Experimental Model Research Articles

Exploring the efficacy of Transcutaneous Auricular Vagus nerve stimulation (taVNS) in modulating local and systemic inflammation in experimental models of colitis

BackgroundCurrent inflammatory bowel disease (IBD) treatments often fail to achieve lasting remission and have adverse effects. Vagus nerve stimulation (VNS) offers a promising therapy due to its anti-inflammatory effects. Its invasive nature, however, has led to the development of non-invasive methods like transcutaneous auricular VNS (taVNS). This study assesses taVNS’s impact on acute colitis progression, inflammatory, anti-inflammatory, and apoptosis-related markers.MethodsMale C57BL/6 mice (11–12 weeks) were used for dextran sulfate sodium (DSS)- and dinitrobenzene sulfonic acid (DNBS)-induced colitis studies. The administration of taVNS or no stimulation (anesthesia without stimulation) for 10 min per mouse began one day before colitis induction and continued daily until sacrifice. Ulcerative colitis (UC)-like colitis was induced by administering 5% DSS in drinking water for 5 days, after which the mice were sacrificed. Crohn’s disease (CD)-like colitis was induced through a single intrarectal injection of DNBS/ethanol, with the mice sacrificed after 3 days. Disease activity index (DAI), macroscopic evaluations, and histological damage were assessed. Colon, spleen, and blood samples were analyzed via qRT-PCR and ELISA. One-way or two-way ANOVA with Bonferroni and Šídák tests were applied.ResultstaVNS improved DAI, macroscopic, and histological scores in DSS colitis mice, but only partially mitigated weight loss and DAI in DNBS colitis mice. In DSS colitis, taVNS locally decreased colonic inflammation by downregulating pro-inflammatory markers (IL-1β, TNF-α, Mip1β, MMP 9, MMP 2, and Nos2) at the mRNA level and upregulating anti-inflammatory TGF-β in non-colitic conditions at both mRNA and protein levels and IL-10 mRNA levels in both non-colitic and colitic conditions. Systemically, taVNS decreased splenic TNF-α in non-colitic mice and increased serum levels of TGF-β in colitic mice and splenic levels in non-colitic and colitic mice. Effects were absent in DNBS-induced colitis. Additionally, taVNS decreased pro-apoptotic markers (Bax, Bak1, and caspase 8) in non-colitic and colitic conditions and increased the pro-survival molecule Bad in non-colitic mice.ConclusionsThis study demonstrates that taVNS has model-dependent local and systemic effects, reducing inflammation and apoptosis in UC-like colitis while offering protective benefits in non-colitic conditions. These findings encourage further research into underlying mechanisms and developing adjunct therapies for UC.

Stard1 promotes cholestatic liver injury and disease progression by sensitizing to bile acid hepatotoxicity.

Cholestatic liver diseases (CLD) are often accompanied by hepatocellular injury, fibrosis, and cirrhosis due to the intracellular accumulation of solutes that cannot be excreted into bile, including bile acids (BAs). These are synthesized in hepatocytes from cholesterol mainly via the classic pathway and in a lower proportion through the mitochondrial acidic pathway. The latter requires STARD1-dependent cholesterol transport to the mitochondrial inner membrane for metabolism, whose contribution to BA-induced hepatotoxicity and CLD is unknown. Here we show that patients with primary biliary cholangitis exhibit increased expression of STARD1 compared to control subjects. Mice with hepatocyte-specific Stard1 deletion (Stard1Δhep) were more resistant to experimental models of complete (bile duct ligation, BDL) and chemical obstructive cholestasis-induced liver injury, inflammation and fibrosis than Stard1f/f mice. Stard1Δhep mice exhibited reduced hepatic BAs and mitochondrial cholesterol accumulation but increased mitochondrial GSH (mGSH) levels following BDL compared to Stard1f/f mice. Pharmacological mGSH depletion sensitized primary mouse hepatocytes to a mix of BAs mimicking the profile seen in Stard1f/f mice after BDL leading to increased inflammatory response and cytotoxicity. These findings highlight a role for STARD1 in cholestatic liver injury and suggest that its targeting may be of relevance for CLD.

The application of artificial neural network (ANN) to validate biosorption of Zn+2 ions onto chemically modified Hevea brasiliensis sawdust

The study examined the biosorption of Zn+2 by chemically modified Hevea brasiliensis sawdust using a batch adsorption system. The influence of concentration, pH, contact time, and adsorbent dose in a batch biosorption process were analyzed and the maximum Zn+2 uptake capacity was found as 36.8 mg/g. The Langmuir and Temkin model was found to be the most accurate when the experimental data were analyzed using the different adsorption isotherms models. This study presents an experimental modeling approach for the biosorption process based on artificial neural networks (ANN). Data from biosorption experiments were utilized to train and validate the ANN model. The ANN model can effectively represent the biosorption process, as demonstrated by the significant correlation (R 2 = 0.998) established between the model and experimental data. A kinetic study revealed that the Zn+2 biosorption was favored by the pseudo-first-order and pseudo-second-order models. The process is feasible, spontaneous, and endothermic, according to thermodynamic study. The H. brasiliensis sawdust underwent chemical carbonization, and its characteristics were examined using energy EDX, BET surface area analyzer, SEM, XRD, and FTIR.

The Effect of Water-Soluble Alpinia Galanga Extract on Sleep and the Activation of the GABAAergic/Serotonergic Pathway in Mice.

Background/Objectives: With increasing interest in plant-based compounds that can enhance sleep quality without the side effects of caffeine, Alpinia galanga (AG) has emerged as a promising herbal supplement for improving mental alertness. This study assessed the impact of water-soluble AG extract on sleep quality; the activity of GABAergic, glutamatergic, and serotonergic receptors; and concentrations of dopamine and serotonin in the brains of mice. Methods: The study employed two experimental models using BALB/c mice to examine the impact of pentobarbital-induced sleep and caffeine-induced insomnia. In the first model, a set of 20 mice was assigned to four groups to assess the effects of pentobarbital (42 mg/kg) or pentobarbital with AG extract on sleep induction, with observations made 45 min post-administration. In the second model, 20 mice were divided into four groups to evaluate the impact of caffeine (25 mg/kg) alone or caffeine with varying doses of AG extract (61.25 or 205.50 mg/kg administered orally) on brain activity along with additional analyses on receptor proteins and neurotransmitters. Results: A higher dose of AG extract (205.50 mg/kg) significantly increased total deep sleep duration compared to the caffeine group (p < 0.0001). Furthermore, this dose extended sleep latency and suppressed GABAergic and glutamatergic receptor activity compared to the lower AG dose (p < 0.05). Additionally, the 205.50 mg/kg dose elevated serotonin and dopamine levels compared to caffeine (p < 0.0001), suggesting improved sleep quality alongside enhanced wakefulness. Conclusions: Our data indicate that a higher dose of AG extract improved sleep latency and duration by regulating GABAergic and glutamatergic receptors through the GABAergic/serotonergic pathway in mice.

Simulation and Analysis of Double Compound CAM Pusher Tool Changing Mechanism for NC Machine Tool

The core mechanism of existing CNC tool changers is typically a single composite cam swinging rod linkage. This setup has two main drawbacks: amplified vibration errors and high impact forces due to the swinging rod. To address these, this study focuses on CNC tool changers, specifically selecting the up-and-down motion follower and proposing a novel double composite cam push rod type mechanism. Solid modeling for both mechanisms was conducted using Creo and Matlab, and theoretical modeling of the contact forces for each follower type was completed. Comparative analysis shows the push rod follower exhibits lower contact forces than the swinging rod. Using the Adams platform, the dynamic characteristics of both mechanisms were analyzed, leading to an optimized design for the push rod type. An experimental model, scaled to 0.5, was built to compare experimental data with simulation results. The findings verify the accuracy of the models, supporting the feasibility of the push rod type tool changer.

Multiscale computational model predicts how environmental changes and treatments affect microvascular remodeling in fibrotic disease.

Investigating the molecular, cellular, and tissue-level changes caused by disease, and the effects of pharmacological treatments across these biological scales, necessitates the use of multiscale computational modeling in combination with experimentation. Many diseases dynamically alter the tissue microenvironment in ways that trigger microvascular network remodeling, which leads to the expansion or regression of microvessel networks. When microvessels undergo remodeling in idiopathic pulmonary fibrosis (IPF), functional gas exchange is impaired and lung function declines. We integrated a multiscale computational model with independent experiments to investigate how combinations of biomechanical and biochemical cues in IPF alter cell fate decisions leading to microvascular remodeling. Our computational model predicted that extracellular matrix (ECM) stiffening reduced microvessel area, which was accompanied by physical uncoupling of endothelial cell (EC) and pericytes, the cells that comprise microvessels. Nintedanib, an Food and Drug Administration-approved drug for treating IPF, was predicted to further potentiate microvessel regression by decreasing the percentage of quiescent pericytes while increasing the percentage of pericytes undergoing pericyte-myofibroblast transition in high ECM stiffnesses. Importantly, the model suggested that YAP/TAZ inhibition may overcome the deleterious effects of nintedanib by promoting EC-pericyte coupling and maintaining microvessel homeostasis. Overall, our combination of computational and experimental modeling can predict and explain how cell decisions affect tissue changes during disease and in response to treatments.

Quantitative assessment of 167Tm isotope production: experimental data analysis and model validation.

167Tm is an effective radioisotope for use in theragnostic purposes. This study aims to find the best alternative routes for producing 167Tm for appropriate cancer therapy and diagnostics. In this study, we comprehensively analyzed production cross-sections of 167Tm and investigated the behavior of these reactions using six different level density models and refined optical model potential (OMP) parameters with the nuclear reaction code TALYS 1.96. Using this code, we estimated the specific activity and production yield for promising production routes. For characterizing the 167Tm isotopes, the energy distribution of 167Tm was also analyzed for each prominent reaction by particle and heavy ion transport code system (PHITS). The maximum specific activity was estimated as 52.6-75.2GBq/g with a production yield of 20.2-32.7GBq/mAh for the route 169Tm(p,x)167Tm. The experimental value was reproduced with good agreement by TALYS adjusting different OMP parameters. 169Tm(p,x)167Tm is the alternative route for producing 167Tm, for which TALYS can reproduce the result more accurately.

Hippocampal damage through foreign body placement in organotypic cultures leads to plastic responses in newly born granule cells.

The dentate gyrus of the hippocampus is a plastic structure that displays modifications at different levels in response to positive stimuli as well as to negative conditions such as brain damage. The latter involves global alterations, making understanding plastic responses triggered by local damage difficult. One key feature of the dentate gyrus is that it contains a well-defined neurogenic niche, the subgranular zone, and beyond neurogenesis, newly born granule cells may maintain a "young" phenotype throughout life, adding to the plastic nature of the structure. Here, we present a novel experimental model of local brain damage in organotypic entorhino-hippocampal cultures that results in the activation of adjacent newly born granule cells. A small piece of filter paper was placed on the surface of the granule cell layer of the dentate gyrus, which evoked a foreign body reaction of astrocytes, along with the activation of local young neurons expressing doublecortin. Forty-eight hours after foreign body placement, the number of doublecortin-immunoreactive cells increased in the subgranular zone in the direct vicinity of the foreign body, whereas overall increased doublecortin immunoreactivity was observed in the granule cell layer and molecular layer of the dentate gyrus. Foreign body placement in the pyramidal layer of the CA1 region evoked a comparable local astroglial reaction but did not lead to an increase in doublecortin-immunoreactive in either the CA1 region or the adjacent dentate gyrus. Seven days after foreign body placement in the dentate gyrus, the increase in doublecortin-immunoreactivity was no longer observed, indicating the transient activation of young cells. However, 7 days after foreign body placement, the number of doublecortin-immunoreactive granule cells coimmunoreactive for calbindin was lower than that under the control conditions. As calbindin is a marker for mature granule cells, this result suggests that activated young cells remain at a more immature stage following foreign body placement. Live imaging of retrovirally green fluorescent protein-labeled newly born granule cells revealed the orientation and growth of their dendrites toward the foreign body placement. This novel experimental model of foreign body placement in organotypic entorhino-hippocampal cultures could serve as a valuable tool for studying both glial reactivity and neuronal plasticity, specifically of newly born neurons under controlled in vitro conditions.

Probiotics Bi-Enzymatic Cascade Repair System for Editing the Inflammatory Microenvironment to Boost Probiotic Therapy in Inflammatory Bowel Disease.

Inflammatory bowel disease presents significant treatment challenges owing to its complex pathology. Although probiotics have shown promise as a therapeutic option, their effectiveness is often limited by low concentrations at sites of inflammation, exacerbated by excessive reactive oxygen species and inflammatory triggers. To address this, an innovative cascade repair system is developed to enhance probiotic therapeutic impact by modulating the intestinal microenvironment. This system uses iMXene's catalytic properties to neutralize reactive oxygen species in the gut and its capacity to deliver the CRISPR/dCas9 gene editing system to activate the NLR family pyrin domain containing 12 genes, helping suppress inflammation. By promoting the colonization of Lactobacillus rhamnosus, the system inhibits inflammation pathways and supports the restoration of a balanced intestinal flora through a cascade repair mechanism. These findings demonstrate significant therapeutic benefits in experimental models, with improvements in the overall well-being of treated mice and effective repair of intestinal inflammation damage. This pioneering approach holds promise for inflammatory bowel disease treatment and opens new avenues for managing other inflammatory conditions, offering valuable insights and guidance for future research into inflammatory diseases.

3D printed respiratory tract enables studies of airborne disease dispersal

The experimental model will help provide a baseline for numerical studies, enable better mask design and improve guidelines for social distancing.

Modeling of fresh paddy aeration on transporting barges in Mekong Delta, Vietnam

The Mekong Delta is regarded as the granary of Vietnam, with an annual production of 24.1 million tons (2023). Currently, fresh paddy is transported directly from farmers’ fields to drying facilities or rice mills. Given the extensive river and canal system in the Mekong Delta, the majority of paddy (up to 92%) is transported by barges. While barge transportation is more costeffective (91,000 VND/ton for a 100 km distance) compared to truck transportation (269,000 VND/ton), it has significant drawbacks, such as a longer transportation time of 3 to 5 days. Moreover, because the paddy is wet during transport, it is prone to discoloration (yellowing), which reduces its quality. To address this issue, this research developed an experimental paddy aeration model as a foundation for implementing aeration on barges during transportation. The model was tested with 1.4 tons of fresh paddy, specifically the DT80 variety, in Cai Lay, Tien Giang province. Key parameters monitored and analyzed included paddy temperature, moisture content, ambient conditions, and grain quality. The model was tested with a specific airflow rate of 129 ± 23 m3/h per ton. The paddy temperature in the aeration model was maintained at 28.8°C, similar to the ambient temperature of 28.3°C. As a result, the whiteness of the aerated paddy was preserved at 3.6% after three days, whereas it decreased from 3.6% to 3.2% in paddy transported without aeration. The application of aeration helped reduce quality losses, particularly discoloration, caused by the high temperatures of fresh paddy during transportation.

Investigating the Expression and Function of HIF-1α in Neocaridina davidi During Embryo Cleavage Stage

Neocaridina davidi, a member of Decapoda within Crustacea, stands out due to its petite stature, robust reproductive capabilities and short molting cycle. Consequently, it has emerged as a valuable experimental model for investigations spanning ecology, development, physiology, and toxicology. Despite the pivotal role of Hypoxia Inducible Factor-1α (HIF-1α) in diverse physiological processes like biological hypoxia stress, cellular growth, and stress resistance, its functionality in crustaceans remains underexplored. Moreover, the role and function of HIF-1α in crustaceans, especially in embryonic stages, have been insufficiently addressed. This study delves into the function of HIF-1α during embryonic development. Initially, the complete sequence of the HIF-1α gene was acquired. Notably, the expression of HIF-1α during the cleavage stage surpassed that of subsequent phases. Subsequently, dsRNAHIF-1α was introduced into sexually mature shrimp, revealing that the inhibitory impact of dsRNA on gene expression in the parental generation could be inherited by the offspring, exerting a specific gene silencing effect in the embryo. The consequences of silencing HIF-1α in embryos manifested as varying degrees of premature division termination, besides, the glycolysis in the embryos was also affected by HIF-1α suppression. These results provide data support for understanding the early embryonic development of crustaceans and HIF-1α functions within it.

Multiple factors on driving load in mountain area at night based on factor analysis.

Different driving environments may lead to increased mental workload and fatigue among drivers, consequently diminishing driving safety. To investigate the impact of various factors on drivers' driving load, this study approaches the issue from three perspectives: external weather environment, road driving environment, and in-vehicle driving environment. Through the experimental modeling of the mountainous road section of Provincial Highway S208 in Chongyang County, Xianning City, and employing simulated driving experiments, various factor combinations were designed to investigate the visual characteristics of drivers. Through factor analysis, a comprehensive examination is conducted on four visual indicators: the rate of change in pupil area, fixation duration, saccade velocity, and saccade angle, to explore the patterns of influence exerted by these factors on the driving load of drivers. The evaluation results of driving workload indicate that the degree of driver distraction decreases when the plant spacing is set at 6 meters and the type of road traffic auxiliary facilities is configured to two. When the traffic flow on the road is zero and no driving sub-mission are present, the driver experiences the minimum level of workload. The findings of this study provide robust theoretical support for nighttime mountain driving safety, contributing to the in-depth exploration of traffic safety theories.

Timing of standard chow exposure determines the variability of mouse phenotypic outcomes and gut microbiota profile.

Standard chow diets influence reproducibility in animal model experiments because chows have different nutrient compositions, which can independently influence phenotypes. However, there is little evidence of the role of timing in the extent of variability caused by chow exposure. Here we measured the impact of different diets (5V5M, 5V0G, 2920X and 5058) and timing of exposure (adult exposure (AE), lifetime exposure (LE) and developmental exposure (DE)) on growth and development, metabolic health indicators and gut bacterial microbiota profiles across genetically identical C57BL/6J mice. Diet drove differences in macro- and micronutrient intake for all exposure models. AE had no effect on phenotypic outcomes. However, LE mice exhibited significant sex-dependent diet effects on growth, body weight and body composition. LE effects were mostly absent in the DE model, where mice were exposed to chow differences only from conception to weaning. Both AE and LE models exhibited similar diet-driven beta diversity profiles for the gut bacterial microbiota, with 5058 diet driving the most distinct profile. However, compared with AE, LE effects on beta diversity were sex dependent, and LE mice exhibited nine times more differentially abundant bacterial genera, the majority of which were inversely affected by 2920X and 5058 diets. Our findings demonstrate that LE to different chow diets has the greatest impact on the reproducibility of several experimental measures commonly used in preclinical mouse model studies. Importantly, weaning mice from different diets onto the same diet for maturation may be an effective way to reduce unwanted phenotypic variability among experimental models.

Phytochemical compounds for treating hyperuricemia associated with gout: a systematic review.

Gout is a prevalent metabolic disorder characterized by increased uric acid (UA) synthesis or decreased UA clearance from the bloodstream, leading to the formation of urate crystals in joints and surrounding tissues. Hyperuricemia (HUA), the underlying cause of gout, poses a growing challenge for healthcare systems in developed and developing countries. Currently, the most common therapeutic approaches for gouty HUA primarily involve the use of allopathic or modern medicine. However, these treatments are often accompanied by adverse effects and may not be universally effective for all patients. Therefore, this systematic review aims to provide a comprehensive outline of phytochemical compounds that have emerged as alternative treatments for HUA associated with gout and to examine their specific mechanisms of action. A systematic search was conducted to identify phytochemicals that have previously been evaluated for their effectiveness in reducing HUA. From a review of > 800 published articles, 100 studies reporting on 50 phytochemicals associated with the management of HUA and gout were selected for analysis. Experimental models were used to investigate the effects of these phytochemicals, many of which exhibited multiple mechanisms beneficial for managing HUA. This review offers valuable insights for identifying and developing novel compounds that are safer and more effective for treating HUA associated with gout.

Myelin Imaging of the Spinal Cord in Animal Models and Patients with Multiple Sclerosis Using [11C]MeDAS PET: A Translational Study.

Multiple sclerosis (MS) is a neurodegenerative disease characterized by demyelinated lesions in the brain and spinal cord. A few clinical studies using PET to image myelin in the brain have been performed, but none investigated the spinal cord. Because clinically relevant motor symptoms are primarily due to spinal cord damage, this translational study evaluated [11C]N-methyl-4,4'-diaminostilbene (MeDAS) as a PET tracer for myelin imaging in the rat and human spinal cord. Methods: [11C]MeDAS PET of the spinal cord was conducted in experimental autoimmune encephalomyelitis, lysophosphatidylcholine, and spinal cord injury animal models of focal demyelination. Then, 6 healthy controls and 11 MS patients were subjected to MRI and [11C]MeDAS PET of the spinal cord between C5 and T6 vertebrae. Regions of interest covering 100%, 60%, and 40% of the diameter of the spinal canal were drawn, and tracer uptake was normalized to the activity in the blood pool, muscle, or injected dose per unit of body weight (SUV). Results: [11C]MeDAS uptake was significantly reduced in spinal cord lesions in all animal models. In humans, tracer uptake was significantly higher in the cervical than the thoracic spinal cord, which corresponds well with the known physiologic rostral-caudal gradient in myelin density. MS patients had significantly lower [11C]MeDAS uptake in the upper spinal cord (C5-T3) than did controls. The [11C]MeDAS PET signal was inversely correlated with the presence of MS lesions in specific sections of the spinal cord. The best differentiation among regions with different myelin density was obtained when the smallest region of interest was used and spinal cord uptake was expressed as SUV. Conclusion: [11C]MeDAS PET shows the potential to quantify myelin density in the spinal cord. It enables detection of physiologic differences in myelin density between spinal cord segments and between MS patients and healthy controls, which warrants further evaluation of this technique.

Heat transfer with MgO nanofluid in laminar flow: experimental study and ANSYS modeling

Heat transfer with MgO nanofluid in laminar flow: experimental study and ANSYS modeling

Evaluation of the redox alteration in Duchenne muscular dystrophy model mice using in vivo DNP-MRI

Duchenne muscular dystrophy (DMD) is a genetic muscular disease and is the most common type of muscular dystrophy in Japan. Noninvasive magnetic resonance imaging (MRI) can be used for follow-up evaluation of myositis and muscular dystrophy, including DMD and inflammation is evaluated based on the increased muscle water as evaluated by T2-weighted MR images. However, in MDM, the redox status has not been evaluated non-invasively during the disease progression. We assessed the inflammation via the redox status in experimental animal disease models using in vivo dynamic nuclear polarization MRI (DNP-MRI) with a redox probe. The current study aimed to evaluate the skeletal muscle of mdx mice, a DMD model, in which muscle fiber necrosis, inflammation, and muscle regeneration were chronically repeated. Results showed that the reduction rate of Carbamoyl-PROXYL (CmP), one of the redox probes, radicals in mdx mice increased compared with that in normal mice. In vitro, more mitochondria or macrophages enhanced the radical form decay reaction by reducing CmP. Due to muscle fiber damage, the mdx mice had a lower mitochondrial concentration in the gastrocnemius muscle than the normal mice. However, the in vivo DNP-MRI results strongly reflected the increased reduction of CmP radicals by macrophages. In conclusion, in vivo DNP-MRI, a noninvasive imaging method is useful for locally evaluating skeletal muscle inflammation.

The effect of caffeic acid phenethyl ester on facial nerve regeneration

Background Caffeic acid phenethyl ester (CAPE), an active component of honey bee propolis, has been demonstrated in animal models and studies to have anti-inflammatory, antioxidant, immunomodulatory, neuroprotective, and cytoprotective properties. Objective We investigated the efficacy of CAPE, which we believe may be therapeutically useful in facial nerve restoration due to its neuroprotective and antioxidant properties. Material and methods 20 Sprague Dawley rats were divided randomly into 4 primary and 2 secondary groups and assigned as control, methylprednisolone, CAPE, CAPE+methylprednisolone groups and the sham and the trauma groups. Except for the sham group, all groups had the left facial nerve crushed. Three weeks after surgery, prospective functional, electrophysiologic, and histologic recovery was assessed. Results The CAPE and CAPE+methylprednisolone group had similar and more significant recovery at Nerve Excitability Thresholds and achieved a significantly faster improvement in histopathological evaluation at the end of three weeks. Conclusions and significance The combination of CAPE and methylprednisolone was found to be efficient in nerve regeneration in an experimental rat facial nerve crush model. Given the strong systemic adverse effects of methylprednisolone, the combination of CAPE may be a good alternative for lowering the dose of methylprednisolone and thereby reducing its negative effects.

Rural parents’ route choices under different modes for school children drop-off and pick-up: a stated choice experiment

ABSTRACT Urbanization and school consolidation in rural areas have led to longer school commuting distances and changes in travel patterns, including increased use of private cars and e-bikes. As a result, greater parental involvement is needed to ensure safe and timely school commutes. This study explored how parents/guardians’ preferences for the built environment affect their route choices under different transportation modes in rural school commutes. Using a stated choice experiment and discrete choice models, the study identified key factors influencing route choices, such as travel distance and built environment characteristics like fewer road intersections, low traffic speeds, presence of sidewalks/bike lanes, road separation provisions, traffic lights, zebra crossings, green plants, and shops when walking, cycling, and riding an e-bike. The findings highlight the need to consider parents’ preferences when planning interventions and policies to enhance rural school travel, promote rural transportation and support diverse transportation modes in rural areas.