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Alzheimer's therapy remains limited by poor drug targeting and multifactorial pathology. The therapeutic potential of SGLT-2 inhibitors like empagliflozin (EGZ) is constrained by poor brain bioavailability. Current study investigates the potential of EGZ-nanostructured lipid carrier (ENLC) for brain delivery via nasal route. The ENLC were prepared using hot melt emulsification technique followed by probe sonication and optimized using Box-Behnken design. ENLC were incorporated into poloxamer 407-chitosan in situ gel (ENPCG) to improve nasal retention, controlled release, and direct brain transport via olfactory and trigeminal uptake. ENPCG demonstrated a sustained drug release of 56.36 ± 3.37 % and enhanced nasal permeation. Nasal kinetics revealed high Cmaxmucosa (48.2 ± 1.42 µg/cm2) relative to plain EGZ-suspension (15.9 ± 0.7 µg/cm2) in goat nasal mucosa. ENPCG significantly improved cognitive memory in sporadic AD model, as confirmed by behavioural, biochemical, and histopathological assessments in Wistar rats. Pharmacokinetic study in Sprague Dawley rats revealed a 4.5-fold increase in AUC0-t of intranasal ENPCG (30.56 ± 0.45 μg/mL*h) relative to intravenous ENLC (6.73 ± 0.15 μg/mL*h). ENPCG showed 95.31 ± 3.89 % drug targeting potential. Furthermore, a strong point-to-point ex vivo-in vivo correlation (R2 = 0.9952) was observed, suggesting a non-invasive potential of ENPCG for translating AD interventions.

Prolonged infusion (extended [EI, 2-4 h] or continuous [CI, 24 h] extended) of beta-lactam antibiotics is considered to have advantages for patients with severe infection compared with intermittent bolus (IB). However, the choice of EI and CI is unclear due to the lack of direct comparison. We aimed to compare the EI and CI of beta-lactams in patients with severe infections using a network meta-analysis method. We systematically searched PubMed, Embase, Web of Science, Cochrane Library, CNKI, Wanfang Database, and Weipu Database for randomized controlled trials (RCTs) comparing EI, CI, or IB with beta-lactams in adults with severe infections. The primary outcome was all-cause mortality. A frequentist network meta-analysis with a random-effects model was performed. Risk of bias was assessed using the Cochrane RoB 2 tool. Thirty-five RCTs (10,627 patients) were included. Risk of bias was moderate to high in most studies. For mortality, EI ranked highest (SUCRA 74.20%) with numerically lower rates versus IB (EI: OR 0.80, 95% CI 0.55-1.17; CI: OR 0.86, 95% CI 0.62-1.02). Both EI and CI significantly improved clinical cure rates versus IB (EI: OR 1.58, 95% CI 1.13-2.23; CI: OR 1.35, 95% CI 1.05-1.85), and EI ranked first (SUCRA 87.72%). For microbiological success, CI ranked highest (SUCRA 83.03%), followed by EI (SUCRA 42.98%) and IB (SUCRA 23.99%), but no significant difference was found. For hospital stay, EI was associated with a reduction of borderline statistical significance (MD -3.49 days, 95% CI -6.79 - -0.08), whereas CI did not show a significant reduction (MD 1.11 days, 95% CI -1.24 - 3.63), and EI ranked best (SUCRA 98.09%). No significant adverse event differences were observed. Subgroup analyses showed variable treatment rankings across categories, with no statistically significant subgroup effects. In patients with severe infections, both EI and CI improved clinical cure versus IB, whereas mortality did not differ significantly. Indirect evidence suggests EI may be more effective than CI in most outcomes except microbiological response. EI trended to shorten hospital stay but the difference was of borderline significance. Considering its practical feasibility, EI appears to be a favorable option based on current evidence. However, this finding is based on indirect evidence and requires confirmation in head-to-head trials. PROSPERO CRD420251242437.

Protopanaxadiol (PPD) is a bioactive ginsenoside with significant anti-inflammatory potential; however, its low natural abundance and dependence on inefficient intestinal microbial bioconversion hinder pharmaceutical development. To overcome these supply and bioavailability constraints, we developed a metabolically engineered rice variety, DJ-PPD, capable of directly biosynthesizing the aglycone PPD. This study investigated the anti-inflammatory and antioxidant mechanisms of DJ-PPD extract in lipopolysaccharide (LPS)-stimulated BV2 cells. DJ-PPD treatment significantly reduced nitric oxide (NO) production, pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), and the expression of iNOS and COX-2. Its efficacy surpassed conventional ginseng extract and was comparable to synthetic PPD (S-PPD). Mechanistically, DJ-PPD inhibited NF-κB, MAPKs, and Akt phosphorylation while activating the NRF2/HO-1 antioxidant pathway. These findings demonstrate that DJ-PPD simultaneously inhibits pro-inflammatory cascades and reinforces intrinsic antioxidant defences. By effectively bypassing the need for gut microbiota metabolism, this genetically engineered rice represents a sustainable, bioavailable, and commercially viable multi-target therapeutic candidate for neuroinflammatory conditions.

Flory-Huggins models underpin phase diagram construction and stability analysis in a wide range of polymer-solute systems, including pharmaceutical solid dispersions, where phase diagrams are routinely used to guide formulation design and stability risk assessment during product development. In practice, the interaction parameter is commonly represented as a temperature-dependent function, χ=χ(T), fitted to experimental data. However, multiple studies and experimental observations suggest that χ may depend on both temperature and composition (ϕ), that is, χ=χ(ϕ,T). Focusing on solid dispersions, here we develop and interrogate a novel composition-dependent Flory-Huggins mathematical framework and quantify the consequences of using χ(ϕ,T) in place of χ(T) when predicting and interpreting phase behaviour. We derive the appropriate chemical potentials and stability conditions required for binodal and spinodal calculations when χ depends on both composition and temperature, and we present generalised criteria for the existence of both upper critical solution temperature (UCST) and lower critical solution temperature (LCST) behaviour. We construct phase diagrams for three solid dispersion systems based on independent experimental data taken from the literature and directly compare predictions obtained using χ=χ(T) and χ=χ(ϕ,T) for each system. We demonstrate that allowing χ to depend on composition can lead not only to substantial quantitative differences, but also to qualitatively different phase behaviour. Finally, we illustrate the implications of these differing phase diagrams by developing a partial differential equation model that enables simulation of microstructural spatiotemporal evolution. The resulting simulations validate the predicted stability landscapes and reveal rich demixing morphologies, including bicontinuous networks and droplet formation.

Diclofenac (DCF) is a frequently detected pharmaceutical pollutant in wastewater and poses ecological risks due to its persistence during conventional treatment. This study investigates the electro-oxidation (EO) of DCF using three commercially available anode materials (Ti/IrO2, Pt/Ti, and mixed metal oxide (MMO)) under varying operational conditions to identify an optimal balance between oxidation capacity and energy efficiency. The effects of current density (2-20 mA/cm2), initial pH (3.5-9), and initial DCF concentration (5-40 mg/L) on degradation performance, kinetics, and specific energy consumption (SEC) were evaluated. Among the tested anodes, Pt/Ti showed the highest overall performance, achieving 71.2% DCF removal under the optimum conditions (10 mA/cm2, pH 7, and initial DCF concentration 10 mg/L). Under these conditions, the SEC was calculated as 19.7 kWh/g DCF (140 kWh/m3), indicating an effective balance between degradation efficiency and energy consumption. Kinetic analysis revealed that while increasing current density significantly enhanced degradation rates, it also led to a rise in energy demand. The initial DCF concentration was found to have a relatively minor effect on degradation kinetics. The Pt/Ti anode demonstrated a stable performance across a wide pH range, with neutral pH providing slightly more favorable conditions. The overall results indicate that Pt/Ti anodes can provide stable electrochemical performance with moderate energy consumption and represent a feasible electrode option for DCF removal in EO-based treatment systems.

The increasing prominence of stereogenic-at-sulfur motifs in drug discovery and catalysis has created a growing demand for versatile synthetic methods. We describe here an Fe(TIBSPDP)-catalyzed enantioselective NH2-amination of sulfenamides to access chiral sulfinamidines. This method accommodates a broad range of sulfenamides, enabling direct access to diverse nitrogenated stereogenic-at-sulfur architectures. The synthetic utility is demonstrated by the efficient preparation of pharmaceutically relevant compounds, such as an aza-analog of drug candidate LY181984 and a PP2A modulator. Moreover, this reaction represents the first example of asymmetric NH2 transfer using the widely employed bioinspired Fe(PDP)-type catalysts.

1,3,4-Oxadiazoles are privileged heterocyclic scaffolds with broad bioactivities, holding significant importance in pharmaceutical and agrochemical discovery. Herein, we report a novel, efficient, and simple synthetic route to 2-amino-1,3,4-oxadiazoles via NaOH-mediated desulfurative cyclization of hydrazides and isothiocyanates under mild reaction conditions. This developed methodology features broad substrate tolerance, excellent yields, operational simplicity, and mild conditions, providing a straightforward pathway for versatile syntheses of valuable 1,3,4-oxadiazole derivative. Consequently, the present reaction opens an alternative path for the preparation of 1,3,4-oxadiazoles via regioselective cyclization hydrazides and isothiocyanates.

Lablab purpureus (hyacinth bean) is a multifaceted legume traditionally integrated into diverse food systems and sustainable agricultural practices, particularly in Asia and Africa. Known for its adaptability to harsh environments, nitrogen-fixing ability, and use as both food and fodder, this crop holds untapped potential for addressing modern nutritional and health challenges. Recent scientific investigations have brought L. purpureus into the spotlight due to its impressive phytochemical profile, which includes phenolics (phenolic acids and flavonoids). These constituents are associated with a wide range of therapeutic properties, such as antioxidant, antimicrobial, anti-inflammatory, antidiabetic, and hepatoprotective activities. Nutritionally, L. purpureus is rich in proteins, essential amino acids, dietary fiber, vitamins, and minerals, making it a valuable candidate for combating malnutrition and enhancing food security, particularly in regions with limited access to diverse food sources. Furthermore, its functional properties suggest promising applications in nutraceutical and pharmaceutical product development. However, despite these attributes, L. purpureus remains largely underutilized and insufficiently represented in mainstream agricultural, food, and biomedical research. This review consolidates and synthesizes current knowledge on the phytochemical composition, nutritional value, and therapeutic potential of L. purpureus, while also identifying critical knowledge gaps and research priorities. By drawing attention to this overlooked legume, the review encourages greater scientific interest and investment in its study and application. As global demand grows for plant-based functional foods and sustainable crop alternatives, L. purpureus presents itself as a viable, eco-friendly, and health-promoting resource worthy of renewed focus and broader utilization across multiple sectors.

Purification in the industry often requires repeated purification of large volume sample using a fixed chromatographic process. Therefore, this study aimed to design an automated chromatographic system which is able to process virtually unlimited sample volume using the minimalism principle. An automated repetitive purification liquid chromatography (ARP-LC) system was developed using active-pump-based sampling and cyclic operational control. The system demonstrated exceptional precision, with injection reproducibility yielding RSD ≤ 1.32% in peak area and retention time stability of ≤ 0.69% RSD. When applied to the purification of coumarins from Cortex Fraxini extract, the system efficiently processed 4 L of the sample, yielding 11 distinct fractions in only seven consecutive runs. Subsequent activity-guided purification identified several high-purity coumarin compounds exhibiting significant antioxidant (EC₅₀ values as low as 5 μg/mL) and antibacterial activities. The ARP-LC system showcases high robustness, scalability, and compatibility with medium- to high-pressure chromatographic modes, providing an efficient and reproducible platform for the large-scale purification of complex natural products with clear implications for pharmaceutical development and industrial applications.

Cyclooxygenase-2 (COX-2) is a key therapeutic target for inflammation-related diseases. Laportea bulbifera, a valued ethnic medicine traditionally utilized by the Miao and Buyi minorities in Guizhou Province, China, has long been used to dispel wind and eliminate dampness, promote blood circulation, and relieve pain and pruritus. While our group previously clarified the anti-inflammatory constituents of its rhizomes, this study represents the first systematic investigation of the aerial parts. The active ethyl acetate fraction was subjected to further purification, yielding 35 phenolic compounds, including seven pairs of enantiomeric lignans and eight previously undescribed compounds. Structural elucidation was achieved by detailed spectroscopic analyses. The majority of these metabolites showed strong inhibitory effects on COX-2 (IC50 values between 0.25 and 14.58 μM) and had notable DPPH radical scavenging activity (IC50 values between 0.93 and 11.77 μM). Moreover, four structurally distinct molecules demonstrated remarkable selectivity toward COX-2 over COX-1, with selectivity indices ranging from 93 to over 240. Kinetic studies further revealed that compounds (+)-6 and 30 function as mixed-type COX-2 inhibitors. Collectively, this study provides the first evidence that the aerial parts of Laportea bulbifera contain COX-2/COX-1 selective inhibitors, thereby supporting its potential for further pharmacological research and ethnomedicine development.

Patient involvement within academic and for-profit medicines and medical technology research and development is happening across organisations and functions. Frameworks exist to conceptualise involvement activities along a spectrum based on patient contributions and their share of voice and decision-making. Examples range from Informing and Education of patients through to Co-design and Co-Production e.g., in research and development. Varying levels of patient expertise profiles also exist based on a spectrum of experience and expertise - from Patient by Experience through to Patient Key Opinion Leader (KOL) - though researchers often lack clarity on the most relevant skills and knowledge of patient partners suited to the task at hand. Here, we propose a conceptual matrix model combining these two concepts into a simple, pragmatic framework intended for patient involvement professionals in research and development, but with broader relevance for other contexts. As authors with a mix of academic, for-profit industry, and patient advocacy backgrounds, we utilised intellectual exchange and consensus formation in three workshops to develop the model. We have developed a conceptual matrix model and applied illustrative case study examples from among our collective experiences and network to demonstrate the model in action. Trends in sample sizes, patient diversity, project longevity, and share of influence exist along the matrix axes. For example, greater numbers and diversity of patients are typically seen at the lower levels of involvement, where there is greater capacity for breadth of involvement. Our aim for this model is to support and encourage patient involvement professionals to better determine appropriate levels of involvement and patient expertise needed for their projects and activities, enabling them to facilitate more appropriate, fair, effective, and sustainable patient involvement. WHAT IS THE MATRIX MODEL?: Universities and for-profit organisations regularly involve patients when they research and develop medicines and medical technologies. Patients can be involved in different types of projects. For example, they can review research plans or patient materials. Different patient involvement activities can be thought of in terms of levels on a spectrum. Patient expertise can also be thought of as levels on a spectrum. Some patients may have expertise in their personal experience. Some patients may have more backgrounds and types of expertise, such as in research processes. So far, these two spectra have been used separately. Researchers may not be aware of the different skills and knowledge that patients may have. So, we took these two spectra – the level of patient involvement and the level of patient expertise – and combined them into a single practical tool using a matrix model (graph). HOW DID WE CREATE IT?: Our group has a mix of expertise, including university researchers, for-profit industry professionals, and patients. We met over three workshops to develop our matrix. We also chose real-life case study examples of patient involvement activities to show how the matrix can be used. HOW CAN IT BE USED?: The matrix is designed for people working in patient involvement at universities and for-profit organisations to consider what levels of involvement and expertise are best for their projects. It might also be helpful for researchers from other types of organisations. We intend for this to create more useful and effective patient involvement.

Targeted radionuclide therapy (TRT) is a cancer treatment method that delivers radiation to specific tumor cells, enabling efficient tumor cell killing. Radionuclides emitting short-range beta or alpha particles have previously been the primary focus. TRT approaches utilizing low-energy electrons, such as Auger and internal conversion electrons, have attracted interest because of their highly localized tumor-killing potential. 134Ce is an imaging surrogate in the form of a 134Ce/134La pair for positron emission tomography (PET) imaging in 225Ac targeted alpha therapy, and it has recently exhibited promising therapeutic properties. This study employs TOPAS-nBio, a Monte Carlo simulation tool, to investigate the radiation damage effects of 134Ce from dosimetric and DNA-scale perspectives. The DNA damage yield analysis incorporates both physical and chemical processes. In the water sphere geometry, the overall dose contribution of 134Ce shows patterns generally similar to those of other Auger-emitting radionuclides, and 134Ce shows damage yields per decay close to those of 161Tb on the DNA scale. Although 134Ce induces fewer DNA double-strand breaks per decay in the nucleus than 125I and 161Tb, it exhibits a higher double-strand break yield when normalized to absorbed dose. Such damage outcome predictions suggest that further research on radionuclide therapy using 134Ce is worthwhile.

Oxeiptosis is a form of regulated cell death that is independent of caspases and is generally considered to be non-inflammatory. This process is triggered by reactive oxygen species and involves the Kelch-like ECH-associated protein 1 (KEAP1)-phosphoglycerate mutase family member 5 (PGAM5)-apoptosis-inducing factor mitochondria-associated 1 (AIFM1) signaling pathway. This review critically assesses the potential of oxeiptosis-related molecules and multi-omics signatures as biomarkers in human cancers, highlighting their analytical validity and future perspectives in the field of translational laboratory medicine. Other factors, such as KEAP1, PGAM5, and AIFM1, are not unique to oxeiptosis but are also involved in the regulation of other forms of cell death, including antioxidant signaling, mitophagy, and apoptosis. Concurrently, AIFM1 may need to be dephosphorylated at serine 116 (Ser116), PGAM5 must be activated, oxidative stress is required, and there must be no activation of caspases, although this pathway profile is not analytically validated. While many composite signatures exhibit hypothesis-generating potential for prognostic stratification, molecular subtyping, and prediction of therapeutic responsiveness, most have not yet been prospectively validated and are at risk of data set overlap, overfitting, algorithm instability, and poor inter-cohort transportability. Preanalytical variability, specimen stability, assay standardization, normalization, inter-platform concordance, and undefined clinical performance thresholds are other factors that limit translation. In summary, the current state of oxeiptosis-related biomarkers is not clinically validated and has a low level of translational readiness. To achieve future progress, standardized definitions of analytic measures, multicenter validation, and prospective clinical utility must be demonstrated.

Ulcerative colitis (UC) is a chronic inflammatory bowel disease with rising global prevalence that substantially impairs quality of life, yet effective therapeutic options remain limited. Non-steroidal anti-inflammatory drugs (NSAIDs) like ibuprofen (IBU) face a therapeutic limitation in UC, as their mechanism exacerbates intestinal injury despite their significant anti-inflammatory efficacy. To overcome this clinical limitation, we designed and synthesized a series of ibuprofen-1,3,4-thiadiazole hybrids (23 compounds in total) and evaluated their anti-inflammatory activities. Notably, compound 20 at 10 mg/kg significantly ameliorated DSS-induced colitis, with efficacy superior to both the parent compound IBU (10 mg/kg) and 5-aminosalicylic acid (5-ASA, 80 mg/kg). Additionally, intervention with compound 20 also restored gut microbiota by modulating gut immunity. Detailed analyses suggested that compound 20 inhibited M1 macrophage polarization and suppressed MAPK-mediated inflammation both in vivo and in vitro. Crucially, compound 20 demonstrated favorable gastrointestinal safety profiles, in contrast to the parent compound IBU. This study underscores the potential clinical relevance of ibuprofen-1,3,4-thiadiazole hybrids as a novel therapeutic strategy for UC with improved gastrointestinal safety profiles.

KDM5B, a member of the KDM5 family of histone demethylases, plays a critical role in transcriptional repression by demethylating H3K4me2/3. It regulates key biological processes such as development, stem cell maintenance, and oncogenesis, and its aberrant expression is implicated in various cancers. Accordingly, KDM5B is considered a promising therapeutic target in cancer drug discovery. In this study, we performed a screening to identify novel inhibitors of KDM5B. Through this screening, we identified a common core scaffold associated with KDM5B inhibitory activity, and subsequent optimization of the side-chain structures led to the identification of a series of compounds with IC50 values ranging from several nanomolar to several micromolar. Evaluation of growth inhibitory activity using the JFCR39 human cancer cell line panel revealed that the final lead compounds, JB-157 and JB-161, exhibited GI50 values in the low micromolar range. In a xenograft model, where the human colorectal cancer cell line HT-29 was implanted into NOD/SCID mice, administration of these compounds resulted in antitumour effects, with JB-161 showing particularly pronounced tumor suppression in a subset of treated animals. Furthermore, combination treatment with in vivo CAR-T cell therapy and JB-161 in C57BL/6J mice was associated with alterations in chromatin accessibility in tumor and immune cell populations. Taken together, these results suggest that JB-161 is a candidate KDM5B inhibitor associated with chromatin accessibility changes and antitumour activity.

Glutarimide-containing Cereblon (CRBN) ligands are critical motifs for PROTACs, molecular glue degraders and next-generation Cereblon E3 ligase modulatory drugs (CELMoDs), which represent promising therapeutic modalities in targeted protein degradation. However, the multistep synthetic routes required to access glutarimide scaffolds continue to present formidable challenges for medicinal chemists, limiting rapid structure-activity relationship (SAR) exploration and late-stage diversification. To streamline access to these privileged motifs, modular and efficient methodologies are still highly desirable. Here, we report a unified organocatalytic synthesis platform for the rapid assembly of diverse glutarimide derivatives from readily available nitrogen heterocycles. Employing a sequence of phosphine-catalysed C-N bond formation, metal-free Giese addition and acid-mediated cyclisation, this approach provides high selectivity, broad functional group tolerance and operational simplicity under conditions amenable to both multigram synthesis and high-throughput parallel synthesis. Using this platform, we rapidly prepare CRBN binder libraries, access control analogues (for example, N‑alkylated glutarimides) and perform late‑stage functionalisation of bioactive molecules. This strategy could offer a transformative solution for the efficient and cost-effective synthesis of CRBN-targeted therapeutics and chemical biology probes, overcoming longstanding synthetic bottlenecks in the field.

B7 family co-inhibitory molecules play a crucial role in maintaining homeostasis and preventing chronic inflammation. However, they also impede antitumor immune responses. Immunoglobulin-like domain-containing receptor 2 (ILDR2) is a B7 family member that modulates T cell activation. Previous studies demonstrated that its blockade synergizes with PD-L1 inhibition in mouse tumor models. Although ILDR2 likely contributes to immune evasion, the precise underlying mechanism remains unknown. Here, we investigated the effect of ILDR2 on the tumor microenvironment (TME) in a mouse squamous cell carcinoma (SCCVII) model. We generated ILDR2-expressing SCCVII cells (ILDR2-SCCVII) through transduction of the extracellular and transmembrane domains of ILDR2. Compared to control vector-transduced cells (Cont-SCCVII), ILDR2-SCCVII cells resulted in accelerated tumor growth and a reduced ratio of activated T cells in the TME 21 days after inoculation. At this late stage, most tumor-infiltrating leukocytes were Ly6C- F4/80+ CD206+ M2-like macrophages. Interestingly, at an earlier time point, we observed the increased infiltration of Ly6C+ F4/80-/+ monocytes-potential macrophage precursors-in ILDR2-SCCVII tumors. Whereas CCL2 expression was enhanced early after inoculation in both groups, its receptor CCR2 was more highly expressed on Ly6C+ F4/80-/+ monocytes specifically in the ILDR2-SCCVII group. Using our in-house anti-mILDR2 monoclonal antibody and ILDR2-Ig, we found that early recruited myeloid cells preferentially expressed ILDR2 and exhibited ILDR2-Ig binding. These results suggest that tumor-expressing ILDR2 interacts with myeloid cells during the early stages of tumor development, inducing premature monocyte accumulation via the CCL2-CCR2 axis. This early shift toward an M2 macrophage-mediated immunosuppressive environment likely accelerates tumor growth.

The interactions between four compounds used as influenza neuraminidase inhibitors-oseltamivir, zanamivir, laninamivir, and peramivir- and influenza virus neuraminidase were analyzed using pair interaction energy decomposition analysis based on fragment molecular orbital (FMO) method, quantitatively elucidating binding characteristics of each inhibitor. Our calculations revealed that structures with common functional groups showed similar binding characteristics, whereas structures with functional groups differing in hydrophilicity/hydrophobicity showed different types of intermolecular interactions for the same amino acid residues. Such analysis is expected to be effective for precise molecular design in structure-based drug design.

The aim of this pilot study was to assess a time-dependent loss of orexin neurons in the unilateral intrastriatal 6-OHDA mouse model and to optimise the immunohistochemistry protocol for formalin-fixed, paraffin-embedded mouse brain tissue. A progressive loss of orexin A positive neurons in the lateral hypothalamus was observed in the lesioned side relative to the non-lesioned side. A significant reduction of orexin A positive neurons in the lateral hypothalamus was observed at 4 weeks with further loss at 6-8 weeks post 6-OHDA induction. Orexin neuron loss in the lateral hypothalamus has been reported in post-mortem Parkinson's disease patients and multiple animal studies, particularly in rats; however, evidence in neurotoxic mouse models remains limited. Orexin A positive neuron loss occurred alongside transient motor deficits, olfactory impairments, subtle cognitive changes and gastrointestinal dysfunction. Additionally, an orexin A immunohistochemistry protocol was successfully established for formalin-fixed, paraffin-embedded mouse brain tissue, enabling reproducible and reliable detection of orexin neurons as an alternative to frozen sections. This pilot study serves as a proof of concept that the unilateral intrastriatal 6-OHDA mouse model can successfully recapitulate the progressive loss of orexin A positive neurons and validates this model's suitably for investigating the longitudinal mechanisms of PD symptoms and pathology.