We acknowledge Drs. Camillo Ricordi, Antonello Pileggi, R. Damaris Molano, Stephan Speier and Daniel Nyqvist for fruitful discussions. We also thank Eleut Hernandez and Diego Espinosa-Heidmann for technical assistance, and Mike Valdes and Margaret Formoso for help with video recording. Byron Maldonado recorded, edited, and produced the final video. Research support was provided by the Diabetes Research Institute Foundation (<a href="http://www.DiabetesResearch.org" target="_blank">www.DiabetesResearch.org</a>), the NIH/NIDDK/NIAID (F32DK083226 to M.H.A.; NIH RO3DK075487 to A.C.; U01DK089538 to P-O.B.). Additional research support to P-O.B was provided through funds from the Karolinska Institutet, the Swedish Research Council, the Swedish Diabetes Foundation, the Family Erling-Persson Foundation, the Family Knut and Alice Wallenberg Foundation, the Skandia Insurance Company Ltd., VIBRANT (FP7-228933-2), Strategic Research Program in Diabetes at Karolinska Institutet, the Novo Nordisk Foundation, and the Berth von Kantzow&#x2019;s Foundation.

The following procedure is performed under the stereoscope in 2 steps, the first step involves loading the islets into the cannula and the second step is the actual transplantation into the ACE. All procedures performed on animals were approved by the institutional animal care and use committee (IACUC) of the University of Miami.
1. Loading Islets in Cannula for Transplantation
<ol>
 <li>Center the islets in culture dish by spinning the dish in narrowing circles.</li>
 <li>Disconnect the cannula from the "reservoir" and place the cannula and connecting tubing on a clean surface. The reservoir can be made out of a 300 &mu;l disposable plastic pipette tip without filter (Figure 1a).</li>
 <li>Flush air bubbles out of the reservoir to ensure continuous stream of islets when aspirating into reservoir. Flushing the reservoir is done by driving forward the hands-free motorized syringe-driver using the foot pedal (Figure 1b, c). This will also make space in the syringe to allow aspiration of the islets into the reservoir (pre-loaded with sterile solution such as saline, PBS or culture media). </li>
 <li>Gently aspirate desired amount of islets into the reservoir. Islets will tend to swirl as they enter the reservoir and will remain together towards the bottom. Aspiration is done by driving backward the motorized syringe-driver using the foot pedal. </li>
 <li>Reconnect the cannula to the reservoir via the connecting tubing. </li>
 <li>Place the cannula tip back in the culture dish and flush the islets out of the reservoir into the tubing then into the cannula. Ensure that islets remain together as you back-fill the tubing/cannula by gently "flicking" (tapping) the tubing (Figure 1d). Stop either before or after all air bubbles ahead of the islets are flushed out the cannula. If not sure, stop as islets enter the back of the cannula as remaining air bubbles ahead of islets can help prevent reflux (backflow) of islets out of the ACE. Will dissipate overnight.
 </li>
 <li>At this stage, you are ready to inject the islets into the ACE (please see next steps). </li>
</ol>
2. Islet Transplantation into the Anterior Chamber of the Eye
<ol>
 <li>Position the anesthetized mouse on a warm pad under stereoscope.</li>
 <li>Place the snout of the mouse into anesthesia "mask" connected to oxygen/isoflurane anesthesia machine. The mask is made out of a 1 ml disposable plastic pipette tip (without filter) and connected to anesthesia tubing through the narrow end (Figure 2a,b).</li>
 <li>Gently retract the eye lids of the eye to be transplanted using the index finger and thumb of your free hand and "pop" the eye out for better exposure and easy access (Figure 2c). This will require some practice to perfect without impeding breathing of the mouse by excessive pressure on the neck or blocking blood flow to the head. </li>
 <li>Using a disposable insulin syringe (29 - 31G) as scalpel, carefully penetrate only the tip in the cornea and make a single lateral incision. Make the incision at midpoint between the apex of the cornea and limbus to minimize reflux of the islets during injection out of the ACE (Figure 2d). </li>
 <li>Carefully insert the cannula (preloaded with islets) through the incision. </li>
 <li>Slowly eject islets out of the cannula and deposit them on top of the iris. To avoid islet reflux due to excessive pressure buildup in the ACE, eject the islets in brief thrusts in as little volume(s) as possible in the quadrant opposite to the incision. This can be further ensured by compacting islets in the tubing while loading the cannula (please see step 1.6).</li>
 <li>Slowly retract the cannula out of the ACE. This is a critical step, especially, if a large volume of islets was injected as islet reflux due to pressure build up inside the ACE may be inevitable. To eliminate/minimize islet reflux, gently rotate the cannula while inside the ACE to release excess pressure through the incision around the cannula. Check for signs of reflux as you attempt to retract the cannula and, if needed, wait until pressure subsides before completely retracting the cannula out of the ACE. </li>
 <li>Rinse the transplanted eye with sterile PBS or saline.</li>
 <li>Inject buprenorphine for post-operative analgesia (0.05-0.1 mg/kg, subcutaneously) for the first 48 hr.</li>
 <li>Apply erythromycin ophthalmic antibiotic ointment to the transplanted eye immediately after transplantation.</li>
 <li>Place the animal back in a warmed cage to allow recovery from anesthesia.</li>
</ol>

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P-O.B. is one of the founders of the biotech company Biocrine, which is going to use the anterior chamber of the eye as a commercial servicing platform. A.C. is on the patent protecting this technology.

Murine pancreatic islets were isolated using collagenase digestion followed by purification on density gradients, as described previously 33. Isolated islets were cultured overnight before transplantation. While this may not be required, it is recommended to allow the islets to recover from the isolation procedure. This is critical when transplantation is performed in diabetic recipients as it will ensure transplantation of surviving/robust islets. 
Transplantation is performed ...

Advances in intravital microscopy have revealed physiological phenomena not predicted by in vitro studies1. This highlights the challenge in translating findings obtained by conventional in vitro methods into the living animal. In the last decade, visualization of tissues in living animals was considerably improved by technological advances in imaging modalities2, 3, 4, 5, 6. This has spurred a need for in vivo imaging approaches with feasible application in experimental animal models to enable longitudinal visualization of target tissues non-invasively. 
Imaging techniques such as magnetic resonance imaging and positron emission tomography or bioluminescence have enabled non-invasive imaging of organs/tissues deep within the body7-8, 9. But these techniques cannot achieve single cell-resolution due to high background signals and low spatial resolution, despite the use of high contrast materials or tissue-specific luminescence4. This was addressed with the advent of two-photon fluorescence confocal microscopy10. Two-photon microscopy enabled intravital imaging studies to visualize and quantify cellular events with unprecedented details11, 12. This has led to the characterization of key biological processes in health and disease13, 14, 15, 16. While pioneering intravital imaging studies have primarily "mimicked" in vivo conditions in excised tissue (e.g. lymph nodes), other studies have used invasive approaches to image exposed target tissues in situ17, 18, 19, 20, 21. Other studies have also used "window chamber models" to circumvent limitations associated with invasive approaches and limited imaging resolution in vivo22, 23, 24, 25. In the window chamber model, a chamber with a transparent window is surgically implanted into the skin at different locations (dorsal or ear skin, mammary fat pad, liver, etc) on the animal (e.g. mouse, rat, rabbit). While this approach clearly enables high-resolution in vivo imaging, it requires an invasive surgery to implant the chamber and may not be able to accommodate longitudinal imaging studies over several weeks or months22.
It was recently demonstrated that combining high-resolution confocal microscopy with a minimally invasive procedure, namely transplantation into the anterior chamber of the eye (ACE) provides a "natural body window" as a powerful and versatile in vivo imaging platform26, 27. Transplantation into the ACE has been used in the last several decades to study biological aspects of a variety of tissues 28, 29, 30; and its recent combination with high-resolution imaging enabled studying the physiology of pancreatic islets with single cell-resolution non-invasively and longitudinally26, 27. This approach was used to study autoimmune responses during development of type 1 diabetes in animal models (unpublished data). It was also used to study pancreatic development, as well as, in studies of kidney function by transplanting into the ACE pancreatic buds or individual renal glomeruli, respectively (unpublished data). A recent report using this approach further demonstrated its application to study immune responses after pancreatic islet transplantation31. Importantly, this study showed that transplantation into the anterior chamber of the eye provides a natural body window to perform: (1) longitudinal, non-invasive imaging of transplanted tissues in vivo; (2) in vivo cytolabeling to assess cellular phenotype and viability in situ; (3) real-time tracking of infiltrating immune cells in the target tissue; and (4) local intervention by topical application or intraocular injection.
Here, we demonstrate how to perform transplantation into the anterior chamber of the eye using pancreatic islets.

<table border="1">
 <tbody>
 <tr>
 <td>Name 				of reagent</td>
 <td>Company</td>
 <td>Catalogue 				number</td>
 <td>Description/Comments</td>
 </tr>
 <tr>
 <td>IsoTHESIA 				(Isoflurane)</td>
 <td>Buttler 				Animal Health Supply</td>
 <td>11695-6775-2</td>
 <td>99.9% 				Isoflurane/ml</td>
 </tr>
 <tr>
 <td>Ketaset (Ketamine 				HCL)</td>
 <td>Fort 				dodge Animal Health</td>
 <td>0856-2013-01</td>
 <td>Alternative 				injectable anesthesia</td>
 </tr>
 <tr>
 <td>Beprenex (Buprenorphine 				HCL)</td>
 <td>Reckitt 				Benckiser Health Care (UK) Ltd.</td>
 <td>12496-075-7-1</td>
 <td>0.3 				mg/ml</td>
 </tr>
 <tr>
 <td>Erythromycin 				Ophthalmic Ointment USP, 0.5%</td>
 <td>Akron</td>
 <td>17478-070-35</td>
 <td>Applied 				prophylactically to transplanted eye </td>
 </tr>
 <tr>
 <td>0.9% 				Sodium Chloride (Saline)</td>
 <td>Hospira 				Inc.</td>
 <td>0409-7983-03</td>
 <td>For iv 				injection. Sterile</td>
 </tr>
 <tr>
 <td>PBS</td>
 <td>Gibco</td>
 <td>10010-023</td>
 <td>1X. 				Sterile</td>
 </tr>
 <tr>
 <td>CMRL 				medium 1066</td>
 <td>Cellgro</td>
 <td>98-304-CV</td>
 <td>Supplemented, 				CIT modification. Preferred media for islets </td>
 </tr>
 </tbody>
 </table>

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.

There are a few parameters that define a "good" transplantation. A good transplantation is one that proceeds without bleeding when making the incision as can be seen in the video. Bleeding is prevented/minimized by penetrating only the tip of the scalpel (needle) into the ACE (Figure 3a). This will also help prevent contact and puncture of the iris. It will also ensure a small incision which will heal very well without causing cloudiness of the cornea over time (Figure 3c, d). Anoth...

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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.

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 ...

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Research

JoVE Journal

Medicine

13.3K vues.  University of Miami Miller School of Medicine. Une nouvelle approche combinant la greffe intraoculaire et la microscopie confocale permet longitudinale, non-invasive imagerie en temps réel avec une seule cellule de résolution au sein des tissus greffés In vivo. Nous démontrons comment transplanter les îlots pancréatiques dans la chambre antérieure de l'œil de la souris.

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

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in functional parameters of key clinical significance. Two-dimensional movies were acquired at high frame rate (8 kHz) and were utilized to estimate high-quality myocardial strain. Two-dimensional elastograms (strain images), as well as strain profiles, were visualized. Results were powerful in quantitatively assessing IR-induced changes in cardiac events including left-ventricular (LV) contraction, LV relaxation and isovolumetric phases of both pre-IR and post-IR beating hearts in intact mice. In addition, compromised sector-wise wall motion and anatomical deformation in the infarcted myocardium were visualized.  The elastograms were uniquely able to provide information on the following parameters in addition to standard physiological indices that are known to be affected by myocardial infarction in the mouse: internal diameters of mitral valve orifice and aorta, effective regurgitant orifice, myocardial strain (circumferential as well as radial), turbulence in blood flow pattern as revealed by the color Doppler movies and velocity profiles, asynchrony in LV sector, and changes in the length and direction of vectors demonstrating slower and asymmetrical wall movement. This work emphasizes on the visual demonstration of how such analyses are performed. 

High-frequency High-resolution Echocardiography: First Evidence on Non-invasive Repeated Measure of Myocardial Strain, Contractility, and Mitral Regurgitation in the Ischemia-reperfused Murine Heart 

High frequency Doppler ultrasound is a novel technology for assessing regional myocardial function. This work presents first evidence demonstrating applicability of this versatile imaging platform for the repeated measure of myocardial strain, dp/dt, and mitral regurgitation in the ischemia-reperfused (IR) murine heart.

Ischemia-reperfusion (IR) was surgically performed in murine hearts which were then subjected to repeated imaging to monitor temporal changes in ...

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

The number of patients with end-stage renal disease, and the number of kidney allograft recipients continuously increases. Episodes of acute cellular allograft rejection (AR) are a negative prognostic factor for long-term allograft survival, and its timely diagnosis is crucial for allograft function 1. At present, AR can only be definitely diagnosed by core-needle biopsy, which, as an invasive method, bares significant risk of graft injury or even loss. Moreover, biopsies are not feasible in patients taking anticoagulant drugs and the limited sampling site of this technique may result in false negative results if the AR is focal or patchy. As a consequence, this gave rise to an ongoing search for new AR detection methods, which often has to be done in animals including the use of various transplantation models.
Since the early 60s rat renal transplantation is a well-established experimental method for the examination and analysis of AR 2. We herein present in addition small animal positron emission tomography (PET) using 18F-fluorodeoxyglucose (FDG) to assess AR in an allogeneic uninephrectomized rat renal transplantation model and propose graft FDG-PET imaging as a new option for a non-invasive, specific and early diagnosis of AR also for the human situation 3. Further, this method can be applied for follow-up to improve monitoring of transplant rejection 4.

Non-invasive Imaging of Acute Allograft Rejection after Rat Renal Transplantation Using 18F-FDG PET

We herein present a rat renal transplantation model to non-invasively assess acute allograft rejection using positron emission tomography with 18F-fluorodeoxyglucose.

The number of patients with end-stage renal  disease, and the number of kidney allograft recipients continuously increases.  Episodes of acute cellular ...

Development of an Audio-based Virtual Gaming Environment to Assist with Navigation Skills in the Blind

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio based cues and set within the context of a video game metaphor, users gather relevant spatial information regarding a building's layout. This allows the user to develop an accurate spatial cognitive map of a large-scale three-dimensional space that can be manipulated for the purposes of a real indoor navigation task. After game play, participants are then assessed on their ability to navigate within the target physical building represented in the game. Preliminary results suggest that early blind users were able to acquire relevant information regarding the spatial layout of a previously unfamiliar building as indexed by their performance on a series of navigation tasks. These tasks included path finding through the virtual and physical building, as well as a series of drop off tasks. We find that the immersive and highly interactive nature of the AbES software appears to greatly engage the blind user to actively explore the virtual environment. Applications of this approach may extend to larger populations of visually impaired individuals.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind.

Audio-based Environment Simulator (AbES) is virtual environment software designed to improve real world navigation skills in the blind. Using only audio ...

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an increased risk of vascular morbidity.&#160;Yet, albeit&#160;the mounting clinical evidence, there is a paucity of clear studies of vascular toxicity and therefore the etiology of a heterogeneous group of vascular/cardiovascular disorders remains to be elucidated. Moreover, the mechanism that may underlie vascular toxicity can completely differ from the principles of chemotherapy-induced cardiotoxicity, which is related to direct myocyte injury. We have established a real-time, in vivo molecular imaging platform&#160;to evaluate&#160;the potential&#160;acute&#160;vascular toxicity of anti-cancer therapies.
We have set up a platform of in vivo, high-resolution molecular imaging&#160;in mice,&#160;suitable for visualizing vasculature within confined organs and reference blood vessels within the same individuals whereas each individual serve as its own control. Blood vessel walls were impaired after doxorubicin administration, representing a unique mechanism of vascular toxicity that may be the early event in end-organ injury. Herein, the method of fibered confocal fluorescent microscopy (FCFM) based imaging is described, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Chemotherapy-induced Vascular Toxicity - Real-time In vivo Imaging of Vessel Impairment

We herein describe the method of fibered confocal fluorescent microscopy (FCFM) based imaging, which provides an innovative mode to understand physiological phenomena at the cellular and sub-cellular levels in animal subjects.

Certain classes of chemotherapies may exert acute vascular changes that may progress into long-term conditions that may predispose the patient to an ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

Encapsulation Thermogenic Preadipocytes for Transplantation into Adipose Tissue Depots

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular weight metabolites in tissues of the treated host to achieve long-term survival. The semipermeable membrane allows engrafted encapsulated cells to avoid rejection by the immune system. The encapsulation procedure was designed to enable a controlled release of bioactive compounds, such as insulin, other hormones, and cytokines. Here we describe a method for encapsulation of catabolic cells, which consume lipids for heat production and energy dissipation (thermogenesis) in the intra-abdominal adipose tissue of obese mice. Encapsulation of thermogenic catabolic cells may be potentially applicable to the prevention and treatment of obesity and type 2 diabetes. Another potential application of catabolic cells may include detoxification from alcohols or other toxic metabolites and environmental pollutants.

Here, we present a protocol for encapsulation of catabolic cells, which consume lipids for heat production in intra-abdominal adipose tissue and increase energy dissipation in obese mice.

Cell encapsulation was developed to entrap viable cells within semi-permeable membranes. The engrafted encapsulated cells can exchange low molecular ...

Extended Time-lapse Intravital Imaging of Real-time Multicellular Dynamics in the Tumor Microenvironment

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and metastasis. Multicellular interactions in the tumor microenvironment can lead to transient events including directional tumor cell motility and vascular permeability. Quantification of tumor vascular permeability has frequently used end-point experiments to measure extravasation of vascular dyes. However, due to the transient nature of multicellular interactions and vascular permeability, the kinetics of these dynamic events cannot be discerned. By labeling cells and vasculature with injectable dyes or fluorescent proteins, high-resolution time-lapse intravital microscopy has allowed the direct, real-time visualization of transient events in the tumor microenvironment. Here we describe a method for using multiphoton microscopy to perform extended intravital imaging in live mice to directly visualize multicellular dynamics in the tumor microenvironment. This method details cellular labeling strategies, the surgical preparation of a mammary skin flap, the administration of injectable dyes or proteins by tail vein catheter and the acquisition of time-lapse images. The time-lapse sequences obtained from this method facilitate the visualization and quantitation of the kinetics of cellular events of motility and vascular permeability in the tumor microenvironment.

This protocol describes the use of multiphoton microscopy to perform extended time-lapse imaging of multicellular interactions in real time, in vivo at single cell resolution.

In the tumor microenvironment, host stromal cells interact with tumor cells to promote tumor progression, angiogenesis, tumor cell dissemination and ...

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 ...

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. To better understand the mechanism behind these surgical procedures, a rat partial liver transplantation (PLTx) model was established for relevant surgical studies. Because of the complexity of the rat PLTx model, a protocol with detailed descriptions is required. An article published previously reported a protocol in which ex vivo hepatectomy was used to achieve 50% rat PLTx. In contrast to this protocol, we introduced a re-arterialized PLTx with an in vivo 70% hepatectomy. An updated vessel-oriented hepatectomy was incorporated into the rat PLTx to refine the microsurgical procedure. The portal veins and hepatic arteries of the left lateral lobe and the median lobe were individually dissected and ligated before removal of the liver parenchyma, thereby decreasing the probability of bleeding in the remnant liver stump. Furthermore, an end-to-side vessel anastomosis between the common hepatic artery and the enlarged proper hepatic artery was introduced to re-arterialize the hepatic artery. By using this end-to-side vessel anastomosis technique, the diameter of the anastomosis was enlarged, thereby decreasing the difficulty of hand suture and maintaining a high rate of anastomotic patency. Moreover, the cuff anastomosis of the infrahepatic vena cava was slightly modified. A section of circumferential liver parenchyma around the vena cava of a recipient was preserved during cuff anastomosis to maintain the three-dimensional shape of the vascular lumen. This section of liver parenchyma was removed after completing the anastomosis. With this modification, the step involving placement of stay sutures was omitted, thereby further shortening the cuff anastomosis time. By using this protocol of rat PLTx, a low liver enzyme level, an intact liver lobular architecture and a high survival rate were achieved after microsurgery.

Re-Arterialized Rat Partial Liver Transplantation with an in vivo Vessel-Oriented 70% Hepatectomy

Here, a protocol involving re-arterialized rat partial liver transplantation is presented. Specifically, 70% liver was resected in vivo by using an updated technique of vessel-oriented hepatectomy. The hepatic artery was reconstructed in an end-to-side manner. The cuff technique was modified to shorten the anastomosis time of the infrahepatic vena cava.

Split liver transplantation and living liver donor liver transplantation were developed in the clinic to utilize liver organs in a more efficient manner. ...