Preclinical Animal Study Research Articles

Evaluation of Dexamethasone-Eluting Cell-Seeded Constructs in a Preclinical Canine Model of Cartilage Repair.

In this 12-month long, preclinical large animal study using a canine model, we report that engineered osteochondral grafts (comprised of allogeneic chondrocyte-seeded hydrogels with the capacity for sustained release of the corticosteroid dexamethasone [DEX], cultured to functional mechanical properties, and incorporated over porous titanium bases), can successfully repair damaged cartilage. DEX release from within engineered cartilage was hypothesized to improve initial cartilage repair by modulating the local inflammatory environment, which was also associated with suppressed degenerative changes exhibited by menisci and synovium. We note that not all histological and clinical outcomes at an intermediary time point of three months paralleled 12-month outcomes, which emphasizes the importance of invivo studies in valid preclinical models that incorporate clinically relevant follow-up durations. Together, our study demonstrates that engineered cartilage fabricated under the conditions reported herein can repair full-thickness cartilage defects and promote synovial joint health and function.

19Fluorine-MRI Based Longitudinal Immuno-Microenvironment-Monitoring for Pancreatic Cancer.

Pancreatic cancer has a poor prognosis. Targeting Kirsten Rat Sarcoma (KRAS) mutation and its related pathways may enhance immunotherapy efficacy. While in vivo monitoring of therapeutic response and immune cell migration remains challenging, Fluorine-19 MRI (19F MRI) may allow noninvasive longitudinal imaging of immune cells. Evaluating the potential of 19F MRI for monitoring changes in the tumor immune microenvironment, in response to combined SHP2/MEK inhibition. Pre-clinical animal study. Murine genetically engineered pancreatic cancer model (N = 20, both sexes). 9.4-T, two-dimensional multi-slice Rapid Acquisition with Relaxation Enhancement sequence. Intravenous injection of 19F-perfluorocarbon (PFC) nanoparticles. Upon tumor detection by conventional 1H MRI screening, 19F MRI was performed in mice 24 hours after PFC nanoparticle administration. Animals were randomly assigned to four treatment groups: allosteric Src-homology-2-containing protein tyrosine phosphatase 2 (SHP2) inhibitor SHP099, the mitogen-activated extracellular signal-regulated kinase 1/2 (MEK1/2) inhibitor Trametinib, the combination of both, or a vehicle control (4 to 6 mice each group), administered every other day per oral gavage. 1H and 19F MRI was repeated 7 days and 14 days later. Pancreatic immune cell infiltrates were analyzed by flow cytometry and multiplex immunohistofluorescence (mIHF) upon sacrifice. Independent t-tests and one-way analysis of variance. 19F MRI revealed continuous decrease of PFC-signals in tumors from vehicle controls (100%, 80%, and 74% on days 0, 7, and 14, respectively), contrasting with stable or increasing signals under KRAS-pathway-directed treatment. MEK inhibition showed 100%, 152%, and 84% and dual SHP2/MEK1/2 inhibition demonstrated signals of 100%, 134%, and 100% on days 0, 7, 14, respectively. mIHF analyses indicated CD11b+ macrophages/monocytes as primary contributors to the observed 19F MRI signal differences. 19F MRI might provide non-invasive longitudinal estimates for abundance and spatial distribution of CD11b+ macrophages/monocytes in pancreatic cancer. 1 TECHNICAL EFFICACY: Stage 2.

Conceptualizing Scaffold Guided Breast Tissue Regeneration in a Preclinical Large Animal Model.

Scaffold-guided breast tissue regeneration (SGBTR) can transform both reconstructive and cosmetic breast surgery. Implant-based surgery is the most common method. However, there are inherent limitations, as it involves replacement of tissue rather than regeneration. Regenerating autologous soft tissue has the potential to provide a more like-for-like reconstruction with minimal morbidity. Our SGBTR approach regenerates soft tissue by implanting additively manufactured bioresorbable scaffolds filled with autologous fat graft. A pre-clinical large animal study was conducted by implanting 100 mL breast scaffolds (n = 55) made from medical-grade polycaprolactone into 11 minipigs for 12 months. Various treatment groups were investigated where immediate or delayed autologous fat graft, as well as platelet rich plasma, were added to the scaffolds. Computed tomography and magnetic resonance imaging were performed on explanted scaffolds to determine the volume and distribution of the regenerated tissue. Histological analysis was performed to confirm the tissue type. At 12 months, we were able to regenerate and sustain a mean soft tissue volume of 60.9 ± 4.5 mL (95% CI) across all treatment groups. There was no evidence of capsule formation. There were no immediate or long-term post-operative complications. In conclusion, we were able to regenerate clinically relevant soft tissue volumes utilizing SGBTR in a pre-clinical large animal model.

A Mechanistic and Preclinical Assessment of BioRestore Bioactive Glass as a Synthetic Bone Graft Extender and Substitute for Osteoinduction and Spine Fusion.

Preclinical animal study. Evaluate the osteoinductivity and bone regenerative capacity of BioRestore bioactive glass. BioRestore is a Food and Drug Administration (FDA)-approved bone void filler that has not yet been evaluated as a bone graft extender or substitute for spine fusion. In vitro and in vivo methods were used to compare BioRestore with other biomaterials for the capacity to promote osteodifferentiation and spinal fusion. The materials evaluated (1) absorbable collagen sponge (ACS), (2) allograft, (3) BioRestore, (4) Human Demineralized Bone Matrix (DBM), and (5) MasterGraft. For in vitro studies, rat bone marrow-derived stem cells (BMSC) were cultured on the materials in either standard or osteogenic media (SM, OM), followed by quantification of osteogenic marker genes ( Runx2, Osx, Alpl, Bglap, Spp1 ) and alkaline phosphatase (ALP) activity. Sixty female Fischer rats underwent L4-5 posterolateral fusion (PLF) with placement of 1 of 5 implants: (1) ICBG from syngeneic rats; (2) ICBG+BioRestore; (3) BioRestore alone; (4) ICBG+Allograft; or (5) ICBG+MasterGraft. Spines were harvested 8 weeks postoperatively and evaluated for bone formation and fusion via radiography, blinded manual palpation, microCT, and histology. After culture for 1 week, BioRestore promoted similar expression levels of Runx2 and Osx to cells grown on DBM. At the 2-week timepoint, the relative ALP activity for BioRestore-OM was significantly higher ( P <0.001) than that of ACS-OM and DBM-OM ( P <0.01) and statistically equivalent to cells grown on allograft-OM. In vivo, radiographic and microCT evaluation showed some degree of bridging bone formation in all groups tested, with the exception of BioRestore alone, which did not produce successful fusions. This study demonstrates the capacity of BioRestore to promote osteoinductivity in vitro. In vivo, BioRestore performed similarly to commercially available bone graft extender materials but was incapable of producing fusion as a bone graft substitute. Level V.

Abstract 5333: NeoMab: A novel platform for fully human therapeutic antibody development

Abstract Antibodies and antibody containing therapeutics, such as multivalent antibodies, chimeric antigen receptors (CARs), and antibody directed conjugates (ADCs), have been proven to be highly prominent in cancer treatment. However, challenges such as immunogenicity stemming from non-human or unnatural sequences and limitations in preclinical animal study systems still impede the rapid development of antibody drugs. To overcome these hurdles, GemPharmatech has created a novel antibody gene humanized model, the NeoMab mouse, to develop fully human antibodies. The NeoMab model was established via large genomic fragment knock-in technology, retaining native mouse constant region encoding genes and regulatory elements, while incorporating human variable gene repertoires into the endogenous loci. Developed in the BALB/c background, the NeoMab mouse carries fully functional MHC molecules, potentially enhancing antigen presentation. These mice sustain a competent immune system with a B cell development and maturation pattern, immune cell profile, and serum immunoglobulin levels comparable to those of BALB/c mice. NeoMab mice exhibit a robust humoral immune response, with a rapid increase in antigen-specific serum titers upon immunization using different antigens, such as recombined protein, plasmids, and overexpressing cell lines. Utilizing human V(D)J genes to encode immunoglobulins at a human-like frequency and diversity, antibodies derived from NeoMab mice demonstrate affinity and functional activity comparable to, or even surpassing, FDA-approved drugs. In conclusion, the NeoMab mouse model in the BALB/c background has been rigorously validated to elicit a robust response to specific antigen immunization, producing antibodies with high affinity, diversity, and functional activity. GemPharmatech's extensive drug-target gene humanized mouse library (comprising over 800 targets) and diverse disease models in oncology and other fields facilitate a seamless connection between in vitro antibody screening and in vivo animal studies. The integration of NeoMab, target humanization animals, and disease models enables GemPharmatech to provide an integrated solution, reducing time and costs while elevating the quality of antibody discovery in the field. Citation Format: Mingkun Zhang, Cunxiang Ju, Song Li, Junchao Peng, Jing Zhao, Xiang Gao. NeoMab: A novel platform for fully human therapeutic antibody development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5333.

Evaluation the Effect of Sonodynamic Therapy with 5-Aminolevulinic Acid and Sodium Fluorescein by Preclinical Animal Study.

Sonodynamic therapy (SDT) is a novel tumor treatment that combines biosafe sonosensitizers and noninvasive focused ultrasound to eradicate solid tumors. Sonosensitizers such as 5-aminolevulinic acid and fluorescein have great potential in tumor treatment. Here, rodent subcutaneous and brain tumor models were used to evaluate the treatment effect of both 5-ALA- and fluorescein-mediated SDT. The subcutaneous tumor growth rates of both SDT groups were significantly inhibited compared with that of the control groups. For intracranial tumors, 5-ALA-SDT treatment significantly inhibited brain tumor growth, while fluorescein-SDT exerted no therapeutic effect in animals. The distribution of fluorescein in the brain tumor region underwent further assessment. Seven days post tumor implantation, experimental animals received fluorescein and were sacrificed for brain specimen collection. Analysis of the dissected brains revealed no fluorescence signals, indicating an absence of fluorescein accumulation in the early-stage glioma tissue. These data suggest that the fluorescein-SDT treatment response is closely related to the amount of accumulated fluorescein. This study reports the equivalent effects of 5-ALA and fluorescein on the treatment of somatic tumors. For orthotopic brain tumor models, tumor vascular permeability should be considered when choosing fluorescein as a sonosensitizer. In conclusion, both fluorescein and 5-ALA are safe and effective SDT sonosensitizers, and the tumor microenvironment and pathologic type should be considered in the selection of adequate sonosensitizers.

Preclinical Animal Study and Pilot Clinical Trial of Using Enriched Peripheral Blood-Derived Mononuclear Cells for Intervertebral Disc Degeneration.

Low back pain (LBP) is a leading cause of long-term disability globally. Intervertebral disk degeneration (IVDD) is mainly responsible for discogenic pain in LBP-affected young patients. There is no effective therapy to reverse disease severity and IVDD progression. This study investigates the effect of human peripheral blood-derived mononuclear cells (PBMCs) on pain relief and life quality improvement in IVDD patients. The enriched monocytes of the PBMCs could differentiate into CD14 and CD206 double-positive M2 macrophages in vitro. Preclinical evidence in rats showed that the transplanted PBMCs exhibited anti-inflammatory and moderate tissue-repair effects on controlling IVDD progress in the rat model. The PBMCs significantly steered the aggrecan and type II collagen expressions and attenuated the pro-inflammatory cytokines in the affected disk. Based on the animal results, 36 patients with chronic low back pain (CLBP) were included in clinical trials. The control group was conservative care only, and the experimental group was platelet-rich plasma (PRP) and PBMCs intradiscal injections. We first confirmed the single lumbar disk causing the discogenic pain by provocative discography or magnetic resonance imaging (MRI). Discogenic LBP participants received one intradiscal injection of autologous PBMCs and followed for 6 months. Our clinical trial showed that patients' LBP and disability were significantly ameliorated after the PBMCs transplantation rather than PRP. These preclinical and pilot clinical studies indicate that intradiscal injection of the enriched PBMCs might be a feasible and potential cell therapy to control pain and disability in IVDD patients.

A preclinical animal study to evaluate the operability and safety of domestic one-way endobronchial valves.

To evaluate the operability and safety of bronchoscopic domestic one-way endobronchial valves (EBV) on animals. Nine pigs were randomly assigned (2:1) to receive domestic one-way EBV (the experimental group, n = 6) and Zephyr® EBV (the control group, n = 3). Routine blood tests, arterial blood gases, and CT scans of the lungs were performed 1 day pre-procedure in addition to 1 week and 1 month post-procedure to assess changes in blood markers and lung volumes. At 1 month post-procedure, the animals were sacrificed, followed by removal of all valves via bronchoscopy. Pathological examinations of critical organs were subsequently performed. A total of 15 valves were placed in the experimental group and 6 valves were placed in the control group, without serious complications. Routine blood tests and arterial blood gas examinations at 1 day pre-procedure, 1 week post-procedure, and 1 month post-procedure did not differ significantly in both groups. No EBV displacement was noted under bronchoscopy, and the valve was smoothly removable by bronchoscope at 1 month post-procedure. At 1 week post-procedure, varying degrees of target lung lobe volume reduction were observed on lung CT in both groups. Lung volume reduction was achieved at 1 month post-procedure in both groups, without significant statistical difference. Although 3 cases in the experimental group and 1 case in the control group developed varying degrees of pneumonia, the inflammatory response did not increase over time during the experimental period. Pathological examination revealed no significant abnormal changes in the critical organs for both groups. Our results demonstrate that domestic EBV is safe and reliable for endobronchial application in general-grade laboratory white pigs. The safety of domestic EBV is similar to that of Zephyr® EBV, with good ease of use and operability. This kind of domestic EBV can meet the safety evaluation requirements for animal testing.

Comparative evaluation of 3D-printed and conventional implants in vivo: a quantitative microcomputed tomographic and histomorphometric analysis

In recent years, 3D-printing technology to fabricate dental implants has garnered widespread attention due to its patient-specific customizability and cost-effectiveness. This preclinical animal study analyzed the radiographic and histomorphometric outcomes of 3D-printed implants (3DIs) placed immediately after extraction and compared them to conventional implants (CIs). 3DIs and CIs of the same dimensions placed immediately were analyzed at 2, 6, and 12 weeks. The micro-computed tomography (micro-CT) analysis revealed statistically significant differences at 2 weeks in favor of 3DIs over the CIs in terms of bone volume/tissue volume (BV/TV), bone surface/bone volume (BS/BV), trabecular bone pattern factor (Tb.Pf), and structure model index (SMI). At 2 weeks, the mean bone-to-implant contact (BIC) of the 3DIs was greater than that of the CIs; the mean bone area fraction occupancy (BAFO) and the number of Haversian canals of the 3DIs showed no statistically significant differences compared to CIs at 2 weeks. At 6 and 12 weeks, there were no statistically significant differences between the 3DIs and CIs in any parameters. Within limitations, in the early stage of extraction socket healing, the 3DIs demonstrated a higher BIC than the CIs, presenting that 3DIs may be a potential option for immediate placement to enhance osseointegration.

Are Absorbable Plates More Resistant to Infection Than Titanium Implants? An Experimental Pre-Clinical Trial in Rabbits.

Background: Infection of orthopaedic implants after internal fixation of bone fractures remains a major complication with occasionally devastating consequences. Recent studies have reported that the use of absorbable materials, instead of metallic ones, may lead to a lower incidence of postoperative infection. In this experimental pre-clinical animal study, we compared the infection rate between absorbable implants consisting of copolymers composed from trimethylene carbonate, L-polylactic acid, and D, L-polylactic acid monomers, and titanium implants after the inoculation of a pathogenic microorganism. Material and Methods: We used an experimental implant-related infection model in rabbits. Sixty animals were randomly and equally divided into two groups. In all animals, the right femur was exposed via a lateral approach and a 2.5 mm two-hole titanium plate with screws (Group A), or a two-hole absorbable plate and screws (Group B), were applied in the femoral shaft. Afterwards, the implant surface was inoculated with Pseudomonas Aeruginosa at a concentration of 2 × 108 CFU/mL. The primary outcome was the comparison of the incidence of developed infection between the two groups. The wound condition was monitored on a daily basis and radiographies were obtained at 12 weeks postoperatively. Infection-related laboratory markers (white blood cell count, erythrocyte sedimentation rate, and C-reactive protein values) were assessed at 3, 6, and 16 weeks postoperatively. Histologic analysis and cultures of tissue samples were also performed to evaluate the presence of infection. Results: Clinical and laboratory signs of infection were evident in 11 rabbits in Group A (36.7%), and 4 in Group B (13.3%). The difference between the groups was statistically significant (p = 0.04). Five animals in Group B (16.7%) had clinical and histologic signs of a foreign-body reaction with significantly elevated CRP and ESR values but no simultaneous presence of infection was identified (p = 0.04). Bone remodelling with thickening of the periosteum and surrounding sclerosis was demonstrated radiologically in animals developing infection or foreign-body reactions. Conclusions: Absorbable plates and screws show lower susceptibility to infection compared to titanium ones. However, their application is associated with foreign-body reaction and the potential need for a second surgical intervention.

Influence of Different Porosities of Titanium Meshes on Bone Neoformation: Pre-Clinical Animal Study with Microtomographic and Histomorphometric Evaluation.

The aim of this study was to evaluate the influence of different types of porosity of titanium meshes on the bone neoformation process in critical defects surgically created in rat calvaria, by means of microtomographic and histomorphometric analyses. Defects of 5 mm in diameter were created in the calvaria of 36 rats, and the animals were randomly treated and divided into the following groups (6 animals per group): NCOG (negative control, only blood clot), TEMG (Polytetrafluoroethylene-PTFE-membrane), SPTMG (small pore titanium mesh), SPMMG (small pore mesh + PTFE), LPTMG (large pore titanium mesh), and LPMMG (large pore mesh + PTFE). After 60 days, the animals were sacrificed, and the bone tissue formed was evaluated with micro-CT and histomorphometry. The data were compared using an ANOVA followed by the Tukey post-test (p ≤ 0.05). The microtomographic results showed that the SPTMG group presented the highest numerical value for bone volume/total volume (22.24 ± 8.97), with statistically significant differences for all the other groups except LPTMG. Considering the histomorphometric evaluation, groups with only porous titanium meshes showed higher values compared to the groups that used the PTFE membrane and the negative control. The SPTMG group presented higher values in the parameters of area (0.44 mm2 ± 0.06), extension (1.19 mm2 ± 0.12), and percentage (7.56 ± 1.45%) of neoformed bone. It was concluded that titanium mesh with smaller pores showed better results and that the association of PTFE membranes with titanium meshes did not improve the outcomes, suggesting a correlation between mesh porosity and underlying bone repair.

MicroRNA profiling in dogs undergoing induced ischemic heart infarction: An experimental study.

MicroRNAs (miRNAs) play an important role in various biological functions. According to many studies, miRNA expression is tissue-specific, strongly controlled throughout embryogenesis, and over- or under-expressed in numerous disorders, including cardiovascular pathologies. This study aimed to screen, characterize, and profile many induced biomarkers (miRNAs) in dog serum before and after experimentally inducing a regional myocardial infarction (MI) by occluding the coronary arteries under general anesthesia. A preclinical experimental animal study recruited 12 healthy canine dogs. The selected canine dogs were anesthetized with 1 mg/kg xylazine and 15 mg/kg ketamine before undergoing femoral arterial catheterization under fluoroscopic supervision. Commercial assay kits were used to purify total RNA and miRNA before the occlusion and 2 h after the occlusion according to the manufacturer's guidelines, and the samples were stored in RNase/DNase-free water at -80°C. Data were analyzed by GraphPad Prism 5.0 software (GraphPad Prism, San Diego, CA) SPSS, and GenEx software (www.multid.se) or (REST V3). Among 325 transcribed genes, 20 were identified in 2 h. After MI, 14 biomarkers were negative, indicating downregulation, and 6 (3-F08, 3-B10, 4-A11, 1-A06, 2-E01, 3-F10) were positive, indicating upregulation. Polymerase chain reaction assay results showed a normalized fold-change in gene expression in the test sample. Fold values >1 represented a biologically significant change. Profiling of miRNAs before and after MI in a dog model revealed upregulation of six previously unidentified biomarkers (3-F08, 3-B10, 4-A11, 1-A06, 2-E01, and 3-F10), indicating various miRNA regulatory patterns.

PO-05-104 FEASIBILITY STUDY OF SINGLE DAY SIMULATION- FREE CARDIAC RADIOABLATION WORKFLOW WITH A PORCINE MODEL

Cardiac radioablation (CRA) is a novel and expanding treatment option for patients with ventricular tachycardia (VT). CRA is a complex process involving interdisciplinary collaboration and requires multiple days between patient imaging and treatment delivery. For patients with active VT, this wait is difficult. Improving efficiency and standardization of CRA workflows is critically important for controllably implementing and assessing this novel treatment approach for research and clinical deployment. We hypothesized that by leveraging state-of-the-art technology within radiotherapy, key steps in the treatment planning and delivery process may be condensed to a single session on the treatment machine. Investigate the feasibility of CRA treatment in a single day, simulation-free workflow with a porcine model. Under an approved animal studies protocol, CRA was performed using a porcine model. In this workflow the animal was brought to radiation oncology under anesthesia under the supervision of a veterinary care team and was placed in the decubitus position on a dedicated research ring gantry linear accelerator platform. The ventilation rate of the animal was set to 8 breaths per minutes. A 6 second cone beam computed tomography scan was completed during exhalation while IV contrast was simultaneously administered. Images were reviewed by a radiation oncologist, medical physicist, and electrophysiologist. Organs at risk as well as the site of the VT target site were contoured. A radiation treatment plan was developed on this data set and reviewed by the treatment team. A CBCT was repeated to confirm alignment of the animal. The animal then received CRA to 25 Gy to planning target volume (PTV) in a single fraction. Simulation-free cardiac radioablation was successfully delivered to a pig in a single session using a commercially available ring gantry linear accelerator. This study demonstrates feasibility of conducting a simulation-free preclinical animal study of CRA using a commercially available linear accelerator and ultimately moves the field closer to same-day cardiac radioablation.

Preclinical animal study of electrospun poly (l-lactide-co-caprolactone) and formulated porcine fibrinogen for full-thickness diabetic wound regeneration

Diabetic foot ulcer is one of the most serious chronic complications of diabetes mellitus. It may lead to amputation of the lower extremities for diabetics. Our study was to evaluate the effect of electrospun poly (L-lactide-co-caprolactone) and formulated porcine fibrinogen (PLCL/Fg) wound dressing on animal wound model. A blend ratio of PLCL/Fg scaffold was 4 (PLCL):1 (Fg). The scanning electron microscopy findings showed that the fibers’ diameter was 122.5 ± 80.3 nm, and the tensile strength was 9.2 ± 0.2 MPa. In-vivo study of the hog normal model demonstrated that PLCL/Fg dressing had better biocompatibility, degradability, and ability to restore the skin's normal structure. We evaluated the wound healing processes in the rat diabetic model by macroscopic observation and histological observation at 1, 2, and 3 post-operation weeks. In our study, the PLCL/Fg group performed better 3 weeks after surgery, in terms of macroscopic healing and scarring. After surgery, the PLCL/Fg group showed better fibroblast accumulation, tissue granulation, and collagen expression than the control group. Topical treatment with PLCL/Fg dressing effectively enhanced wound healing in both normal and hyperglycemic conditions, suggesting that it may possess wound-healing potential.

Glucocorticoid- β-adrenoceptors interactions in the infralimbic cortex in acquisition and consolidation of auditory fear memory extinction in rats

This study investigated the interactive effect of glucocorticoid and β-adrenoceptors in the infralimbic (IL) cortex on the acquisition and consolidation of fear extinction in rats' auditory fear conditioning (AFC) task. On day 1, rats underwent habituation for 9 min (12 tones, 10 s, 4 kHz, 80 dB, without footshock). On day 2 (conditioning), rats received 3 mild electrical footshocks (US; 2 s, 0.5 mA) paired with the auditory-conditioned stimulus (CS; tone: 30 s, 4 kHz, 80 dB). On days 3–5 (Ext 1–3), rats received 15 tones with no footshock in the test box. Intra-IL injection of corticosterone (CORT, 20 ng/0.5 μl per side) before Ext 1 and after Ext 1–2, respectively, facilitated the acquisition and consolidation of fear memory extinction. Intra-IL injection of the β2-adrenoceptor agonist clenbuterol (CLEN, 50 ng/0.5 μl per side) inhibited, but the β-adrenoceptor antagonist propranolol (PROP, 500 ng/0.5 μl per side) enhanced the facilitatory effects of CORT on fear memory extinction. CORT injection before the acquisition of fear extinction increased p-ERK levels in the IL. Co-injection of CORT with CLEN increased, but PROP decreased p-ERK activities. CORT injection after the consolidation of fear extinction increased p-CREB in the IL. Co-injection of CORT with CLEN increased, but PROP reduced p-CREB activities. Our findings show that corticosterone facilitates the acquisition and consolidation of fear memory extinction. GRs and β-adrenoceptors in the IL jointly regulate fear memory extinction via ERK and CREB signaling pathways. This pre-clinical animal study may highlight the effect of GRs and β-adrenoceptors of the IL cortex in regulating fear memory processes in fear-related disorders such as PTSD.

Development of DNA aptamers for visualization of glial brain tumors and detection of circulating tumor cells

Here, we present DNA aptamers capable of specific binding to glial tumor cells invitro, exvivo, and invivo for visualization diagnostics of central nervous system tumors. We selected the aptamers binding specifically to the postoperative human glial primary tumors and not to the healthy brain cells and meningioma, using a modified process of systematic evolution of ligands by exponential enrichment to cells; sequenced and analyzed ssDNA pools using bioinformatic tools and identified the best aptamers by their binding abilities; determined three-dimensional structures of lead aptamers (Gli-55 and Gli-233) with small-angle X-ray scattering and molecular modeling; isolated and identified molecular target proteins of the aptamers by mass spectrometry; the potential binding sites of Gli-233 to the target protein and the role of post-translational modifications were verified by molecular dynamics simulations. The anti-glioma aptamers Gli-233 and Gli-55 were used to detect circulating tumor cells in liquid biopsies. These aptamers were used for in situ, exvivo tissue staining, histopathological analyses, and fluorescence-guided tumor and PET/CT tumor visualization in mice with xenotransplanted human astrocytoma. The aptamers did not show invivo toxicity in the preclinical animal study. This study demonstrates the potential applications of aptamers for precise diagnostics and fluorescence-guided surgery of brain tumors.

Corticosterone injection into the infralimbic prefrontal cortex enhances fear memory extinction: Involvement of GABA receptors and the extracellular signal-regulated kinase

This study investigated the interactive effect of glucocorticoid and Gamma-aminobutyric acid (GABA) receptors in the Infralimbic (IL) cortex on fear extinction in rats' auditory fear conditioning task (AFC).Animals received 3 conditioning trial tones (conditioned stimulus, 30 s, 4 kHz, 80 dB) co-terminated with a footshock (unconditioned stimulus, 0.8 mA, 1 s). Extinction testing was conducted over 3 days (Ext 1–3) after conditioning. Intra-IL injection of corticosterone (CORT, 20 ng/0.3 µl/side) was performed 15 min before the first extinction trial (Ext 1) which attenuated auditory fear expression in subsequent extinction trials (Ext 1–3), demonstrating fear memory extinction enhancement. Co-injection of the GABAA agonist muscimol (250 ng/0.3 µl/side) or the GABAB agonist baclofen (250 ng/0.3 µl/side) 15 min before corticosterone, did not significantly affect the facilitative effects of corticosterone on fear extinction. However, co-injection of the GABAA antagonist bicuculline (BIC, 100 ng/0.3 µl/side) or the GABAB antagonist CGP35348 (CGP, 100 ng/0.3 µl/side) 15 min before corticosterone, blocked the facilitative effects of corticosterone on fear extinction. Moreover, extracellular signal-regulated kinase (ERK) and cAMP response element-binding (CREB) in the IL were examined by Western blotting analysis after the first extinction trial (Ext 1) in some groups. Intra-IL injection of corticosterone increased the ERK activity but not CREB. Co-injection of the bicuculline or CGP35348 blocked the enhancing effect of corticosterone on ERK expression in the IL.Glucocorticoid receptors (GRs) activation in the IL cortex by corticosterone increased ERK activity and facilitated fear extinction. GABAA or GABAB antagonists decreased ERK activity and inhibited corticosterone's effect. GRs and GABA receptors in the IL cortex jointly modulate the fear extinction processes via the ERK pathway. This pre-clinical animal study may highlight GRs and GABA interactions in the IL cortex modulating fear memory processes in fear-related disorders such as post-traumatic stress disorder (PTSD).

Eugenol and its liposome-based nano carrier reduce anxiety by inhibiting glyoxylase-1 expression in mice.

The most common form of psycho-social dysfunction is anxiety with depression being related closely without any age bar. They are present with combined state of sadness, confusion, stress, fear etc. Glyoxalase system contains enzyme named glyoxalase 1 (GLO1).It is a metabolic pathway which detoxifies alpha-oxo-aldehydes, particularly methylglyoxal (MG). Methylglyoxal is mainly made by the breakdown of the glycolytic intermediates, glyceraldehyde-3-phosphates and dihydroxyacetone phosphate. Glyoxylase-1 expression is also related with anxiety behavior. A casual role or GLO-1 in anxiety behavior by using viral vectors for over expression in the anterior cingulate cortex was found and it was found that local GLO-1 over expression increased anxiety behavior. The present study deals with the molecular mechanism of protective activity of eugenol against anxiolytic disorder. A pre-clinical animal study was performed on 42 BALB/c mice. Animals were given stress through conventional restrain model. The mRNA expression of GLO-1 was analyzed by real time RT-PCR. Moreover, the GLO-1 protein expression was also examined by immunohistochemistry in whole brain and mean density was calculated. The mRNA and protein expressions were found to be increased in animals given anxiety as compared to the normal control. Whereas, the expressions were decreased in the animals treated with eugenol and its liposome-based nanocarriers in a dose dependent manner. However, the results were better in animals treated with nanocarriers as compared to the compound alone. It is concluded that the eugenol and its liposome-based nanocarriers exert anxiolytic activity by down-regulating GLO-1 protein expression in mice.

Targeting proteostasis of the HEV replicase to combat infection in preclinical models

Appropriate treatment options are lacking for hepatitis E virus (HEV)-infected pregnant women and immunocompromised individuals. Thus, we aimed to identify efficient anti-HEV drugs through high-throughput screening, validate them invitro and invivo (in a preclinical animal study), and elucidate their underlying antiviral mechanism of action. Using appropriate cellular and rodent HEV infection models, we studied a critical pathway for host-HEV interactions and performed a preclinical study of the corresponding antivirals, which target proteostasis of the HEV replicase. We found 17 inhibitors that target HEV-HSP90 interactions by unbiased compound library screening on human hepatocytes harboring an HEV replicon. Inhibitors of HSP90 (iHSP90) markedly suppressed HEV replication with efficacy exceeding that of conventional antivirals (IFNα and ribavirin) invitro. Mechanistically, iHSP90 treatment released the viral replicase ORF1 protein from the ORF1-HSP90 complex and triggered rapid ubiquitin/proteasome-mediated degradation of ORF1, resulting in abrogated HEV replication. Furthermore, a preclinical trial in a Mongolian gerbil HEV infection model showed this novel anti-HEV strategy to be safe, efficient, and able to prevent HEV-induced liver damage. In this study, we uncover a proteostatic pathway that is critical for host-HEV interactions and we provide a foundation from which to translate this new understanding of the HEV life cycle into clinically promising antivirals. Appropriate treatment options for hepatitis E virus (HEV)-infected pregnant women and immunocompromised patients are lacking; hence, there is an urgent need for safe and effective HEV-specific therapies. This study identified new antivirals (inhibitors of HSP90) that significantly limit HEV infection by targeting the viral replicase for degradation. Moreover, these anti-HEV drugs were validated in an HEV rodent model and were found to be safe and efficient for prevention of HEV-induced liver injury in preclinical experiments. Our findings substantially promote the understanding of HEV pathobiology and pave the way for antiviral development.

A New Animal Model of Laryngeal Transplantation.

Only three laryngeal transplants have been described in the literature to date, and none of the techniques has enabled a completely satisfactory functional result to be obtained. This article presents a new model of laryngeal transplantation, with quality of revascularisation of the transplant being the principal objective and optimisation of the various steps of the procedure, with the integration of a new reinnervation technique as a secondary objective. We present a preclinical animal study. Three pig larynges removed in vivo underwent allotransplantation according to the same protocol. The quality of the revascularisation was examined immediately after the surgery as well as by endoscopy for one animal on the fourth day after the operation. The mean time of cold ischaemia was 3 h 15 min. The anaesthetic tolerance of the pigs was excellent. Revascularisation was achieved and judged to be excellent for the three transplants immediately after the operation and the endoscopy performed for one pig on the fourth day after the operation confirmed this result. The anatomical similarities also enabled the application and integration of an innovative technique of laryngeal reinnervation into the various phases of the operation. We describe a reliable and reproducible animal model for laryngeal transplantation. Its application in humans can be envisaged.