<meta charset="utf8"></head><body>Monitoramento em tempo real da dinâmica celular em transplante de pulmão de camundongo

<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

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.

<meta charset="utf8"></head><body>Autor em destaque: Avançando na imunologia do transplante de pulmão por meio de imagens intravitais

Microscopia intravital de dois fótons do pulmão de camundongo transplantado

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

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

Research

JoVE Journal

Medicine

Intravital Two-Photon Microscopy of the Transplanted Mouse Lung

634 visualizações.  Washington University in St. Louis. O objetivo de nossa pesquisa é entender os mecanismos imunológicos subjacentes à lesão de isquemia e reperfusão e as respostas inflamatórias e tolerogênicas após o transplante pulmonar. Queremos estudar as interações dinâmicas entre as células doadoras e receptoras. Atualmente fazemos uso de várias técnicas cirúrgicas e de imagem no estudo da imunologia do transplante de pulmão em camundongos.Isso inclui transplante e retransplante orto...

Vídeo: Autor em destaque: Avançando na imunologia do transplante de pulmão por meio de imagens intravitais

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.

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.