Morphological features of "neosplenic" tissue
The first group of rats was autopsied after 25 days of the surgery, no DPSS was observed in their abdominal cavities. However, on day 30 after the operation, two out of the four rats showed two foci of DPSS each. These foci appeared as dark cherry-coloured round formations with a diameter of approximately 1.0 mm. Subsequently, in all the remaining time periods (47, 57, 63, 79, 85, 99, and 128 days after surgery), foci of post-splenectomy splenosis were found in all the experimental animals, except for one rat in the group at day 63.
The number of splenic foci ranged from 1 to 12 (M = 2.6) for every rat (Fig. 3). The sizes of the foci varied from 1.0 mm to 7×5 mm. The average length of the splenic foci was 1.7 mm.
The majority of DPSS foci were commonly located on the greater omentum (47.2%), followed by the serous membrane of the stomach (19.4%), serous membrane of the colon (16.6%), and root of the mesentery (2.7%). Other less frequent locations included the parietal peritoneum, splenic bed, skin suture area, adhesions, and surface of the ovary (ranging from 1.3–6.9%) (Fig. 4).
The histological examination of the splenic nodules (presumed DPSS foci) revealed the presence of "neosplenic" tissue with a cell composition characteristic of the spleen (Fig. 5), hemosiderin granules, reticular-elastic stroma, and a clearly defined thin fibrous capsule of the nodules. However, the typical histological architecture of the spleen was not preserved in the DPSS nodules. Underdeveloped venous sinuses were commonly observed in the majority of the foci. Most of the nodules displayed a prominent presence of white pulp components, including a high number of plasma cells. These plasma cells are known to be active B-lymphocytes that produce antibodies, suggesting potential functional activity of the tissue. All splenic nodules exhibited a diffuse type of blood supply.
Stages of development of foci of post-splenectomy splenosis in an experiment
Comparing the histological pattern of the splenic nodules at various time points following the operation, a sequential progression of changes was observed. At 30 days post-modelling of DPSS foci, the nodules exhibited a distinct thin fibrous capsule infiltrated by polymorphonuclear leukocytes and components of splenic tissue, along with a significant presence of hemosiderin granules (Fig. 5). By day 47 after the operation, there was a notable reduction in the number of hemosiderin granules, accompanied by an increase in the accumulation of lymphoid cells at different stages of maturation. By day 57 after the operation, giant cells resembling megakaryocytes were evident in nearly all preparations, highlighting that hematopoiesis in the rat spleen occurs throughout their lifespan.
From 63 days after the surgical modelling, groups of lymphoid cells began to organize into white pulp follicles around small arteries within the nodules. However, there were no more than 2–3 clusters per nodule. Subsequently, the proliferation of lymphoid cells and the development of white pulp were observed. However, the formation of red pulp was not consistently evident throughout all stages of nodule formation. In 90% of the histological samples taken at 63 days, venous sinuses were irregularly distributed and occupied a maximum of 10–15% of the tissue area.
Functional indicators of "neosplenic tissue" activity
The immune function of the spleen can be assessed using various indicators, including the phagocytic index, phagocytic number, completeness index of phagocytosis, opsonic index, and the sizes of active phagocytes. The spleen plays a crucial role in regulating phagocytosis at different stages, and these indicators provide insights into its involvement in immune response and defense mechanisms.
The phagocytic index represents the ratio of leukocytes engaged in phagocytosis to the total number of leukocytes. Reference values typically fall within the range of 75–95%. In the study, the phagocytic index, which reflects the percentage of active phagocytes, was within the normal range (median 86%) in the group of rats with splenosis after 30 days. However, after splenectomy, the indicator did not reach normal values during the same timeframe (median 68%). After 100 days following the surgical intervention, the phagocytic index in both compared groups returned to normal levels (Fig. 6).
The phagocytic number represents the ratio of ingested microorganisms to the number of leukocytes that have undergone phagocytosis, and the reference range is typically 5–10. In the group with modelled DPSS at 30 days, the average number of bacteria inside the phagocytes was normal, with 6 bacteria (± 2). However, after 30 days of splenectomy, the phagocytic number decreased to 4 (± 1). By the hundredth day, the indicators in both groups were almost equalized. Under microscopy, a noticeable difference was observed in the number of active phagocytes and their phagocytic capacity between the group after splenectomy and the group with modelled splenosis at 30 days (Fig. 6).
Following the calculation of the main phagocytic indices, the opsonic index was determined. The opsonic index represents the ratio of the phagocytic index in the experimental group to that in the control group, indicating the level of humoral attack on bacterial cells that enhances subsequent phagocytosis. Normal values for this parameter typically exceed 90%. The study revealed that all groups with DPSS and 100 days after splenectomy exhibited normal values for the opsonic index. However, in the early stages after splenectomy, the index was significantly below normal levels (Fig. 6).
The size of flattened active phagocytes provides an indirect indication of their chemotaxis degree. In the experimental groups, the highest average values were observed in rats with splenomegaly 100 days after surgery. The smallest range of sizes was observed in splenectomized animals in the early postoperative period. The sizes of rat phagocytes 30 days after splenectomy were comparable to those of non-stimulated cells, suggesting a lack of significant activation or changes in size during this period.
Morphological examination of erythrocytes in a peripheral blood smear, including the identification of degenerative forms (codocytes, spherocytes, stomatocytes) and erythrocytes with intracellular inclusions (Howell-Jolly bodies), provides insights into the filtration process within the red pulp sinuses of the spleen. According to the erythrocytogram findings, the most significant poikilocytic deviations were observed in codocytes (target-shaped erythrocytes), with occasional instances of acanthocytosis (one case after splenectomy). On the 30th day of the experiment, the number of codocytes in the group of rats without a spleen was four times higher than the normal range. By the 100th day, the average number of codocytes in the experimental groups reached 22 per 1000 erythrocytes (Fig. 7).
In both analyzed groups, starting from 30 days after surgery, the number of Howell-Jolly bodies exceeded the normal range. However, in rats after splenectomy, this indicator was more than twice as high as the other group from day 50 onwards (Fig. 7). These findings indicate the inadequate utilization of degenerative forms of erythrocytes by splenic nodules, likely due to insufficient amounts of red pulp in their composition and disrupted histological architecture.