A Treadmill-Based Mouse Model for Investigating Hot Flashes with Continuous Skin Temperature Monitoring

摘要

This study establishes a mouse model of menopausal hot flashes by combining ovariectomy with exercise-induced thermogenesis and continuous high-resolution tail skin temperature monitoring.

摘要

Hot flashes significantly impair the quality of life in menopausal women, yet the underlying neural mechanisms and effective therapies remain poorly understood. A major challenge in this field has been the lack of reliable animal models and non-invasive methods for continuous skin temperature monitoring. Here, a mouse model combining ovariectomy with exercise-induced thermogenesis was developed to study hot flashes. Using a telemetric logger secured by a 3D-printed tail sleeve, tail skin temperature was continuously monitored in a stress-free state. Additionally, infrared cameras provided a complementary method to monitor heat production and dissipation, allowing for a more comprehensive assessment of body temperature. Using this model, ovariectomized mice were found exhibiting higher skin temperature increases and lower core body temperature, effectively mimicking the hot flash phenotype observed in menopause. This approach establishes a precise model for hot flash monitoring and will be valuable for researchers in thermoregulation, neuroscience, and clinical menopausal studies.

引言

Hot flashes are among the most prevalent and disruptive symptoms of menopause, significantly impairing quality of life¹. These episodes are characterized by a sudden surge in skin temperature followed by a transient drop in core body temperature², often accompanied by increased heart rate, anxiety, and dizziness³. Despite their profound impact, effective treatments remain limited⁴, underscoring the need for a deeper understanding of their underlying mechanisms.

A critical step in studying hot flashes is the development of reliable animal models that accurately replicate thermoregulatory dysfunction. Currently, the primary approach involves ovariectomy (OVX), which induces altered skin and core body temperatures⁵. However, these changes are often inconsistent, as they can be confounded by stress or ambient temperature fluctuations⁶. Moreover, capturing sporadic hot flash-like events remains challenging. Alternative methods employ pharmacological agents (e.g., senktide, capsaicin, tamoxifen, or naloxone) to trigger acute cutaneous vasodilation in rodents^7,8,9. While these compounds produce rapid, measurable skin temperature increases, their invasive administration and dose-dependent effects complicate the interpretation of thermoregulatory responses. Furthermore, it remains unclear whether drug-induced vasodilation fully recapitulates the physiological conditions of natural hot flashes. Thus, a more refined and reliable animal model is urgently needed.

Exercise is a well-known modulator of body temperature and has been linked to hot flash induction in humans¹⁰. Some studies have leveraged forced exercise in mice to mimic heat dissipation patterns resembling hot flashes^11,12,13. Although this approach provides a non-invasive means to provoke temperature fluctuations, monitoring skin temperature in active mice, particularly via infrared thermography, is technically challenging. Additionally, a comprehensive assessment of hot flash-like symptoms requires continuous, long-term recording of both tail (skin) and core body temperatures.

To overcome these limitations, an optimized mouse model was developed combining ovariectomy with treadmill-induced exercise to reliably evoke hot flash-like thermoregulatory responses. In the experiment, C57BL/6J mice from 8-10 weeks were housed under standard conditions (12:12 light-dark cycle, 22 °C ±1 °C, 50% ± 10% humidity). Furthermore, a telemetric temperature logger secured by a 3D-printed tail sleeve was designed to enable stress-free, continuous tail temperature monitoring in freely moving mice. Infrared thermography was also incorporated to provide complementary data on heat production and dissipation. This model will serve as a valuable tool for investigating the mechanisms of hot flashes and evaluating potential therapeutic interventions.

方案

These methods were designed for mouse models. Mice studies were approved by the Institutional Animal Care and Use Committee of Fudan University (2021JS0040). Prior to the implementation of the protocol, animals were housed in conformance with the Guide for the Care and Use of Laboratory Animals. In this study, 8- to 10-week-old female C57BL/6J mice, weighing 20-23 g, were housed under standard conditions (12:12 h light-dark cycle, 22 °C ± 1 °C, 50% ± 10% humidity). The details of the reagents and the equipment used are listed in the Table of Materials.

1. Experimental preparation

1. Prepare ten 8-10 week-old female mice housed under standard conditions (12:12 light-dark cycle, 22 °C ±1 °C, 50% ± 10% humidity) with ad libitum food and water.
2. Randomly allocate mice into OVX and sham groups (n = 5/group). Grouped house to help with the synchronization of estrous cycles.
3. Treadmill protocol setup
   1. Open the treadmill control software "TSE TreadMill (USB) 5.1.7_2" on the connected computer (Supplementary Figure 1).
   2. Initialize a new experimental file by selecting File > Initialize a New File from the menu bar.
   3. Program the running protocol:
      1. Set the first segment: 0-20 s acceleration from 0-0.17 m/s.
      2. Set the second segment: 20-150 s constant speed at 0.17 m/s.
      3. Set the third segment: 150-170 s acceleration from 0.17-0.25 m/s.
      4. Set the final segment: 170-600 s constant speed at 0.25 m/s^10,12.
         NOTE: Do not use electrical stimulation in this experiment to avoid extra stress.

2. Estrous cycle monitoring

1. Smear vaginal swabs on a staining slide daily at the same time each day for 8 consecutive days by flushing with 100 µL of sterile PBS using a pipette.
2. Air-dry samples on slides for 24 h in a fume hood.
3. Briefly dunk the samples in 100% methanol at room temperature.
   CAUTION: Methanol is toxic by inhalation, ingestion, or skin contact. Use in a well-ventilated fume hood, wear nitrile gloves, and avoid sparks/flames.
4. Dilute commercial 20× Giemsa dye to 1× using PBS.
5. Stain  the methanol fixed slideswith 1× Giemsa in the dark for 1-2 h.
6. Examine the vaginal cytology to determine the estrous stages based on these studies^14,15.
7. Proceed with experiments when the mice are in proestrus (Figure 2B).
   NOTE: Handle mice gently to avoid stress-induced cycle disruption. Avoid deep insertion of pipette tips to prevent pseudo-pregnancy.

3. Ovariectomy surgery

1. Record the weight of the mice and anesthetize the mice with isoflurane. Use a heating pad to maintain the animal's body temperature during the operation.
2. Shave the hair on the dorsolateral region bilaterally before the hind legs and disinfect with 70% ethanol and iodine alternatively for 3 times.
3. Make a 1 cm long paravertebral incision on the skin and use a blunt-tipped instrument to separate the skin from the underlying muscle tissue. Then, make the incision in the muscle wall. Expose the ovarian fat pads with cotton sticks.
4. Ligate ovarian blood vessels with sutures. Excise ovaries with surgical scissor¹⁶.
5. Close the incision in layers, muscle, and skin with non-absorbable sutures. Clean skin around the surgical site gently with saline to remove any dried blood or residual antiseptic scrub.
6. For sham controls, cut a piece of the fat pad without touching the ovaries.
7. Monitor the health of mice daily for 7 days post-operation.
   NOTE: Ensure the mice are kept on a heating pad at around 35 °C throughout the anesthesia process.

4. Core temperature logger implantation

1. Logger Programming and Initialization (Supplementary Figure 2).
   1. Insert the temperature logger vertically into the docking station.
   2. Click on Recorder > Connect in the software menu bar.
   3. Select Edit > New Measurement Seq. Def. from the menu bar.
   4. Choose Multiple Record Intervals from the recording mode options.
   5. Set parameters: Start date: [YYYY-MM-DD HH:MM], End date: [YYYY-MM-DD HH:MM], Recording interval: 5 min.
   6. Add the intervals setting to the sequence.
   7. Click on Recorder > Start New Measurement Sequence.
2. Under anesthesia, shave and sanitize the skin of the abdomen.
   NOTE:This abdominal temperature logger implantation procedure can be performed along with OVX surgery to avoid multiple surgeries.
3. Make a 2 cm midline skin incision approximately 1 cm below the diaphragm.
4. Extend the incision along the linea alba to open the peritoneal cavity.
5. Gently insert the core temperature logger into the abdominal cavity along the sagittal plane.
   NOTE: Proper placement is critical for accurate temperature readings. Avoid superficial positioning near the skin, as ambient temperature may interfere with measurements.
6. Suture the abdominal incision using 5-0 non-absorbable sutures.
7. Disinfect the abdomen with iodine solution.
   NOTE: Monitor the animal closely during recovery. Maintain a warm environment using a heating pad until the mice are fully awake.

5. Tail-mounted temperature logger attachment

1. Logger Programming and Initialization (Refer to step 4.1 procedure, modifying the recording interval to 2 min).
2. Clean the tail with an ethanol swipe. Disinfect the 3D sleeve with 70% ethanol and fasten the temperature logger inside the sleeve.
3. Apply fast-curing adhesive to the sleeve. Position the logger 1 cm from the tail base (Figure 1A). Make sure the detection face of the logger is in close contact with the skin. Ensure the logger is fixed on the ventral side of the tail to minimize movement burden.
4. Hold it in place with pressure for 2 min. Use brief anesthesia if needed for precise placement.
   NOTE: Monitor for tail swelling or logger detachment post-placement daily. A properly attached device could stay well for at least 3 days.

6. Treadmill running and data collection

1. Acclimate mice to the treadmill during the light phase for 3 days using the protocol in step 1.3.
   1. Place mice individually on the stationary treadmill belt. Allow 10 min for environmental exploration and habituation.
   2. Execute the protocol:
      1. Click on the Measure button. Click on Start to initialize the treadmill.
      2. Select Start Profile to begin the programmed running protocol.
2. Attach the tail temperature logger 24 h before experiments for good habituation (see step 5).
3. Treadmill running on the light phase of experimental day:
   1. Record baseline temperature for 30 s using an infrared camera 10 min prior to exercise. The core temperature is recorded simultaneously using the implanted abdominal temperature logger.
   2. Run mice on the treadmill per the protocol in step 1.3.
      NOTE: Ensure mice run continuously for 600 s. If mice stop running, gently encourage continued movement using a hairbrush.
   3. After treadmill running, transfer mice to clean cages. Measure tail temperature via infrared thermography at 0 min, 5 min, 10 min, and 15 min post-exercise.

7. Data analysis

1. For infrared and core temperature data:
   1. Calculate baseline as the average pre-exercise temperature.
   2. Plot ΔT (post-exercise temperature-baseline) for each time point.
2. For tail temperature logger data:
   1. Use the 10-min pre-exercise average as baseline.
   2. Compute ΔT at 2 min intervals for 15 min post-exercise.

结果

Development of a reliable thermoregulatory monitoring system

To establish a reliable model for measuring core and skin temperature, a custom 3D-printed tail sleeve was designed to securely attach a temperature logger 1 cm from the base of the tail (Figure 1A), enabling continuous and precise measurement of heat dissipation during exercise-triggered hot flashes.

讨论

This study establishes a practical and quantitative mouse model for investigating menopausal hot flashes, addressing a critical gap in the field. The integration of ovariectomy with exercise-induced thermogenesis successfully recapitulates core features of human hot flashes, namely, a rapid rise in tail skin temperature followed by a decline in core body temperature¹⁸. This model combines and extends the strengths of existing approaches by amplifying temperature variations and allowing precise tem...

材料

本文使用的材料清单

姓名	公司	目录编号	评论
动物植入式温度记录仪	星奥迪	DST纳米-T
C57BL/6J 小鼠	宝石药业科技	N000013
吉姆萨染色液	碧奥泰姆	C0133
红外热图像	变异CAM	VC HD 云台 980
跑步机系统	谢	Treadmill_6M