A Method to Study the Impact of Chemically-induced Ovarian Failure on Exercise Capacity and Cardiac Adaptation in Mice

The overall goal of this procedure is to accelerate the natural process of follicular atresia in the ovaries, thereby resulting in ovarian failure, depletion of estrogen, and a non cycling female, a condition that closely mimics menopause. This is accomplished by first administering for 15 to 20 consecutive days, the occupational chemical for vinyl cycline dioxide or VCD, which has been shown to specifically target and destroy small primary and primordial ovarian follicles by accelerating their natural process of atresia. The second step is to determine esra cycle and ovarian cyclicity by vaginal cytology beginning approximately six weeks after the start of VCD injections.

15 to 20 days of persistent dste or non cycling must be observed by vaginal cytology to confirm loss of cycling and ovarian failure. The final step is to implement an intervention that may be impacted by menopause or the transition to menopause, such as voluntary cage wheel exercise, and cardiac adaptation. Ultimately, voluntary cage wheel activity and cardiac adaptation is used to show whether menopause decreases cage wheel activity.

The main advantage of this technique over existing methods, such as ectomy, is that ectomy is a surgical procedure that results in the sudden depletion of estrogen, which is unlike the natural progressive loss of estrogen as occurs during the pre to postmenopausal transition. Implications of this technique extend towards therapy of metabolic and cardiovascular disease in postmenopausal women because postmenopausal women are particularly more susceptible to metabolic syndrome and its associated comorbidities including cardiovascular disease relative to premenopausal women. Visual Demonstration of this method is critical as the vaginal cytology steps are difficult to learn because care must be taken to execute the proper smear technique and identification of the EST cycle.

Four, vinyl cycline dioxide or VCD is stored at minus 20 degrees Celsius. To prepare VCD for the injection protocol, add point 587 milliliters of VCD to a clean dosing vial and bring the final volume to 10 milliliters with sesame oil cover and seal with param and mix by inverting gently. Store at four degrees Celsius for up to seven days.

To begin the VCD injection protocol, weigh two mice at two months of age for 20 consecutive days, administer daily intraperitoneal injections of room temperature VCD at a dose of 160 milligrams per kilogram, using a 27 gauge needle during the injection period. Try to alternate the sites of injection to reduce the chances of local infection in a similar manner. Inject control mice with the vehicle solution sesame oil at a volume of 2.5 milliliters per kilogram after the injection period is complete.

Monitor the esra cycle daily by obtaining vaginal smears. One of the factors critical for the success of this study is obtaining high quality vaginal smears for cytology determination. Restrain mice using the dorsum scruff method.

Insert an eye dropper with 0.2 milliliters of sterile saline into the vaginal orifice to a depth of 0.5 centimeters. Mix the injected saline three times with the eyedropper, and then place one drop into a blank slide. Immediately evaluate each sample microscopically using a standard light microscope at 100 x magnitude to validate ovarian failure.

Subsequently, the VCD induced menopausal mice are used for the exercise experiments. The cage wheel apparatus consists of an 11.5 centimeter diameter wheel with a five centimeter wide running surface equipped with a digital magnetic counter that is activated by wheel rotation. Before each wheel running, test calibrate the cage wheel according to the manufacturer's guidelines.

Measure the inner diameter of the running wheel. Calculate the circumference and enter this value into the digital counter as wheel size to mount the wheel to the cage, slide the wheel. Stand through the top wiring of the animal cage.

Secure the wheel stand to the cage wiring with binder clips. The clips should be on the top of the wiring away from the animal. Feed the computer portion and any extra cable through the wiring of the cage.

Place the computer an excess cable out of reach of the animal. This may be accomplished by placing a medium-sized plastic weigh dish on top of the wiring of the cage to hold the computer an excess cable. Next, attach the computer portion of the digital magnetic counter to the wheel stand.

Using duct tape, attach the free magnet of the digital magnetic counter to the central bar on the same side as the magnet cable to the computer. Again, taping sufficiently to protect the magnet from chewing damage. The magnet should be positioned so that each rotation of the wheel is recorded by the computer without any interference or resistance to complete wheel rotation.

Attach the running wheel to the wheel. Stand by inserting the wheel stand posts into the mounting holes on the central bars of the wheel. Make sure the wheel is able to rotate freely.

The setup should be monitored daily for chewing damage and repaired as necessary for the experiment inbred seven month old C 57, black six and B six C3 F1 female mice from a given litter are randomly assigned to either the exercise regimen or the sedentary control. How's experimental animals individually? In a 47 by 26 by 14.5 centimeter cage with unencumbered access to a cage wheel for 28 days, control animals are housed together in a cage without a wheel.

Give all animals water and standard hard rodent chow. Add lido manually record daily values for the distance ran and total time spent running given by the magnetic counter record distance and time values within the same one hour timeframe every day for accurate and consistent values throughout the 28 day duration of the exercise period. After each day's recording, clear the magnetic counter to ensure accurate readings the following day.

At the end of the specific exercise period, the body weights of euthanized animals are recorded prior to tissue harvest. To exercise the heart, make a medial incision on the ventral side, open the diaphragm and visualize the heart rapidly. Excise the heart at the base and place in cold phosphate buffered saline to sufficiently wash out residual ventricular blood Trim the heart of excess tissue, weigh the whole heart and snap freeze in liquid nitrogen if desired.

Collect additional excise tissues wash with a modified ice cold phosphate buffered saline solution and snap freeze and isop pentane cooled in liquid nitrogen. In this study, ovarian failure was induced by VCD treatment and confirmed by microscopic examination of vaginal smears. The stage in the esra cycle is determined by the proportion of epithelial cells, quantified epithelial cells and leukocytes in PROEs nucleated epithelial cells predominate.

Here the arrows indicate nucleated cells during estro cells are predominantly un nucleated quantified squamous epithelial cells. Maestros is characterized by a mix of cell types with a predominance of leukocytes. Diastrous is dominated by leukocytes.

The estro cycle in mice typically lasts four days before VCD injected. Animals were considered acyclic after 15 days of persistent diastrous. This figure shows the average length in days of the est extra cycle of vehicle and VCD injected groups.

One cycle is measured from the first day in ES to the first day of the next period. In ES vaginal cytology began approximately six weeks following the onset of injections. It was observed that the ESTRO cycles of VCD injected animals became irregular compared to vehicle injected animals, and after 12 estro cycles, cytology of all VCD injected animals no longer indicated cycling at the completion of the VCD injection protocol, H and e staining of ovarian tissue cross-sections revealed significant atrophy in VCD treated ovarian tissue compared to controls.

When viewed at 20 x magnification at 200 magnification, a complete depletion of primary and primordial follicles was observed in the VCD treated tissue. In contrast to the vehicle treated tissue, the effect of VCD induced ovarian failure on exercise performance was tested in seven month old VCD and vehicle treated mice from two strains daily cage wheel exercise values were recorded for time and distance for each exercised animal throughout a four week exercise period. This figure shows the weekly averages of daily running values for distance, time, and speed.

For C 57, black six mice treated with either vehicle or VCD, significant differences were not observed in exercise performance measured by daily average running distance and time spent on the wheel between VCD and vehicle treated groups in either the C 57, black six or B six C3 F1 mouth strain. This indicates the impact of VCD induced ovarian failure On voluntary cage wheel exercise transcends at least two mouth strains following the duration of the voluntary cage exercise protocol. Body morphometrics were recorded and the hearts were rapidly excised and weighed consistent with previous observations.

Cardiac hypertrophy in response to cage wheel exercise measured by absolute heart weight and heart weight normalized to tibial length was observed. Although the absolute heart mass and heart weight to tibial length ratio were significantly greater in vehicle and VCD treated mice when compared to sedentary counterparts, there were no measurable differences in cardiac hypertrophy between control and VCD induced ovarian failure. Mice following the voluntary cage wheel exercise, finally, VCD and vehicle treated mice were subjected to forced treadmill running to determine both high and low intensity exercise capacities.

In the high intensity protocol, the average belt speed was increased stepwise every 10 minutes until the mice showed signs of exhaustion. On average, the maximal speed of vehicle treated mice was 26.7 plus or minus 0.95 meters per minute, and the maximal speed of VCD treated mice was 28 plus or minus 1.4 meters per minute before exhaustion. Lower intensity running at 20 meters per minute was used to assay for endurance capacity.

On average, the maximal time of vehicle treated mice was 28.3 plus or minus 1.3 minutes, and the maximal time of VCD treated mice was 29.7 plus or minus 1.4 minutes before exhaustion. No significant differences in high or low intensity Exercise capacity was found between vehicle and VCD treated groups indicating that VCD induced ovarian failure had no effect on mouse exercise capacity. After its development, this technique paved the way for researchers in the fields of cellular, molecular, and physiological sciences to explore the role of menopause and rodents.

After watching This video, you should have a good understanding of how to perform the proper VCD injections, execute the vaginal cytology, and implement a physiological intervention.