The Expression And Function Of TLR4 After Spinal Cord Compression Injury In The Rats

The functional restoration after spinal cord injury has always been the challenge in the field of neuroscience. Nowadays due to the fast development of construction and transportation, the incidence of SCI is increasing. SCI often cause various lifelong disabilities of the patients which result in enormous suffering and burden of the family and society. In order to reduce the suffering and raise the living standards of SCI patients, the study of SCI pathophysiology and development of effective treatment to enhance the recovery of SCI are becoming the main tasks for neuroscience scientists. With more and more study in this area, the pathological sequelae following acute SCI are divided into two broad chronological events: the primary injury and the secondary injury caused by the first phase. And the secondary injury leads to even greater injury several times more than the primary injury. Therefore, the study of the mechanism of the secondary and how to relieve the secondary injury are the common goals for the neuroscientists.Studies have identified a number of interrelated processes that are thought to contribute to secondary damage after SCI, including alterations in microvascular perfusion, free radical generation and lipid peroxidation, inflammation, necrotic and apoptotic cell death and dysregulation of ionic homeostasis (Tator, 1998). And imflammation play a very important role in the pathophysiology process of secondary injury, while it's mechanism is not very clear. Innate immunity responses are soon induced by cell damage and toxicity after SCI, and resident microglia is the key component of innate immunity. In CNS resting microglia exhibit a characteristic ramified morphology and are responsible for immune surveillance. Microglia become readily activated in response to CNS injuries or to immunological stimuli (Kreutzberg, 1996; Liu and Hong, 2003; Streit, et al., 1988, 1999) and undergo dramatic morphologic alterations because of activation, changing from resting ramified microglia into activated amoeboid microglia. In addition, activated microglia may release a variety of soluble factors, which are pro-inflammatory in nature and potentially cytotoxic (Table 1). Further, surface molecules, such as complement receptors and major histocompatibility complex molecules, are also upregulated when microglia is activated (Graeber, et al., 1988; Oehmichen and Gencic, 1975). In 2003 Lehnardt reported that TLR4 is mainly expressed in microglia, and play a key role in the innate immunity and adaptive immunity in CNS (Lehnardt, 2003). Nowadays studies pay more attention the role of TLRs in SCI, but the results are inconsistent by using TLRs gene mutation (Kigerl, 2007; Lehnardt, 2003; Schonberg, 2007). In order to identify the relationship between TLRs and secondary injury after SCI, we will eaplore the expression change of TLR4 in microglia after SCI in the first part, and then study the roles TLRs played in SCI in the second part.In the first part, we studied TLR4 express in microglia at different time point and found that TLR4 may play an important role in the secondary injury and TLR4 expression maybe associated with the compromise of blood-spinal cord barrier. Spinal cord compression injury was made at T8 segment in 96 adult male Sprague-Dawleys rats, and the rats were randomly divided into 6 groups. Half of the sham and injured animals were fixed by perfusion of 4% paraformaldehyde, Two cm of the spinal cord blocks with compressed site in center were then removed at 0 h, 3 h, 24 h, 72 h and 7 d after injury. Frozen sections were made for H-E and immunofluoresent staining to detect the injury area and the expression of TLR4 in the spinal cord. The distribution of TLR4 positive cells and the injury area and the extravasated IgG were compared as well. The number of TLR4 positive microglial cells was counted by using Image-pro plus software. We found that TLR4 was mainly expressed in microglial cells and the expression started to increase between 3 h and 24 h and peaked at 72 h after the spinal cord compression injury, then descended at 7d, according to the number of TLR4-positive microglia. The distribution of TLR4 immunoreactive product was consistent with the area of injury and that of extravasated IgG. The other half of sham and injured animals were anaesthetized at 0 h, 3 h, 24 h, 72 h, 7 d, and then decapitated to take the spinal cord samples. Then we extracted the total RNA to do TLR4 RT-PCR and real-time PCR. The results showed that mRNA of TLR4 also peaked at 72h, and then dramatically descended at 7d. The time course of TLR4 expression is coincidence with the change of spinal cord injury area, which may suggest that TLR4 expression in microglia may play an important role in the secondary injury.The second part we studied the roles of TLRs in SCI secondary injury. We also used the compression injury made at T8 segment in 16 adult male Sprague-Dawleys rats. The rats were divided into two groups randomly, and then we inject 5ul MyD88 inhibitor peptide (MIP) solution and 5ul CP solution separately. The injury area was covered with absorbable gelatin sponge contained 5ul the MIP solution and 5ul CP solution separately. Half of the animals in each group were fixed by perfusion of 4% paraformaldehyde, and two cm of the spinal cord blocks with compressed site in center were then removed 7 d after injury. Frozen sections were made for H-E and immunofluoresent staining. The other half of animals in each group were anaesthetized at 7 d, and then decapitated to take the spinal cord samples to do the experiments of western blot to detect the expression of P-P38 MAPK, TNF-α, IL-1 and caspase-3. During the experiments we utilized BBB score and beam-walking tests to detect the functional recovery after compression injury. The results show that MIP may promote the functional recovery by inhibiting produces of inflammatory factors inducing cell apoptosis. The results suggest that TLRs play a important role in secondary injury after SCI, and inhibit TLRs signal pathway at early time may reduce secondary injury.In a word, we proved the expression changes of TLR4 after spinal cord compression injury in the protein level and mRNA level, which may take part in the pathophisiology of secondary injury through MyD88 signal pathway. After given MIP, functional recovery was promoted and cell apoptosis was reduced. This may provide some evidences for further study and treatment.