| Platelets are stored at 22 degrees C, and are strictly limited to 5 days in view of storage lesion and the risk of bacterial contamination. The harsh storage conditions and short shelf life have caused platelets in short supply for a long time, at the same time it was also not conducive to the timely supply of platelet products to remote areas, disaster site and military conflict areas. Cryopreserved platelets(CPPs) were valid for up to 2 years, had smaller risk of bacterial contamination, so can ease the problem of insufficient platelet supply to a large extent. Although the in vivo recovery rate of CPPs after infusion was about 50% of fresh platelets, the function improving platelet count was weak, a large number of basic and clinical studies have indicated that CPPs have significant immediate hemostasis function. Therefore, CPPs are more suitable for hemostasis therapy after coagulation dysfunction due to trauma hemorrhage and surgical bleeding.CPPs have a broad application prospect in the treatment of trauma hemorrhagic shock because of its outstanding advantages. At present, the Dutch and American military have implemented and deployed a deep frozen(-80°C) inventory of CPPs, the Paul Ehrlich Institute in Germany has also given their approval for the use of CPPs in military operations. However, CPPs has not been approved by the competent authority of other country or regions as a formal blood product with the exceptions of France, CPPs were used only for autologous platelet transfusion treatment and in primary hospitals in our country. The main reasons were that adverse reaction monitoring after CPPs infusion has not been complete and the risk assessment of clinical application were insufficient.As we all know, in addition to the function of hemostasis, platelets also mediate inflammation and immune regulation. Clinical studies have shown that platelet transfusion is more likely to cause transfusion related acute lung injury(TRALI) than other blood components(such as red blood cells and plasma), at the same time, platelets playe an important role in ischemia reperfusion injury(IRI) of liver, lung and other tissues. In the earlier period of liver ischemia, platelets and kupffer cell adhesion, quickly gathered in the liver tissue, and through the activation of Kupffer cells to promote the release of lysosomal enzymes, induced liver sinusoids endothelial cell apoptosis, mediated neutrophil adherence and aggregation, aggravate liver injury. The CPPs infusion may also cause tissue inflammatory injury, especially after a massive loss of blood in a two-hit theory.The effects of CPPs on the tissue inflammatory injury have not been reported. The lack of safety data seriously restricted the popularization and application of CPPs. BALB/C mouse have characteristics of strong reproductive ability, sensitive to external stimuli, suitable for the study of immunology and molecular mechanism. This study aims to establish hemorrhagic shock model with BALB/C mouse, simulate clinical conditions, carry out cryopreserved platelets transfusion experiments, comprehensive evaluate tissue inflammatory injury after hemorrhagic shock and cryopreserved platelets infusion through serum liver and renal function indexes, tissue inflammatory factors, immunohistochemistry and pathological analysis. Provide reference data for the adverse reaction and its prevention and treatment after cryopreserved platelet transfusion. PartⅠEstablishment of extraction and storage methods of mouse plateletsIt is a prerequisite to establish a stable mouse platelet extraction and storage methods for carrying out the experiment of CPPs infusion and investigate the influence of CPPs to tissue inflammatory injury with hemorrhagic shock. Mouse blood was collected by cardiac puncture under ventilator supplies conditions, anticoagulated with 14% citrate phosphate dextrose adenine – 1(CPDA-1). Then filtered with x30 filter, Centrifuged at 300 g for 8 minutes to obtain upper rich platelet plasma(PRP). PRP were storaged at 22 degrees or-80 degrees respectively according to the conventional method and the Dutch military method. According to the characteristics of mouse platelets in vivo and in vitro, Platelets stored at 22 degrees less than 1 hour were defined as fresh platelets(FPs), Platelets stored at 22 degrees for 16 hours were defined as liquidpreserved platelets(LPPs), Platelets stored at-80 degrees for 48 hours were defined as CPPs. Evaluate the quality changes of platelets in the process of extraction and storage by means of blood cell count, blood gas analysis and thrombelastograph.Results show: The success rate of collectting mouse blood under ventilator supplies conditions by cardiac puncture was 93.6%, 1 ml of blood was obtained per mouse average. 14%CPDA-1 have good anticoagulant effects, there was no coagulation phenomenon. The filtering rate of white blood cells(WBC) in mice whole blood was up to(85.15 + 6.06) %, and had no effect on red blood cell(RBC) and platelet(PLT) count. After centrifugation, the overall residual rates of RBC and WBC were(5.14 + 2.78) % and(0.15 + 0.05) %, the PLT extraction rate was(79.42 + 14.92) %, and the count value was(479.75 + 61.22) ×109/L. That can meet the requirement of storage, infusion test. Compared with fresh platelets, the 16-hour-old liquid-preserved platelets have no significant difference in RBC, WBC, PLT and the average volume of platelet(MPV), did not appear cell aggregation or rupture phenomenon; have no significant difference in R, MA and Angle of the thrombus elastic figure detection, blood coagulation state was normal. p H, base excess(BE), bicarbonate concentration(c HCO3-) decreased, blood lactate concentration(c Lac) increased, consistented with the process of energy consumption, accumulation of lactic acid, p H decline trend in platelet storage, capable of representing liquid-preserved platelets metabolic characteristics. The 48-hour-old Cryopreserved platelets have no significant difference in PLT; WBC, RBC and MPV were significantly elevated, which may be related to the small amount of WBC and RBC cells in the plasma, and the intracellular volume expansion effect of DMSO on the platelets; had no significant changes in p H, BE, c HCO3-, platelet metabolic activity was inhibited in low temperature environment; R, MA and Angle of the thrombus elastic figure detection significantly decreased, procoagulant activity increased, decreased blood clot strength. Indicating that the activity of coagulation was increased and the blood clot strength reduced.Summary: We have established a stable method for the extraction and storage of mouse platelets. The 16-hour-old liquid-preserved platelets and the 48-hour-old Cryopreserved platelets can represent two ways of storaged platelets in blood cell count, blood gas analysis and blood coagulation function and meet the next infusion experiment requirements. PartⅡ Study of Cryopreserved platelets transfusion on tissue inflammatory injury in mice with hemorrhagic shockThe hemodynamic changes of 30% hemorrhagic shock mouse models are close to the clinical, which are suitable for the study on metabolism, pathophysiology, survival and treatment strategies of hemorrhagic shock. 30% hemorrhagic shock mouse models were established and divided into three groups: Fresh group, Old group and Frozen group. After one hour, the mice were infused with FPs, LPPs or CPPs the same amount of blood loss, detect the serum liver and renal function before and 6 hours after infusion, observe the pathological changes of liver, lung and kidney 6 hours after infusion to investigate the effect of three kinds of platelet transfusion on tissue inflammatory injury of hemorrhagic shock in mice.Results show: Before platelet transfusion, three groups of Hemorrhagic shock mice have not significant difference in alanine amino transferase(ALT), aspartate amino shift enzyme(AST), blood urea nitrogen(BUN) and serum creatinine(CREA) content. After 6 hours of platelet transfusion, the levels of ALT and AST in the three groups were significantly increased, and the contents of CREA and BUN were significantly decreased. The content of ALT and AST in Frozen group was significantly higher than that in Old group, There was no significant difference in the content of BUN and CREA between the three groups.After 6 hours of platelet transfusion, liver tissue pathological analysis showed that: In the Fresh group, a small amount of inflammatory cell infiltration was mainly scattered in the sinus gap; In the Old group, the inflammatory cells were scattered in the gap, the local hepatic tissue was dilated, and the isolated inflammatory lesions appeared; In the Frozen group, the inflammatory cells were scattered in the gap, the local hepatic tissue was dilated, the isolated inflammatory lesion or the focal necrosis of the liver cells appeared. The pathology score difference of liver between three groups is remarkable, tissue inflammatory injury of liver in Frozen group is the most serious. Lung tissue pathological analysis showed that: In the Fresh group, the structure was clear, no obvious change; In the Old group, a small amount of inflammatory cell infiltration were seen in interstitial lung tissue, and the local alveolar septum was slightly thickened; In the Frozen group, a small amount of inflammatory cell especially neutrophils infiltration were seen in Interstitial lung tissue, the local alveolar septum was slightly thickened, the alveolar lumen was slightly reduced. The pathology score difference of lung between three groups is remarkable, tissue inflammatory injury of lung in Frozen group is the most serious. Renal tissue pathological analysis showed that: In the Fresh group, the structure was clear, no obvious change; In the Old and Frozen group, a small amount of inflammatory cell infiltration were seen in local interstitial kidney tissue, lymphocytes and mononuclears being prevailed. There was no significant difference of the pathology score between the three groups.Summary: Compared with FPs and LPPs, CPPs can aggravate the inflammatory injury of liver and lung in mice with hemorrhagic shock; the effect of three kinds of platelets on renal injury was not obvious. The sensitivity of different tissues to the inflammatory injury of platelets was different. Part Ⅲ Study on the mechanism of Cryopreserved platelets transfusion aggravatting liver inflammatory injury of hemorrhagic shock miceDetect the expression of inflammatory cytokines and chemokines(TNF-α、IL-1β、IL-6、MIP-2) and the content of MPO in liver homogenate, combined with the results of immunohistochemical staining of macrophage specific antibody F4/80 in paraffin sections of liver tissues, investigate the effect of CPPs on inflammatory injury of liver tissue and its possible mechanism.Results show: The number of F4/80 positive cells in Frozen group was significantly higher than that in Fresh group and Old group, the number of F4/80 positive cells in Old group was also higher than that in Fresh group; The content of TNF-α, IL-1, IL-6 and MIP-2 in Frozen group was higher than that in Fresh group, the content of TNF-a in Frozen group was higher than that in Old group, the content of IL-6 in Old group was higher than that in Fresh group. The number and activity of kupffer cell in Frozen and Old group is elevated than that in Fresh group, Frozen group is the highest. The content of MPO in Frozen group was significantly higher than that in Fresh and Old group, there was no significant difference in MPO content between Fresh and Old groups. Compared with Fresh and Old group, there are more neutrophils adhesion and aggregation in the liver of Frozen group.Summary: Compared with FPs and LPPs, CPPs promoted the aggregation and activation of KC and Neutrophils in the liver, the mechanism of hepatic injury aggravated by CPPs in hemorrhagic shock mouse may be related to the aggregation and activation of KC and Neutrophils. PartⅣ Study on the mechanism of Cryopreserved platelets transfusion aggravatting lung inflammatory injury of hemorrhagic shock miceDetect the expression of inflammatory cytokines and chemokines(TNF-α、IL-1β、IL-6、MIP-2) and the content of MPO in lung homogenate, combined with the results of immunohistochemical staining of macrophage specific antibody F4/80 and Neutrophil specific antibody Ly-6G in paraffin sections of lung tissues, investigate the effect of CPPs on inflammatory injury of lung tissue and its possible mechanism.Results show: The number of Ly-6G positive cells in Frozen group was significantly higher than that in Fresh group and Old group, the number of Ly-6G positive cells in Old group was also higher than that in Fresh group; The content of MPO in Frozen group was significantly higher than that in Old group and Fresh group, there was no difference between Old and Fresh groups. These results indicate that: Frozen and Old group have more number of neutrophils than Fresh group, Frozen group has the most number of neutrophils, with high degree of maturity and strong activity. Frozen and Old group have more number of F4/80 positive cells than Fresh group, but there were no differences in Frozen and Old groups. The content of TNF-α, IL-1 and IL-6 in Frozen group were significantly lower than those in the other two groups, The content of IL-1b in Old group was higher than that in Fresh group. These results indicate that: Fresh and Old group have more number of macrophages, but the activity of macrophages in Frozen group was lower.Summary: Compared with FPs and LPPs, CPPs promoted the infiltration of neutrophils in the lung, and the content of neutrophil infiltration was consistent with the degree of lung pathological injury, suggesting that lung injury induced by CPPs may be related to neutrophil infiltration. Compared with FPs, LPPs and CPPs promoted the aggregation of macrophages in the lung.To sum up, this study established a technology platform for the extraction and storage of mouse platelets, investigated the effects of three different kinds of platelets(FPs, LPPs and CPPs) on tissue inflammatory injury of hemorrhagic shock in mice. Results show:(1) Compared with FPs and LPPs, CPPs can aggravate the inflammatory injury of liver and lung in mice with hemorrhagic shock; the effects of three kinds of platelets on renal injury was not obvious. The sensitivity of different tissues to the inflammatory injury of platelets was different.(2) There may be differences in the mechanisms of the effects of CPPs on the liver and lung inflammatory injury. Neutrophils and macrophages are all important factors to tissue inflammatory injury. It was found that the infiltration of neutrophils in the lung and the aggregation of macrophages in the liver may respectively be the key factor to their inflammatory injury after CPPs infusion. So the corresponding preventing measures should be made. |
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