Dual in vivo PET ex vivo FACS cell tracking of neutrophils – first results in a mouse model of Alzheimer disease

AbstractBackgroundPressurised intraperitoneal aerosol chemotherapy (PIPAC) is a novel technique of intraperitoneal chemotherapy devoted to unresectable peritoneal metastasis (PM). The first results obtained with PIPAC in preclinical models of colon cancer are presented here.MethodsIn vitro, PIPAC (normotherm oxaliplatin at 0.028 mg/mL for 10 min at 1.6 bars) and HIPEC (hyperthermic oxaliplatin at 0.14 mg/mL for 30 min) were compared using the apoptosis and proliferation assay on two colon cancer cell lines (LS 174 and CT 26); ex vivo tumours from an orthotopic mouse model of PM and non-tumour peritoneum from a patient treated according to the two modalities were assessed, investigating the percentage of penetration of oxaliplatin in the tumour and oxaliplatin concentration below the peritoneum. In vivo, a mouse model of colon (CT 26) PM was used to create a PIPAC model (same modalities) for the comparison of IV oxaliplatin (at 5 mg/mL).ResultsIn vitro, the rate of apoptotic and proliferative cells as well as the level of oxaliplatin penetration in tumour nodes was higher in PIPAC groups with less systemic passage through the peritoneum. In vivo, in the colon PM mouse model, the peritoneal cancer index (PCI) was decreased to the same level using PIPAC or IV oxaliplatin. Systemic passage was lower in the PIPAC group.ConclusionsPIPAC with low-dose oxaliplatin is efficient in both in vitro and in vivo models of colon PM. Lower concentrations of chemotherapy are needed in PIPAC to achieve the same effect as IV chemotherapy on PCI. With a very low systemic oxaliplatin passage, this technique of drug delivery seems to be as effective as IV delivery for PM control.

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A novel mouse model based on intersectional genetics enables unambiguous in vivo discrimination between plasmacytoid and other dendritic cells and their comparative characterization

10.1101/2022.01.07.475382 ◽

2022 ◽

Author(s):

Michael Valente ◽

Nils Collinet ◽

Thien-Phong Vu Manh ◽

Karima Naciri ◽

Gilles Bessou ◽

...

Keyword(s):

Dendritic Cells ◽

Steady State ◽

Mouse Model ◽

Single Cell ◽

Ex Vivo ◽

High Specificity ◽

Type I ◽

Specific Expression ◽

Physiological Functions

Plasmacytoid dendritic cells (pDC) were identified about 20 years ago, based on their unique ability to rapidly produce copious amounts of all subsets of type I and type III interferon (IFN-I/III) upon virus sensing, while being refractory to infection. Yet, the identity and physiological functions of pDC are still a matter of debate, in a large part due to their lack of specific expression of any single cell surface marker or gene that would allow to track them in tissues and to target them in vivo with high specificity and penetrance. Indeed, recent studies showed that previous methods that were used to identify or deplete pDC also targeted other cell types, including pDC-like cells and transitional DC (tDC) that were proposed to be responsible for all the antigen presentation ability previously attributed to steady state pDC. Hence, improving our understanding of the nature and in vivo choreography of pDC physiological functions requires the development of novel tools to unambiguously identify and track these cells, including in comparison to pDC-like cells and tDC. Here, we report successful generation of a pDC-reporter mouse model, by using an intersectional genetic strategy based on the unique co-expression of Siglech and Pacsin1 in pDC. This pDC-Tomato mouse strain allows specific ex vivo and in situ detection of pDC. Breeding them with Zbtb46GFP mice allowed side-by-side purification and transcriptional profiling by single cell RNA sequencing of bona fide pDC, pDC-like cells and tDC, in comparison to type 1 and 2 conventional DC (cDC1 and cDC2), both at steady state and during a viral infection, revealing diverging activation patterns of pDC-like cells and tDC. Finally, by breeding pDC-Tomato mice with Ifnb1EYFP mice, we determined the choreography of pDC recruitment to the micro-anatomical sites of viral replication in the spleen, with initially similar but later divergent behaviors of the pDC that engaged or not into IFN-I production. Our novel pDC-Tomato mouse model, and newly identified gene modules specific to combinations of DC types and activations states, will constitute valuable resources for a deeper understanding of the functional division of labor between DC types and its molecular regulation at homeostasis and during viral infections.

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Abstract 1767: Feasibility of In Vivo Cardiac Stem Cell Tracking, by SPECT and PET Imaging, Using the Sodium-iodide Symporter as Reporter Gene

Circulation ◽

10.1161/circ.116.suppl_16.ii_374 ◽

2007 ◽

Vol 116 (suppl_16) ◽

Author(s):

John Terrovitis ◽

Keng Fai Kwok ◽

Riikka Läutamaki ◽

James M Engles ◽

Andreas S Barth ◽

...

Keyword(s):

Stem Cell ◽

Reporter Gene ◽

Cell Tracking ◽

Ex Vivo ◽

Small Animal ◽

Cell Labeling ◽

Sodium Iodide Symporter ◽

Cell Injection

Background. Stem cells offer the promise of cardiac repair. Stem cell labeling is a prerequisite to tracking cell fate in vivo . Aim. To develop a reporter gene that permits in vivo stem cell labeling. We examined the sodium-iodide symporter (NIS), a protein that is not expressed in the heart, but promotes cellular uptake of 99m Tc or 124 I, thus permitting cell tracking by SPECT or PET imaging, respectively. Methods. The human NIS gene ( h NIS) was expressed in rat cardiac derived stem cells (rCDCs) using lentivirus driven by the CAG or CMV promoter. NIS function in transduced cells was confirmed by in vitro 99m Tc uptake. Eleven rats were injected with 1 or 2 million rCDCs intramyocardially immediately after LAD ligation; 6 with CMV-NIS and 5 with CAG-NIS cells. Dual isotope SPECT imaging was performed on a small animal SPECT/CT system, using 99m Tc for cell detection and 201 Tl for myocardial delineation, 24 hrs after cell injection. PET was performed on a small animal PET scanner using 124 I for cell tracking and 13 NH 3 for myocardial delineation, 48hrs after cell injection. Contrast Ratio (CR) was defined as [(signal in the cells)-(signal in blood pool)]/signal in blood pool. High resolution ex vivo SPECT scans of explanted hearts (n=3) were obtained to confirm that in vivo signal was derived from the cell injection site. The presence of h NIS mRNA was confirmed in injected hearts after animal sacrifice (n=2), by real-time RT-PCR. Results. NIS expression in rCDCs did not affect cell viability/proliferation (p=0.718, ctr vs NIS). In vitro 99m Tc uptake was 6.0±0.9% vs 0.07±0.05, without and with perchlorate (specific NIS blocker), respectively. NIS-transduced rCDCs were easily visualized as spots of 99m Tc or 124 I uptake within a perfusion deficit in the SPECT and PET images. CR was considerably higher when cells were transduced by the CMV-NIS virus in comparison to the CAG-NIS virus (70±40% vs 28±29%, p=0.085). Ex vivo small animal SPECT imaging confirmed that in vivo 99m Tc signals were localized to the injection sites. PCR confirmed the presence of h NIS mRNA in injected hearts. Conclusion. NIS expression allows non invasive in vivo stem cell tracking in the myocardium, using both SPECT and PET. This reporter gene has great potential for translation in future clinical applications.

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Development and Optimization of a Fluorescent Imaging System to Detect Amyloid-β Proteins: Phantom Study

Biomedical Engineering and Computational Biology ◽

10.1177/1179597218781081 ◽

2018 ◽

Vol 9 ◽

pp. 117959721878108 ◽

Cited By ~ 2

Author(s):

David Tes ◽

Karl Kratkiewicz ◽

Ahmed Aber ◽

Luke Horton ◽

Mohsin Zafar ◽

...

Keyword(s):

Alzheimer Disease ◽

Imaging System ◽

Ex Vivo ◽

Amyloid Β ◽

The United States ◽

Fluorescent Imaging ◽

Fluorescent Properties ◽

In Vivo Experiments ◽

The Brain

Alzheimer disease is the most common form of dementia, affecting more than 5 million people in the United States. During the progression of Alzheimer disease, a particular protein begins to accumulate in the brain and also in extensions of the brain, ie, the retina. This protein, amyloid-β (Aβ), exhibits fluorescent properties. The purpose of this research article is to explore the implications of designing a fluorescent imaging system able to detect Aβ proteins in the retina. We designed and implemented a fluorescent imaging system with a range of applications that can be reconfigured on a fluorophore to fluorophore basis and tested its feasibility and capabilities using Cy5 and CRANAD-2 imaging probes. The results indicate a promising potential for the imaging system to be used to study the Aβ biomarker. A performance evaluation involving ex vivo and in vivo experiments is planned for future study.

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Video-rate multimodal multiphoton imaging and three-dimensional characterization of cellular dynamics in wounded skin

Journal of Innovative Optical Health Sciences ◽

10.1142/s1793545820500078 ◽

2020 ◽

Vol 13 (02) ◽

pp. 2050007

Author(s):

Joanne Li ◽

Madison N. Wilson ◽

Andrew J. Bower ◽

Marina Marjanovic ◽

Eric J. Chaney ◽

...

Keyword(s):

Cell Tracking ◽

Skin Diseases ◽

Ex Vivo ◽

Three Dimensional ◽

Biological Information ◽

Cellular Dynamics ◽

Multiphoton Imaging ◽

Cellular Events ◽

Analysis Platform

To date, numerous studies have been performed to elucidate the complex cellular dynamics in skin diseases, but few have attempted to characterize these cellular events under conditions similar to the native environment. To address this challenge, a three-dimensional (3D) multimodal analysis platform was developed for characterizing in vivo cellular dynamics in skin, which was then utilized to process in vivo wound healing data to demonstrate its applicability. Special attention is focused on in vivo biological parameters that are difficult to study with ex vivo analysis, including 3D cell tracking and techniques to connect biological information obtained from different imaging modalities. These results here open new possibilities for evaluating 3D cellular dynamics in vivo, and can potentially provide new tools for characterizing the skin microenvironment and pathologies in the future.

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P1-488: In vivo monitoring of mitochondrial dysfunction in an Alzheimer disease mouse model

Alzheimer s & Dementia ◽

10.1016/j.jalz.2008.05.1071 ◽

2008 ◽

Vol 4 ◽

pp. T362-T363

Author(s):

Hong Xie ◽

Laura A. Borrelli ◽

Brian J. Bacskai

Keyword(s):

Alzheimer Disease ◽

Mouse Model ◽

Mitochondrial Dysfunction ◽

In Vivo Monitoring

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Comparison of In Vivo and Ex Vivo MRI for the Detection of Structural Abnormalities in a Mouse Model of Tauopathy

Frontiers in Neuroinformatics ◽

10.3389/fninf.2017.00020 ◽

2017 ◽

Vol 11 ◽

Cited By ~ 13

Author(s):

Holly E. Holmes ◽

Nick M. Powell ◽

Da Ma ◽

Ozama Ismail ◽

Ian F. Harrison ◽

...

Keyword(s):

Mouse Model ◽

Ex Vivo ◽

Structural Abnormalities

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Predilection for developing a hematogenous orthopaedic implant-associated infection in older versus younger mice

Journal of Orthopaedic Surgery and Research ◽

10.1186/s13018-021-02594-0 ◽

2021 ◽

Vol 16 (1) ◽

Author(s):

John M. Thompson ◽

Alyssa G. Ashbaugh ◽

Yu Wang ◽

Robert J. Miller ◽

Julie E. Pickett ◽

...

Keyword(s):

At Risk ◽

Staphylococcus Aureus ◽

Mouse Model ◽

Ex Vivo ◽

Kirschner Wire ◽

Age Groups ◽

Orthopaedic Implant ◽

X Ray ◽

Risk Patients

Abstract Background The pathogenesis of hematogenous orthopaedic implant-associated infections (HOIAI) remains largely unknown, with little understanding of the influence of the physis on bacterial seeding. Since the growth velocity in the physis of long bones decreases during aging, we sought to evaluate the role of the physis on influencing the development of Staphylococcus aureus HOIAI in a mouse model comparing younger versus older mice. Methods In a mouse model of HOIAI, a sterile Kirschner wire was inserted retrograde into the distal femur of younger (5–8-week-old) and older (14–21-week-old) mice. After a 3-week convalescent period, a bioluminescent Staphylococcus aureus strain was inoculated intravenously. Bacterial dissemination to operative and non-operative legs was monitored longitudinally in vivo for 4 weeks, followed by ex vivo bacterial enumeration and X-ray analysis. Results In vivo bioluminescence imaging and ex vivo CFU enumeration of the bone/joint tissue demonstrated that older mice had a strong predilection for developing a hematogenous infection in the operative legs but not the non-operative legs. In contrast, this predilection was less apparent in younger mice as the infection occurred at a similar rate in both the operative and non-operative legs. X-ray imaging revealed that the operative legs of younger mice had decreased femoral length, likely due to the surgical and/or infectious insult to the more active physis, which was not observed in older mice. Both age groups demonstrated substantial reactive bone changes in the operative leg due to infection. Conclusions The presence of an implant was an important determinant for developing a hematogenous orthopaedic infection in older but not younger mice, whereas younger mice had a similar predilection for developing periarticular infection whether or not an implant was present. On a clinical scale, diagnosing HOIAI may be difficult particularly in at-risk patients with limited examination or other data points. Understanding the influence of age on developing HOIAI may guide clinical surveillance and decision-making in at-risk patients.

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Mitochondrial augmentation of CD34+ cells from healthy donors and patients with mitochondrial DNA disorders confers functional benefit

npj Regenerative Medicine ◽

10.1038/s41536-021-00167-7 ◽

2021 ◽

Vol 6 (1) ◽

Author(s):

Elad Jacoby ◽

Moriya Ben Yakir-Blumkin ◽

Shiri Blumenfeld-Kan ◽

Yehuda Brody ◽

Amilia Meir ◽

...

Keyword(s):

Mitochondrial Dna ◽

Mouse Model ◽

Ex Vivo ◽

Hematopoietic Stem ◽

Functional Benefit ◽

Syngeneic Mouse Model ◽

Multisystemic Disease ◽

Stem And Progenitor Cells ◽

Disease Modifying

AbstractMitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.

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Ultrasonography validation for early alteration of diaphragm echodensity and function in the mdx mouse model of Duchenne muscular dystrophy

PLoS ONE ◽

10.1371/journal.pone.0245397 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0245397

Author(s):

Antonietta Mele ◽

Paola Mantuano ◽

Adriano Fonzino ◽

Francesco Rana ◽

Roberta Francesca Capogrosso ◽

...

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Mouse Model ◽

Ex Vivo ◽

Eccentric Contraction ◽

Invasive Technique ◽

Mdx Mouse ◽

Early Stages ◽

Over Time

The mdx mouse model of Duchenne muscular dystrophy is characterized by functional and structural alterations of the diaphragm since early stages of pathology, closely resembling patients’ condition. In recent years, ultrasonography has been proposed as a useful longitudinal non-invasive technique to assess mdx diaphragm dysfunction and evaluate drug efficacy over time. To date, only a few preclinical studies have been conducted. Therefore, an independent validation of this method by different laboratories is needed to increase results reliability and reduce biases. Here, we performed diaphragm ultrasonography in 3- and 6-month-old mdx mice, the preferred age-window for pharmacology studies. The alteration of diaphragm function over time was measured as diaphragm ultrasound movement amplitude. At the same time points, a first-time assessment of diaphragm echodensity was performed, as an experimental index of progressive loss of contractile tissue. A parallel evaluation of other in vivo and ex vivo dystrophy-relevant readouts was carried out. Both 3- and 6-month-old mdx mice showed a significant decrease in diaphragm amplitude compared to wild type (wt) mice. This index was well-correlated either with in vivo running performance or ex vivo isometric tetanic force of isolated diaphragm. In addition, diaphragms from 6-month-old dystrophic mice were also highly susceptible to eccentric contraction ex vivo. Importantly, we disclosed an age-dependent increase in echodensity in mdx mice not observed in wt animals, which was independent from abdominal wall thickness. This was accompanied by a notable increase of pro-fibrotic TGF-β1 levels in the mdx diaphragm and of non-muscle tissue amount in diaphragm sections stained by hematoxylin & eosin. Our findings corroborate the usefulness of diaphragm ultrasonography in preclinical drug studies as a powerful tool to monitor mdx pathology progression since early stages.

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