Monitoring Intra-hepatocellular Ca²⁺ Dynamics In Hemorrhagic Shock Rats With MEMRI

Background and Objective:Hemorrhagic shock?HS?is a form of hypovolemic shock.Rapid and severe hemorrhage in a short time can lead to a sharp decrease in effective circulating blood volume,insufficient microcirculation perfusion of important organs and tissues,and obstruction of oxygen transfusion at cellular level.If treatment is not timely,it will eventually develop into multiple organ dysfunction syndrome?MODS?or multiple organ failure?MOF?,or even death.Liver is a vulnerable target organ in the early stage of HS.Liver dysfunction will further accelerate the progress of the disease and aggravate the condition.Damage caused by various causes triggers intracellular Ca²⁺overload in many types of cells.Ca²⁺can not only act as an independent factor,but also play an important role in the damage of many other factors.Early and accurate assessment of liver function in HS is helpful to guide relevant clinical treatment,and to evaluate therapeutic effect and prognosis.Because of limitations of serological examination and conventional imaging in evaluating the severity of early hepatic dysfunction in HS,a new method is urgently needed to evaluate noninvasively hepatic function in HS in vivo at early stage.At present,there are many methods to detect intracellular Ca²⁺overload,but most of them are used in experimental research and belong to invasive examination.In addition,the degree of intracellular Ca²⁺overload in multiple organ injury can not be detected simultaneously.Magnetic resonance imaging?MRI?is widely used in clinical work and scientific research because of its superior spatial and soft tissue resolution.Manganese-enhanced magnetic resonance imaging?MEMRI?,a new technique of MRI,was first proposed by Lauterbur.It is mainly applied in the study of anatomy and function of the central nervous system and myocardial ischemia.Based on the significant biological characteristics of Mn²⁺,MRI ion tracing of Mn²⁺mimicing Ca²⁺is a new technique which is most likely to detect intracellular Ca²⁺overload in vivo at early stage.The main purpose of this study is to establish HS rat models at different time points,to obtain the changes of longitudinal relaxation rate of liver with MEMRI,and to combined with the absolute manganese content in liver tissue and pathological results,which can be used to evaluate the degree of intracellular Ca²⁺overload and liver injury comprehensively and to provide a non-invasive method and reliable imaging technique for quantitative assessment of liver function.Study Methods:Part I:Optimum MnCl₂ infusion dose and scanning delay time using MEMRI in rat liver1.Sprague Dawley?SD?male rats without specific pathogen free?SPF?were selected to prepare fresh rat serum by abdominal aorta blood sampling,which was used to prepare rat serum samples containing different Mn²⁺concentrations.2.Prepare the target concentration of 10 mmol/L MnCl₂ solution,and rat serum and saline samples containing different Mn²⁺concentration.The gradients of eight different Mn²⁺concentration in each group were 0-0.8 mmol/L in turn.3.Rat serum and saline samples were scanned by 7.0-T small animal magnetic resonance imaging instrument.The images of T1-weighted imaging?T1WI?and T1-mapping were obtained.T1 values of each sample were measured,and then relationship of Mn²⁺concentration-MRI T1 values and relationship Mn²⁺concentration-longitudinal relaxation in rat serum and saline samples were fitted.4.Analyze and compare the changes of T1WI signal intensity,Mn²⁺concentration-MRI T1 value and Mn²⁺concentration-longitudinal relaxation rate in rat serum and saline samples with different Mn²⁺concentration,and explore the interaction between Mn²⁺and protein in rat serum.5.Different doses of MnCl₂ solution were injected into SD rats.T1-mapping was performed with 7.0-T small animal MRI instrument at different time points of scanning delay,and T1 values were measured quantitatively.MnCl₂ dose-variation of longitudinal relaxation rate??R1?curve and Mn²⁺-time clearance curve in rat liver were obtained.Part II:Evaluation of intra-hepatocellular Ca²⁺alterations with MEMRI in hemorrhagic shock rats at different time points1.Hemorrhagic shock rat models at different time points were established by common carotid artery catheterization and rapid bloodletting.2.T1-mapping were performed on the upper abdomen of rats before MnCl₂ injection with 7.0-T small animal magnetic resonance imaging instrument,and the baseline T1 value of liver was measured.After MnCl₂ injection into the external jugular vein,the enhanced T1value of liver was measured again,and the variation of longitudinal relaxation rate of liver was calculated.3.Inductively coupled plasma mass spectrometry?ICP-MS?was used to measure the content of Mn²⁺in liver tissue of rats before and after MnCl₂ infusion.Liver tissues of rats were obtained immediately by perfusion and fixation after MRI scanning.liver tissues were used to measurement of Mn²⁺content and pathological examination.4.According to the pathological results,combined with the variations of the longitudinal relaxation rate of the liver??R1?and the corresponding changes of Mn²⁺content(?Mn²⁺),the intracellular Ca²⁺overload and the degree of liver injury were evaluated.Study Results:Part I:Optimum MnCl₂ infusion dose and scanning delay time using MEMRI in rat liver1.When the concentration of Mn²⁺is 0,the signal of saline is low,and the signal of rat serum is medium.With the increase of Mn²⁺concentration,the signal of T1WI in saline and rat serum increases gradually,and the signal of T1WI in rat serum rises faster.2.The T1 values of rat serum with the same concentration of Mn²⁺were lower than those of saline?P<0.001?.There were significant differences in T1 values between serum and saline samples with different Mn²⁺concentrations?all P<0.001?.With the increase of Mn²⁺concentration,T1 values gradually decreased,and there were significant differences in T1values between any two samples with different concentrations?all P<0.001?.3.Concentration of Mn²⁺was approximately linear with relaxation rate in rat serum and saline solution.With the increase of Mn²⁺concentration,the relaxation rate of the corresponding samples also increases approximately linearly.When ambient temperature was22?,the R of Mn²⁺in rat serum was 5.068 mmol^-1s^-1,which was significantly higher than that of Mn²⁺in saline(3.863 mmol^-1s^-1).4.Relaxation enhancement coefficients??^*?of rat serum samples with different concentrations of Mn²⁺were greater than 1 compared with normal saline.5.The optimum dose of MnCl₂ infusion was 10?mol/kg BW and scanning delay time was 5 minutes when the liver of SD rats was scanned by 7.0-T small animal MRI instrument.Part II:Evaluation of intra-hepatocellular Ca²⁺alterations with MEMRI in hemorrhagic shock rats at different time points1.The hepatic T1 values of hemorrhagic shock rats at different time points were measured by MEMRI and T1-mapping.There was no significant difference in the hepatic T1values before the administration of MnCl₂ solution in each group?F=0.107,P=0.99?.With hemorrhagic shock time prolonging,the color of hepatic T1-mapping imaging gradually deepened and the value of hepatic T1 gradually decreased in each group rats.There were significant differences in the value of hepatic T1 in each group?F=1156.102,P<0.001?.2.Mn²⁺content in liver of hemorrhagic shock rats at different time points was measured by ICP-MS.With the prolongation of hemorrhagic shock time,Mn²⁺content in liver gradually increased,and there were significant differences among groups?F=68.332,P<0.001?.3.With the prolongation of shock time,the variations of liver longitudinal relaxation rate and corresponding changes of Mn²⁺content in liver were increased in hemorrhagic shock rats at different time points.Pearson correlation analysis showed that there was a linear strongly positive correlation between?R1 and?Mn²⁺?r=0.968,P<0.001?.4.Pathological examinations showed that there were no obvious abnormal changes in liver tissue in control group,sham-operated group and HS-0 min group.With the prolongation of shock time,slight disorders of hepatic lobule structure,hepatic sinus occlusion and stenosis,edematous degeneration of hepatocytes,vacuolation formation and interstitial inflammatory cell infiltration appeared in different degrees in HS-30 min group,HS-60 min group and HS-90 min group,and the degree of injury was gradually aggravated.Study Conclusion:1.Mn²⁺can shorten the T1 value of rat serum and saline medium,and the concentration of Mn²⁺has a linear relationship with the longitudinal relaxation rate.Longitudinal relaxation of Mn²⁺in rat serum is much more effective than that of saline.The interaction between Mn²⁺and serum biomacromolecules?such as albumin?can shorten the time of hydrogen proton relaxation around biomacromolecules.2.The optimum dose of MnCl₂ infusion was 10?mol/kg BW and scanning delay time was 5 minutes when the liver of SD rats was scanned by 7.0-T small animal MRI instrument.3.With the prolongation of hemorrhagic shock time in rats,the T1 value of liver gradually decreases,and the Mn²⁺content gradually increases.There is a strong correlation between the variations of longitudinal relaxation rate of liver and corresponding changes of Mn²⁺content of liver.Therefore,?R1 can be used as a quantitative maker to evaluate the degree of Ca²⁺overload in hepatocytes of hemorrhagic shock.4.Combined with the results of routine imaging,ICP-MS and histopathology,MEMRI can detect the evidence of liver injury earlier than routine pathological examination.Mn²⁺mimicing Ca²⁺MRI ion tracing can detect intracellular Ca²⁺overload early in vivo.