Role And Mechanism Of Toll-Like Receptor4-mediated Signal Pathway Following Optic Nerve Injury In Mice

Nerve regeneration studies of central nervous system (CNS) have been a hot anddifficult problem in medical research. As a part of CNS, the optic nerve exhibits specialphysiological characteristics. Studying visual functional recovery following optic nerveinjury, acts as an important role to CNS diseases and nerve injury. Currently, a large numberof studies have focused on the promotion of neural regeneration, predominantly through theremoval of inhibitory factors that prevent axonal regeneration following injury. Despitehaving obtained some success from these studies, there remain additional issues thatprevent nerve regeneration following injury. Research shows that inflammation plays animportant role in the process of secondary brain damage in CNS injury.We had found a variety of cytokines, such as tumor necrosis factor (TNF) andinterleukin-6(IL-6), chemokines, and adhesion molecules that are involved in theinflammatory process, which may increase brain damage. However, current research hasnot yet unraveled the mechanisms, which still need allow for the progression towardsclinical studies. It is speculated that TLR4may serve as a trigger point in the acuteinflammation response that activates the pathogenic process of injury. In recent years, therole and mechanism of toll-like receptors expressed innate immune cells of organisim (eg,microglia) are concerned in inflammatory reaction. Innate immune functions in CNS areperformance through microglia, and inflammatory cytokine release is mediatedTLR4-NF-κB signal pathway. Initially, TLRs were thought to mediate cellular responsesthat were considered to be a pro-inflammatory host defense response, primarily as a way toeliminate invading pathogens within the CNS. However, studies have confirmed that TLRsalso play significant roles in all kinds of diseases of the absence of exogenous pathogens.TLR4can be activated both by exogenous and endogenous ligands, such as LPS, HSP60,70, necrosis cell debris and so on. When nerve was damaged, HSP60and70was foundaround axon injury. These results suggest, as infection, trauma may also increase expression of microglia TLR4by some mechanism, which involved in the regulation of inflammatoryresponse following optic nerve injury.Hashimot found TLRs are part of an evolutionarily conserved family of type Itransmembrane glycoprotein receptors1988ago. TLRs are the receptor protein family withhomology as the Drosophila Toll protein, which not only involve in the process ofembryonic development of Drosophila, but also play an important role in immune defenseof adult Drosophila. In1997and1998, Medazhitov and Rock cloned out another Tollhomologue, now known as the TLR4protein.Initially, TLRs were thought to mediate cellular responses that were considered to be apro-inflammatory host defense response. However, studies have confirmed that TLRs alsoplay significant roles in CNS diseases, such as Alzheimer’s disease, multiple sclerosis,stroke, and other neurodegenerative diseases. TLRs mainly expressed microglia andastrocyte in CNS. When microglia was activated, TLRs were rapid emergence. Study foundwhen optic nerve was injuried, microglia was activated rapidly. Therefore, when optic nerveis damaged, studying expression change, significance and mechanism of the inflammatorychain cascade reponse in microglia TLR4of retinal and optic nerve, for betterunderstanding the mechanism of the retina and optic nerve secondary damage followingoptic nerve injury, is very imptortant.Study had confirmed the TLR4signal pathway includes the MyD88-dependent andnon-dependent signaling pathway (the TRIF channel). After nerve injury, TLR4by which asignal pathway transfer signal is activated is unknown. Maybe, it is possible to play aspecific role each signal pathways activated TLR4.Previous studies thought nuclear factor-κB (NF-κB) was the core of regulationinflammatory response through activation of a variety of physical and biochemical factors,induced expression of a variety of inflammatory factors and enzymes, mediatedinflammatory response formation, development and aggravation. TLR4is the receptor ofLPS, early phase which identifys invading G-bacteria, starts inflammatory response byLPS－TLR4－NF-κB signaling. Now considering TLR4－NF-κB signaling is also originof some diseases in CNS. Then, after optic nerve injury, whether inflammtion is mediatedby TLR4-MyD88-NF-κB signalling, and produce of inflammatory factors IL-6, TNF and soon, results in inflammatory damage of the retina, optic nerve, it is not reported yet. So, the purpose of this study focused to microglia TLR4expression change,significance and regulation of signalling. Through comparison study between TLR4genemutant mice and wild mice following optic nerve crush, identifying role and mechanism ofTLR4about regeneration or repair, these establishs expermental basis for treatment opticnerve damage.The main results and conclusions of the study are as followed:1. Optic nerve expressed TLR4following optic nerve crush in mice. With timeincrease, TLR4expression gradually increased in retina and optic nerve, reached peakingabout2weeks and kept high level about3,4weeks. The results suggested TLR4-mediatedaseptic inflammation plays an important role following optic nerve injury in mice.2. After optic nerve injury in mice, microglia was activated rapidly. The marker Mac1expression of microglia activation increased sharply in the retina and optic nerves, thengradually decreased slowly and remain at a high level in3,4weeks.3. After optic nerve injury in mice, the expression of MyD88molecules graduallyincreased in optic nerve. The protein and mRNA expression of MyD88reached high levelsabout2and3weeks in optic nerve, but TRIF had not change significantly. The same time,NF-κB, IL-6and TNF-αexpression gradually increased in optic nerve, and reach at highlevel about2,3weeks after injury. These results suggested TLR4signalling is mainlymediated by MyD88-NF-κB signal pathway transfering following optic nerve injury.4. After optic nerve injury in mice, through the TLR4gene mutantand wild-type mice comparison study, early phase, expression of GAP-43protein is higherin the TLR4gene mutant mice than wild-type, and apoptosis of retinal ganglion cells isrelatively small. The results suggest TLR4gene mutant mice have a protective effecton the optic nerve following optic nerve damage in early phase.