Abstract:

Objective To observe the pathological characteristics of hepatocyte damage, local inflammation response, and the composition of cyst fibrin in liver of Echinococcus granulosus infected sheep during the cyst formation at different stages. Methods Livers with significant E. granulosus cysts were collected from slaughtered Altai sheep in an abattoir in Urumqi City, Xinjiang, China. From the sampled livers, cysts were separated and assigned into the groups of development stage, formation stage and mature stage according to the morphological features of the cysts. The cysts of all groups and the lesion tissues at the junction with healthy liver tissues were used to prepare paraffin sections. Liver tissues from healthy sheep were used as the control. The morphological features and changes of the hepatocytes and liver tissues around the cysts were observed by hematoxylin-eosin (HE) staining, and the fibrosis process of the cyst wall at different formation stages was observed by Masson staining. Transmission electron microscope was used to observe the ultrastructural changes of hepatocytes. Immunohistochemical staining was used to observe the cysts and surrounding tissues at different cyst-formation stages for the changes of inflammatory cells CD3⁺ T lymphocytes (CD3⁺), CD25⁺ regulatory T cells (CD25⁺), CD56⁺ NK cells (CD56⁺), CD14⁺ monocyte macrophages (CD14⁺) and basophilic granulocytes CD63 (CD63), as well as the changes of expression of fibrotic proteins collagen type I (COL1), COL3, alpha-smooth muscle actin (a-SMA), calcium-binding protein A4 (S100A4), matrix metalloproteinase 2 (MMP2) and tumour necrosis factor-alpha (TNF-α), and vascular endothelial growth factor receptor-3 (VEGFR-3). One-way ANOVA analysis was used for comparison between groups, and the LSD-t test was used for pairwise comparison. Results The HE staining results showed that the liver exhibited significant inflammatory reaction bands and inflammatory cell clusters in the cyst wall during the encapsulation development stage. The inflammatory cells spread to the liver parenchyma in the periphery of the cyst, and the hepatocytes around the encapsulation atrophied, degenerated, and damaged. During the formation of the encapsulation, the cyst wall became thinner, the number of inflammatory cells decreased, the inflammatory reaction bands became thinner, the encapsulation cavity enlarged. In the maturation stage, fibrous tissue proliferation occurred in the encapsulation wall to form the cuticle, and the number of inflammatory cells decreased. The outer wall of the cyst, known as the stratum corneum, was formed by the proliferation of fibrous tissue. The number of inflammatory cells decreased, resulting in a significant reduction in the inflammatory response. Masson’s staining showed that a large amount of fibrous tissue was produced in the periphery of the cyst wall during the period of cyst development and showed extensive growth into the surrounding liver tissue. The cyst grew and developed during the period of cyst formation, the inflammatory response was attenuated, the fibrous tissue of the cyst wall matured, and a dense, fibrous cyst wall was formed during the period of cyst maturation. Transmission electron microscopy showed that as the cyst developed and formed, the mitochondria of the hepatocytes gradually enlarged and increased in number, and the lipid droplets in the hepatocytes increased in number and size. Immunohistochemical staining showed that as the cyst increases, the inflammatory response zone on the cyst wall becomes thinner, the range of positive expression of inflammatory cells decreases, and the expression level decreases. The mean optical density of the CD3⁺ inflammatory cells at the cyst developmental stage, cyst formation stage, and cyst maturation stage were 0.171 ± 0.009, 0.132 ± 0.009, 0.120 ± 0.006 (F = 1.640, P > 0.05); CD25⁺ cells were 0.302 ± 0.012, 0.174 ± 0.009, and 0.080 ± 0.005 (F = 49.051, P < 0.01); CD56⁺ cells were 0.219 ± 0.008, 0.209 ± 0.009, and 0.118 ± 0.004 (F = 126.411, P < 0.01), CD14⁺ cells were 0.140 ± 0.027, 0.096 ± 0.012, 0.090 ± 0.017 (F = 3.954, P > 0.05); CD63 cells were 0.318 ± 0.007, 0.096 ± 0.013, 0.086 ± 0.011 (F = 307.442, P < 0.01). The positive expression range and expression level of fibroin increased, and the mean optical density of COL1 were 0.139 ± 0.029, 0.157 ± 0.022, 0.186 ± 0.014 (F = 2.136, P > 0.05); COL3 were 0.109 ± 0.014， 0.144 ± 0.008， 0.206 ± 0.008 (F = 42.116, P < 0.01); α-SMA were 0.255 ± 0.008, 0.283 ± 0.009, 0.301 ± 0.022 (F = 5.106, P < 0.05); MMP2 were 0.155 ± 0.002, 0.172 ± 0.011, 0.185 ± 0.008 (F = 7.853, P < 0.05); S100A4 were 0.210 ± 0.012, 0.248 ± 0.004, 0.258 ± 0.007 (F = 18.137 3, P < 0.05). TNF-α was expressed in the surrounding tissue of the cyst, the positive expression range increased, and the expression level increased. the mean optical density of TNF-α were 0.115 ± 0.016, 0.263 ± 0.003, 0.267 ± 0.006 (F = 145.627, P < 0.01). VEGFR-3 VEGFR-3 was expressed in the cyst wall, with the highest expression during the formation of the cyst, and the mean optical density at 3 stages were 0.248 ± 0.009, 0.357 ± 0.045, 0.268 ± 0.004 (F = 9.423, P < 0.05). The mean optical density values in the cyst phase were higher than those in healthy livers (all P < 0.01). Conclusion As the cyst develops, the cyst and its surrounding liver tissue show varying degrees of hepatocellular damage, cyst wall structural changes and fibrotic response, enhanced liver mitochondrial metabolism, lipid metabolism affected, as well as the cyst-surrounding inflammatory cytokines and liver fiber seen in the inflammatory zone of cyst wall.

Key words: Echinococcus granulosus, Cyst, Pathology, Inflammatory cell, Fibrosis, Sheep