Effect of Maternal Aerobic Exercise on Myocardial and Cerebral Ischemia/Reperfusion Injury in Offspring of Hypertensive Rats

Summary

This research aims to assess whether maternal aerobic exercise during pregnancy in spontaneously hypertensive rats can reduce the susceptibility of offspring to ischemia/reperfusion injury. To achieve this, surgical models and physiological assessments are applied to the offspring.

Abstract

Hypertension-mediated organ damage (HMOD) affects vital organs such as the heart and brain, leading to adverse outcomes and posing a serious threat to human health. Maternal exercise helps promote fetal growth and development by improving the intrauterine environment, thereby reducing disease risk and enhancing offspring health. This study investigates whether maternal exercise during pregnancy can reduce the susceptibility of the offspring's heart and brain to ischemia/reperfusion (I/R) injury linked to hypertension, in order to evaluate the protective potential of maternal exercise as an early-life intervention. Pregnant normotensive Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) were randomly assigned to either a sedentary (p-WKY-SED, p-SHR-SED) or exercise (p-WKY-EX, p-SHR-EX) group for the duration of their gestation. A maternal swimming exercise model was established during pregnancy. Three-month-old (3M) offspring were selected as research subjects. During this period, blood pressure was monitored, and myocardial ischemia/reperfusion injury (MI/RI) and middle cerebral artery occlusion (MCAO) surgeries were performed to induce ischemia/reperfusion (I/R) injury. Maternal exercise during pregnancy significantly reduced infarct size in both male and female offspring of hypertensive rats following MI/RI and MCAO. In the MI/RI model, infarct size was reduced by 60.9% in males and 55.9% in females. In the MCAO model, infarct size was reduced by 31.0% in males and 35.9% in females. Additionally, maternal exercise improved post-myocardial infarction (MI) cardiac function in male offspring and decreased their overall susceptibility to ischemic injury in both the heart and brain. In the context of hereditary hypertension during pregnancy, maternal aerobic exercise has been shown to confer significant protective effects on adult offspring by reducing I/R injury in the heart and brain. These findings highlight the potential of maternal exercise as an effective non-pharmacological strategy for early-life intervention to reduce susceptibility to HMOD.

Introduction

The global incidence and mortality of cardiovascular diseases (CVD) continue to rise, hypertension, a key risk factor for CVD, also shows a steadily increasing global prevalence¹. Hypertension-mediated organ damage (HMOD) frequently affects the heart and brain, leading to left ventricular hypertrophy, myocardial infarction, and stroke². These complications are driven by vascular remodeling, endothelial dysfunction, and impaired organ perfusion.

In 1986, Barker et al. introduced the "Developmental Origins of Health and Disease" (DOHaD) theory³, linking the intrauterine environment to an offspring's susceptibility to chronic diseases in adulthood. In cases of maternal hypertension, fetuses are often exposed to ischemia and hypoxia⁴, which can impair normal cardiac development and predispose offspring to early-onset hypertension, cardiovascular disease, and metabolic dysfunction in adulthood⁵. Exercise, as a key non-pharmacological intervention, has received growing attention. The beneficial effects of maternal exercise during pregnancy on offspring include improved placental blood flow⁶, enhanced autonomic regulation⁷, increased insulin sensitivity⁸, and better glucose homeostasis⁹. Compared to pharmacological or dietary interventions, maternal exercise is non-invasive, broadly applicable, and poses minimal risk to both mother and fetus. Among various exercise modalities, swimming is particularly suitable in rodent models due to its low injury risk, better stress control, and reproducibility. While previous studies show that maternal exercise improves cardiovascular function in hypertensive offspring, its ability to confer direct organ protection against ischemic injury remains unclear. This is the first study to evaluate maternal aerobic exercise as a protective intervention against cardiac and cerebral ischemia/reperfusion (I/R) injury in hypertensive offspring.

This study aims to investigate the impact of maternal exercise on target organ outcomes in the offspring of Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Three-month-old (3M) offspring are selected as research subjects and subjected to myocardial ischemia/reperfusion injury (MI/RI) and middle cerebral artery occlusion (MCAO) procedures. Our data provide valuable insights into how maternal exercise ameliorates target organ I/R susceptibility in the offspring of hypertensive dams.

Protocol

All experimental procedures were approved by the Animal Ethics Committee of Beijing Sport University (Approval No. 2022034A). Rats were housed individually at the Beijing Sport University animal facility under controlled conditions (temperature: 22-24 °C; humidity: 45%-55%; 12-h light/dark cycle). Specific pathogen-free (SPF) 11-week-old female and 12-week-old male SHR, along with age-matched normotensive WKY rats, were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (License No. SYXK [Beijing] 2021-0053) for breeding purposes. The reagents and the equipment used for this study are listed in the Table of Materials.

1. Experimental animals and grouping

Pregnant rats were provided a standard rodent breeding diet and water ad libitum. All rats delivered spontaneously, and their offspring were weaned onto a standard rodent diet. For mating, female and male rats of the same strain (WKY or SHR) were co-housed at a 1:1 ratio. The presence of a vaginal plug and sperm in a vaginal smear was used to designate gestation day 1 (GD1). Upon confirmation of pregnancy, rats were randomly assigned to four groups: normotensive sedentary (p-WKY-SED), normotensive exercise (p-WKY-EX), hypertensive sedentary (p-SHR-SED), or hypertensive exercise (p-SHR-EX). Experimental analyses were performed on rats at gestation day 21 (GD21), fetuses at embryonic day 21 (ED21), and 3M male and female offspring from each group.

2. Maternal aerobic exercise procedure

Prior to mating, female rats were acclimatized to the housing facility for one week, followed by a 5-day water adaptation period involving 15 min of daily exposure to 10 cm deep water maintained at 34-35 °C. Following successful mating, rats in the exercise group began a structured swimming regimen. The protocol commenced with a 4-day adaptation phase in 40 cm deep water (34-35 °C), where the daily swimming duration was progressively increased from an initial 20 min by 10 min each day. From day 5 until GD20, these rats swam for 60 min per day, 6 days per week. To control for potential stress from water immersion, rats in the sedentary groups were placed in shallow water (10 cm depth, 34-35 °C) for the same duration without engaging in active swimming¹⁰.

3. Measurement of blood pressure

Systolic blood pressure (SBP), diastolic blood pressure (DBP), and mean arterial pressure (MAP) were measured in 3M offspring rats (both male and female) using the CODA Monitor tail-cuff system under conscious and resting conditions. All measurements were conducted in a quiet environment between 8:00 AM and 12:00 PM at an ambient temperature of approximately 25 °C.
The heating pad was turned on 1 h in advance and set to 37-38 °C to ensure the device reached the required temperature before measurement. An appropriately sized restrainer was selected based on the body weight of each rat, and the animal was gently guided into the restrainer. After fixation, the rat was placed on the heating pad for 5 min to allow sufficient blood flow to the tail. An appropriately sized occlusion cuff (O-Cuff) and volume pressure recording cuff (VPR-Cuff) were selected based on the size of the rat's tail. During operation, the O-Cuff was carefully positioned approximately 1 cm from the base of the tail, followed by placement of the VPR-Cuff over the tail. After the 5-min acclimation period, blood pressure measurements were initiated while the rat remained conscious and at rest. Each rat underwent five acclimation cycles followed by ten consecutive measurement cycles to record blood pressure data. Throughout the entire procedure, animals were closely monitored and promptly removed from the restrainer upon completion of the measurement protocol.

4. MI/RI surgery in rats

Rats were fasted for 12 h prior to surgery, with free access to water. Body weight was measured before anesthesia. Anesthesia was induced using 5% isoflurane and maintained with 2% isoflurane during the procedure. Each rat was placed in a supine position and secured on a heating pad set to 37 °C.

Oral endotracheal intubation was performed using a 16 G catheter, which was gently inserted into the trachea and secured in place. The catheter was then connected to an integrated anesthesia and ventilation system. Ventilation parameters were set to a respiratory rate of 70 breaths per minute, a tidal volume of  8 mL/kg, and an inspiratory-to-expiratory ratio of 1:2.

Electrodes were subcutaneously inserted into the limbs of the rat according to standard lead placement: black for the right forelimb, green for the right hindlimb, and red for the left hindlimb. The electrodes were connected to a biological function experimental system and bio-amplifier, and electrocardiographic (ECG) monitoring was performed using LabChart software.

The chest hair of the rat was shaved, and the surgical site was disinfected with alcohol. An incision was made in the skin on the left side of the sternum corresponding to the heart. Blunt dissection was performed to expose the ribs. A chest retractor was inserted between the third and fourth ribs to open the thoracic cavity. The pericardium was carefully torn and separated to fully expose the heart. The accompanying great cardiac vein was used as a landmark. Approximately 1-2 mm below the left atrial appendage, an 8-0 suture was passed through the epicardial surface to ligate the left anterior descending (LAD) coronary artery. A small wooden tube was placed at the knot site, and a slipknot was tied to occlude blood flow. The chest cavity was then rapidly closed. Successful ligation was confirmed by ST-segment elevation on the electrocardiogram and visible cyanosis in the ischemic myocardial region.

After 45 min of occlusion, the slipknot was loosened, and the wooden tube was removed to initiate reperfusion. Air was expelled from the thoracic cavity, which was then closed and sutured. The surgical site was disinfected with povidone-iodine, completing the procedure.

A MI/RI model was thus established following 24 h of reperfusion. Once spontaneous breathing resumed, the endotracheal tube was removed, and the rat was returned to a clean cage.

5. Echocardiography

Echocardiographic assessment was performed using a small-animal color Doppler ultrasound imaging system equipped with an L22-8K3 high-frequency linear array probe (center frequency: 15 MHz). Rats were anesthetized with 5% isoflurane for induction and maintained under 2% isoflurane throughout the procedure. Each rat was placed in the supine position and secured on the surgical platform, with the temperature maintained at 37 °C. Respiratory status was continuously monitored, and the isoflurane flow rate was adjusted as needed to avoid overly deep or shallow anesthesia, which could compromise the accuracy of cardiac ultrasound measurements.

After shaving the chest area, an appropriate amount of ultrasound coupling gel was applied. The ultrasound probe was positioned over the left anterior chest wall to obtain M-mode echocardiographic images from the parasternal long-axis and short-axis views of the left ventricle. For each parameter, the average value of six consecutive cardiac cycles was calculated. Cardiac function was assessed by measuring the left ventricular internal diameter in systole (LVIDs), left ventricular internal diameter in diastole (LVIDd), fractional shortening (FS), and ejection fraction (EF).

6. MCAO surgery in rats

Rats were fasted for 12 h before surgery but had free access to water. The MCAO model was established using an optimized Zea-Longa suture method. Anesthesia was induced with 5% isoflurane and maintained with 2% isoflurane. Once muscle relaxation was observed and respiration and heart rate stabilized, the rats were placed in a supine position and secured on a thermostatically controlled heating pad.

The cervical region of the rat was routinely shaved and disinfected. A midline incision slightly to the left of the neck was made, and the muscle layers were bluntly dissected to expose and isolate the left common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA). The ECA was ligated with fine suture thread, and the CCA and ICA were temporarily clamped with artery clips. An incision was made in the ECA, and the suture embolus was inserted into the CCA through the ECA opening. After transecting the ECA, it was gently pulled downward to align with the ICA, facilitating a straight path for embolus insertion. The embolus was carefully advanced into the intracranial segment of the ICA. Insertion was stopped upon encountering slight resistance, indicating proper positioning. At this point, the embolus was inserted 18-20 mm from the bifurcation of the ECA and ICA, corresponding to the pre-marked depth, with the tip occluding the origin of the middle cerebral artery (MCA).

The diameter of the embolus tip was selected based on the rat's body weight: 0.31-0.32 mm for rats <200 g, and 0.38-0.40 mm for rats weighing 281-330 g. After confirming correct placement, the embolus was secured in place, artery clips were removed, the skin incision was sutured, and the surgical site was disinfected with povidone-iodine.

Successful insertion of the suture was confirmed by a reduction of more than 50% in blood perfusion on the affected side. After 2 h of ischemia, the embolus was withdrawn to initiate reperfusion. A cerebral ischemia-reperfusion injury model was established after 24 h of reperfusion.

Neurological deficits in rats were evaluated 24 h after MCAO using the Zea Longa scoring system. The scoring criteria were as follows (Table 1).

Only rats with Zea Longa scores of 1 to 3 were included in the subsequent analyses. Rats with scores of 0 or 4 were excluded due to the absence of effective occlusion or excessive brain injury, respectively.

7. Laser speckle contrast imaging (LSCI)

Rats were fasted for 12 h prior to the experiment but had free access to water. Anesthesia was induced with 5% isoflurane and maintained at 2% isoflurane.

The rats were then placed in a prone position and secured in a stereotaxic apparatus. After shaving and disinfecting the scalp, a midline incision was made along the sagittal suture to expose the skull. Connective tissue and periosteum on the skull surface were carefully removed. While preserving the integrity of the dura mater, both sides of the skull were thinned using a cranial drill until the bone became transparent. A cranial window measuring 8 mm × 15 mm was created, allowing cortical blood vessels to be clearly visualized under a microscope.

The LSCI system was activated, and the device position and focal length were adjusted until the cranial window was clearly visualized. A laser light source with a wavelength of 780 nm was used. The illumination area was captured using a charge-coupled device with an exposure time of 15 ms and a frame rate of 15 frames per second. Continuous monitoring was performed for 15-30 seconds to obtain images with a resolution of 1280 × 960 pixels. The system subsequently converted the original speckle images into blood flow maps and quantitative perfusion data.

Baseline cerebral blood flow in the MCA supply regions on both hemispheres was first recorded prior to ischemia. The scalp was then sutured and disinfected before initiating the MCAO procedure. Cerebral blood flow in both hemispheres was continuously monitored at immediate post-occlusion, 2 h, and 24 h following ischemia.

During the entire monitoring period, anesthesia was maintained with isoflurane, and the heating pad was regulated to keep the rat's body temperature constant at 37 °C. Data acquisition began once the animal exhibited stable respiration and heart rate.

8. 2,3,5-triphenyltetrazolium chloride (TTC) staining

2% (w/v) TTC solution was prepared by dissolving 0.2 g of TTC powder in 10 mL of phosphate-buffered saline (PBS, pH 7.4). The solution was transferred into a foil-wrapped centrifuge tube, vortexed thoroughly to ensure complete dissolution, and then placed in a water bath preheated to 37 °C.

After 24 h of reperfusion, rats were anesthetized with 5% isoflurane and euthanized via exsanguination through the abdominal aorta. The heart and brain were immediately harvested and rinsed thoroughly with 10 mL of PBS. Tissues were snap-frozen at −40 °C for 30 min, then embedded in molds and sectioned coronally from anterior to posterior at a thickness of 2-3 mm per slice. The slices were incubated in 2% TTC solution. Heart slices were incubated in a 37 °C water bath protected from light for 10 min, while brain slices were incubated under the same conditions for 25 min. Slices were turned every 5 min during incubation to ensure even staining. After staining, slices were rinsed three times with 10 mL of PBS and fixed in 10 mL of 10% formalin for 24 h.

After 24 h of fixation, tissue slices were removed and blotted gently with gauze to remove excess moisture. Each slice was placed on a transparent glass plate, and both the front and back sides were scanned. In the scanned images, white regions indicated infarcted (ischemic) tissue, while red regions represented viable (non-ischemic) tissue. Image analysis was performed using ImageJ software to calculate the infarct area ratio of both myocardial and cerebral slices. Myocardial infarction area (%) = (Infarct area / Total myocardial area) × 100%; Cerebral infarction area (%) = (Infarct area on the MCAO side / Total area of the ipsilateral hemisphere) × 100%.

9. Safety notes and hazardous material handling

Isoflurane was handled inside a fume hood to prevent inhalation exposure and was stored in a cool, well-ventilated area. Formalin and TTC were handled using nitrile gloves and face masks to minimize skin contact and inhalation risk. TTC was recognized as photosensitive and therefore stored and used in foil-wrapped containers.

The surgical area was disinfected with 75% ethanol, and all instruments were autoclaved at 121 °C for 20 min prior to use. Operators wore sterile gloves and gowns to maintain aseptic conditions throughout procedures.

Post-surgical monitoring was performed every 6 h for 24 h to ensure animal welfare. Animals exhibiting severe distress, such as continuous trembling or lack of food and water intake, were immediately euthanized in accordance with institutional ethical guidelines.

All TTC and formalin waste was collected in labeled biohazard containers and disposed of following the institutional hazardous waste management protocol. Animal tissues were incinerated through the university's designated biowaste facility. Contaminated surgical materials were autoclaved before disposal.

10. Statistical analysis

All data were presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 8 software. Differences among the four groups were assessed using two-way analysis of variance (two-way ANOVA; factors: hypertension and exercise). Comparisons between male and female offspring were evaluated using a two-tailed unpaired Student's t-test. P value < 0.05 was considered statistically significant.

Results

Maternal exercise during pregnancy improves fetal body weight in hypertensive rats and reduces blood pressure in male offspring

To investigate the effects of maternal exercise on developmental outcomes, fetal and adult offspring parameters were assessed. At ED21, no significant differences in fetal body length were observed among the four groups (Figure 1B). However, fetal body weight was significantly lower in the p-SHR-SED group compared to the ...

Discussion

This study provides strong evidence that maternal exercise during pregnancy in hypertensive rats conferred significant protection against ischemic injury to the heart and brain of adult offspring, with sex-specific differences in the degree of protection. This study found that maternal exercise reduced myocardial and cerebral infarct volumes in both male and female offspring. However, improvements in blood pressure and post-ischemic cardiac function were observed only in males, reflecting the intricate relationship ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10% Neutral Buffered Formalin	Sigma-Aldrich	R04586	TTC
2,3,5-Triphenyltetrazolium chloride	Sigma-Aldrich	T8877	TTC
75% Ethanol	Shandong Hualu Pharmaceutical Co., LTD	N/A	Surgery
Biopotential amplifier	AD Instruments	PowerLab	MI/RI surgery
Brain Matrix	Shenzhen RWD Life Technology Co., LTD	68716	TTC
Circular water bucket	Naliya	N/A	Rats swimming
Constant temperature water bath	Senxin	DK-S12	TTC
Electric Forced-Air Drying Oven	Shanghai Yiheng Scientific Instruments Co.,LTD	DHG-9620A	Surgery
Experimental Surgical Instrument	Shenzhen RWD Life Technology Co., LTD	SP0001-G	Surgery
Hair removal cream	Veet	N/A	Surgery
Hairdryer	Panasonic	EH-WNE5H	Post-swimming care
Heart mold	Shenzhen RWD Life Technology Co., LTD	68720	TTC
Integrated small animal anesthesia and ventilation system	Kent Scientific	Somnosuite	Surgery
Isoflurane	Shenzhen RWD Life Technology Co., LTD	R510-22-10	Surgery
Laser Speckle Blood Flow Imaging System	Shenzhen RWD Life Technology Co., LTD	RFSLI ZW/RFLSI III	MCAO surgery
Mating cage	Zhongke Life Science	SS3	Rats mating
MCAO Monofilament Suture	Shenzhen RWD Life Technology Co., LTD	MSRC42B200PK50	MCAO surgery
Mini Handheld Cranial Drill and Drill Bit	Shenzhen RWD Life Technology Co., LTD	78001/78063	MCAO surgery
Non-invasive Tail-cuff Blood Pressure Monitoring System	Kent Scientific	CODA Monitor	Blood Pressure Monitoring
Phosphate-Buffered Saline	Thermo Fisher Scientific	10010023	TTC
Physiological function experimental system	Chengdu TME Technology Co.,LTD	PL-3504	MI/RI surgery
Physiological Saline Solution (0.9% NaCl)	Shandong Hualu Pharmaceutical Co., LTD	N/A	Surgery
Povidone-iodine	Shandong Hualu Pharmaceutical Co., LTD	N/A	Surgery
Rat tracheal intubation tube	Zhongke Life Science	16G	MI/RI surgery
Razor Blades	Gillette	Q31/0115000301C004	TTC
Rodent breeding feed	Beijing Huafukang Biotechnology Co., LTD	1032	Pregnant rats feeding
Rodent maintenance feed	Beijing Huafukang Biotechnology Co., LTD	1022	Offspring rearing
Scanner	EPSON	Model V19II	TTC
Small animal ECG electrode	Yuyan Instruments	IX-BIO8	MI/RI surgery
Small animal ultrasound imaging system	Cona	SiliconWave	MI/RI surgery
Standard Stereotaxic Frame	Shenzhen RWD Life Technology Co., LTD	68002	MCAO surgery
Sterile cotton balls	Shandong Hualu Pharmaceutical Co., LTD	N/A	Surgery
Sterile gauze	Kufu Medical Instrument	N/A	Surgery
Suture needle with thread	Jinheng	5-0	Surgery
Suture needle with thread for ligation	Ningbo Lingqiao	3/8 circle, 8-0	Surgery
Thermostatic heating pad for small animals	Changsha Maiyue Instrument Co.,LTD	M5200	Surgery
Towels	Grace	N/A	Post-swimming care
Ultrasound coupling gel	Haisihainuo	N/A	MI/RI surgery
Vertical Autoclave	Xiamen Zhiwei Instrument Co.,LTD	FD50E	Surgery
Vortex mixer	Scientific Industries	Vortex-Genie 2	TTC
Water heater	Haier	EC6001-Q6S	Rats swimming
Weight Scale	Electronlc Acale	JM.A10001	Body weight measurement
Wistar Rats	Vital river Laboratory Animal Technology Co., LTD	N/A	Experiment