Expetimental Study On Megakaryocytophagia And Its Mechanism In Rat With Severe Burn

The interaction between megakaryocytes and polymorphonuclear neutrophils PMNs (PMNs) has been documented in the bone marrow of dogs and rats with irradiation,severe burn or burn-blast combined injury in our previous study.The phenomenon that PMNs invading into and destructing megakaryocytes is designated megakaryocytophagia.In above injuries megakaryocytophagia is one of the causes of thrombocytopenia,which is a key problem in treating severe trauma and may lead to bleeding,infection and MODS.The mechanism of megakaryocytophagia is still not elucidated.Much more attentions.have been paid to investigate the phenomenon of PMNs and other blood cells locating in megakaryocytes.Of this phenomenon the characteristics, mechanism and significance are under disputing.Different explanations are proposed as the megakaryocytes can migrate and engulf other cells,the cells inside megakaryocytes are mistaken overlaying cells,the relationship between megakaryoeytes and cells inside is symbiotic and independent,and etc.In animal with thrombocytopenic purpura, megakaryocytes proliferated with PMNs migrating inside the cytoplasm of megakaryocytes,and platelets were observed in the peripheral PMNs or attached to the cell membrane.The phenomenon of PMNs locating inside megakaryocytes occurs more often in.Mouse transfected with TPO may develope analogous human idiomyelofibrosis, and 10%increases the number of megakaryocyte-accommodated blood cells,which are PMNs with morphological changes,accompanied by thrombocytopenia and hemorrhage. The high expression of P-selectin in megakaryocytes could cause PMNs selectively invading into megakaryocytes as observed in bone marrow samples from idiomyelofibrosis patients.The frequency increased up to as high as 16%for PMNs locating inside murine megakaryocytes with low GATA-1 expression.We have confirmed that in bone marrow from dogs with irradiation,severe burn and burn-blast combined injury megakaryocytes were destroyed by PM-Ns by light microcope, electron microscope and cytochemistry.In our viewpoint,megakaryocytophagia is the main reason for thrombocytopenia,anemia,hemorrhage and infection.To clarify the mechanism of megakaryocytosis,we adopted rat model with 30%Ⅲdegree(full thickness burn) TBSA bum to screen megakaryocytophagia in bone marrow and detected the gene expressions of transcription factor that regulate megakaryocytes proliferation/ differentiation and of growth factors that affect at the different stages of megakaryocytes maturation.NAP-2,which secreted by megakaryocytes and possessed special chemotaxis effect on PMNs is focused.The transwell assay was adopted to test immigration of bone marrow megakaryocytes and PMNs from normal and severe burnt rats.The main methods, techniques and results are shown as followings:1.Megakaryocytes at different maturing stages were observed in histogical section of bone marrow from normal rat.Megakaryocytophagia was not found.However,after burns megakaryocytes degeneration,partial cytolysis,degenerated megakaryocytes enclosed and destroyed by PMNs in a way of worm-eclipsing leaf were obvious.PMNs were confirmed located inside megakaryocytes by labeling with CD-15 and NAP-2 respectively.2.PF-4 was highly expressed in the cytoplasm and at the membrane of normal amatured megakaryocytes.The expression of PF-4 in megakaryocytes declined dramatically at 24 h after burn injury and recovered to certain extent but still weaker than normal control at 15d.NAP-2 normally expressed in both membrane and cytoplasm of megakaryocytes was suppressed in expression at 24h after burn and showed similar recovery pattern as PF-4.The expression of TPO and IL-3 decreased in megakaryocytes in 24 h after burn and did not improve at 15 d as compared with normal control.The expressions of TGF-β1 and P-selectin became weaker after burn (24h and 15d).The expression of GATA-1 in megakaryocytes increased at 24h and decreased at 15d as compared with normal control.NF-E2 was expressed in normal marrow cell,but lowly expressed after burn(24h and 15d).3.After burn the surface ofmegakaryocytes displayed irregular coral-like shapes,no more smooth and spherical shapes under scanning electron microscope.The megakaryocytes turn to be nucleus shrunk,organells reduced and focal necrosis in cytoplasm after burn by transmission electron microscope.Pseudopodia of neutrophils were seen inside the born marrow megakaryocytes,which have pyknosis and chromatin margination. 4.There were much more pseudopodia of PMNs from rat 24h after burn comparing with normal control.In ink phagocytosis assay,the rate of ink-positive PMNs in rat after burn was 64.35%,much higher than normal control(15.17%).5.Megakaryocytes and PMNs isolated from rat bone marrow of normal and after bum rats were co-incubated in Transwell system(PMNs in insert and megakaryocytes in lower chamber) in order to observe their migration abilities.Neutrophils migrated significantly towards megakaryocytes when both types of cell or only magekaryocytes isolated from burn rats were used comparing to the test with both types of cells or only megakaryocytes were from normal rats.Adding NAP-2 antibody to the insert could not inhibit the migration but calcium channel inhibitor Verapamil could.Conclusion:The cytoplasm of megakaryocyte after bums(30%TBSA,full thickness bum) degenerated partially,irregular coral-like surface,nucleus shrunk,organells reduced and focal necrosis in cytoplasm,pyknosis and.chromatin margination.PMNs were chemoattracted to the vicinity of megakaryocytes.Degenerated megakaryocytes enclosed and destroyed by PMNs in a way of worm-eclipsing leaf.The expression of megakaryocyte-specific proteins,growth factors(both positive and negative factors) and transcription factor all reduced.The chemotaxis on PMNs towards megakaryocytes was observed by transwell assay in vitro.The increased ink cytophagosis of PMNs means the cells were active after burn.The megakaryocytophagia phenomenon after burn injury is mainly caused by two factors:the function damage and ultrastructure degeneration of megakaryocytes,and the increased engulfment ability of PMNs.