The Research On Forensic Body Fluid Identification Of Hypoglycemic Brain Injury Caused By Insulin Overdose

【Background】In forensic pathology practice,it is more challenging to identify insulin overdose as the cause of death.Because of the insidious nature of insulin overdose cases and the absence of specific histopathological findings,the diagnosis of insulin overdose death is currently aided mainly by case investigation and insulin toxicological analysis tests,but the stability and feasibility of insulin detection are still insufficient due to postmortem biochemical alterations of insulin,therefore,there is an urgent need to find stable molecular markers and reliable alternative examination materials for insulin overdose forensic pathology tests.In this study,based on the previous research of our group,we initially investigated m Glu R4(glutamate receptor metabotropic 4),PEX1(peroxisomal biogenesis factor 1)and PEX5(peroxisomal biogenesis factor 5)in serum and cerebrospinal fluid as the molecular markers of insulin overdose.factor 5)as molecular markers of insulin excess in body fluids and the mechanisms by which these molecules enter serum and cerebrospinal fluid,as well as a preliminary assessment of the feasibility of insulin,C-peptide and glucose detection in serum,cerebrospinal fluid,vitreous fluid,pericardial fluid and urine.【Objectives】1.To study the patterns of m Glu R4,PEX1 and PEX5 in serum and cerebrospinal fluid of rats with hypoglycemic brain injury caused by insulin overdose and to preliminarily evaluate the potential value of the above molecules in serum and cerebrospinal fluid as molecular markers of insulin overdose in body fluids.2.To study the change pattern of brain water content in rats with hypoglycemic brain injury caused by insulin overdose and to preliminarily analyze the possible pathways of m Glu R4,PEX1 and PEX5 into serum and cerebrospinal fluid.3.To study the expression pattern of insulin,C-peptide and glucose in serum, cerebrospinal fluid,vitreous fluid,pericardial fluid and urine of non-diabetic patients who died without insulin and to preliminarily evaluate the feasibility of detecting insulin,C-peptide and glucose in the above body fluids.【Methods】1.The experimental groups were control group,acute insulin overdose group,24 hours of resuscitation group and 7 days of resuscitation group.Adult male Sprague Dawley rats were used to construct an insulin overdose induced hypoglycemic brain injury rat model by referring to Auer’s method and making some modifications to the model.2.The levels of m Glu R4,PEX1 and PEX5 in serum and cerebrospinal fluid of rats were measured by enzyme linked immunosorbent assay(ELISA),and the expression of m Glu R4,PEX1 and PEX5 in brain tissue of rats was verified by immunohistochemistry;the expression of m Glu R4,PEX1 and PEX5 in brain tissue of rats was verified by hematoxylin-eosin staining and The brain edema in rat brain was assessed by hematoxylin-eosin staining and water content detection of brain tissues in various parts,and the possible pathways of m Glu R4,PEX1 and PEX5 into serum and cerebrospinal fluid were preliminarily analyzed by immunohistochemical staining of GFAP.3.The expression of insulin,C-peptide and glucose in postmortem serum,cerebrospinal fluid,vitreous fluid,pericardial fluid and urine of non-diabetic patients who died without insulin was detected by chemiluminescence microparticle immunoassay(CMIA),and the preliminary assessment of the expression of insulin,C-peptide and glucose in serum,cerebrospinal fluid,vitreous fluid,pericardial fluid and urine.vitreous fluid,pericardial fluid and urine for the detection of insulin,C-peptide and glucose.【Results】1.mGluR4: In serum,the expression of mGluR4 was decreased in the acute group and the 24-hour recovery group compared with the control group;in cerebrospinal fluid,the expression of m Glu R4 was increased in the 24-hour recovery group and the 7-day recovery group compared with the control group.PEX1: In serum,the expression was increased in the acute and 24-hour recovery groups compared with the control group;in cerebrospinal fluid,the expression was increased in the acute,24-hour recovery and 7-day recovery groups compared with the control group.PEX5: In serum,the expression was increased in the acute and 24-hour recovery groups compared to the control group;in cerebrospinal fluid,the expression was increased in the 24-hour recovery group compared to the control group.2.Histopathological and brain water content tests confirmed that the rats in the acute and 24-hour recovery groups had significant brain edema.Immunohistochemical staining of GFAP showed that there was damage to astrocytes,characterized by damage to spines and astrocyte pedicles near small blood vessels,and similar changes were observed in the area around the third ventricle,tentatively indicating that there was damage to the blood-brain barrier in insulin-overdosed rats,and m Glu R4,PEX1 and PEX5 might be excreted into the serum and cerebrospinal fluid by this means.3.The insulin levels in the postmortem serum and pericardial fluid samples of non-diabetic deceased rats without insulin were all below the detection limit or located in the lower range of clinical serum reference values,and the insulin levels in cerebrospinal fluid and vitreous fluid were mostly below the detection limit or located near the detection limit;the mean insulin/C-peptide molar ratio in serum,cerebrospinal fluid,vitreous fluid,pericardial fluid and urine was less than 1.0;the glucose levels in all body fluids fluctuated widely.The levels of glucose in each body fluid fluctuated widely.【Conclusion】1.The present study demonstrated that the levels of mGluR4,PEX1 and PEX5 in serum and cerebrospinal fluid have potential value as diagnostic markers of insulin overload.2.Hypoglycemic brain injury leads to more severe brain edema and blood-brain barrier damage,which may be the mechanism for the changes of the above molecules in serum and cerebrospinal fluid.3.The determination of insulin,C-peptide and insulin/C-peptide ratio in postmortem serum,cerebrospinal fluid,vitreous fluid and pericardial fluid is of greater value for the diagnosis of insulin overdose cases,and the glucose concentration in each postmortem body fluid fluctuates more and has less diagnostic value.