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/6 (B6) mice are transplanted into sex- and age-matched B6 recipients. B6.LysM-GFP reporter mice are used as recipients for select experiments to visualize neutrophil infiltration into lung grafts. Recipient mice can be imaged immediately following a lung transplant.
<ol>
	<li>Re-anesthetize mice with intraperitoneal administration of ketamine (80&#8722;100 mg/kg) and xylazine (8&#8722;10 mg/kg). Administer sustained-release buprenorphine (0.5-1.0 mg/kg) subcutaneously for additional pain control. Confirm adequate depth of anesthesia with a paw pinch. 
	NOTE: If time between lung transplantation and planned intravital imaging is less than two hours, can redose anesthetic with 50% of initial dose of ketamine.</li>
	<li>Remove the hair from the entire left chest using an electric clipper and wipe the chest to remove the excess clipped hairs.</li>
	<li>Apply non-medicated ophthalmic ointment to the eyes of the mouse to prevent corneal drying.</li>
	<li>Re-intubate the mouse orotracheally with a 20 G angiocatheter, as previously described21.</li>
	<li>Ventilate with room air at a rate of 120 breaths/min and tidal volume of 0.5 cc.</li>
	<li>Deliver 1% isofluorane endotracheally during the entire procedure.</li>
	<li>To visualize blood vessels during imaging, inject 20 &#181;L of 655 nm nontargeted quantum dots (q-dots) in 50 &#181;L of phosphate-buffered saline intravenously at least 5 min prior to imaging.</li>
</ol>
2. Surgical preparation of left lung for imaging
<ol>
	<li>Place the mouse in a right lateral decubitus position (see Figure 1A).</li>
	<li>Disinfect the chest skin with a mixture of 0.75% iodine and 70% ethanol, three times.</li>
	<li>Re-open the left thoracotomy (from mouse lung transplant) through the third intercostal space (see Figure 1A).</li>
	<li>Remove a portion of the skin and soft tissue over the left thoracotomy site, measuring 1.5 cm x 1.5 cm (see Figure 1B).</li>
	<li>Prior to rib resection, first clamp the ribs for ~10 s to thrombose any microvasculature to achieve hemostasis (see Figure 1C, D).</li>
	<li>Resect portions of the 3rd to 5th ribs to create an imaging window large enough to extricate the lung (~0.8 cm craniocaudally and ~1.0 cm anteroposteriorly, Figure 1D, E).</li>
	<li>Stop any additional bleeding from the rib edges with electrocautery. 
	NOTE: It is critical to achieve hemostasis in the cut edge of the ribs to avoid excessive blood loss during the imaging period.</li>
	<li>Place a small bump (4 cm x 4 cm gauze folded longitudinally 3 times) under the right chest (see Figure 1E).</li>
	<li>Using cotton swabs, elevate the lung graft out of the chest and place the lower aspect of the lung onto a 1 cm x 3 cm strip of saline-soaked gauze (see Figure 1F). 
	NOTE: This strip of saline-soaked gauze prevents sliding of the lung, maintains a moist environment for the lung, protects the lung from the sharp edges of the resected ribs, and separates the lung from heart motion. Once the circumferential imaging chamber is applied over the lung (Figure 1G, H, described in section 3), there should be minimal heat and fluid loss from the open thoracic cavity during the imaging period.</li>
</ol>
3. Imaging chamber preparation 
NOTE: An imaging chamber is custom-built (see Figure 2A). This imaging chamber consists of a base plate and a top plate between which the mouse is placed and secured in place with spring-loaded bolts on either side (Figure 2B). The top plate contains a circular cutout measuring ~2 cm in diameter. A correspondingly sized black O ring is placed into this opening on the front side of the top plate, which will protect the objective lens (Figure 2C). A 24 mm x 50 mm coverglass is adhered to the back of the top plate using high vacuum grease, which will create a watertight seal. This coverglass will serve as the imaging window by adhering to the lung below and holding the imaging media (i.e., water) above. Ensure that no vacuum grease gets within the circular imaging window. The coverglass will be replaced for each new imaging experiment. The base plate is heated to 35-37 &#730;C using a thermocouple temperature probe (Figure 2A).
<ol>
	<li>Place the intubated mouse onto the base plate of the imaging chamber with the open left chest facing up (see Figure 1G, H). Secure the mouse with silk tape over the face, front paws, and tail (see Figure 1H and Figure 2A).</li>
	<li>Apply glue to the bottom side of the cover glass attached to the top plate (grey circle in Figure 2C, bottom), leaving a 0.8-1.0 cm glue-free circle in the center. 
	NOTE: There is a tradeoff between the size of the glue-free circle and the amount of motion artifact; thus, it is recommended that the circle does not exceed 1.0 cm in diameter.</li>
	<li>Lower the top plate until the coverglass makes contact with the lung, with the glue touching the lung surface (see Figure 1G). 
	NOTE: The left lung will be glued to the coverglass with a clean, glue-free circle in the middle, which is the area that will be imaged.</li>
	<li>Hold the coverglass against the lung and inflate the lung for 1-2 s so that the area of glue adheres to the surrounding tissue. Accomplish lung inflation by occluding the outflow tubing from the mouse endotracheal tube to the ventilator. 
	NOTE: Do not inflate the lung for longer than 1-2 seconds, as this may cause excessive barotrauma.</li>
	<li>Back off the top plate slightly to reduce pressure on the lung tissue. Ensure that the area of the lung within the imaging window does not move with ventilation.</li>
	<li>Secure both ends of the top plate by securing the thumb nut over each bolt (see Figure 2B).</li>
</ol>
4. Two-photon imaging 
NOTE: A fixed-stage, upright microscope with a 20x or 25x &#62;1.0 numerical aperture (NA) water immersion objective should be used for intravital microscopy. Below is the setup used in this study for a B6 to B6.LysM-GFP left lung transplant with 655-nm q-dot blood vessel-labeling. When applying this protocol, the setup of the microscope, lasers, and dichroic filters can be adapted based on the needs of the specific experiment and the fluorescent reporters used.
<ol>
	<li>Place the entire imaging chamber with the intubated recipient mouse (B6 left lung transplanted into B6.LysM-GFP with 655-nm q-dots) onto the microscope stage (see Figure 2A).</li>
	<li>Use dichroic filters of wavelengths 480 nm, 560 nm, and 635 nm, which will appropriately separate the signals from the GFP reporter, q-dots, and second harmonic generation (SHG) signal (445 nm) generated by collagen. These filters can be adapted based on the specific experiment. 
	NOTE: The SHG signal generated by collagen demarcates dark crater-like structures, which represent alveolar airspaces (see white arrows in Figure 3).</li>
	<li>Set the titanium-sapphire femtosecond pulsed laser to 890 nm (within the near-infrared range) for excitation. Use the lowest possible laser power to achieve a sufficient signal. 
	NOTE: The laser settings should be adjusted for the specific experiment to maximize signal separation.</li>
	<li>Apply ~1 mL of water to completely cover the coverglass on the top plate, which will be kept in place by the O-ring and the vacuum grease seal of the cover glass to the top plate (see Figure 1G and Figure 2C).</li>
	<li>Use the 20x &#62;1.0 NA water immersion objective on the microscope. Ensure that the objective lens is adapted based on the specific experiment. 
	NOTE: Larger NA allows for more light collection, higher contrast, and more detailed images. Water immersion objectives are preferred as water or buffer is more compatible with biological tissues.</li>
	<li>Lower the objective of the microscope into the water and focus on the surface of the lung.</li>
	<li>Activate the base plate heater and maintain the temperature at 35-37 &#730;C. 
	NOTE: If there are concerns about the inability to maintain tissue temperature or adequate perfusion to the lung, two additional resisters can be attached to warm the top plate in addition to the base plate (see Supplementary Figure 1).</li>
	<li>Acquire image stacks with acquisition software of choice. 
	NOTE: Ensure that the room is completely dark during acquisition, which may involve the use of blackout curtains/blinds and strategic covering of all light sources.</li>
</ol>
5. Video acquisition
NOTE: The following parameters can be adapted based on the specific experiment. Steps 5.1-5.3 describe the specific parameters used for the B6 to B6.Lysm-GFP murine left lung transplant, which can be used as a reference guide.
<ol>
	<li>Using a video-rate bidirectional scanner, set the x-y scan dimensions to 512 x 512 pixels.</li>
	<li>Using an xyz stepper unit, acquire time-lapse recordings of a 21-step z-stack with 10-frame averaging per step. Each stack takes about 20 s to acquire, which includes the time required for frame-averaging, the stepper motor to move and check its position, and a 100 ms delay. 
	NOTE: The bidirectional scanner on the microscope used in this study operates at a video rate of 30 frames/s. This may differ depending on the specific microscope setup.</li>
	<li>Acquire 80 consecutive z-stacks to obtain time-lapse imaging of leukocyte migration within the tissue parenchyma. This takes about ~27 min at 20 s/stack. 
	NOTE: The stack size, frame-averaging, laser power, and total length of the time-lapse recording should be minimized to prevent excessive laser exposure to the tissue. If fluorescent reporters used are bright (i.e., GFP) and laser power is low, then repeating a 21-step z-stack with a 10-frame average, 512 x 512-pixel scan, at 20 s intervals for ~27 min is well tolerated by the mouse. If longer periods of imaging are desired, the acquisition time per stack should be increased to keep the total laser exposure the same (i.e., 1 min per stack over ~1.5 h). These settings will need to be optimized based on the fluorescent reporters used.</li>
	<li>Euthanize the mouse at the end of image acquisition. To euthanize, anesthetize the mouse with ketamine (80&#8722;100 mg/kg) and xylazine (8&#8722;10 mg/kg), followed by subsequent cervical dislocation. Typical imaging preparations will easily maintain mouse viability for up to 2 h. 
	NOTE: To extend this period, additional fluid supplementation, anesthesia with isoflurane, and attention to temperature regulation may be required.</li>
	<li>Complete video rendering with Imaris. Other imaging software (such as ImageJ) can also be used. 
	NOTE: Images may be processed post-acquisition using contrast enhancement, smoothing, and channel arithmetics to remove channel crosstalk.</li>
</ol>

Real-Time Monitoring of Cellular Dynamics in Mouse Lung Transplantation

The authors report no relevant disclosures.

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.

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

Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

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.

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 ...

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

Research

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Medicine

834 Views.  Washington University in St. Louis. 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. 

Video: Author Spotlight: Advancing Lung Transplant Immunology Through Intravital Imaging

A Simple Method of Mouse Lung Intubation

A simple procedure to intubate mice for pulmonary function measurements would have several advantages in longitudinal studies with limited numbers or expensive animal. One of the reasons that this is not done more routinely is that it is relatively difficult, despite there being several published studies that describe ways to achieve it. In this paper we demonstrate a procedure that eliminates one of the major hurdles associated with this intubation, that of visualizing the trachea during the entire time of intubation. The approach uses a 0.5 mm fiberoptic light source that serves as an introducer to direct the intubation cannula into the mouse trachea. We show that it is possible to use this procedure to measure lung mechanics in individual mice over a time course of at least several weeks. The technique can be set up with relatively little expense and expertise, and it can be routinely accomplished with relatively little training. This should make it possible for any laboratory to routinely carry out this intubation, thereby allowing longitudinal studies in individual mice, thereby minimizing the number of mice needed and increasing the statistical power by using each mouse as its own control.

This paper describes a striaghforward and efficient method of intubating mice for pulmonary function measurements or pulmonary instillation, that allows the mice to recover and be studied at later times. The procedure involves an inexpensive fiberoptic light source that directly illuminates the trachea.

A  simple procedure to intubate mice for pulmonary function measurements would  have several advantages in longitudinal studies with limited numbers or  ...

Noninvasive Intratracheal Intubation to Study the Pathology and Physiology of Mouse Lung

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. With the increasing availability of knockout and transgenic derivatives, together with a vast amount of genetic information, mice provide one of the best models to study the molecular mechanisms underlying the pathology and physiology of lung diseases. Inhalation, intranasal instillation, intratracheal instillation, and intratracheal intubation are the most widely used techniques by a number of investigators to administer materials of interest to mouse lungs. There are pros and cons for each technique depending on the goals of a study. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented. This technique was applied to administer bleomycin to mouse lungs as a model to study pulmonary fibrosis.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular disease and for developing therapeutic intervention. Here a noninvasive intratracheal intubation method that can directly deliver exogenous materials to mouse lungs is presented.

The use of a model that mimics the condition of lung diseases in humans is critical for studying the pathophysiology and/or etiology of a particular ...

An Orthotopic Glioblastoma Mouse Model Maintaining Brain Parenchymal Physical Constraints and Suitable for Intravital Two-photon Microscopy

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology rely on the injection of a suspension of glioma cells into the brain parenchyma following incision of the dura-mater. Whereas the cells have to be injected superficially to be accessible to intravital two-photon microscopy, superficial injections fail to recapitulate the physiopathological conditions. Indeed, escaping through the injection tract most tumor cells reach the extra-dural space where they expand abnormally fast in absence of mechanical constraints from the parenchyma.
Our improvements consist not only in focally implanting a glioma spheroid rather than injecting a suspension of glioma cells in the superficial layers of the cerebral cortex but also in clogging the injection site by a cross-linked dextran gel hemi-bead that is glued to the surrounding parenchyma and sealed to dura-mater with cyanoacrylate. Altogether these measures enforce the physiological expansion and infiltration of the tumor cells inside the brain parenchyma. Craniotomy was finally closed with a glass window cemented to the skull to allow chronic imaging over weeks in absence of scar tissue development.
Taking advantage of fluorescent transgenic animals grafted with fluorescent tumor cells we have shown that the dynamics of interactions occurring between glioma cells, neurons (e.g. Thy1-CFP mice) and vasculature (highlighted by an intravenous injection of a fluorescent dye) can be visualized by intravital two-photon microscopy during the progression of the disease.
The possibility to image a tumor at microscopic resolution in a minimally compromised cerebral environment represents an improvement of current GBM animal models which should benefit the field of neuro-oncology and drug testing.

We have established a cortical orthotopic glioblastoma model in mice for intravital two-photon microscopy that recapitulates the biophysical constraints normally at play during the growth of the tumor. A chronic glass window replacing the skull above the tumor enables the follow-up of the tumor progression over time by two-photon microscopy.

Glioblastoma multiforme (GBM) is the most aggressive form of brain tumors with no curative treatments available to date.
Murine models of this pathology ...

Intravital Video Microscopy Measurements of Retinal Blood Flow in Mice

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

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

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

Diagnostic Ultrasound Imaging of Mouse Diaphragm Function

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive surgical procedures. Although this is an effective method of assessing in vitro diaphragm activity, it involves non-survival surgery. The application of non-invasive ultrasound imaging as an in vivo procedure is beneficial since it not only reduces the number of animals sacrificed, but is also suitable for monitoring disease progression in live mice. Thus, our ultrasound imaging method may likely assist in the development of novel therapies that alleviate muscle injury induced by various respiratory diseases. Particularly, in clinical diagnoses of obstructive lung diseases, ultrasound imaging has the potential to be used in conjunction with other standard tests to detect the early onset of diaphragm muscle fatigue. In the current protocol, we describe how to accurately evaluate diaphragm contractility in a mouse model using a diagnostic ultrasound imaging technique.

Diagnostic ultrasound imaging has proven to be effective in diagnosing various respiratory diseases in human and animal subjects. We demonstrate a comprehensive ultrasound protocol utilized by Dr. Zuo's lab to analyze diaphragm kinetics specifically in mouse models. This is also a non-invasive research technique which can provide quantitative information on mouse respiratory muscle function.

Function analysis of rodent respiratory skeletal muscles, particularly the diaphragm, is commonly performed by isolating muscle strips using invasive ...

A Murine Model of Myocardial Ischemia-reperfusion Injury through Ligation of the Left Anterior Descending Artery

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms at work during MI and developing effective therapeutic approaches requires methodology to reproducibly simulate the clinical incidence and reflect the pathophysiological changes associated with MI. Here, we describe a surgical method to induce MI in mouse models that can be used for short-term ischemia-reperfusion (I/R) injury as well as permanent ligation. The major advantage of this method is to facilitate location of the left anterior descending artery (LAD) to allow for accurate ligation of this artery to induce ischemia in the left ventricle of the mouse heart. Accurate positioning of the ligature on the LAD increases reproducibility of infarct size and thus produces more reliable results. Greater precision in placement of the ligature will improve the standard surgical approaches to simulate MI in mice, thus reducing the number of experimental animals necessary for statistically relevant studies and improving our understanding of the mechanisms producing cardiac dysfunction following MI. This mouse model of MI is also useful for the preclinical testing of treatments targeting myocardial damage following MI.

We introduce a surgical method to induce experimental ischemia/reperfusion (I/R) injury to simulate myocardial infarction (MI) in mouse models that allows for more clarity in positioning of the ligation on the left anterior descending artery (LAD) to increase the reproducibility of MI experiments in mice.

Acute or chronic myocardial infarction (MI) are cardiovascular events resulting in high morbidity and mortality. Establishing the pathological mechanisms ...

Acute Brain Trauma in Mice Followed By Longitudinal Two-photon Imaging

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no effective treatment for it yet. Limitations of currently used animal models impede understanding of the pathology mechanism. Multiphoton microscopy allows studying cells and tissues within intact animal brains longitudinally under physiological and pathological conditions. Here, we describe two models of acute brain injury studied by means of two-photon imaging of brain cell behavior under posttraumatic conditions. A selected brain region is injured with a sharp needle to produce a trauma of a controlled width and depth in the brain parenchyma. Our method uses stereotaxic prick with a syringe needle, which can be combined with simultaneous drug application. We propose that this method can be used as an advanced tool to study cellular mechanisms of pathophysiological consequences of acute trauma in mammalian brain in vivo. In this video, we combine acute brain injury with two preparations: cranial window and skull thinning. We also discuss advantages and limitations of both preparations for multisession imaging of brain regeneration after trauma.

Acute brain trauma is a severe injury that has no adequate treatment to date. Multiphoton microscopy allows studying longitudinally the process of acute brain trauma development and probing therapeutical strategies in rodents. Two models of acute brain trauma studied with in vivo two-photon imaging of brain are demonstrated in this protocol.

Although acute brain trauma often results from head damage in different accidents and affects a substantial fraction of the population, there is no ...

Implantation of Fibrin Gel on Mouse Lung to Study Lung-specific Angiogenesis

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and repair damage in simple tissues in the orthopedic and periodontal fields. However, attempts to regenerate the structures and functions of more complex three-dimensional (3D) organs such as lungs have not been very successful because the biological processes of organ regeneration have not been well explored. It is becoming clear that angiogenesis, the formation of new blood vessels, plays key roles in organ regeneration. Newly formed vasculatures not only deliver oxygen, nutrients and various cell components that are required for organ regeneration but also provide instructive signals to the regenerating local tissues. Therefore, to successfully regenerate lungs in an adult, it is necessary to recapitulate the lung-specific microenvironments in which angiogenesis drives regeneration of local lung tissues. Although conventional in vivo angiogenesis assays, such as subcutaneous implantation of extracellular matrix (ECM)-rich hydrogels (e.g., fibrin or collagen gels or Matrigel - ECM protein mixture secreted by Engelbreth-Holm-Swarm mouse sarcoma cells), are extensively utilized to explore the general mechanisms of angiogenesis, lung-specific angiogenesis has not been well characterized because methods for orthotopic implantation of biomaterials in the lung have not been well established. The goal of this protocol is to introduce a unique method to implant fibrin gel on the lung surface of living adult mouse, allowing for the successful recapitulation of host lung-derived angiogenesis inside the gel. This approach enables researchers to explore the mechanisms by which the lung-specific microenvironment controls angiogenesis and alveolar regeneration in both normal and pathological conditions. Since implanted biomaterials release and supply physical and chemical signals to adjacent lung tissues, implantation of these biomaterials on diseased lung can potentially normalize the adjacent diseased tissues, enabling researchers to develop new therapeutic approaches for various types of lung diseases.

Recapitulation of the organ-specific microenvironment, which stimulates local angiogenesis, is indispensable for successful regeneration of damaged tissues. This report demonstrates a novel method to implant fibrin gels on the lung surface of living mouse in order to explore how the lung-specific microenvironment modulates angiogenesis and alveolar regeneration in adult mouse.

Recent significant advances in stem cell research and bioengineering techniques have made great progress in utilizing biomaterials to regenerate and ...

Mouse Pneumonectomy Model of Compensatory Lung Growth

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to disability or death. Injury-based animal models of lung repair and regeneration are limited by injury-specific responses making it difficult to differentiate changes related to the injury response and injury resolution from changes related to lung repair and lung regeneration. However, use of animal models to identify these repair and regeneration signaling pathways is critical to the development of new therapies aimed at improving pulmonary function following lung injury. The mouse pneumonectomy model utilizes compensatory lung growth to isolate those repair and regeneration signals in order to more clearly define mechanisms of alveolar re-septation. Here, we describe our technique for performing mouse pneumonectomy and sham pneumonectomy. This technique may be utilized in conjunction with lineage tracing or other transgenic mouse models to define molecular and cellular mechanism of lung repair and regeneration.

Mouse pneumonectomy is a commonly employed model of compensatory lung growth. This procedure can be used in conjunction with lineage tracing or transgenic mouse models to elucidate underlying mechanisms.

In humans, disrupted repair and remodeling of injured lung contributes to a host of acute and chronic lung disorders which may ultimately lead to ...

Real-time X-ray Imaging of Lung Fluid Volumes in Neonatal Mouse Lung

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting and sustaining this phenotype is critically important in normal alveolar growth and gas exchange throughout life. Several in vitro studies have characterized the role of key regulatory proteins, signaling molecules, and steroid hormones that can influence the rate of lung fluid clearance. However, in vivo examinations must be performed to evaluate whether these regulatory factors play important physiological roles in regulating perinatal lung liquid absorption. As such, the utilization of real time X-ray imaging to determine perinatal lung fluid clearance, or pulmonary edema, represents a technological advancement in the field. Herein, we explain and illustrate an approach to assess the rate of alveolar lung fluid clearance and alveolar flooding in C57BL/6 mice at post natal day 10 using X-ray imaging and analysis. Successful implementation of this protocol requires prior approval from institutional animal care and use committees (IACUC), an in vivo small animal X-ray imaging system, and compatible molecular imaging software.

We present a protocol to assess the rate of alveolar fluid clearance or pulmonary edema in neonatal mouse lung using X-ray imaging technology.

At birth, the lung undergoes a profound phenotypic switch from secretion to absorption, which allows for adaptation to breathing independently. Promoting ...