Name: longitudinal in vivo imaging and quantification of human pancreatic islet grafting and contributing host cells in the anterior eye chamber
Uploaded: 2020-06-11T14:30:00.000+00:00
Duration: 9 min 7 s
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 transplantation of human islets10.
1. Islet preparation for transplantation
<ol>
	<li>Culture human islets in CMRL 1066 supplemented with 10 mM HEPES, 2 mM L-glutamine, 50 &#956;g/mL gentamycin, 0.25 &#956;g/mL fungizone, 20 &#956;g/mL ciproxfloxacin, 10 mM nicotinamide (NIC), and 10% heat-inactivated human serum at 37 &#176;C in 5% CO2 and humidified air until transplantation, as described previously12. 
	NOTE: Islets should be free of exocrine tissue and not touch each other in culture. Exocrine tissues appear translucent.</li>
	<li>On the day of transplantation, transfer culture media containing the islets to a new Petri dish using an aspirator tube assembly connected to a pulled glass capillary. 
	NOTE: Alternatively, use a 200 &#181;L pipette. Coloring the back of the Petri dish helps make the islets more easily distinguishable under the stereo microscope.</li>
	<li>Using a stereo microscope, pick ~20&#8211;40 islets per transplantation and transfer to a 1.5 mL tube. Fill the tube to the top with culture media from the incubator.</li>
	<li>Seal the tubes with paraffin film and store on ice until transplantation. Prepare an appropriate amount for the number of transplantations performed.</li>
	<li>Alternatively, ensure a CO2 incubator is available in the surgery room to keep islets in culture and pick them immediately prior to each transplantation.</li>
</ol>
2. Preparation of transplantation equipment and surgery table
NOTE: All surgical tools should be autoclaved, and the surgery table and instruments disinfected with 70% alcohol.
<ol>
	<li>Connect a stereotaxic head holder to anesthesia via a nose mask and turn on the heating pad.</li>
	<li>Connect a gastight Hamilton syringe to polyethylene tubing and a blunt end eye cannula. 
	NOTE: It is recommended to fill all parts with PBS before assembly. Check for trapped air bubbles and remove if present.</li>
	<li>Attach the Hamilton syringe tightly to the table (Figure 1a) or a movable base (Figure 1e) and attach the tubing to the stereo microscope, with cannula hanging down (i.e., waiting position). 
	NOTE: Use surgical tape, because it is easy to remove and reattach.</li>
	<li>Prepare a 1 mL syringe connected to a 30 G needle filled with 0.1 mg/kg buprenorphine solution.</li>
	<li>Prepare a syringe with sterile PBS. Alternatively, use a pipette.</li>
	<li>Set aside a clean wake-up cage with heating lamp.</li>
</ol>
3. Anesthesia and positioning of recipient mice for surgery
NOTE: All animals were bred and maintained in a pathogen-free environment at the animal facilities at Lund University.
<ol>
	<li>Anesthetize the mouse in a chamber filled with 40% O2/60% N2/3% isoflurane and transfer the anesthetized mouse to the head holder platform on a warm heating pad (Figure 1a). Check for the lack of pedal reflexes. 
	NOTE: Isoflurane anesthesia is the preferred method of anesthesia for fast recovery after surgery. The microscope room must be properly ventilated to use isoflurane.</li>
	<li>Place the snout of the mouse into the anesthesia mask connected to 40% O2/60% N2/0.9%&#8211;1.5% isoflurane anesthesia machine. Use the thumb and finger to lift the head up slightly and fasten it using the metallic pieces on the sides. Ensure that the earpieces fix the head directly below the ears. Inject 0.1 mg/kg buprenorphine solution subcutaneously on the back of the mouse. 
	NOTE: Buprenorphine is used as an analgesic.</li>
	<li>Tilt the head so that the eye to be operated on is facing upwards and is close to the researcher.</li>
	<li>Gently retract the eyelids of the eye to be transplanted using blunt forceps, pop the eye out, and loosely fix with a pair of tweezers. Ensure that the tips of the tweezers are covered with a polythene tube attached to the head holder platform (Figure 1a, insert).</li>
	<li>Always keep both eyes wet by applying a droplet of sterile PBS onto the eye.</li>
	<li>Transfer the human islets from the sealed 1.5 mL tube (section 1) to a Petri dish with sterile PBS and make sure that the islets are close to each other to minimize the amount of cell culture media transferred (Figure 1c).</li>
	<li>Pick up ~20&#8211;30 islets in the eye cannula connected via polythene tubing to the Hamilton syringe. 
	NOTE: Take up as little liquid as possible with the islets.</li>
	<li>Hang the tubing upside down and attach to the stereo microscope (Figure 1d). Tape the tubing carefully to let the islets sink to the end of the tube toward the cannula.</li>
</ol>
4. Transplantation procedure
NOTE: This method has been previously described for the transplantation of mouse islets6. A slightly modified procedure is presented here.
<ol>
	<li>Pinch the pads on the hind legs to make sure that the mouse is asleep.</li>
	<li>Tighten the forceps restraining the eye without disrupting the blood flow and apply a droplet of sterile PBS onto the eye.</li>
	<li>Using a 25 G needle as a scalpel, bevel upwards, carefully penetrate only half of the tip in the cornea and make a single lateral incision. Make the hole in an upward angle; the hole will seal more easily after the transplant (Figure 1f).</li>
	<li>Carefully lift up the cornea with the cannula preloaded with islets and slowly apply islets in the eye. Avoid insertion of the cannula into the anterior chamber to prevent damage of the iris, but rather push carefully against the corneal opening (Figure 1g).&#160;&#160;Slowly retract the cannula from the ACE. 
	NOTE: Aim for an injection volume of 3&#8211;8 &#181;L. If the volume is too large, it will expose the eye to unnecessarily high intraocular pressure and may result in reflux of the injected islets out of the anterior chamber.</li>
	<li>When facing difficulties with insertion of the islets due to increased pressure in the eye chamber, enlarge the incision site by reinforcing the lateral incision site and reapply islets. 
	NOTE: Occasionally, introduced air bubbles can be used as space holders.</li>
	<li>Apply eye gel to the eye, loosen the eye-restraining forceps and leave the mouse on isoflurane in the same position for 8&#8211;10 min to let the islets set.</li>
	<li>Remove the forceps holding the eyelid and put the eyelid back to its normal position.</li>
	<li>Remove the mouse from the head holder and transfer it to a wake up cage.</li>
	<li>When the mouse is awake and moving, transfer it back to the original cage and keep in the animal housing until scanning (at least 5 days are recommended).</li>
</ol>
5. Imaging of implanted human islets by 2-photon microscopy
NOTE: Taking overview images of the eye using a fluorescence stereoscopic microscope (Figure 2a&#8211;c) 4&#8211;5 days after transplantation prior to 2-photon imaging is recommended to localize the islets of interest. Avoid restraining the eye too tightly this early after transplantation. Use 2-photon imaging 6&#8211;7 days posttransplantation.
<ol>
	<li>Start the image acquisition software (see Table of Materials). In the &#8220;Laser&#8221; menu activate the Mai Tai laser (Power &#8220;ON&#8221;) and in the &#8220;Light Path&#8221; menu set the wavelength to 900 nm and apply a minimal transmission laser power starting with 5%&#8211;10% laser power (use sliders). 
	NOTE: While scanning, adjust the laser power as needed.</li>
	<li>Set green, orange, and red channels. Collect emission light simultaneously onto three nondescanned detectors (NDD) using a dichronic mirror (LBF 760) and emission filter information as follows: Red/Angiosense 680, 690&#8211;730 nm; Green/Autofluorescence, 500&#8211;550 nm; and Orange/Tomato, 565&#8211;610nm (Figure 2d).</li>
	<li>Place the head holder stage onto the motorized microscope stage and connect the gasmask to the tubing of the anesthesia machine and tubing connected to ventilation system. Turn on the heating pad.</li>
	<li>Anesthetize the recipient mouse, transfer to the head holder platform, restrain the eye for imaging, and administer buprenorphine solution as described above (steps 3.1&#8211;3.5).</li>
	<li>Adjust isoflurane vapors as needed. A breath rate of ~55&#8211;65 breaths per minute (bpm) indicates optimal anesthesia. If anesthesia is too deep, the rate will be &#60;50 bpm with heavy breathing or gasping; if too light, the rate will be &#62;70 bpm with superficial breathing. Carefully monitor mice during anesthesia by visual inspection every 15 min. 
	NOTE: Anesthetization varies from mouse to mouse, between mouse strains, and as time under anesthesia progresses13.</li>
	<li>Administer enough eye gel onto the eye as an immersion liquid between the cornea and the lens, allowing it to slowly accumulate (Figure 2f, insert). Avoid air bubbles. 
	NOTE: Side illumination with a flexible metal hose lamp is recommended to adjust the focus and localize islet grafts.</li>
	<li>To visualize blood vessels, administer 100 &#181;L of the imaging agent (e.g., Angiosense 680) intravenously into the tail vein using a disposable insulin 30 G syringe.</li>
	<li>In &#8220;Acquisition mode&#8221; adjust the frame size to 512 x 512 and the scan speed. 
	NOTE: Slower scans (i.e., increasing dwell time) will improve the signal-to-noise ratio.</li>
	<li>In the &#8220;Channels&#8221; menu adjust Master Gain for each PMT in Volts to amplify the signal until an image is seen on screen in the Live scanning mode. The higher this value, the more sensitive the detector becomes to signal and noise. 
	NOTE: Preferably, keep values between 500&#8211;800 V.</li>
	<li>In the &#8220;Z-stack&#8221; menu define the beginning and the end of the z-stack by manually moving the focus to the top of the islet graft. Save position by selecting &#8220;Set First&#8221;. Move to the last bottom plane that can be focused in the islet graft and save position by selecting &#8220;Set Last&#8221;. Use a z-step size of 2 &#181;m.</li>
	<li>Collect the final image stack by clicking the &#8220;Start Experiment&#8221; tab and save as 8-bit CZI (i.e., Carl Zeiss format) file.</li>
</ol>
6. Imaging of implanted human islets by confocal microscopy
NOTE: The total volume, morphology, and plasticity of transplanted islets can be assessed by monitoring the in vivo scattering signal in a separate scan (i.e., separate track) by detection of laser backscatter light10.
<ol>
	<li>Take out the main beam splitter (i.e., LBF Filter) and in the &#8220;Light Path&#8221; dialog set up a separate track for confocal imaging. Choose the Argon laser with wavelength of 633 nm and detection at the same wavelength as the laser light. Z-stacks are acquired with a step size of 2&#8211;3 &#181;m for backscatter light signal.</li>
	<li>Readjust the z-stack settings to make sure to record the whole islet (see step 5.10).</li>
	<li>Acquire the image stack and save as 8 bit CZI file.</li>
</ol>
7. Image analysis
NOTE: Commercial software (see Table of Materials) was used for this step.
<ol>
	<li>Removing islet autofluorescence (Figure 3b)
	<ol>
		<li>In the &#8220;Image processing&#8221; tab choose &#8220;Channel arithmetic&#180;s&#8221; and type &#8220;ch1-ch2&#8221;. This creates a new channel 4 (ch 4); rename as &#8220;Vasculature&#8221;. 
		NOTE: The Green/Autofluorescence channel is subtracted from Red/Angiosense channel.</li>
		<li>Repeat the previous step and type &#8220;ch3-ch2&#8221; to create a new channel (ch 5); rename as &#8220;Tomato (all)&#8221;. 
		NOTE: The Green/Autofluorescence channel is subtracted from the Orange/Tomato channel.</li>
	</ol>
	</li>
	<li>Defining islet mask by manual drawing (Figure 3c)
	<ol>
		<li>Create a new surface (blue symbol) and in the wizard choose &#8220;Edit manually&#8221;. Keep the pointer in &#8220;Select&#8221; mode and in 3D view unclick &#8220;Volume&#8221; (under Scene) to visualize sections.</li>
		<li>For easier islet border discrimination, activate all channels, including ch 1&#8211;ch 3. 
		NOTE: The Orange/Tomato channel is useful to define islet borders by the Tomato capsule signal. Alternatively, increase channel intensities to use multichannel islet autofluorescence and detector background signal as guidance.</li>
		<li>In the &#8220;Drawing&#8221; tab choose &#8220;Contour&#8221; and click &#8220;Draw&#8221; to start drawing contours around the islet border starting in slice position 1.</li>
		<li>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 &#8220;Create surface&#8221; tab. Usually it is enough to draw contours every 10th slice.</li>
	</ol>
	</li>
	<li>Segmentation of &#8220;Islet vasculature&#8221; and &#8220;Islet tomato&#8221; fluorescence using islet mask (Figure 3d).
	<ol>
		<li>Choose the previously defined &#8220;Islet mask&#8221; object, go to the editing tab (pencil symbol), and click the &#8220;Mask all&#8221; tab, which opens a new window.</li>
		<li>Choose the previously named channel &#8220;Vasculature&#8221; (ch 4) in the channel selection dropdown menu and activate options &#8220;Duplicate channel before applying mask&#8221;, &#8220;Constant inside/outside&#8221;, and set voxels outside surface to &#8220;0.000&#8221;, which creates a new channel; rename as &#8220;Islet vasculature&#8221; (ch 6).</li>
		<li>Repeat steps 7.3.1 and 7.3.2 and choose the previously created channel &#8220;Tomato (all)&#8221; (ch 5) in the channel selection drop down menu to create the new channel; rename as &#8220;Islet tomato&#8221; (ch 7).</li>
	</ol>
	</li>
	<li>Surface rendering of islet vasculature (Figure 3e)
	<ol>
		<li>Create a new surface in the &#8220;Scene&#8221; menu and in the wizard choose &#8220;Automatic creation&#8221;.</li>
		<li>Set the source channel to previously created &#8220;Islet vasculature&#8221; (ch 6) and choose background subtraction. Automatic threshold estimation can be adjusted if needed. Compare to the responding fluorescence channel (e.g., by blending in/out the newly created surface tab). Proceed in the wizard.</li>
		<li>Optionally, use filters. For example, choose &#8220;Volume&#8221; and adjust the filter (yellow) in the window, which can remove selected surface objects. Finish the wizard and name the new surface object &#8220;Islet vasculature&#8221;.</li>
	</ol>
	</li>
	<li>Segmentation of &#8220;Islet tomato vasculature&#8221; fluorescence signal (Figure 3f)
	<ol>
		<li>In the previously created &#8220;Islet vasculature&#8221; surface object, go to editing tab and click the &#8220;Mask all&#8221; tab, which opens a new window.</li>
		<li>Choose the previously named channel &#8220;Islet tomato&#8221; (ch 7) in the channel selection drop down menu and set voxels outside surface to &#8220;10.000&#8221;, which creates the new channel; rename as &#8220;Islet tomato vasculature&#8221; (ch 8).</li>
	</ol>
	</li>
	<li>Segmentation of &#8220;Tomato capsule&#8221; fluorescence signal (Figure 3g)
	<ol>
		<li>Choose &#8220;Channel Arithmetic&#8217;s&#8221; in the &#8220;Image processing&#8221; tab and type &#8220;ch7-ch8&#8221;, creating the new channel; rename as &#8220;Tomato capsule&#8221; (ch 9). 
		NOTE: The &#8220;Islet tomato vasculature&#8221; fluorescence signal is subtracted from the total &#8220;Islet tomato&#8221; fluorescence signal.</li>
	</ol>
	</li>
	<li>Surface rendering of &#8220;Islet tomato vasculature&#8221; and &#8220;Tomato capsule&#8221; (Figure 3h)
	<ol>
		<li>Follow step 7.4, and in the wizard choose source channels &#8220;Islet tomato vasculature&#8221; (ch 8) or &#8220;Tomato capsule&#8221; (ch 9) to create new surface objects accordingly.</li>
	</ol>
	</li>
	<li>Surface rendering of total islet surface (Figure 3i)
	<ol>
		<li>Open the islet backscatter file and create a new surface.</li>
		<li>In the wizard, choose &#8220;Automatic creation&#8221; and define &#8220;Region of interest&#8221;. 
		NOTE: &#34;Region of interest&#34; is used to separate the signals of multiple islets and to define the depth of the islet to be analyzed (e.g., top 75 &#181;m).</li>
		<li>In &#8220;Absolute intensity&#8221; adjust threshold if needed. The surface object can be clicked on or off to cross-check with corresponding channel intensity. Close the wizard.</li>
	</ol>
	</li>
	<li>Quantification (Figure 3j)
	<ol>
		<li>Select a created surface object in the &#8220;Scene&#8221; menu and go to the &#8220;Statistics&#8221; tab.</li>
		<li>To retrieve detailed volume data in the selected surface object choose the &#8220;Detailed&#8221; tab and select &#8220;Specific values&#8221; and &#8220;Volume&#8221; from dropdown menu. To retrieve a total volume value of the selected surface object, go to the &#8220;Detailed&#8221; tab and choose &#8220;Average values&#8221;.</li>
	</ol>
	</li>
</ol>

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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PET/MRI+enables+simultaneous+in+vivo+quantification+of+beta-cell+mass+and+function.">PET/MRI enables simultaneous in vivo quantification of beta-cell mass and function.</a> Theranostics. 10 (1), 398-410 (2020).</li><li>Wang, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+of+Allogeneic+Islet+Grafts+in+Nonhuman+Primates+Using+MRI.">Monitoring of Allogeneic Islet Grafts in Nonhuman Primates Using MRI.</a> Transplantation. 99 (8), 1574-1581 (2015).</li><li>Gotthardt, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+quantification+of+beta+cells+by+PET+imaging:+why+clinical+implementation+has+never+been+closer.">Detection and quantification of beta cells by PET imaging: why clinical implementation has never been closer.</a> Diabetologia. 61 (12), 2516-2519 (2018).</li><li>Joosten, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+Pancreatic+beta+Cell+Mass+in+Vivo+with+Exendin+SPECT+during+Hyperglycemia+and+Severe+Insulitis.">Measuring the Pancreatic beta Cell Mass in Vivo with Exendin SPECT during Hyperglycemia and Severe Insulitis.</a> Molecular Pharmaceutics. 16 (9), 4024-4030 (2019).</li><li>Virostko, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+in+mouse+models+quantifies+beta+cell+mass+in+the+pancreas+and+after+islet+transplantation.">Bioluminescence imaging in mouse models quantifies beta cell mass in the pancreas and after islet transplantation.</a> Molecular Imaging and Biology. 12 (1), 42-53 (2010).</li></ol>

The authors have nothing to disclose.

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&amp;eacute;</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?), 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>&lt;strong&gt;Head holder&lt;/strong&gt;</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>&lt;strong&gt;Human islet culture media&lt;/strong&gt;</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>&lt;strong&gt;2-Photon/confocal microscope&lt;/strong&gt;</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>&lt;strong&gt;Stereomicroscope (Flourescence)&lt;/strong&gt;</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 ...

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

5.1K Views.  Lund University. 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.

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

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 ...

Murine Pancreatic Imaging Using Implanted Stabilized Window

Stabilized Window for Intravital Imaging of the Murine Pancreas

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have high morbidity and mortality. Intravital imaging (IVI) is a powerful technique enabling the high-resolution imaging of tissues in both healthy and diseased states, allowing for real-time observation of cell dynamics. IVI of the murine pancreas presents significant challenges due to the deep visceral and compliant nature of the organ, which make it highly prone to damage and motion artifacts. 
Described here is the process of implantation of the Stabilized Window for Intravital imaging of the murine Pancreas (SWIP). The SWIP allows IVI of the murine pancreas in normal healthy states, during the transformation from the healthy pancreas to acute pancreatitis induced by cerulein, and in malignant states such as pancreatic tumors. In conjunction with genetically labeled cells or the administration of fluorescent dyes, the SWIP enables the measurement of single-cell and subcellular dynamics (including single-cell and collective migration) as well as serial imaging of the same region of interest over multiple days. 
The ability to capture tumor cell migration is of particular importance as the primary cause of cancer-related mortality in PDAC is the overwhelming metastatic burden. Understanding the physiological dynamics of metastasis in PDAC is a critical unmet need and crucial for improving patient prognosis. Overall, the SWIP provides improved imaging stability and expands the application of IVI in the healthy pancreas and malignant pancreas diseases.

Author Spotlight: Revolutionizing Pancreatic Disease Understanding Through Advanced Intravital Imaging 

We present a protocol for the surgical implantation of a stabilized indwelling optical window for subcellular-resolution imaging of the murine pancreas, allowing serial and longitudinal studies of the healthy and diseased pancreas.

The physiology and pathophysiology of the pancreas are complex. Diseases of the pancreas, such as pancreatitis and pancreatic adenocarcinoma (PDAC) have ...

Cancer Research

In situ Quantification of Pancreatic Beta-cell Mass in Mice

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic beta-cell proliferation and islet growth is particularly challenging. We have developed a method to capture the distribution of beta-cells in the intact pancreas of transgenic mice with fluorescence-tagged beta-cells with a macro written for ImageJ (rsb.info.nih.gov/ij/). Following pancreatic dissection and tissue clearing, the entire pancreas is captured as a virtual slice, after which the GFP-tagged beta-cells are examined. The analysis includes the quantification of total beta-cell area, islet number and size distribution with reference to specific parameters and locations for each islet and for small clusters of beta-cells. The entire distribution of islets can be plotted in three dimensions, and the information from the distribution on the size and shape of each islet allows a quantitative and qualitative comparison of changes in overall beta-cell area at a glance.

In situ Quantification of Pancreatic Beta-cell Mass in Mice

The following protocol outlines the process of pancreatic dissection for virtual slice imaging, and the subsequent quantification of all GFP-tagged beta-cells in the entire pancreas.

Tracing changes of specific cell populations in health and disease is an important goal of biomedical research. The process of monitoring pancreatic ...

Biology

Intraportal Transplantation of Pancreatic Islets in Mouse Model

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation site in experimental islet transplantation is the kidney capsule. However, as less is known about the interaction of pancreatic islets with blood constituents, it also makes sense to utilize the portal vein approach in experimental islet transplantation.
This protocol demonstrates an intraportal islet transplantation technique in NMRI nude mice. Streptozotocin (180 mg/kg) is injected intraperitoneally to induce hyperglycemia in recipient mice. They are considered as diabetic at a non-fasting blood glucose level greater than 20 mmol/L. One day prior to transplantation, mouse pancreatic islets are isolated from the donor pancreas by collagenase digestion; a minimum of 350 islets are utilized per diabetic recipient. Depending upon the islet isolation yield, two or more donor mice are utilized per recipient. After overnight culture at 37 &#176;C, islets are administered into the recipient liver via the portal vein. After surgery, the mice are protected in red Makrolon houses and observed until are awake. This protocol maintains glycemic control for 120 days in syngeneic mice and 15 days in allogeneic mice.

Pancreatic islet transplantation is a way to achieve normoglycemia in type 1 diabetes. This article focuses on the transplantation technique through the intraportal route to maintain normal glucose levels in diabetic mice.

Pancreatic islet transplantation to reduce hyperglycemia is highly successful in rodents with chemically-induced diabetes. The most common transplantation ...

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging study, with an abdominal imaging window, enabled visualization of the cellular dynamics in abdominal organs in vivo. However, due to the soft sheet-like architecture of the mouse pancreas that can be easily influenced by physiologic movement (e.g., peristalsis and respiration), it was difficult to perform stabilized longitudinal in vivo imaging over several weeks at the cellular level to identify, track, and quantify islets or cancer cells in the mouse pancreas. Herein, we describe a method for implanting a novel supporting base, an integrated pancreatic intravital imaging window, that can spatially separate the pancreas from the bowel for longitudinal time-lapse intravital imaging of the pancreas microstructure. Longitudinal in vivo imaging with the imaging window enables stable visualization, allowing for the tracking of islets over a period of 3 weeks and high-resolution three-dimensional imaging of the microstructure, as evidenced here in an orthotopic pancreatic cancer model. With our method, further intravital imaging studies can elucidate the pathophysiology of various diseases involving the pancreas at the cellular level.

Stabilized Longitudinal In Vivo Cellular-Level Visualization of the Pancreas in a Murine Model with a Pancreatic Intravital Imaging Window

In vivo high-resolution imaging of the pancreas was facilitated with the pancreatic intravital imaging window.

Direct in vivo cellular-resolution imaging of the pancreas in a live small animal model has been technically challenging. A recent intravital imaging ...

A Method for Islet Transplantation to the Omentum in Mouse

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet infusion is far from ideal and therefore, the omentum is re-emerging as a potentially valuable site for islet transplantation. This experiment requires the isolation of high quality islets and the implantation of the islets to the diabetic recipients. Transplantation to the omentum requires surgical steps that can be better demonstrated visually. Here, the detailed steps for this procedure are presented. Two methods of mixing the isolated islets with hydrogel before placing the mixture into the omental pouch of diabetic mice are described here. Different hydrogels are used for the different conditions. Blood glucose levels of diabetic mouse recipients of syngeneic islets in the omentum were monitored for up to 35 days. Some animals were sacrificed after 14 days to perform immuno-histochemical analysis. This pre-clinical transplantation approach can be used as preliminary data leading up to translation to clinical transplantation.

A method for the omental transplantation of islets in a mouse is introduced. The isolated islets are mixed with hydrogel and the mixture is placed into the omental pouch of a diabetic mouse. Then, the blood glucose is monitored, and immuno-histochemical analysis is performed.

Islet transplantation has been proposed to be a potential treatment for type 1 diabetes. Recent compelling evidence indicates that intravascular islet ...

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult mice for in vivo longitudinal real-time monitoring of thymocytes&#180; dynamics within a vascularized thymus segment. Following the transplantation, laser scanning microscopy (LSM) through the cornea allows in vivo noninvasive repeated imaging at cellular resolution level. Importantly, the approach adds to previous intravital T-cell maturation imaging models the possibility for continuous progenitor cell recruitment and mature T-cell egress recordings in the same animal. Additional advantages of the system are the transparency of the grafted area, permitting macroscopic rapid monitoring of the implanted tissue, and the accessibility to the implant allowing for localized in addition to systemic treatments. The main limitation being the volume of the tissue that fits in the reduced space of the eye chamber which demands for lobe trimming. Organ integrity is maximized by dissecting thymus lobes in patterns previously shown to be functional for mature T-cell production. The technique is potentially suited to interrogate a milieu of medically relevant questions related to thymus function that include autoimmunity, immunodeficiency and central tolerance; processes which remain mechanistically poorly defined. The fine dissection of mechanisms guiding thymocyte migration, differentiation and selection should lead to novel therapeutic strategies targeting developing T cells.

Real Time In Vivo Tracking of Thymocytes in the Anterior Chamber of the Eye by Laser Scanning Microscopy

The goal of the protocol is to show longitudinal intravital real-time tracking of thymocytes by laser scanning microscopy in thymic implants in the anterior chamber of the mouse eye. The transparency of the cornea and vascularization of the graft allows for continuously recording progenitor cell recruitment and mature T-cell egress.

The purpose of the method being presented is to show, for the first time, the transplant of newborn thymi into the anterior eye chamber of isogenic adult ...

Bioluminescent Monitoring of Graft Survival in an Adoptive Transfer Model of Autoimmune Diabetes in Mice

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease is the transplantation of stem cell-derived beta cells. Genetic modifications, however, may be necessary to protect the transplanted cells from persistent autoimmunity. Diabetic mouse models are a useful tool for the preliminary evaluation of strategies to protect transplanted cells from autoimmune attack. Described here is a minimally invasive method for transplanting and imaging cell grafts in an adoptive transfer model of diabetes in mice. In this protocol, cells from the murine pancreatic beta cell line NIT-1 expressing the firefly luciferase transgene luc2 are transplanted subcutaneously into immunodeficient non-obese diabetic (NOD)-severe combined immunodeficient (scid) mice. These mice are simultaneously injected intravenously with splenocytes from spontaneously diabetic NOD mice to transfer autoimmunity. The grafts are imaged at regular intervals via non-invasive bioluminescent imaging to monitor the cell survival. The survival of mutant cells is compared to that of control cells transplanted into the same mouse.

This protocol describes a straightforward and minimally invasive method for transplanting and imaging NIT-1 cells in non-obese diabetic (NOD)-severe combined immunodeficient mice challenged with splenocytes purified from spontaneously diabetic NOD mice.

Type 1 diabetes is characterized by the autoimmune destruction of the insulin-producing beta cells of the pancreas. A promising treatment for this disease ...

Noninvasive Intraocular In Vivo Imaging of Engrafted Liver Spheroids in Mice

In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until now, optical imaging of the liver in situ is possible by intravital imaging, which offers high-resolution imaging at the cellular level but cannot be performed multiple times and, therefore, longitudinally in the same animal. Noninvasive imaging methods, such as bioluminescence, allow repeated imaging sessions on the same animal but do not achieve cell resolution. To address this methodology gap, we have developed a platform for noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. In the workflow described in this study, primary mouse liver spheroids are generated in vitro and transplanted into the anterior chamber of the eye of recipient mice, where they engraft on the iris. The cornea acts as a natural body window through which we can image the engrafted spheroids by conventional confocal microscopy. The spheroids survive for months in the eye, during which the cells can be studied in contexts of health and disease, as well as being monitored in response to different stimuli over repeated imaging sessions using appropriate fluorescent probes. In this protocol, we provide a breakdown of the necessary steps to implement this imaging system and explain how to best harness its potential.

Author Spotlight: Harnessing Mouse Eye Chambers for Noninvasive Liver Spheroid Studies

Here, we describe a platform that allows noninvasive in vivo imaging of liver spheroids engrafted in the anterior chamber of the mouse eye. The workflow spans from generating spheroids from primary liver cells to transplantation into the mouse eye and in vivo imaging at cellular resolution by confocal microscopy.

Biomedical studies of the liver in mammals are hindered by the lack of methods for in vivo noninvasive longitudinal imaging at cellular resolution. Until ...

Grafting Pancreatic Islets: A Method to Implant Pancreatic Islets into the Anterior Chamber of the Mouse Eye for In Vivo Imaging

Source: Nilsson, J.et. al.&#160;<a href="https://jove.unicartagenaproxy.elogim.com/v/61234/" target="_blank">A Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the Anterior Eye Chamber</a>. J. Vis. Exp. (2020).
This video describes the protocol for grafting human pancreatic islets into the anterior chamber of the mouse&#8217;s eye to monitor revascularization of human islet grafts in vivo. It also helps to understand the contribution of recipient versus donor cells in promoting the encapsulation and vascularization of the graft.

Szczepienie wysp trzustkowych: metoda wszczepiania wysp trzustkowych do przedniej komory oka myszy w celu obrazowania in vivo

Source: Nilsson, J.et. al.&#160;A Longitudinal In Vivo Imaging and Quantification of Human Pancreatic Islet Grafting and Contributing Host Cells in the ...

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

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In Vivo Imaging of Liver Spheroids Engrafted in the Anterior Chamber of the Mouse Eye

Szczepienie wysp trzustkowych: metoda wszczepiania wysp trzustkowych do przedniej komory oka myszy w celu obrazowania in vivo