EB+F exhibited elevated V.O2, V.CO2, and heat production in the light and dark cycle compared to Intact:oil, while ORX:EB only exhibited elevated heat production compared to ORX:oil. Similarly, Intact:EB+F and ORX:EB moved more than their counterparts during the dark cycle; however, only Intact:EB+F exhibited elevated wheel running. Tolerance tests (i.p.) found that glucose clearance was augmented in ORX:EB mice while insulin-induced glucose clearance was lower in Intact:EB+F compared to Intact:oil but modestly elevated by EB in ORX mice. These data, along with suppressed fasting blood glucose in ORX:EB mice, suggest that glucose homeostasis is differentially modulated by GAHT paradigms in mice. Analysis of hypothalamic, hepatic, adipose, and intestinal gene expression is currently underway. Collectively, these data, generated by using a translationally-relevant preclinical animal model, are the first to establish that metabolism is differentially affected by both steroid treatments and ORX and suggesting that the long-term metabolic effects of GAHT are dependent on the type of treatment. The underlying mechanisms and sites of action remain to be investigated. Presentation: 6/3/2024