Here we present an effective method to investigate the antifibrotic activity of intravenously infused human mesenchymal stromal cells obtained from the whole umbilical cord following the induction of lung injury by an endotracheal injection of bleomycin in C57BL/6 mice. This protocol can be easily extended to the preclinical testing of other therapeutics.
Pulmonary fibrosis is a hallmark of several human lung diseases with a different etiology. Since current therapies are rather limited, mouse models continue to be an essential tool for developing new antifibrotic strategies. Here we provide an effective method to investigate in vivo antifibrotic activity of human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) in attenuating bleomycin-induced lung injury. C57BL/6 mice receive a single endotracheal injection of bleomycin (1.5 U/kg body weight) followed by a double infusion of hUC-MSC (2.5 x 105) into the tail vein, 24 h and 7 days after the bleomycin administration. Upon sacrifice at days 8, 14, or 21, inflammatory and fibrotic changes, collagen content, and hUC-MSC presence in explanted lung tissue are analyzed. The injection of bleomycin into the mouse trachea allows the direct targeting of the lungs, leading to extensive pulmonary inflammation and fibrosis. The systemic administration of a double dose of hUC-MSC results in the early blunting of the bleomycin-induced lung injury. Intravenously infused hUC-MSC are transiently engrafted into the mouse lungs, where they exert their anti-inflammatory and antifibrotic activity. In conclusion, this protocol has been successfully applied for the preclinical testing of hUC-MSC in an experimental mouse model of human pulmonary fibrosis. However, this technique can be easily extended both to study the effect of different endotracheally administered substances on the pathophysiology of the lungs and to validate new anti-inflammatory and antifibrotic systemic therapies.
Pulmonary fibrosis is a progressive pathological process characterized by the excessive deposition of extracellular matrix components, mainly type I collagen, in the lung interstitium, leading to impaired lung function. It is the hallmark of several human lung diseases with a different etiology and represents a poor clinical prognostic factor. Since current therapies are rather limited1, mouse models continue to be an essential tool both for the further investigation of the pathogenic mechanisms influencing the onset and the progression of the disease and for developing new antifibrotic strategies2,3.
To date, the administration of bleomycin has been the most commonly applied model of experimentally induced pulmonary fibrosis4. Beside multiple delivering methods (including intravenous, intraperitoneal, subcutaneous, and inhalational), intratracheal or endotracheal injections of bleomycin have emerged as the most frequently used routes4,5. The method that we describe herein has been developed to avoid the scalding effect of bleomycin on the tracheal mucosa. In fact, by exteriorizing the trachea and visualizing it through an operating microscope, it is possible to achieve the instillation of the entire volume of bleomycin solution directly into the lower airway without any spills in the upper airway. When the required surgical expertise and instrumentation are available, this method allows for the safe, robust, and reproducible induction of lung inflammation and fibrosis, as reported below.
All animal care and experimental procedures were approved by the Italian Ministry of Health (authorization n. 456/2016-PR) and performed according to the Declaration of Helsinki conventions.
1. Mice
2. Endotracheal injection of bleomycin
3. Tail vein infusion of human umbilical cord mesenchymal stromal cells
4. Organ explant and tissue processing
Lung injury was induced by a single endotracheal injection of 1.5 U/kg body weight of bleomycin sulfate in 100 µL of sterile saline. Control animals received an endotracheal injection of an equal volume of saline. Two shots of hUC-MSC (2.5 x 105 in 200 µL of sterile saline) were infused into the mouse tail vein, 24 h and 7 days after the bleomycin administration. Control animals received an intravenous infusion of an equal volume of sterile saline. Mice were sacrificed for lung explant and tissue processing at days 8, 14, and 21 after the bleomycin administration (Figure 1).
We demonstrated that a direct instillation of bleomycin into the mouse trachea allowed a rapid diffusion down to the lungs, resulting in extensive inflammation, progressive fibrosis, and a distortion of their normal architecture, consistently with prior studies11. Lung histopathological changes were assessed by hematoxylin-eosin (H&E) and picrosirius red staining10, and fibrosis was confirmed by an increased hydroxyproline content and collagen deposition (Figure 2). Inflammatory changes in tissue were assessed by a histological scoring system based on the inflammatory infiltration around bronchioles, bronchi, and blood vessels, and interstitial pneumonia observed in hematoxylin-eosin stained lung sections10. Following the bleomycin injection, the Ashcroft score of lung sections progressively increased from a mean value of 1.5 at day 8 to a mean value of 4.5 at day 14 and of 6.5 at day 2110. The double infusion of hUC-MSC into the mouse tail vein largely attenuated bleomycin-induced lung injury, with significant reduction, although not complete abrogation, of both the inflammatory infiltration and the extent of fibrosis at each time point (Figure 2). Immunostaining with specific antibodies10 showed that infused hUC-MSC rapidly and effectively reached mouse lungs, although only a few cells were detected, with a decreasing number from day 8 to day 21 (Figure 3). As previously reported12, these data suggest a rapid dislocation of the cells from the site of injury, despite their prolonged protective effect. Immunohistochemistry (IHC) staining of hUC-MSC was performed, also in the saline-treated samples, but no cell could be detected, given the absence of inflammatory foci attracting hUC-MSC.
Figure 1: Schematic of the experimental protocol. (A) Mice received a single endotracheal (e.t.) injection of 1.5 U/kg body weight of bleomycin to induce lung injury (day 0). (B) A double intravenous (i.v.) infusion of 2.5 x 105 human mesenchymal stromal cells obtained from whole umbilical cord (hUC-MSC) was performed 24 h (day 1) and 7 days (day 7) after the bleomycin administration. (C) A timeline of the injections and moments of sacrifice is shown here. Mice groups were sacrificed at days 8, 14, and 21 after the bleomycin administration (i.e., 24 h, 7 days, and 14 days after the second hUC-MSC infusion, respectively). This figure has been modified from Moroncini et al.10. Please click here to view a larger version of this figure.
Figure 2: hUC-MSC downregulate bleomycin-induced lung inflammation and fibrosis. (A and B) Representative microscopic images (10x magnification) of hematoxylin-eosin (H&E) and picrosirius red staining of lung sections obtained from C57BL/6 mice, 21 days after the endotracheal injection of sterile saline (saline) or bleomycin (bleomycin), the latter also followed by an intravenous infusion of hUC-MSC (bleomycin+hUC-MSC) or sterile saline (bleomycin+saline). The saline controls demonstrated normal lung architecture. Widespread inflammatory infiltrates were observed 21 days after the bleomycin injury, with a severe distortion of the lung architecture and the formation of fibrotic foci. Bleomycin-induced alterations were significantly attenuated by the hUC-MSC treatment but not by saline. (C) Hydroxyproline content at days 8, 14, and 21 in the lungs of C57BL/6 mice that received the aforementioned treatments. The results are the mean ± SD (n = 8 per group), expressed as a percentage of the value obtained from endotracheal saline-treated mice and are representative of three independent experiments. *P < 0.05, **P < 0.01, compared to bleomycin-treated mice. (D) Mouse Col1A1 expression levels in whole lung mRNA obtained at days 8, 14, and 21 from C57BL/6 mice that received the aforementioned treatments. The results are the mean ± SD (n = 5 per group) and are representative of three independent experiments performed in triplicate. *P < 0.05, **P < 0.01, compared to bleomycin-treated mice. This figure has been modified from Moroncini et al.10. Please click here to view a larger version of this figure.
Figure 3: Detection of hUC-MSC in lung tissue. (A and B) Representative microscopic images (200x and 400x magnification, respectively) of immunostaining with anti-HLA-1 antibody of lung sections obtained from C57BL/6 mice receiving endotracheal bleomycin followed by intravenous hUC-MSC. The red arrows indicate positive-stained hUC-MSC. (C) Human GAPDH assessed by quantitative real-time polymerase chain reaction (PCR) assay in whole mRNA extracted from cultured hUC-MSC prior to infusion (infused hUC-MSC) or from lung tissue at days 8, 14, and 21 of C57BL/6 mice receiving endotracheal bleomycin followed by intravenous hUC-MSC (bleomycin+hUC-MSC). The results are the mean ± SD (n = 5 per group) and are representative of three independent experiments performed in triplicate. Of note, the source of human GAPDH transcript in this experimental protocol can be provided exclusively by the intravenously infused hUC-MSC. This figure has been modified from Moroncini et al.10. Please click here to view a larger version of this figure.
Endotracheal administration is the preferential route for delivering exogenous agents into the lungs. Since several years, the direct injection of bleomycin into the trachea has been widely used to induce pulmonary fibrosis13 and, recently, more advanced, noninvasive techniques have been developed to accomplish this14,15,16.
The method described here provides several meaningful benefits over some potential limitations. Injection of the trachea requires a surgical intervention, carrying with it the potential for complications caused by the procedure itself, together with the need for deep animal sedation. Therefore, good preparation and practice in perfecting the procedure are needed. Besides, to minimize mouse suffering, it is imperative to set the appropriate dose of anesthetic according to the mouse strain and to the individual response and to maintain a rigorous observation of the animal sedation state. Nevertheless, we observed a very low rate of mortality and optimal animal recovery from anesthesia. Ketamine and xylazine can be used for anesthesia, as well as tribromoethanol. However, in mice, the effective dose of ketamine and xylazine is close to the lethal dose; thus, they can easily induce a respiratory arrest. Conversely, tribromoethanol dosing can be easily adjusted and is, thus, a preferable anesthetic agent. Following the endotracheal injection of bleomycin into the trachea, we did not observe any adverse effects. The mice were free from fever and no signs of inflammation or infection were observed around the trachea and the skin wound. Therefore, there was no need for antibiotic prophylaxis. Moreover, the use of an operating microscope ensures a high confidence of success by allowing the operator to accurately monitor the correct placement of the needle into the mouse trachea prior to the instillation, thus minimizing the risk of damaging it.
The endotracheal injection of bleomycin results in a potent inflammatory and fibrotic response in both lungs and can be seen as a robust method to generate experimental mouse models of human interstitial lung diseases (ILD). However, as previously documented7, the fibrotic response to bleomycin in mice is strain-dependent and gender- and age-related. Therefore, it is critical to the success of the protocol to find the tolerable dose of bleomycin in every experimental setting. Female mice were used in this study because the main interest in this research was interstitial lung disease associated with systemic sclerosis, which is a disease largely prevalent in young adult females. Three- to four-month-old mice were chosen because this is the age at which they just enter the adult phase (mice attain sexual maturity at 8-12 weeks of age)17. Thus, they are considered to be young adult mice and are preferable over younger animals, since lung fibrosis is not common in very young individuals. They are also preferable over older animals since previous studies18 have demonstrated that aged mice exhibited alterations in the lung fibroblast phenotype, leading to an increased susceptibility to disrepair and fibrosis after lung injury, which could represent a possible bias in the experimental model presented here.
Tail vein infusion is a simple, reliable, and noninvasive way to ensure the rapid and effective delivery of drugs to the bloodstream. It can be easily performed with simple medical equipment, short manual training, and reduced costs.
The experimental protocol described here, modified from previously published studies19,20,21, exists of a double intravenous infusion of 2.5 x 105 hUC-MSC to enhance cell engraftment into the mouse lungs and their therapeutic effect. In fact, since the procedure is nontraumatic, it can be repeated in the same animal, but a period of 7 days between two consecutive injections is recommended, to allow the reparation of eventual vasal wounds. Moreover, we used isoflurane inhalation to anesthetize the C57BL/6 mice during the procedure, to avoid tail vein injury in case of sudden animal movements.
In conclusion, this protocol has been successfully applied to efficiently induce pulmonary fibrosis in C57BL/6 mice and to validate the in vivo antifibrotic effect of hUC-MSC. This method can also be used for administering drugs or agents other than bleomycin into the airway, in order to generate different experimental lung disease models.
The authors have nothing to disclose.
This work was supported by a grant RF-2011-02352331 from Ministero Italiano della Salute (to Armando Gabrielli).
C57BL/6 mice | Charles River | Jax Mice Stock n. 000664 | |
2,2,2-Tribromoethanol (Avertin) | Sigma-Aldrich | T48402 | |
Barraquer Micro Needle Holder | Lawton | 62-3755 | |
Bleomycin sulfate | Sigma-Aldrich | B1141000 | |
Bürker chamber | Brand | 718905 | |
Culture Flasks | EuroClone | ET7076 | |
Disposable razors | Unigloves | 4080 | |
Dissecting Forceps | Aesculap Surgical Instruments | BD311R | |
DPBS | Gibco | 14190-144 | |
Heating pad | 2Biological Instruments | 557023 | |
Isoflurane Vet | Merial Italia | N01AB06 | |
Operating Microscope | Carl Zeiss | Model OPM 16 | |
TrypLE Select Enzyme | Gibco | 12563-029 | |
Vannas Micro Scissors | Aesculap Surgical Instruments | OC498R | |
Vicryl Plus 4/0 Absorbable Suture, FS-2 needle 19 mm | Ethicon | VCP392ZH |