Murine Spinotrapezius Model to Assess the Impact of Arteriolar Ligation on Microvascular Function and Remodeling

Peripheral vascular disease affects approximately 12 to 14%of the population and is especially common amongst tobacco users and individuals with diabetes, obesity, hypertension, advanced stage, or atherosclerosis. The goal of this surgery is to produce a model for skeletal muscle ischemia by ligating one of three feeder arteries to the coddle half of the mirroring spino trapezius muscle. This animation depicts generalized location of these arteries and indicates the intended ligation site in response to ischemia.

Vascular tissue will undergo changes characteristic of arterio agenesis depending on the mouse strain. This includes formation of bridging collaterals or increasing vessel tortuosity. We'll also show you how to perform a functional vasodilation procedure on the spinal trapezius using intra vital microscopy.

This involves exposing the muscle carefully, placing the stimulating electrodes with their power source and controller, and then setting up the imaging device. Last, we'll show how to excise the muscle and describe a fixation process suitable for immunochemical staining and imaging. Hi, my name is Shane Pierce Kotler and I'm an associate professor in the Department of Biomedical Engineering at the University of Virginia in Charlottesville.

Hi, I'm Kyle Martin. I am a grad student in Shane's lab and I will be performing in the spinal trapezius ligation model. Our lab studies microvascular growth and remodeling, particularly in the settings of ischemic disease such as heart disease, peripheral arterial disease and stroke.

We developed this mirroring spinal trapezius ligation model to study how localized obstruction of arterial blood flow generates structural adaptations in downstream microvascular networks. We view this model as being complimentary to the well-established and widely used hind limb ischemia model in that it offers an on FFAs whole network view of arter, neurogenesis and angiogenesis in a very thin muscle that doesn't require histological sectioning for analysis. Because this muscle is so thin and because the ligation creates a reproducible reduction in blood flow, we can visualize the effects of arterial ligation across the entire microvascular bed and with a single cell level of resolution.

After Kyle takes us through the step, step-by-step procedure to perform the spino trapezius ligation model, our collaborators at Cal Poly will show us how they use this model to study functional vasodilation. Once we see that, we will bring you back to the University of Virginia and harvest the spine trapezius, which needs to be done prior to imaging and staining. Hello, my name is Trevor Cardinal.

I'm an assistant professor in the Department of Biomedical Engineering at Cal Poly State University in San Luis Obispo, California, And my name is Josh Cutz. I'm a master's student in biomedical engineering at Cal Poly in San Luis Obispo. Primary interest of our research laboratory is in understanding the impact of chronic ischemia as occurs with peripheral VAs disease on the functional capabilities of the resistance vasculature.

So after the spinal trapezius feed artery ligation surgery is performed, Josh will demonstrate the functional vasodilation procedure used in our lab. In our lab, we use the functional vasodilation procedure to examine the effect of arterial occlusion on vascular reactivity and blood flow control Instruments. To prepare beforehand include 10 knot location suture, a o closing suture, a needle holder, iris scissors, standard forceps, spring scissors, and a bent micro probe.

After preparing the surgical instruments anesthetize the mouse, here we use the display formula and dosage. Confirm anesthetization by toe, pinch reflex test, then apply protective IGEL using an electric razor shape, a large patch of fur from the animal's back. Although the spinal trapezius ligation only requires a small area to be cleared near the shoulder blade, the spinal trapezius tissue harvest to be performed within a few days will require a much larger cleared surface.

Rush away excess fur, then apply hair removal cream following the manufacturer's instructions. Wash the back with a wet sponge. Then apply three sets of alternating alcohol Betadine wipes ending with Betadine odine.

Under good lighting conditions, the best incision site can be found by transdermal identification of the dorsal fat pad. Since the feeder artery transverses eventually through the spinal trapezius muscle at abruptly, the coddle fat pad border tent the skin in this location and bring iris scissors in directly from above perpendicular to the animal's dorsal and cut to make a three to five millimeter linear incision. If lighting or skin pigmentation do not permit transdermal identification.

Place the incision five millimeters coddle to the bony prominence of the shoulder blade, extend the incision through the skin layers and expand laterally as needed to find the spinal trapezius. Dissect overlying fascia to access the ligation site. If a surface vessel is damaged and hemorrhages as in this case, allow the bleeding time to stop and apply pressure if needed, suse with saline to prevent desiccation.

The artery indicated lies along the dorsal aspect of the spinal trapezius and then transverses through the muscle and into the ventral fat pad. Find the lateral edge of the spinal trapezius and reflect it, lifting it over so as to almost be inverted. This reveals its ventral aspect and the artery transversal site dissect the ventral fat pad which obscures the vessel.

Be careful with forceps pressure to avoid crush injury. Here the surgeon indicates the spina trapezius artery to be ligated. Note the presence of vein pairs vasodilator such as shorter acting adenosine, or longer acting papin may be applied.

In this case, the artery lies between two other vessels as indicated by the arrows. One is similar in size to the artery and would be seen to lie more laterally if the muscle were not reflected while the other is much larger and would lie more immediately. The two ligation sites are marked by ai.

The first ligature is placed in the downstream direction relative to the second. This ensures that the vessel is still filled with blood and visible during placement of the second ligature. A indicates the region to be transected.

Notice the location B, where the artery and its paired vein entered the ventral fat pad outlined by C.This lets us know that the second smaller vessel is a vein. Also note the spinal art trapezius muscle itself outlined by D.A few pointers for discriminating the artery from vein are provided in the accompanying text protocol. But briefly, our preferred method is to obstruct blood flow by applying pressure with a micro probe.

If the vessel is an artery flow will be obstructed in the downstream direction towards the reflected muscle applying. The technique here rules out the larger vessel. If needed, use the 80 degree bent micro probe to separate the artery from the other vessels.

This is done by piercing through the connective tissue underneath the vessel and making short lateral movements to expand a gap. Until the probe can be easily passed through the suture needle may now be more easily threaded beneath the artery. In this case, a small vein was nicked causing minor bleeding.

This vessel lies far enough away from the ischemic zone that it should have minimal impact. On the procedure, tie a single surgeon's knot and prepare the next suture. Again, passing through the pre-made gap and tying another surgeon's knot a few millimeters upstream of the previous ligature.

Keep in mind that the muscle is reflected and flow direction may be opposite of expected. After both ligatures have been placed, look for evidence of an obstructed red blood cell column. If flow hasn't been stopped, another ligature is needed.

Otherwise transect the vessel between ligatures, noting if any bleeding occurs in this case the ligation was successful and no arterial hemorrhaging is seen. An alternative method employs a single ligature and transection in the downstream direction. See accompanying literature for when this method is preferred.

Restore the muscle to its original orientation, reposition the displaced adipose tissue and fascia and close with adot non-resorbable suture. Finally, place the mouse in a heated recovery cage under observation and analgesic for spino trapezius intra vital microscopy. The mouse is first anesthetized with isof fluorine in an induction chamber and then connected to continuous flow isof fluorine through a nose cone.

Insert the rectal temperature probe and set thermo controller to 35 degrees C instruments to prepare beforehand include iris scissors, standard forceps, spring scissors, and a moistened PBS swab. Make a skin incision at the coddle end of the spinal trapezius using iris scissors and standard patent forceps. Extend the incision cranial to the fat pad, creating a horseshoe incision and cover the skin flap with plastic wrapping to prevent desiccation blunt, dissect the subcutaneous connective tissue with fine forceps to maximize visibility.

Place stimulating electrodes, keeping them as close together as possible to minimize the size of current field, position them at the coddle end of the exposed muscle just lateral to the spine. Anchor using clay. Perform a test stimulation to confirm electrode placement using a power lab data acquisition system, stimulus, isolator and lab chart software set to square waves of 200 microsecond duration, two milliamp amplitude and delivered at one hertz.

Cover the exposed muscle with plastic wrap again. To avoid desiccation, allow 30 minutes per vessel diameters to equilibrate and then capture your image or video. Position the intravital microscope over the spinal trapezius muscle to view vasculature architecture.

If using an immersion lens, place a drop of PBS between the objective and plastic wrap starting above the main arterial, which is the largest vessel visible. Manipulate the stage in the XY plane to locate the vessel of interest. Stimulate the muscle with square waves of the previous duration and amplitude, but delivered at eight hertz and for 90 seconds immediately after stimulation.

Image the vessel and continue to do so every minute until it has returned to resting diameter. Data analysis can be performed in real time with video calipers or offline with image analysis software. Repeat the procedure from initial incision to imaging using the contralateral muscle.

When finished. Euthanize the mouse following standard A CUC protocol for the spina trapezius tissue harvest. We'll need iris scissors standard forceps and spring scissors.

Anesthetize the mouse as outlined previously. Begin by making incision a few millimeters cranial to the mouse's shoulder blade. Expand the incision laterally and then coate on both sides.

The goal is to expose the spinal trapezius muscle, which extends from T three to L four after making the incision. Skin may be separated from the back by either gentle pulling or careful cutting of the connective tissue, holding it in place, super fused with saline to prevent desiccation and aid with tissue handling. Use blunt dissection to remove dorsal adipose tissue and expose the spinal trapezius muscle.

Similarly, remove ventral adipose tissue. After reflecting the muscle, the animal's right-sided spinal t trapezius is identified here. Next, remove as much overlying fascia as possible.

This part of the procedure can be tedious, but as important in order to prevent artifacts during microscopy. Once the connective tissue has been removed, excised the tissue as shown by first cutting the lateral edge roughly parallel to the dorsum and moving in a cranial to coddle direction. Then cut transversely across the most cranial extent and finally along the medial border, nearest the spine to collect the contralateral muscle.

Follow the same procedure. First removing dorsal adipose tissue, then ventral adipose tissue, and finally overlying fascia while cutting. You may find it easier to switch instruments to the less dominant hand.

Excised tissue may be fixed and washed following the procedure outlined here after the animal has been euthanized. According to an improved A CUC protocol. See accompanying literature for details on an alternative perfusion fixation procedure.

Here we show a labeled diagram of the reflected spini trapezius muscle. After exposing the feed artery letters indicate regions as previously. Keep in mind that each animal has a unique anatomy.

This particular mouse is considered rather difficult for this procedure due to the proximity of the two other vessels. A more typical animal is shown here. Note the relatively large separation of the indicated artery from the para vein just above it and the absence of a third vessel.

Here we see a confocal microscope montage of a ligated right-sided spinal trapezius muscle that has been stained for smooth muscle alpha actin. Under ischemic conditions, there has been remodeling in the indicated region downstream of the ligation site. These changes can be quantified with image analysis software, which we use here to measure tortuosity as artery path length to core distance ratio.

Some sample data shown here indicates a statistically significant result for increased vessel tortuosity compared to contralateral control. We also demonstrate functional measurements of vessel reactivity using the MicroVision medical software a VA on an offline dark field microscope. Video vessel diameters are measured before stimulation and after stimulation and then compared to each other as a percent change.

Sample data shown here indicates a statistically significant result for increased vessel diameter for terminal arterials after stimulation. Hi, I'm Alexander Ell and I'm the undergrad in the Pierce Scholer lab who edited this video. Together, we've just shown you how to create a model for skeletal muscle ischemia in the mirroring spin trapezius muscle.

Then we demonstrated a functional vasodilation procedure suitable for measuring vascular reactivity and blood flow control. Last we showed how to excise the muscle to prepare for fixation suitable for immunochemical staining and imaging. On behalf of my collaborators in San Luis Obispo and here in Charlottesville, I'd like to thank you for watching and wish you goodbye and good luck in your research endeavors.