Pulmonary emphysema is a form of COPD which reported by World Health Organization (WHO) as the third most common cause of death worldwide in 2020 [1]. In Egypt, it is the sixth leading cause of death according to American CDC. Recently, as environmental pollution has become more severe and more women have taken up smoking, its incidence has increased year by year [2]. It is an incurable disease with permanent progressive disability and impairment, representing a huge economic and social burden worldwide [3,4]. It is associated with progressive severe inflammation and airway blockage characterized by bronchitis and /or emphysema which is abnormal permanent enlargement of airspaces distal to the terminal bronchioles, accompanied by destruction of their walls [5]. No definitive drugs are currently available for reversing the progression of COPD, but only restrictive options to alleviate the symptoms and manage exacerbation [6]. Stem cell therapy has attracted considerable attention as it has the potential to be applied in COPD and other respiratory disease treatment [7]. It has the ability to repair damaged cells as well as regenerating worn out cells [8]. BM-MSCs therapy is promising in repairing the damage from cigarette smoke induced emphysema and elastase induced emphysema [9-11]. They used BM-MSCs intravenously to evaluate their therapeutic effect in an emphysematous model [10,11] but the intra-tracheal route of administration of stem cells is not applied a lot in researches. Antunes et al [9] reported that intra-tracheal administration might result in greater retention of stem cells in target tissue and might have greater reduction of alveolar hyperinflation than intravenous route, as it has a direct pathway of delivery. Ascorbate or vitamin C has been the subject of much debate largely because of its antioxidant property. Ascorbate inhibits both oxidation and nitration of lung proteins and improves vascular endothelial growth factor levels in lungs [12]. Also, it is required for elastin and collagen synthesis [13]. Moreover, it suppresses alveolar septal apoptosis in cigarette smoke induced emphysema. Ascorbate may provide a new therapeutic strategy for COPD treatment in human [12]. Elastase induced emphysema is a simple method that mimics pulmonary emphysema in humans [14]. It acts as protease and destroys lung parenchyma which is the main pathological abnormality in emphysema [15]. A single dose of elastase is enough to produce histopathological changes of emphysema after 3 weeks [14]. So, the present work was designed to explore the effects of intratracheal mesenchymal stem cells versus oral ascorbate on the lung tissue in a model of elastase induced emphysema using light and electron microscopic techniques.

Chemicals

Porcine Pancreatic Elastase was derived from Sigma-Aldrich (USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin-streptomycin solution, phosphate buffer saline (PBS) and 0.25% trypsin were purchased from (Lonza Bioproducts, Belgium).

Animals

Swiss albino mice, 24 adult females, weighing approximately 25-35 gm, obtained from National Research Center for experimental animals, Cairo, housed under standardized conditions, away from any stressful stimuli with 12 hr day/night cycle and free access to food and water, for one week. The research was proceeded after approval from the research ethics committee, faculty of medicine, Suez Canal University was obtained.

Experimental groups

The mice were divided randomly into 4 groups, each of 6 mice. Group I (Control group): Each mouse received 0.5 ml intranasal saline, as a single dose at the first day. Then after 21 days they were sacrificed.

Group II (Elastase group) animals received single intranasal PPE 200 units/kg, to induce emphysema. After 21 days they were sacrificed.

Group III (BM-MSCs treated Group): After 21 days from emphysema induction, each mouse was injected by BM-MSC 5 x 10⁵ cells in 50 µl PBS introduced intra-tracheally.

Group IV (Ascorbate treated Group): After 21 days from emphysema induction, each mouse received ascorbate in a dose of 100 mg/kg/mouse. dissolved in purified water given orally using a syringe with long blunt nozzle, once daily for 21 days.

The cells were isolated from bone marrow of young male albino rats and injected after 9 days of culture.

Stem Cells Isolation

The male rats (4-8 weeks) were euthanized by cervical dislocation and soaked in 70% ethanol for 2 minutes [16]. An incision around the hind limbs was made where they attach to the trunk. Limbs were stored on ice in DMEM supplemented with 1% penicillin/streptomycin waiting for further dissection under the hood [17]. Muscles, ligaments, and tendons were removed, then the tibias and femurs were transferred to culture dish with DMEM and penicillin/streptomycin on ice. The dish was transferred into the biosafety cabinet using proper sterile technique to cut the two ends just below the end of the marrow cavity. Five ml of complete media was withdrawn with a syringe and inserted into the bone cavity to ?ush the marrow out over the dish until the bones became pale [16].

Seeding and Culture

The dish was then incubated at 37 ? in a 5% CO2 incubator after removing all bone pieces. Cell viability was maximized by ensuring that processing time had not exceeded 30 minutes from animal death [16]. Cell viability was assessed by using 0.1 ml of trypan blue with an equal volume of cell suspension and 0.8 ml of complete media [18].

Stem Cell Characterization, Passaging and Harvesting

By day 3, non-adherent cells were removed, media was changed every 24- 72 hrs. MSCs are characterized by their spindle shape and their capacity of adhesion to plastic flasks [19]. When cells reached 70-90 % confluency, they were washed with PBS twice to remove the residual media and then digested with 2.5 mL of 0.25% trypsin. The bottom of the plate was flushed with complete media and the cells transferred to Falcon tube, centrifuged at 800g for 5 minutes, then the pellet of cells was seeded. Cell count and cell viability assessment were done using hemocytometer and typan blue, and so the cell density was adjusted as per required number and volume for the experiment, using BM-MSCs 5 x 10⁵ cells in 50 µl PBS introduced intra-tracheally per animal [9].

Processing Of Mice Lungs

At the end of the experiment (on the 42^nd day), mice were anesthetized by diethyl ether, their chests were opened and lungs of each animal were removed.

• One part of the lung was fixed in 10% buffered neutral formalin and processed to 4 µm paraffin sections for histological stainings:
• Haematoxyline and eosin (H&E), Orcein and Masson’s trichrome stains.
• The other part of the lung was dissected and fixed in 2.5% gluteraldehyde for transmission electron microscope examination (TEM).
• PCR of SRY gene: to detect MSCs engraftment in lung tissue of female mice (for group III only).

Histopathological Examination

The H&E stain; lung general architecture was examined and imaged × 40 or ×100 for necrotic cells, airway spaces enlargement, loss of inter-alveolar septa, septal thickening with inflammatory infiltrate and erythrocytes extravasation. Mean linear intercept (MLI) was carried out according to Dunnil method; by measuring five sections per animal and thirty captures per group. Ten horizontal lines and eleven vertical lines were drawn on captured views of H&E stain and printed to count the intercepts and traverses of lung tissue with each grid line then calculated by the following equation: [20]

Lm= (N) (L)/M

L= the length of traverses of the airspace

N= the number of traverses

M= the sum of all intercepts

Orcein stain: for elastic fibers demonstration in the walls of bronchioles, blood vessels and inter-alveolar septa.

Masson’s trichrome stain: for collagen fibers demonstration.

Quantitative assessment, of the mean color area percentage of elastic and collagen fibers, was done using Image J software.

Using the Statistical Package for Social Sciences (SPSS 20 software). One way analysis of variance (ANOVA) test and Tukey HSD Post Hoc tests were used. The differences between the studied groups were only statistically significant when P value < 0.05.

H&E stained sections of group I showed normal spongy structure of thelung; the septa were covered by the alveolar epithelium made up of two cell types. Type I pneumocytes which had flattened dense nuclei and attenuated cytoplasm beyond the perinuclear region. Type II pneumocytes were rounded in shape, had large vesicular rounded nuclei and foamy cytoplasm. Macrophages were seen ccasionally with their irregular nuclei (Figure 1 A). Sections from group II showed areas of; septal loss with appearance of widely spaced alveoli alternating with adjacent areas of septal thickening, interstitial eodema and mononuclear cellular infiltration. Hemosiderin laden macrophages were also evident. Type I pneumocytes appeared normal, but there was significant increase in the percentage of necrotic type II pneumocytes with karyolytic and pyknotic nuclei. (Figure 1, B1 & B2). Groups III& IV showed statistically significant improvement in the histopathological changes in general and in the percentage of necrotic pneumocytes type II in specific (Figure 1, C & D) (Figure 1 E). There was a significant increase in the MLI in group II compared to control group. Group IV showed significant decrease in the MLI compared to group II, but, a non-significant decrease in group III was evident (Figure 1 F).

Orcein stained sections in the lung of group I revealed dark brown, thin, long and regular elastic fibers, in the lamina propria of bronchioles, elastic lamina of the blood vessels and in the inter-alveolar septa (Figure 2A). Group II showed disruption and fragmentation of elastic fibers in the septa, bronchioles and blood vessels (Figure 2B). Groups III& IV showed nearly normal distribution of dark brown elastic fibers, compared to control (Figure 2C, 2D). Mean color area percentage of elastic fibers in orcein stained sections was significantly decreased in group II compared to group I with a non-significant increase in both groups III &IV compared to group II (Figure 2E).

 Masson’s trichrome stained sections in the lungs of group I revealed minimal green collagen fibers. They were detected in the interalveolar septa, around the bronchioles and the blood vessels (Figure 3A). Group II showed statistically significant increase in the amount of collagen fibers around the bronchioles and blood vessels, compared to control sections (Figure 3B). Group III revealed nearly normal distribution of collagen fibers (Figure 3C). While, Group IV showed significant increased compared to group I (Figure 3D & 3E).

Transmission electron microscopic results

Ultrathin sections of group I showed clear alveolar space lined with two types of alveolar cells; type I and type II pneumocytes. Alveolar macrophages were found in the alveolar lumen (Figure 4A). Group II revealed edematous fluid in the alveolar spaces, cellular debris, extravasated erythrocytes, alveolar macrophages and other inflammatory cells. Alveolar walls lined by type II pneumocytes with shrunken heterochromatic nuclei. Numerous erythrocytes within the capillary lumen were also observed (Figure 4B). Group III& IV revealed improvement compared to group II. Alveolar spaces were clear lined by type I and type II pneumocytes. Alveolar macrophages and some inflammatory cells were observed occasionally (Figures 4C, 4D).

Type II pneumocyte in Group I was much larger and rounded, had large rounded vesicular nucleus with dispersed chromatin and prominent nucleolus. Its cytoplasm had ovoid membrane bound lamellar bodies containing dense lamellae and dense mitochondria. There were numerous short microvilli projecting from their free surfaces into the alveolar lumen (Figure 5A). Group II, Type II pneumocyte revealed features of damage including shrinkage of its nucleus with loss of its chromatin pattern. Its cytoplasm contained empty membrane bound lamellar bodies and numerous cytoplasmic vacuoles. Few mitochondria were dense whereas others were swollen or ruptured with loss of their cristae. Moreover, microvilli were lost from the free surface (Figure 5B). Group III& IV, Type II pneumocytes regained their normal appearance as their nuclei showed normal pattern of chromatin distribution, their cytoplasm contained many dense mitochondria with slight damage to some of them and partially empty lamellar bodies. Moreover, short microvilli appeared projecting from their free surfaces (Figure 5C & 5D).

Alveolar macrophage in group I had large indented nucleus and cytoplasmic vacuoles (Figure 6A). In group II, Alveolar macrophages were abundantly found in alveolar spaces. Their cytoplasm contained many secondary lysosomes, phagocytic vacuoles and many phagocytosed hemosiderin particles (Figure 6B). Group III & IV; some alveolar macrophages contained numerous secondary lysosomes, phagocytic vacuoles and some hemosiderin particles (Figure 6C, 6D).

Characteristics of MSC in Culture

At day 1 of plating, the cells were rounded with 30-40 % confluency (Figure 7A). The adherent cells were fusiform and elongated, with central nuclei and cytoplasmic prolongations. They adhered to the culture plate by day 3 and reached 50-70 % confluence within 7∼10 days (Figure 7B).

The PCR product was amplified from the lung tissue of female mice subjected to BM-MSCs therapy (group III). 100% of the lung tissue samples were taken from group III, were positive for the presence of Sry gene (Figure 7B), indicating homing of our injected stem cells into the lung tissue of animals.

Figure 1: Histopathological changes of the lung tissue among the four groups [H&E x 1000].

A, a section in control mouse showing type I pneumocytes (P1) with flattened nuclei and attenuated cytoplasm. Type II pneumocytes (P2) contain relatively large rounded nuclei with foamy cytoplasm. Alveolar macrophages (M) have irregular nuclei. B1, section in elastase group, magnification x 400 areas of lost interalveolar septa (L), areas of thickened interalveolar septa (T) and mononuclear cellular infiltration (*). Interstitial eodema (green arrows) and hemosidrin laden macrophages (black arrow) with their brown granules are shown. B2, showing normal appearance of (P1), necrotic (P2) in the form of pyknotic (K), karyolytic (L) nuclei with eosinophilic cytoplasm. Areas of septal loss (arrows) are shown. C, BM-MSC treated group with normal (P1) and (P2). Few areas with septal loss (arrow) compared to control group. D, Ascorbate treated mouse with (P1, P2 and M) nearly similar to control group. Some areas are showing septal loss (arrows). E, Pie graph showing means of the percentage of necrotic P2 among different experimental groups. P value was 0.0001 among all groups F, Comparison of Mean linear intercept (MLI) among different experimental groups: Data are shown as mean ± SD. *significant compared to group I, ^significant compared to group II and ¶significant compared to group III.

Figure 2: Orcein stained elastic fibers of the lung tissue in the four groups [x 400].

A, a section in control mouse showing the elastic fibers as continuous, regular dark brown fibers, in the lamina propria of a bronchiole (B), in the internal elastic lamina of a blood vessel (BV) and in the inter- alveolar septa (arrows). B, a section in elastase group showing decrease of elastic fibers (green arrows) in the interalveolar septa. Fragmentation (yellow arrows) of elastic fibers in the wall of (BV) and (B) are also shown. C, BM-MSC treated group and D, ascorbate treated group showing nearly normal distribution of elastic fibers (arrows) compared to control group. E, mean color area percentage of brown elastic fibers among different experimental groups: Data are shown as mean ± SD. P value <0.05 is considered significant. *significant compared to group I.

Figure 3: Masson’s trichrome stained collagen fibers of the lung tissue in the four groups [x 400].

Figure 3: A, a section in control mouse showing minimal collagen fibers appearing green in the inter- alveolar septa (arrow), around a bronchiole (B) and around a blood vessel (BV). B, showing increase in the amount of collagen fibers (green) in elastase group, around the bronchioles (B) and the blood vessels (BV). C, BM-MSC treated mouse showing nearly normal distribution of collagen fibers (green bundles) compared to control group. D, ascorbate treated mouse showing markedly increased collagen fibers (green bundles) in the inter-alveolar septa (arrow), around a bronchiole (B) and around a blood vessel (BV) compared to control group. E, Mean color area percentage of collagen fibers by Masson trichrome’s stain among different experimental groups: Data are shown as mean ± SD. P value <0.05 is considered significant. *significant compared to group I, ^significant compared to group II and

^¶significant compared to group III.

Figure 4: Ultrastructural changes in the interalveolar sepa in the four groups [Magnification X 1200].

A, a section in control group showing the alveolar space (AS) lined by, type I pneumocyte (P1) with attenuated cytoplasm, type II pneumocytes (P2) contain rounded vesicular nuclei and lamellar bodies. Alveolar macrophage (M) with indented nucleus and capillary lumen (C) containing erythrocytes. B, elastase group showing shrinkage of (P2) nuclei. Alveolar macrophage (M), other inflammatory cells (IC), erythrocytes (E), edematous fluid (EF) and cellular debris (D) in the alveolar space. C, BM-MSC treated group showing clear (AS) lined by (P1) and (P2). Alveolar macrophage (M), some inflammatory cells (IC) and erythrocytes within the capillaries (C) are also shown. D, ascorbate treated group with clear alveolar spaces (AS) lined by (P1) with flattened nucleus and narrow cytoplasm. Type II pneumocyte (P2) containing euchromatic nucleus and lamellar bodies. Alveolar macrophage (M) with irregular nucleus and erythrocytes within capillary lumen (C) are also shown.

Figure 5: Ultrastructural changes on the pneumocyte type II among the four groups [Magnification X 3000].

A, a section in control group showing type II pneumocyte (P2) with large rounded, slightly irregular, vesicular nucleus and prominent nucleolus. Its cytoplasm contains characteristic lamellar bodies (L) containing dense lamellae and dense mitochondria (M). Short microvilli (arrow) project from the free surface of the cell into the alveolar space. B, elastase group shows type II pneumocyte with shrinkage of its nucleus and loss of normal chromatin pattern. Emptiness of most of its lamellar bodies (L), some mitochondria (M) are swollen or ruptured and its microvilli (arrows) are lost. Cytoplasmic vacuoles (V) are also shown. C, BM-MSCs treated group showing type II pneumocyte contains large rounded euchromatic nucleus with a prominent nucleolus. Its cytoplasm contains lamellar bodies (L) with few lamellae and dense mitochondria (M). Short microvilli (arrow) are shown projecting from its free surface. D, ascorbate treated group shows type II pneumocyte with large rounded nucleus. Its cytoplasm contains partially empty lamellar bodies (L) and dense mitochondria (M). Numerous short microvilli (arrows) are shown projecting from its free surface into the alveolar lumen.

Figure 6: Ultrastructural changes on the of alveolar macrophages among the four groups [Magnification X 3000].

A, control lung macrophage (M) has large indented nucleus and cytoplasmic vacuoles (arrows), an erythrocyte is shown within the capillary lumen (C). B, elastase group shows macrophage (M) containing large irregular nucleus with clumped peripheral heterochromatin, secondary lysosomes (L), phagocytic vacuoles (V) and numerous phagocytosed particles (P). Cellular debris (D) are shown in the alveolar space. C, BM-MSC treated lung shows alveolar macrophage (M) with large nucleus, secondary lysosomes (L), phagocytic vacuoles (V) and phagocytosed particles (P). D, ascorbate treated lung shows macrophage (M) with large nucleus, secondary lysosomes (L), cytoplasmic vacuoles (V) and pseudopodia (PP) from its free surface.

A, Phase contrast photomicrography of 30-40 % confluent rat MSCs at day 1 of plating showing rounded cells (arrow) with few erythrocytes (arrow head). B, Phase contrast photomicrography of 60- 70 % confluent rat BM-MSCs after 9 days of plating showing fibroblastic morphology in the form of spindle shaped cells (arrow head). C, photomicrograph of gel electrophoresis showing PCR-amplified Sry gene from the DNA of lung tissue of MSCs group at day 42. Lane 1: is the size (molecular weight) marker. Lane 2: negative control for the gene. Lane 3: positive control for the gene. Lane 4- 8: positive samples from the lung tissue of group III.

Emphysema induced by elastase is an interesting and advantageous simple method that mimics pulmonary emphysema in humans [21]. In this study, we administered Porcine Pancreatic Elastase, its mechanism of injury is not clearly known, however, Craig et al [22] suggest that a direct effect of elastase induction causes about 20% of lung injury, while the rest of tissue injury happens due to be the consequence of host inflammatory response. This work revealed histopathological changes of emphysematous group, were detected by light microscopic examination and confirmed ultrastructurally by transmission electron microscope. There were enlargement of airspaces as a striking feature, loss of inter-alveolar septa, septal thickening with inflammatory infiltrate, edematous fluid and erythrocytes extravasation which occurred after 21 days of elastase injection. Longhini-dos-Santos et al [21] and Kim et al [11] show similar results of alveolar destruction in mice subjected to elastase. They explain injury to the immediate effect of proteolytic action of elastase on lung parenchyma, causing rapid significant enlargement of airspaces. Inflammatory infiltration and interstitial edema in the lung tissue were in agreement with the results of Antunes et al [9] and Chen et al [23]. This could be due to the instant effect of elastase on lung tissue leading to recruitment of neutrophils followed by macrophages along the process of injury and hemorrhage within the lungs [21]. Elastase has vasodilator effect on capillaries causing leakage of inflammatory cells, erythrocytes and fluid from the capillaries, narrowing of airway lumen with consequent trafficking of inflammatory cells and accumulation of mononuclear cellular infiltration [24, 25]. While the brown hemosiderin granules in the lung tissue could be resulted from the congestion of blood vessels followed by extravasation of erythrocytes. Alveolar macrophages phagocytose them, so become filled with brownish granules of hemosiderin [26]. The significant increase in necrotic type II pneumocytes, compared to other experimental groups, were in accordance with the study results of Kuethe et al [27]. They report that one of the histological abnormalities in emphysematous rats is necrosis to type II pneumocytes. Our findings were confirmed ultrastructurally, as type II pneumocytes had shrunken dark nuclei with loss of their normal chromatin pattern, few swollen mitochondria, emptiness of their lamellar bodies and absence of their short microvilli from their free surfaces with consequent appearance of cellular debris in the narrow alveolar lumen. Similar ultrastructural findings were also obtained by Antunes et al [9]. Although type I pneumocytes appeared unaffected in our TEM findings, Mohi El-Din et al [28] find that type I pneumocytes are mainly affected exhibiting nuclear changes as indentation, lysis and pyknosis but this might be attributed to their different emphysema model which was induced by lipopolysaccharide (LPS). Kosmider et al [29] mention that type II pneumocytes are capable of proliferation after the damage of the alveolar epithelium to restore type I pneumocyte. Ghosh et al [5] demonstrate that lung injury was due to oxidative damage and inflammatory process. Moreover, Sato and Seyama [30] state that inflammatory cells, such as macrophages and neutrophils are the most endogenous generators of oxidants, their role initially is protective sparing surrounding structures from damage, however, if this process prolonged, oxidative stress happen. Kosmider et al [29] detect low levels of key regulators of antioxidant defense system in type II pneumocytes, which render their ability to synthesize, secrete, and recycle surfactant. This explains the empty lamellar bodies leaving empty cytoplasmic vacuoles and loss of regular lamellar pattern observed in our TEM results. In our study, Mitochondrial swelling and loss of their cristae in type II pneumocyte injury may be caused by oxidative injury, which results in cellular degeneration and necrosis with appearance of the nuclear changes as indentation, lysis and pyknosis and these were in agreement with Soliman et al and Satoand Seyama [30,31]. In the current study, BM-MSCs and ascorbate treated groups showed improved lung architecture with less widely spaced alveoli and septal loss, slight monocellular infiltration, minimal interstitial edema, decrease of hemosiderin granules and hemosiderin laden macrophages and less degenerative changes of type II pneumocytes. In addition, the quantitative analysis of BM-MSC group revealed that the percentage of necrotic type II pneumocytes were significantly lower than group II. These findings were confirmed ultrastructurally by TEM that showed type II pneumocytes had regained their normal appearance with preservation of their rounded vesicular nuclei and prominent nucleoli, appearance of numerous short microvilli and the lamellar bodies showed normal dense secretions of surfactant. These finding were in agreement with the findings of Antunes et al [9]. There was decrease in the inflammatory cells in the lung tissue compared to group II as MSCs have an immuno-modulatory role in reducing the neutrophilic infiltration. MSCs increase the IL-4 secretion, inhibit the production of TNF-α (proinflammatory mediator) and elevate the anti-inflammatory response [28] Furthermore, MSC administration decrease the proportion of macrophages in the bronchoalveolar lavage (BAL) of emphysematous rat models and may have increased the M2/M1 macrophage ratio in BAL (where M1 is pro-inflammatory whereas M2 is an anti-inflammatory) [32] In addition, Yamazato et al [33] mention that macrophages favor differentiation into alternatively activated regulatory M2 profile. So, MSC administration alleviate lung airway inflammation, by down-regulating cyclooxygenase-2 which mediates prostaglandin E2 production, possibly through the effect on alveolar macrophages [32]. MSC-treated group showed reduction in hemorrhage and edema. These could be attributed to the preservation of endothelial and epithelial tissue integrity, mediated by MSCs [28]. Less widely spaced alveoli and septal loss observed in this group, compared to emphysematous group, were similar to that of Cruz et al [34] who detect decrease of apoptotic cells by BM-MSCs inhibiting caspase-3 expression. BM-MSCs can enhance cell proliferation by migration and localization near injured tissues [28] and release soluble factors such as IL-10, hepatocyte growth factor, adrenomedullin (paracrine factor) that promotes alveolar and vascular regeneration. In addition, MSCs also release keratinocyte growth factor which recruits endogenous bone marrow cells into the lungs to be incorporated there, bearing the markers of alveolar epithelial cells and capillary endothelial cells. Both Huh et al and Cruz et al [34,35] suggest that the repair activity of MSCs is due to the paracrine effect rather than stem cell engraftment as they found out most of donor cells have disappeared. In contrast to their suggestion, in our study, there was engraftment of BM-MSCs in the lung tissue of female mice (group III) proved by the presence of SRY gene of the male donors in the lungs of all female recipients. By PCR technique, Longhini-dos-Santos et al [21] prove migration and engraftment of BM-MSCs to emphysematous lungs as they were systemically injected intravenously. BM-MSCs may be progenitors to type II pneumocytes or may be capable of serving as type II pneumocytes that can divide to be type I pneumocytes to regenerate the lung tissue. Moreover, MSCs are thought to differentiate into endothelial cells by the effect of basic fibroblast growth factor which also promotes angiogenesis and improving ischemia usually seen in human emphysema [2].Ascorbate treatment revitalized the lung structure through amelioration of oxidative stress and alveolar cell apoptosis. This was confirmed by the light microscopic results of type II pneumocytes showing less damage (cellular necrosis) and were confirmed by our TEM results which showed type II pneumocytes regained their normal appearance and partially empty lamellar bodies. Inflammatory infiltration was reduced in the lung tissue, this observation was in accordance with Ghosh et al [5] who explain that ascorbate strongly inactivates p-benzoquinone which claimed to initiate oxidative damage and is accompanied by inflammation and apoptosis leading to destruction of alveolar cells. Thereby ascorbate prevents the initiation of pathogenesis of emphysema. Reduced spacing of alveoli and septal loss that were observed in this group may be due to the role of ascorbate in reducing the inflammatory process by suppressing proinflammatory protein expressions like the major lung protease, MMP- 9 [36]. The histopathological improvement of this group was evident by the quantitative analysis of type II pneumocytes revealing that the mean percentage of necrotic type II pneumocytes was significantly lower than group II. This was explained by Koike et al who report that ascorbate treatment subsides oxidative stress and alveolar septal cell apoptosis and restored the concentration of VEGF in the BAL and in the lungs back to normal. These observations of Koike et al [12] support the speculation that VEGF which is a growth factor affecting the function and survival of endothelial cell that might explain reduced hemorrhage and edematous fluid within lung tissue of mice of group IV. They also add that oxidative stress generated in COPD causes septal apoptosis, which disrupts the VEGF signaling that in turn, causes more oxidative damage. In the present work, Orcein stained sections showed marked decrease and fragmentation of elastic fibers in the interalveolar septa, the walls of the bronchioles and the blood vessels of emphysematus group. This was confirmed by a significant decrease in mean color area percentage of elastic fibers performed by image J software compared to control group. Our findings coincided with the study of Cruz et al [34] and was explained by Longhini-dos-Santos et al [21] and Oliveira et al [37] who report that degradation of elastin (by elastase) resulted in elastin fragments which act as chemo-attractants for recruitment of neutrophils and macrophages. These cells release matrix metalloproteases, thus promoting elastic fiber rupture mainly by MMP9, which has greater elastolytic capacity than elastase itself. This is followed by restructuring process of disorganized elastic fibers deposition which happens in the chronic phase of elastase induction that results in loss of the lung tissue elastic recoil. BM-MSCs and ascorbate treated groups showed reappearance of elastic fibers in the interalveolar septa and in the walls of bronchioles and blood vessels like control. This was confirmed by the significant increase in mean color area percentage of elastic fibers in both groups compared to emphysematous group. Our results were consistent with Kruk et al [38] who demonstrate that BM-MSCs express considerably higher levels of collagen Iα1 and elastin than MSCs isolated from lung or adipose tissue. Furthermore, Ozsvar et al [39] report that collagen I elevates MSCs viability and enhances their repair activity. They also add that elastin fragments promote MSCs differentiation into epithelial cells and endothelial cells. BM-MSCs exhibit a mature phenotype characterized by contractility and migrative tendencies similar to healthy rat smooth muscle cells, and capable of tropoelastin (precursor) synthesis and assembly of a fibrous, highly crosslinked elastic matrix [40]. On the other hand, ascorbate inhibits the action of proteases such as MMP-9, so minimizing elastin degradation with consequent preservation of the extracellular matrix (ECM) [36]. The Masson’s trichrome stained sections of emphysematus group showed moderate increase in the connective tissue fibers around the bronchioles and around the blood vessels whereas decreased in the interalveolar septa, compared to control group. The mean color area percentage of collagen fibers was significantly higher than control group. Our results coincided with the study of Taguchi et al [41]. The increased collagen deposition in this group, was explained by Mohi El-Din et al [28] who report that the production of inflammatory cytokines by inflammatory cells such as TNF- α and IL-1 mediate fibrotic lung injury. Moreover, Oliveira et al [37] explain that new collagen fibers are resynthesized, in an attempt to repair the damaged lung but they are relatively weak, disorganized and can rupture, so reduce overall tissue stiffness. They also observe a time lag between elastic fiber deterioration and collagen deposition, by fibroblasts, which are in a quiescent state during equilibrium controlling the extracellular matrix size. However, after injury, in an attempt to restore homeostasis, these cells become activated and convert into myofibroblasts, thus secreting collagen fibers and participating in the inflammatory response. BM-MSCs treated groups showed nearly normal distribution of collagen fibers in the interalveolar septa, around the bronchioles and around the blood vessels compared to emphysematous group. This was confirmed by significant decrease of the mean color area percentage of collagen fibers detected by image J software. One of the explanations was that, MSCs block the production of TGF-β, which is a potent inducer of collagen production by fibroblasts and myofibroblasts [34]. Mohi El-Din et al [28] mention that MSCs also reduce the production of TNF-α and IL- that mediate fibrotic lung injury. Another explanation of reduced collagen fiber levels could be attributed to alveolar macrophages, which increased collagen uptake during an intervention to alleviate pulmonary fibrosis [42]. In ascorbate treated group, sections showed marked increase of collagen fibers in the interalveolar septa, around the bronchioles and around blood vessels, compared to other experimental groups. It was even higher than that of emphysematous mice. This was confirmed by significant increase in the mean color area percentage of collagen fibers detected by image J software. These findings were supported by the results of Koike et al [12] who treat SMP30-KO mice with ascorbate in a dose of 1.5 g/L/day for 60 days. Their study shows that more than 2-fold increases of collagen I and IV mRNA than the corresponding emphysematous mice. They attribute their findings to the fact that ascorbate has a primary role in collagen synthesis as it promotes the activity of prolyl hydroxylase and increases the mRNA transcripts of collagen. In contrast, Ghosh et al [5] observe reduction in the collagen deposition in the ascorbate treated guinea pigs with a dose of 30 mg/kg/day for 56 days. We attribute our results to the type of experimental animals, as we used mice, duration, or ascorbate action that might be dose dependent.

To sum up, in the present study, the elastase model of emphysema in mice had produced emphysematous changes as expected and hypothesized, giving almost the same emphysematous picture as other emphysematous animal models. The curative effect of BM-MSCs therapy was more apparent than ascorbate supplementation, especially regarding the marked collagen content restoration to normal levels, which were disappointingly overexpressed in ascorbate group. Due to ascorbate mechanism of action that may rely on dose, calling for further investigations of ascorbate in several doses to find the optimum dose for emphysema treatment.

Elastase induced severe lung damage which developed to emphysematous changes and is associated with inflammatory reaction that had led to marked histological and ultrastructural destructive changes in the lung tissue of the mice particularly type II pneumocytes. Administration of BM- MSCs therapy showed marked improvement of these harmful effects better than ascorbate administration. Therefore, BM-MSC therapy might be used as a therapeutic agent for restoration of lung architecture in emphysematous patients.

Funding Informations

The study was totally funded through the authors.

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