Retro-orbital Injection Research Articles

Targeting Neurons with Functional Oxytocin Receptors: A Novel Set of Simple Knock-In Mouse Lines for Oxytocin Receptor Visualization and Manipulation.

The neuropeptide oxytocin (Oxt) plays important roles in modulating social behaviors. Oxt receptor (Oxtr) is abundantly expressed in the brain and its relationship to socio-behavioral controls has been extensively studied using mouse brains. Several genetic tools to visualize and/or manipulate Oxtr-expressing cells, such as fluorescent reporters and Cre recombinase drivers, have been generated by ES-cell based gene targeting or bacterial artificial chromosome (BAC) transgenesis. However, these mouse lines displayed some differences in their Oxtr expression profiles probably because of the complex context and integrity of their genomic configurations in each line. Here, we apply our sophisticated genome-editing techniques to the Oxtr locus, systematically generating a series of knock-in mouse lines, in which its endogenous transcriptional regulations are intactly preserved and evaluate their expression profiles to ensure the reliability of our new tools. We employ the epitope tagging strategy, with which C-terminally fused tags can be detected by highly specific antibodies, to successfully visualize the Oxtr protein distribution on the neural membrane with super-resolution imaging for the first time. By using T2A self-cleaving peptide sequences, we also induce proper expressions of tdTomato reporter, codon-improved Cre recombinase (iCre), and spatiotemporally inducible Cre-ERT2 in Oxtr-expressing neurons. Electrophysiological recordings from tdTomato-positive cells in the reporter mice support the validity of our tool design. Retro-orbital injections of AAV-PHP.eB vector into the Cre line further enabled visualization of recombinase activities in the appropriate brain regions. Moreover, the first-time Cre-ERT2 line drives Cre-mediated recombination in a spatiotemporally controlled manner on tamoxifen (TMX) administration. These tools thus provide an excellent resource for future functional studies in Oxt-responsive neurons and should prove of broad interest in the field.

Abstract 11220: Cardiac Bridging Integrator 1 Gene Therapy Rescues Diabetic Cardiomyopathy in Mice

Introduction: Diabetes is a major cause of heart failure with preserved ejection fraction (HFpEF), which has limited diagnostic tools and therapeutic options. Abnormal remodeling in the t-tubule calcium handling cardiac bridging integrator 1 (cBIN1)-microdomains has emerged as key to the pathophysiology of failing cardiomyocytes, which can be targeted for cardiac functional improvement. It remains unclear whether cBIN1 replacement therapy is effective in restoring cardiac function in diabetic HFpEF. Hypothesis: We hypothesized that cBIN1 gene therapy rescues diastolic failure in mice with diabetic cardiomyopathy. Methods: 9-wks old male and female db/db mice that received retro-orbital injection of 1x10 11 vg of AAV9 transducing GFP or cBIN1 were compared to non-diabetic control db/m littermates injected with AAV9-GFP (N = 16 mice per group). Mice were terminated 8-wks later for tissue acquisition, imaging, as well as biochemical analysis. Echocardiography, exercise tolerance, and plasma cBIN1 score (CS, an inverse index of plasma cBIN1 concentration) were obtained before and after gene therapy. Results: db/db mice developed diastolic failure with reduced E/A, increased E/e’, exercise intolerance, and cardiac hypertrophy with reduced tissue cBIN1 levels and corresponding elevations in plasma CS. Supporting our hypothesis, AAV9-cBIN1 normalized E/A and E/e’ and improved exercise tolerance in db/db mice, which can be distinguished by plasma CS as an inverse marker of cBIN1-microfolds and cardiomyocyte remodeling (Figure 1). Preservation of cBIN1-microdomains in db/db cardiomyocytes reorganized functional SERCA2a and rescued mitochondrial respiration with diminished oxidative stress. Conclusions: Exogenous cBIN1 normalizes lusitropic function of diabetic hearts through microdomain-organized effective calcium handling and mitochondrial respiration. cBIN1 gene therapy is a new and effective method for guided treatment of diabetic HFpEF.

Treatment with DC/AML Fusion Vaccine and CD3xCD123 Bi-Specific T-Cell Engager (CD123-CODV-TCE) for Treatment of Acute Myeloid Leukemia

Treatment with DC/AML Fusion Vaccine and CD3xCD123 Bi-Specific T-Cell Engager (CD123-CODV-TCE) for Treatment of Acute Myeloid Leukemia

Haemostatic effect of adding tranexamic acid to emicizumab prophylaxis in severe haemophilia A: A preclinical study.

Patients with severe haemophilia have impaired haemostatic response, delayed clot formation and fibrin clots that are vulnerable to fibrinolysis. Emicizumab is a bispecific antibody that mimics activity of activated factor VIII (FVIII) and increases haemostatic capacity to the level of moderate-to-mild haemophilia, thereby used for prophylaxis. Regardless of the impressive clinical performance of emicizumab, breakthrough bleeds may still occur. We aimed to study, in FVIII knockout mice (FVIII-KO), whether haemostasis is improved with the addition of tranexamic acid (TxAc) to emicizumab. FVIII-KO mice received prophylaxis with emicizumab or emicizumab+TxAc before trauma. FVIII-KO mice were given emicizumab 1.5mg/kg via IV injection. A second retro-orbital IV injection containing human FIX and FX (both 100U/kg) was given 24h later and 5min before the tail amputation or knee trauma. After trauma-induced knee joint bleeding, magnetic resonance imaging (MRI), and histological analysis were used to compare haemostatic efficacy of the two prophylactic strategies. Thrombin generation (TG) was measured and clots obtained with TG experiment were analysed by scanning electron microscopy. In FVIII-KO mice, blood loss after tail clip was lower after prophylaxis with emicizumab+TxAc compared to emicizumab. MRI results and histological analysis of knee joints showed that the addition of TxAc significantly decreased joint bleeding. Fibrin fibre diameters of mice treated with emicizumab only was thicker than those who received combined prophylaxis with emicizumab+TxAc. Our results suggest a potential benefit of TxAc when used in combination with emicizumab in prophylactic settings, especially in patients presenting breakthrough bleeds.

ENGRAFTMENT POTENTIAL OF CD34+ CELLS FROM SEVERE APLASTIC ANEMIA PATIENTS EXPANDED WITH UM171

Immune aplastic anemia (AA) is a hematological disease characterized by pancytopenia and bone marrow (BM) hypocellularity. It is caused by an immune attack against hematopoietic stem and progenitor cells (HSPC). Its treatment is based on allogeneic BM transplantation and immunosuppressive therapy. More recently, the addition of eltrombopag showed that the expansion of hematopoietic precursors is a therapeutic alternative. Fares et al. demonstrated that the small molecule UM171 is capable of expanding the most primitive CD34 + cell compartment of umbilical cord blood (UCB) ex vivo. Our research group showed that UM171 is also capable of effectively expanding immature subpopulations of CD34 + cells from patients with immune AA. The aim of this study was to evaluate the engraftment capacity of CD34 + cells from patients with AA expanded with UM171 in a murine xenotransplantation model. CD34 + cells were purified from either bone marrow of one patient or one UCB unit using immunomagnetic labeling with human CD34 MicroBeads, MS columns, and a magnetic separator. Cells were cultured for seven days in medium supplemented with cytokines and 35 nM UM171 or 0.1% DMSO (control) and then transplanted via retro-orbital injection into pre-conditioned 8-to-12-week-old female NSG-SGM3 (NSGS) mice. The progeny of 3×10 4 UCB CD34 + cells or 8.250 AA patient CD34 + cells was transplanted per mouse (four mice per experimental condition). The conditioning regimen consisted of two intraperitoneal injections of 12.5 mg/kg busulfan administered 48 and 24 hours before transplantation. Human hematopoietic cells were monitored in the murine BM by flow cytometry at week 20 post-transplantation. BM cells were collected from the two femurs and the two tibias of each animal and treated twice with red cell lysis, labeled with APC-Cy7 anti-mouse and PerCP-Cy5.5 anti-human CD45 antibodies, then analyzed on a flow cytometer using the FlowJo V.10 software. We found that both progenies of UCB and AA BM CD34 + cells cultured with UM171 showed an improved reconstitution potential in mice BM 20 weeks after transplant compared to DMSO, which was demonstrated by the detection of higher percentages of human CD45 + cells in the mice BM (UCB: UM171 = 10,07% ±2,29 vs DMSO = 3,73% ±1,99; AA BM: UM171 = 0,095% ±0,075 vs DMSO = 0,042% ±0,002; data represent mean ±Standard Error). Differences were not significant. We wondered whether the UM171 treatment would impact the engraftment capacity of the in vitro expanded CD34 + cells. The enhanced engraftment capacity enabled by UM171 could allow for immune AA patients to be treated with a novel strategy, autologous transplantation of their own CD34 + expanded cells. The present study indicates that UM171 could be a promising candidate drug for treating patients with immune AA. More replicates (patients and controls) are required to firmly establish the benefit of UM171 in conferring repopulation potential to ex vivo expanded CD34 + cells from AA patients. In initial experiments, in vitro treatment with UM171 appears to increase the engraftment of CD34 + cells isolated from UCB and from the BM of immune AA patients. These results need to be confirmed in additional experiments.

Abstract P329: Exosomes Derived From Podoplanin Positive Cells Alter The Cellular Composition Of Healthy Mouse Hearts

Fibrosis and blood hypoperfusion stimulated by paracrine signals enhances the ventricular dysfunctionafter myocardial infarction (MI). We have earlier reported that within 2 days post-MI a cohort ofpodoplanin (PDPN) positive cells populate injured heart and enhance inflammatory response by physicalinteractions with monocytes. Here we explored whether exosomes from these cells could independentlyalter healthy heart physiology and structure. PDPN+ cells were isolated 2 days after MI, culture expandedand activated with TNFα and Angiotensin II. Exosomes derived from activated PDPN+cells conditionedmedia (PDPN+exo) were used in vitro for the treatment of mouse cardiac endothelial cells (mCECs) andmouse fibroblast (3T3) and in vivo for the treatment of healthy mouse hearts. In vitro, PDPN+exoinfluenced the phenotype of mCECs, stimulating their lineage into lymphatic endothelial cells andfacilitated fibroblasts transition to myofibroblast. Characterization of the protein content of PDPN+exoshowed high concentration of Notch receptors and γ-Secretase, suggesting these cellular transitions maydepend on exosome-mediated Notch translocation and cleavage. In fact, after exosomes treatmentcleaved notch (NICD) translocated in the nuclei of mCECs and 3T3 as early as 1h of treatment and eitherHes-1 or Hey-1, major transcription factors activated by NICD were enhanced within 2d of treatment.Using DAPT, a γSecretase inhibitor, notch cleavage was inhibited, and no phenotype switching in responseto exosome treatment was observed. In vivo, PDPN+exo were injected into the left ventricle of healthymouse hearts followed by boosters delivered by retro-orbital vein injection. Treated mice developed anextended epicardial fibrosis with a subsequent impairment in the contractility and increase of the enddiastolic and systolic volumes. The fibrotic area was characterized by vessels double positive toendothelial and lymphatic endothelial markers, and infiltrating CD45+ cells. Podoplanin positive cellsrepresent 80% of the scar’s cells of a chronic infarcted myocardium and the specific exosomes cargo highlyinfluence the lineage of cardiac cells altering the biology of endothelial cells and fibroblasts which mayfacilitate adverse remodeling.

Abstract 29: Acute Systemic Treatment With Sphingosine -1- Phosphate Receptor 1 Agonist Diminishes Sex-difference In Renal Function Seen In Intrauterine Growth Restricted Mice.

IUGR is a risk factor for the early development of cardiorenal diseases in life. We previously showed that our IUGR mouse model exhibits sex differences in blood pressure (BP) and kidney functions as males have elevated BP and impaired kidney function while females are not. Sphingosine-1-Phosphate Receptor 1 (S1PR1) is reported to be involved in developing and progressing several cardiorenal diseases. We found that acute activation of S1PR1 transiently reduced BP in male IUGR; however, the effects of SIPR1 on renal function in IUGR are still unknown. We hypothesize that S1PR1 plays a role in the sex differences of impaired kidney function in IUGR. Here we investigated the acute effects of a specific S1PR1 agonist (SEW2178) on kidney function in IUGR offspring generated through placental insufficiency. Methods: C57BL IUGR or control offspring were obtained from the Reduced Uterine Perfusion Pressure (RUPP) or sham surgeries on the 13 th day of gestation. At 6 months, nephron number was measured by analyzing kidney histology while Glomerular Filtration Rate (GFR) was measured using FITC-Anulin decay after retroorbital injection. Mice were treated with 3mg/kg SEW2871 i.p before GFR measurement. Results: Both male and female IUGR had a decrease in nephron number compared to same-sex control (371.8± 28.8 vs 284.0± 11.3, P<0.01, male control vs IUGR) and (333.6±13.3 vs 256± 13.0, P<0.05, female control vs IUGR). Furthermore, IUGR impaired kidney function in male but not female mice as indicated by reduced GFR (8.9 ±0.6 vs 13.9±1.1 μL/min/kg BW, P<0.05, IUGR vs control males) and (12.3±0.9 vs 15.9 ±1.9 L/min/kg BW, control females vs IUGR). Administration of SEW2871 diminished the difference of GFR between male control and IUGR (9.5±0.6 vs 9.7±1.1 μL/min/kg BW, P> 0.05 control vs IUGR). While in females, both IUGR and control had a similar response to SEW2178 (7.4±1.0 vs 10.6 ±1.9 μL/min/kg BW, control vs. IUGR). Conclusion: IUGR impairs renal function in male offspring but not in females, indicating sex differences in fetal programming of kidney disease. Male and female IUGR offspring respond differently to acute systemic activation of S1PR1. Further studies are required to investigate the kidney-specific and cell-specific of S1PR1 activation/inhibition.

Shape effect of cerium oxide nanoparticles on mild traumatic brain injury

The catalytic performance and therapeutic effect of nanoparticles varies with shape. Here, we investigated and compared the therapeutic outcomes of ceria nanospheres (Ceria NSs) and ceria nanorods (Ceria NRs) in an in vivo study of mild traumatic brain injury (mTBI). In vivo TBI was induced in a mouse model of open head injury using a stereotaxic impactor. Outcomes including cytoprotective effects, cognitive function, and cerebral edema were investigated after retro-orbital injection of 11.6 mM of ceria nanoparticles. Ceria nanoparticles significantly reduced fluoro-jade B (FJB)-positive cells and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells, and restored mRNA levels of superoxide dismutase 1 (SOD1) and SOD2. They also decreased the cyclooxygenase-2 (COX-2) expression compared with the untreated control group. Comparing the two nanomaterials, Ceria NRs showed less stable and high-energy (100) and (110) planes, which increased the number of active sites. The Ce3+/Ce4+ molar ratio of Ceria NRs (0.40) was greater than that of Ceria NSs (0.27). Ceria NRs (0.059 ± 0.021) appeared to exhibit better anti-inflammatory effect than Ceria NSs (0.133 ± 0.024), but the effect was statistically insignificant (p = 0.190). Ceria nanoparticles also improved cognitive impairment following mTBI compared with the control group, but the effect did not differ significantly according to the nanoshape. However, Ceria NRs (70.1 ± 0.5%) significantly decreased brain water content compared with Ceria NSs (73.7 ± 0.4%; p = 0.0015), indicating a more effective reduction in brain edema (p = 0.0015). Compared with Ceria NSs, the Ceria NRs are more effective in alleviating cerebral edema following in vivo mTBI.

1: Fat Grafting Improves Dorsal Skin Fibrosis after Radiation in an Engrailed-1 Mouse Model

Purpose:Fat grafting is known to rejuvenate and improve fibrosis in irradiated skin, but the underlying mechanisms behind these effects are poorly understood. We have previously identified the Engrailed-1 fibroblast sub-population as having a role in post-natal dorsal skin fibrosis in mice. Using a radiation model to the scalp of Engrailed-1 mice, we sought to better understand the regenerative effects of fat grafting in irradiated tissue.Methods:Adult (60-day old) En1Cre;R26mTmG transgenic mice (n=5) underwent total body irradiation with 9 Gy for immunodepletion. Mice were then immediately reconstituted with 2 million nucleated bone marrow cells from donor NSG(NOD.CB17-Prkdcsscid/J) mice via retro-orbital injections. Reconstitution was confirmed with fluorescence-activated cell sorting (FACS) 2 weeks after whole body irradiation. Mice scalps were then irradiated with 5 Gy every other day for 12 days (30 Gy total) and allowed to recover for 4 weeks to facilitate fibrotic conversion. Irradiated scalps were grafted with 200μL of fresh human lipoaspirate. Graph retention was measured in-vivo for 8 weeks using Micro-Computed Tomography Scanner (Bruker SkyScan 1726™) and scalp skin was harvest for histology.Results:Two weeks post bone-marrow transplant, >90% of circulating hematopoietic cells (CD45+) cells in En1Cre;R26mTmG reporter mice were non-fluorescent, signifying successful reconstitution. Volumetric analysis of fat grafting in-vivo was performed using 3-dimensional reconstruction. At 8 weeks post-grafting, 4 of the 5 grafts demonstrated >50% graft retention, confirming successful grafting. Histological sections of scalp skin 8 weeks post-grafting demonstrated significantly less En1+GFP cells compared to non-grafted scalp skin, signifying less presence of En1+ fibroblasts. Furthermore, histology demonstrated significantly less dermal thickening, epidermal thinning, and collagen deposition and disorganization in fat grafted irradiated scalp skin compared to non-grafted scalp skin.Conclusion:Fat grafting mitigates radiation-induced fibrosis in scalp skin by decreasing collagen deposition, remodeling collagen formation, decreasing epidermal thinning, and reducing presence of pro-fibrotic fibroblast sub-populations.

QS8: Fat Grafting Depletes Profibrotic Prrx1-positive Fibroblasts In Irradiated Skin And Mitigates Radiation-induced Groin Contracture

Purpose:Contracture is a long-term complication of radiotherapy (RT) that results from pathologic fibrosis of soft tissue in the radiation field. Fat grafting can regenerate fibrotic soft tissue by decreasing collagen density and reorganizing collagen fiber networks. Fibroblasts are the predominant cell involved in extracellular matrix (ECM) synthesis, and dermal fibroblasts possess distinct embryonic origins. We recently identified a profibrotic fibroblast in the mouse ventral dermis marked by embryonic expression of paired related homeobox 1 (Prrx1). Here we sought to investigate how fat grafting alters Prrx1-positive subpopulation distribution as well as functional contractures.Methods:Adult Prrx-1Cre;R26mTmG reporter mice (n=9) underwent whole body lethal irradiation with 9 Gy for hematopoietic depletion. Mice were immediately reconstituted with 2 million nucleated bone-derived cells from donor NSG (NOD.CB17-Prkdcsscid/J) mice via retro-orbital sinus injection. The success of reconstitution and immunodepletion was assessed by fluorescence-activated cell sorting (FACS) analysis of peripheral blood. After 4 weeks, 30 Gy was delivered to the right hindlimb in five fractionated doses to generate limb contracture. The irradiated, contracted limb was then grafted with 200 μl fresh human lipoaspirate and limb extension was measured over the subsequent 8 weeks, at which point skin was harvested for assessment of fibroblast subtypes for FACS and immunofluorescence. A group of mice with radiation-induced groin contracture did not undergo fat grafting and served as the control group.Results:FACS analysis indicated successful immunodepletion and engraftment by 3 weeks post bone marrow transplantation. At one month following groin irradiation, mice had developed significant right hind limb contracture with significantly reduced limb extension (****p≤0.0001). Histologically this was paralleled by thickening of the dermis, and substantial expansion of the fibrogenic Prrx-1-positive fibroblast subpopulation. While human fat graft volume retention was reduced over 8 weeks following implantation, this was associated with significantly improved in limb extension. The skin overlying the grafted fat showed reduced collagen density, as indicated by trichrome staining, as well as a reduction in the fibrogenic Prrx-1-positive fibroblast subpopulation by immunofluorescence imaging, as compared to the control mice.Conclusion:Here we show that fat grafting improves the extensibility of irradiated and contracted hind limbs and reverses radiation-induced skin fibrosis by both reducing the collagen content and by altering the composition of dermal fibroblast subpopulations. Specifically, fat grafting results in a depletion of the Prrx-1-positive fibroblast subpopulation. Further elucidating how this profibrotic fibroblast subpopulation is involved in ventral surface soft tissue fibrosis will facilitate development of novel strategies to treat/prevent debilitating late side-effect of radiotherapy.

Biocompatibility Assessment Of DNR-Peg-PLL-PLGA Nanoparticles In Vitro and In Vivo

The present study was designed to evaluate the biocompatibility of DNR-PEG-PLL-PLGA as nanoparticles which provide the evidence before clinical application. We applied some tests to assess the safety of DNR-PEG-PLL-PLGA nanoparticles. There was low cytotoxicity of the PEG-PLL-PLGA nanoparticles in vitro when detected by the MTT assay. Cell apoptosis and intracellular accumulation of PEG-PLL-PLGA were determined by FCM assay. The apoptosis induced by nanoparticles and the fluorescence intensity of intracellular DNR demonstrated that DNR-PEG-PLL-PLGA could be taken up by L929 cells, in which DNR was persistently released. Hemolysis test and micronucleus (MN) assay were carried out to estimate the nanoparticles that they were no blood toxicity and no genotoxicity. DNR-PEG-PLL-PLGA nanoparticles were injected into mice through tail vein to calculate the median lethal dose (LD50), the results showed that they had a wide safe scale. The pathologic changes of heart, liver, spleen, lung and kidney were observed in nanoparticles treated mice. The influence of DNR-PEG-PLL-PLGA to mice hepatic and renal functions were measured by extracting the blood by retro orbital injection, they were no significant difference compared with the control group. These results clearly illustrated that DNR-PEG-PLL-PLGA appear to be highly biocompatible and safe nanoparticles that are suitable for further application in the treatment of tumor. Disclosures:No relevant conflicts of interest to declare.

Abstract LB239: CryoVizTM and photoacoustic imaging for assessment of tumor vasculature in mouse models of pancreatic tumors

Abstract Introduction: Assessing the tumor vasculature in models of pancreatic tumors can improve the characterization of these models, which can then support the eventual assessments of anti-cancer treatments. In this study, we sought to relate hypoxia, vascular perfusion, and vascular patency using synergistic imaging methods. In vivo Multispectral Optoacoustic Tomography (MSOT; also known as photoacoustic imaging) can measure %sO2 a relative measurement of oxy- vs. deoxy-hemoglobin in tumors. In vivo Dynamic Contrast Enhanced (DCE) MSOT can measure vascular perfusion. CryoVizTM imaging, a serial-sectioning-and-block-face-imaging technique, provides high-resolution 3D microscopic color anatomy and sensitive fluorescence making it ideal for assessing vascular perfusion by imaging ICG uptake and vascular patency by imaging Dylight488 lectin uptake. Methods: We performed MSOT and CryoVizTM imaging with subcutaneous tumor mouse models of SU.86.86 and MIA PaCa-2 pancreatic cancer. We performed MSOT with 21% O2 and 100% O2 carrier gas, which measures %sO2 (% oxyhemoglobin/total hemoglobin) as a biomarker of hypoxia. We performed DCE MSOT with 50 nmol indocyanine green (ICG), which measures Kapp (uptake rate from blood to tumor tissue) - a biomarker of vascular perfusion. To assess microvessel patency, we injected Dylight488 lectin via retro-orbital injection into the mice 3 min before euthanasia to enable CryoVizTM visualization of functional blood vessels. Subsequently, mice were euthanized, embedded in optimal cutting temperature (OCT) gel and flash-frozen in liquid nitrogen, and then imaged on the CryoVizTM. We acquired brightfield color anatomy, Dylight488 lectin (visible) fluorescence (ex: 460-490 nm, em>510nm) and ICG (NIR) fluorescence images (ex: 747/11 nm, em: >790nm) with an in-plane resolution of 10.232µm and a section thickness of 40 µm. Results: The in vivo MSOT analysis indicated that the MIA PaCa-2 model was more hypoxic than the SU.86.86 model, based on lower %sO2 measurements with both carrier gases. The in vivo DCE MSOT analysis indicated that the MIA PaCa-2 model had greater vascular perfusion than SU.86.86, based on a higher Kapp value. CryoVizTM images showed higher levels of lectin and ICG fluorescence in MIA PaCa-2 as compared to SU.86.86, indicating greater vascular patency and perfusion in the former. A strong lectin signal was observed in the injection site (retro-orbital sinuses), and in the renal medulla. ICG was also found to be retained in the axillary lymph node in both MIA PaCa-2 and SU.86.86, and in the kidney/adrenal glands of the MIA PaCa-2 mouse. Conclusion: Together, these results confirmed that the MIA PACa-2 model had higher hypoxia, vascular perfusion, and vascular patency, demonstrating the relationship between hypoxia and angiogenesis. This study reveals the power of 3D CryoVizTM imaging as a gold standard validation tool in cancer imaging. Citation Format: Madhusudhana Gargesha, Shreya Goel, Bryan Scott, Mark D. Pagel, Debashish Roy. CryoVizTM and photoacoustic imaging for assessment of tumor vasculature in mouse models of pancreatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB239.

Retro-orbital injection of FITC-dextran combined with isolectin B4 in assessing the retinal neovascularization defect

BackgroundA reliable and effective method is required to deliver agent that can aid the in vivo imaging of retinal vessels. The aim of the present study was to evaluate retro-orbital (RO) injection of fluorescein-labeled isothiocyanate dextran (FITC-dextran) as a method of demonstrating retinal neovascularization (NV) and avascular areas in oxygen-induced retinopathy (OIR) mice.MethodsDifferent concentrations of FITC-dextran were used to compare the efficacy of this agent in perfusing the retinal vessels. Hematoxylin–eosin (HE) staining was used to evaluate the safety of RO injection. The vitreous blood vessels and extent of NV were assessed in P17 OIR mice using FITC-dextran and compared with the corresponding measurements obtained following isolectin B4 staining or the combination of both methods.ResultsThe fluorescence of small vessels and neovascular tufts could be observed clearly following RO injection of 0.05 ml of 25 mg/ml or 50 mg/ml FITC-dextran. No visible damage to tissues adjacent to the injection site was discovered. Vitreous blood flow was gradually reduced from P0 to P5 and eventually disappeared in P17 OIR mice, as demonstrated by FITC-dextran perfusion. The retinal NV areas assessed by isolectin B4 were larger than those assessed by FITC-dextran, but the retinal avascular areas were smaller. The combination of both methods could conduce to distinguish non-functional blood vessels.ConclusionsRO injection of FITC-dextran combined with isolectin B4 is an effective, optimal method for assessing the NV area and avascular area.

Organic Cation Transporter 3 Mediates Tissue Uptake and Accumulation of meta-iodobenzylguanidine (mIBG)

Radioiodinated meta-iodobenzylguanidine (131I-mIBG) is utilized as a targeted radiotherapy for neuroendocrine cancers.131I-mIBG selectively accumulates in the cancer cells through uptake by the norepinephrine transporter. However, whole-body scintigraphy also shows extensive mIBG uptake and accumulation in several normal tissues (e.g. salivary glands, liver, heart) via mechanisms that are not fully understood. The accumulation of radioactive 131I-mIBG in these tissues is associated with a range of adverse reactions and tissue damages due to the unnecessary radiation exposure. We hypothesize that the efficacy and safety of 131I-mIBG can be improved by selectively inhibiting mIBG uptake in healthy tissues without affecting its uptake in tumor cells. The goal of this study is to elucidate the impact of organic cation transporter 3 (OCT3) on the in vivo pharmacokinetics andtissue distribution of mIBG using a mouse model with genetic deletion of Oct3. Additionally, we conducted in vitro studies in HEK293 cell lines stably transfected with mouse Oct3 (mOct3) and human OCT1-3 (hOCT1-3) to determine the uptake kinetics of mIBG and dose-dependent inhibition of OCT1-3-mediated uptake of mIBG. The in vitro kinetic analysis revealed similar apparent affinities in the uptake of mIBG by hOCT3 and mOct3 (Km of 14.5 ± 7.1 and 29.4 ± 3.4 µM, respectively), suggesting minimal species difference in mIBG transport by OCT3. Interestingly, hOCT3-mediated uptake of mIBG was inhibited by crizotinib (IC50 of 2.1 ± 0.3 µM) and crizotinib was 6.7-fold and 18-fold more potent towards hOCT3 than for hOCT2 and hOCT1, respectively. For the in vivo pharmacokinetic studies, full concentration-time profiles were determined to calculate mIBG exposure, defined as area under the concentration-time curve (AUC), in the plasma and each analyzed tissue. After retro-orbital injection (10 mg/kg), mIBG exhibited extensive accumulation in all tested tissues with exposures ranging from 4 to 90-fold higher than the plasma AUC in Oct3+/+ mice. In contrast, deletion of Oct3 significantly reduced mIBG exposure in the salivary glands by approximately 40%. The exposure of mIBG in the skeletal muscle, heart, and the lungs were also significantly reduced in Oct3-/- mice, demonstrating an important role of Oct3 in mediating uptake and accumulation of mIBG in various healthy tissues. On the other hand, the plasma concentration-time profiles of mIBG were similar between the Oct3+/+ and Oct3-/- mice with no significant changes in systemic AUC and other pharmacokinetic parameters. Additionally, no changes were observed between the two groups in the kidney, the main route of elimination for mIBG, suggesting that Oct3 does not contribute to the renal elimination of mIBG. In summary, we demonstrated that OCT3-mediated uptake of mIBG can be selectively inhibited and that blocking this pathway leads to decreased mIBG uptake and accumulation in normal tissues. Furthermore, these findings support inhibition of OCT3 as a viable clinical strategy to reduce 131I-mIBG accumulation and toxicity in healthy tissues in cancer patients undergoing radiotherapy.

Surgery Performed Under Propofol Anesthesia Induces Cognitive Impairment and Amyloid Pathology in ApoE4 Knock-In Mouse Model.

Background: Postoperative cognitive dysfunction (POCD) following anesthesia and surgery is a common and severe complication, especially in elderly patients. A pre-existing cognitive impairment may impart susceptibility to further cognitive dysfunction; the mechanism remains unclear. We hypothesized that the specific impacts of anesthesia and surgery on individuals with preclinical Alzheimer’s disease (AD) may render them more susceptible to an increase in the risk of cognitive impairment. The aim of this study was to compare the cognitive impairment between normal adult mice and those with preclinical AD after propofol anesthesia and surgery.Methods: We performed abdominal surgery in cognitively pre-symptomatic, 5-month-old male mice with sporadic AD (apolipoprotein E4 allele, ApoE4-KI) and age-matched (C57BL/6J) controls. Propofol anesthesia (170 mg/kg) was induced via retro-orbital injection over 2 h. Morris water maze (MWM) and Y-maze tests were conducted 2 days before and 2, 4, and 7 days after surgery. The mean escape latencies and spontaneous alternation percentages were the major outcomes. Neuronal apoptosis in hippocampal sections was evaluated using the terminal dUTP nick-end labeling (TUNEL) assay. Hippocampal amyloid beta (Aβ) levels were assessed via quantitative immunohistochemistry (IHC).Results: The control mice exhibited increased mean escape latencies of MWM at postoperative 2 and 4, but not at day 7; ApoE4-KI mice exhibited such increases at postoperative days 2, 4 and 7. Significant differences between ApoE4-KI and control mice in terms of the mean escape latencies were evident at days 2 and 7 (both P < 0.05). However, performance on a non-hippocampal memory tasks (Y-maze test) did not differ. More TUNEL-positive neurons were evident in the hippocampal CA3 region of ApoE4-KI mice at postoperative days 2 and 4, but not at day 7 compared to the control group (both P < 0.05). IHC revealed significantly elevated Aβ deposition in the hippocampal CA3 region of ApoE4-KI mice at postoperative days 4 and 7 compared to control mice (both P < 0.05).Conclusions: Propofol anesthesia followed by surgery induced persistent changes in cognition, and pathological hippocampal changes in pre-symptomatic, but vulnerable AD mice. It would be appropriate to explore whether preclinical AD patients are more vulnerable to POCD development.

Simple methodology to visualize whole-brain microvasculature in three dimensions.

.Significance: To explore brain architecture and pathology, a consistent and reliable methodology to visualize the three-dimensional cerebral microvasculature is beneficial. Perfusion-based vascular labeling is quick and easily deliverable. However, the quality of vascular labeling can vary with perfusion-based labels due to aggregate formation, leakage, rapid photobleaching, and incomplete perfusion.Aim: We describe a simple, two-day protocol combining perfusion-based labeling with a two-day clearing step that facilitates whole-brain, three-dimensional microvascular imaging and characterization.Approach: The combination of retro-orbital injection of Lectin-Dylight-649 to label the vasculature, the clearing process of a modified iDISCO+ protocol, and light-sheet imaging collectively enables a comprehensive view of the cerebrovasculature.Results: We observed increase in contrast-to-background ratio of Lectin-Dylight-649 vascular labeling over endogenous green fluorescent protein fluorescence from a transgenic mouse model. With light-sheet microscopy, we demonstrate sharp visualization of cerebral microvasculature throughout the intact mouse brain.Conclusions: Our tissue preparation protocol requires fairly routine processing steps and is compatible with multiple types of optical microscopy.

Biodistribution of Transplanted Hematopoietic Precursor Cells Injected Through Different Administration Routes in Newborn Mice.

Hematopoietic stem cell transplantation has been studied for several decades now, mostly as a treatment for malignancies and hematological diseases but also for genetic metabolic disorders. Since many diseases that could be potentially treated with this approach develop early in life, studies of cell transplantation in newborn mice are needed, especially for gene therapy protocols. However, the small size of pups restricts the possibilities for routes of administration, and those available are normally technically challenging. Our goal was to test different routes of administration of Lin- cells in 2-day-old mice: intraperitoneal, intravenous through temporal vein (TV), and intravenous through retro-orbital (RO) sinus. Routes were evaluated by their easiness of execution and their influence in the biodistribution of cells in the short (48 h) and medium (30 days) term. In either 48 h or 30 days, all three routes presented similar results, with cells going mostly to bone marrow, liver, and spleen in roughly the same number. RO injection resulted in quick distribution of cells to the brain, suggesting better performance than the others. Rate of failure was higher for the TV route, which was also the hardest to execute, whereas the other two were considered easier. In conclusion, TV was the hardest to perform and all routes seemed to demonstrate similar results for cell biodistribution. In particular, the RO injection results in quicker biodistribution of cells to the brain, which is particularly important in the study of genetic metabolic disorders with a neurological component.

In vivo base editing rescues Hutchinson-Gilford progeria syndrome in mice.

SummaryHutchinson-Gilford progeria syndrome (HGPS) is typically caused by a dominant-negative C•G-to-T•A mutation (c.1824 C>T, G608G) in LMNA, the nuclear lamin A gene. This mutation causes RNA mis-splicing that produces progerin, a toxic protein that induces rapid aging and shortens lifespan to ~14 years1–4. Adenine base editors (ABEs) perform targeted A•T-to-G•C base pair conversion with minimal byproducts and without requiring double-strand DNA breaks or donor DNA templates5,6. Here, we describe the use of an ABE to directly correct the pathogenic HGPS mutation in cultured progeria patient-derived fibroblasts and in a mouse model of HGPS. Lentiviral delivery of ABE to patient-derived fibroblasts results in ~90% correction of the pathogenic allele, mitigation of RNA mis-splicing, reduced progerin levels, and correction of nuclear abnormalities. Unbiased off-target DNA and RNA analysis did not detect off-target editing activity in treated patient-derived fibroblasts. In transgenic mice homozygous for the human LMNA c.1824 C>T allele, a single retro-orbital injection of adeno-associated virus 9 (AAV9) encoding the ABE resulted in substantial, durable correction of the pathogenic mutation (~20-60% across various organs 6 months post-injection), restoration of normal RNA splicing, and reduction of progerin protein. In vivo base editing rescued vascular pathology, preserving vascular smooth muscle cell counts and preventing adventitial fibrosis. A single ABE AAV9 injection at P14 improved animal vitality and greatly extended median lifespan from 215 to 510 days. These findings support the potential of in vivo base editing to treat HGPS, and other genetic diseases, by directly correcting the root cause of disease.

Exosome-mediated delivery of an anti-angiogenic peptide inhibits pathological retinal angiogenesis.

Background: Pathological angiogenesis is the hallmark of many vision-threatening diseases. Anti-VEGF is a primary treatment with substantial beneficial effects. However, such agents require frequent intravitreal injections. Our previous work established a method for effectively modifying exosomes (EXOs) for loading therapeutic peptides. Here, we used this system to load the anti-angiogenic peptide KV11, aiming to establish an EXO-based therapy strategy to suppress neovascularization in the retina.Methods: Using an anchoring peptide, CP05, we linked KV11 to endothelial cell (EC) derived EXOs, yielding EXOKV11. We tested the delivery efficiency of EXOKV11 via two commonly used ocular injection methods: retro-orbital injection and intravitreal injection. Deploying an oxygen-induced retinopathy (OIR) model and a VEGF injection model, we tested the effects of EXOKV11 on neovascular formation, EC proliferation, and vascular permeability. In vitro experiments were used to test the mechanism and to analyze the effects of EXOKV11 on EC proliferation, migration, and sprouting.Results: By using the EXO loading system, KV11 was more efficiently delivered to the blood vessels of the mouse retina via retro-orbital injection. In both OIR model and VEGF injection model, EXOKV11 was more effective than KV11 alone in inhibiting neovascularization and vessel leakage. The therapeutic effect of retro-orbital injection of EXOKV11 was comparable to the intravitreal injection of VEGF-trap. Mechanistically, KV11 alone inhibited VEGF-downstream signaling, while EXOKV11 showed a stronger effect.Conclusions: We used EXOs as a carrier for intraocular delivery of KV11. We showed that KV11 itself has an anti-angiogenic effect through retro-orbital injection, but that this effect was greatly enhanced when delivered with EXOs. Thus, this system has the potential to treat proliferative retinopathy via retro-orbital injection which is a less invasive manner compared with intravitreal injection.

AAV9 transduction mediated by systemic delivery of vector via retro-orbital injection in newborn, neonatal and juvenile mice.

Adeno-associated virus (AAV)-based gene therapy is gaining popularity owing to its excellent safety profile and effective therapeutic outcomes in a number of diseases. Intravenous (IV) injection of AAV into the tail vein, facial vein and retro-orbital (RO) venous sinus have all been useful strategies to infuse the viral vector systemically. However, tail vein injection is technically challenging in juvenile mice, and injection at young ages (≤ postnatal day-(P)21) is essentially impossible. The temporal or facial vein is localized anterior to the ear bud and is markedly visible in the first couple of days postnatally. However, this method is age-dependent and requires a dissecting microscope. Retro-orbital injection (ROI), on the other hand, is suitable for all murine ages, including newborn and older mice, and is relatively less stressful to animals compared to tail vein injection. Although many reports have shown ROI as an effective route of AAV delivery, herein we aim to highlight and summarize the methods and benefits of ROI. To capture the full spectrum of transduction efficiency mediated by ROI, we transduced the editing-dependent reporter mice (Ai9 Cre reporter mice) with the AAV9 vector, which targets a wide range of peripheral tissues with exceptional brain tropism. We also provide a comprehensive description of the ROI technique to facilitate viral vector administration without complications.