The research project was funded under New Investigator grant from the National Medical Research Council (NMRC), Singapore. The team will like to acknowledge the research training provided to Dr Agrawal at Institute of Ophthalmology (IoO), University College London (UCL) under National Medical Research Council (NMRC) Overseas Research Training Fellowship from November 2012 till October 2014 under mentorship of Prof. David Shima. Dr. Agrawal acquired the concept and skills for labelling the cells and live imaging in Dr. Shima&#39;s lab. The team will hence like to acknowledge supervision and guidance during the training fellowship from Prof. David Shima, Pro.f Kenith Meissner, Dr. Peter Lundh and Dr. Daiju Iwata.

The animal protocols used in this study were approved by the Institutional Animal Care and Use Committee of SingHealth, Singapore and are in accordance to the guidelines of the Association for Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research.
1. Labeling of Erythrocytes and Leukocytes with Fluorescent Dyes
<ol>
	<li>Preparation of reagents
	<ol>
		<li>Prepare ICG (1.5 mg/mL) by dissolving 3 mg of ICG in 1800 &#181;L of sterilized distilled water. Add 200 &#181;L of 10x phosphate buffered saline (PBS).</li>
		<li>Prepare 40% 1x PBS by adding 4 mL of 1x PBS to 6 mL of sterilized distilled water. Prepare 1% bovine serum albumin (BSA) in PBS by adding 100 mg of BSA to 10 mL of 1x PBS. Mix well until the BSA is dissolved.</li>
	</ol>
	</li>
	<li>Isolation of erythrocytes and leukocytes using density gradient centrifugation
	<ol>
		<li>Anesthetize the mice (wild type C57BL/6) using a combination of ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (0.5 mg/kg) (according to schedule 1, under ARVO guidelines). Confirm anesthesia by pedal withdrawal reflex (i.e., toe pinch).</li>
		<li>Slowly insert the 29 gauge needle (attached to a 1 mL syringe) at a costal angle directed towards the heart. To confirm that the needle is inside the heart, withdraw the plunger slightly and check if blood is withdrawn into the syringe. Exsanguinate 0.8 - 1 mL of blood directly from the heart.</li>
		<li>Immediately transfer the blood into an ethylene diamine tetra acetic acid (EDTA) (1.8 mg/mL of blood) containing blood collection tube and mix gently to prevent coagulation of the blood.</li>
		<li>Euthanize the mice by administering pentobarbital (80 mg/kg) by intraperitoneal injection.</li>
		<li>Layer the whole blood on top of a polysucrose and sodium diatrizoate solution (1:1 ratio; density 1.077 g/mL) in a 2 mL microcentrifuge tube and centrifuge (450 x g, 30 min) to allow the separation of the leukocytes and erythrocytes from the plasma.</li>
		<li>Remove and discard the supernatant (plasma) using a pipette. Carefully transfer the buffy coat (leukocytes) and erythrocytes (red blood cell layer) into new and separate tubes using pipettes.</li>
	</ol>
	</li>
	<li>ICG (1.5%) labeling of erythrocytes
	<ol>
		<li>Wash the erythrocytes with 1x PBS to remove any contaminants. Resuspend the erythrocytes in 1x PBS (1:1) to make 50% hematocrit. Aliquot 0.5 mL of 50% hematocrit (~ 250 &#181;L packed erythrocytes) into microcentrifuge tubes. Centrifuge the erythrocytes (750 x g, 5 min) and discard the supernatant.</li>
		<li>Add 200 &#181;L of 40% 1x PBS to the pelleted erythrocytes, mix well and incubate at room temperature for 5 min. Add 100 &#181;L of ICG (1.5 mg/mL) to the erythrocyte suspension, mix gently by inverting the tube, and incubate at room temperature for 5 min. Add 300 &#181;L of 1x PBS and incubate at 37 &#176;C in a shaker incubator for 60 min.</li>
		<li>Centrifuge the carrier erythrocytes (750 x g, 3 min) and resuspend in 1 mL 1x PBS.</li>
		<li>Wash the cells ~ 3 - 5 times with 1x PBS until the supernatant is clear (step 1.3.3). After the last wash, decant the supernatant and add 1% BSA PBS (1:1) to the labeled pre-swelled erythrocytes to achieve a 50% hematocrit (1.25 x 108 cells/mL) of ICG labeled erythrocytes.</li>
	</ol>
	</li>
	<li>Sodium fluorescein (1%) labeling of leukocytes
	<ol>
		<li>After separating the buffy coat from the blood using density gradient centrifugation (from step 1.2.6), wash the cells once with 10 mL of 1x PBS by centrifuging at 450 x g for 10 min to remove any contaminants. Resuspend the pellet in 900 &#181;L of 1x PBS.</li>
		<li>Add 100 &#181;L of 10% sodium fluorescein to 900 &#181;L of the leukocyte suspension and incubate at room temperature for 2 min. Wash the labeled cells three times with 10 mL 1X PBS by centrifuging (450 x g, 10 min). Resuspend the cells into 100 &#181;L 1x PBS.</li>
	</ol>
	</li>
</ol>
2. Live Imaging of Fluorescently Labeled Cells
<ol>
	<li>Live-cell imaging of ICG labeled erythrocytes
	<ol>
		<li>To prepare cells for injection via tail vein or retro-orbital route, dilute the 50% hematocrit of ICG labeled erythrocytes by 10-fold and 50-fold with 1x PBS to make 5% and 1% hematocrit cells, respectively.</li>
		<li>Place the mouse under the infrared light (250W intensity) for 5 min to let the tail vein dilate. Anesthetize the mouse with ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (0.5 mg/kg) through intraperitoneal injection (here, according to schedule 1, under ARVO guidelines).</li>
		<li>Dilate each pupil of the eyes with a drop of 0.5% tropicamide and 2.5% phenylephrine hydrochloride.</li>
		<li>Place the contact lens over one eye for imaging. Use ophthalmic gel as a medium for imaging and to prevent dryness of the cornea. Fix the confocal laser scanning ophthalmoscopy (SLO) camera with +25 diopter lens to compensate for the refraction of the mouse eye.</li>
		<li>Place the mouse onto the SLO imaging platform. Ensure that the cornea of the mouse is facing straight towards the optical head of the SLO machine.</li>
		<li>Switch on the imaging module. Using the associated imaging module software, click on the &#34;New Patient&#34; button and add animal identification details such as mouse ear tag number, date of birth, percentage of fluorescent dye and labeled cell type.
		<ol>
			<li>Position the optic nerve of the animal at the center of the imaging screen by maneuvering the imaging module. Using the associated imaging module software, click on the &#34;Acquire&#34; button to take the infrared (IR) fundus images with the 30&#176; or 15&#176; angle view setting. Ensure that the center of the retina, the pupil and the optical path of the laser are aligned to achieve a best quality image. 
			NOTE: The optical path is aligned by maneuvering the joystick to obtain the sharpest image and best lighting exposure.</li>
		</ol>
		</li>
		<li>Take the ICG fundus video.
		<ol>
			<li>Turn on the ICG filter (790 nm), set the camera to high speed mode with the 30&#176; or 15&#176; angle view and set the ICG intensity at 85 for all readings for standardization. On the control panel, click on the &#34;Acquire&#34; button to take the video (baseline control) at 8.8/15 frames/s for 1 min.</li>
		</ol>
		</li>
		<li>Load 100 &#181;L of ICG labeled erythrocytes into an insulin syringe attached to a 30 gauge needle.
		<ol>
			<li>For the injection via the tail vein route, insert the needle bevel up into the tail vein. Confirm the intravenous access by checking the backflow of the blood into the syringe and inject the labeled cells into the circulation.</li>
			<li>For the injection via the retro-orbital route, insert the needle adjacent to the eye into the retro-orbital space. Confirm the retro-orbital access by checking the backflow of the blood into the syringe and inject the labeled cells into the circulation.</li>
		</ol>
		</li>
		<li>After the injection, reposition the mouse and take the IR fundus images (see step 2.1.6.1) and video using the ICG filter (see step 2.1.7.1). The labeled cells in the retinal circulation can be visualized immediately after the injection of the cells.</li>
		<li>After acquiring the video frames, extract .tiff images of the video sequences by choosing the .tiff format when exporting the video using the SLO viewing module software. Save the files in a desired folder.</li>
		<li>Remove the contact lens, place it over the other eye, and complete the imaging as described in steps 2.1.4 - 2.1.7.</li>
		<li>After imaging, place the anesthetized animal under the infrared light (250W) to maintain the body temperature until it recovers from anesthesia. When the animal is fully recovered, place the mouse back into the holding cage.</li>
	</ol>
	</li>
	<li>Live-cell imaging of 1% sodium fluorescein labeled leukocytes
	<ol>
		<li>Prepare the mouse and set-up the instrument as described in section 2.1.</li>
		<li>Position the mouse and take the IR fundus images as described in steps 2.1.4 - 2.1.6.
		<ol>
			<li>Obtain the fluorescein angiogram of the mouse using the fluorescein filter (488 nm) with the high-speed camera mode with the 30&#176; or 15&#176; angle view and fluorescein intensity at 85 for all readings for standardization. Record the video (baseline control) at 8.8/15 frames/s (see step 2.1.7.1). 
			NOTE: Here, multiple tracks of videos (1 min videos) were acquired at different time intervals.</li>
		</ol>
		</li>
		<li>Inject 100 &#181;L labeled leukocytes into the mouse through the tail vein or retro-orbital injection as described in section 2.1.8.</li>
		<li>Immediately after the injection, position the mouse and take the IR fundus images (see steps 2.1.5 and 2.1.6) and the fluorescein angiogram of the mouse (see section 2.2.2).</li>
		<li>Observe the labeled cells in the retina or choroidal blood vessels by adjusting the focal point of the scanning laser by turning the focusing knob on the imaging module.</li>
		<li>Acquire the video frames and images using SLO viewing module software (see step 2.1.10) and save it in a desired folder.</li>
		<li>After the procedure, follow step 2.1.12 for care of animals.</li>
	</ol>
	</li>
	<li>Image analysis by MtrackJ software
	<ol>
		<li>Open the image sequence in ImageJ by clicking on &#34;File&#34;. Select &#34;Import&#34; and choose &#34;Image sequence&#34;, select the desired image folder, and click &#34;Open&#34;. A pop up window called &#34;Sequence options&#34; will appear on the screen. Click &#34;Ok&#34;; the image sequence will open.</li>
		<li>Set the frame interval of the image video by clicking on &#34;Image&#34; and select &#34;Properties&#34; for velocity measurements. Here, it is 8.8 frames/s.</li>
		<li>Open the MTrackJ plugin by selecting &#34;Plugins&#34;. Select &#34;Tracking&#34; and then select &#34;MtrackJ&#34;. A menu should appear.</li>
		<li>To adjust the tracking settings, select &#34;Tracking&#34; (under menu) and check the box &#34;Move to next time frame index after adding point&#34;.</li>
		<li>Track the first cell.
		<ol>
			<li>Locate one cell and track the same cell in subsequent frames. On the menu click &#34;Add&#34; to add a new track.</li>
			<li>Hover the cursor over the image (+ sign) and click on the appropriate cell. 
			NOTE: MTrackJ will automatically jump to the next time frame and add a small circle, marking the position of the first point in the trajectory.</li>
			<li>Continue clicking on the current position of the cell while moving through the frames of the image sequence. 
			NOTE: Occasionally, the circles marking the previous points in the trajectory interfere with the ability to view the current location. If this occurs, proceed to step 2.3.5.3.1.
			<ol>
				<li>Change the display option for the trajectory by clicking on &#34;Displaying&#34; in the menu and selecting the option &#34;display only track points at current time&#34;. To see all the trajectories, de-select this option.</li>
			</ol>
			</li>
			<li>Continue clicking until the trajectory of the first cell is seen. Then save the track by clicking &#34;Save&#34;.</li>
		</ol>
		</li>
		<li>Track the second cell.
		<ol>
			<li>In the menu, click &#34;Add&#34; to start a new track. Repeat steps 2.3.5.1 - 2.3.5.4 for tracking the new cell. Click on &#34;Save&#34; to save the second track.</li>
			<li>Continue these steps for all trackable cells observed. After tracking the cells, click on &#34;Measure&#34; and &#34;OK&#34; in the menu tab to get the results. 
			NOTE: A results window opens up automatically, which can be saved and opened in a spreadsheet for further analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose. No financial or conflict of interest.

Studying the hemodynamics in the retinal and choroidal circulation is vital for understanding the pathophysiology of many ocular diseases. The blood flow dynamics in the retinal circulation can be studied by Fourier-domain optical coherence tomography (FD-OCT), laser speckle flowgraphy (LSFG) and retinal oximetry. Although these methods use different approaches to study the total blood flow in the retinal circulation40,41,42<sup...

Studying the flow dynamics of the blood cells in the retinal and choroidal circulation is imperative to understanding the pathogenesis of potentially vision-threatening ocular diseases and other ocular inflammatory conditions. However, the conventional angiography techniques, which involve the binding of fluorescent dyes to plasma proteins, do not provide any information regarding the dynamics of the erythrocytes or leukocytes1. The erythrocyte retinal flow dynamics are important for studying metabolically efficient circulation in the retina, and the leukocyte flow dynamics, for understanding the cell migration, recognition, adhesion and destruction in various inflammatory conditions2. There are several fluorescent molecules used in the identification and characterization of various cell types3. The hemodynamics of the blood cells can be measured by staining them with the appropriate fluorescent dyes and applying the proper imaging techniques4.
The presence of inflammatory responses in intraocular diseases like age-related macular degeneration (AMD) and diabetic retinopathy (DR) involve the accumulation of lymphocytes in the diseased area5,6. Tracking the immune cells in the tissues can help understand the complex events involved in the mechanism of disease pathogenesis. Radioactive isotopes like 51Cr and 125I were used as cell tracers in early studies. These dyes are toxic and affect the cell viability. Although the radioactive markers 3H and 14C are less toxic to the cells, due to their lower emission energies, it is difficult to detect their signals in the system7,8. A number of fluorochrome dyes were introduced to overcome the potential problems associated with radioactive markers and track lymphocyte migration in vitro using fluorescent microscopy and flow cytometry9,10. Hoechst 33342 and thiazole orange are DNA binding fluorescent dyes, which are used to track lymphocytes in vivo. Hoechst 33342 binds to AT-rich regions in the DNA, is membrane permeable, retains fluorescent signals for 2 - 4 days and is resistant to quenching9,10. The disadvantages of Hoechst 33342 and thiazole orange are the inhibition of lymphocyte proliferation11 and the short half-life, respectively9.
Calcein-AM, fluorescein diacetate (FDA), 2&#8242;,7&#8242;-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM), 5-(and-6)-carboxyfluorescein diacetate (CFDA), and 5-(and-6)-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) are the cytoplasmic fluorescent dyes used for lymphocyte migration studies. However, FDA, CFDA and CFDA-AM have lower retention in the cells9. BCECF-AM reduces the proliferative response and influences the chemotaxis and superoxide production9,12. Calcein-AM is a fluorescent dye and useful for short-term in vivo lymphocyte migration studies. It emits strong fluorescent signals, does not interfere with most of the cellular functions and retains fluorescent signals for up to 3 days12,13. Fluorescein isothiocyanate (FITC) and carboxyfluorescein diacetate succinimidyl ester (CFDA-SE) are covalent coupling fluorescent dyes, which are used for lymphocyte migration studies. FITC exhibits no effect on the cell viability and has a stronger affinity with B lymphocytes than T lymphocytes14,15. The CFDA-SE labeled lymphocytes can be tracked in vivo for more than 8 weeks and up to 8 cell divisions9,16. C18 DiI (1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindocarbocyanine perchlorate), DiO (3,3&#39;-dioctadecyloxacarbocyanine perchlorate), Paul Karl Horan (PKH)2, PKH3, and PKH26 are membrane-inserting fluorescent lipophilic carbocyanine dyes used to label leukocytes and erythrocytes. C18 Dil and DiO exhibit higher signals when incorporated into the cell membrane and are relatively non-toxic12,17. PKH2, PKH3 and PKH26 labeled cells exhibit a good retention of the fluorescent signals with less toxicity18,19,20,21,22. However, PKH2 down regulates the CD62L expression and reduces the lymphocyte viability23.
Most of the above-mentioned studies have been performed for tracking the lymphocyte migration and proliferation in the lymphatics and studying the labeled erythrocytes in the non-ocular circulation. There are very few studies applying the labeling techniques to study the blood cells in the ocular circulation. Application of scanning laser ophthalmoscopy (SLO) has a great advantage in studying the labeled cells in the retinal and choroidal circulation in vivo by fundus angiography24. There are several fluorescent dyes, such as ICG, acridine orange, FITC, sodium fluorescein, and CFDA that are used to study the leukocytes in the retinal circulation by SLO25,26,27,28,29,30,31,32,33,34. The phototoxicity and carcinogenicity of acridine orange26,27, the interference of FITC with the cellular activity, and the requirement of an intravascular contrast agent for the resolution of the retinal and choroidal blood vessels limits their application in in vivo animal experiments29. Sodium fluorescein and ICG are non-toxic, approved by the Food and Drug Administration, and safe for testing on humans32,35. Most of the flow dynamic studies are related to the labeling of the leukocytes or erythrocytes and its visualization in the retinal and choroidal blood vessels36,37,38,39. Here, we describe a standardized protocol of ICG labeling of the erythrocytes, sodium fluorescein labeling of the leukocytes, and tracking the visualized labeled cells in the mouse retinal circulation using SLO.

<table><tbody><tr><td>Cardiogreen polymethine dye (Indocyanine green)</td><td>Sigma Aldrich</td><td>12633-50MG</td><td></td></tr><tr><td>Fluorescein 100 mg/mL</td><td>Novartis</td><td>U1705A/H-1330292</td><td></td></tr><tr><td>10x Phosphate-buffered saline (PBS) Ultra Pure Grade</td><td>1st BASE</td><td>BUF-2040-10X1L</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>A7906-100G</td><td></td></tr><tr><td>Microtainer tubes with K2E (K2EDTA) - EDTA concentration - 1.8 mg/mL of blood</td><td>BD, USA</td><td>REF 365974</td><td></td></tr><tr><td>Histopaque 1077 solution</td><td>Sigma Aldrich</td><td>10771</td><td></td></tr><tr><td>Centrifuge 5810 R</td><td>Eppendorf</td><td>05-413-401</td><td></td></tr><tr><td>Microcentrifuge tubes 2 mL</td><td>Axygen</td><td>MCT-200-C-S</td><td></td></tr><tr><td>Vortex mixer</td><td>Insta BioAnalytik pte. ltd</td><td>FINE VORTEX</td><td></td></tr><tr><td>Shaker incubator</td><td>Lab Tech</td><td></td><td></td></tr><tr><td>Ceva Ketamine injection (Ketamine hydrochloride 100 mg/mL)</td><td>Ceva</td><td>KETALAB03</td><td></td></tr><tr><td>ILIUM XYLAZIL-20 (Xylazine hydrochloride 20 mg/Ml)</td><td>Troy Laboratories PTY. Limited</td><td>LI0605</td><td></td></tr><tr><td>1% Mydriacyl 15 mL (Tropicamide 1%)</td><td>Alcon&amp;nbsp;Laboratories, Inc. USA</td><td>NDC&amp;nbsp;0998-0355-15</td><td></td></tr><tr><td>2.5% Mydfrin 5 mL (Phenylephrine hydrochloride 2.5%)</td><td>Alcon&amp;nbsp;Laboratories, Inc. USA</td><td>NDC&amp;nbsp;0998-0342-05</td><td></td></tr><tr><td>Terumo syringe with needle 1 cc/mL Tuberculin</td><td>Terumo (Philippenes) Corporation, Philippines</td><td>SS-01T2613</td><td></td></tr><tr><td>Vidisic Gel 10 G</td><td>Dr. Gerhard Mann, Chem.-Pharm, Fabrik Gmbh, Berlin, Germany</td><td></td><td></td></tr><tr><td>Alcohol swabs</td><td>Assure medical disposables</td><td>7M-004-L-01</td><td></td></tr><tr><td>Confocal laser scanning angiography system (Heidelberg Retina Angiograph 2)</td><td>Heidelberg Engineering, GmbH, Heidelberg, Germany</td><td></td><td></td></tr><tr><td>Hiedelberg Spectralis Viewing Module software, v4.0</td><td>Heidelberg Engineering, GmbH, Heidelberg, Germany</td><td></td><td></td></tr><tr><td>Fluorescent microscope</td><td>ZEISS</td><td>Model: axio imager z1</td><td></td></tr></tbody></table>

fluorescent dye labeling of erythrocytes and leukocytes for studying the flow dynamics in mouse retinal circulation

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, diabetic retinopathy, age-related macular degeneration (AMD) and other ocular inflammatory conditions. It may also help to monitor the therapeutic responses in the eye. The proper labeling of the blood cells, coupled with live-cell imaging of the labeled cells, allows for the investigation of the flow dynamics in the retinal and choroidal circulation. Here, we describe the standardized protocols of 1.5% indocyanine green (ICG) and 1% sodium fluorescein labeling of mice erythrocytes and leukocytes, respectively. Scanning laser ophthalmoscopy (SLO) was applied to visualize the labeled cells in the retinal circulation of C57BL/6J mice (wild type). Both methods demonstrated distinct fluorescently labeled cells in the mouse retinal circulation. These labeling methods can have wider applications in various ocular disease models.

Erythrocytes labeled with 1.5% ICG were visualized in the retinal circulation of C57BL/6J mice (wild type). Both 1% and 5% hematocrit of 1.5% ICG labeled erythrocytes were distinguishable in the retinal circulation. However, the individual labeled cells were more clearly visualized with 1% hematocrit of 1.5% ICG labeled erythrocytes (Figure 1). In 5% hematocrit, due to the large number of labeled cells in the retinal vessels, it was not possible to mark the i...

Watch this Scientific Journal Video about Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation at JoVE.com

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

Live-cell imaging of the labeled blood cells in ocular circulation can provide information about inflammation and ischemia in diabetic retinopathy and age-related macular degeneration. A protocol to label blood cells and image the labeled cells in the retinal circulation is described.

The retinal and choroidal blood flow dynamics may provide insight into the pathophysiology and sequelae of various ocular diseases, such as glaucoma, ...

fluorescent-dye-labeling-erythrocytes-leukocytes-for-studying-flow

Research

JoVE Journal

Medicine

12.4K Views.  Tan Tock Seng Hospital.  Live-imaging van de gelabelde bloedcellen in de oculaire circulatie kan informatie verschaffen over ontsteking en ischemie bij diabetische retinopathie en leeftijdgerelateerde macular degeneratie. Een protocol om bloedcellen te labelen en de gemarkeerde cellen in de retinale circulatie af te beelden is beschreven. 

Video: Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

Intravital Microscopy of the Mouse Brain Microcirculation using a Closed Cranial Window

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, inflammatory and metabolic conditions, using in vivo fluorescence microscopy. A closed cranial window is placed over the left parieto-occipital cortex of the mice. Local microcirculation is recorded in real time through the window using epi and fluorescence illumination, and measurements of vessels diameters and red blood cell (RBC) velocities are performed. RBC velocity is measured using real-time cross-correlation and/or fluorescent-labeled erythrocytes. Leukocyte and platelet adherence to pial vessels and assessment of perfusion and vascular leakage are made with the help of fluorescence-labeled markers such as Albumin-FITC and anti-CD45-TxR antibodies. Microcirculation can be repeatedly video-recorded over several days. We used for the first time the close window brain intravital microscopy to study the pial microcirculation to follow dynamic changes during the course of Plasmodium berghei ANKA infection in mice and show that expression of CM is associated with microcirculatory dysfunctions characterized by vasoconstriction, profound decrease in blood flow and eventually vascular collapse.

Intravital microscopy to follow temporal and spatial hemodynamic and inflammatory events in the pial microcirculation.

This experimental model was designed to assess the mouse pial microcirculation during acute and chronic, physiological and pathophysiological hemodynamic, ...

Neuroscience

Flow Cytometric Analysis of Mitochondrial Reactive Oxygen Species in Murine Hematopoietic Stem and Progenitor Cells and MLL-AF9 Driven Leukemia

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from healthy mice as well as mice with AML driven by MLL-AF9. Specifically, we describe a two-step cell staining process, whereby healthy or leukemia BM cells are first stained with a fluorogenic dye that detects mitochondrial superoxides, followed by staining with fluorochrome-linked monoclonal antibodies that are used to distinguish various healthy and malignant hematopoietic progenitor populations. We also provide a strategy for acquiring and analyzing the samples by flow cytometry. The entire protocol can be carried out in a timeframe as short as 3-4 h. We also highlight the key variables to consider as well as the advantages and limitations of monitoring ROS production in the mitochondrial compartment of live hematopoietic and leukemia stem and progenitor subpopulations using fluorogenic dyes by flow cytometry. Furthermore, we present data that mitochondrial ROS abundance varies among distinct healthy HSPC sub-populations and leukemia progenitors and discuss the possible applications of this technique in hematologic research.

We describe a method for using multiparameter flow cytometry to detect mitochondrial reactive oxygen species (ROS) in murine healthy hematopoietic stem and progenitor cells (HSPCs) and leukemia cells from a mouse model of acute myeloid leukemia (AML) driven by MLL-AF9.

We present a flow cytometric approach for analyzing mitochondrial ROS in various live bone marrow (BM)-derived stem and progenitor cell populations from ...

Cancer Research

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered perfusion can aid research into the mechanisms of retinal pathologies. Intravital video microscopy of fluorescent tracers can be used to measure vascular diameters and bloodstream velocities of the retinal vasculature, specifically the arterioles branching from the central retinal artery and of the venules leading into the central retinal vein. Blood flow rates can be calculated from the diameters and velocities, with the summation of arteriolar flow, and separately venular flow, providing values of total retinal blood flow. This paper and associated video describe the methods for applying this technique to mice, which includes 1) the preparation of the eye for intravital microscopy of the anesthetized animal, 2) the intravenous infusion of fluorescent microspheres to measure bloodstream velocity, 3) the intravenous infusion of a high molecular weight fluorescent dextran, to aid the microscopic visualization of the retinal microvasculature, 4) the use of a digital microscope camera to obtain videos of the perfused retina, and 5) the use of image processing software to analyze the video. The same techniques can be used for measuring retinal blood flow rates in rats.

Intravital microscopy can be used in animals to visualize and measure retinal vascular diameters, bloodstream velocities, and total retinal blood flow.

Alterations in retinal blood flow can contribute to, or be a consequence of, ocular disease and visual dysfunction. Therefore, quantitation of altered ...

Tracking Mouse Bone Marrow Monocytes In Vivo

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal environment. Biological activities of immune cells mainly rely on their motility capacities. Blood monocytes have short half-life in the bloodstream; they originate in the bone marrow and are constitutively released from it. In inflammatory condition, this process is enhanced, leading to blood monocytosis and subsequent infiltration of the peripheral inflammatory tissues. Identifying the biomechanical events controlling monocyte trafficking from the bone marrow towards the vascular network is an important step to understand monocyte physiopathological relevance. We performed in vivo time-lapse imaging by two-photon microscopy of the skull bone marrow of the Csf1r-Gal4VP16/UAS-ECFP (MacBlue) mouse. The MacBlue mouse expresses the fluorescent reporters enhanced cyan fluorescent protein (ECFP) under the control of a myeloid specific promoter 1, in combination with vascular network labelling. We describe how this approach enables the tracking of individual medullar monocytes in real time to further quantify the migratory behaviour within the bone marrow parenchyma and the vasculature, as well as cell-to-cell interactions. This approach provides novel insights into the biology of the bone marrow monocyte subsets and allows to further address how these cells can be influenced in specific pathological conditions.

Tracking Mouse Bone Marrow Monocytes In Vivo

Monocytes are key regulators of innate immunity and play a critical role in the renewal of the peripheral mononuclear phagocytic system and in case of inflammation. This manuscript describes the procedure of real time imaging of the mouse calvaria bone marrow to study the monocyte mobilisation mechanism.

Real time multiphoton imaging provides a great opportunity to study cell trafficking and cell-to-cell interactions in their physiological 3-dimensionnal ...

Immunology and Infection

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, including phagocytosis of damaged synapses or cells, debris, and/or invading pathogens. Impaired phagocytic function has been implicated in the pathogenesis of diseases such as Alzheimer's and age-related macular degeneration, where amyloid-&#946; plaque and drusen accumulate, respectively. Despite its importance, microglial phagocytosis has been challenging to assess in vivo. Here, we describe a simple, yet robust, technique for precisely monitoring and quantifying the in vivo phagocytic potential of retinal microglia. Previous methods have relied on immunohistochemical staining and imaging techniques. Our method uses flow cytometry to measure microglial uptake of fluorescently labeled particles after intravitreal delivery to the eye in live rodents. This method replaces conventional practices that involve laborious tissue sectioning, immunostaining, and imaging, allowing for more precise quantification of microglia phagocytic function in just under six hours. This procedure can also be adapted to test how various compounds alter microglial phagocytosis in physiological settings. While this technique was developed in the eye, its use is not limited to vision research.

Assessing Retinal Microglial Phagocytic Function In Vivo Using a Flow Cytometry-based Assay

Microglial phagocytosis is critical for the maintenance of tissue homeostasis and inadequate phagocytic function has been implicated in pathology. However, assessing microglia function in vivo is technically challenging. We have developed a simple but robust technique for precisely monitoring and quantifying the phagocytic potential of microglia in a physiological setting.

Microglia are the tissue resident macrophages of the central nervous system (CNS) and they perform a variety of functions that support CNS homeostasis, ...

Identification of a Murine Erythroblast Subpopulation  Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation.&#160;We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.
We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation.&#160;Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry.&#160;Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters &#8220;aspect ratio&#8221; of a cell in the bright-field channel that aids in the recognition of elongated cells and &#8220;delta centroid XY Ter119/Draq5&#8221; that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate.&#160;The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.

The present protocol describes a novel method of identifying a population of enucleating orthochromatic erythroblasts by multi-spectral imaging flow cytometry, providing a visualization of the erythroblast enucleation process.

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not ...

Biology

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...

Dynamic Measurement and Imaging of Capillaries, Arterioles, and Pericytes in Mouse Heart

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion pressure. However, it is difficult to monitor the dynamic changes of the coronary arterioles and the capillaries in the whole heart, primarily due to its motion and non-stop beating. Here we describe a method that enables monitoring of arterial perfusion rate, pressure and the diameter changes of the arterioles and capillaries in mouse right ventricular papillary muscles. The mouse septal artery is cannulated and perfused at a constant flow or pressure with the other dynamically measured. After perfusion with a fluorescently labeled lectin (e.g., Alexa Fluor-488 or -633 labeled Wheat-Germ Agglutinin, WGA), the arterioles and capillaries (and other vessels) in right ventricle papillary muscle and septum could be readily imaged. The vessel-diameter changes could then be measured in the presence or absence of heart contractions. When genetically encoded fluorescent proteins were expressed, specific features could be monitored. For examples, pericytes were visualized in mouse hearts that expressed NG2-DsRed. This method has provided a useful platform to study the physiological functions of capillary pericytes in heart. It is also suitable for studying the effect of reagents on the blood flow in heart by measuring the vascular/capillary diameter and the arterial luminal pressure simultaneously. This preparation, combined with a state-of-the-art optic imaging system, allows one to study the blood flow and its control at cellular and molecular level in the heart under near-physiological conditions.

Presented here is a protocol to study the coronary microcirculation in living murine heart tissue by ex vivo monitoring of the arterial perfusion pressure and flow that maintains the pressure, as well as vascular tree components including the capillary beds and pericytes, as the septal artery is cannulated and pressurized.

Coronary arterial tone along with the opening or closing of the capillaries largely determine the blood flow to cardiomyocytes at constant perfusion ...

Measurement of Strial Blood Flow in Mouse Cochlea Utilizing an Open Vessel-Window and Intravital Fluorescence Microscopy

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining endocochlear potential, ion transport, and fluid balance. Different forms of hearing disorders are reported to involve abnormal microcirculation in the cochlea. Investigation of how cochlear blood flow (CoBF) pathology affects hearing function is challenging due to the lack of feasible interrogation methods and the difficulty in accessing the inner ear. An open vessel-window in the lateral cochlear wall, combined with fluorescence intravital microscopy, has been used for studying CoBF changes in vivo, but mostly in guinea pig and only recently in the mouse. This paper and the associated video describe the open vessel-window method for visualizing blood flow in the mouse cochlea. Details include 1) preparation of the fluorescent-labeled blood cell suspension from mice; 2) construction of an open vessel-window for intravital microscopy in an anesthetized mouse, and 3) measurement of blood flow velocity and volume using an offline recording of the imaging. The method is presented in video format to show how to use the open window approach in mouse to investigate structural and functional changes in the cochlear microcirculation under normal and pathological conditions.

An open vessel-window approach using fluorescent tracers provides sufficient resolution for cochlear blood flow (CoBF) measurement. The method facilitates the study of structural and functional changes in CoBF in mouse under normal and pathological conditions.

Transduction of sound is metabolically demanding, and the normal function of the microvasculature in the lateral wall is critical for maintaining ...

Monitoring Dynamic Growth of Retinal Vessels in Oxygen-Induced Retinopathy Mouse Model

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), proliferative diabetic retinopathy (PDR), and retinal vein occlusion (RVO). Most OIR studies observe retinal neovascularization at specific time points; however, the dynamic vessel growth in live mice along a time course, which is essential for understanding the OIR-related vessel diseases, has been understudied. Here, we describe a step-by-step protocol for the induction of the OIR mouse model, highlighting the potential pitfalls, and providing an improved method to quickly quantify areas of vaso-obliteration (VO) and neovascularization (NV) using immunofluorescence staining. More importantly, we monitored vessel regrowth in live mice from P15 to P25 by performing fluorescein fundus angiography (FFA) in the OIR mouse model. The application of FFA to the OIR mouse model allows us to observe the remodeling process during vessel regrowth.

This protocol describes a detailed method for the preparation and immunofluorescence staining of mice retinal flat mounts and analysis. The use of fluorescein fundus angiography (FFA) for mice pups and image processing are described in detail as well.

Oxygen-induced retinopathy (OIR) is widely used to study abnormal vessel growth in ischemic retinal diseases, including retinopathy of prematurity (ROP), ...

Fluorescent Dye Labeling of Erythrocytes and Leukocytes for Studying the Flow Dynamics in Mouse Retinal Circulation

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