الفهرس 
Anatomical and histological aspects of the mammalian lung.Only 14 pages are availabe for public view
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Abstract
Leflunomide is an oral, disease-modifying drug that is used in the treatment of rheumatoid arthritis. Leflunomide reduces inflammation in the joints that is responsible for both the symptoms of rheumatoid arthritis and the destruction of joints. This reduces symptoms as well as the progressive deformities of the joints caused by the arthritis. Leflunomide reduces inflammation by suppressing the activity of immune cells responsible for the inflammation. Leflunomide suppresses immune cells by inhibiting dihydroorotate dehydrogenase, an enzyme that is necessary for the production of DNA and RNA. Without DNA and RNA the immune cells cannot multiply or function. Because of its unique and different mechanism of action, leflunomide is of value when added to other medications used for treating rheumatoid arthritis.
The risk of interstitial lung disease in patients with rheumatoid arthritis treated with leflunomide have been appearing recently. So an intial pulmonary function tests and periodic ones should be done for patients receiving leflunomide. Any disturbances in these tests or appearance of symptoms of interstitial lung disease, leflunomide treatment must be stopped. N-acetylcysteine is the acetylated variant of the amino acid L-cysteine and is widely used in medicine. Its clinical applications arised from its ability to support the body’s antioxidant and nitric oxide systems. So, coadministration of N-acetylcysteine with leflunomide treatment is highly recommended to minimize harmful effect of leflunomide on lung.
The present study was conducted for studying the histological, immunohistochemical and ultrstructural changes in rat lung following leflunomide administration and the possible protective effect of N- acetyl cysteine.
MATERIALS & METHODS
Sixty (60), adult male albino rats weighing nearly (100- 120) gm were used in the present study. They were divided into 4 main groups:
1-Control groups:-
Group I (Control group): It was composed of 20 rats were further subdivided into 2 equal subgroups; each one was composed of 10 rats. The first: kept without any treatment and served as control for all experimental groups. The second: given 1 ml saline [vehicle of leflunomide]/ day orally for 4 weeks and served as control for leflunomide treated group. At the end of the period of the experiment (4 weeks), the animals in each subgroup were sacrified.
Group II (N- acetyl cysteine treated group): It was composed of 10 rats that were treated with N- acetyl cysteine at a dose of 200 mg/ kg body weight daily for 4 weeks using modified plastic syringe. N- acetyl cysteine was dissolved in 1 ml distilled water to produce the desired concentration. At the end of the period of the experiment (4 weeks), the animals were sacrified.
Group III (Leflunomide treated group):
It was composed of 20 rats were treated with leflunomide in the form of tablets at dose of 10 mg / kg. Each tablet containing 10 mg leflunomide was dissolved in 1 ml saline and given orally by modified plastic syringe for 4 weeks.This group III is further subdivided into two subgroups: Subgroup IIIa: (Rats sacrified four (4) weeks after treatment with leflunomide). Subgroup IIIb: (Rats sacrified two (2) weeks after arrest of leflunomide treatment for four (4) weeks).
Group IV (Leflunomide & N-acetyl cysteine treated group: It was composed of 10 rats that were treated with both leflunomide & N- acetyl cysteine at dose similar to that in leflunomide treated and N- acetyl cysteine treated groups respectively. At the end of the period of the experiment (4 weeks), the animals were sacrified. At the end of the period of the experiment, and then animals were anaesthetized, lungs were extracted and prepared for the following studies:
1. Histological studies: using haematoxylin & eosin stain and Mallory trichrome stain. 2. Immunohistochemical study: using Caspase-3 immunostaining. 3. Electron Microscopic (EM) Study. 4. Morphometric study and statistical analysis.
from Hx& E sections of different experimental groups (control and treated groups) the number of pneumocyte type II was counted and the thickness of the inter-alveolar septa was measured. Then, stastistical
analysis for these data was done in the form of mean and standard deviation.
RESULTS & DISCUSSION
The following data have been observed in the present study:
I- GENERAL RESULTS:
By observations throughout the experiment, animals in control group and N- acetyl cysteine treated group were in a good general condition and showed a normal behaviour, activity, eating and growth. No abnormalities were observed. On the other hand, those in leflunomide treated group showed decreased activity and appetite but those in leflunomide & N- acetyl cysteine treated group showed little changes in comparsion to animals treated with leflunomide only. Animals in recovery groups showed some improvement in their activity and appetite.
II- MACROSCOPIC EXAMINATION:
Four weeks after treatment, lungs obtained from control rats and N- acetyl cysteine treated rats showed the normal homogeneous glistening bright red appearance. On the other hand, lungs obtained from leflunomide treated rats for four weeks showed dark opaque red appearance (patches of haemorrhage) but leflunomide & N- acetyl cysteine treated rats for four weeks showed bright red appearance and small areas of haemorrhage.Two weeks after arrest of treatment, lungs obtained showed little haemorrhage.
III-MICROSCOPIC EXAMINATION:
Light Microscopic Studies:
A) Haematoxylin & Eosin:
Group I (Control group):
The control rats showed the well known histological picture of rat lung. Lung sections showed numerous alveoli with thin alveolar walls. An alveolus may open into alveolar sacs, alveolar ducts or a respiratory bronchiole. An alveolus was lined by pneumocyte type І which was flattened with thin membrane like cytoplasm and pneumocyte type ІІ with large central rounded nucleus and dome shaped surface facing the alveolar lumen. There were alveolar macrophages in the alveolar lumen. Bronchiole was detected in some sections showed normal mucosal lining (cubodial ciliated epithelium) and circular layer of smooth muscle fibers.
Group II (N- acetyl cysteine treated group):
Four weeks after treatment with N- acetyl cysteine, there was normal lung architecture detected by patent alveoli, normal bronchiole and normal blood vessel .Patent alveoli were lined by flattened pneumocyte type І and rounded pneumocyte type II. Group III (Leflunomide treated group): Subgroup IIIa: (Rats sacrified 4 weeks after treatment with leflunomide) There were varying degrees of pathological changes after treatment with leflunomide, which were in the form of: thickening of pulmonary interstitium by inflammatory cells and inflammatory
exudates with extensive interstitial haemorrhage. These changes led to collapse of some alveoli & thickened interalveolar septa and subsequent compensatory overexpansion of neighboring alveoli with destroyed alveolar walls. This is compatible with emphysematous changes as leflunomide induces recurrent pulmonary inflammation which damages & eventually destroys the alveolar walls, creating large air spaces (emphysematous changes). The influx of inflammatory cells is explained by cytotoxic lung injury as a result of direct injury to pneumocytes or the alveolar capillary endothelium.
There were interstitial and alveolar haemorrhage represented by numerous Red Blood Cells (RBCs) filling the pulmonary interstitium & alveolar lumen. This occurs as a result of increased vascular endothelial permeability due to hypoxia and leak of macromolecule associate with extravasation of protein to the alveolar space. Also, pulmonary vascular congestion was detected in the form of dilated congested blood vessels and attributed to vasodilator substances released by blood vessels into the blood stream.
Numerous alveolar macrophages were demonstrated containing haemosiderin granules in their cytoplasm. This could be explained by breakdown of red blood cells followed by phagocytosis of the released iron pigment by the pulmonary alveolar macrophages.
There was hyperplasia of pneumocyte type II. This is considered as attempt to regenerate the damaged alveolar cells as pneumocytes type II are the progenitors of pneumocytes type I.
There were cellular debris & haemorrhage in the lumen of the bronchioles. Also, there was partial shedding of bronchiolar mucosa and epithelial proliferation in some areas of mucosa. Subgroup IIIb (Rats sacrified 2 weeks after arrest of leflunomide treatment for 4 weeks)
There was persistence of histopathological changes including dilated congested pulmonary vessels, minimal inflammatory cellular infiltrate & minimal haemorrhage. These results are contribuated to very long half-life (approximately 2 weeks) of leflunomide.
Group IV (Leflunomide & N-acetyl cysteine treated group:
Simultaneous administration of N-acetyl cysteine to leflunomide treated rats resulted in improvement in general condition of the treated rats. Also, lung sections showed remarkable improvement in histological pictures in the form of reduced inflammatory cell infiltrate, minimal interstitial haemorrhage and reduced alveolar destruction.This could be explained by the ability of N-acetyl cysteine to act as a free radical scavenger and aprecursor of the antioxidant reduced glutathione. Also, N- acetyl cysteine has the capacity to inhibit various inflammatory elements related to oxidative stress.
B) Toluidine Blue:
Group I (Control group):
There were patent alveoli lined by peumocyte type І & peumocyte type II. Also, there were alveolar macrophages in the lumen. The mucosa of the bronchiole showed the cuboidal ciliated cells & the
non ciliated cells (Clara cell) with dome- shaped apical surface.
Group II (N- acetyl cysteine treated group):
There were normal patent alveoli lined by pneumocyte type І with Flattened nucleus & pneumocyte type II with rounded nucleus.
Group III (Leflunomide treated group): Subgroup IIIa: (Rats sacrified 4 weeks after treatment with leflunomide)
There was thickening of pulmonary interstitium by inflammatory cells. Also, pneumocyte type II appeared with apparent increase in number with vacuolated cytoplasm but peumocyte type I appeared normal with flattened nucleus.There were numerous mast cells with purple granules (metachromatic granules) & congested capillaries with packed RBCs showing rouleaux appearance. Subgroup IIIb: (Rats sacrified 2 weeks after arrest of leflunomide treatment for 4 weeks) There were patent alveoli, numerous mast cells & congested capillaries.
Group IV (Leflunomide & N-acetyl cysteine treated group:
There were patent alveoli & apparent increase of pneumocyte type II in number.C) Mallory Trichrome Technique (M.T.)
In both group I and group II, there was minimal amount of collagen fibers (tinge of blue color) in the pulmonary interstitium, surrounding the blood vessel and around the adventitia of the bronchioles. While in
subgroup IIIa (rats sacrified 4 weeks after treatment with leflunomide), there was excess deposition of collagen fibers at the peribronchiolar and perivascular areas as well as around some alveoli forming patchy areas of pulmonary fibrosis. This explained by fibroblast activity brought to the irritated area and starts to lay down collagen fibers replacing severely damaged epithelial cells. In subgroup IIIb (rats sacrified 2 weeks after arrest of leflunomide treatment for 4 weeks), there was excess collagen in the pulmonary interstitium and the peribronchiolar & perivascular areas. But in group IV (leflunomide & N-acetyl cysteine treated group), there was minimal collagen fibers in the peribronchiolar & perivascular areas and the pulmonary interstitium. (D)Immunohistochemical study (caspse-3 immunostaining) Both groups I and II showed negative immune reaction to caspase-3 protein in the inter-alveolar septa and weak reaction in alveolar epithelial cells. While subgroup IIIa (rats sacrified 4 weeks after treatment with leflunomide) showed intense positive immune reaction to caspase-3 protein in thickened inter-alveolar septa, infiltrating cells and alveolar epithelial cells. This is due to activation of caspase 3 enzymes by leflunomide, these enzyme are involved in apoptosis. Subgroup IIIb (rats sacrified two 2 weeks after arrest of leflunomide treatment for 4 weeks) showed moderate positive immune reaction to caspase-3 protein in thickened inter-alveolar septa, infiltrating cells and alveolar epithelial cells. But group IV (leflunomide & N-acetyl cysteine treated group) showed mild positive immune reaction to caspase-3 protein in inter-alveolar septa, infiltrating cells and alveolar epithelial cells.
II. Electron Microscopic Results:
Group I (Control group):
Examination of ultrathin sections collected from control rats showed pneumoeyte type I containing large oval nucleus with peripheral condensed chromatin and thin attenuated cytoplasm. Pneumocyte type II was rounded with large rounded central vesicular nucleus with prominent nucleolus .Its cytoplasm showed mitochondria, and lamellar bodies with concentric lamellae of secretory material (surfactant). Its surface showed short apical microvilli. Alveolar macrophage appeared with kidney shaped indented nucleus. Its cytoplasm demonstrated multiple lysosomes. It had multiple pseudopodia on the surface .Also, blood capillaries were seen lined by normal flattened endothelial cell and contains blood.
Group II (N- acetyl cysteine treated group):
Examination of ultrathin sections collected from rats treated with N- acetyl cysteine for 4 weeks showed there were no pathological changes and the alveoli were seen normal similar to that in control group. The alveoli lined by normal pneumocyte type I & pneumocyte type II. Pneumocyte type I appeared with large oval nucleus & thin cytoplasm. Pneumocyte type II had large rounded nucleus & short apical microvilli. The cytoplasm of Pneumocyte type II contained numerous mitochondria, cisternae of rER and multiple lamellar bodies.
Group III (Leflunomide treated group): Subgroup IIIa: (Rats sacrified 4 weeks after treatment with leflunomide)
Examination of ultrathin sections collected from rats treated with leflunomide for four weeks showed varying degrees of pathological changes. There were degeneration of pneumocytes type I & type II. There were hyperplasia of pneumocytes type II and loss of characteristic lamellar pattern of their lamellar bodies leaving empty vacuoles. This occurred as a result of injury to pneumoctyes type II reducing their ability to synthesize, secrete and recycle surfactant. Also, swollen ballooned mitochondria demonstrated due to as a result of inhibition of dihydroorotate dehydrogenase by leflunomide.
In addition, there were massive haemorrage and congestion of alveolar blood capillaries which are filled with red blood cells and some neutrophlils & lined by irregular endothelial cell. Alveolar macrophages were also demonstrated. They showed electron dense bodies & numerous mitochondria in their cytoplasm. As macrophages are involved in the phagocytosis of apoptotic and necrotic cells that have undergone cell-death as degenerated pneumocytes. Many fibroblasts were found & appeared having irregular nucleus with peripheral chromatin and deposit excess collagen fibers. These fibers were present around fibroblast, different alveolar cells& pulmonary intersitium. Subgroup IIIb: (Rats sacrified 2 weeks after arrest of leflunomide treatment for 4 weeks)
Examination of ultrathin lung sections obtained from rats two weeks after arrest of leflunomide treatment for 4 weeks showed that no
restoration of the normal histological structure of the alveoli. Pneumocyte type I appeared flattened & surrounded by collagen fibers deposited by fibroblasts .Pneumocytes type II showed irregular lamellar bodies and disturbed microvillous border. Alveolar space showed alveolar macrpphage with its characteristic pseudopodia, congested blood capillaries (which lined by normal endothelial cell & others with abnormal one) and extravasted red blood cells.
Group IV (Leflunomide & N-acetyl cysteine treated group:
Examination of ultrathin lung sections obtained from rats treated with leflunomide & N-acetyl cysteine showed a great reduction in pathological changes demonstrated in leflunomide treated rats. Patent alveoli lined by normal flattened pneumocytes type I and pneumocytes type II. pneumocytes type II had short apical microvilil , their cytoplasm demonstrated numerous mitochondria with intact cristae and lamellar bodies with regular lamellar pattern . Blood capillaries lined by regular flattened endothelial cell & contained red blood cells. Alveolar macrophage appeared with its characteristic pseudopodia in alveolar space.
In conclusion, it was found that treatment with leflunomide resulted in considerable histological and ultrastructural changes on lung. Coadministration of N-acetyl cysteine with leflunomide was greatly reduced these changes providing a good protection of lung.
Overall, it is evident that treatment with leflunomide has numerous
hazardous effects on lung. So, strict follow up and coadministration of N-acetyl cysteine are highly recommended. Also, further studies using other protective agents are highly recommended