Mouse Models Of Pulmonary Fibrosis
Teena Ruiter
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive disease of the lung. How to build a typical human mimicking animal model has been a challenge. Thus, to reveal the mechanism and to make it useful for IPF clinical treatment, a different type of mice model and inspection methods are used to evaluate which one is applicable for the study of IPF.
PET/CT of BLM-receiving mice showed an increase in fibrotic consolidations and an increase in non-aerated lung area in BLM-treated mice compared with that in controls. TGF-b1, TNF-a, IL-6, GM-CSF in BALF and serum. PAI-1, HYP in the lung tissue of mice were significantly different in each BLM groups than those in the controls. The results of Masson staining in mice indicate that the lung tissues of all BLM received groups, the intratracheal groups, the intravenous groups, and the intraperitoneal groups have a higher degree of pulmonary septal thickening and collagen fiber consolidation compare to saline control. Picro-Sirius staining results are consistent with the results of Masson staining. Compared with the saline control group, the ratio of Col 1/Col 3 was significantly increased in each BLM group. TEM results found that in BLM group, type I alveolar epithelial cells were degenerated. Exfoliated endothelial cells were swelling, and type II alveolar epithelial cells were proliferated, the shape of the nucleus was irregular, and some tooth-like protrusions were seen.
With three different methods of animal model construction, high dose of each show more compliable, and BLM can successfully induce animal models of pulmonary fibrosis, however, certain differences in the fibrosis formation sites of them three, and tail vein injection of BLM induced PF model is closer to the idiopathic pulmonary interstitial fibrosis.
Thus, to reveal the mechanism and to make it useful for clinical treatment for IPF, this study aimed to use a different type of IPF animal model and use many inspection methods to evaluate which one is more applicable for the study of IPF. Direct aspiration into the lungs and systemic delivery of BLM in mice was induced in pulmonary fibrosis. In the past, the rather commonly used intratracheal injection model fibrosis lesions were mainly distributed around the bronchi and bronchioles, which was inconsistent with the IPF lesions mainly distributed under the pleura [18]. And the incidence of complications such as intratracheal asphyxia and pulmonary infection was high. In recent years, the mouse model of low-dose multiple tail vein injections of BLM has been used to observe the efficacy of anti-pulmonary fibrosis drugs. This study compared tail vein injections of BLM with the intraperitoneal injection model and the intratracheal injection model to explore the lungs differences of each model and the characteristics of fibrosis.
All the mice were anesthetized with an intraperitoneal injection of 1% pentobarbital (0.15 ml/20 g), and tracheostomy was conducted, as previously described [21], with a standard catheter. All the mice were tracheostomized and placed in a forced pulmonary function testing (PFT) system (Buxco, NY, USA) with FinePointe and FinePoint Control Panel software in the supine position on the bed within the plethysmograph, which simulated clinical pulmonary testing routinely performed on humans.
BALF was collected by endotracheal intubation with 300 μl ice-cold PBS twice and then centrifuged at 500 g for 10 min at 4 C. The supernatants were used for further ELISA. The total cells were resuspended with 50 μl PBS and counted via the Mindray BC-5000Vet automated hematology analyzer (Mindray, Shenzhen, CHN). (See Fig. 6).
The enzyme-linked immunosorbent assay (ELISA) kits in this study determined TGF-β1, TNF-α, IL-6 and GM-CSF antigen in BALF and Serum. The PAI-1 and HYP antigen in lung tissue were determined by using their ELISA kit.
Histopathological Examination For histological analysis, pulmonary tissues were fixed with 4% paraformaldehyde and embedded in paraffin. The paraffin blocks were cut at 5 μm using a microtome. Sections stained against Masson Trichrome [14]. Collagen deposition of the sections were observed and assessed with an optical microscope and quantified with Image-Pro Plus6.0 software. PSR staining and quantification [22,23,24] were used in this test. The tissue sections were deparaffinized and rehydrated in a graded ethanol series and then incubated for 1 h in PSR staining. The stained sections were analyzed by using an ECLIPSE Ci-L/Ci-E Clinical Upright Microscope (Nikon; magnification, 200) with a linear polarizer. To avoid missing any details, the filter was tilted to an angle between 0 and 90 until the birefringence became evident and the background became completely black. The focus was then corrected once more [23]. The halogen lamp intensity and exposure time were constant within each image. Under polarized light, Collagen type 1(also known as collagen alpha, Col1A1, and alpha-1 type 1 collagen, Col 1) appeared to red or yellow with strong birefringence, while Collagen type 3 (Col 3) was green with weak birefringence. The areas of Col 1 and 3 staining were analyzed by using Image Pro Plus 6.0 (Media Cybernetics, Inc.).
Pulmonary function changed in each group. The parameters included in pulmonary function test analysis are presented in Fig. 5. The FRC (functional residual capacity), PEF (Peak Expiratory Flow), Cchord (Chord compliance), and VC (Vital capacity) showed a down-regulated trend in all BLM groups, compared to those in the saline control. The pulmonary function was decreased in all model groups. Pulmonary function of intratracheal administration of BLM decreased seriously among these three groups.
The counts of inflammatory cells in BALF of each group were presented in Fig. 6. In this study TGF-β1, TNF-α, IL-6, and GM-CSF antigen in BALF and serum were determined by using theirs ELISA kits; The PAI-1 and HYP antigen in lung tissue were also determined by using ELISA kit. ELISA results showed that compared with that of the saline control group, the total number of BALF cells were significantly different among groups (Fig. 7). Inflammation was most serious in intratracheal administration of the BLM group. TGF-β1, TNF-α, IL-6, and GM-CSF in BALF and serum of the mice were significantly different in BLM groups than those in the saline control groups (Figs. 7 and 8). The contents of HYP and PAI-1 in the lung tissue of the mice in the BLM group were significantly increased compared to those in the saline control group (Fig. 9).
TEM images found type I alveolar epithelial cell degeneration, disintegration and shedding, endothelial cell swelling, type II alveolar epithelial cell proliferation, abundant microvilli on the free surface of the cell, vacuole-like transformation of lamellar corpuscles in the cell, irregular nucleus morphology, partly visible Jagged bumps. The base membrane was thickened, partly thinner, uneven, and integrity was destroyed. The interstitial fibroblasts increased, the intracellular procollagen increased, the rough endoplasmic reticulum expanded, and the collagen fibers were stacked in a large amount in the interstitial, and which were arranged in a crisscross pattern. Alveolar septum macrophages increased. See Fig. 12.
BLM is clinically used to treat a variety of neoplastic diseases, and its main adverse reaction is to induce severe PF [27]. In 1970, some scholars tried BLM in the treatment of 237 cancer patients and found that BLM would damage the skin, lungs, and mucous membranes [28]. In 1972, Halnan et al. [29] used 103 cases of cancer patients treated by BLM to undergo clinical observation, with one patient died of pneumonia. In 1973, Bedrossian et al. [30] performed an electron microscope observation on ultra-thin sections of lung tissue of patients who died of BLM. The results showed that type I alveolar epithelial cells decreased, and type II alveolar epithelial cells proliferated, and vascular endothelial cells and immune cells were damaged. Infiltration of alveoli, large amounts of collagen deposition, interstitial edema, diffuse alveolar, and pleural fibrosis, etc., were similar to clinical IPF lesions found in the past. In 1974, Adamson et al. [31] first adopted repeated intraperitoneal injections of BLM. An animal model method of rat lung interstitial fibrosis was established. Because BLM-induced pulmonary fibrosis (bleomycin-induced pulmonary fibrosis, BPF) has the most similar histopathological changes to human PF. The animal model of BLM replication is currently the most popular method for studying PF.
At present, the more mature method recognized is transtracheal administration [32, 33]. Most of the animals used are rats and mice. The common supplies are C57BL/6 mice, SD rats, and Wistar rats. The pathological changes caused by the model are similar to those in the clinic. The drug enters the lungs through tracheal perfusion or atomization inhalation. The former has higher technical requirements, while the latter makes the drug evenly distributed in the body, but the model requires special equipment and is less used. Tracheal perfusion administration requires shorter anesthesia time than rapid nasal administration, which is simple and fast, causing less animal damage, but requiring high of technical proficiency for the operator. Intraperitoneal injection administration allows BLM to absorb blood from the abdominal cavity to the lungs, playing a role in producing IPF. The use of this method of the model can reduce the degree of fibrosis caused by differences in surgical operations, with simple operation, low animal mortality, the more uniform distribution of lesions, but the number of administrations and the doses are larger. The modeling time is longer, with high cost, and it has certain limitations [34]; tail vein injection is mainly used for the preparation of mouse IPF models.
In BPF animal models, its typical feature is alveolar epithelial cell damage. The early-stage manifests as acute inflammation, including alveolar epithelial cell injury, inflammatory cell infiltration, and the release of inflammatory mediators. In the subacute stage, the proliferation and differentiation of fibroblasts and the expression of pro-fibrotic cytokines will appear in the lesion area; there will be a large amount of extracellular matrix deposition and fibrotic lesions [35]. The BPF mouse model and found that the lungs of the model group showed varying degrees of alveolar structural damage, inflammatory cell infiltration, and a large amount of collagen-based extracellular matrix deposition [36]. The gene expression test results showed that mouse lung tissue interleukin, collagen gene connective tissue growth factor, and transforming growth factor were all increased to varying degrees compared with those in the normal control group, which was consistent with the histopathological changes of IPF.
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