Standard Animal Model Research Articles

In Situ Dual-targeted Drug Delivery System for Alleviating Imaging and Pathological Damage in Septic Arthritis.

Septic arthritis is a severe disease that damages articular cartilage and triggers a strong inflammatory response. Current treatments mainly depend on systemic antibiotics and lack effective intra-articular therapies, as well as standardized animal models, and precise detection methods. In this study, we present a drug delivery system responsive to the bacterial microenvironment for targeted inflammation control, along with an effective method for monitoring changes in septic arthritis in SD rats. This system consists a core with pH-sensitive metal-organic frameworks ZIF-8 loading anti-inflammatory drugs indomethacin and a shell with hybrid cell membranes from macrophages (MM) and platelets (PM), refer as MP@ZIF-8@IN. This system, which diverges from traditional treatments, enhances drug utilization, prolongs local retention, and allows for spontaneous release at the treatment site, thereby enabling the exclusive intra-articular treatment of septic arthritis. The drug delivery system inhibits the NF-κB pathway, reduces oxidative stress, and regulates macrophage polarization, preventing cartilage destruction. Additionally, in this standardized animal model utilizing the knee joints of SD rats, we have developed musculoskeletal ultrasound and magnetic resonance imaging for time-based monitoring, thus overcoming the limitation of conventional methods, which are unsuitable for soft tissue analysis. Our findings advance therapeutic strategies for septic arthritis and encourage further application of visualization techniques in related fields. STATEMENT OF SIGNIFICANCE: This study presents significant advancements in the treatment and understanding of septic arthritis. Our customized drug delivery system targets bacteria and macrophages, ensuring long-time drug retention and enhanced inflammation control, all while reducing reliance on antibiotics-an important step toward addressing antibiotic resistance. Additionally, we have refined septic arthritis animal models to establish clearer guidelines for intervention timing, grounded in clinical symptoms and imaging data. This addresses a critical gap in current research and offers a practical framework for future therapeutic approaches.

A Fused Membrane-Camouflaged Biomimetic Nanosystem for Dual-Targeted Therapy of Septic Arthritis.

Due to the inherent aseptic and enclosed characteristics of joint cavity, septic arthritis (SA) almost inevitably leads to intractable infections and rapidly progressing complex pathological environments. Presently, SA faces not only the deficient effectiveness of the gold-standard systemic antibiotic therapy but also the scarcity of effective localized targeted approaches and standardized animal models. Herein, an ingenious multifunctional nanosystem is designed, which involves the methylation of hyaluronic acid (HA), copolymerization with DEGDA, loading with vancomycin (VAN), and then coating with fused macrophage-platelet membrane (denoted as FM@HA@VAN). Upon intra-articular administration, FM@HA@VAN nanoparticles exhibit sustained retention and selectively targeting to infected sites, leveraging macrophage-mediated inflammation homing and platelet-directed bacteria targeting. The acidic microenvironment triggers responsive release of vancomycin, leading to potent bactericidal effects. Subsequently, the exposed HA@VAN nanoparticles are efficiently internalized by activated macrophages, releasing HA to alleviate oxidative stress and achieve chondroprotection by inhibiting pro-inflammatory cytokines, neutralizing ROS and upregulating macrophage M2 polarization. In vivo model and experiments confirm the efficacy of this dual-targeting antibacterial approach, demonstrating its precision in eradicating bacterial infections and alleviating associated pathological processes, including synovial hyperplasia and cartilage erosion. The dual-targeting therapeutic nanosystem, coordinated with fused-membranes, holds promise for enhancing the treatment efficacy of SA.

Development of a unique crosslinked glycosaminoglycan for soft tissue repair: Treatment of interstitial cystitis/bladder pain syndrome.

Chemical modification of naturally derived glycosaminoglycans (GAGs) expands their potential utility for applications in soft tissue repair and regenerative medicine. Here we report the preparation of a novel crosslinked chondroitin sulfate (~200 to 2000 kilodaltons) that is both soluble in aqueous solution and microfilterable. We refer to these materials as "SuperGAGs." One can further conjugate these materials with diverse capture agents to further modify polymer properties and add new capabilities. A representative material (GLX-100) demonstrated durable restoration of bladder impermeability in a gold standard animal model of Interstitial Cystitis/Bladder Pain Syndrome (IC/BPS). Histologic examination of the animal bladders treated with a GLX-100 SuperGAG conjugated to biotin as a reporter demonstrated that the residence time of GLX-100 is superior to chondroitin sulfate (a product that is currently used for clinical treatment of patients with IC/BPS). As expected, this novel crosslinked GAG biopolymer was restricted to the luminal surface of the bladder wall. In this communication we describe a simple and versatile synthesis of a crosslinked glycosaminoglycan (GAG) biopolymer for soft tissue repair. Chondroitin sulfate (~12 kD) was crosslinked to form a water soluble and microfilterable polymer with approximately 200 to 2000 kD molecular weight. The synthesis presented here allows for control of molecular weight while avoiding formation of an extended block gel. Moreover, the procedure enables further chemical modification of the SuperGAG through the selection of a capture agent. A set of agents have been used, demonstrating the preparation of a family of SuperGAGs with diverse capabilities. We can optimize polymer properties, adjust adherence to various tissues, add reporters, and engage the biochemistry of surrounding tissues with peptides and other bioactives.

A Review on Reliable and Standardized Animal Models to Study the Pathogenesis of Schmallenberg Virus in Ruminant Natural Host Species.

In the late summer of 2011, the Netherlands reported a cluster of reduced milk yield, fever, and diarrhea in dairy cattle. In March 2012, congenital malformations appeared, and Schmallenberg virus (SBV) was identified, becoming one of the few orthobunyaviruses distributed in Europe. Initially, little was known about the pathogenesis and epidemiology of these viruses in the European context, so assumptions were largely extrapolated from related viruses and other regions worldwide. To study SBV's pathogenesis and its ability to cross the placental barrier, standardized and repeatable models that mimic clinical signs observed in the field are essential. This review discusses some of the latest experimental designs for infectious disease challenges involving SBV, covering infectious doses, routes of infection, inoculum preparation, and origin. Special attention is given to the placental crossing associated with SBV.

Searching for Old and New Small-Molecule Protein Kinase Inhibitors as Effective Treatments in Pulmonary Hypertension-A Systematic Review.

Treatment options for pulmonary arterial hypertension (PAH) have improved substantially in the last 30 years, but there is still a need for novel molecules that can regulate the excessive accumulation of pulmonary artery smooth muscle cells (PASMCs) and consequent vascular remodeling. One set of possible candidates are protein kinases. The study provides an overview of existing preclinical and clinical data regarding small-molecule protein kinase inhibitors in PAH. Online databases were searched from 2001 to 2023 according to PRISMA. The corpus included preclinical studies demonstrating alterations in at least one PH-related parameter following chronic exposure to an individual protein kinase inhibitor, as well as prospective clinical reports including healthy adults or those with PAH, with primary outcomes defined as safety or efficacy of an individual small-molecule protein kinase inhibitor. Several models in preclinical protocols (93 papers) have been proposed for studying small-molecule protein kinase inhibitors in PAH. In total, 51 kinase inhibitors were tested. Meta-analysis of preclinical results demonstrated seralutinib, sorafenib, fasudil hydrochloride, and imatinib had the most comprehensive effects on PH with anti-inflammatory, anti-oxidant, and anti-proliferative potential. Fasudil demonstrated more than 70% animal survival with the longest experimental period, while dasatinib, nintedanib, and (R)-crizotinib could deteriorate PAH. The substances targeting the same kinases often varied considerably in their activity, and such heterogeneity may be due to the variety of causes. Recent studies have addressed the molecules that affect multiple networks such as PDG-FRα/β/CSF1R/c-KIT/BMPR2 or FKBP12/mTOR. They also focus on achieving a satisfactory safety profile using innovative inhalation formulations Many small-molecule protein kinase inhibitors are able to control migration, proliferation and survival in PASMCs in preclinical observations. Standardized animal models can successfully reduce inter-study heterogeneity and thereby facilitate successful identification of candidate drugs for further evaluations.

Developing a Carotid Pseudoaneurysm Model in Swine.

Traumatic carotid artery pseudoaneurysms (PSAs) represent a vascular anomaly with potential for serious complications, including stroke. Traditional treatments involve endovascular stenting, which may not be ideally suited to otherwise healthy vessels that have the potential to remodel. Given the limitations of smaller animal models in replicating human vasculature and the need for improved treatment modalities, this study introduces a novel swine model for the creation and evaluation of carotid PSAs. The PSAs were created from bovine dura and anastomosed in an end-to-side fashion to bilateral carotid arteries. The PSAs were radiologically followed up post-operatively and were harvested at 3 and 10 days for histological analysis. Pseudoaneurysms were successfully created in both animals without intra-operative or immediate post-operative complications. Radiological analysis showed well-perfused PSAs with intra-aneurysmal turbulence, hemodynamically mimicking human carotid artery PSAs. There was no evidence of thrombus or arterial stenosis. Histological examinations revealed thrombus maturation and tight anastomosis of the PSA sac with the native artery. This PSA swine model offers a replicable, cost-effective, and easily implemented tool with the potential to advance carotid PSA management and educational efforts in vascular surgery. The model presented in this methodology paper allows for a standardized PSA animal model allowing for the standardization of a clinical entity which is frequently heterogenenous in morphology. This can allow for the development of new therapies or provide a ground truth for computer modelling for further in silico study.

Placental microphysiological systems: new advances on promising platforms that mimic the microenvironment of the human placenta.

One of the most complex human physiological processes to study is pregnancy. Standard animal models, as well as two-dimensional models, lack the complexity and biological relevance required to accurately study such a physiological process. Recent studies have focused on the development of three-dimensional models based on microfluidic systems, designated as placental microphysiological systems (PMPSs). PMPS devices provide a model of the placental barrier through culturing relevant cell types in specific arrangements and media to mimic the in vivo environment of the maternal-fetal circulation. Here, recent developments of PMPS models for embryo uterine implantation, preeclampsia evaluation, and toxicological screening are presented. Studies that use bioprinting techniques are also discussed. Lastly, recent developments in endometrium microphysiological systems are reviewed. All these presented models showed their superiority compared to standard models in recapitulating the biological environment seen in vivo. However, several limitations regarding the types of cells and materials used for these systems were also widely reported. Despite the need for further improvements, PMPS models contribute to a better understanding of the biological mechanisms surrounding pregnancy and the respective pathologies.

A scoping review of preclinical intensive care unit-acquired weakness models.

Animal models focusing on neuromuscular outcomes are crucial for understanding the mechanisms of intensive care unit-acquired weakness (ICU-AW) and exploring potential innovative prevention and treatment strategies. To analyse and evaluate preclinical ICU-AW models. We manually searched five English and four Chinese databases from 1 January 2002, to 1 February 2024, and reviewed related study references. Full-text publications describing animal models of muscle weakness and atrophy in critical illness were included. Detailed information about model types, animal species, sex, age, induction methods, outcome measures, drawbacks and strengths was extracted from each included study. A total of 3,451 citations were initially retrieved, with 84 studies included in the final analysis. The most frequently studied animal model included rodents (86.9%), 64.3% of which were male animals. ICU-AW animal models were mostly induced by comprehensive intensive care unit (ICU) interventions (38.1%) and sepsis (51.2%). Most studies focused on limb muscles (66.7%), diaphragm muscles (21.4%) or both (9.5%). Reported outcomes primarily included muscular pathological changes (83.3%), electrophysiological examinations of muscles (57.1%) and animal grip strength (16.6%). However, details such as animal age, mortality data, experimental design, randomisation, blinding, sample size and interventions for the experimental group and/or control group were inadequately reported. Many preclinical models are used to study ICU-AW, but the reporting of methodological details is often incomplete. Although current ICU animal models can mimic the characteristics of human ICU-AW, there is no standard model. Future preclinical studies should develop a standard ICU-AW animal model to enhance reproducibility and improve scientific rigor in exploring the mechanisms and potential treatment of ICU-AW.

Establishment and evaluation of a stable and reliable rat model of peritoneal adhesions

BackgroundIn this study, we aimed to establish a stable and standardized animal model of peritoneal adhesions. MethodsForty-eight male Sprague–Dawley rats were randomly divided (n = 12 each) into blank control, classic cecum sidewall, ischemic button, and cecum–sidewall suture groups. The modified American Fertility Society adhesion score was used on postoperative day 7 to evaluate adhesions. Sixty male Sprague–Dawley rats were used to dynamically observe the adhesion characteristics of cecum–sidewall ischemic injury suture model at different time points (n = 60, randomly divided into groups a–e with 12 rats each). The modified American Fertility Society and Zühlke histologic scoring systems, hematoxylin-eosin staining, Masson staining, and computed tomography of the abdomen were used to evaluate adhesions on postoperative days 1, 3, 5, 7, and 14. ResultsNo peritoneal adhesions were observed in the blank control group on postoperative day 7. In the classic cecum sidewall group, 8 rats had inconsistent adhesions, which had a modified American Fertility Society adhesion score of 2.25 ± 1.96. All rats in the ischemic button and cecum–sidewall suture groups developed significant adhesions with modified American Fertility Society scores of 3.08 ± 1.31 and 4.67 ± 0.78, respectively. When the modified American Fertility Society score was used, statistically significant differences were observed between the classic cecum sidewall groups and cecum–sidewall suture groups and between the ischemic button groups and cecum–sidewall suture groups. All animals in groups a–e developed adhesions; adhesion scores increased gradually with time. ConclusionsThe cecum–sidewall ischemic injury suture model is a stable and standardized animal model of peritoneal adhesions.

Dysregulation of persistent inward and outward currents in spinal motoneurons of symptomatic SOD1-G93A mice.

Persistent inward currents (PICs) and persistent outward currents (POCs) regulate the excitability and firing behaviours of spinal motoneurons (MNs). Given their potential role in MN excitability dysfunction in amyotrophic lateral sclerosis (ALS), PICs have been previously studied in superoxide dismutase 1 (SOD1)-G93A mice (the standard animal model of ALS); however, conflicting results have been reported on how the net PIC changes during disease progression. Also, individual PICs and POCs have never been examined before in symptomatic ALS. To fill this gap, we measured the net and individual PIC and POC components of wild-type (WT) and SOD MNs in current clamp and voltage clamp during disease progression (assessed by neuroscores). We show that SOD MNs of symptomatic mice experience a much larger net PIC, relative to WT cells from age-matched littermates. Specifically, the Na+ and Ca2+ PICs are larger, whereas the lasting SK-mediated (SKL) POC is smaller than WT (Na+ PIC is the largest and SKL POC is the smallest components in SOD MNs). We also show that PIC dysregulation is present at symptom onset, is sustained throughout advanced disease stages and is proportional to SOD MN cell size (largest dysregulation is in the largest SOD cells, the most vulnerable in ALS). Additionally, we show that studying disease progression using neuroscores is more accurate than using SOD mouse age, which could lead to misleading statistics and age-based trends. Collectively, this study contributes novel PIC and POC data, reveals ionic mechanisms contributing to the vulnerability differential among MN types/sizes, and provides insights on the roles PIC and POC mechanisms play in MN excitability dysfunction in ALS. KEY POINTS: Individual persistent inward currents (PICs) and persistent outward currents (POCs) have never been examined before in spinal motoneurons (MNs) of symptomatic amyotrophic lateral sclerosis (ALS) mice. Thus, we contribute novel PIC and POC data to the ALS literature. Male SOD MNs of symptomatic mice have elevated net PIC, with larger Na+ and Ca2+ PICs but reduced SKL POC vs. wild-type littermates. Na+ PIC is the largest and SKL POC is the smallest current in SOD cells. The PIC/POC dysregulation is present at symptom onset. PIC dysregulation is sustained throughout advanced disease, and is proportional to SOD MN size (largest dysregulation is in the largest cells, the most vulnerable in ALS). Thus, we reveal ionic mechanisms contributing to the vulnerability differential among MN types/sizes in ALS. Studying disease progression using SOD mice neuroscores is more accurate than using age, which could distort the statistical differences between SOD and WT PIC/POC data and the trends during disease progression.

Comparative study on the establishment efficacy of four types of animal models of rectovaginal fistula in rabbits

Various surgical methods have so far been developed for treating rectovaginal fistula (RVF), each with its own advantages and disadvantages. The lack of standardized animal models of RVF is a major reason for the failure to establish a unified and effective surgical method for the treatment of RVF. This study aimed to explore the feasibility of an RVF animal model by magnetic compression and compare it with the traditional modeling method. Thirty-two female Japanese white rabbits were randomly divided into four groups: A, B, C, and D, based on how the rectovaginal septum was treated. The operation time, intraoperative blood loss, and model success rate of each group were determined. The experimental animals were euthanized 2 weeks after the operation. Their rectovaginal septum specimens were obtained. RVF was observed by the naked eye. The fistula size was measured. Histological changes of fistula were observed by hematoxylin and eosin and Masson staining. All rabbits completed the RVF model and survived 2 weeks after the operation. Groups A and B had no bleeding, while groups C and D had < 0.5 mL of bleeding. The magnet detached in 4–6 days in group A, while it remained in place for 2 weeks after surgery in group B. Only one group D rabbit had a plastic hose for 2 weeks after surgery. The RVFs of groups A and C healed by themselves. In group B, the fistula was well formed. In group D, fistula healing was observed in three animals and the diameter of the fistulas was only 2.82–4.64 mm in the other four animals. Groups B and D had a scar on the inner surface of fistulas. Our study shows that the magnetic compression technique based on the T-shaped magnet is a highly useful method to establishing a continuous and stable RVF model in rabbits.

Maternal genetic effects throughout the life of the dam in Pirenaica beef cattle. a random regression model approach

In beef cattle, dams play a crucial role in shaping the pre- and postnatal environment for the growth of their offspring. Acknowledging the substantial impact of maternal influence on the early development of calves, researchers utilize maternal animal models. These models take into account both maternal genetic and permanent environmental effects, operating under the assumption that these influences remain constant throughout the productive life of the cow. Nevertheless, it cannot be ruled out that these genetic and environmental effects may evolve throughout the lifespan of the cows. Therefore, this study aims to describe the changes in genetic and environmental maternal effects over the productive lifespan of cows. To accomplish this goal, we utilized random regression models, incorporating the age of the dam effect, maternal genetic effects, and environmental permanent effects using Legendre orthogonal polynomials. Additionally, the analytical model incorporated a covariate to adjust for the calf’s age at recording, a two-level sex effect, a random herd-year-season effect, and an additive direct genetic effect associated linked to the calf. The dataset comprised information from dams aged between 2 and 16 years, resulting in a final database that comprised weight records of 58 332 calves from 21 673 dams. The average weight at 90 days was 135.0 ± 39.3 kg, and the mean age of the dam at calving was 7.03 ± 3.41 years. We evaluated models incorporating 2, 3, 4, 5, and 6 orthogonal polynomials alongside the standard maternal animal model. Afterward, we selected the model with five orthogonal polynomials based on the Akaike Information Criteria. The Restricted Maximum Likelihood estimates within this model indicated a direct heritability of around 0.50, and a maternal heritability ranging between 0.15 and 0.25, exhibiting a consistent increase between 4, 5 to 13 years. The genetic correlation estimates between direct and maternal genetic effects remained stable at approximately −0.55 across the lifespan of the cows. Furthermore, maternal genetic correlations between different ages of the dam decreased to around 0.7 for more distant age points. The maternal permanent correlations were notably lower, occasionally even reaching negative values, suggesting variability in environmental influence on maternal effects over the productive lifespan of the cow. Finally, the model enables the prediction of breeding values for the maternal genetic effects of the cow across its lifespan, providing opportunities for innovative selection strategies on the maternal side.

ZNF717 or PABPC1 Gene Variants Contribute to Congenital Esophageal Atresia by Interfering with Normal Esophageal Growth

Congenital esophageal atresia (EA) is an abnormality induced by the incomplete differentiation of the foregut in infants, and is frequently accompanied by tracheoesophageal fistula (TEF). Our understanding of the pathogenesis of EA-TEF is limited, additionally, there is still a lack of standard animal or cell models for in vitro EA-TEF investigation. Therefore, we analyzed esophageal tissue samples from 10 children with EA-TEF via Exome sequencing (ES) to identify gene variants. And esophageal organoid units (EOUs) were established as an in vitro model of EA by culturing esophageal tissues from Adriamycin-challenged rats. The ES results indicated 11 mutated genes, including the frameshift variants of ZNF717 and PABPC1. The EA organoids expressed the esophageal marker proteins CK13 and CK4 and showed a significantly slower rate of growth and dysplasia of cell development. In EA organoids, the transcription of SOX2, ZNF717, and PABPC1 was downregulated at varying levels, while NOGGIN transcription was markedly upregulated. Furthermore, when siRNA-ZNF717 or siRNA-PABPC1 was transfected into normal esophageal organoids, the proliferation of esophageal cells was significantly decreased. In conclusion, we found that normal ZNF717 and PABPC1 expressions are essential to the esophageal development, whereas the variant or deficiency of these genes might lead to EA-TEF.

Rodents' visual gamma as a biomarker of pathological neural conditions.

Neural gamma oscillations (indicatively 30-100Hz) are ubiquitous: they are associated with a broad range of functions in multiple cortical areas and across many animal species. Experimental and computational works established gamma rhythms as a global emergent property of neuronal networks generated by the balanced and coordinated interaction of excitation and inhibition. Coherently, gamma activity is strongly influenced by the alterations of synaptic dynamics which are often associated with pathological neural dysfunctions. We argue therefore that these oscillations are an optimal biomarker for probing the mechanism of cortical dysfunctions. Gamma oscillations are also highly sensitive to external stimuli in sensory cortices, especially the primary visual cortex (V1), where the stimulus dependence of gamma oscillations has been thoroughly investigated. Gamma manipulation by visual stimuli tuning is particularly easy in rodents, which have become a standard animal model for investigating the effects of network alterations on gamma oscillations. Overall, gamma in the rodents' visual cortex offers an accessible probe on dysfunctional information processing in pathological conditions. Beyond vision-related dysfunctions, alterations of gamma oscillations in rodents were indeed also reported in neural deficits such as migraine, epilepsy and neurodegenerative or neuropsychiatric conditions such as Alzheimer's, schizophrenia and autism spectrum disorders. Altogether, the connections between visual cortical gamma activity and physio-pathological conditions in rodent models underscore the potential of gamma oscillations as markers of neuronal (dys)functioning.

Development of the standard mouse model for human bacterial vaginosis induced by Gardnerella vaginalis.

Bacterial vaginosis (BV) is a polymicrobial syndrome characterized by a diminished number of protective bacteria in the vaginal flora. Instead, it is accompanied by a significant increase in facultative and strict anaerobes, including Gardnerella vaginalis (G. vaginalis). BV is one of the most common gynecological problems experienced by reproductive age-women. Because an ideal and standard animal model for human BV induced by G. vaginalis is still underdeveloped, the main objective of this study was to develop a mouse model for human BV induced by G. vaginalis to demonstrate the clinical attributes observed in BV patients. A total of 80 female ICR mice were randomly assigned to 4 groups and intravaginally inoculated with different doses of G. vaginalis: NC (uninfected negative control), PC1 (inoculated with 1 × 105 CFU of G. vaginalis), PC2 (inoculated with 1 × 106 CFU of G. vaginalis) and PC3 (inoculated with 1 × 107 CFU of G. vaginalis). The myeloperoxidase (MPO) activity and serum concentrations of cytokines (IL-1β, IL-10) in mice administered with G. vaginalis were significantly higher than those of the control group. Gross lesion and histopathological analysis of reproductive tract of mice inoculated with G. vaginalis showed inflammation and higher epithelial cell exfoliation compared to the control group. In addition, vaginal swabs from the mice inoculated with G. vaginalis showed the presence of clue cells, which are a characteristic feature of human BV. Altogether, our results suggested that G. vaginalis is sufficient to generate comparable clinical attributes seen in patients with BV.

Trends in research on the efficacy of herbal medicines for particular matter-induced asthma focused on Pubmed

Objectives: This study aimed to review the experimental research trends in asthma caused by particulate matter to conduct further researches, especially in clinical trials.Methods: We searched for the meaningful literature using medicinal herb for asthma through the Pubmed databases.Results: Finally, six studies were finally selected. These studies showed recent trends, from 2018 to 2021 and conducted mostly in South Korea. The type of fine dust and the method of inducing asthma are different for each paper, but the effective mechanism is relatively common. It was commonly confirmed that the ratio and number of eosinophils, th2 cells and related cytokines are decreased in BALF and lung tissue by administration of medicinal herb.Conclusions: Although the method of inducing asthma using fine dust has not yet been standardized, it is thought that more meaningful evaluation results can be derived if a standardized animal model is established in the future. Nevertheless, based on the results that herbal medicine is effective for particular matter induced asthma, it is expected that it will be the basis for expanding into future clinical studies.

Intrinsic pulmonary sealing, its mechanisms and impact on validity and translational value of lung sealant studies: a pooled analysis of animal studies.

No validated and standardized animal models of pulmonary air leakage (PAL) exist for testing aerostatic efficacy of lung sealants. Lack of negative control groups in published studies and intrinsic sealing mechanisms of healthy animal lungs might contribute to a translational gap, leading to poor clinical results. This study aims to address the impact of intrinsic sealing mechanisms on the validity of PAL models, and investigate the conditions required for an ovine model of PAL for lung sealant testing. An ovine acute aerostasis model was developed, consisting of a bilateral thoracotomy with lesion creation, chest tube insertion and monitoring of air leaks using digital drains (≥80 minutes), under spontaneous respiration. Healthy mixed-breed adult female sheep were used and all in vivo procedures were performed under terminal anesthesia. Superficial parenchymal lesions were tested post-mortem and in vivo, extended lesions including bronchioles (deep bowl-shaped and sequential lung amputation lesions) were tested in vivo. Experiment outcomes include air leakage (AL), minimal leaking pressure (MLP) and histology. Two post-mortem (N=4 superficial parenchymal lesions) and 10 in vivo experiments (N=5 superficial parenchymal and N=16 lesions involving bronchioles) were performed. In contrast to the post-mortem model, superficial parenchymal lesions in vivo showed less air leak [mean flow ± standard deviation (SD): 760±693 vs. 42±33 mL/min, P=0.055]. All superficial parenchymal lesions in vivo sealed intrinsically within a median time of 20 minutes [interquartile range (IQR), 10-75 minutes]. Histology of the intrinsic sealing layer revealed an extended area of alveolar collapse below the incision with intra-alveolar hemorrhage. Compared to superficial parenchymal lesions in vivo, lesions involving bronchioles induced significantly higher air leak post-operatively (normalized mean flow ± SD: 459±221 mL/min, P=0.003). At termination, 5/9 (55.6%) were still leaking (median drain time: 273 minutes, IQR, 207-435 minutes), and intrinsic sealing for the remaining lungs occurred within a median of 115 minutes (IQR, 52-245 minutes). Lung parenchyma of healthy sheep shows a strong intrinsic sealing mechanism, explained pathologically by an extended area of alveolar collapse, which may contribute to a translational gap in lung sealant research. A meaningful ovine model has to consist of deep lesions involving bronchioles of >⌀1.5 mm. Further research is needed to develop a standardized PAL model, to improve clinical effectiveness of lung sealants.

Contributed Session II: In vivo imaging of immune cell activity in primate retina after photoreceptor ablation.

The non-human primate (NHP) is the gold standard animal model for preclinical development of gene and cell based therapies for vision restoration. However, the ocular immune response to these interventions remains poorly understood. We conducted a proof of concept study using offset aperture adaptive optics scanning light ophthalmoscopy (AOSLO) to visualize cellular-scale changes in the primate retina following photoreceptor (PR) ablation. Ultrafast 730nm laser exposure at 26.6 - 32.5 J/cm2 was used to create six lesions in four NHPs. Offset aperture images focused on retinal vascular layers were collected with an offset distance of ~10 Airy Disk Diameters from 15 minutes up to three hours after PR ablation. We observed putative immune cells in and around vessels supplying the lesioned areas. Consistent with previous findings in murine models, cells within vessels adhered to the inner wall, exhibited crawling behavior, and had a diameter ranging from ~9.3 - 11.5 µm. Additionally, we observed the emergence of cellular-scale structures above the PR layer that originated in the center of the lesion 15 minutes post-insult and gradually radiated outward. Vascular perfusion was maintained in these regions. Our data suggest that offset aperture imaging offers cellular-scale, label free, in vivo assessment of the retinal response to insult in NHPs and could be employed to advance our understanding of the ocular immune response provoked by disease and therapeutic interventions.

Advancing Perfusion Models: Dual-Vessel Ex Vivo Rat Liver Perfusion Based on a Clinical Setup.

Normothermic ex vivo liver machine perfusion (NEVLP) has been developed to address the increasing organ shortage in liver transplantation, through optimal preservation, assessment, and conditioning of grafts from extended criteria donors. There remains a need to establish simple and standardized animal models that simulate clinical NEVLP to test novel therapies. Liver grafts from 36 Sprague-Dawley rats were perfused for 6 h in a dual-vessel NEVLP system with a Dulbecco's modified Eagles medium-based perfusate supplemented with rat plasma and erythrocytes. Varying doses of the clinically used vasodilator epoprostenol, Kupffer cell inhibitor glycine, and a Steen™-based perfusate were assessed. Perfusion pressures and bile production were recorded, and perfusate was analyzed for transaminase secretion. Tissue samples were evaluated histologically, and levels of cytokines and 8-Isoprostane were measured. Increasing levels of epoprostenol and the addition of glycine resulted in a stepwise decrease of transaminase secretion and improved bile production. Steen further decreased transaminase release and interleukin 1 beta levels. Liver grafts perfused with the optimized Steen-based protocol exhibited lowest levels of oxidative stress and best-preserved liver integrity. In conclusion, epoprostenol seemed to ameliorate liver function and prevent cellular damage beyond its vasodilatory effect, with glycine acting synergistically. The anti-inflammatory and antioxidative properties of Steen further improved the outcome of perfusion. Our rodent NEVLP system may be used to rapidly test new agents for the pharmacologic conditioning of livers and help translate findings from bench-to-bedside.

Toward the use of novel alternative methods in epilepsy modeling and drug discovery.

Epilepsy is a chronic brain disease and, considering the amount of people affected of all ages worldwide, one of the most common neurological disorders. Over 20 novel antiseizure medications (ASMs) have been released since 1993, yet despite substantial advancements in our understanding of the molecular mechanisms behind epileptogenesis, over one-third of patients continue to be resistant to available therapies. This is partially explained by the fact that the majority of existing medicines only address seizure suppression rather than underlying processes. Understanding the origin of this neurological illness requires conducting human neurological and genetic studies. However, the limitation of sample sizes, ethical concerns, and the requirement for appropriate controls (many patients have already had anti-epileptic medication exposure) in human clinical trials underscore the requirement for supplemental models. So far, mammalian models of epilepsy have helped to shed light on the underlying causes of the condition, but the high costs related to breeding of the animals, low throughput, and regulatory restrictions on their research limit their usefulness in drug screening. Here, we present an overview of the state of art in epilepsy modeling describing gold standard animal models used up to date and review the possible alternatives for this research field. Our focus will be mainly on ex vivo, in vitro, and in vivo larval zebrafish models contributing to the 3R in epilepsy modeling and drug screening. We provide a description of pharmacological and genetic methods currently available but also on the possibilities offered by the continued development in gene editing methodologies, especially CRISPR/Cas9-based, for high-throughput disease modeling and anti-epileptic drugs testing.