https://orcid.org/0000-0003-4609-499X
Abstract
Background and aim: Pulmonary hypertension (PH) is a serious and even fatal disorder with limited treatment strategies. The hypoxia-induced pulmonary hypertension (HPH) rat model is commonly used in this field. While the HPH rat model has strong predictability and repeatability, the model is a chronic model, making it time-consuming, costly, and complicated and limiting the progress of the experiments. Currently, there is no uniform international standard for the HPH model. Our study aimed to find a relatively effective and efficient HPH modeling protocol. Methods: We established HPH rat models with different total hypoxia periods and different daily hypoxia times, and assessed different hypoxia modeling modes in multiple dimensions, such as haemodynamics, right ventricular (RV) hypertrophy, pulmonary arterial remodeling, muscularization, inflammation, and collagen deposition. Results: Longer daily hypoxia time resulted in higher mean pulmonary arterial pressure (mPAP)/right ventricular systolic pressure (RVSP) and more obvious RV hypertrophy, as well as more severe pulmonary arterial remodeling and muscularization, regardless of the total period of hypoxia (3- or 4-week). Moreover, pulmonary perivascular macrophages and collagen deposition showed daily hypoxia time-dependent increases, both in 3- and 4-week hypoxia groups. Conclusion: Our findings showed that the 3-week continuous hypoxia mode was a relatively efficient way to reduce the time needed to induce significant disease phenotypes, which offered methodological evidence for future studies in building HPH models.
Introduction
Pulmonary hypertension (PH) is a progressive and even fatal disease mainly characterized by pulmonary arterial remodeling and collagen deposition, leading to elevated mean pulmonary artery pressure and right ventricular hypertrophy.Citation1–4 Currently, PH is still a devastating disorder with limited therapeutic options. According to the etiology, PH is separated into 5 recognized groups, of which PH due to lung diseases and/or hypoxia is the third group.Citation5,Citation6 Chronic hypoxia exposure has been linked to the development of PH in patients with chronic respiratory diseases, such as chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis.Citation7 The most notable histological change in hypoxia-induced pulmonary hypertension (HPH) is pulmonary vascular remodeling, represented by the remodeling of the pulmonary arterioles. The reasons for this manifestation may be hypoxia-induced pulmonary artery endothelial dysfunction, smooth muscle proliferation, perivascular collagen deposition, and inflammation.Citation4,Citation8–10 As the vascular compliance decreases, the vascular wall hardens and the arterial pressure rises, eventually leading to severely raised right ventricular pressure and lethal right heart failure.Citation11 Therefore, in this study, elevated pulmonary artery pressure, right ventricular systolic pressure, vascular remodeling, collagen deposition, and inflammatory cell infiltration were considered indicators of the severity of PH.
PH animal models act as a cornerstone of PH research. The HPH animal model is one of the most common models in this field and has widely recognized advantages.Citation12–14 However, the hypoxia mode for making HPH models lacks a unified standard. In the majority of experimental protocols, mice or rats are placed in a hypoxic environment for at least 21 days.Citation15–19 We found that the total period of hypoxia (21, 28, or more days) and the daily hypoxia time (continuous or intermittent) were not uniform across different papers.Citation15,Citation16,Citation19–21 It is unknown how much different total and daily hypoxia durations affect the various phenotypes of the HPH animal model. Therefore, it is urgent to find a unifying and efficient model protocol under the premises that the pathophysiological indicators meet the requirements of the HPH model, the modeling time is minimized, and experimental resource consumption is reduced.
Here, we established HPH rat models through different hypoxia modes and compared multiple disease indicators in each group, aiming to find the most efficient model.
Materials and methods
Animal model and experimental groups
Sprague-Dawley male rats (6–8 w, 180–220 g) were obtained from the Laboratory Animal Center of Wenzhou Medical University, (Zhejiang, China), and raised in the specific-pathogen-free (SPF) facility in Laboratory Animal Center of Wenzhou Medical University. The rats were given free access to food and water and housed in a 12 h/12 h light-dark cycle and a temperature of 20–24 °C and 55–65% humidity. Rats were randomly assigned to 6 groups: 3w8h group, 3w16h group, 3w24h group, 4w8h group, 4w16h group, 4w24h group. Briefly, every hypoxia-treated group were placed into a cabin, which can automatically detect the O2 concentration and maintain FiO2 at 10%. The normoxia control group was exposed to room air. All the rats were in a normobaric atmosphere. All experiments in this study were approved by the Animal Ethics Committee of Wenzhou Medical University and performed in accordance with the ARRIVE guidelines and the Guide for the Care and Use of Laboratory Animals.
Haemodynamic analyses
After hypoxia exposure, HPH model rats were anesthetized with 20% urethane (1 mL/100 g, i.p.). After successful anesthesia, two polyethylene catheters that were prefilled with heparin were inserted into the right ventricle and left carotid artery, respectively. Then, the right ventricular systolic pressure (RVSP), mean pulmonary arterial pressure (mPAP) and mean carotid arterial pressure (mCAP) were collected as previously reported,Citation4,Citation22 and then analyzed through the pressure measurement system (PowerLab 8/35 multichannel biological signal recording system, AD Instruments, AUS).
Right ventricular hypertrophy measurement
After haemodynamic data collection, rats were sacrificed by exsanguination and their hearts were dissected out. The hearts were cut along the edge of the interventricular septum and RV to divide hearts into two parts: the RV and the left ventricle (LV) plus interventricular septum (S). Each part was weighed separately. The heart hypertrophy indices, the ratio of the RV to (LV + S) weights, was calculated to assess the degree of RV hypertrophy.
Pulmonary arterial remodeling and collagen deposition measurement
The upper lobe of the right lung of the rat was fixed in 4% paraformaldehyde, and de-hydrated in a graded ethanol series, embedded in paraffin. The samples were cut into 5 μm sections for hematoxylin-eosin staining. Optical microscope was used to observe the remodeling of pulmonary arteries in each group. After the imaging, we randomly selected pulmonary arterioles with a diameter of 50–100 μm and calculated the ratios of the pulmonary artery wall area to the total area (WA/TA) and the wall thickness to the total thickness (WT/TT) with Image-Pro Plus 6.0 (Media Cybernetics, Bethesda, USA) to reflect the degree of pulmonary arterial remodeling. The sections were processed by Masson’s Trichrome to assess the degree of pulmonary arterial collagen deposition. After stained, sections in each group were observed under a microscope.
Immunohistochemical analyses
After blocking, the paraffin sections were incubated primary antibodies against α-smooth muscle actin (α-SMA) (Santa Cruz Biotechnology, USA, 1:50) or CD68 (Santa Cruz Biotechnology, USA, 1:50) or proliferating cell nuclear antigen (PCNA) (Santa Cruz Biotechnology, USA, 1:50). After overnight incubation with primary antibodies, slides were washed with phosphate buffered saline and incubated with secondary HRP-linked antibodies (Biosharp, CHN, 1:100). Lung tissues, incubated with 1% bovine serum albumin to replace the specific primary antibody, acted as negative controls. Imaging was assessed by microscopy and the positive staining was analyzed with Image-Pro Plus 6.0. We evaluated the degree of pulmonary arterial muscularization by calculating the ratio of the α-SMA-positive area to the total vessel area.
Statistical analysis
Statistical analyses were performed with GraphPad Prism 6.0 (GraphPad Software, CA, USA). All data are expressed as the mean ± standard error of mean (SEM). Comparisons between two groups were analyzed by Student’s t-test, and multiple comparisons were analyzed by one-way analysis of variance (ANOVA) with Bonferroni correction. P values of <0.05 were considered statistically significant.
Results
Longer daily hypoxia leads to a higher mPAP/RVSP
We performed haemodynamic analyses on rats in each group. As shown in , we found that the RVSP and mPAP of all hypoxia-treated rats significantly increased when compared with those of the N group, but there was no significant difference in mCAP among the groups. In both the 3w groups and the 4w groups, the mPAP and RVSP increased with increasing daily hypoxia time. However, there was no significant difference in RVSP or mPAP between the 3w24h group and the 4w24h group.
Figure 1. Representative images of RVSP waves, mPAP waves and mCAP waves (a) were obtained by an invasive catheterization procedure. RVSP (b; n = 6), mPAP (c; n = 6), and mCAP (d; n = 6) values for each group were analyzed. The data are presented as the mean ± SEM. #P < 0.05, ##P < 0.01 vs. the N group, *P < 0.05, **P < 0.01.
The longer the daily hypoxia is, the more RV hypertrophy there is
We evaluated RV hypertrophy in each group to reflect the degree of damage to the RV caused by hypoxia. As shown in , except for the 3w8h group, the RV/(LV + S) and RW/BW in the other hypoxia groups were higher than those in the N group. In both the 3w groups and the 4w groups, the RV/(LV + S) and RW/BW increased with increasing daily hypoxia time. There was no significant difference in RV/(LV + S) or RW/BW between the 3w24h group and the 4w24h group with the highest degree of RV hypertrophy.
Figure 2. The ratio of the RV to the (LV + S) weight was calculated to assess the degree of RV hypertrophy in each group. [RV/(LV + S)] (a; n = 6) and (RV/BW) (b; n = 6) values for each group were analyzed. The data are presented as the mean ± SEM. #P < 0.05, ##P < 0.01 vs. the N group, *P < 0.05, **P < 0.01.
![Figure 2. The ratio of the RV to the (LV + S) weight was calculated to assess the degree of RV hypertrophy in each group. [RV/(LV + S)] (a; n = 6) and (RV/BW) (b; n = 6) values for each group were analyzed. The data are presented as the mean ± SEM. #P < 0.05, ##P < 0.01 vs. the N group, *P < 0.05, **P < 0.01.](/cms/asset/449a00bd-457d-4581-a3af-906a16bc7975/ielu_a_2148016_f0002_b.jpg)
Longer daily hypoxia leads to more severe pulmonary arterial remodeling
The WT/TT and WA/TA ratios in each group were calculated to quantify the degree of pulmonary arterial remodeling. As shown in , the WT/TT and WA/TA ratios in each hypoxia group were more obvious than those in the N group. In both 3w and 4w groups, the WT/TT and WA/TA ratios increased with increasing daily hypoxia time. However, there was no significant difference in the WT/TT or WA/TA ratio between the 3w24h group and the 4w24h group.
Figure 3. Lung tissue sections were stained with hematoxylin-eosin, and the representative morphological structures of the pulmonary arteries in each group are shown (a; scale bar indicates 50 μm). (WT/TT) (b; n = 5, 3 vessels per rat) and (WA/TA) (c; n = 5, 3 vessels per rat) values were analyzed. The data are presented as the mean ± SEM. #P < 0.05, ##P < 0.01 vs. the N group, *P < 0.05, **P < 0.01.
Continuous hypoxia leads to more pulmonary arterial muscularization and proliferation
The protein α-SMA is strongly expressed in pulmonary arterial smooth muscle cells (PASMCs) and is regarded as a marker of PASMCs. The degree of pulmonary arterial muscularization reflects the proliferation of PASMCs. We evaluated the degree of pulmonary arterial muscularization by calculating the ratio of the α-SMA-positive area to the total vessel area. As shown in , muscularization was significantly increased after hypoxia exposure. Muscularization increased gradually as the daily hypoxia time increased in both the 3w and 4w groups. There was no difference in muscularization between the 3w24h group and the 4w24h group. PCNA expression of PASMCs reflects the proliferation of pulmonary arterioles. As shown in , the percentage of PCNA-positive PASMCs was increased after hypoxia exposure. Compared with the other intermittent hypoxia groups, the continuous group showed the most significant increase in the percentage of PCNA-positive PASMCs, no matter in the 3w groups or 4w groups. There was no difference between the 3w24h group and the 4w24h group in PCNA expression.
Figure 4. The expression of α-SMA in pulmonary arteries in each group was assessed by immunohistochemistry (a; scale bar indicates 50 μm). The ratio of the α-SMA-positive area to the total vessel area was analyzed (b; n = 5, 3 vessels per rat). The data are presented as the mean ± SEM. #P < 0.05, ##P < 0.01 vs. the N group, *P < 0.05, **P < 0.01.
Figure 5. The expression of PCNA in each group was assessed by immunohistochemistry (a; scale bar indicates 50 μm). The percentage of PCNA-positive PASMCs was calculated and analyzed (b; n = 5, 3 vessels per rat). The data are presented as the mean ± SEM. #P < 0.05, ##P < 0.01 vs. the N group, *P < 0.05, **P < 0.01.
Continuous hypoxia leads to more macrophages around pulmonary vessels
CD68 is a highly glycosylated transmembrane protein that is currently the most widely used macrophage marker. The number of CD68-positive cells around pulmonary vessels in each group was calculated to reflect the inflammatory infiltration. As shown in , except for the 3w8h group, CD68-positive cells around pulmonary vessels increased after hypoxia exposure. In both the 3w and 4w groups, the number of CD68-positive cells significantly increased in the continuous hypoxia group compared with the intermittent hypoxia group. There was no difference in the infiltration of CD68-positive cells between the 3w24h and the 4w24h group.
Figure 6. The expression of CD68 in each group was assessed by immunohistochemistry (a; scale bar indicates 50 μm). The number of CD68-positive cells around pulmonary vessels in each group was counted and analyzed (b; n = 5, 3 vessels per rat). The data are presented as the mean ± SEM. #P < 0.05, ##P < 0.01 vs. the N group, *P < 0.05, **P < 0.01.
Longer daily hypoxia leads to more collagen around pulmonary arterioles
Paraffin sections in each group were processed by Masson’s Trichrome to determine the collagen composition around the pulmonary arterioles. As shown in , hypoxia markedly increased the amount of collagen around these arterioles. A significant amount of collagen staining was seen in the continuous hypoxia groups. There was no significant difference between the 3w24h group and the 4w24h group.
Figure 7. The degree of collagen deposition (blue) around the pulmonary arterioles in each group was evaluated by Masson’s Trichrome staining (a; scale bars indicate 50 μm). The ratio of the collagen area to the total area of the recording area was calculated to reflect the degree of collagen deposition (b; n = 5, 3 vessels per rat). The data are presented as the mean ± SEM. #P < 0.05, ##P < 0.01 vs. the N group, *P < 0.05, **P < 0.01.
Discussion
PH is a fatal and progressive disorder characterized by increased pulmonary vascular resistance, leading to RV failure and death.Citation23 Pulmonary vascular remodeling, inflammation, endothelial dysfunction, aberrant PASMC proliferation, and collagen deposition are the major pathobiological features of PH that cause its progression.Citation24 According to the etiological classification, HPH is the third group of PH and is mainly related to hypoxia caused by chronic lung diseases.Citation5,Citation6
At present, there is no perfect preclinical model that completely recapitulates human PH. Among the multiple types of PH model, chronic hypoxia induces the most stable response to disease induction with regard to elevated haemodynamic parameters, RV hypertrophy and wall thickening.Citation14 The HPH model often stands out when studying the third group of PH because of its similarity to the pathophysiological changes in chronic human lung diseases. Due to the strong reproducibility and sensitivity of the response, the exposure of rats to hypoxia is a common approach to study HPH,Citation7,Citation13 as first formally proposed in 1976.Citation25 However, there is no uniform international standard for the HPH model. We found that in some papers,Citation15–18 researchers tended to choose continuous hypoxia exposure, while other researchers tended to use the intermittent hypoxia mode.Citation19–21,Citation26 As to the total period hypoxia, 3 weeks,Citation15,Citation16,Citation19 4 weeks,Citation17,Citation26 and longer periodsCitation18 have been adopted. It appears that most of the reported hypoxia modes successfully establish HPH animal models, but the pathophysiological differences between these models remain unknown. In general, haemodynamic changes, RV hypertrophy, vascular structural changes, pulmonary arterial muscularization, inflammatory cell infiltration, and pulmonary collagen deposition are the main phenotypes that researchers are concerned about in HPH animal models. In this study, we compared the phenotypes mentioned above in rats exposed to different hypoxia protocols.
We established HPH rat models from different total hypoxia periods and different daily hypoxia times, aiming to find a relatively effective and efficient HPH modeling mode. The results showed that given the same length of hypoxia, haemodynamic indices (RVSP, mPAP), RV hypertrophy indices (RV/(LV + S), RW/BW), pulmonary arterial remodeling indices (WT/TT, WA/TA), muscularization, the number of CD68-positive cells and the area of collagen staining increased with increasing daily hypoxia time, which indicated that the length of daily hypoxia plays a key role in the severity of HPH models. Compared with 8-h or 16-h hypoxia mode, the 24-h continuous hypoxia mode induced a more severe HPH model. The continuous hypoxia model showed higher RVSP and mPAP and more severe RV hypertrophy arterial remodeling, muscularization, inflammation, and collagen deposition. When the daily hypoxia time was fixed, we found that increasing the total period of hypoxia had little effect on the various indicators in these rats. That is, both 3- and 4-week hypoxia can successfully establish an HPH rat model, and when the daily hypoxia time is fixed, there is little difference in the degree of hypoxia-related changes caused by the two hypoxia periods. However, some indicators suggested a more severe phenotype in a longer period of daily intermittent hypoxia, such as α-SMA expression and PCNA expression. Individual parameters cannot reflect the overall quality of the model, hence multiple indicators were taken into consideration for better interpretation of establishment of the murine pulmonary hypertension model. Therefore, we believe that the daily hypoxia time (continuous or intermittent) has a greater impact on HPH rat model than the total period of hypoxia (3w or 4w).
Ma et al.Citation21 compared rat HPH models of different altitudes and hypoxia durations. Both our study and their study were concerned about HPH rat models created by different protocols, but they focused more on altitude-related HPH, in which atmospheric pressure is a crucial factor. They also did not study the differences between the continuous mode and the intermittent mode. We focused more on the HPH phenotypes of different time modes (continuous or intermittent, 3- or 4-week) under normal atmospheric pressure, which is more similar to the etiology of human chronic lung disease that develops at sea level. In addition, we were mainly concerned with finding a more efficient and time-saving model mode.
Our findings showed that the 3-week continuous hypoxia mode was a relatively efficient way to reduce the time needed to induce significant disease phenotypes. Our work can provide other researchers in this field methodological evidence with which to build HPH models. Through this continuous hypoxia mode, researchers can obtain high-quality HPH models with obvious disease characteristics in a shorter modeling period. However, this study has several limitations. First, the groups of shorter hypoxia duration are lacking. Whether lower FiO2 can further reduce the modeling period or not, on the premise of tolerance in rats, remains to be elucidated. Second, more noninvasive tests are needed, such as echocardiography and pulmonary angiography, which will be carried out gradually in our further research. Third, although no rats died due to hypoxia, the rats of our study were healthy adult wild-type. It is noteworthy that some studies involve further biological treatment, such as knockout of key genes and injection of toxic drugs, which will reduce the tolerance of animals to hypoxia.
Ethical approval
All experiments in this study were approved by the Animal Ethics Committee of Wenzhou Medical University and performed in accordance with the ARRIVE guidelines and the Guide for the Care and Use of Laboratory Animals.
Acknowledgement
The authors thank the staff of Experimental Animal Center of Wenzhou Medical University for their guidance and help.
Disclosure statement
The authors report there are no competing interests to declare.
Data availability statement
The datasets generated during and/or analyzed during the current study are not publicly available, but are available from the corresponding author on reasonable request.
Additional information
Funding
References
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