Postnatal Changes of Lung Structure in Albino Rats after Aluminum Chloride Exposure and Possible Protective Role of Omega 3 View PDF

Aluminium is 8% of metallic element in addition, the third most common metal. Naturally it presents in environment as silicate, oxides and hydroxides as well as combined with elements as sodium and fluoride, the aluminium is flexible metal and silver-white in colour, in our bodies it is poorly absorbed and well eliminated, if absorption occurs the distribution mainly in brain, kidney, bone, testes, and liver [1]. In the industry, the aluminium powders are used in paints and pigments, and insert in fuel industry. The Aluminium oxides are used as food and the production of refractories, ceramics, electrical insulators, glass and heat resistant fibres. In medical part, the Aluminium is used in pharmaceutical and personal care product. [2]. Aluminium is added to drinking water in water purification as aluminium sulphate. In pharmaceutical industry many medications containing aluminium as topical solutions or as drugs such as aspirin, analgesic, antipyretic, and antacids. Aluminium is also found in infant formulae human and cow milk [3]. Baxter R, et al. (2008) mentioned that aluminium hydroxide is prescribed as medication to lower plasma phosphorus levels in kidney failure patients [1]. In addition, dentists use aluminium silicate as component of dental cement. Lastly the aluminium chloride enters rubber industry as lubricants, food preservatives, and cosmetics. Omega-3 are polyunsaturated fatty acids and are divided into 2 classes, Omega 3 (n-3) and Omega6 (n-6). Therefore, PUFAs is called essential fatty acids that act as energy substrates and integral membrane components. They play vital role in the preservation of nervous system [4]. In addition to ant inflammation, proresolving lipid mediators, reduction of triglycerides, antithrombotic and ant arrhythmic effects. In addition to fatty acid synthesis gene expression and up-regulate gene expression in FA oxidation [5]. The lungs represent the major part of respiratory system. It consists mainly of spongy tissue and conducting air tubules called bronchi and bronchioles. The spongy tissue takes place between air and blood are forming alveolar duct and alveoli [6]. The lung development has been divided into: saccular stage, embryonic stage, canalicular stage, pseudo glandular stage, and alveolar stage. The alveolar stage is split in two and a 6^th stage was defined as the stage of micro vascular maturation. These stages are a specific developmental milestone [7-9]. Many studies have proved that Aluminum has toxic effects on several organs. Buraimoh AA, et al. (2013) suggested that Aluminum Chloride has effects on the lung histology of Wister rats, which were eminent in the form of hemorrhage and congested blood vessels [10].

Materials

Chemicals: The chemical substances used are (1) Aluminum chloride anhydrous extra pure (Al Cl3) was obtained from Research-Lab Fine Chemicals Industries, India, through El-Goumhouria Company Medicines, in the form of silvery white crystals with a molecular weight of 133.34. Aluminum chloride powder was dissolved using water. A dose of aluminum chloride was 475mg/kg B.W. [10]. Each rat received 1 ml distilled water containing76 mg aluminum chloride orally. (2) Omega 3 was taken as soft gels tablet contain 1000 mg yellow in color made in USA the dose of Omega 3 is 20 mg per kg dissolved in corn oil orally. (3) Corn oil to dissolve 0mega 3 obtained from market.

Animals: 48 virgin female albino rats and 24 male albino rats of 160-200gm were obtained from Zagazig University. Temperature was kept at 25~30?. A cycle of twelve hours of Light and twelve hours of darkness was maintained throughout this study. All animals received food and tap water ad libtium.

Determination of pregnancy

Animals were mated overnight, after that separated, and examined in the following morning for sperm. Vaginal smears stained with Papanicolaou stain were taken every day. When smear was positive, the first day of pregnancy was considered. The females with negative vaginal smear were also isolated and re kept again with males. The process was repeated until all females became pregnant.

Classification of groups

The length of gestation for animals averaged 22days. 48 pregnant female rats were divided to 4 groups of twelve each. Pups were sacrificed at 14^th and 21th days postnatal.

Group I (negative control group): This group contained 12 pregnant rats which were not received any treatment and were given water and balanced diet by gastric intubation. The offspring of these pregnant rats are subdivided into 2 subgroups

• Subgroup Ia14PND: contains 6 mothers with their pups.
• Subgroup Ib21PND; contains 6 mothers with their pups

Group II (Positive control group): Each rat gavaged orally with Omega 3 (20 mg/kg b.w) dissolved in corn oil once daily [11]. The offspring of these pregnant rats are subdivided into 2 subgroups

• Subgroup IIa14PND: contains 6 mothers with their pups.
• Subgroup IIb21PND; contains 6 mothers with their pups

Group III (Aluminum chloride-treated group): Each rat was given orally aluminum chloride (475 mg/l (76 mg/kg BW), orally) dissolved in distilled water [12]. The offspring of these pregnant rats are subdivided into 2 subgroups

• Subgroup IIIa14PND: contains 6 mothers with their pups.
• Subgroup IIIb21PND; contains 6 mothers with their pups

Group IV (Aluminum chloride and Omega 3 group): Rat’s gavage orally with Omega 3 (20 mg/kg) and aluminum chloride (475 mg/l equivalent to 76 mg/kg BW, orally). The offspring of these pregnant rats are subdivided into 2 subgroups

• Subgroup IVa14PND: contains 6 mothers with their pups.
• Subgroup IVb21PND; contain 6 mothers with their pups.

Pups were taken and separated to be sacrificed on day 14and 21 after parturition. Pups were anesthetized to avoid stress. Histopathological examination of lung tissue was done. Samples are rinsed and homogenized (10 % ) in Tris-HCl.

Histopathological Examination

Samples fixed in Bouin’s solution, then dehydrated in alcohols, cleared in xylene and embedded in paraffin. The slides stained by hematoxylin and Eosin and Masson's Trichrome [13].

Immunohistochemical Study

Immunohistochemical reactions were carried out on sections of the lung using an antibody against ASMA marker to muscle specific protein around epithelial tube [14]. A mouse anti alpha muscle (1A4, Sigma, USA) diluted as supplied (1:800) and designed for the specific localization of ASMA in paraffin sections. They were delivered from DAKO life trade Egypt. A biotinylated secondary anti-immunoglobulin capable of binding and streptavidinbiotin enzyme complex since both primary antibody and antibody of the label are produced in the same animal species. Sections were mounted on positively charged glass slides. The slides were incubated at 37? overnight for accurate adhesion of the sections to the slides. Slides placed in 60 C of 13 to 18 h. It was transferred into three changes of fresh xylene for 10 minutes each change. Slides were transferred into graded ethanol (two changes of 100%, two changes of 95%, two changes of 70% and two changes of 50%), 3 minutes or each change. The pH (7.2) of the buffer was checked with each run. The primary antibody replaced by buffer was included for each specimen, stained with Mayer's hematoxylin and was first noted to determine the amount of specific staining seen in the examined sections.

Morphometric Study

Non- overlapping five images were randomly taken from each section. At least three randomly selected sections were analyzed per animal (3 animals from each group) on a light microscope using a colored camera. The total area and the terminal segment area were then measured in pixels using (Image analyzer, LEICA DN500). The measurements are performed on H, E, and Masson Trichrome sections. The mean values for each parameter in different groups were determined.

Statistical Analysis

The data obtained from the image analyzer were analyzed by student's t test using analysis program SPSS V.15. with a p value less than 0.05 (the level of significance). The following statistical methods were used for analysis of results of the present study [15]:

• Image analysis and morphometry was performed by ImageJ (FIJI) software. For the sections stained by Hematoxylin and eosin. The area percentage of collagen was assessed in sections stained by Masson’s Trichrome. As well as, the area percentage of α-SMA expression was assessed in sections treated with α-SMA.
• The collected data were computerized and statistically analyzed using Graph Pad Prism 5.01. Quantitative data were expressed as (Standard deviation).
• Differences between mean values of experimental groups are tested with ANOVA. Turkey's multiple test was carried out as post hoc test of ANOVA.

These Examinations are conducted for the groups.

Group I (Negative control group)-Light microscopic examination

Subgroup Ia: Age 14^th postnatal

Hematoxylin and Eosin (H&E): Examination of H&E stained sections from rat lung at 14th post-natal day revealed that the rat lung was formed (Figure 1). The wall of bronchioles consists of ciliated cuboidal epithelium (Figure 2). Primitive alveoli were lined by flattened I pneumocytes and cuboidal II pneumocytes (Figure 3).

Masson's trichrome stain: Masson’s trichrome stained is showed ordinary amount of collagen fibers in the wall of the bronchioles and bronchial vessels. In addition, the surrounding lung tissue demonstrates minimal amount (Figure 4).

α SMA Immunostaining: Immunoperoxidase technique for α SMA showed a population of round α SMA –positive cells a in addition to the slender α SMA-positive cells in primitive septa (Figure 5).

Subgroup Ib: Age 21^th day postnatal

Hematoxylin and Eosin (H&E): Examination of H & E of lung at 21^th postnatal day revealed architecture of alveoli. It was separated by apparently thin. (Figure 6). The double capillary layers in primary and secondary septa are transformed (Figure 7). The terminal bronchiole is lined by simple columnar epithelium and surrounded by thin layer of smooth muscle (Figure 8).

Masson's trichrome stain: Masson’s trichrome of 21^th DPN showing average amount of collagen fibers of the bronchi and bronchial vessels. Surrounding lung tissue demonstrates scattered amount of collagen fibers in the interalveolar septa. (Figure 8).

α SMA Immunostaining: Immunoperoxidase technique for α SMA showed a round α SMA-positive cells interstitial cells in the primary septa are disappeared (Figure  9).

Group II

Subgroup IIa: Age 14^th postnatal

Hematoxylin and Eosin (H&E): Sections of lung at 14^th postnatal day group treated with Omega 3 was formed mainly of medium sized bronchiole with the lining respiratory epithelium and bronchial vessels, air saccules, secondary septa and primitive alveoli. (Figure 10).

Masson’s Trichrome stain: Masson’s trichrome showed ordinary amount of collagen fibers of the wall of the bronchioles and bronchial vessels. In addition, the surrounding lung tissue demonstrates (Figure  11).

α SMA Immunostaining: Immunohistochemical stained sections showing prominent of α-SMA in the wall of the bronchi. In addition, α-SMA–positive cells in the primitive alveolar septa are demonstrated (Figure 12).

Subgroup II b: Age 21^th postnatal

Hematoxylin and Eosin (H&E): The respiratory portion of the lung was formed mainly of medium sized bronchiole with healthy lining respiratory epithelium and bronchial vessels, air saccules with thin interalveolar septa, secondary septa and primitive alveoli (Figure 13).

Masson’s Trichrome stain: Masson’s trichrome explain ordinary amount of collagen fibers it the wall of the bronchi and bronchial vessels. In addition, the surrounding lung tissue demonstrates non-visible collagen fiber (Figure 14).

α SMA Immunostaining: Immunohistochemical stained sections showing average of α-SMA in the wall of the bronchi. In addition, α-SMA–positive in the interstitial tissue of immature alveoli and other α-SMA–positive cells are demonstrated (Figure 15).

Group III (Aluminum chloride-treated group)

Subgroup III a: Age 14^th postnatal

Hematoxylin and eosin (H&E): Examination of H and E of lung at 14^th postnatal day showing bronchiole with sever destruction of the mucosal lining and the nearby bronchial vessel. In addition, para-bronchial inflammatory infiltration and multiple sacculations are seen in the lung tissue. Also dark stained nuclei and vacuolated cells are seen (Figure 16). Other section showing large highly congested bronchial vessel (CBV). As well as, the surrounding lung tissue shows prominent thick interalveolar septa and variably sized sacculations (Figure 17). Also bronchial vessels (BV), with sever congested, thickened walls are seen. In addition, the alveoli demonstrated high degree of sacculations, interstitial infiltration, exudation and consolidation (Figure 18). Also saccules (S), collapsed alveoli with dark stained nuclei, intra-bronchiolar hemorrhage and exudate are observed. (Figure 19).

Masson’s Trichrome stain: Masson’s showing very high content of collagen of bronchioles. The surrounding lung tissue demonstrates abundant collagen fibers in the inter-alveolar septa (Figure  20).

α SMA Immunostaining: Immunohistochemical stained sections showing abundant expression of α-SMA in the wall of the bronchi. As well as, multiple alveoli with thick interalveolar septa and sacculations displaying highly prominent expression of α-SMA are noticed in several areas of the alveolar tissue (Figure 21).

Subgroup IIIb: Age 21^th postnatal

Hematoxylin and Eosin (H&E): Examination of H and E of lung showing congested bronchial vessel and small bronchioles with intra-bronchiolar hemorrhage and exudate. The surrounding lung tissue displays multiple foci of massive intra-alveolar hemorrhage, interstitial hemorrhage and exudate (Figure 22). Other section-showing medium sized bronchiole and the nearby bronchial vessel. In-between inflammatory infiltration focus and multiple foci of interstitial infiltration are prominent (Figure 23). Sever destruction of epithelial lining (arrow heads) is seen. Other sections showing multiple foci of peri-bronchiolar inflammatory infiltration destroying the wall of the bronchiole, discrete foci of interstitial infiltration and consolidation, some air sacculations and prominent thick inter-alveolar septa are found (Figure 24).

Masson’s Trichrome stain: Masson’s showing higher content collagen fiber in wall of bronchioles, bronchial vessels and peri-bronchial infiltration. In addition, the surrounding lung tissue demonstrates abundant collagen fibers in the inter-alveolar septa (Figure  25).

α SMA Immunostaining: Immunohistochemical stained sections showing abundant expression of α-SMA in the wall of the bronchi. As well as, multiple alveoli with thick interalveolar septa and sacculations displaying highly prominent expression of α-SMA in the primitive septa and interstitial tissue (Figure 26).

Group IV (Aluminum chloride and Omega 3 group)

Subgroup IVa: Age 14^th postnatal

Hematoxylin and eosin (H&E): Examination showing multiple small bronchioles and bronchial vessel surrounded by abundant lung tissue with air saccules and primitive alveoli. However, multiple foci with interstitial infiltration, extravasations and exudation are still seen. In addition, destruction of epithelial lining is still present and alveolar surrounding is improved. (Figure 27).

Masson’s Trichrome stain: Masson’s showing average amount of collagen fibers in the wall of the bronchi and bronchial vessels. However, the surrounding lung tissue displayed abundant amount of collagen fiber more than average (Figure 28)

α SMA Immunostaining: Immunohistochemical stained sections showing ordinary expression of α-SMA in the wall of the bronchi. As well as, α-SMA–positive cells (Figure 29).

Subgroup IVb: Age 21^th postnatal

Hematoxylin and eosin (H&E): Examination of H & E showing 2 bronchioles with sloughed respiratory mucosa, congested small bronchial and parenchymal vessels. In addition, multiple air saccules and primitive alveoli with average septal thickness. However, foci of intra-alveolar infiltration are discrete. (Figure 30).

Masson’s Trichrome stain: Masson’s showing the common amount of collagen fibers in the wall of the bronchi and bronchial vessels. As well as, the surrounding lung tissue displayed average amount of collagen fibers. (Figure  31).

α SMA Immunostaining: Immunohistochemical stained sections showing ordinary expression of α-SMA in the wall of the bronchi. As well as, α-SMA–positive cells in the interstitial tissue of immature alveoli. (Figure  32).

Morphometrical Results

Omega 3 and Al Cl3 groups were compared to the control group. However, Al Cl3+Omega 3 group was compared to Al Cl3 group (Table 1).

Table 1: (14 PND) Morphometry table: Statistical comparison of different morphometric parameters in different studied groups at the age 14 PND.

Where: SD = Standard Deviation; *(highly significant); *(moderately significant); *(mildly significant); Ns: Not significant; and *p = Significant.

The results were statistically significant when P <0.05. Different stages of significance were considered. High significance (***) when P value < 0.001, Moderate significant (**) at 0.01 >P value >0.001 and low significance (*) when 0.05 >P value >0.01 (Table 2).

Table 2: (21 PND) Morphometry table: Statistical comparison between mean values of different morphometric parameters in different studied groups at the age 21PND.

The AL is the 3^rd metal in the earth [16]. That naturally presents in water (e.g. ponds, lakes and streams). So that, human is exposed to a little amount of AL from drinking water [17]. The current study was make to detect Aluminum chloride effect, beside its mechanism of action and the possible protective role of Omega 3. The rat was chosen as it is considered to be the first animal to be domesticated for strictly scientific purposes and become a species of choice because of metabolic similarities, as well as their small size and short life span. In the current study embryos were exposed to Aluminum chloride from the 14th day of pregnancy until day14 after delivery. This age corresponds to lung development which has been divided into 5 stages. The present work revealed diffuse affection of the lung tissue and loss of the normal alveolar architecture in response to aluminum administration. Riihimäki and Aitio (2012) declared that lung epithelium may be a site for the accumulation of aluminum and a surface for its uptake into lung tissues and access to the systemic circulation [6].

In the current study, the control group at age 14^th day postnatal shows air saccules and secondary septa that appear as finger like projection which divided air space to primitive alveoli this was similar to what. Bolle I, et al. (2007) and Joshi and Kotecha (2007) said [8,18].

In addition there were several characteristic features shown in the lung at this age group as respiratory epithelium, ciliated cells and Clara cells. This description was in accordance with Ross MH, et al. (2009) [19]. Also, the surrounding lung tissue show developing alveoli, secondary septa, primary septa, capillary network, flattened I pneumocytes and cuboidal II pneumocytes. This description was in accordance with Tomashefski JF, et al. (2008) [20]. These septa contain double capillary layer. This was in agreement with Prodhan P, et al. (2002), Roth-Kleiner M, et al. (2005) and Joshi & Kotecha (2007), who stated that the formation of the pulmonary alveoli as shown in the present study was initiated by developing of secondary septa from the pre- existed primary septa [21,22, and 8]. These secondary septa subdivided. All the primary and secondary septa explained a double capillary network. The interalveolar walls contain only a capillary layer.

Masson's trichrome stained sections show ordinary amount of bronchial vessels, the surrounding lung tissue demonstrates minimal amount of collagen fibers. This description was in accordance with Ahmed RR, et al. (2009) who found that minimal amount of collagen fibers was concentrated mainly around bronchus and blood vessels [23]. α-SMA immunohistochemical stained sections showed prominent expression of α-SMA in the wall of the bronchi as well as, α-SMA–positive cells and other α-SMA–positive cells are demonstrated. This description was in accordance with Ibáñez-Vea M, et al. (2018) who demonstrated the lung compartment specific expression of α-SMA positive cells [24]. Lastly α-SMA is a useful marker for smooth muscle because of its consistent appearance in bronchiolar smooth muscle. We could succeed in clarifying the early differentiation and later distribution of vascular smooth muscle cells (Mitchell et al., 1990). In the current study, the control group at age 21th day postnatal shows bronchiolar wall with the lining respiratory epithelium and bronchial vessels, air saccules, secondary septa and primitive alveoli with thin interalveolar septa. This description was in accordance with Ross and Pawlina (2009) who said that the surrounding lung tissue show alveoli lined by squamous pneumocytes I cell, cuboidal pneumocytes II cell and secondary septa with single central capillary layerz . The double capillary layers inside the primary and secondary septa are transformed to a single capillary layer, this agreed with Schittny JC and Burri PH (2007) who stated that the essence of this stage is the restructuring [9]. Masson's trichrome stained sections show average amount of collagen fibers. In addition, the surrounding lung tissue demonstrates scarce amount of collagen fibers. This description was in accordance with Prokopakis E, et al. (2013) who noticed the presence of minimal amount of collagen in alveolar wall, bronchi, bronchial vessels, the interstitial tissue of immature alveoli and the developing secondary septa [25]. This description was in accordance with Warburton D, et al. (2005) who stated that the round α-SMA-positive [26]. From our knowledge, no previous research was done on postnatal Aluminum chloride affection of rat lung. Therefore, we will compare our results with adult rat lung. In aluminum chloride exposed rats at age 14 PND (H&E) stained sections show rat lung in the saccular stage of development. The air saccules were lined largely by squamous I pneumocytes and the II pneumocytes were interspersed singly among I pneumocytes. These findings agreed with Zhuang T, et al. (2010) who reported that bronchopulmonary dysplasia (BPD) remains a major cause for morbidity [27]. Aluminum chloride exposed rats at 21 day postnatal showed diffuse affection. Alveoli were seen collapsed in many areas with thickening of the interalveolar septa. In addition, some air passages filled with vacuolated acidophilic exudate could be detected. Some alveoli containing desquamated cells and other alveoli are interconnected. Cellular proliferation especially in pneumocytes II was seen. In addition, some pneumocytes II cells were seen extruded in alveolar. Extravasations of RBCs and hemorrhage were also seen in some sections. This agreed with Wu L, et al. (2011) who reported proliferation of pneumocytes II with pale vacuolated cytoplasm [28]. Buraimoh AA, et al. (2013) as well observed blood vessels engorged with blood in aluminum subjected rats [9].

On light microscopic examination, the treated sections of the lung showed some histological changes that were at variance with those obtained in control. There was evidence of increased number of neutrophils and macrophages within the airspace, airway and blood vessels in Aluminum chloride resaved newborn pups compared with control section. Tian S, et al. (2009) stated that the present lesions in the lungs were not restricted on the vascular congestion but were associated with numerous areas of bronchopneumonia characterized by an excess of bronchiolar, peribronchiolar and alveolar infiltrations of mononuclear cells [29].

Al has been reported to cause oxidative stress of reduced glutathione [30]. Al Kahtani MA (2010) suggested that aluminum was able to induce oxidative stress in tissues which is described by EL-Dermerdash FM (2004) who stated that aluminum chloride generates reactive oxygen [31,32]. Buraimoh AA, et al. (2013) suggested that AlCl (Aluminum Chloride) detrimental effects on the histology of the lungs [9]. With an increase of malondialdehyde, hydrogen peroxide, and protein carbonyls levels [33-35]. Aluminum chloride exposed rats treated with Omega 3 at 14 and 21 days post-natal by (H&E) stain, showing a marked improvement in the histological pattern of the lun.

The results of the study revealed that aluminum chloride-induced histological and immunohistochemical abnormalities in the lung of postnatal albino rats. Omega 3 improved the changes associated with Aluminum chloride administration. This study supports the usage of Omega 3 as protective against the toxic effects of Aluminum chloride.

According to the results, we recommend a reduction in the rate of exposure to aluminum chloride as much as possible. Trying to find a substitute for aluminum in the treatment of drinking water. Reducing the rate of occupational exposure of workers to aluminum compounds in different industries. The use of O3 compounds to reduce the toxic effects caused by exposure to aluminum chloride.

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