INTERNATIONAL JOURNAL OF VETERINARY AND ANIMAL MEDICINE (ISSN:2517-7362)

Objectives: This study aimed to evaluate the histopathological impact of platelet rich plasma (PRP) in treatment of lung fibrosis induced by amiodarone drug.

Materials and Methods: The rats were divided into three groups (n=15). The first group used as control group. The rats in group 2 were injected (i.p.) daily with amiodarone drug at (80 mg/ kg. bwt) for three weeks, then injected (24 hour after last dose of amiodarone) (i.p.) with phosphate buffer saline (PBS) (0.5 ml/ kg.bwt) two times weekly for three weeks. Group 3 were injected (i.p.) daily with amiodarone drug at (80 mg/ kg. bwt) for three weeks, plus (i.p.) injection with platelet rich plasma (PRP) at dose (0.5ml/kg.bwt) (24 hour after last dose of amiodarone injection) two time for three weeks. The animals were examined during the experiment and sacrificed at 4th, 5th, and 6th week of the experiment.

Results: amiodarone resulted in significant lung tissues alteration as interstitial pneumonia and fibroblast proliferation. Group 3 revealed significant reduction in inflammatory lesion and tissues fibrosis.

Conclusions: This study concluded that platelet rich plasma has remodeling effects against damaged effect of amiodarone in albino rats.

Amiodarone; Platelet rich plasma; Lung fibrosis; Rats

Pulmonary fibrosis is a progressive and fatal lungs disease characterized by fibroblasts proliferation and deposition of extracellular matrix in lungs tissues. It consider the end stage of a wide range of lung inflammatory conditions. It initiated after collagen diseases, scleroderma [1], viral infection, rheumatoid arthritis, radiotherapy, inorganic substances as (silica, asbestos) and developed as an adverse effect of some drugs as bleomycin and amiodarone [2]. Pulmonary fibrosis is the most common interstitial lungs disease affecting over five million individuals worldwide with limited therapeutic options and mean survival time about three years [3]. It induced through different factors contributing the development and persistence of the disease including genetic factors, chronic lungs injury, aging, oxidative stress, and impaired healing process [4]. There is no effective treatment reported for pulmonary fibrosis except lung transplantation [5].

Amiodarone is one of the most common iodine-containing compound used for treatment of a wide variety of disease as cardiac arrhythmias [6], ventricular tachycardia, ventricular fibrillation [7], left ventricular dysfunction and heart failure [8]; In otherwise, it causes many adverse effects in lungs as pulmonary toxicity, chronic interstitial pneumonia, diffuse alveolar damage [9] and life-threating pulmonary fibrosis [10].

The use of biologic therapy in treatment of various diseases has increased significantly over the last ten years specifically, platelet-rich plasma (PRP). PRP is a modern treatment strategy with worldwide recognition. It was introduced in the 1950s and is currently used in many branches of medicine [11]. PRP is an autologous blood derivatives with high platelets concentration in a small volume of plasma and considered an alternative treatment for several diseases as, it is low-cost human by product. It decreases the chances of adverse effects and rejection [12]. PRP was used in cardiac surgery, pediatric surgery, gynecology, urology, plastic surgery and ophthalmology [13]. In addition to, oral, maxillofacial surgery and dermatology [14]. It also used in wound healing process as it accelerate repairing of the damaged tissues [15]. 

Drug

Amiodarone produced by Alexandria Company for Arab company for pharmaceuticals and medicinal plant (MEPACO – MEDIFOOD), Enshas El Raml, Sharkeya, Egypt. The drug was stored in dry place at temperature below 30°C. Each 3ml ampoule containing 150 mg amiodarone hydrochloride, benzyl alcohol, polysorbate 80 and water for injection [16].

Animals and experimental design

Animals: Experimental design and animal handling procedures were approved by the Research Ethical Committee of the Faculty of Veterinary Medicine, South Valley University, Qena, Egypt. Fifty five male Wistar albino rats (each weighing 180-200 gm and two months old) were purchased from the Animal House Facility of the Egyptian Organization for Biological Products and Vaccines (VACSERA), Helwan, Cairo, Egypt, and maintained in clean environment. All animals were allowed to acclimatize in plastic cages (5 animals/cage) inside a wellventilated room for one week prior to the experiment. The animals were maintained under standard conditions (temperature of 23 ± 3ºC, relative humidity of 60–70%, and a 12 hour light/dark cycle), fed a diet of standard commercial pellets, and given water ad libitum.

Experimental design: Forty five adult male Wistar albino rats were divided into 3 groups (n=15). Group (1) the rats were injected intraperitoneal (i.p.) daily with phosphate buffered saline (PBS) at 0.5 ml/rat for 6 weeks and used as a control group. Group (2) the rats were injected (i.p.) daily with amiodarone drug at (80 mg/ kg. bwt) (0.5 ml /rat) for three weeks, then injected (24 hour after last dose of amiodarone) (i.p.) with (PBS) (0.5ml/kg.bwt) twice weekly until the end of the experiment [17]. Group (3) the rats were injected (i.p.) daily with amiodarone drug at (80 mg/ kg. bwt) (0.5 ml drug/rat) for three weeks, plus (i.p.) injection with platelet rich plasma at dose (0.5ml/kg.bwt) (24 hour after last dose of amiodarone injection) twice weekly until the end of the experiment [18].

Preparation of platelet rich plasma (PRP)

Ten age-matched healthy male Wistar rats were used as PRP donors. The whole blood of rats was drawn through median eye canthus veins and mixed with 3.2% sodium citrate at a blood/ citrate ratio of 9/1, centrifuged at 1000 r.p.m for 10 minutes at room temperature and the supernatant was separated and centrifuged again at 800 r.p.m for 10 minutes at room temperature then the above two third was discard and the remaining third (sediment) was used as PRP. The average platelet in PRP was evaluated using a Sysmex XT-1600i system. The platelet count was 800×103 platelets/μL [19].

Tissues samples and histopathological examination

All animals in all groups were examined daily along the experiment and clinical signs and mortalities were recorded. The rats in all groups were sacrificed at 4th ,5th and 6th week from the beginning of the experiment. Lungs were collected from all sacrificed animals and fixed directly in 10% neutral buffered formalin for histopathology examinations. Sections about 5µm thickness were prepared and stained with Harries hematoxylin and eosin stain for histopathological examination [20] and Masson’s trichrome stain for detection of collagen fiber in lungs tissues [21].

Histological and histochemical results

Hematoxylin and eosin stain

Group (1) (control group): Examination of lung sections of control albino rats revealed normal typical histological architecture (Figure 1a).

Group (2) (amiodarone treated group): Lungs at 4th week of the experiment revealed desquamated epithelium and cell debris inside bronchiole lumen with peribronchiolar mononuclear inflammatory cells infiltration (Figure 1b) alveoli showed emphysematous parts and disruption in their wall. Figure (1c, d and e) showed lungs at 5th and 6th week of the experiment massive fibrous tissue proliferation scattered in lung tissue, in addition to, periartirial fibrillation in pulmonary vasculature.

Group (3) (platelet rich plasma treated group): After 4th week of (PRP) injection lungs sections showed interalveolar septa thickening with fibrous tissue proliferation in between bronchioles (Figure 2a). Partial regaining of the normal lung structure, patent alveoli, alveolar sacs, and thin interalveolar septa but with mildly congested blood capillaries and less inflammatory cells infiltration was found. Bronchioles appeared with patent lumen and mildly thickened surrounding layer with less peribronchiolar cellular infiltrations were seen but some bronchioles revealed intraluminal desquamated epithelial cells at 5th and 6th week of the experiment respectively (Figure 2b,c).

Masson’s trichrome stain

Minimal collagen fibers were found around bronchioles, and blood vessels of lung sections of control rats (Figure 3a). However, groups 2 showed extensive collagen fibers deposition appeared blue in color in the interalveolar septa, peribronchial and around blood vessels (Figure 3b,c). After PRP injection, moderate to mild collagen fibers deposition was observed (Figure 3 d,e).

Amiodarone is an iodinated class III antiarrhythmic drug widely used in treatment of many forms of life-threatening cardiac arrhythmia and sudden cardiac death [22]. However, its use related to many side effects involving different organs as lungs causing pulmonary complications [23]. Lung fibrosis is a lethal pathological process worldwide and has limited therapeutic options [24]. Platelet rich plasma widely used nowadays in various medicinal fields as bone defects [25] and tissue regeneration [26]. As it contains growth factors it plays role in angiogenesis and tissue regeneration [27].

In our work, amiodarone administrated group showed obliteration of the lumen of bronchioles, detachment of the bronchiolar lining epithelium this result was agreed with [28] who referred that to direct toxic effect of amiodarone on the bronchioles mucosa and injury of bronchiolar epithelium as well as its inflammatory reactions. The highly significant increase in collagen fibers percent reported by [29,30] that found increased collagen deposition in amiodarone injected rats was attributed to amiodarone immune and inflammatory mechanisms that lead to fibroblasts stimulation and collagen deposition.

Other others suggested that to amiodarone oxidative stress contributes overexpression of fibrogenic cytokines such as connective tissue growth factor these factors stimulate the differentiation and proliferation of fibroblasts from epithelial and endothelial cells with increased synthesis and deposition of collagen [31].

After PRP treatment (group 3), there was moderate improvement with return of the normal lung structure. Thin interalveolar septa with few inflammatory cellular infiltrations were observed with a highly significant decrease in collagen fibers in comparison with amiodarone-injected group. These results could be explained by the ability of PRP to secrete many cytokines and growth factors that have essential role in tissue regeneration, healing, reducing inflammation and fibrosis reduction (Villela and Santos 2010).

It could be concluded that amiodarone has adverse effects on lung tissue. Platelet rich plasma proved moderate improvement in lung injury and decrease pulmonary fibrosis caused by amiodarone drug and further biochemical and immunohistochimical studies should be evaluated.

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