The Effect And Mechanism Of Spleen-derived Inflammatory Response On Rats With Severe Traumatic Brain Injury

Backgroud and ObjectiveTraumatic brain injury (TBI) is the major cause of death of adolescents and young adults over the world, and has a significant impact on the quality of life afterwards. TBI Patients with Glasgow Coma Scale(GCS)≤8 are recognized as severe TBI, showing a high mortality rate of 20-50%. Athough there comes highly improved medical facilities, examing technology, and much more mature guidline for clinical treatment, the outcome of severe TBI hasn't significantly changed over the past several decades. It's very significant for us to explore effective therapeutics to improve the survival rate and quality of life for those with severe TBI.Besides the severity of primary head injury, second brain injury is a key factor for the prognosis of TBI patients. Blood Brain Barrier (BBB) bankrupt, polymorphonuclear neutrophils (PMNs) infiltrating to the injured site, local immune cells activation including microglia and astrocytes, systemic and local proinflammatory chemokines and cytokines involvement and so on are all accounting for the secondary brain injury, which easily lead to the fatal brain edema. Intractable brain edema is a leading cause for the death of severe TBI.Nowadays, studies on the mechamism of brain edema formation after TBI are mainly focused on the brain, little is known about how peripheral organs responde after TBI, nor the effect on secondary brain injury. Recently, a series of studies indicate that systemic inflammatory response syndrome (SIRS) occurs with a high incidence in the patients of TBI, especially severe TBI. Interleukin 1β(IL-1β), tumor necrosis factor-α(TNF-α), IL-6 etc. are increased systematically. As we all known, IL-1β, TNF-α, and IL-6 play very important role in secondary brain injury, so there is a very good chance of the peripheral immune system are involved in the pathological process of brain edema formation. Spleen is the largest immune organism in human body, it could response to a variety of stress stimuli, a large amount of immune cells would be activated, and then release a lot of cytokines, chemokines, and so on, including IL-1β, TNF-α, and IL-6. These imformation implied that the spleen may respond after TBI by releasing stored immune cells and proinflammatory cytokines into bloodstream, which then infiltrate the brain and promote secondary brain damage and brain edema.This study is composed of three parts to testify the hypothesis. Part I consists of severe TBI rat model establishment, mortality, brain water content, and cognitive function were determined in rats of Sham, TBI, and TBI&SPX (splenectomy was performed after TBI) groups. In part II, chief pro-inflammatory factors including IL-1βand TNF-αwas tested in spleen after TBI, IL-1β, TNF-α, IL-6 and IL-10 content in serum and the mRNA expression in brain were also measured in all three groups. In part III, in vitrol experiments were performed to explore the molecular mechanism of how spleen afterTBI effect on PC12 and BV-2 cells. All three parts of this study was to determine the role of spleen-derived inflammatory response in secondary brain injury.Material and MethodIn vivo and in vitrol experiments were included:1. To establish a severe TBI Sprague Dawley rat model using a modified Feeney'method;2. Mortality and brain water content were detected in Sham, TBI, and TBI&SPX groups;3. Cognitive function was determined by Morris Water Maze of rats in Sham, TBI, and TBI&SPX groups;4. IL-1βand TNF-αcontent was measured in the rat spleen at different time points after TBI;5. IL-1β, TNF-α, IL-6, and IL-10 content in serum were detected after surgery in all groups by ELISA;6. IL-1β, TNF-α, IL-6, and IL-10 mRNA expression level in injuried brain were also determined using RT-PCR in all three groups;7. p38 MAPK, ERK, and NF-κB were tested in injuried brain by western blot in Sham, TBI, and TBI&SPX groups;8. Flow cytometry was applied to determine the effect of spleen after TBI on the apoptotic rate of PC12 cels (substitution for neurons);9. Western blot was used to determine the effect of spleen after TBI on p38 MAPK, ERK, and JNK expression of BV-2 cells, and Laser scanning confocal microscope was applied to observe NF-κB activation in BV-2 with or without spleen stimuli.Results1. The mortality of severe TBI SD rats model is 35.42%, which is similar to the death rate of human with severe TBI;2. Splenectomy immediately after TBI could significantly decrease the mortality of SD rats, and effectively lessen the brain water content;3. Rats in TBI&SPX group have better space memory and learning function than those in TBI group;4. After TBI, spleen responded in a very short time, and release a larger mount IL-1βand TNF-α;5. Splenectomy could effectively decrease IL-1β, TNF-α, and IL-6 in serum, and also their mRNA expression in injuried brain;6. Splenectomy could effectively down-regulate the expression of p38 MAPK, ERK, and NF-κB protein, especially on p38 MAPK;7. Apoptosis of PC12 cells significantly increased when stimulated with the supernatant of rat spleen after TBI, while IL-1 receptor antagonist (IL-1RA) could reverse this effect;8. Supernatant of rat spleen after TBI could promote MAPK signal pathway in BV-2 cells, of which p38 MAPK was the most prominent; it could also induce NF-κB activation and move into nucleus;9. IL-1RA is useful for alleviate spleen's effect in BV-2 cells.Conclusions1. Splenectomy immediately after severe TBI in rats could effectively decrease the mortality, alleviate brain edema, and promote cognitive functions;2. TBI could promote IL-1βand TNF-αrelease in spleen; Splenectomy could effectively decrease IL-1β, TNF-α, and IL-6 content in serum, and also inhibit their mRNA expression in injuried brain;3. Spleen-derived inflammatory substance after severe TBI could activate MAPK and NF-κB in brain;4. Spleen after severe TBI could activate MAPK and NF-κB in BV-2 cells, and increase apoptosis of PC12 cells.