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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Chapters in this video

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

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

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

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

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

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

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

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

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

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