Effects Of Hypertonic Sodium Chloride Hydroxyethyl Starch Injection On Oxygen Free Radicals And Histopathological Change Of Brain Tissue In Dog Of Acute Intracranial Hypertension And Hemorrhagic Shock

Craniocerebral trauma, one of the most serious traumas in clinical, can cause a high mortality when associated with hemorrhagic shock. In hemorrhagic shock patients, application of volume therapy to maintain blood pressure may cause cerebral edema. Subsequently, a great deal of oxygen free radicals produced in ischemic-reperfusion could produce irreversible damage of brain cells. In the other hand, application of dehydrate and diuretic to treat cerebral edema and decrease intracranial pressure would reduce effective blood volume and aggravate hemorrhagic shock.Recently, an increasing attention has been paid on volume therapy and management during early stage of hemorrhagic shock associated with craniocerebral trauma. Some studies have confirmed the resuscitation effect of small capacity hypertonic saline solution and its combined solutions through animal experiment and clinical trial. Although investigators have found hypertonic saline solution can reduce ICP through animal experiment, there is no solid proof in clinical practice. But hypertonic saline, in which concentration of NaCl is less than 7.5%, is reported rarely.In this study, we designed a dog acute intracranial hypertension and hemorrhagic shock model to observe the effects of hypertonic sodium chloride hydroxyethyl starch 40 injection (HSH) on circulating blood volume, ICP and cerebral edema and oxygen free radicals. Furthermore, we evaluated the application value of HSH in cerebral protection by detecting the contents of malondialdehyde (MDA), the activity of superoxide dismutase (SOD) and the change of cerebral tissues.Materials and methods1. Animals and groupsThirty adult mongrel healthy dogs, male or female, weighing 10-20 kg, were provided by Laboratory Animal Center of Southern Medical University. All the animal were randomly divided into 6 groups, they are group HES, group RL, group HS and group HSH4, group HSH8, group HSH12. Each group consisted of 5 dogs.2. Model and resuscitationAnesthesia was induced with venous injection of 3% pentobarbital (1 ml/kg), followed by endotracheal intubation and mechanical ventilation. Adjusted respiratory parameters (RR=15bpm-30 bpm, Vt =250ml-430ml) to keep ETCO₂ changing between 32-37 mmHg. Intravenous infusion of vecuronium, Inhalation of 1% Isoflurane and oxygen maintains anesthesia. Incise right jugular vein and insert catheter (18 G) to monitor CVP. Left femoral artery incision was for monitoring arterial blood pressure. Indwell catheter after cystostomy. A catheter with balloon was placed in epidural cavity through an 10mm burr hole at the parietal bone and then fixed. Another catheter was placed in right parietal lobe through a 2-mm burr hole at the parietal bone and then connected to a piezometric catheter for monitoring ICP. Adjust zero point to parallel with left external auditory canal. Water was inject into the balloon, making the ICP 10 mmHg higher than the basic pressure and maintain the pressure. Kept MAP at near 40 mmHg by bloodletting through right femoral artery in 15 minutes and maintain for 60 minutes. Thus the model of acute intracranial hypertension and hemorrhagic shock was accomplished. Then resuscitation followed. All the dogs were resuscitated with either Ringer-Lactates solution (RL, three times as the amount of blood loss), hydroxyethyl starch (HES, equivalent to the amount of blood loss), 7.5 %NaCl (HS, 6 ml/kg), HSH (4 ml/kg, 8 ml/kg, 12 ml/kg). All kinds of liquid were infused in 20 minutes. After resuscitation for 4 hours, brain tissues of temporal lobe were taken for detecting MDA and SOD, and morphologic observation.3. Observation indexes3.1 Mean arterial pressure, cerebral perfusion pressure, intracranial pressure, central vein pressure were monitored consecutively.3.2 Data of MAP and ICP was record at baseline (T1), after making model (T2), 20 minutes after resuscitation (T3), 40 minutes after resuscitation (T4), 1 hour after resuscitation (T5), 2 hours after resuscitation (T6), 3 hours after resuscitation (T7), and 4 hours after resuscitation (T8).3.3 Detection of contents of MDA and activity of SOD: After resuscitation of 4 hours, the cranium was opened and brain tissue was removed for detecting. Both of the kits were provided by Nanjing Jiancheng Biology Engineering Institute. Contents of MDA and activity of SOD were measured based on the test kit instruction.3.4 Histopathological study: The brain tissues were removed and fixed in a 4% Formalin solution for 24 hours, desiccated by ethanol and embedded in paraffin. The histopathological changes of brain were observed by microscope according to HE dyeing.3.5 Ultrastructural examination of brain tissue: Cut the brain tissue of cerebral cortex into small pieces of 1mm³, for sample preparation and then electron microscope examination.4. Statistical analysis Results were expressed as mean±standard deviation ((x|-)±s). First, Test of homogeneity of variances was analyzed. Then one-way analysis of variance (ANONA) was used to evaluate the basic status data and contents of oxygen free radicals, and analysis of variance of repeated measure data was used for comparisons between groups. Post Hoc multiple comparisons were analyzed by using SNK test. SPSS 13.0 were used to analyze the data. Differences were accepted as statistically significant when P values were less than 0.05.Result1. In mean weight of dogs, size of water balloon, volume of blood loss, baseline of intracranial pressure, intracranial pressure after balloon inflation. There was no statistical significance among the groups (P>0.05).2. Changes of MAP and ICP after resuscitationMAP increased markedly after resuscitation in all groups(compare with MAP before resuscitation , F= 133.096, .P<0.001). There was no statistic significance among the groups (P> 0.05). But it responded most fast in group HSH and reached a peak in 20 minutes after the recovery. MAP decreased remarkably after two hours in group HS (P <0.01), but the other three groups could maintain until the end of experiment. There was interaction-effect between time and liquid(F= 2.727, P= 0.001).There was no statistical significance in ICP among groups before resuscitation (P>0.05). After resuscitation, there was statistical significance in each group(F= 133.096, P<0.001). ICP decreased significantly in group HS, group HSH4, group HSH8 and group HSH12, but increased after 2 hours in group HSH4. ICP in group HS maintained the level of less than nomal. ICP increased significantly after resuscitation in group RL and group HES.3. MDA, SOD After resuscitation for 4 hours, MDA contents of brain tissue in group HSH, group HS and group HSH was significantly lower than that in group RL, associated with increase of SOD activity(P<0.05). Significant changes were observed in group HSH8 and group HSH12, and there is statistical significance when compared with group HS, group RL and group HES.4. Histopathology of brain tissueLight microscopic findings in group HS included reduced size of cells, dark-dyeing nuclear and enlarged space around the cells. Brain tissue of group HES and group RL swelled severely and local constitution lysis appeared. HSH improves the structure of brain tissue, revealed by less edema and exudation and no necrosis was found.5. Changes of ultra-structure in palliumObserving pallium in group HES by transmission electron microscope: The integrity of some neuron was damaged, mitochondrion was swelling and vacuolization, Golgi body was swelling obviously.The integrity of several neuron was damaged in group HS. Vacuolization appeared intracytoplasm. Other changes were apparent gaps among cells, apparently edged chromatin and less mitochondrion.In group RL, neuron was swelling obviously, some of the cell envelope damaged and the cell organ disappeared. The cell nucleus and Golgi body were swelling. Mitochondrion swelled, cristae disappeared and degranulated.In group HSH, the damage became less, which mainly laid in that form of neuron and nuclei was almost normal, chromatin was mainly composed of euchromatin; swelling and vacuolization of mitochondrion became less, and Golgi body was less swelling.Conclusion 1. HSH can resuscitate hemorrhagic shock effectively.2. HSH can reduce ICP of intracranial hypertension after traumatic brain injury in dog.3. HSH can reduce oxygen free radicals levels in brain tissue and relieve tissue ischemia-reperfusion injury.4. According histopathological study and ultrastructural examination, HSH can relieve edema of brain tissue in acute intracranial hypertension and hemorrhagic shock of dog.