The overall goal of this procedure is to perform hind limb unloading or hu in mice with a simpler, more straightforward method that minimizes stress for the animal. This tailing method successfully induces hind limb atrophy without compromising overall health and body weight. The first step is to implant a tail ring into an adult mouse.
It is important that body weight and health observations are recorded throughout the study. The hind limb unloading apparatus is then assembled and placed into a cage where the unloading will take place. Once the animal has recovered, it is placed in the apparatus for the unloading procedure.
Ultimately, results can be obtained through measurements of both body and muscle mass that showed muscular atrophy in the hind limbs of unloaded mice. The main advantage of this technique over the existing methods, like the Maori Holton taping technique, are that the skin of the tail is always visible. Mice are not able to get out of the suspension apparatus or play with the ring and are quicker to recover and maintain a stable body weight.
This method can help answer key questions in the musculoskeletal physiology fields, also in gravitational and space biology, such as how to prevent skeletal muscle atrophy and bone loss after prolonged periods of physical inactivity, bed rest, limb mobilization, microgravity, neuromuscular disease, and with advancing age. We first had this idea when we experienced complications with the Maury Holden tail taping technique. We consistently lost animals and experimental data due to mice escaping from the hind limb apparatus Demonstrating this technique.
Today are Andre's Ferrara, senior Research Associate from my laboratory In assisting Andres is Jacqueline Chrissy, a doctoral student in the lab, and Andrew Dunn, an undergraduate student also in the lab. Begin by sterilizing the surgical area, anise the Ty, an adult mouse with isof fluorine, and then record the weight of the mouse and place it on a heating pad. Prepare the tail for surgery by scrubbing with chlorhexidine solution.
Start counting from the end of the body hair at the base of the tail to locate the fifth, sixth, or seventh intervertebral space between two bony bumps on the tail. Use a hemostat and a 20 5G needle to make a pilot hole between two vertebrae into the intervertebral space. Remove the needle and insert a steel suture.
Cut to a 10 centimeter length through the hole. Five centimeters of suture should protrude from either side of the tail. Place the tip of your thumb onto the tail and use your free hand to wind the steel suture around your thumb.
Twist the ends to form a big loop. The wound or circular part should be plus or minus three millimeters long. Clamp one strand of suture with a hemostat and bend it into a smaller loop, the end of which is attached as closely as possible to the twist on the original large loop.
Cut off excess wire and bend the twisted wire back. Stop any excess bleeding with silver nitrate sticks. Allow the animal to recover for five to seven days prior to hind limb unloading or hu after surgery.
The miser house singly to prevent the tail rings from getting entangled. Layer one inch wired by three-eighths thick plated slotted steel bar over the top of the cage and tape it down. Add another washer and wing nut, but do not fully tighten them to allow for adjustments to be made later.
Insert the machine screw through the center hole of the bobin to prevent the bobin from falling off. Screw a nylon lock nut to the end of the machine.Screw. The bobin should swivel freely.
Insert the snap hook from a fishing swivel through the lower part of the bobin. Use pliers to form an S-shaped hook from a paperclip. Insert one end of the S-shaped hook through the hole of the fishing swivel and pinch it shut with the pliers.
Place a U-shaped wire parallel to the machine screw on the inside of the cage and bend the ends over the side. To support a full water bottle, insert a water bottle into the U-shaped wire with a spigot facing down, but not touching the bottom. Secure the water bottle to the cage with duct tape.
Bend a two inch binder clamp open, leaving enough tension to hold a few pieces of food without crushing the pellets prior to the hind limb unloading procedure. Record the weight of the mouse to use for comparisons. Later, insert the open end of the S-shaped hook through the smaller ring protruding from the tail.
Adjust the height of the mouse by tightening or loosening the lower wing nut on the machine.Screw. The lower extremities should not be weightbearing. Once the optimal height is achieved, tighten the upper wing nut, reposition the water bottle a new shaped wire so that the mouse cannot climb onto the spigot with its hind limbs.
To measure food consumption during the study, weigh pellets prior to placing them into the binder clamp. Provide about eight to 10 grams to allow for the consumption of about five to six grams of chow per day. Per mouse position, the binder clamp with food on the bottom of the cage so that the mouse cannot climb onto it and tape it down with duct tape.
Using two by two inch Gores loosely. Wrap the tail onto the fish swivel. Wrap a small piece of tail around the gores securing the gores to the tail.
This step is important to prevent the tail from drooping downward leading to blood pulling in the tip and the tail becoming necrotic. Repeat the same procedure for an additional animal on the other side of the cage. When housing two animals per cage space them so that they can almost touch each other.
In the middle of the cage. I add a small amount of cob bedding to the cage, making sure that there is not enough for the mouse to use to form a mound. After the hind limb unloading procedure, remove the animal from the apparatus and remove the tail ring.
Using a wire cutter record the body weight and return the animal to its home cage for recovery. After a period of recovery, the animals wait again and the soleus muscle and adrenal masses are measured. Body weights prior to the tailing surgery were not significantly different from those five days after surgery and prior to hu.
Although hind limb unloading for 14 and 28 days resulted in a small but non-significant loss in body mass, the mice fully recovered to their pre HU body weights. After three days of recovery. Food consumption for ambulatory control, mice and HU mice is shown here.
Average daily food intake was not different in HU mice over 28 days compared to controls. Significant soleus muscle atrophy is observed following HU for 14 and 28 days, indicating an effective procedure after three days of recovery. Sous mass was significantly greater than both of the HU groups, but still significantly less than ambulatory control animals.
Both the left and right adrenal glands were excised and weighed to provide an indicator of animal stress. As seen here, the average weights were comparable between control and HU mice Once mastered, the tailoring surgery can be completed within five to 10 minutes per mouse, and the cage set up can be completed in 15 minutes Following this procedure. Other methods like mice, Hein, limb reloading and recovery from disuse atrophy may be performed in order to answer additional questions such as how the muscle and bone systems can recover and respond to these prolonged periods of disuse.
Now that you've seen this video, you should have a good understanding of how to perform hind limb unloading in mice. This technique permits the study of multiple tissues and systems such as cardiovascular, musculoskeletal, and interment under loading and reloading conditions, and using a variety of mouse models such as knockouts or knockins.
