Name: longitudinal in vivo imaging and quantification of human pancreatic islet grafting and contributing host cells in the anterior eye chamber
Uploaded: 2020-06-11T14:30:00
Description: Watch this Scientific Journal Video about Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber at JoVE.com

This study was supported by the Swedish Research Council, Strategic Research Area Exodiab, Dnr 2009-1039, the Swedish Foundation for Strategic Research Dnr IRC15-0067 to LUDC-IRC, the Royal Physiographic Society in Lund, Diabetesf&#246;rbundet and Barndiabetesf&#246;rbundet.

The Regional Ethics Committee in Lund, Sweden, approved the study according to the Act Concerning the Ethical Review of Research Involving Humans. Animal experiments were performed in strict accordance with the Swedish ethics of animal experiments and approved by the ethics committees of Malm&#246; and Lund. 6 to 8-week-old immunodeficient NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/- (NOD.ROSA-tomato.Rag2-/-) recipient mice were used as recipients for tra...

<ol>
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A method is presented to study the human pancreatic islet cell grafting process by observing the involvement of recipient and donor tissue. After a minimal invasive surgery implanting human islets into the anterior chamber of an immunodeficient mouse eye, the mouse recovers quickly within minutes after surgery. The procedure is performed on one eye. Generally, from 5&#8211;7 days postimplantation onwards the cornea is sufficiently healed to perform intravital imaging.
In this protocol, the qua...

Diabetes mellitus describes a group of metabolic diseases characterized by elevated levels of blood glucose as results of insufficient insulin production from loss or dysfunction of pancreatic islet beta cells, often accompanied by insulin resistance. Type 1 (T1D) and type 2 diabetes (T2D) are complex diseases in which the progressive dysfunction of the beta cells causes disease development. T1D is precipitated by an autoimmune attack on the beta cells, while T2D is considered to be driven by metabolic factors, albeit with increasing evidence of low-grade systemic inflammation1. Transplantation of donor human islets, particularly to T1D patients, offers the potential for providing physiological glycemic control. However, a shortage of tissue donors and poor islet engraftment has prevented islet transplantation to become a mainstream therapeutic option. A substantial proportion of the functional islet graft is lost in the immediate posttransplantation period (24&#8211;48 h) due to the hypoxic, in&#64258;ammatory, immunogenic host environment2,3. To evaluate the efficiency of intervention methods for the improvement of islet survival, continuous monitoring of such transplantations is necessary.
In vivo techniques to image and track the fate of transplanted human pancreatic islets after transplantation still remains a challenge for diabetes research4,5. To date, noninvasive imaging techniques, including positron emission tomography (PET), magnetic resonance imaging (MRI), or ultrasound (US) show potential for the quantification and functional evaluation of transplanted islets in experimental conditions5. However, given the small islet sizes, quantitative measurements by those modalities suffer from insufficient resolution. The anterior chamber of the eye (ACE) as a transplantation site for observation is a promising noninvasive imaging solution offering effectively higher spatial resolution and frequent monitoring over long time periods6. This method has been successfully exploited to study mouse islet biology (reviewed in Yang et al.7), autoimmune immune responses8, as well as human islet grafting9,10.
Here the ACE transplantation method is combined with a 2-photon imaging approach to investigate the dynamics of the human pancreatic islet engraftment process by continuous and repeated recordings on individual islet grafts for up to 10 months after transplantation. The multiphoton imaging properties of greater imaging depths and reduced overall photobleaching and photo damage overcome the imaging limitations of confocal microscopy11. Quantification of fluorescent imaging involves several stages, including islet sample preparation, islet transplantation, image acquisition, image filtering to remove islet noise or background, segmentation, quantification, and data analysis. The most challenging step is usually partitioning or segmenting an image into multiple parts or regions. This could involve separating signal from background noise, or clustering regions of voxels based on similarities in color or shape to detect and label voxels of a 3D volume that represents islet vasculature, for example. Once segmented, statistics such as object volume sizes are typically straightforward to extract. Provided is a method for the quantification and extraction of the imaging data, such as segmentation and data visualization. Particular attention is paid to the removal of autofluorescence in human islets and distinction between islet vasculature and islet capsule forming recipient cells.

<table>
	<tbody>
		<tr>
			<td>Anasthesia machine, e.g. Anaesthesia Unit U-400</td>
			<td>Agnthos</td>
			<td>8323001</td>
			<td>used for isofluran anasthesia during surgery and imaging</td>
		</tr>
		<tr>
			<td>-induction chamber 1.4 L</td>
			<td>Agnthos</td>
			<td>8329002</td>
			<td>connect via tubing to U-400</td>
		</tr>
		<tr>
			<td>-gas routing switch</td>
			<td>Agnthos</td>
			<td>8433005</td>
			<td>connect via tubing to U-400</td>
		</tr>
		<tr>
			<td>AngioSense 680 EX</td>
			<td>Percin Elmer</td>
			<td>NEV10054EX</td>
			<td>imaging agent for injection, used to image blood vessels in human islet grafts</td>
		</tr>
		<tr>
			<td>Aspirator tubes assemblies</td>
			<td>Sigma</td>
			<td>A5177-5EA</td>
			<td>connect with pulled capillary pipettes for manual islet picking</td>
		</tr>
		<tr>
			<td>Buprenorphine (Temgesic) 0.3mg/ml</td>
			<td>Schering-Plough Europ&#233;</td>
			<td>64022</td>
			<td>fluid, for pain relief</td>
		</tr>
		<tr>
			<td>Capillary pipettes</td>
			<td>VWR</td>
			<td>321242C</td>
			<td>used together with Aspirator tubes assemblies</td>
		</tr>
		<tr>
			<td>Dextran-Texas Red (TR), 70kDa</td>
			<td>Invitrogen</td>
			<td>D1830</td>
			<td>imaging agent for injection</td>
		</tr>
		<tr>
			<td>Eye cannula, blunt end , 25 G</td>
			<td>BVI Visitec/BD</td>
			<td>BD585107</td>
			<td>custom made from Tapered Hydrode lineator [Blumenthal], dimensions: 0.5 x 22mm (25G x 7/8in) (45&#8304;), tip tapered to 30 G (0.3mm)</td>
		</tr>
		<tr>
			<td>Eye gel</td>
			<td>Novartis</td>
			<td></td>
			<td>Viscotears, contains Carbomer 2 mg/g</td>
		</tr>
		<tr>
			<td>Hamilton syringe 0.5 ml, Model 1750 TPLT</td>
			<td>Hamilton</td>
			<td>81242</td>
			<td>Plunger type gas-tight syringe for islet injection</td>
		</tr>
		<tr>
			<td>Head holder</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Head holding adapter</td>
			<td>Narishige</td>
			<td>SG-4N-S</td>
			<td>assemled onto metal plate</td>
		</tr>
		<tr>
			<td>-gas mask</td>
			<td>Narishige</td>
			<td>GM-4-S</td>
			<td></td>
		</tr>
		<tr>
			<td>-UST-2 Solid Universal Joint</td>
			<td>Narishige</td>
			<td>UST-2</td>
			<td>assemled onto metal plate</td>
		</tr>
		<tr>
			<td>-custom made metal plate for head-holder assembly</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Dumont #5, straight</td>
			<td>Agnthos</td>
			<td>0207-5TI-PS or 0208-5-PS</td>
			<td>attached to UST-2 (custom made)</td>
		</tr>
		<tr>
			<td>Heating pad, custom made</td>
			<td></td>
			<td></td>
			<td>taped to the stereotaxic platform</td>
		</tr>
		<tr>
			<td>Human islet culture media</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-CMRL 1066</td>
			<td>ICN Biomedicals</td>
			<td></td>
			<td>cell culture media for human islets</td>
		</tr>
		<tr>
			<td>-HEPES</td>
			<td>GIBCO BRL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-L-glutamin</td>
			<td>GIBCO BRL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Gentamycin</td>
			<td>GIBCO BRL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Fungizone</td>
			<td>GIBCO BRL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Ciproxfloxacin</td>
			<td>Bayer healthcare AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Nicotinamide</td>
			<td>Sigma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Image analysis software</td>
			<td>Bitplane</td>
			<td></td>
			<td>Imaris 9</td>
		</tr>
		<tr>
			<td>Image Aquisition software</td>
			<td>Zeiss</td>
			<td></td>
			<td>ZEN 2010</td>
		</tr>
		<tr>
			<td>Infrared lamp</td>
			<td>VWR</td>
			<td>1010364937</td>
			<td>used to keep animals warm in the wake-up cage</td>
		</tr>
		<tr>
			<td>Isoflurane Isoflo</td>
			<td>Abott Scandinavia/Apotek</td>
			<td></td>
			<td>fluid, for anesthesia</td>
		</tr>
		<tr>
			<td>Needle 25 G (0.5 x 16mm), orange</td>
			<td>BD</td>
			<td>10442204</td>
			<td>used as scalpel</td>
		</tr>
		<tr>
			<td>Petri dishes, 90mm</td>
			<td>VWR</td>
			<td>391-0440</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Photon/confocal microscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-LSM7 MP upright microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Ti:Sapphire laser Tsunami</td>
			<td>Spectra-Physics, Mai Tai</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-long distance water-dipping lens 20x/NA1.0</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-ET710/40m (Angiosense 680)</td>
			<td>Chroma</td>
			<td>288003</td>
			<td></td>
		</tr>
		<tr>
			<td>-ET645/65m-2p (TR)</td>
			<td>Chroma</td>
			<td>NC528423</td>
			<td></td>
		</tr>
		<tr>
			<td>-ET525/50 (GFP)</td>
			<td>Chroma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-ET610/75 (tomato)</td>
			<td>Chroma</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-main beam splitter T680lpxxr</td>
			<td>Chroma</td>
			<td>T680lpxxr</td>
			<td>Dichroic mirror to transmit 690 nm and above and reflect 440 to 650 nm size 25.5 x 36 x 1 mm</td>
		</tr>
		<tr>
			<td>Polythene tubing (0.38mm ID, 1.09 mm OD)</td>
			<td>Smiths Medical Danmark</td>
			<td>800/100/120</td>
			<td>to connect with Hamilton syringe and eye canula</td>
		</tr>
		<tr>
			<td>Stereomicroscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Model SMZ645, for islet picking</td>
		</tr>
		<tr>
			<td>Stereomicroscope (Flourescence)</td>
			<td></td>
			<td></td>
			<td>for islet graft imaging</td>
		</tr>
		<tr>
			<td>-AZ100 Multizoom</td>
			<td>Nikon</td>
			<td></td>
			<td>wide field and long distance</td>
		</tr>
		<tr>
			<td>-AZ Plan Apo 1x</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-AZ Plan Apo 4x</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-AZ-FL Epiflourescence with C-LHGFI HG lamp</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-HG Manual New Intensilight</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-Epi-FL Filter Block TEXAS RED</td>
			<td>Nikon</td>
			<td></td>
			<td>contains EX540-580, DM595 and BA600-660</td>
		</tr>
		<tr>
			<td>-Epi-FL Filter Block G-2A</td>
			<td>Nikon</td>
			<td></td>
			<td>(EX510-560, DM575 and BA590)</td>
		</tr>
		<tr>
			<td>-Epi-FL Filter Block B-2A</td>
			<td>Nikon</td>
			<td></td>
			<td>(EX450-490, DM505 and BA520)</td>
		</tr>
		<tr>
			<td>-DS-Fi1 Colour Digital Camera (5MP)</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1-ml, Omnitix</td>
			<td>Braun</td>
			<td>9161406V</td>
			<td>for Buprenorphine injection, used with 27 G needle</td>
		</tr>
		<tr>
			<td>Surgical tape</td>
			<td>3M</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

longitudinal in vivo imaging and quantification of human pancreatic islet grafting and contributing host cells in the anterior eye chamber

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology have been developed and utilized in vivo, they are limited in terms of allowing single cell resolution and continuous longitudinal recordings. Because of the transparency of the cornea, the anterior chamber of the eye (ACE) in mice is well suited to study human and mouse pancreatic islet cell biology. Here is a description of how this approach can be used to perform continuous longitudinal recordings of grafting and revascularization of individual human islet grafts. Human islet grafts are inserted into the ACE, using NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mice as recipients. This allows for the investigation of the expansion of recipient versus donor cells and the contribution of recipient cells in promoting the encapsulation and vascularization of the graft. Further, a step-by-step approach for image analysis and quantification of the islet volume or segmented vasculature and islet capsule forming recipient cells is outlined.

Non-labeled human islets were transplanted into the ACE of 8-week-old female NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-(NOD.ROSA-tomato.Rag2&#8722;/&#8722;) recipient mice. To prevent human tissue rejection, immunodeficient Rag2 knockout mice were chosen as recipients. In these transgenic mice, all cells and tissues expressed a membrane-targeted tomato fluorescence protein (mT) that allows clear identification of the recipient and the donor tissue. Repe...

Watch this Scientific Journal Video about Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber at JoVE.com

Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber

The goal of this protocol is to continuously monitor the dynamics of the human pancreatic islet engraftment process and the contributing host versus donor cells. This is accomplished by transplanting human islets into the anterior chamber of the eye (ACE) of an NOD.(Cg)-Gt(ROSA)26Sortm4-Rag2-/-mouse recipient followed by repeated 2-photon imaging.

Imaging beta cells is a key step towards understanding islet transplantation. Although different imaging platforms for the recording of beta cell biology ...

The ACE is an optimal imaging sights. It supports to repeat it in long-term in vivo imaging of islet cells in order to study their function and viability in a grafting situation. It can also be extended to study other aspects under physiological and pathological conditions in real time.The advantage of the ACE technique lies in the fact that the same individual human pancreatic islets can be followed noninvasively with sub-cellular resolution for an extended period of time. Success rates of beta cell replacement therapy continue to improve. However, evaluating the efficiency of islet grafting and survival in vivo, remains still challenging.This method could help assess and optimize the outcomes of islet transplantations. This approach is not restricted to pancreatic islets only. It is also well-suited to study tissues affected.For example, in diabetic complications, like kidney glomeruli or liver pieces, just to name a few. To begin place the mouse on a warm heating pad position it in the head holder and put on the nose mask. Use the thumb and index finger to lift the head slightly.Fasten it with the metallic pieces on the sides. Making sure that the ear pieces fix the head directly below the ears. Subcutaneously inject buprenorphine solution into the back of the mouse.Gently retract the eyelids of the eye to be transplanted using blunt forceps. Pop the eye out and loosely fix it with a pair of tweezers covered with a polythene tube. After transferring the eyelids to a Petri dish with PBS, pick up approximately 20 to 30 islets in the eye cannular.Using a 25 gauge needle level upwards, carefully insert the tip into the cornea and make a single lateral incision. Carefully lift the cornea with the cannula preloaded with islets, and slowly release the islets into the eye. Slowly retract the cannula and apply eye gel to the eye.After 10 minutes, remove the forceps, holding the eyelid and put the eye back to its normal position. For imaging of implanted human islets with two photon microscopy, place the head holder platform under the microscope and administer eye gel onto the eye as an immersion liquid between the cornea and the lens. Position the eye under the objective and image the implanted human islets as described in the text manuscript.To remove the autofluorescence, go to the image processing tab, choose channel arithmetics and type channel one minus channel two. This creates a new channel four. Rename it vasculature.Repeat this process and type channel three minus channel two to create a new channel five and rename it tomato all. To define the islet manually create a new surface and choose edit manually in the wizard. Keep the pointer in select mode and in 3D view.And click volume to visualize sections. In the drawing tab choose contour and click draw to start drawing contours around the islet border, starting in slice position one. Then move to a new slice position and repeat drawing contours.Finish with the last slice on the top of the islet and end by clicking the create surface tab. Next segment, islet vasculature and islet tomato fluorescence using islet mask. Choose the previously defined islet mask object, go to the editing tab and click the mask all tab, which opens a new window.Choose the vasculature channel in the channel selection dropdown menu and activate options duplicate channel before applying mask. Constant inside, outside and set voxels outside surface to 0.000 which creates a new channel six. Rename this channel islet vasculature.Repeat the previous steps and choose the tomato all in the channel selection dropdown menu, to create the new channel, rename it, islet tomato. Then create a new surface in the scene menu and choose automatic creation in the wizard. Set the source channel to the previously created islet vasculature and choose background subtraction.Optionally use filters. For example choose volume and adjust the filter in the window, which can remove selected service objects. Finish the wizard and name the new surface object, islet vasculature.In the islet vasculature service object go to the editing tab and click the mask all tab, which opens a new window. Choose the islet tomato channel in the channel selection dropdown menu and set voxels outside surface to 10.000 which creates a new channel. Rename this channel islet tomato vasculature.To segment tomato capsule fluorescent signal choose channel arithmetics in the image processing tab and type channel seven minus channel eight creating the new channel. Rename it tomato capsule. Create a new surface as previously described and choose source channels, islet tomato vasculature or tomato capsule in the wizard.Then perform background subtraction. Open the islet backscatter file and create a new surface. In the wizard, choose automatic creation and define region of interest.If needed, adjust the threshold in absolute intensity. The surface object can be clicked on or off, to crosscheck with corresponding channel intensity. When finished close the wizard.Finally, perform quantification, select a created surface object in the scene menu, and go to the statistics tab. To retrieve detailed volume data, choose the detailed tab and select specific values and volume from the dropdown menu. To retrieve total volume value go to the detailed tab and choose average values.This protocol was used to transplant non labeled human islets into the interior chamber of the eye of eight-week old female red fluorescent recipient mice. Interactive imaging software was then used to extract quantitative data from the images. In vivo fluorescens imaging, the human islet grafts was compromised by significant interference from tissue autofluorescence which was not observed in mice islet grafts.To improve the signal to noise ratio, the autofluorescence channel was subtracted. Then the islet boundary called islet mask and the corresponding channels were defined manually. Finally, the segmentation of the tomato signal into the eyelid vasculature and eyelid capsule and final surface rendering was used to extract quantitative data.Here as a representative longitudinal imaging session of the same human islet graft at two weeks, two months, five months and eight months post transplantation. MIP images of originally recorded raw data, processed images after islet autofluorescence removal and a segmented islet objects are shown. When performing the eyelid injection, make sure to preload the islets in a small volume and use flexible twin light arms on your stereo microscope, to highlight the eye chamber space in order to successfully inject the islets into the ACE.Following the in vivo imaging the eyes can be fixed and further investigated for antibody staining and conventional histology. This technique is very useful for functional studies of individual human islets in real time and under physiological conditions, especially in the context of vascularization, viability and metabolic studies.

longitudinal-vivo-imaging-quantification-human-pancreatic-islet

Research

JoVE Journal

Medicine

Subretinal Injection of Gene Therapy Vectors and Stem Cells in the Perinatal Mouse Eye

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy and stem cell transplantations have become key therapeutic tools for treating blindness resulting from retinal degenerative diseases. Several forms of autologous transplantation for age-related macular degeneration (AMD), such as iris pigment epithelial cell transplantation, have generated encouraging results, and human clinical trials have begun for other forms of gene and stem cell therapies.2 These include RPE65 gene replacement therapy in patients with Leber's congenital amaurosis and an RPE cell transplantation using human embryonic stem (ES) cells in Stargardt's disease.3-4 Now that there are gene therapy vectors and stem cells available for treating patients with retinal diseases, it is important to verify these potential therapies in animal models before applying them in human studies. The mouse has become an important scientific model for testing the therapeutic efficacy of gene therapy vectors and stem cell transplantation in the eye.5-8 In this video article, we present a technique to inject gene therapy vectors or stem cells into the subretinal space of the mouse eye while minimizing damage to the surrounding tissue.

This surgical technique illustrates the injection of gene therapy vectors and stem cells into the subretinal space of the mouse eye.

The loss of sight affects approximately 3.4 million people in the United States and is expected to increase in the upcoming years.1 Recently, gene therapy ...

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

Intravital imaging has emerged as an indispensable tool in biological research. In the process, many imaging techniques have been developed to study different biological processes in animals non-invasively. However, a major technical limitation in existing intravital imaging modalities is the inability to combine non-invasive, longitudinal imaging with single-cell resolution capabilities. We show here how transplantation into the anterior chamber of the eye circumvents such significant limitation offering a versatile experimental platform that enables non-invasive, longitudinal imaging with cellular resolution in vivo. We demonstrate the transplantation procedure in the mouse and provide representative results using a model with clinical relevance, namely pancreatic islet transplantation. In addition to enabling direct visualization in a variety of tissues transplanted into the anterior chamber of the eye, this approach provides a platform to screen drugs by performing long-term follow up and monitoring in target tissues. Because of its versatility, tissue/cell transplantation into the anterior chamber of the eye not only benefits transplantation therapies, it extends to other in vivo applications to study physiological and pathophysiological processes such as signal transduction and cancer or autoimmune disease development.

Transplantation into the Anterior Chamber of the Eye for Longitudinal, Non-invasive In vivo Imaging with Single-cell Resolution in Real-time

A new approach combining intraocular transplantation and confocal microscopy enables longitudinal, non-invasive real-time imaging with single-cell resolution within grafted tissues in vivo. We demonstrate how to transplant pancreatic islets into the anterior chamber of the mouse eye.

Intravital imaging has emerged as an indispensable tool in biological research.  In the process, many imaging techniques have been developed to study ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, the role of beta-cell dysfunction in type 2 diabetes has been clearly established 2. Research progress has required methods for the isolation of pancreatic islets. The protocol of the islet isolation presented here shares many common steps with protocols from other groups, with some modifications to improve the yield and quality of isolated islets from both the wild type and diabetic Leprdb (db/db) mice. A live-cell 2-photon imaging method is then presented that can be used to investigate the control of insulin secretion within islets.

Leprdb Mouse Model of Type 2 Diabetes: Pancreatic Islet Isolation and Live-cell 2-Photon Imaging Of Intact Islets

We present here a protocol for the isolation of islets from the mouse model of type 2 diabetes, Leprdb and details of a live-cell assay for measurement of insulin secretion from intact islets that utilizes 2 photon microscopy.

Type 2 diabetes is a chronic disease affecting 382 million people in 2013, and is expected to rise to 592 million by 2035 1. During the past 2 decades, ...

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest improved outcome measures in both experimental and clinical studies. Although the BMMSC are implanted into the tendon lesion in large numbers (usually 10 - 20 million cells), only a relatively small number survive (&#60;10%) although these can persist for up to 5 months after implantation. This appears to be a common observation in other species where BMMSC have been implanted into other tissues and it is important to understand when this loss occurs, how many survive the initial implantation process and whether the cells are cleared into other organs. Tracking the fate of the cells can be achieved by radiolabeling the BMMSC prior to implantation which allows non-invasive in vivo imaging of cell location and quantification of cell numbers.
This protocol describes a cell labeling procedure that uses Technetium-99m (Tc-99m), and tracking of these cells following implantation into injured flexor tendons in horses. Tc-99m is a short-lived (t1/2 of 6.01 hr) isotope that emits gamma rays and can be internalized by cells in the presence of the lipophilic compound hexamethylpropyleneamine oxime (HMPAO). These properties make it ideal for use in nuclear medicine clinics for the diagnosis of many different diseases. The fate of the labeled cells can be followed in the short term (up to 36 hr) by gamma scintigraphy to quantify both the number of cells retained in the lesion and distribution of the cells into lungs, thyroid and other organs. This technique is adapted from the labeling of blood leukocytes and could be utilized to image implanted BMMSC in other organs.

In Vivo Imaging and Tracking of Technetium-99m Labeled Bone Marrow Mesenchymal Stem Cells in Equine Tendinopathy

This protocol describes the radiolabeling of equine mesenchymal stem cells and their implantation into tendon injuries in the horse in order to determine cell survival and tissue distribution using gamma scintigraphy.

Recent advances in the application of bone marrow mesenchymal stem cells (BMMSC) for the treatment of tendon and ligament injuries in the horse suggest ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular plasticity or damage occurring overtime and in relation to neuronal activity or blood flow. In vivo two-photon microscopy allows the study of the structural and functional plasticity of large cellular units in the living brain. In particular, the thinned-skull window preparation allows the visualization of cortical regions of interest (ROI) without inducing significant brain inflammation. Repetitive imaging sessions of cortical ROI are feasible, providing the characterization of disease hallmarks over time during the progression of numerous CNS diseases. This technique accessing the pial structures within 250 &#956;m of the brain relies on the detection of fluorescent probes encoded by genetic cellular markers and/or vital dyes. The latter (e.g., fluorescent dextrans) are used to map the luminal compartment of cerebrovascular structures. Germane to the protocol described herein is the use of an in vivo marker of amyloid deposits, Methoxy-O4, to assess Alzheimer&#39;s disease (AD) progression. We also describe the post-acquisition image processing used to track vascular changes and amyloid depositions. While focusing presently on a model of AD, the described protocol is relevant to other CNS disorders where pathological cerebrovascular changes occur.

Longitudinal In Vivo Imaging of the Cerebrovasculature: Relevance to CNS Diseases

This manuscript describes a procedure to track the remodeling of the cerebrovasculature during amyloid plaque accumulation in vivo using longitudinal two-photon microscopy. A thinned-skull preparation enables the visualization of fluorescent dyes to assess the progression of cerebrovascular damage in a mouse model of Alzheimer&#39;s disease.

Remodeling of the brain vasculature is a common trait of brain pathologies. In vivo imaging techniques are fundamental to detect cerebrovascular ...

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon dioxide and waste products. The impairment of microvascular vasomotion might be a crucial step in the development of microcirculation-related diseases. In addition, the highly vascularized pancreatic islet is adapted to support endocrine function. In this respect, it seems possible to infer that the functional status of pancreatic islet microvascular vasomotion might affect pancreatic islet function. Analyzing the pathological changes of the functional status of pancreatic islet microvascular vasomotion may be a feasible strategy to determine contributions that pancreatic islet microcirculation makes to related diseases, such as diabetes mellitus, pancreatitis, etc. Therefore, this protocol describes using a laser Doppler blood flow monitor to determine the functional status of pancreatic islet microvascular vasomotion, and to establish parameters (including average blood perfusion, amplitude, frequency, and relative velocity of pancreatic islet microvascular vasomotion) for evaluation of the microcirculatory functional status. In a streptozotocin-induced diabetic mouse model, we observed an impaired functional status of pancreatic islet microvascular vasomotion. In conclusion, this approach for assessing pancreatic islet microvascular vasomotion in vivo may reveal mechanisms relating to pancreatic islet diseases.

Laser Doppler: A Tool for Measuring Pancreatic Islet Microvascular Vasomotion In Vivo

Pancreatic islet microvascular vasomotion regulates islet blood distribution and maintains the physiological function of islet &#946; cells. This protocol describes using a laser Doppler monitor to determine the functional status of pancreatic islet microvascular vasomotion in vivo and to assess the contributions of pancreatic islet microcirculation to pancreatic-related diseases.

As a functional status of microcirculation, microvascular vasomotion is important for the delivery of oxygen and nutrients and the removal of carbon ...

Human Fetal Blood Flow Quantification with Magnetic Resonance Imaging and Motion Compensation

Magnetic resonance imaging (MRI) is an important tool for the clinical assessment of cardiovascular morphology and heart function. It is also the recognized standard-of-care for blood flow quantification based on phase contrast MRI. While such measurement of blood flow has been possible in adults for decades, methods to extend this capability to fetal blood flow have only recently been developed.
Fetal blood flow quantification in major vessels is important for monitoring fetal pathologies such as congenital heart disease (CHD) and fetal growth restriction (FGR). CHD causes alterations in the cardiac structure and vasculature that change the course of blood in the fetus. In FGR, the path of blood flow is altered through the dilation of shunts such that the oxygenated blood supply to the brain is increased. Blood flow quantification enables assessment of the severity of the fetal pathology, which in turn allows for suitable&#160;in utero&#160;patient management and planning for postnatal care.
The primary challenges of applying phase contrast MRI to the human fetus include small blood vessel size, high fetal heart rate, potential MRI data corruption due to maternal respiration, unpredictable fetal movements, and lack of conventional cardiac gating methods to synchronize data acquisition. Here, we describe recent technical developments from our lab that have enabled the quantification of fetal blood flow using phase contrast MRI, including advances in accelerated imaging, motion compensation, and cardiac gating.

Here we present a protocol for measuring fetal blood flow rapidly with MRI and retrospectively performing motion correction and cardiac gating.