Simulating High-Altitude Hypoxic Conditions Delays Wound Healing In Rats

Summary

This study developed and preliminarily validated a rat model of delayed wound healing under high-altitude hypobaric hypoxia conditions.

Abstract

High-altitude hypobaric conditions may cause prolonged wound hypoxia and impaired healing, yet standardized models remain scarce. In this study, we developed a wound delayed healing model and preliminarily validated it by simulating high-altitude hypoxia using a controlled hypobaric system. Rats were housed at simulated altitudes of 3000 m (air pressure: 70.1 kPa, partial oxygen pressure: 14.7 kPa) to 8000 m (air pressure: 35.6 kPa, partial oxygen pressure: 7.5 kPa) to determine safety thresholds, with oxidative stress and skin hypoxia assessed. Results showed increased mortality risk at simulated 6000 m altitude, with tolerance observed below 5000 m. Elevated altitudes were associated with worsening oxidative stress and increased skin hypoxia; exhibited altitude-dependent delays in wound healing, reduced perfusion, suppressed collagen remodeling, and exacerbated inflammatory responses. Despite upregulation of vascular regenerative factors, microvascular density decreased. Overall, this study developed and preliminarily validated a rat model suitable for investigating mechanisms and interventions in high-altitude hypoxia-induced chronic wounds.

Introduction

Skin wound repair presents a widespread and significant clinical challenge. From acute trauma to various types of non-healing wounds, these conditions impose a persistent burden on healthcare systems and patients' quality of life^1,2,3,4,5,6. Wound healing is not a singular event but a highly coordinated, overlapping multistage process: inflammatory cells, stromal cells, keratinocytes, and the microvascular system collectively rebuild barriers and remodel tissues under the joint regulation of the extracellular matrix and diverse signaling networks^7,8,9,10. Given the energy-intensive nature of repair and its extreme sensitivity to the internal environment, alterations in systemic metabolism and physiological states—such as heightened oxidative stress, immune dysregulation, and impaired microcirculatory perfusion—can disrupt the normal repair cascade at multiple points, leading to prolonged inflammation, inadequate regeneration, and abnormal remodeling^{9,11,12,13,14,15}.

Oxygen supply is a key factor limiting wound repair processes, participating in energy metabolism and cell migration/proliferation while also modulating angiogenesis and inflammatory responses through hypoxia signaling pathways^16,17,18. Transient hypoxia in the early wound phase stabilizes hypoxia-inducible factor and initiates adaptive repair programs. However, when hypoxia becomes excessive or persistent, although angiogenesis signals may be compensatorily upregulated, they may fail to translate into effective microvascular reconstruction. Meanwhile, macrophage phenotype switching and inflammatory regression become more prone to imbalance, forming a vicious cycle of "hypoxia-inflammation-revascularization deficiency"^19,20,21,22. This is particularly relevant in high-altitude environments: reduced atmospheric pressure lowers inspired oxygen partial pressure, inducing persistent hypoxia that heightens risks of impaired wound healing and infection-related complications^23,24,25,26.

Exposure to high altitudes triggers a marked hypoxic stress response in humans²⁷. High-altitude environments can lead to persistent tissue hypoxia, inadequate perfusion, and inflammatory responses at wound sites²⁸. These pathophysiological changes may impair the healing process by promoting excessive reactive oxygen species, activating proteases, and inducing cellular senescence, while simultaneously disrupting the function of mesenchymal stem cells^29,30. Studies directly evaluating the effects of high-altitude exposure on human skin wound healing remain limited. Therefore, the use of hypobaric hypoxic animal models can help validate the impact of altitude-related factors on wound repair.

Previous animal models at fixed altitudes have reported delayed wound healing; however, the altitude-dependent effects, the safety threshold for prolonged exposure to hypobaric conditions, and direct evidence of local skin hypoxia remain unclear²⁶. Furthermore, there is a lack of systematic assessment of healing impairments, which has limited research into the underlying mechanisms and the identification of potential intervention targets³¹. In this study, we developed and preliminarily validated a skin wound model based on a hypobaric control system to simulate high-altitude hypobaric hypoxia under controlled conditions. Employing a graded simulated altitude exposure strategy, we first defined the safety margins for sustained hypobaric exposure and assessed the biological effects of hypobaric hypoxia through systemic indicators. Crucially, we further validated in vivo that the hypobaric environment indeed caused localized skin oxygen supply limitation. Subsequently, we demonstrated that sustained hypobaric hypoxia delays wound closure in an altitude-dependent manner, accompanied by histological alterations and key pathway abnormalities. Overall, this study developed and preliminarily validated an animal model platform for chronic wounds associated with high-altitude hypoxia, enabling mechanism elucidation and systematic evaluation of subsequent therapeutic strategies³².

Protocol

All experimental procedures involving animals were reviewed and approved by the ethics committees of the University of Electronic Science and Technology of China (No. 106142025091534170) and the Army Medical University (No. AMUWEC20265509). The graphical workflow for the experimental design and analysis involved in this study is shown in Figure 1. Additionally, the reagents and equipment employed in this study are listed in the Table of Materials.

Experimental animals and ethical statement
This study utilized male Sprague-Dawley (SD) rats, purchased from Chengdu Dashuo Laboratory Animal Co., Ltd., China. All animals underwent a 7-day acclimatization period under standard conditions prior to experimentation.

Inclusion and Exclusion Criteria
Animal inclusion criteria were as follows: male SD rats, 6 to 8 weeks of age, body weight 200–250 g, appearing healthy and exhibiting normal behavior, and free of skin lesions or infections.

Animal exclusion criteria were as follows: wounds showing obvious infection following wound modeling; wounds with non-standard healing or lacerations; death during the experiment or accidental death due to anesthesia.

Data point exclusion criteria were as follows: wound images with extensive scabbing, hair obstruction, or uneven illumination; laser speckle contrast imaging (LSCI) images showing motion artifacts; tissue sections exhibiting obvious wrinkles, fragmentation, or missing target areas; serum samples showing obvious hemolysis; Immunofluorescence (IF) or immunohistochemistry (IHC) images with overexposure or incorrect antibody localization

Handling of exclusions:
For all excluded animals or data points, valid data were re-obtained through supplementary experiments to ensure that the final valid sample size for each group was N ≥ 3 for all endpoint analyses.

Hypobaric control system and simulated high-altitude hypoxia environment
The simulated high-altitude hypoxic environment was established using a hypobaric control system. This system allows precise regulation of cabin air pressure to reproduce atmospheric pressure corresponding to different altitudes, thereby lowering ambient oxygen partial pressure and inducing controlled hypoxic exposure (Figure 2A).

Cabin pressure was adjusted to simulate altitudes of 3000 m, 4000 m, 5000 m, 6000 m, 7000 m, and 8000 m, with detailed parameters provided in Table 1. Throughout the experiment, SD rats were housed in groups according to their experimental allocation. During cabin operation, internal pressure, temperature, and humidity were continuously monitored to ensure that, apart from pressure, all environmental conditions remained comparable to those of standard laboratory housing. The housing environment maintained a temperature of 22–24 °C, relative humidity of 50%–60%, a 12-h light/dark cycle, and provided free access to standard chow and drinking water.

Safety assessment of hypobaric exposure and survival analysis
To evaluate the safety margins for sustained hypobaric exposure, rats were individually numbered and randomly assigned to experimental groups using a computer-generated random number sequence (the RAND function in Excel). The allocation was performed by an investigator not involved in subsequent animal care, data collection, or outcome assessment. Based on reference animal studies involving exposure to high-altitude gradients, the sample size for each group was set at 12³³. Rats were divided into a normoxic control group (500 m above sea level) and simulated altitude groups at 3000 m, 4000 m, 5000 m, 6000 m, 7000 m, and 8000 m. Rats in each group were continuously housed in their respective environments for 30 days. Every 2 days, the hypobaric control system is briefly restored to sea-level conditions for 30 min to allow for cage cleaning and the provision of food and water. During decompression and recompression, the rate of pressure variation is maintained at approximately 300 m/min to minimize the risk of barotrauma. During this period, the general condition of all rats and any mortality events were observed and recorded daily. Survival curves were plotted using the Kaplan-Meier method, and log-rank tests were performed to compare survival differences between groups.

Detection of serum oxidative stress-related markers
On day 21 of hypobaric exposure, venous blood samples were collected from rats under anesthesia. After standing at room temperature, serum was separated by centrifugation (1,000 × g for 15 min at 4 °C) and stored at -80 °C for subsequent use. Serum levels of lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were measured using corresponding assay kits.

Assessment of hypoxic conditions in skin tissue
The hypoxia detection kit was used to assess hypoxia in skin tissue. In brief, on days 4 and 7 of hypobaric exposure, rats were intraperitoneally injected with pimozide (60 mg/kg); the rats were euthanized 1 h after injection, and skin tissue from the back was collected for analysis. The detailed euthanasia procedure is as follows: According to the American Veterinary Medical Association's guidelines on euthanasia, the animal was placed in an induction chamber and induced anesthesia using oxygen containing 4–5% isoflurane. Once the animal has lost consciousness, the isoflurane concentration is reduced to 2–3% and maintained for at least 10 min. Before performing cervical dislocation as a confirmatory method, it must be confirmed that the heartbeat and breathing have ceased. Tissue samples were cryopreserved in liquid nitrogen and stored at -80 °C for subsequent use. Sections were prepared using a cryostat microtome. IF staining was performed to label hypoxic regions within the tissue.

All fluorescence images were acquired using an automated slide scanner. The fluorescence parameters settings were as follows: DAPI (excitation wavelength 365 nm ± 10 nm, emission wavelength 440 nm ± 40 nm), Cy3 (excitation wavelength 542–566 nm, emission wavelength 579–611 nm), and Cy5 (excitation wavelength 628 nm, emission wavelength 692 nm). Hypoxia signals were quantitatively analyzed using ImageJ software.

Full-thickness skin wound model in rats
To assess alterations in skin wound healing under sustained hypobaric conditions, rats were first grouped and acclimated to their respective environments before wound modeling. Rats were individually numbered and randomly assigned to either the normoxic control group or the hypobaric exposure groups simulating altitudes of 3000 m, 4000 m, and 5000 m using a computer-generated random number sequence (the RAND function in Excel). The randomization was performed by an investigator who was not involved in the subsequent wound creation, postoperative care, or outcome assessment. The detailed parameters for each group are as follows: the normoxic control group was located at 500 m above sea level (atmospheric pressure: 101 kPa, partial oxygen pressure: 21 kPa); the 3000 m group (70.1 kPa, 14.7 kPa); the 4000 m group (61.6 kPa, 13.1 kPa); and the 5000 m group (54 kPa, 11.3 kPa).

After the 7-day adaptation period, SD rats were anesthetized and maintained under anesthesia via inhalation of 2% isoflurane. Simultaneously, a local infiltration injection of 5% lidocaine containing epinephrine (dilution ratio: 1:100,000) is administered to the marked surgical site to provide perioperative analgesia and hemostasis. Using a biopsy punch (with a 10-mm-diameter circular blade at the base), create a skin incision. Press the biopsy punch against the rat's dorsal skin, then gently rotate it so that the circular blade gradually cuts through the skin until it reaches the superficial subcutaneous fascia. Upon removing the biopsy punch, a regular circular skin incision approximately 10 mm in diameter will be formed.

Following wound creation, rats in each group were returned to and maintained within their original environments until the experimental endpoint, ensuring that the entire wound healing process proceeded under the predetermined oxygen conditions.

Macroassessment of wound healing process
Photographs of the wound were taken at days 0, 2, 4, 6, 8, 10, 12, and 14 post-wound formation under uniform lighting and consistent imaging distance. Wound area was measured using Image J. Briefly, wound images obtained at each time point were imported into Image J, calibrated according to the scale bar, and the wound margins were manually outlined to determine the wound area³⁴. Wound healing was calculated as follows: Wound healing (%) = (Initial wound area − Current wound area)/Initial wound area × 100%.

Assessment of wound microcirculation
On the 7th day of wound healing, microcirculatory blood perfusion in the wound area was evaluated using an LSCI system. Prior to the examination, rats were anesthetized via inhalation with 2% isoflurane and secured to a temperature-controlled operating table to maintain body temperature. The dorsal wound area was fully exposed, and the laser speckle probe was positioned vertically approximately 20 cm above the wound. The probe power was set to 110 mW, the sampling frequency to 30 Hz, and the exposure time to 3 seconds. Under dark conditions, acquire a blood flow perfusion pseudocolor image of the wound area. Use the LSCI software to manually outline the wound area on the blood flow perfusion pseudocolor image; the software will then automatically calculate the blood flow perfusion value for that area over a 3-s period (in arbitrary units [A.U.]). Finally, export the perfusion values to an appropriate software for plotting.

Histological staining and collagen remodeling analysis
Animals were euthanized at designated time points, and intact wound and surrounding skin tissues were harvested, fixed, dehydrated, paraffin-embedded, and sectioned. Hematoxylin and eosin (HE): used to evaluate wound width, re-epithelialization extent, and regenerated epidermal thickness; Masson's trichrome staining: used to assess collagen fiber deposition and calculate collagen volume fraction; Sirius red staining: used to distinguish collagen type I (COL I) and collagen type III (COL III) under polarized light microscopy and calculate the COL I/COL III ratio.

Immunohistochemical staining
Paraffin sections underwent dewaxing, rehydration, and antigen retrieval before IHC or IF staining. Primary antibodies were incubated overnight at 4 °C, followed by the addition of corresponding secondary antibodies. Specific antibody information and corresponding dilution are provided in the Table of Materials. IHC sections were developed with DAB, while IF sections used fluorescent secondary antibodies with DAPI counterstaining for nuclei^35,36. All images were acquired under identical conditions and quantified using ImageJ software.

Statistical analysis
All data are presented as mean ± standard deviation. Statistical analyses were carried out using GraphPad Prism software. For comparisons among multiple groups at a single time point, one-way analysis of variance (ANOVA) was applied, and when a significant overall difference was detected, Tukey's multiple comparison test was used for subsequent intergroup analyses. For wound healing data assessed across multiple time points, two-way ANOVA was performed with treatment group and time included as independent factors to evaluate main effects as well as their interaction. Post-hoc analyses were likewise conducted using Tukey's multiple comparison test. To further assess the statistical power, a post-hoc power analysis was conducted in the G*Power software based on the primary endpoint (wound healing at day 14).

Survival analysis employed the Kaplan-Meier method to plot survival curves, with log-rank tests used to compare survival differences between groups. For pairwise comparisons among multiple groups, the Bonferroni correction was applied to control the risk of Type I errors from multiple comparisons. All statistical tests were two-tailed, with P < 0.05 considered statistically significant.

All outcome assessments, including wound area measurements, histological and immunohistochemical quantitative analyses, and LSCI analyses, were performed by a single investigator who was blinded to the study group assignments.

Results

Validation of safety margins for persistent hypoxia induced by low pressure
Kaplan–Meier survival analysis demonstrated a gradual downward shift in survival curves as simulated altitude increased, with mortality events first observed in the 6000 m group (Figure 2B). Pairwise comparisons with Bonferroni correction revealed no statistically significant differences in survival rates between the control group and the 6000 m to 8000 m groups. By day 30, the final survival rat...

Discussion

High-altitude conditions impair the wound healing process by inducing tissue hypoxia, reduced blood perfusion, and inflammatory responses, which in turn promote excessive production of reactive oxygen species, protease activation, cellular senescence, and dysfunction of mesenchymal stem cells^27,28,29,30. Given the current limitations in human research on the effects of high altitude, the develo...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Automated slide scanner	Olympus	VS200	Tissue Section Scanning
Bovine serum albumin (BSA)	Servicebio	GC305010	Blocking reagent for IHC/IF
CD206 antibody	Proteintech	18704-1-AP	Detection of M2 macrophages (Dilution 1:200)
CD31 antibody	Abcam	ab182981	Detection of microvessel density (Dilution 1:800)
CD86 antibody	HuaBio	ER1906-01	Detection of M1 macrophages (Dilution 1:200)
CK19 antibody	HuaBio	ET1601-6	Evaluation of re-epithelialization (Dilution 1:200)
DAB Substrate Kit	ZSGB-BIO	ZLI-9018	Chromogenic substrate for IHC staining
FITC-conjugated goat anti-rabbit IgG	Servicebio	GB22303	Fluorescent secondary antibody for IF (Dilution 1:100)
G*Power	University of Duisburg-Essen, Germany	www.gpower.hhu.de	Calculation of Statistical Power
GraphPad Prism software	GraphPad Software	https://www.graphpad.com/	Statistical analysis and graph generation
Hematoxylin staining solution	J&K Scientific	LM10N13	H&E staining
HIF-1α antibody	Bioss	BS-0737R	Detection of hypoxia-related signaling (Dilution 1:100)
HRP-conjugated goat anti-rabbit IgG	Servicebio	GB23303	Secondary antibody for IHC (Dilution 1:100)
Hypobaric control system	Shanghai Yuyan Instruments Co., Ltd.	LP-1500	Used to simulate graded high-altitude hypobaric hypoxia
Hypoxyprobe-1 Kit	Hypoxyprobe Inc.	HP1-100	Used for in vivo hypoxia detection
IF555 Tyramide signal amplification kit	Servicebio	G1233	Used for enhanced fluorescence signal amplification
IL-10 antibody	HuaBio	HA722032	Detection of anti-inflammatory cytokine (Dilution 1:100)
IL-1β antibody	HuaBio	HA723965	Detection of pro-inflammatory cytokine (Dilution 1:100)
ImageJ software	National Institutes of Health (NIH), USA	https://imagej.net/ij/download.html	Image quantification
Isoflurane	RWD Life Science Co., Ltd.	R510-22-16
Ki-67 antibody	HuaBio	HA721115	Detection of keratinocyte proliferation (Dilution 1:400)
Laser speckle blood flow imaging system	RWD Life Science Co., Ltd.	RFLSI ZW	Assessment of  blood perfusion
LDH Assay Kit	Nanjing Jiancheng Bioengineering Institute	A005-1-2	Serum LDH measurement
Masson’s trichrome staining kit	Servicebio	G1006-100ML	Collagen deposition analysis
MDA assay kit	Nanjing Jiancheng Bioengineering Institute	A003-1-1	Lipid peroxidation assessment
Sirius red staining kit	Servicebio	G1078-100ML	Collagen I/III differentiation
SOD assay kit	Nanjing Jiancheng Bioengineering Institute	A001-1-1	Serum SOD activity measurement
TNF-α antibody	HuaBio	ER65189	Detection of pro-inflammatory cytokine (Dilution 1:200)
VEGF-A antibody	HuaBio	ET1604-28	Detection of angiogenesis-related signaling (Dilution 1:200)