Iraqi Journal of Veterinary Sciences

Introduction

After cytokine stimulation of the hypothalamus-pituitary-adrenal, corticosteroids are naturally formed in the adrenal cortex of vertebrates (1,2). It has an essential role in controlling various biological processes involving immune responses, metabolism, reproduction, cell development, and proliferation (3,4). Prednisolone is a synthetic steroid named 11,17,21 trihydroxypregna-1,4-diene-3,20-dione that is chemically clarified. It is used to treat many inflammatory diseases, for example, rheumatoid arthritis, systemic lupus erythematosus, and many other inflammatory diseases (5). The corticosteroid action connects the intracellular glucocorticoid receptor, which is then transferred to the nucleus where the receptor-ligand complex binds to specific glucocorticoid-response elements on DNA, thus activating genes that mediate glucocorticoid responses (6,7). These have potent anti-inflammatory effects and reduce the causes of swelling in joints and other organs and the pain (8). Cyclophosphamide is the most widely used to remedy cancer, autoimmune disease, marrow, and blood transplantation (9,10). It is a medication that has a broad extent of clinical applications, especially in autoimmune disorders such as rheumatoid arthritis and vasculitis, and cancer treatment (11). It is an alkylating nitrogen agent composed of C₇H₁₅C_l2N₂O₂P (6). The target organs are the causes of different side effects on the gastrointestinal, liver, lungs, heart, urinary bladder, and reproductive system (12). Moreover, Chemotherapy alters the fecal microbiota in patients, reducing species richness and total bacterial capacity (13). Some studies dealt with the use of different concentrations of cyclophosphamide for different periods to know its toxic effects on cells and identify its harmful effect on different tissues. Bhat et al. (14) found many histological changes in the liver when rats were dosed for 7 and 28 days with low and high concentrations of cyclophosphamide.

Materials and methods

 Healthy male Swiss albino mice were used for the study. Seventy-five adult male albino mice (Mus musculus) with an average weight of 25±2 g and ages between 8-12 weeks. Animals were housed in polycarbonate boxes and maintained under standard laboratory conditions in the animal house of the Department of Biology, Mustansiriyah University. Kept for adaptation for ten days before starting the experimental under the controlled temperature conditions of 25°C and 12 hrs light /12 hrs dark cycle. The animals were provided with pellets and tap water for feeding and drinking. 

Ethical approve

Informed consent according to the Declaration of Helsinki was obtained from the ethics committee of the College of Science, Mustansiriyah University Ref. No. BCSMU/0721/0001.

Study groups

The animals were divided into three groups of 25 animals at random. The G1 group, which considers as control, received orally distilled water only for 30 days. According to Shafi (15) G2 group which orally given 0.1mg/Kg of Prednisolone 20 mg (Julphar Company, Gulf pharmaceutical industries, Ras AL Khaimah, UAE) administration for 30 days while the G3 group which orally given 0.1 mg/Kg of Cyclophosphamide 50 mg, Baxter, mfg. Lic. No.186) administration for 30 days. After 24 hours of the last administration, the mice were sacrificed by chloroform for several minutes. The organs were removed, and some small specimens were taken for histological techniques.

Histological examination and grading

In 10% formalin solution, the liver, kidneys, and small intestinal tissues were preserved for 24 hrs and washed with 70% ethanol, tissues were then dehydrated using 70% to 100% alcohol series and embedded in paraffin. Blocks of paraffin were cut at 5 µm using a rotating microtome, spread on glass slides, and then dried. Slides were observed under a light microscope following staining with hematoxylin and eosin stain and mounted with mount media according to Suvaran et al. (16), and a percentage scale determined histopathological grading.

Statistical analysis

The Chi-square test values of P

Results 

Effect of Prednisolone and Cyclophosphamide on the liver tissue

Microscopic finding of the liver was summarized in (Table 1). Normal morphological findings were seen in the control of all the liver tissues except 8% showed degeneration in epithelium cells (Figure 1). Salient microscopical findings in this study, as observed in (Figure 2-7) statistical analysis, were an increase in grade 1 of G2 in all categories. On the other hand, statistical analysis was an increase in grade 1 of G3 were necrosis, cellular inflammation, and vascular congestion and hemorrhage, while increased in grade 2 were fibrosis, degeneration in epithelium cells, when treated mice with 0.1 mg/kg body weight of cyclophosphamide and prednisolone for 30 days.

Table 1: Histopathological grading of liver tissues in prednisolone, cyclophosphamide, and control groups

P-value: P>0.05 between control and tested groups, each group total number=25 mice

Figure 1: Crosse section in the normal mice liver architecture shows appearance of hepatocytes (H), a branch of portal vein (BPV), branch of the hepatic artery (BHA), and a branch of the bile duct (BBD) (H&E, X10).

Figure 2:Cross section in the liver of the treated mice shows the necrotic zone (N) in the liver lobe (H &E, X10).

Figure 3:Cross section in the liver of the treated mice shows periportal and septal fibrosis (F) with the aggregate of inflammatory cells (H &E, X10).

Figure 4:Cross section in the liver of the treated mice shows the aggregate of inflammatory cells (IC) (H &E, X10).

Figure 5:Cross section in the liver of the treated mice shows hydropic degeneration (HD) in hepatocytes (H &E, X10).

Figure 6:Cross section in the liver of the treated mice shows congestion of blood vessels (C) (H &E, X4).

Figure 7:Cross section in the liver of the treated mice shows haemorrhage in sinusoids (red colour) (H &E, X10).

Effect of prednisolone and cyclophosphamide on the kidneys tissue

Microscopic examination of the kidney tissue revealed the glomeruli and renal tubules (Figure 8). From table 2 and figure 9-13, it is found that kidney damage significantly occurs in treated groups compared to the control group. Our study found that treated mice with 0.1 mg/kg body weight of cyclophosphamide and prednisolone for 30 days statistical analysis of G2 and G3 were an increase in grade 1 of all categories.

Table 2: Histopathological grading of kidney tissues in prednisolone, cyclophosphamide, and control groups

P-value: P>0.05 between control and tested groups, each group total number=25 mice

Figure 8: Cross section in the normal mice kidney architecture shows appearance of therenal corpuscles (glomerulus and Bowman’s space), PCT = proximal convoluted tubule, and DCT = distal convoluted tubule (H&E, X4).

Figure 9:Cross section in the kidney of the treated mice shows necrosis epithelial cells and hemorrhage between convoluted tubules (H &E, X10).

Figure 10:Cross section in the kidney of the treated mice shows fibrin (F) deposition between tubules (H &E, X10).

Figure 11:Cross section in the kidney of the treated mice shows inflammatory cells infiltration around Bowman's capsule (IC) (H &E, X10).

Figure 12:Cross section in the kidney of the treated mice shows hydropic degeneration (HD) in epithelial cells of the collecting tubule (H &E, X40).

Figure 13:Cross section in the kidney of the treated mice shows congestion of blood vessel (C) and haemorrhage (H) in the interstitial space between the tubules (H &E, X10).

Effect of prednisolone and cyclophosphamide on the small intestinal tissue

Gross examination of the small intestine obtained from the control group revealed normal appearance in all sections (Figure 14). Our study observed histological alterations in small intestinal tissue between the prednisolone, cyclophosphamide, and controls group in table 3 and figure 15-18 statistical analysis were an increase in grade 1 of G2 in all category. On the other hand, statistical analysis was an increase in grade 1 of G3 were necrosis, cellular inflammation, degeneration in epithelium cells, and vascular congestion and hemorrhage, while fibrosis increased in grade 2 when treated mice with 0.1 mg/kg body weight of cyclophosphamide and prednisolone for 30 days.

Table 3: Histopathological grading of small intestine tissues in prednisolone, cyclophosphamide, and control groups

P-value: P>0.05 between control and tested groups, each group total number=25 mice

Figure 14: Crosse section in the normal small intestine mice architecture appearance normal villi (V) with epithelial cells of the intestinal mucosa (H&E, X10).

Figure 15:Cross section in the small intestine of the treated mice shows fibrin deposition (F) in the mucosa and increased fibers of the submucosa (FS) (H &E, X40).

Figure 16:Cross section in the small intestine of the treated mice shows inflammatory cells infiltration in villi (IC) (H &E, X10).

Figure 17:Cross section in the small intestine of the treated mice shows haemorrhage in villi (arrows) (H &E, X10).