This work is supported by grants from NIH 1P01AI11650 and the Foundation for Barnes-Jewish Hospital. We thank the In Vivo Imaging Core at Washington University School of Medicine.

All animal handling procedures were conducted in compliance with the National Institutes of Health Care and Use of Laboratory Animals guidelines and approved by the Institutional Animal Care and Use Committee at Washington University School of Medicine.
1. Anesthesia and intubation
NOTE: Orthotopic mouse left lung transplant is performed, as previously described20,21. Lungs from 20-25 g C57BL/...

Real-Time Monitoring of Cellular Dynamics in Mouse Lung Transplantation

Two-photon excitation was first described in her doctoral thesis by Maria G&#246;ppert-Mayer in 1931, who later won the Nobel Prize in Physics for describing the nuclear shell structure22,23. Traditional fluorescence microscopy relies on single-photon excitation, with excitation wavelengths that are shorter and higher energy than emission wavelengths. In contrast to single-photon microscopy, two-photon microscopy involves simultaneous excitation by two photons, e...

Lung transplantation is the final option for many patients suffering from end-stage pulmonary disease; however, long-term survival after lung transplantation is poor compared to other solid organ transplants. Survival at 5 years is only ~60%-70%1, compared to 80%-90% in hearts2 and 85%-90% in kidneys3. Many complications after lung transplantation, such as primary graft dysfunction, antibody-mediated rejection, and chronic lung allograft dysfunction, are due to the host immune response to the allograft. For example, our group has shown that neutrophils are rapidly recruited into the lung allograft following transplant-induced ischemia-reperfusion injury and form dynamic clusters surrounding blood monocytes4. Crosstalk between donor and recipient cells is responsible for deleterious alloimmune responses5,6,7, and the ability to study these dynamic cell interactions in a live animal model is invaluable.
Two-photon microscopy allows for high-resolution intravital imaging for depths up to several hundred microns with minimal photobleaching of tissues8,9. It is used in a variety of tissues and anatomic sites, including the neocortex10,11, skin12,13, and kidney14,15. More recently, it has been adapted to non-static organs such as the lung and heart4,16,17. In this protocol, we describe a technique to image stabilized, ventilated, and perfused pulmonary grafts following murine orthotopic left lung transplantation. A key benefit of the transplant model is the ability to genetically manipulate the donor and recipient separately. Individual cell populations can be visualized with transgenic mouse strains with knock-in fluorescent protein expression, adoptive transfer of fluorescently-labeled cells5, or intravenous injection of fluorescently-labeled antibodies to bind cell-specific markers4,16,17,18.
In order to stabilize the lung during imaging, this protocol involves gluing the lung to a coverglass, while other groups have described suction stabilization using a custom-made reversible vacuum device19. Our protocol has several advantages, including a larger area of imaging and relative ease of setup using commonly available materials in a microscopy lab (including coverglass and glue). Since this gluing technique constrains the lung at the upper surface, it is expected to decrease ventilatory motion and allow for deeper imaging. This intravital imaging technique allows for detailed observation of immune cell behavior and interactions in real-time, which contributes to the study of immune mechanisms that regulate inflammatory versus tolerogenic responses following lung transplantation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.75% povidone-iodine</td>
			<td>Aplicare</td>
			<td>NDC 52380-0126-2</td>
			<td>For disinfectant</td>
		</tr>
		<tr>
			<td>1-inch 20G IV catheter</td>
			<td>Terumo</td>
			<td>SROX2025CA</td>
			<td>For endotracheal tube (ETT)</td>
		</tr>
		<tr>
			<td>1-inch silk tape</td>
			<td>Durapore</td>
			<td>3M ID 7100057168</td>
			<td>To secure mouse in position</td>
		</tr>
		<tr>
			<td>20x water immersion long objective lens</td>
			<td>Olympus</td>
			<td>N20X-PFH</td>
			<td></td>
		</tr>
		<tr>
			<td>3M Vetbond glue</td>
			<td>Medi-Vet.com</td>
			<td>10872</td>
			<td>To glue coverglass to lung</td>
		</tr>
		<tr>
			<td>655 nm non-targeted quantum dots</td>
			<td>ThermoFisher</td>
			<td>Q21021MP</td>
			<td>For labeling of blood vessels</td>
		</tr>
		<tr>
			<td>70% ethanol</td>
			<td>Sigma Aldrich</td>
			<td>EX0281</td>
			<td>For disinfectant</td>
		</tr>
		<tr>
			<td>Argent High Temp Fine Tip Cautery Pen</td>
			<td>McKesson</td>
			<td>231</td>
			<td></td>
		</tr>
		<tr>
			<td>Black O ring (2 cm)</td>
			<td>Hardware store</td>
			<td>N/A</td>
			<td>For custom-built imaging chamber</td>
		</tr>
		<tr>
			<td>Bolt (2)</td>
			<td>Hardware store</td>
			<td>N/A</td>
			<td>For custom-built imaging chamber</td>
		</tr>
		<tr>
			<td>Brass thumb nut (2)</td>
			<td>Hardware store</td>
			<td>N/A</td>
			<td>For custom-built imaging chamber</td>
		</tr>
		<tr>
			<td>Buprenorphine 1.3 mg/mL</td>
			<td>Fidelis Animal Health</td>
			<td>NDC 86084-100-30</td>
			<td>For pain control</td>
		</tr>
		<tr>
			<td>Chameleon titanium-sapphire femtosecond pulsed laser</td>
			<td>Coherent</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glass (24 mm x 50 mm)</td>
			<td>Thomas Scientific</td>
			<td>1202F63</td>
			<td>For custom-built imaging chamber</td>
		</tr>
		<tr>
			<td>Curved mosquito clamp (1)</td>
			<td>Fine Science Tools</td>
			<td>13009-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Dual channel heater controller</td>
			<td>Warner Instruments</td>
			<td>TC-344B</td>
			<td></td>
		</tr>
		<tr>
			<td>Fine scissors (1)</td>
			<td>Fine Science Tools</td>
			<td>15040-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixed-stage upright microscope</td>
			<td>Olympus</td>
			<td>BX51WI</td>
			<td></td>
		</tr>
		<tr>
			<td>Gauze (cut to 1 cm x 3 cm)</td>
			<td>McKesson</td>
			<td>476709</td>
			<td>To place under left lung</td>
		</tr>
		<tr>
			<td>High vacuum grease</td>
			<td>Dow Corning</td>
			<td>N/A</td>
			<td>To adhere coverglass onto top plate</td>
		</tr>
		<tr>
			<td>Isoflurane 1%</td>
			<td>Sigma Aldrich</td>
			<td>26675-46-7</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Ketamine hydrochloride 100 mg/mL</td>
			<td>Vedco</td>
			<td>NDC 50989-996-06</td>
			<td>For anesthesia</td>
		</tr>
		<tr>
			<td>Metal sheet (3 cm x 7 cm)</td>
			<td>Hardware store</td>
			<td>N/A</td>
			<td>For custom-built imaging chamber</td>
		</tr>
		<tr>
			<td>Pointed cotton-tipped applicators</td>
			<td>Solon</td>
			<td>56225</td>
			<td>To manipulate lung and for blunt dissection</td>
		</tr>
		<tr>
			<td>Power Pro Ultra clipper</td>
			<td>Oster</td>
			<td>078400-020-001</td>
			<td></td>
		</tr>
		<tr>
			<td>Puralube Vet eye ointment</td>
			<td>Medi-Vet.com</td>
			<td>11897</td>
			<td>To prevent eye dessiccation</td>
		</tr>
		<tr>
			<td>Small animal ventilator</td>
			<td>Harvard Apparatus</td>
			<td>55-0000</td>
			<td></td>
		</tr>
		<tr>
			<td>Straight forceps (1)</td>
			<td>Fine Science Tools</td>
			<td>91113-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Three channel shutter driver</td>
			<td>Uniblitz</td>
			<td>VMM-D3</td>
			<td>Resonant scanner</td>
		</tr>
		<tr>
			<td>x.y.z optical stepper motor</td>
			<td>Prior Scientific</td>
			<td>OptiScan II</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine 20 mg/mL</td>
			<td>Akorn</td>
			<td>NDC 59399-110-20</td>
			<td>For pain control</td>
		</tr>
	</tbody>
</table>

intravital two-photon microscopy of the transplanted mouse lung

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by crosstalk between donor and recipient cells. A better understanding of these processes relies on the use of preclinical animal models and is aided by an ability to study intra-graft immune cell trafficking in real-time. Intravital two-photon microscopy can be used to image tissues and organs for depths up to several hundred microns with minimal photodamage, which affords a great advantage over single-photon confocal microscopy. Selective use of transgenic mice with promoter-specific fluorescent protein expression and/or adoptive transfer of fluorescent dye-labeled cells during intravital two-photon microscopy allows for the dynamic study of single cells within their physiologic environment. Our group has developed a technique to stabilize mouse lungs, which has enabled us to image cellular dynamics in na&#239;ve lungs and orthotopically transplanted pulmonary grafts. This technique allows for detailed assessment of cellular behavior within the vasculature and in the interstitium, as well as for examination of interactions between various cell populations. This procedure can be readily learned and adapted to study immune mechanisms that regulate inflammatory and tolerogenic responses after lung transplantation. It can also be expanded to the study of other pathogenic pulmonary conditions.

Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

The goal of our research is to understand the immune mechanisms underlying ischemia reperfusion injury and the inflammatory and tolerogenic responses after lung transplantation. We want to study the dynamic interactions between donor and recipient cells. We currently make use of several surgical and imaging techniques in the study of lung transplant immunology in the mouse.These include orthotopic left lung transplantation and retransplantation, left pulmonary hilar clamping, non-invasive positron emission tomography, and two-photon intravital imaging. Our research reveals unique immune responses in transplanted lungs. Using advanced imaging techniques, we discovered that recipient monocytes promote neutrophil infiltration in lung allografts via IL-1 beta.We also found that FOXP3 regulatory T cells cluster within these allografts and protect against antibody mediated rejection. Our technique is easy to set up, with materials readily found in a microscopy lab, such as tissue glue and cover glasses. Additionally, it provides a larger field of view compared to other techniques.Our lab is interested in understanding mechanisms that induce and maintain lung allograft tolerance. Intravital imaging will allow us to visualize interactions between immune cells during rejection and tolerance.

After 1 h of cold ischemic storage at 4 &#730;C, we orthotopically transplanted the left lung from a B6 mouse into a B6.LysM-GFP mouse4, and then intravital two-photon imaging was performed, as described above. We performed imaging at two time points post-transplant - 2 h (Figure 3A) and 24 h (Figure 3B). Blood vessels are labeled in red by the q-dots injected immediately prior to imaging. Additionally, we can visualize monocytes that hav...

Read this Scientific Article Protocol about Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging at JoVE.com

Here, we present a protocol to intravitally image the transplanted mouse left lung using two-photon microscopy. This represents a valuable tool for studying cellular dynamics and interactions in real-time following murine lung transplantation.

Complications after lung transplantation are largely related to the host immune system responding to the graft. Such immune responses are regulated by ...

intravital-two-photon-microscopy-of-the-transplanted-mouse-lung