Using single-nucleus multiomic profiling of the marmoset midbrain, we develop and benchmark enhancer-AAVs to selectively access dopamine (DA) neurons in wild-type animals without combinatorial systems. In vivo candidate screenings identified one highly specific DA enhancer, cjDAE8, in both mice and marmosets. To overcome low-level off-target (leaky) expression, a common limitation for enhancer AAVs, we engineered next-generation AAV backbones that strengthen expression while minimizing leakiness. Quantitative histology comparing natural versus antibody-amplified fluorescence defined AAV doses to achieve high labeling efficiency with greater than 90-95% DA-neuron specificity across species and injection routes. We further demonstrate applications of DA-enhancer-AAVs for (i) retrograde targeting of projection-defined DA populations in marmoset, (ii) fiber-photometric recording of divergent DA-axonal dynamics in striatal subregions, and (iii) optogenetic VTA-DA self-stimulation in mice. Our results establish a resource for cross-species DA targeting and two practical guidelines: backbone context critically shapes enhancer performance, and antibody-amplified readouts rigorously assess specificity.

The current understanding of diabetic kidney disease (DKD) has significant gaps regarding the underlying pathogenesis. In this study, we aimed to characterize the temporal progression of DKD using a state-of-the-art mouse model of hypertension-accelerated disease, integrating kidney biomarker analysis, histopathology, and glomerular transcriptomic profiling. Female diabetic db/db mice received a single intravenous dose of adeno-associated virus-mediated renin overexpression (ReninAAV, week 5) and underwent uninephrectomy (UNx, week 4). db/db UNx-ReninAAV mice were terminated at weeks 1, 4, 8, and 12 (n = 7-8 per group). Female db/m mice were used as healthy controls. Study endpoints included plasma and urine biochemistry, glomerulosclerosis scoring, quantitative kidney histology, and RNA sequencing of glomeruli isolated using laser-capture microdissection. db/db UNx-ReninAAV mice developed progressive albuminuria (from week 4) and glomerulosclerosis (from week 8). A pathway analysis of clustered gene regulations revealed broad glomerular transcriptome perturbations with signatures of increased extracellular matrix (ECM) turnover from week 8 and early onset of metabolic dysfunction. Markers of glomerular cell types and injury exhibited temporal regulation over the course of DKD, with early and sustained downregulation of endothelial markers, heterogeneous regulation of podocyte markers, and significant mesangial and parietal epithelial aberrations. Furthermore, the upregulation of cell injury markers confirmed progressive glomerular injury in the model. The db/db UNx-ReninAAV mouse model exhibits distinct temporal dynamics in glomerular cell markers, metabolic dysregulation, ECM remodeling, and injury. Together, these results highlight the utility of the db/db UNx-ReninAAV model as a relevant preclinical platform for studying progressive DKD.

MARTHA - Combining gaze into deep learning for fully quantitative human testicular histology analysis.

Background: Brain injury is a major cause of death and disability after cardiac arrest (CA). Quantitative histology enables the assessment of neuronal damage in preclinical models and CA patients. However, manual quantification of labeled neurons is time-consuming and variable, posing a significant challenge to the comprehensive assessment of the brain injury severity and neuroprotective therapy's efficacy. Hypothesis: Machine learning (ML) approach can identify and quantify neuronal damage from brain histological images of swine CA models with comparable accuracy to human raters. Methods: We developed a swine CA model to simulate out-of-hospital CA with 5 or 10 minutes of untreated ventricular fibrillation. Following 24 hours of standardized post-CA care, the animals were euthanized by transcardial perfusion with 4% paraformaldehyde. The brains were post-fixed, cryoprotected, and cryosectioned. Coronal sections (20 μm) containing the caudate putamen were stained with Fluoro-Jade C to label injured neurons. Three blinded human raters quantified Fluoro-Jade C-positive neurons in 136 images from 15 animals. These images were split into training (n=54), validation (n=27), and testing (n=55) sets for ML model development. We compared transfer learning models including VGG16, MobileNetV2, DeepLabv3+, and SegFormer. Model performance was evaluated on individual cells via precision, recall, and F1-score, then by comparing cell counts to the human raters for the best performing model. Results: Human raters showed strong reliability in image-wise counts of Fluoro-Jade C neurons with an average pairwise correlation coefficient of R=0.936. The SegFormer model demonstrated the best performance, with a test-set R=0.989 compared to neurons identified by 2 of 3 human raters, or an average R=0.967 when compared to each rater individually (Figures 1 and 2). On an individual cell level, the model yielded a precision of 0.789, a recall of 0.709, and an F1-score of 0.747 (Table 1). Conclusions: We developed and validated a reliable automated ML approach to quantify neuronal damage after CA in a swine model. Future studies will focus on validating the ML models for other brain regions, other stainings, and application in quantitative histology for CA patients.

Fibroblast growth factor 21 (FGF21) analogs have significant therapeutic potential in metabolic dysfunction-associated steatohepatitis (MASH) but limited body weight effects in patients with MASH. This study investigated the effect of combined treatment with the FGF21 analog zalfermin and the glucagon-like peptide-1 receptor agonist semaglutide on body weightand plasma and liver biochemistry and histology in a mouse model of MASH. Amylin liver nonalcoholic steatohepatitis diet-induced obese-MASH mice with biopsy-confirmed MASH and fibrosis were administered (subcutaneous [SC], daily [QD]) vehicle, zalfermin (0.05 or 0.2 mg/kg), semaglutide (3 or 120 µg/kg), or zalfermin 0.05 mg/kg + semaglutide 3 µg/kg for 8 weeks (n = 11-12 per group). Vehicle-dosed (SC, QD) chow-fed mice served as normal controls (n = 10). Pre- to post-liver biopsy histology was compared for within-subject evaluation of changes in non-alcoholic fatty liver disease Activity Score (NAS), fibrosis stage, and quantitative histology. Additional endpoints included plasma/liver biochemistry and liver RNA sequencing. Combined low-dose zalfermin and semaglutide treatment resulted in super-additive body weight loss (-18%) vs. individual low-dose monotherapies (zalfermin, -6%; semaglutide, -4%) and was equally effective as high-dose zalfermin monotherapy (-16%) and semaglutide (-15%). Low-dose combination therapy promoted greater benefits on transaminases, total cholesterol and triglycerides, NAS, steatosis, and inflammation vs. individual low-dose monotherapies and high-dose semaglutide, and high-dose zalfermin was as effective as the low-dose combination therapy on most endpoints. Combination treatment reduced gene expression markers of fibrosis to a greater degree than monotherapies. In conclusion, combined low-dose zalfermin and semaglutide, as well as high-dose zalfermin, resulted in beneficial effects on body weight and biochemical and histological endpoints, supporting the clinical development of zalfermin as therapy for patients with MASH.

Fibroblast growth factor 21 (FGF21) analogs have significant therapeutic potential in metabolic dysfunction–associated steatohepatitis (MASH) but limited body weight effects in patients with MASH. This study investigated the effect of combined treatment with the FGF21 analog zalfermin and the glucagon-like peptide-1 receptor agonist semaglutide on body weight and plasma and liver biochemistry and histology in a mouse model of MASH. Amylin liver nonalcoholic steatohepatitis diet-induced obese-MASH mice with biopsy-confirmed MASH and fibrosis were administered (subcutaneous [SC], daily [QD]) vehicle, zalfermin (0.05 or 0.2 mg/kg), semaglutide (3 or 120 µg/kg), or zalfermin 0.05 mg/kg + semaglutide 3 µg/kg for 8 weeks (n = 11–12 per group). Vehicle-dosed (SC, QD) chow-fed mice served as normal controls (n = 10). Pre- to post-liver biopsy histology was compared for within-subject evaluation of changes in non-alcoholic fatty liver disease Activity Score (NAS), fibrosis stage, and quantitative histology. Additional endpoints included plasma/liver biochemistry and liver RNA sequencing. Combined low-dose zalfermin and semaglutide treatment resulted in super-additive body weight loss (−18%) vs. individual low-dose monotherapies (zalfermin, −6%; semaglutide, −4%) and was equally effective as high-dose zalfermin monotherapy (−16%) and semaglutide (−15%). Low-dose combination therapy promoted greater benefits on transaminases, total cholesterol and triglycerides, NAS, steatosis, and inflammation vs. individual low-dose monotherapies and high-dose semaglutide, and high-dose zalfermin was as effective as the low-dose combination therapy on most endpoints. Combination treatment reduced gene expression markers of fibrosis to a greater degree than monotherapies. In conclusion, combined low-dose zalfermin and semaglutide, as well as high-dose zalfermin, resulted in beneficial effects on body weight and biochemical and histological endpoints, supporting the clinical development of zalfermin as therapy for patients with MASH.

Consensus position statements for the standardized application of histological grading and staging systems in MASH clinical trials.

Background: Necrotizing enterocolitis (NEC) is a life-threatening condition characterized by necrosis of the gastrointestinal tract caused by hypoperfusion and hypoxia-induced inflammation. Surgical treatment often requires resection, with high morbidity and mortality. Intestinal tissue engineering using absorbable biomaterials represents a potential alternative. Small intestinal submucosa (SIS) is a biodegradable extracellular matrix (ECM) scaffold that may facilitate regeneration of the native tissue. Objectives: The aim of our study is to investigate the regenerative potential of SIS in a rat model with multiple gastrointestinal defects. Methods: In rats, after a midline laparotomy, an approximately 1 cm full-thickness incision was performed on the anterior gastric wall, on the antimesenteric side of the small and large intestine, each covered with an SIS patch. After three weeks, the graft sites and adjacent fragments were harvested and fixed in 10% neutral buffered formalin. Cross-sections of the grafted area were processed and stained with hematoxylin and eosin for histologic analysis. Results: Among the fifteen Wistar rats used in the study, the survival rate was 80% (12/15). Macroscopic examination of the abdominal cavity after the second surgery showed no complications. Adhesions were present in 92% (11/12). Histological examination demonstrated complete mucosal coverage in all stomach samples, nine of the small intestine, and ten of the large intestine. Mild fibrosis with minimal inflammatory infiltrates predominated. Ulceration with granulation tissue replacement was observed in three small intestine samples. Foreign body reactions were restricted to suture sites. Conclusions: In this multifocal injury model, SIS integrated effectively and supported early regenerative healing across gastric, small-intestinal, and colonic sites at 3 weeks. These data support further studies with longer follow-up, quantitative histology and functional assessment, and evaluation in neonatal-relevant large animal models to determine translational potential for NEC surgery.

Disruptions in synaptic transmission and plasticity are early hallmarks of Alzheimer’s disease (AD). Endosomal trafficking, mediated by the retromer complex, is essential for intracellular protein sorting, including the regulation of amyloid precursor protein (APP) processing. The VPS35 subunit, a key cargo-recognition component of the retromer, has been implicated in neurodegenerative diseases, with mutations such as L625P linked to early-onset AD. Despite growing evidence for retromer dysfunction in AD, its role in synaptic pathology and neuroinflammation remains incompletely understood. Here, we investigate the acute molecular effects of retromer stabilization in the 5xFAD mouse model of AD using the pharmacological chaperones R55 and R33, previously identified to enhance VPS35 stability. Following intracranial stereotaxic injections, we performed transcriptomic profiling, quantitative histology, and immunohistochemistry to assess synaptic function, neuroinflammation, and endosomal trafficking. Our findings reveal that retromer stabilization reverses multiple AD-associated molecular changes. R55 treatment significantly reduced Aβ-related pathology, normalized synaptic gene expression, and restored long-term potentiation (LTP)-associated pathways, including Gria1 (AMPA receptors), Grip1, and semaphorin/plexin signaling. Additionally, retromer stabilization counteracted dysregulated calcium signaling by modulating Ryr2 and L-type calcium channel expression. Beyond synaptic effects, we observed broad transcriptional and structural changes in the endosomal system. Notably, R55 treatment decreased VPS13 family gene expression, implicated in membrane contact site regulation, while increasing RAB7 levels, suggesting enhanced late-endosomal recycling. VPS35-positive vesicles were redistributed away from the nucleus, indicating restored intracellular trafficking dynamics. In the neuroinflammatory domain, retromer stabilization modulated microglial activation, shifting towards a profile characterized by balanced pro-inflammatory (Il1, Nfkb2) and anti-inflammatory (Il4r, Il13ra1, Stat6) markers, consistent with disease-associated microglia (DAM) phenotypes. Together, these findings demonstrate that retromer dysfunction contributes to key AD pathologies, including synaptic dysfunction and neuroinflammation, and that pharmacological retromer stabilization can restore cellular homeostasis. Given that 5xFAD mice lack direct VPS35 mutations, our results suggest that retromer-targeting strategies may be applicable to both familial and sporadic AD, offering a promising therapeutic avenue for modifying disease progression.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40478-025-02096-8.

BackgroundDiabetic keratopathy (DK) is a common ocular complication of diabetes, with its progression closely linked to autophagy regulation. This study aims to explore the role of long non-coding RNAs (lncRNAs) in modulating autophagy during diabetic pathogenesis, focusing on lncRNA general transcription factor IIIC subunit 1 (GTF3C1) and its potential as a therapeutic target for diabetic corneal neuropathy (DCN).MethodsHigh-throughput sequencing identified dysregulated lncRNAs in the trigeminal ganglia of diabetic mice. Functional validation included mechanistic studies on lncRNA GTF3C1, miR-542-3p, and autophagy-related targets. Autophagy activity, corneal nerve density, and epithelial healing were quantified using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence, and histology in diabetic models.ResultslncRNA GTF3C1 was significantly downregulated in diabetic trigeminal ganglion (TG). It functioned as a molecular sponge for miR-542-3p, alleviating its repression on GABA type A receptor-associated protein (GABARAP) and phosphatase and tensin homolog (PTEN), thereby enhancing autophagy activity. This process promoted corneal nerve fiber regeneration and epithelial wound healing in diabetic mice.ConclusionsOur findings highlight lncRNA GTF3C1 as a critical regulator of autophagy in diabetic corneal nerves, offering a potential diagnostic and therapeutic target for DCN. This study provides molecular insights into the pathogenesis of DCN and lays the groundwork for future clinical strategies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s40662-025-00448-y.

TSPO participates in the pathogenesis of diabetic periodontitis related to mitophagy.

Unravelling the role of environmentally realistic concentrations of titanium dioxide nanoparticles in altering gametogenesis and gonadal health of female Pacific oysters (Magallana gigas): a histopathological approach.

Introduction and Objective: The GLP-1: GIP receptor dual agonist Tirzepatide has demonstrated therapeutic efficacy on clinical endpoints in a recent phase 2 clinical trial in patients with metabolic-dysfunction associated steatohepatitis (MASH) and liver fibrosis. The present study aimed to evaluate therapeutic efficacy of tirzepatide on clinical endpoints and outcome in the GAN diet-induced obese (DIO) mouse model of MASH with advanced fibrosis and hepatocellular carcinoma (HCC). Methods: Male GAN DIO-MASH-HCC mice with liver biopsy-confirmed NAFLD Activity Score (NAS ≥5) and advanced fibrosis (stage F3) received (SC, QD) vehicle (n=15) or tirzepatide (10 nmol/kg, n=17) for 14 weeks. Tumor histopathological evaluation was performed by an expert pathologist. Histopathological scoring was performed. Additional endpoints included blood and liver biochemistry and quantitative liver histology. Results: GAN DIO-MASH-HCC mice demonstrated progressive tumor burden over the 14-week study period. Tirzepatide completely prevented progression in tumor burden and reduced histological marker for hepatocellular proliferation (Ki67). Concurrently, tirzepatide improved hallmarks of MASH, including hepatomegaly, transaminases (ALT/AST), plasma and liver lipids (TG/TC), in addition to histopathological ≥2 point improvement in NAS. In agreement, tirzepatide reduced quantitative histological markers of steatosis and inflammation (CD45, galectin-3). Tirzepatide did not improve fibrosis stage, albeit reduces clinical-derived plasma fibrosis markers (TIMP-1, PIIINP) and histological marker for hepatic stellate cell activation (α-SMA), indicating anti-fibrogenic action. Conclusion: This is the first study to demonstrate that tirzepatide reduces tumor burden and improves clinical biomarkers and histopathological endpoints for NAFLD Activity Score in a preclinical translational diet-induced obese mouse model of MASH-driven HCC with advanced fibrosis. Disclosure M. Feigh: None. N.O. Eskesen: Employee; Gubra. J. Nøhr-Meldgaard: None. S.E. Pors: None. H.H. Hansen: None.

Introduction and Objective: The present study aimed to investigate the hepatoprotective effects of OPK-88006, a novel GLP-1/glucagon receptor agonist, in the GAN diet-induced obese and biopsy-confirmed mouse model of MASH with liver fibrosis. The late-stage clinical candidates semaglutide and survodutide for the treatment of patients with MASH and liver fibrosis, were included for comparison. Methods: Male GAN DIO-MASH mice with NAFLD Activity Score (NAS≥5) and moderate/advanced fibrosis (stage F2-F3) were administered once daily subcutaneous vehicle, semaglutide (30 nmol/kg), survodutide (15) nmol/kg) and OPK-88006 (20 nmol/kg) for 12 weeks. Histopathological scoring was performed. Other terminal endpoints included quantitative liver histology, blood and liver biochemistry in addition to RNAsequencing-Bioinformatic analysis. Results: GAN DIO-MASH mice demonstrated vehicle-corrected weight loss of 12%, 24% and 26% for OPK-88006, semaglutide and survodutide treatment, respectively. All treatments improved hepatomegaly, plasma transaminases and plasma lipids levels, albeit only OPK-88006 induced 2-point statistically significant improvement in NAS. In agreement, OPK-88006 reduced quantitative liver histology on steatosis and inflammation (galectin-3). Treatments reduced markers for fibrosis (TIMP-1 PIIINP) and hepatic stellate activation (α-SMA), indicating effect on fibrogenesis. Finally, all treatments induced significant gene regulation and suppressed genes involved in extracellular matrix and inflammation pathways. Conclusion: OPK-88006 treatment improved metabolic, biochemical and histopathological parameters of MASH, including hepatic transcriptomic profile, in the GAN DIO-MASH mouse model. Notably, OPK-88006 treatment improved NAFLD Activity Score superior to late-stage clinical candidates semaglutide and survodutide, introducing OPK-88006 as a promising treatment for MASH. Disclosure J. Hsiao: None. L. Moschcovich: None. M. Gerzon-Zakar: None. A. Rivkin: None. S.E. Pors: None. M.R. Madsen: None. T. Cruz: Employee; OPKO Health. M. Feigh: None.

Investigating the tissue modifications occurring as a consequence of tumour development is an important goal in preclinical medical research, as it can provide a better understanding of the mechanisms behind its origin and spread. Tumor microenvironment has a supportive role in cancer development and can be exploited as a therapeutic target to prevent and contrast metastatic spread, which usually leads to a poor prognosis. In this work, a colorectal cancer model of liver metastasis is used to perform proof-of-concept quantitative investigations of the changes occurring in murine liver tissue due to the formation of metastases. X-ray phase contrast imaging performed with synchrotron radiation was used to obtain high resolution and contrast on soft tissues with minimum sample preparation and a large field of view on a 3D volume. A pixel size of 3 µm, and 0.7 µm have been used. to visualize and quantify liver microvasculature, referred to as sinusoids, and to identify significant morphological differences between control and metastatic tissues. A reorganization of the hepatic tissue, characterized by increased vascularization around the metastatic lesions coupled with a significant reduction in the sinusoidal network in the distal liver parenchyma was observed. X-ray findings are also supported by conventional histology, proving X-ray phase contrast imaging as an informative complementary technique.

A hallmark of Alzheimer's disease (AD) is the extracellular aggregation of toxic amyloid-beta (Aβ) peptides in form of plaques. Here, we identify netoglitazone, an antidiabetic compound previously tested in humans, as an Aβ aggregation antagonist. Netoglitazone improved cognition and reduced microglia activity in a mouse model of AD. Using quantitative whole-brain three-dimensional histology (Q3D), we precisely identified brain regions where netoglitazone reduced the number and size of Aβ plaques. We demonstrate the utility of Q3D in preclinical drug evaluation for AD by providing a high-resolution brain-wide view of drug efficacy. Applying Q3D has the potential to improve pre-clinical drug evaluation by providing information that can help identify mechanisms leading to brain region-specific drug efficacy.

Su1931: DEVELOPMENT AND VALIDATION OF A DEEP LEARNING FRAMEWORK FOR QUANTITATIVE HISTOLOGICAL ANALYSIS IN INFLAMMATORY BOWEL DISEASE (IBD)

Big mechanically-active culture systems (BigMACS) are promising to stimulate, control, and pattern cell and tissue behaviours with less soluble factor requirements. However, it remains challenging to predict if and how distributed mechanical forces impact single-cell behaviours to pattern tissue. In this study, we introduce a tissue-scale finite element analysis framework able to correlate sub-cellular quantitative histology with centimetre-scale biomechanics. Our framework is relevant to diverse BigMACS, including media perfusion, tensile-stress, magnetic, and pneumatic tissue culture platforms. We apply our framework to understand how the design and operation of a multi-axial soft robotic bioreactor can spatially control mesenchymal stem cell (MSC) proliferation, orientation, differentiation to smooth muscle, and extracellular vascular matrix deposition. We find MSC proliferation and matrix deposition to positively correlate with mechanical stimulation but cannot be locally patterned by soft robot mechanical stimulation within a centimetre scale tissue. In contrast, local stress distribution was able to locally pattern MSC orientation and differentiation to smooth muscle phenotypes, where MSCs aligned perpendicular to principal stress direction and expressed increased α-SMA with increasing 3D Von Mises Stresses from 0 to 15 kPa. Altogether, our new biomechanical-histological simulation framework is a promising technique to derive the future mechanical design equations to control cell behaviours and engineer patterned tissue.

In ischemic stroke due to large vessel occlusion, cerebral blood flow is impaired by thromboemboli, which can originate from various sources in the body. A better understanding of thromboembolus composition can improve our understanding of the underlying pathophysiology and potentially guide improvement of prevention strategies. The aim of this study therefore was to perform a large-sample multi-parameter quantitative histological analysis of retrieved thromboemboli from stroke patients. Thromboemboli (n=501) were collected from thrombectomy-treated ischemic stroke patients at AZ Groeninge Hospital (Kortrijk, Belgium) and CHU Lille (Lille, France). Stroke etiology was determined by the treating stroke specialist using the TOAST classification system (Trial of ORG 10172 in Acute Stroke Treatment). Extensive histological analysis, blinded to stroke etiology, was performed for key thrombus constituents including red blood cells (RBCs), platelets, fibrin, von Willebrand Factor, leukocytes, citrullinated histone H3 (as a marker for neutrophil extracellular traps), and extracellular (ex)DNA. Quantitative histology results were linked to etiology. Compared with large-artery atherosclerotic thromboemboli, cardioembolic thromboemboli contained significantly fewer RBCs and significantly more platelets, fibrin, leukocytes, and exDNA. Interestingly, cryptogenic thromboemboli contained relatively low amounts of RBCs and high amounts of platelets, similar to cardioembolic thromboemboli. A multivariable logistic regression model indicated that it is difficult to predict stroke etiology based on the individual thrombus composition. Based on a large sample cohort of stroke thromboemboli, we found that cardiac thromboemboli contained fewer RBCs and more platelets than atherosclerotic thromboemboli and that cryptogenic thromboemboli have a similar composition to cardiac thromboemboli in terms of RBCs and platelets.

Diagnosis of Fibrotic Interstitial Lung Diseases Based on the Combination of Label-Free Quantitative Multiphoton Fiber Histology and Machine Learning.