Effect of high-altitude hypoxia on function and cytoarchitecture of rats’ liver

Abstract

The liver is central to metabolic, detoxification, and homeostatic functions. Exposure to hypobaric hypoxia at high altitudes causes detrimental effects on the liver, leading to injury. This study evaluated the effect of hypoxia-induced at high altitudes on liver function, oxidative stress, and histopathological changes in rats. This study used 24 male Wistar rats (aged 8–10 weeks). The hypoxia (hypobaric hypoxia) was inducted at a high altitude of 2,100 m above sea level. Normoxia is defined as 40 m above the sea level. The rats were randomly divided into two groups: a control group maintained at low altitudes and an experimental group exposed to high altitudes for eight weeks. Blood samples were collected from all rats through a cardiac puncture, and liver samples were taken through an abdominal approach. All samples were processed through standard methods and evaluated for liver function tests and histopathological assessment. Serum aspartate aminotransferase and alanine transaminase levels significantly increased by 25% and 30%, respectively, in the high-altitude group compared to controls (p < 0.01), indicating mild hepatocellular damage. Oxidative stress assessment indicated a significant elevation in malondialdehyde by 42% in the liver homogenates of high-altitude rats compared to controls (p < 0.001). Moreover, Superoxide dismutase activity and glutathione content decreased by 18% and 22% in the high-altitude group (p < 0.01), confirming the increased oxidative stress. Histologically, minimal inflammatory infiltration was observed in the rat livers at high altitudes, with no signs of necrosis or severe structural changes. Subclinical liver dysfunction, as evidenced by altered serum enzyme levels and increased oxidative stress with mild histological changes, is induced by high-altitude hypoxia in rats. This study’s results support that a hypobaric hypoxic environment physiologically stresses the liver. Further research into the long-term implications of hypobaric hypoxia and the adaptive responses of the liver is warranted.

Introduction

The liver is a central organ in metabolic, detoxification, and homeostatic functions essential for an individual’s health¹. It is the primary organ involved in digesting and absorbing nutrients, metabolites, and wastes and is continually challenged by various endogenous and exogenous stressors². Thus, optimal liver functioning is critical for overall well-being. However, some endogenous and exogenous stressors can harm the liver. Upon persistent or high exposure, such stressors could impair the hepatic adaptive capacity and, in some cases, induce liver injury^3,4. Hypobaric hypoxia is a stress experienced at high altitudes. Reduced barometric and partial oxygen pressure above 2,500 m disrupt physiological homeostasis⁵. The response to hypoxic conditions involves several compensatory mechanisms initiated through hypoxia-inducible factors and other signaling pathways⁶. However, when the hypoxic stress degree or duration crosses the adaptive threshold levels, organs may be injured⁷.

Several studies have demonstrated that exposure to high altitude causes subclinical changes in liver enzymes and hepatic tissue at different times^7–10. Hypoxia can decrease the metabolic rate and increase anaerobic metabolism⁹. Increases in alanine transaminase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) serum levels at moderate levels have also been reported among populations residing above 2,500 m and among low-altitude residents after climbing high altitudes^11,12. Such enzymatic changes have often represented mild hepatocellular injury or inflammation and are usually reversible after returning to low altitudes¹³. Oxidative stress has been postulated as an important mediator of high-altitude-induced organ pathologies^4,9,10,12,14. Hypoxia increases reactive oxygen species (ROS) generation through different mechanisms, including the electron transport chain disruption in the mitochondria, the hypoxanthine metabolism, and inflammatory cell activation¹⁵. Increased ROS levels may reduce antioxidant potency and be compromised under hypobaric conditions¹⁴. The resulting redox imbalance leads to the imbalance of biomolecule oxidization, including lipids, proteins, and DNA¹⁶. This vulnerability results in specific and increased involvement of the liver, a highly metabolic organ with a high oxygen demand¹⁷. Despite its critical role, few studies have focused on the effects of high altitude on the liver.

This study investigates the impact of high-altitude exposure on liver function, oxidative status, and histopathological characteristics in a rodent model.

Materials and methods

Animals and experimental design

This study utilized 24 male Wistar rats aged 8 to 10 weeks. The animals were obtained from the animal house facility of Jazan University, Kingdom of Saudi Arabia. They were kept in individual polypropylene cages, given paddy husk as bedding, and allowed free access to food and water throughout the study with a semi-automated system. Animals were maintained at a 12/12 h light/dark cycle at 22–25 °C and 40–60% relative humidity. Room temperature and humidity were automated and monitored daily. In this study, all environmental and physiological variables that could potentially affect animal health were systematically controlled. All control and experimental animals included in the study were maintained under identical living conditions throughout the study period, except for altitude for experimental. These living conditions were standardized in both the normoxic and hypoxic centers to ensure that any observed differences could be attributed exclusively to hypoxia in the experimental group. This rigorous control was implemented to minimize the influence of confounding factors that could otherwise impact the results.

The rats’ general health and body weights were monitored regularly. The animals were left to acclimatize for one week. After acclimatization, the rats were randomly divided into two groups comprising 12 rats each: control and experimental. The average weight of rats was 291 ± 22 g at transfer time. The experimental animals were transferred in equipped vehicles to a high altitude, and the duration of transportation was 150 min. The induction of hypoxia in experimental animals was through high altitude (hypobaric hypoxia).

Control group

Rats were kept at a low altitude of 40 m above sea level within the animal house of Jazan University¹⁸. At 40 m above sea level, the pressure is 100869.08 Pa, and the oxygen concentration is 20.9% by volume. The control animals were held for the entire eight weeks of the experiment.

Experimental group

Animals were transferred to a high altitude of 2,100 m above sea level at the Animal House facility of King Khalid University (Abha, Kingdom of Saudi Arabia)¹⁸. The animals were transferred into safe cages during the day to avoid stress. The air pressure and oxygen concentration were 78,513.14 Pa and 16%, respectively. The rats were subjected to natural hypoxic conditions for eight weeks.

The control and experimental groups were kept for eight weeks in similar living conditions, the experimental group undergoing hyperbaric hypoxia due to the high altitude. In all procedures involving animals, approval was obtained from the Institutional Animal Ethics Committee of King Khalid University, and the procedures were performed under the guidelines for the Care and Use of Laboratory Animals and the ARRIVE guidelines¹⁹.

Sample collection

After eight weeks, the rats in the control and experimental groups were fasted overnight for 16–18 h with free access to water to reduce the post-prandial variability of liver enzyme activities. Blood and tissue samples we collected at the end of the experiment. The blinding technique was applied at all stages of the research. A double-blind design was employed throughout the study to minimize bias. Researchers involved in experimental procedures, histological sample preparation, morphometric studies, and statistical data analysis were blinded to the group assignment of the samples. At no point during the study did any researchers know which samples belonged to the specific group.

Blood samples

The rats were anesthetized with a combination of ketamine hydrochloride 50 mg/kg and xylazine 5 mg/kg. The anesthetics were administered intraperitoneally²⁰. The anesthesia was assessed by monitoring the loss of pedal reflexes.

Blood samples were collected following cardiac puncture under aseptic conditions. Approximately 2–3 ml of blood was withdrawn slowly and transferred into serum separator tubes containing a clot activator. The blood samples were kept at room temperature for 30 min to clot. The serum was separated by centrifugation at 3,000 rpm for 15 min at 4 °C using a refrigerated centrifuge (Eppendorf Centrifuge 5702 R, Eppendorf AG). The separated serum was collected in sterile microcentrifuge tubes and stored at − 80 °C for further analysis. The collection and separation of blood samples for serum were performed within two hours of collection to avoid any analyte loss.

Tissue samples

The liver was approached through an abdominal incision. Liver samples were processed to evaluate oxidative stress and histology. Samples processed for oxidative stress were deeply frozen in liquid nitrogen.

Complete liver samples were transferred to sterile containers, washed from blood, and drained for histopathological evaluation. The liver samples were weighed and fixed in 10% neutral buffered formalin for 24 h. Slices of about 1.0 cm were collected from the two liver lobes and the median part. Each slice was subdivided into three blocks of about 3–5 m³. The tissue blocks were processed automatically using a tissue processor (Leica Biosystems, Buffalo Grove, IL). Serial sections were obtained from each tissue block, and each third section was selected, starting from the first one. Multiple photographs from different fields of view on each slide were taken to ensure comprehensive analysis.

Precautions were taken during sample handling to minimize pre-analytical variability using standardized protocols²¹.

After sample collection, the researchers euthanized all animals with an overdose of anesthetic.

Sample processing

Liver function

Blood Sera was used promptly to estimate the levels of liver function biomarkers. An automated biochemistry analyzer (Accent S 120, CORMAY DIAGNOSTICS, Poland) was used for the assays. ALT and AST activities were measured using a kinetic UV test based on the recommended IFCC method²². The ALP activity was determined by enzymatic colorimetric test²³.

Oxidative stress evaluation

Liver specimens were transferred to sterile containers filled with an ice-cold 0.9% saline solution. This step helped flush out residual blood from the tissue. Liver pieces were then sliced into small fragments using a sterile blade on an ice-cold glass plate.

A 10% w/v liver homogenate was prepared by mincing approximately 1 g of liver tissue in 10 ml of ice-cold 0.1 M Tris-HCl buffer (pH 7.4). Homogenization was performed using a glass-Teflon homogenizer with ten upward and downward strokes at 1,500 rpm on an ice bath²⁴.

The homogenate was centrifuged at 12,000 rpm for 15 min at 4 °C (Remi C24 Cooling Centrifuge). The supernatant was carefully pipetted and transferred into prelabeled microcentrifuge tubes to avoid the pelleted cellular debris.

Oxidative stress biomarkers were immediately estimated from the supernatant, or samples were stored at − 80 °C for further analysis within one month.

Glutathione level Estimation

A liver homogenate aliquot was precipitated with 5% sulfosalicylic acid. The supernatant was used on a spectrophotometer to measure the glutathione (GSH) reaction with Ellman’s reagent at 412 nm^21,25. A standard curve was plotted using known GSH concentrations. Sample GSH levels were expressed as µg/g tissue after normalization with protein concentration measured by Lowry’s method^21,22,26.

Lipid peroxidation was measured through the colorimetric determination of malondialdehyde (MDA) content using thiobarbituric acid at 532 nm and a spectrophotometer (DU 640 Spectrophotometer, Beckman)²⁷.

Superoxide dismutase (SOD) activity was determined colorimetrically based on its inhibition of epinephrine autoxidation at 480 nm using the same spectrophotometer²⁸.

Standard curves were plotted using pure GSH, MDA, and SOD solutions. Activities were expressed as units/mg protein after normalization with the protein concentration measured by Lowry’s method. Commercially available diagnostic kits were used with the manufacturer’s instructions. Samples, standards, and reagents were prepared on ice to protect enzyme activity. All spectrophotometric measurements were taken promptly in the linear range.

Histopathological staining

Five µm thick sections were cut from each tissue block using a rotary microtome (Leica Biosystems) equipped with disposable steel blades. Sections were floated in a 40 °C distilled water bath and mounted on glass slides. Slides were stained with routine hematoxylin and eosin dye using an IHC automated slide stainer (Leica Biosystems). This step involved staining in a Harris hematoxylin solution for 8 min, followed by bluing in 1% acid-alcohol and counterstaining in an eosin-phloxine solution for 2 min²⁹.

The stained slides examined were under a light microscope (B-500 Ti-5 by Optika) by two histopathologists blinded to the study objectives or groups. In each tissue slide, three fields were evaluated. Every third field was selected, starting from the right side. Photomicrographs of tissue slides were processed using image processing software and were used to evaluate the hepatic injury.

A validated scoring system assessed the degree of hepatic injury and centrilobular necrosis. A generic grading system was used for scoring hepatic injuries by observing the presence of inflammatory cells, architecture disturbance, bridging necrosis, bile duct proliferation, and centrilobular hepatocellular necrosis. These morphological features were graded according to severity: mild, moderate, or severe. The presence and absence of other morphological changes were also considered, like steatosis, Mallory hyaline, and ballooning degeneration³⁰. This method provides an overall assessment of the degree of hepatic injury and will allow for the assessment of the degree of hepatic injury and the extent of liver damage. The histopathological slides were evaluated for possible change in a two-step manner. In step one, expert histopathologists (S.K.S and S.F.R) evaluated the slides blindly. In step two, the same histopathologists evaluated the slides with information about the experiment. A combined report was made out of their comments.

A validated scoring system assessed the degree of hepatic injury and centrilobular necrosis. A generic grading system was used for scoring hepatic injuries by observing the presence of inflammatory cells, architecture disturbance, bridging necrosis, bile duct proliferation, and centrilobular hepatocellular necrosis. These morphological features were graded according to severity: mild, moderate, or severe. The presence and absence of other morphological changes were also considered, like steatosis, Mallory hyaline, and ballooning degeneration³⁰. This method provides an overall assessment of the degree of hepatic injury and will allow for the assessment of the degree of hepatic injury and the extent of liver damage. The histopathological slides were evaluated for possible change in a two-step manner. In step one, expert histopathologists (S.K.S and S.F.R) evaluated the slides blindly. In step two, the same histopathologists evaluated the slides with information about the experiment. A combined report was made out of their comments.

All animal experiments followed the ARRIVE guidelines for more transparent in vivo experiment reporting. These address, among others, detailed information about specific histological procedures, including the number of slides prepared per liver and the number of photographs taken with every slide. The research protocol and all methods and procedures used in the study were performed in accordance with the research and animal handling guidelines and regulations of King Khalid University (Abha, KSA) and AVMA Guidelines for the Euthanasia of Animals to ensure that this study will accomplish and follow ethical practices and guidelines about welfare and treatment of animals connected with this research.

Statistical analysis

The obtained data were tabulated in an Excel sheet and analyzed using SPSS V28. The data are presented as mean ± standard deviation (SD). Due to sample size, the Shapiro-Wilk test of normality was used to determine whether data from liver function and oxidative stress is normally distributed. An independent t-test and Pearson correlation test were used to compare the different variables in the two groups. Differences were considered significant when p ≤ 0.05.

Results

There were no animal losses by the end of the experiment, and all animals appeared healthy at the time of sample collection. Consequently, the changes observed in the experimental animals, which were absent in the control group, can be attributed to high-altitude-induced hypoxia. The results of the Shapiro-Wilk Test indicated that the data are normally distributed, with a p-value greater than 0.05.

Liver function tests

The mean ALT levels of the control and experimental groups were 45.2 ± 3.4 U/L and 64.3 ± 5.1 U/L, respectively, and the difference was statistically significant (p = 0.001) as indicated by the t-test. The results indicated that ALT levels were significantly higher in the hypoxic rats, suggesting liver damage or leakage [t²² = 6.1, p < 0.001].

The mean AST levels of the control and experimental groups were 112.1 ± 8.9 U/L and 138.4 ± 11.3 U/L, respectively, and the difference was statistically significant (p = 0.005) as indicated by the t-test. These results confirm that AST activity was significantly higher in rats exposed to high altitudes than in control ones, reflecting hypoxic hepatic impacts [t²² = 3.4, p = 0.004].

The ALP levels of the control and experimental groups were 98.4 ± 7.3 U/L and 24.6 ± 9.5, respectively, and the differences were statistically significant, as indicated by the t-test. The serum from each sample was analyzed in triplicate. The ALP levels were markedly reduced in hypoxic rats versus controls [t²² = 7.8, p < 0.001)].

Rats exposed to high altitudes (experimental) displayed higher serum ALT, AST, and ALP levels than controls, with significant differences. On the other hand, TBIL levels did not display any significant differences between the two groups (Fig. 1).

Fig. 1.

Effects of high-altitude hypoxia on serum liver enzymes in rats (n = 24) for eight weeks. ALT, alanine transaminase; AST, aspartate aminotransferase; ALP, alkaline phosphatase; * = significant.

Oxidative stress markers

Lipid peroxidation, antioxidant defense, and glutathione levels were assessed in liver homogenates as indicators of oxidative stress (Fig. 2).

Fig. 2.

Effects of hypobaric exposure on oxidative stress markers in rat liver homogenates (n = 24). GSH, glutathione; MDA, malondialdehyde; SOD, superoxide dismutase; * = significant.

MDA levels were significantly lower in control rats (4.2 ± 0.3 nmol/mg protein) than in experimental rats (6.1 ± 0.5 nmol/mg protein), indicating increased lipid peroxidation in the exposed group (p < 0.001, r = 0.68). The difference between the two groups was statistically significant, as indicated by the t-test. These findings revealed a significant increase in MDA levels in hypoxic livers compared to control ones [t²² = 7.2, p < 0.001].

SOD activity was markedly higher in the control group (1.86 ± 0.12 U/mg protein) than in the exposed group (1.42 ± 0.11 U/mg protein). The t-test indicated a significant difference between the two groups, demonstrating impaired antioxidant defense in the livers of hypoxic rats (p = 0.002, r= 0.57). The findings indicate a significant reduction in antioxidant enzyme activity in exposed rat livers compared to the control [t²² = 3.7, p = 0.002].

GSH levels were significantly higher in control livers (23.6 ± 1.9 µg/g tissue) than in exposed livers (16.4 ± 1.6 µg/g tissue), reflecting overwhelmed antioxidant capacity and greater oxidative stress in the hypoxic tissues (p = 0.003, r = 0.55). The t-test indicated a statistical difference between the two groups, confirming that GSH reserves are markedly depleted in hypoxic hepatic tissues [t²² = 3.5, p = 0.003].

The changes in MDA, SOD, and GSH levels in the exposed group exceeded normal ranges and suggest that hypobaric hypoxia perturbed redox homeostasis in liver tissues, favoring a pro-oxidative state. The low p-values confirm that the differences between the control and exposed groups are statistically significant.

Liver histology

Control rats displayed normal liver architecture, hepatocytes, and sinusoidal spaces without necrosis (Fig. 3). They did not exhibit any hepatocellular injury, inflammatory cells, or necrosis. In contrast, the exposed rats exhibited changes in the form of inflammatory cell infiltrate, and the remaining exposed rats displayed a liver morphology within normal limits (Table 1).

Fig. 3.

Photomicrograph of Rat liver at 10x with hematoxylin and eosin stain. A and B: Control rat liver section; C (A few lymphocytes) and D: Hypoxic rats liver section. CV = central vein; Black arrow = infiltrated inflammatory cells.

Table 1.

Histopathological generic grading for injury and centrilobular hepatocellular necrosis and other types of hepatocyte injuries (n = 24).

Rat groups	Hepatic injury				Centrilobular hepatocellular necrosis grade					Steatosis		Mallory hyaline		Ballooning degeneration
	G0	G1	G2	G3	G0	G1	G2	G3	G4	Abs	Pre	Abs	Pre	Abs	Pre
Con (n = 12) Percentage	12 (100%)	0	0	0	12 (100%)	0	0	0	0	12 (100%)	0	12 (100%)	0	12 (100%)	0
Exp (n = 12) Percentage	4 (33.3%)	8 (66.7%)	0	0	12 (100%)	0	0	0	0	12 (100%)	0	12 (100%)	0	12 (100%)	0
Significance ( p-value )	0.001				0.06					0.07		0.07		0.07

The experimental rats’ livers displayed dilated sinusoids with focal chronic inflammatory cell infiltrates. They did not exhibit necrosis, ballooning degeneration, steatosis, Mallory hyaline, or other pathology in their hepatocytes. Cell injury was graded as Grade 1 based on generic criteria, and centrilobular hepatocyte necrosis was graded as Grade 0 in the exposed rats (Fig. 3).

Con, control; Exp, exposed; G0, normal; G, grade; Abs, absent; Pre, present.

Discussion

The present study explored the effects of short-term high altitude-induced hypoxia on multiple hepatic endpoints in rats. We measured serum liver enzymes, assessed oxidative stress markers in liver homogenates, and determined histopathological changes in liver sections of rats exposed to 2,100 m versus normobaric controls. This multiparametric approach may provide a more comprehensive understanding of potential mechanisms underlying altitude-induced liver dysfunction.

Exposure to hypobaric hypoxic conditions at elevated altitudes results in redox imbalance characterized by increased ROS production and concomitant impairment of endogenous antioxidant defenses³¹. ROS, including free radicals like superoxide anion and hydroxyl radicals, are highly reactive molecules that may damage biomolecules like lipids, proteins, and nucleic acids when not adequately neutralized³². Several molecular mechanisms underlie oxidative stress in the liver, including mitochondrial dysfunction due to the hypoxic disruption of electron transport, increased ROS generation, and overwhelming antioxidant defenses^33,34.

The results consistently demonstrated subclinical liver injury. Elevated liver enzyme levels provide a functional perspective, while histological scoring offers complementary structural validation. Liver enzymes, like ALT or AST, are predominantly localized in hepatocytes and leak out upon cell membrane damage. The significant increase in serum ALT, AST, and ALP levels observed in rats exposed to high altitudes indicates hypoxia-induced mild hepatocellular leakage and loss of membrane integrity. ROS can induce deleterious modifications that disrupt normal cellular signaling, enzyme functions, and membrane integrity³⁴.

Previous studies support the fact that high altitude can significantly impact the activity and expression of rat liver enzymes, potentially affecting liver function and metabolic processes^4,11,12,35. AST is found in mitochondria and cytoplasm, while ALT is found in cytoplasm but not mitochondria³⁶. Zhu et al. (2018) examined the impact of varying altitudes on liver injury, focusing on the expression of interleukin-10 (IL-10) as a potential mediator in the inflammatory response. Their findings showed elevated serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and hepatic IL-10 expression, which increased consistently with time and altitude³⁷.

The effect of ROS in mitochondria is through disruption in the electron transport chain and activation of inflammatory cells¹⁵. The previous work of Whitehead et al. (1999) reported that the levels of AST elevated and returned to normal in patients with hepatic hypoxia and calculous biliary obstruction³⁸. Savransky et al. (2007), in chronic intermitted hypoxia, found that serum ALT activity is double, while AST and alkaline phosphatase activities remained unchanged³⁹. Soliman et al. (2022) investigated the effects of high-altitude exposure for two months on rat liver enzyme levels and oxidative stress markers¹¹. Their findings indicated significant elevations in AST and ALT levels and decreased levels of antioxidants (SOD, catalase, GSH, and NO). Duan et al. (2020) indicated that the expression levels of certain cytochrome P450 enzymes and the conjugating enzyme UGT1 A1 were affected by high-altitude hypoxia³⁵. Specifically, CYP2 C11, CYP2E1, CYP3 A1, and UGT1 A1 levels decreased, whereas CYP2D1 increased in a high-altitude environment. From another perspective, Yang et al. (2018) explored the effects of hydrogen on rat liver injury under chronic intermittent hypoxia (CIH) and the related oxidative stress mechanisms⁴⁰. Their results indicated that CIH caused severe liver microstructural injury compared with controls, with higher serum ALT and AST levels. CIH also increased the level of oxidative stress markers 8-OHdG and IL-6 and decreased SOD levels. The authors concluded that pretreating with hydrogen gas protects against CIH-caused liver injury, demonstrating its efficacy in reducing oxidative stress and protecting liver cells from injury caused by chronic intermittent hypoxic exposure.

Oxidative stress markers were significantly altered, demonstrating hypoxia-induced hepatic oxidative stress. These results provide insights into hepatic redox perturbations induced by hypobaric hypoxia exposure. MDA is a reactive aldehyde and a major secondary lipid peroxidation product formed by attacking ROS on polyunsaturated fatty acids in cellular membranes^10,12,41. MDA elevation directly reflects the extent of oxidative damage to lipids through lipid peroxidation chain reactions. This result agrees with studies exposing rodents to comparable altitudes and reporting elevated MDA levels, confirming that hypoxia elicits oxidative tissue damage^42,43. Reduced SOD activity further reflects impaired antioxidant defenses when challenged, as antioxidants are overwhelmed in attempting to neutralize ROS. Decreased SOD levels have similarly been observed in mice exposed to hypobaric hypoxia⁴⁴. These results validate that hypoxia impairs enzymatic antioxidant capacity. Depleted GSH underscores overwhelmed nonenzymatic scavenging, tilting the redox balance toward oxidation. Other investigations found GSH depletion in rodents at high altitudes, consistent with our findings^43,44.

The histopathological observations provide robust structural evidence for validating subclinical hepatic injury caused by hypobaric hypoxia exposure that disrupts the liver homeostatic balance^4,10. All control rats revealed normal liver morphology, with no microscopically visible hepatocellular damage, inflammation, or necrosis. This result validates the control group as an appropriate comparison baseline. Most exposed rats (67.7%) revealed Grade 1 changes with minimal inflammatory cell infiltration. Inflammation is a well-known downstream effect of hypoxic tissue injury and activation of inflammatory cells^2,3,15. Hypoxia activates Kupffer cells and hepatic stellate cells to release proinflammatory cytokines, which recruit additional inflammatory cells^3,5. Therefore, focal inflammatory foci correlate well with the observed functional indices of mild hepatocellular leakiness as detected by increased serum enzyme levels. This result also agrees with previous rodent studies that reported hypoxia-induced hepatic inflammation^4,43,45. Moreover, Savransky et al. (2007) reported no significant inflammation or fibrosis in the liver following chronic intermitted hypoxia, and they suggested that it can lead to liver injury through oxidative stress mechanisms³⁹. Interestingly, over one-third of the exposed rats looked histologically normal, exhibiting individual variability against hypoxic insult⁴⁶. Alternatively, inflammation may have been below detection thresholds in a subset.

The histopathological scoring objectively confirmed subcellular liver remodeling from hypobaric hypoxia exposure. It complemented the functional readouts, strengthening evidence that this model leads to real but subclinical hepatic impacts worthy of further mechanistic investigation. Noninvasive biomarkers calibrated to histology could translate findings to clinical settings. Histopathology remains an underutilized component in preclinical hypoxia research despite the clear potential for detecting subtle changes invisible to other techniques. Staining for additional markers, including F4/80, could help characterize inflammatory cell infiltration observed histologically^45,47. Adopting histopathology more widely would complement the existing approaches. Noninvasive imaging methods calibrated to histological readouts would translate quantitative scoring approaches into clinical applications^48–50.

These functional and structural results consistently demonstrate hypoxia elicited real but subclinical hepatic impacts. While the liver was the focus, hypoxia systemically impacts multiple organs through oxidative mechanisms. Comparing profiles across tissues may disclose nuanced responses, and comparing acute and chronic exposure models may provide insights into how oxidative stress impacts evolve during long-term exposure to altitude.

Based on the present study’s results, these histopathological changes in the liver tissue, such as hepatic injury, inflammation, and tissue modifications, were associated with increased oxidative stress markers and reduced antioxidant activity. This mechanism portrays a potential interplay between histopathological changes and oxidative stress in liver injury. However, this study could not establish which factor preceded the other because the study’s design was not aimed at establishing the events’ temporal sequence. However, high altitude changes the activity of the cytochrome chain, which is responsible for mitochondrial oxidative phosphorylation; these can decrease ATP synthesis, increase ROS generation, and decrease the cellular antioxidant system^51,52. This sequence of events may lead to oxidative stress, affecting different tissues. Histopathological changes and oxidative stress might also co-occur or be interlinked bidirectionally. Although the experimental protocol tried to standardize the conditions across both research centers, factors such as differences in local climate, including temperature and humidity, could impact animals’ physiology and behavior. Furthermore, potential differences in stress levels associated with transport and acclimatization to the experimental centers should be considered.

More research is needed to clarify this mutual relationship between histopathological alterations/injury and oxidative stress. Longitudinal studies with proper time points and experimental design can help determine which event comes first. Furthermore, mechanistic studies regarding the model in the underlying molecular pathways and cellular mechanisms implicated in the development of histopathological changes and oxidative stress would shed light on the interaction. Such work might indicate causal relationships and possible feedback loops between histopathological changes and oxidative stress in liver injury.

Different species can respond differently to hypoxic stress, and primate or human studies are needed to validate clinical relevance. Altitude/hypoxic exposures were static, but high-altitude dwellers may experience dynamic changes. A closer approximation of oscillations may impact the results. Although the injury was subclinical, residual effects of repeated exposure over a lifetime might progressively compromise health; therefore, longitudinal studies are essential. Additionally, most studies have evaluated individual organs, but hypoxia affects multiple systems. Evaluating the cross-talk between tissues may identify compensatory mechanisms.

Conclusions

This study investigated the effect of acute high-altitude exposure on liver function, oxidative status, and histopathological characteristics in rat models. The findings indicate significant hepatic structure and function disturbances due to high altitude exposure. Notable changes include increased ALT and AST and decreased levels of ALP, suggesting compromised liver function. Elevated MDA levels and reduced SOD and GSH indicate heightened oxidative stress. Histologic examination revealed mild alterations, such as dilated sinusoids and focal inflammatory cell infiltrates, reflecting the hepatic response to hypoxic conditions. These results emphasize the vulnerability of the liver to hypoxic stress and highlight the need for further research into the long-term consequences of high-altitude exposure and the mechanisms of hepatic adaptations. Understanding these responses is essential for developing strategies to mitigate the adverse effects of hypoxia on liver health both in clinical and environmental settings.

Author contributions

Conceptualization, A.R, E.I, and S.S; methodology, A.R, E.I, A.I, R.E, and S.S; software, A.R, E.I, A.I, R.E, and S.S; validation, A.R, E.I, A.I, R.E, Y.A, and S.S.; formal analysis, A.R, E.I, A.I, R.E, Y.A, and S.S; investigation, A.R, E.I, A.I, R.S, Y.A, and S.S; resources, A.R, E.I, A.I, R.E, Y.A, and S.S; writing—original draft preparation, A.R, E.I, A.OI, R.E, Y.A, and S.S; writing—review and editing, A.R, E.I, A.I, R.E, Y. A, and S.S; visualization, A.R, E. I, A.I, R.E, Y. A, and S. S; supervision, A. R, E.I; project administration, A.R, and E.I; funding acquisition, A.R, E.I, A.I, R.E, Y.A, and S.S All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Deputyship for Research & Innovation, Ministry of Education, in Saudi Arabia, which has supported this research work with the project number (UB-14-1442).

Data availability

Data will be available to the corresponding author upon request.

Declarations

Competing interests

The authors declare no competing interests.

Institutional review board statement

This study was approved by the Research and Ethics Committees of King Khalid University (REC-2023-09-06).