Study On The Early Effect Of Normal Saline, Hypertonic Saline Or Hydroxyethyl Starch Resuscitation With Hemorrhagic Shock On The Treg, Th1/Th2 And Tc1/Tc2

ObjectiveTo evaluate the early effect of normal saline (NS), hypertonic saline (HTS) or hydroxyethyl starch (HES) resuscitation with hemorrhagic shock on the body immune system, we established rat model of hemorrhagic shock, resuscitated with normal saline, hypertonic saline or hydroxyethyl starch respectively and then detected the early variation (about 2.5 hours after resuscitation) of Treg, Th1/Th2 and Tc1/Tc2 in peripheral blood and spleen by flow cytometry.MethodsThe rat models of hemorrhagic shock were induced by controlled bleed through carotid artery. According to the emergency treatment process, we divided the experimental process to four successive stages:bleeding stage, early fluids resuscitation stage, blood transfusion stage and observation stage. Except sham and control group, NS, HTS and HES group were designed according to the fluids used in early fluids resuscitation stage. Each group has 7 male rats. The rats in control group only accept anesthesia, and the rats in sham group only accept anesthesia, carotid artery and femoral vein cannulation, and heparinization, with no other operation like bleed and resuscitation. The time from anesthesia to the end of the experimental was same in each group. All the rats were sacrificed in the end by draw off blood through carotid artery except the rats in sham group, they were sacrificed by bleed through cardiac puncture. The peripheral blood monouclear cells and spleen cell suspension were separated in sterile environments. The number of (CD4+CD25+Foxp3+) Treg were detected by three colour flow cytometry. The peripheral blood sample and spleen cell suspension were cultured respectively in RPMI1640 which contained PMA, Ionomycin, Monensin and fetal bovine serum for 4 hours in 37℃and 5% CO2. After then the variation of Th1/Th2 and Tc1/Tc2 in PBMC and spleen cell suspension were detected by three colour flow cytometry.Results1. The establishing of rats modelsThe rat hemorrhagic shock models resuscitation with NS, HTS and HES were established successfully. All three fluids have immediately treatment effect, increased mean arterial blood pressure rapidly, and all could maintain the MAP to the end of observation stage.2. The results of Treg in peripheral blood and spleen of different groupsFor peripheral blood, compared to control group, the number of Treg in sham group were increased and this showed statistically significant difference (P=0.031). The number of Treg in NS, HTS and HES group were all bellowed the control and sham group, all showed statistically significant difference (for control group: P=0.023,0.02,0.032; for sham group:P<0.001, respectively) For spleen, the numbers in control, sham and HTS group were almost in the same line with no statistically significant differences (P> 0.05). The numbers in NS and HES groups were all bellowed control and sham groups, but only have statistically significant differences when compared to sham group (P=0.032,0.028, respectively).3. The results of Thl/Th2 and Tcl/Tc2 in in peripheral blood and spleen of different groupsFor Thl and Th2 cells in peripheral blood, the Thl/Th2 in sham group had a little more than control but showed no statistically significant difference (P>0.05). The Th1/Th2 in NS, HTS and HES all lower than control and sham, but only had statistically significant differences with sham (P=0.012,0.025,0.008, respectively).For Tc1and Tc2 cells in peripheral blood, only the Th1/Th2 in HTS group had a increased tendency, however, this had no statistical significance compared to other groups (P>0.05). The differences in other four groups were small and no statistical significances (P>0.05) too.For Thl and Th2 cells in spleen, the Th1/Th2 in sham was higher than control with statistical significance (P=0.016). Compared to the control, the Th1/Th2 in HTS increased significantly and showed statistical significance (P<0.001); the Th1/Th2 in HES also showed a increased tendency but had statistically significant difference (P=0.047), no differences was observed in NS group (P>0.05). Compared to the sham, the ratio in NS group decreased significantly (P=0.012). Although the ratio in HTS group was higher than sham, this had no statistical significance (P>0.05). No differences was observed between sham and HES group (P>0.05) too.For Tc1 and Tc2 cells in spleen, the Tc1/Tc2 in sham was higher than the control but no significances (P>0.05). Compared to the control and sham, the Tc1/Tc2 in HTS increased significances (P<0.001, P=0.002, respectively), no differences were observed between NS, HES and control, sham (P>0.05).ConclusionCompared to NS or HES resuscitation, HTS could impact the spleen immune system faster and significantly, such as increased the number of Treg cells and enhanced the ability of special lymphocyte transform to Thl or Tcl cells respectively in spleen. Further study should be performed to research the mechanisms and the effect on the body.