IACUC Research Articles

VPS35 Interacts with Dopamine Transportor and Disrupts Dopamine Transporter Recycling Pathway

The exact pathophysiological role of vacuolar protein sorting 35 ortholog (VPS35) in Parkinson's disease (PD) is unknown. Here, we have identified dopaminergic transportor (DAT) as an interacting partner of VPS35. Mutant VPS35 D620N and DAT interaction causes a decrease in membrane DAT by disrupting DAT transport from an early endosome to a recycling endosome via weaker binding to FAM21. The in vitro interactions are further validated in the VPS35-D620N transgenic C. elegans and mice with the AAV-mediated overexpression of VPS35-D620N, which displayed key features of human PD. Our findings identified a novel pathway linking endosomal recycling as a mechanistic link with VPS35 associated PD that could potentially unravel new therapeutic targets. Funding Statement: The study was partly supported by the grants from: the National Natural Science Foundation of China (No. 81671102, 81873751 ); the National Key Research and Development Program of China, Stem Cell and Translational Research (No. 2017YFA0105104); Guangdong provincial science and technology plan project (No. 2016B030230002, 2017A040406007); Guangdong Provincial Key Laboratory for Diagnosis and Treatment of Major Neurological Diseases; The Southern China International Cooperation Base for Early Intervention and Functional Rehabilitation of Neurological Diseases (2015B050501003); Guangdong Provincial engineering Center for Major Neurological Disease Treatment. Declaration of Interests: The authors declare no competing financial interests. Ethics Approval Statement: Mice were maintained in accordance with institutional guidelines, and all protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the First Affiliated Hospital, Sun Yat-sen University, China.

Targeting the ERβ/Angiopoietin-2/Tie-2 Signaling-Mediated Angiogenesis with the FDA-Approved Anti-Estrogen Faslodex to Increase the Sunitinib Sensitivity in RCC

Sunitinib has been used as the first-line therapy to suppress the metastatic clear cell Renal Cell Carcinoma (ccRCC) as it could function via suppressing the tumor growth and angiogenesis. Yet most ccRCC tumors may still regrow due to the development of sunitinibresistance, and detailed mechanisms remain unclear. Here, we found the estrogen receptor β (ERβ) in RCC cells might function via increasing the endothelial cell tube formation to alter the sensitivity to sunitinib treatment. Mechanism dissection revealed that ERβ could function via transcriptional regulation with direct binding to the promoter to increase the cytokine Angiopoietin-2 expression in the ccRCC cells. The up-regulated Angiopoietin-2 could then increase the Tie-2 phosphorylation in the endothelial Huvec cells to promote the angiogenesis tube formation that results in increasing the endothelial cell resistance to sunitinib treatment. Preclinical study using an in vivo mouse RCC model also confirm in vitro data. Targeting this newly identified ERβ/Angiopoietin2/Tie-2 signaling-increased angiogenesis tube formation with the FDAapproved anti-estrogen, ICI 182,780 (Faslodex) may help in the development of a novel combined therapy with sunitinib to better suppress the ccRCC progression. Funding Statement: This work was partially supported by the URMC Urology research fund and the George Whipple Professorship Endowment. Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: The Institutional Animal Care and Use Committee (IACUC) review board at University of Rochester Medical Center approved the studies.

Serum Citrulline Levels Exhibit Circadian Variation and Fluctuations in Relation to Food Intake in Mice.

BackgroundPlasma citrulline is a nonessential amino acid synthesized almost exclusively by enterocytes of the small intestine. Correlation between post-operative plasma citrulline levels and remnant small bowel mass and function has led to its use as a simple biomarker of functional intestinal mass in adults and children, with a proposed prognostic value for achieving enteral autonomy in the setting of intestinal failure. While it is standard to measure fasting levels in humans, the optimal timing of blood draws in relation to food intake and time of day has not been standardized and is poorly studied in animal models. We hypothesize that serum citrulline levels vary both throughout the day and with relation to food intake in mice.MethodsSerum citrulline levels were measured from 12 C57BL/6 mice (6 - 9 weeks old) in the mornings and evenings, either after ad libitum food intake or after 8 - 12 h of access to water only. Blood draws for each experimental set-up were performed 1 week apart according to our Institutional Animal Care and Use Committee (IACUC) guidelines for acceptable blood collection volumes for survival procedures. At the end of the experiment, mice were euthanized and small intestine was harvested for morphometric measurements. Group means were compared using Student’s t-test with significance assumed for P < 0.05.ResultsAfter withholding chow for 8 - 12 h, serum citrulline levels were significantly greater in the morning compared to levels drawn in the evening. When mice were allowed ad libitum chow, there was no significant difference in serum citrulline levels drawn in the morning compared to the evening. There was no difference detected in villus height across the different experimental conditions.ConclusionsSerum citrulline levels exhibit circadian variation and fluctuations in relation to food intake in mice, without apparent concurrent changes in enterocyte mass. There was no diurnal variation of serum citrulline levels in fed mice whereas fasted mice had significantly higher levels of serum citrulline in the morning compared to the evening. These findings underscore the paramount importance of consistent sample collection strategies in the setting of translational research.

Effect of intermittent hyperoxia on stem cell mobilization and cytokine expression.

The best known form of oxygen therapy is hyperbaric oxygen (HBO) therapy, which increases both concentration and atmospheric pressure. HBO supports tissue regeneration and is indicated in an increasing number of pathologies. Less known but still showing some promising effects is normobaric oxygen (NBO) therapy, which provides some advantages over HBO including eliminating barotrauma risk, increased ease of administration and a significant cost reduction. However, still little is known about differences and similarities in treatment effects between HBO and NBO. Therefore we tested whether NBO induces a biological response comparable to HBO with a focus on stem progenitor cell mobilization and changes in serum cytokine concentration. We randomly assigned Sprague-Dawley rats into an NBO treatment group (n = 6), and a room air control group (n = 6). The NBO treatment group was exposed to 42% oxygen for 2 hours a day for 10 days. The room air group was concurrently kept at 20.9% oxygen. The frequency and number of stem progenitor cells in peripheral blood were analyzed by flow cytometry. Plasma cytokine expression was analyzed by cytokine array enzyme linked immunosorbent assay. All analyses were performed 24 hours after the final exposure to control for transient post treatment effects. The NBO treatment group showed an increase in circulating CD133+/CD45+ stem progenitor cell frequency and number compared to the room air control group. This rise was largely caused by CD34– stem progenitor cells (CD133+/CD34–/CD45+) without changes in the CD34+ population. The plasma cytokine levels tested were mostly unchanged with the exception of tumor necrosis factor-α which showed a decrease 24 hours after the last NBO exposure. These findings support our hypothesis that NBO induces a biological response similar to HBO, affecting serum stem progenitor cell populations and tumor necrosis factor-α concentration. The study was approved by Institutional Animal Care and Use Committee (IACUC) of the University of Wisconsin, Madison, WI, USA (approval No. M005439) on June 28, 2016.

Laboratory Guidelines for Animal Care.

Animal research is a controversial subject because of the ethical and moral implications of using unwilling research subjects in potentially painful or distressful procedures usually ending in euthanasia. As such, it must be conducted in a compassionate and responsible manner geared toward maximizing the animals' quality of life prior to and during experimentation. Because of its contentious nature, the conduct of animal research is highly regulated at the federal, state, city, and institutional levels. It is essential that researchers acquire a thorough knowledge of the procedures to be conducted as well as a working knowledge of the regulations. This will maximize humane care of research animals and prevent potentially negative or detrimental interactions with groups opposed to using animals in biomedical research. Perhaps the best way to attain these goals is to avoid inadvertent instances of noncompliance with their research protocol or applicable regulations. Regulatory noncompliance can also have serious negative consequences on investigators' research careers ranging from temporary suspension of their protocols to loss of funding and revocation of principal investigator status and associated privileges. To minimize such adverse outcomes, it is advised that researchers build positive and collaborative relationships with key institutional players such as the veterinary staff, the Institutional Animal Care and Use Committee (IACUC), and top administrators. Guidance is provided regarding the appropriate handling of regulatory noncompliance.

Salvianolic Acid a Protects Optic Nerve from Ischemia–Reperfusion Injury &lt;i&gt;via&lt;/i&gt; Inhibiting Astrocytes and ROCK Activation

Backgrounds: Ischemia/Reperfusion (I/R) injury induces apoptosis of retinal ganglion cells (RGCs) and optic nerve, which supposed to be mechanism of acute high IOP in glaucoma. Salvianolic Acid A is a potent natural anti- I/R monomer while little is known about its neuro-protecting effects. The present study was performed to investigate the underlying mechanism of Sal A in alleviating I/R injury, especially focusing on astrocytes inactivation and Rho-kinase inhibition. Methods: SD mice were divided into 3 groups: Control, I/R and I/R+Sal A group. A 60-min ischemic model was administered by raising the IOP, followed by a reperfusion period till 3 weeks. Retina and optic nerve morphology, RGC apoptosis and glial cells activation were detected, the underlying neuro-protective mechanisms of Sal A were also explored. Results: Sal A attenuated I/R injury by reducing RGC apoptosis and optic nerve degeneration. I/R activated astrocytes ,reduced βⅢ-Tubulin expression and retina thickness, which were also antagonized by Sal A.Meanwhile Sal A prohibits I/R-induced optic nerve degeneration via inactivating mapk and Rock1/2. Conclusions: The above results suggested that Sal A prohibits glaucomatous neuropathy induced by I/R injury and is indicative of potential drug activity. Funding Statement: This work was supported by Chinese National Natural Science Foundation (81674027). Declaration of Interests: The authors report no conflicts of interest. Ethics Approval Statement: This study was approved by the institutional Animal Care and Use Committee (IACUC) at the medical academy of Shanghai Jiao Tong University. All animal experiments were performed in accordance with the guidelines of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Deletion of &lt;i&gt;OSBPL2&lt;/i&gt; in Auditory Cells Increases Cholesterol Biosynthesis and Derives Reactive Oxygen Species Production by Inhibiting AMPK Activity

Background: Oxysterol binding protein like 2 (OSBPL2) was identified as a novel causal gene for autosomal dominant nonsyndromic hearing loss. The function of OSBPL2 as a lipid-sensing regulator in multiple cellular processes suggested that OSBPL2 might play an important role in the regulation of cholesterol-homeostasis, which was essential for inner ear. Methods: The Osbpl2-knockout (KO) and control OC1 auditory cells were subjected to RNA sequencing (RNA-seq) and gene pathway analysis. The regulatory mechanism of OSBPL2 in cholesterol synthesis and its cytological effects were investigated in Osbpl2-KO OC1 cells and osbpl2b-KO zebrafish. Finding: RNA-seq-based analysis suggested that OSBPL2 was implicated in cholesterol biosynthesis and AMPK signaling pathway. Osbpl2/osbpl2b-KO resulted in a reduction of AMPK activity and up-regulation of Srebp2/srebf2, Hmgcr/hmgcr and Hmgcs1/hmgcs1, the key effectors in AMPK signaling pathway. OSBPL2 was also found to interact with ATIC, the key activator of AMPK. The level of total cholesterol (TC) and reactive oxygen species (ROS) in OC1 cells or zebrafish inner ear were both increased in Osbpl2/osbpl2b-KO mutants and the mitochondrial damage was detected in Osbpl2-KO OC1 cells. Interpretation: This study uncovered the regulatory role of OSBPL2 in cellular cholesterol biosynthesis, which was essential in cholesterol-homeostasis. OSBPL2 deficit led to the promotion of cholesterol biosynthesis by inhibiting AMPK activity and resulted in intracellular ROS production. These founds might contribute to the deep understanding of the pathogenesis of OSBPL2 mutation in ADNSHL. Funding Statement: This research was supported by the grants from the National Natural Science Foundation of China (31571302, 81771000), the Key Research and Development Program of Jiangsu Province (Social Development: BE2016762) and the Key Project of Science and Technology Innovation of Nanjing Medical University (2017NJMUCX001) to XC; the grants from China Postdoctoral Science Foundation (2016M600431) and Jiangsu Planned Projects for Postdoctoral Research Funds (1601071B) to JY. Declaration of Interests: The authors state that they have no conflict of interests to declare. Ethics Approval Statement: Zebrafish were maintained using standard procedures and used in accordance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) of the Nanjing Medical University.

Opioid Use Potentiates the Virulence of Hospital Acquired Infection, Increases Systemic Bacterial Dissemination and Exacerbates Gut Dysbiosis in a Murine Model of Citrobacter Rodentium Infection

Background: Opioid analgesics are frequently prescribed in the United States and worldwide. However, serious side effects such as addiction, immunosuppression and gastrointestinal symptoms limit their use. It was recently demonstrated that morphine treatment results in a significant disruption in gut barrier function, leading to an increased translocation of gut commensal bacteria. Further studies have indicated distinct alterations in the gut microbiome and metabolome following morphine treatment, contributing to the negative consequences that are associated with opioid use. However, it is unclear how opioids modulate gut homeostasis in the context of a hospital acquired bacterial infection. Methods: In the current study, a mouse model of C. rodentium infection was used to investigate the role of morphine in the modulation of gut homeostasis in the context of a hospital acquired bacterial infection. Citrobacter rodentium is a natural mouse pathogen that models intestinal infection by enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) and causes attaching and effacing lesions and colonic hyperplasia. Findings: Morphine treatment resulted in 1) the promotion of C. rodentium systemic dissemination, 2) an increase in the expression of the virulence factors of C. rodentium colonization in intestinal contents, 3) altered gut microbiome, 4) damaged integrity of gut epithelial barrier function, 5) inhibition of the C. rodentium-induced increase in goblet cells, and 6) dysregulated IL-17A immune response. Interpretation: This is the first study to demonstrate that morphine promotes pathogen dissemination in the context of an intestinal C. rodentium infection, indicating that morphine modulates the virulence factor-mediated adhesion of pathogenic bacteria and induces disruption of mucosal host defense during C. rodentium intestinal infection in mice. This study demonstrates and further validates a positive correlation between opioid drug use/abuse and an increased risk of infections, suggesting that the overprescription of opioids may increase the risk of the emergence of pathogenic strains and should therefore be prescribed cautiously. Therapeutics that are directed at maintaining gut homeostasis during opioid use may reduce the comorbidities associated with opioid use for pain management. Funding Statement: This work was supported by the NIH grants R01 DA043252, R01 DA037843, R01 DA044582, and R01 DA047089 to SR. The funding agencies had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: All animals were maintained in specific-pathogen-free facilities, and all procedures were approved by the University of Minnesota Institutional Animal Care and Use Committee (IACUC). The IACUC protocol number was 1203A11091. All procedures were conducted in line with the guidelines set forth by the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

A REDCap application that links researchers, animal facility staff and members of the IACUC in animal health monitoring.

Research studies involving animal experimentation are regulated by the Institutional Animal Care and Use Committee (IACUC). To this end, the IACUC must integrate the information provided by the investigators of each preclinical study and the veterinarians from the animal facility in order to monitor and approve the process. Using a paper-based system to collect animal health and welfare data is a common, albeit time-consuming practice, prone to transcription and reading errors, not to mention inconvenient for veterinarians and investigators wishing to make timely and collaborative decisions when animal welfare is at risk. We created a web-based monitoring system focused on animal health with the potential to improve animal welfare. The data management system is based on REDCap software, which enables data integration in order to offer a solution for animal welfare assessment. The proposed scheme includes key indicators of general health status, such as environment, physical/nutritional information, and behavioral parameters during animal breeding and experimentation, as important components of animal welfare. In addition, the system facilitates communication of this information among researchers, animal facility staff, and the IACUC. REDCap is available to non-profit organizations, and may be adapted and replicated by institutions interested in and responsible for animal care, and used in research. REDCap is an excellent tool for promoting good practices that benefit experimental animal health.

Animal Research on Campus: Reflections on My Experience in the Field

In this essay, I discuss my experience as a member of my university’s Institutional Animal Care and Use Committee (IACUC). Using Blum’s (2016) model of experiential learning (or “learning in the wild”), I reflect on the connected processes of reading, thinking, seeing, hearing, smelling, talking, and listening that were the basis for my education about the use of animals for research on university campuses. In conclusion, I suggest that faculty members, staff members, and students have an obligation to understand, and work to change, the lives of the animals who exist among us.

The Role of the Basement Plate in Physeal Bar Formation.

Physeal fractures and resultant physeal bars can pose significant problems in skeletal development for the injured growing child. Although now well-recognized, only a small body of experimental literature covering this problem is available. The goal of this study was to help further develop an understanding of the different regions of the physis and the way in which each region responds to injury/fracture. This Institutional Animal Care and Use Committee (IACUC)-approved study assessed bar formation using radiologic and histologic methods and measured leg lengths of skeletally immature rats. The right tibia was used as the control to measure leg length discrepancy (LLD), and the left tibia received either a fracture only (F), an epiphyseal scrape (ES), an epiphyseal drilling procedure (ED), or metaphyseal drilling (MD). Radiographs and LLD measurements were obtained at postoperative days 0, 21, and 56. A significant LLD was present at day 56 in the ED group (P=0.01). Radiographic identification of bars showed significant evidence of bar formation for the ES and ED groups at 21 days and the ED group at 56 days (P<0.05). Histologic examination showed a high incidence of histologic physeal bar formation in the ES, ED, and MD groups at 21 and 56 days. Findings showed that the physis was able to continue to grow following an injury to the physis' hypertrophic region. MD produced little effects with few physeal bars and little LLD. By postoperative day 56, ED animals showed greater LLD than ES animals. Penetration of the basement plate was more likely to lead to bar formation/growth retardation than was ablation of the epiphyseal region of the physis (including resting cells). Data presented here provides insight into the importance of different regions of the physis and its repair/continued growth after physeal fracture. We suggest that a better understanding of the physiological cause of physeal arrest after physeal fracture will be important for the development of treatments to prevent physeal arrest or to treat physeal arrest after it occurs.

Prevention of Stomach Carcinoma Spread by Induces Bioenergetic Dysfunction in UCP2-Negative Gastric Cancer Cells with Hydrogen Sulfide

Background: Metabolite profiling stratifies gastric cancer (GC) cells into subtypes with distinct sensitivities to metabolic inhibitors. Hydrogen sulfide (H2S), an obvious characteristic is selectively suppressing cancer spread. However, it intrigues broad interest to explore the clinical benefits of cancer therapy, with the current understanding of molecular mechanisms of H2S which remains unknown. Methods: Transcriptome analysis identified that H2S inhibited uncoupling protein 2 (UCP2)-negative GC cells spread in cells cultures and animal models. The levels of bioenergetic function and proton leak was assayed by real-time bioenergetic analyzer. Metabolomics profiling revealed that H2S disturbed palmitic acid beta-oxidation to inhibitory of mitochondrial ATP (mitoATP) synthesis. Liquid chromatograph-mass spectrometer (LC-MS) and Chromatin Immunoprecipitation (ChIP) evaluated H2S promoted POZ/BTB family protein-33 (ZBTB33) nuclear translocation. Results: H2S promotes ZBTB33 nuclear translocation, and up-regulates UCP2 expression at the level of gene and protein in UCP2-negative GC cells. H2S triggers proton leak and turn off the metabolic switch that is essential for GC spread. Functional analysis showed repressed mitoATP synthesis, correlated negatively with ROS-mTOR signaling pathway, activated Casp8, and induced cell death in UCP2-negative GC cells following overexpression of UCP2. H2S reinforces that UCP2 catalyzes respiratory chain uncoupling by an H -assisted mechanism. Interpretation: Our preclinical work showed UCP2-negative GC cells into subtypes with distinct sensitivities to lipid inhibitor in drug discovery for screening chemopreventive agents against GC. We additionally demonstrate that the expression of UCP2 in GC served as a prognostic biomarker and negatively correlated with recurrence of de novo GC after curative treatment. Funding Statement: This work was supported by the Strategic Priority Research Program of the Science and Technology Commission of Shanghai (1312JC1408402 for Zhirong Wang). Declaration of Interests: No potential conflicts of interest were disclosed. Ethics Approval Statement: This animal study was conducted in accordance with the rules and regulations of the Institutional Animal Care and Use Committee (IACUC) at the Department of Laboratory Animal Science, Fudan University (Shanghai, P.R. China). Informed consent was approved by the Ethics Committees of Tongji Hospital and all subjects gave written informed consent.

Describing the Use of Animals in Animal-Assisted Intervention Research

ABSTRACTCurrently, there has been growing attention to animal welfare in animal-assisted interventions (AAI). However, there is a lack of scrutiny regarding what steps to take to ensure animal wellbeing during AAI research. The Institutional Animal Care and Use Committee (IACUC) reviews, monitors, and approves all research involving animal subjects in order to ensure an animal’s ethical and humane treatment. However, AAI publications rarely report IACUC approval. The purpose of this critical review was to determine the frequency of IACUC approval and to report the descriptions of animal use in AAI research publications. In the 139 articles reviewed, 14 (10%) studies reported attaining IACUC approval; 4/139 (3%) reported an adverse outcome on at least one animal used in the study. The publications inconsistently reported the training or certification and veterinary care of the animals. Lacking IACUC approval in AAI studies indicates that the field underutilizes this method of monitoring animal health and welfare. The IACUC should assess, approve, and monitor research protocols involving animals prior to conducting every study to safeguard AAI animal welfare.

Hyperglycemia-Triggered ATF6-CHOP Pathway Modulates Inflammatory Responses by β-Catenin Signaling in Liver Ischemia/Reperfusion Injury

Background: Hyperglycemia is an adverse risk factor for hepatic ischemia reperfusion injury (IRI); however, the underlying mechanism remains unclear. Herein, we addressed the question of whether and how hyperglycemia triggered endoplasmic reticulum (ER) stress and affected liver IRI. Methods: Diabetic patients and streptozotocin (STZ)-induced diabetic mice were involved in vivo. Bone marrow-derived macrophages (BMDMs) were used in vitro. Findings: The ER stress-ATF6-CHOP pathway was specifically activated in liver tissues and Kuppfer cells (KCs) from diabetic patients and STZ-induced diabetic mice. The ER stress inhibitor, 4-phenylbutyrate (PBA), attenuated hyperglycemia-related liver IRI, and decreased TLR4-related pro-inflammatory responses. Furthermore, CHOP gene knockout (CHOP-/-) had markedly reduced hyperglycemia-related liver IRI and inflammatory responses. Importantly, expression of β-catenin, was effectively inhibited, accompanied by activation of ATF6-CHOP signaling in diabetic mice, and was almost restored in liver tissues from PBA-treated or CHOP-/- diabetic mice. Furthermore, β-catenin small interfering RNA (siRNA) targeting KCs abrogated the CHOP-/--related protective effects against hyperglycemia-related liver IRI. High glucose (HG) specifically activated ATF6-CHOP signaling, inhibited β-catenin expression, and enhanced TLR4-related inflammatory responses in BMDMs in vitro; these effects were partly reversed in PBA- or CHOP siRNA-treated/CHOP-/- BMDMs. However, the anti-inflammatory functions of CHOP-/- were almost abolished by β-catenin siRNA in BMDMs under HG conditions. The above results were furthermore confirmed using a chimeric mice carrying WT or CHOP-/- BMDMs by BMDMs transplantation. Interpretation: This study revealed hyperglycemia specifically triggers ER stress-ATF6-CHOP signaling, inhibits β-catenin activity, promotes inflammation, and exacerbates liver IRI, which might provide the rationale for novel therapeutic strategies aimed at managing diabetic-related liver surgery. Funding: This study was supported by the Foundation of Jiangsu Collaborative Innovation Center of Biomedical Functional Materials, the Priority Academic Program Development of Jiangsu Higher Education Institutions, the National Natural Science Foundation of China (81400650, 814700901, 81273261, and 81270583), and Basic research program-Youth Fund Project of Jiangsu Province (BK20140092). Declaration of Interest: All authors declare no conflict of interests. Ethical Approval: Informed consent was obtained from all participants, and the study was approved by the local ethics committee of Nanjing Medical University. The animal protocol was approved by the Institutional Animal Care and Use Committee (IACUC) of Nanjing Medical University (Protocol Number IACUC-1702001).

Vaccinia Virus-Mediated IL-24 Gene Expression Inhibits the Growth of Human Breast Cancer by Inducing Apoptosis

Breast cancer is one of the most leading causes of cancer-related death in the world. Short survival, recurrent disease, chemotherapy-resistant and highly aggressive clinical course make it looking forward to novel targeted therapeutic strategies. Oncolytic vaccinia virus is an attractive candidate for cancer therapy due to its tumor cell-specific replication causing lysis of infected tumor cells as well as a delivery vector to overexpress therapeutic transgenes. Interleukin-24 (IL-24) is a novel tumor suppressor cytokine that selectively induces apoptosis in a wide variety of tumor types, including breast cancer. In present study, a targeted oncolytic vaccinia virus armed with IL-24 gene (VG9-IL-24) was constructed to evaluate its antitumor effects both in vitro and in vivo. We observed VG9-IL-24 efficiently infected and selectively eradicated breast cancer cells with no significant cytotoxicity to normal cells. VG9-IL-24 induced increased number of apoptotic cells and blocked breast cancer cells in the G2/M phase of the cell cycle. Western blotting results indicated that VG9-IL-24-mediated apoptosis was related to PI3K/β-catenin signaling pathway. In MDA-MB-231 xenograft mouse model, VG9-IL-24 delayed tumor growth and improved survival. Our findings provided documentation that VG9-IL-24 may be an innovative therapy for breast cancer with tumor-specific lysing and apoptosis-inducing. Funding: This work was supported by grants from the National Natural Science Foundation of China (81703061). The funding source was used for data collection and analysis. Declaration of Interest: None. Ethical Approval: The animal experiment was approved by the Institutional Animal Care and Use Committees (IACUC) of Jiangsu Institute of Nuclear Medicine (JSINM2010007).

IRF3 Deficiency Promotes Retinal Senescence and Age Related Synaptic Modeling

Age-related retinal degeneration is caused by irreversible physiological process in aged people. Interferon-related factor 3 (IRF3) stimulates cell-autonomous IFN-β expression in response to age related double-stranded DNA break. However, in this study, IRF3 deficiency promotes the retinal aging process. We examined the retinas of both young adult and old WT and IRF3-/- mice, found remarkable differences of age-related phenotypes between them, including synaptic morphological changes, functional ERG alteration, increased protein level of senescence-related markers. Moreover, when IRF3 deleted, nuclear p-γH2A.X deposition and senescence-associated β-galactosidase (SA-β-gal) staining were increased in retinal layers of IRF3-/- mice, respectively. This phenomenon was blocked in the presence of G10 (an agonist of STING) administration intravitreously. In terms of H2O2 induced neuronal senescence modeling, IRF3 deletion caused increased SA-β-gal and p16INK4a expression, however, rescued by G10 administration, in vitro. In addition, extracellular signal-regulated kinases (ERK) signaling molecules increased significantly when IRF3 deleted, which may contribute to IRF3-deficiency accelerated retina senescence in both young adult and old IRF3-/- mice. Together, these results identify that IRF3 deficiency aggravate the retinal synaptic aging phenotypes of both young and old mice, promote the retinal senescence and suggest the essential role of IRF3 in maintaining the retina youthfulness. Funding: This research was supported by the National Natural Science Foundation of China (No. 81570840, No. 81371006, No. 81200926), Academician Led Science and Technological Innovation of Chongqing (cstc2017jcyj-yszxX0006) and Research Foundation of Dept. Ophthalmology in Daping Hospital, AMU (No. 9-2543). Declaration of Interest: The authors declared that there is no conflict of interest. Ethical Approval: The Institutional Animal Care and Use Committee (IACUC) of the Army Medical University approved all studies using animals.

Effect of Eylea on recombinant AAV2-VEGF-induced choroidal neovascularization

Objective To evaluate the effect of Eylea (aflibercept) on recombinant adeno-associated virus 2(AAV2)-vascular endothelial growth factor (VEGF)-induced choroidal neovascularization (CNV). Methods Two normal cynomolgus monkeys were used in this study.Recombinant AAV2-VEGF was delivered by subretinal injection (60 μl/eye, 7.0×1011 IU/ml) into both eyes for each cynomolgus monkey.Both eyes of one animal were treated with single intravitreal injection of Eylea (50 μl/eye, 40 mg/ml) 3 weeks after AAV2-VEGF injection, and the two eyes of the other animal were untreated.Optical coherence tomography(OCT), electroretinography (ERG), fluorescein angiography (FFA) and ocular photography were conducted.The experiment was approved by the Institutional Animal Care and Use Committee (IACUC) at JOINN Laboratories (Suzhou) (IACUC serial number: ACU15-1112). Results OCT showed that subretinal hyperreflective material, retinal edema, choroid edema and local pigment epithelial detachment were found after injection of recombinant AAV2-VEGF.All of those symptoms were relieved after treated with Eylea, but aggravated after 4 weeks treated with Eylea.FFA showed that the area of fluorescein leakage expanded obviously after 2 weeks treated with AAV2-VEGF, and it was observed at the whole of fundus posterior pole at 5 weeks after injection.After 1 week treated with Eylea, the area of fluorescein leakage decreased obviously, but increased gradually after 4 weeks treated with Eylea.ERG results showed that the amplitude of ERG decreased gradually after injection of recombinant AAV2-VEGF.After intravitreal injection of Eylea, the amplitude of ERG increased gradually, but it decreased again after 4 weeks treated with Eylea. Conclusions Injection of recombinant AAV2-VEGF is capable of inducing CNV and pathological retinal in cynomolgus monkey.Eylea can prevent the development of clinically relevant CNV, but the effect just lasts for a period. Key words: Choroidal neovascularization; Cynomolgus monkey; Retinopathy; Eylea; Recombinant AAV2-VEGF

Friday, September 28, 2018 4:05 PM–5:05 PM abstracts: basic science of spinal fusion: 233. Opioids delay healing of spinal fusion: a rabbit posterolateral lumbar fusion model

BACKGROUND CONTEXT Opioid use is prevalent for management of pre- and postoperative pain in patients undergoing spinal fusion. There is evidence that opioids downregulate osteoblasts in-vitro, and one previous study found that morphine delays the maturation and remodeling of callus in a rat femur fracture model. However, the effect of opioids on healing of spinal fusion has not been investigated before. Isolating the effect of opioid exposure in humans would be limited by the numerous confounding factors that affect fusion healing. Therefore, we have used a well-established rabbit model to study the process of spinal fusion healing that closely mimics humans. PURPOSE To study the effect of systemic opioids on the process of healing of spinal fusion in a rabbit posterolateral spinal fusion model. STUDY DESIGN/SETTING Preclinical animal study. PATIENT SAMPLE A total of 24 adult New Zealand white rabbits. OUTCOME MEASURES Assessment of fusion was done by manual palpation, plain radiographs, micro-computed tomography (microCT), and histology. METHODS A total of 24 adult New Zealand white rabbits were studied in two groups after approval from the Institutional Animal Care and Use Committee (IACUC). The opioid group (n=12) received four-weeks preoperative and six-weeks postoperative transdermal fentanyl. Serum fentanyl levels were measured just before surgery and four-weeks postoperatively to ensure adequate levels. The control group (n=12) received only perioperative pain control as necessary. All animals received a bilateral L5-L6 posterolateral spinal fusion using iliac crest autograft. Animals were euthanized at the six-week postoperative time point for analysis. RESULTS A total of 12 animals in control group and 11 animals in the opioid group were available for analysis at the end of six weeks. The fusion scores on manual palpation, radiographs, and microCT were not statistically different. Three-dimensional microCT morphometry found that the fusion mass in the opioid group had a lower bone volume (p=.09), lower trabecular number (p=.02) and higher trabecular separation (p=.02) as compared to control. Histological analysis found areas of incorporation of autograft, and unincorporated graft fragments in both groups. In the control group, there was remodeling of de-novo woven bone to lamellar organization with incorporation of osteocytes, formation of mature marrow, and relative paucity of hypertrophied osteoblasts lining new bone. Sections from the opioid group showed formation of de-novo woven bone, and hypertrophied osteoblasts seen lining the new bone. There were no sections showing lamellar organization and development of mature marrow elements in the opioid group. Less dense trabeculae on microCT correlated with histological findings of relatively immature fusion mass in the opioid group. CONCLUSIONS Systemic opioids led to an inferior quality fusion mass with delay in maturation and remodeling at six-weeks in this rabbit spinal fusion model. These preliminary results lay foundation for further research to investigate underlying cellular mechanisms, temporal fusion process, and dose-duration relationship of opioids responsible for our findings.

IACUC is in the wrong.

IACUC is in the wrong.

"Who is on my side?" When the investigator feels that the IACUC is working against them.

"Who is on my side?" When the investigator feels that the IACUC is working against them.