Experimental Study And The Mechanism Of Facial Nerve Recovery Through Toll-Like Receptor2 (TLR2)/Nuclear Factor Kappa B (NF-?B) Signaling Pathway

Background and objectiveFacial paralysis is a common disease with an increasing morbidity around the world.It may be caused by radiation therapy,trauma,systemic infection,metabolic disorders,acute or chronic otitis media,and many other causes.Peripheral lesions are more common than central lesions.The etiology of peripheral facial paralysis is unknown,but autoimmune diseases have been postulated as a possible path mechanism.Meanwhile,peripheral facial paralysis could lead to serious consequences,and therefore,it is necessary to investigate the pathogenesis and treatment methods.Toll-like receptors(TLRs)are a large family whose members play a decisive role in innate immunity and inflammation.And 10 types of TLRs have been found in humans which called from TLR1 to TLR10.More recently,studies on TLRs have focused on the nervous system.The most widely investigated TLRs were TLR2 and TLR4.Microarray analysis detected TLR2,TLR4,TLR5,and TLR9 in cultured murine cortical neurons.Several autoimmune diseases,chronic inflammation,and infectious diseases are known to be closely related to excessive immunity according to the activation of TLR2 and TLR4,the case in point is,TLR1/TLR2 and TLR2/TLR6 heterodimers and arteriosclerosis in ischemic brain injury while TLR4/TLR6 heterodimers in Alzheimer's disease.Pam3CSK4 was a TLR2 agonist that could specifically activate TLR2 and rapidly triggers a variety of anti-microbial immune responses through the induction of pro-inflammatory cytokines.TLR2 could trigger several cellular responses,including NF-?B activation and inflammatory cytokines and chemokines secretion.Signals activates NF-?B by TLR2 and its downstream molecular adapter protein in the TLR domain,myeloid differentiation factor88,and TNF receptor-associated factor 6,leading to the inflammatory cytokines like interleukin-6.Nuclear factor-kappa B(NF-?B),as a major transcription factor in the inflammatory response,can enhance the expression of pro-inflammatory mediators and enzymes.The inhibition of NF-?B target genes is of great importance to avoid the control of infection and the development of autoimmune-related inflammation.In response to a variety of stimuli including viral and bacterial pathogens,cytokines and the cytoplasm NF-?B pathway can be activated.It has been reported that TLR2 can activate NF-?B and induce the production of inflammatory cytokines and chemokines in many cell types,including macrophages,monocytes,neutrophils,glial and neuronal cells,epithelial cells,and keratinocytes.Besides,TLR2 and 4/NF-?B signaling pathways also play a key role in innate and acquired immune responses to acute lung injury.Co relational studies have been conducted on peripheral facial paralysis in our institute over the past decade,for example,we have proved that NF-?B plays a primal role in coordinating inflammatory responses and participates in the process of facial nerve repair after it was infected by HSV-1.The obvious immune response will be observed around the injured nerve.Thus,we supposed that facial nerve injury might stimulate TLR2,activates the pro-inflammatory NF-?B pathway,and promotes production of pro-inflammatory cytokines,and ultimately,facial paralysis was aggravated in the paralyzed animal model.In this study,facial nerve surgery was performed by us in rats to set up a paralyzed animal model with intraperitoneal injection of Pam3CSK4.We tried to explore the molecular mechanism that facial nerve injury and recovery through TLR2/NF-?B signaling pathway in vivo.Research methods1.Experimental animals and groupingFemale Wister rats aged 3 weeks(each weighing 60-80g)were purchased from Shandong University Animal Centre.All rats were matched in terms of age,gender,and weight and bred and group-raised in standard box cages in a thermoregulated environment(ambient temperature of 26?),with access to standard laboratory diet and water.The routine care procedures and laboratory procedures have been approved by the Animal Ethics Committee.The procedure was performed under general anesthesia with 10%chloral hydrate.Rats were divided into four groups as follows:Group 1(n=39)received facial nerve surgery only,which was done to buckle the facial nerve trunk using a trumpet clamp for 20s.Group 2(n=21)was the normal control group with no treatment.Rats in group 3(n=9)comprised the sham operation group,with facial nerve trunk exposure with no injury.Rats in group 4(n=27)received facial nerve surgery as well as intraperitoneal injection of Pam3CSK4(20 ?g/each)to active the TLR2/NF-?B signaling pathway.2.Behavior testingThe blink reflex,the motion of the vibrissae and the motion of the nose were assessed 1,4,7,10,13 and 16 days after operation to evaluate facial nerve function.The 5-mL syringe needle was placed 2 cm away from the eyes of the rats,and the blink reflex was observed immediately:0 was not closed;1 is delayed closure;2 is completely closed.The motion of vibrissae in 30s was calculated as follows:0 for no motion,1 for effective movement inferior,and 2 for effective movement indistinguishable.The nose motion was observed carefully and categorized as follows:0 for clearly deviated,1 for slightly deviated,and 2 for deviated.A total score of less than 3 could determine facial paralysis in rats.3.Morphological MeasurementsSamples of the facial nerve and the brainstem(dissected and contained for morphological measurements),with details as follow:(1)Transmission electron microscopy(TEM):On day 7 after the surgery,the facial nerve trunk was collected carefully and preserved in 2.5%glutaraldehyde and 4%paraformaldehyde.Later these specimens were immobilized in 1%osmium tetroxide and embedded in EPON-812 epoxy resin.The facial nerves were cut into 5-?m slices and observed by TEM.(2)Immunofluorescence staining(IFS):Each sample was washed three times with PBS and fixed with 4%paraformaldehyde.The dissected brainstem was immersed in gradient sucrose solutions(15%,20%,and 30%in PBS and OCT gel at 4? overnight),rapidly frozen,and sliced into 7-?m-thick coronal sections.The brainstem sections were incubated with primary antibodies(NF-?Bp65=1:500,TLR2=1:500,and Tuj-1=1:1,000),and then incubated with DyLight 488 and DyLight 647-labeled secondary antibodies.After final washing with PBS,the specimens were imaged using a Leica confocal microscope.4.Quantitative real-time PCRIn this study,each 25?L PCR volume consisted of 12.5?L SYBR Green PCR,0.5?L forward primer(10?M),0.5?L reverse primer(10?M),0.5?L template,and 11?L sterilized distilled water.The primers used for TLR2 were 5'-TCCATGTCCTGGTTGACTGG-3'(forward)and 5'-GGAATCCTGCTCGCTGTA GG-3'(reverse).The primers used for NF-?B p65 were 5'-AGGACTGCCGGGATG GCTTCTAT-3'(forward)and 5'-GGTCTGGATGCGCTGGCTAATGG-3'(reverse).The primers used for ?-actin were 5'-TGTCACCAACTGGGACGATA-3'(forward)and 5'-GGGGTGTTGAAGGTCTCAAA-3'(reverse).38 cycles were performed with the amplificated conditions:pre-denaturation at 94? for 5 min,denaturation at 94? for 40s,annealing at 59.1?(TLR2)/63.6?(NF-?B p65)for 40s,extension at 72? for 90s,and final extension at 72? for 10min.The different primers' suitable annealing temperature was pretesting through 12 holes staircase temperature.To assess the adequacy of the cDNA and the efficiency of the RT-PCR system,murine?-actin primers were used as RT-PCR control under the same condition.Data were analyzed by 2-??Ct method.5.Western BlotThe brainstems were extracted and washed three times with ice-cold PBS.Protein concentrations were determined by BCA method.And a total of 20 ?g of the protein was isolated on 8%SDS-PAGE gels.After electrophoresis,the separated proteins were electrically transferred into nitrocellulose filter membranes and blocked in 5%nonfat dried milk containing 1×TBST for lh at room temperature.Subsequently,the membranes were incubated overnight with primary antibodies at 4?.The primary antibody was diluted to 1:2,000,and the secondary antibody was diluted to 1:1,000 with ?-Actin probe used as an internal control.Antibodies were visualized using the ECL kit.Study results1.Establishment of Facial Paralysis Rat Model by Facial Nerve SurgeryFacial nerve surgery was performed on rats by clamping the facial nerve trunk on the left side for 20s using trumpet clamp to establish a facial paralysis animal model.At 1,4,7,10,13,and 16 days after the surgery,the score of the blink reflex,the motion of vibrissae,and the motion of nose were analyzed to assess the function of facial nerves.The rat model of facial paralysis was successfully constructed.The blink reflex result revealed that the eyelid of rat with facial paralysis exhibited poor closure on the left side.Results of the motion of vibrissae showed that the vibrissae of rat with facial paralysis exhibited weakened movement on the left side.Results of the motion of nose revealed that the nose tip of facial paralysis rat exhibited.After facial nerve trunk surgery,all rats showed facial paralysis,with a total score of 0.Facial nerve function recovered at the 4 and 7 days,but the score was still less than 3.The facial nerve function recovered obviously on the 10th and 13th days,and the score was improved.The facial nerve function score was approach 6 at the 16 days.2.Morphological Changes(1)Morphological Appearances of Facial Nerve TrunkMorphological changes of the facial nerve trunk were evaluated 7 days after surgery.The TEM findings revealed that the fiber diameter of the facial nerve was shortened and the organelle was swollen and demyelinated.The IF results demonstrated that the axons were stained with Tuj-1(red)and the myelin was stained with S-100(green).Compared to that in the control group,degenerated nerves were found in the operation group,manifested as disorderly distributed smaller fibers with demyelination and swollen perineurium.(2)Morphological Appearances of BrainstemThe results of immunofluorescence staining were observed under the confocal microscope.The fluorescence activity was measured using the number of cells or the luminous intensity of green light(DyLight 488).Compared with the matched group,the fluorescence activities of both NF-?Bp65 and TLR2 were significantly increased in the operation group.3.mRNA Expressions of TLR2 and NF-?Bp65 were Increased in Facial Paralysis RatsThe results of RT-PCR revealed that the mRNA expression of TLR2 was retained in the brainstem of the control group,as well as in the sham group,and at the same time,there was a significant increase in TLR2 production in the operation group.Similarly,the mRNA expressions of NF-?Bp65 were significantly increased in the operation group(compared to those in the control and sham groups)(P<0.05).4.Protein Expressions of TLR2 and NF-?Bp65 were Upregulated in the Paralyzed RatWestern blotting was performed to determine the protein expressions of TLR2,NF-?Bp65 in the brainstem taken ?-actin as internal reference.As protein band showed,the protein expression of TLR2(89 kDa)and the protein expression of NF-?Bp65(65 kDa)was significantly higher than that of the control group after surgery which was also upregulated after the facial nerve surgery.The results of RT-PCR and western blotting together indicated that both TLR2 and NF-?Bp65 were activated in rats with facial paralysis,thus indicating that the TLR2/NF-?Bp65 signaling pathway might be involved in the process of facial paralysis.Rats were subjected to facial nerve surgery and the intraperitoneal injection of Pam3CSK4 to activate the TLR2/NF-?B signaling pathway.As illustrated in histogram,the expression of TLR2 protein in Pam3CSK4-operation group was extremely higher than that in the operation group,and the protein expression of NF-?Bp65 was also upregulated dramatically after the intraperitoneal injection of Pam3CSK4.5.Facial Nerve Recovery was Delayed After Pam3CSK4 Injection Along with the Facial Nerve SurgeryFacial nerve surgery and intraperitoneal injection of Pam3CSK4 were performed on rats.Rats injected intraperitoneally with Pam3CSK4 exhibited facial nerve dysfunction in the behavior test,which was consistent with the impairment caused by facial nerve surgery.The behavior test showed that the average recovery time of the Pam3CSK4-operation group was significantly longer than that of the operation group,with a delay of 2-3 days.The average recovery time of the motion of vibrissae was the shortest,that of the motion of the nose was the longest(peak 10 days,P<0.05).ConclusionsIn this study,we found that the expressions of NF-?Bp65 and TLR2 were increased significantly in the brainstem of facial nerve paralyzed rats after the facial nerve surgery.The facial nerve dysfunction of rats caused by the facial nerve surgery was exacerbated by co-treatment of Pam3CSK4,while the expression of NF-?Bp65 and TLR2 in Pam3CSK4-operation group was both significantly higher than that in the operation group.TLR2 may be proved to be an innovative gene target and a novel idea for the clinical applications of facial paralysis immunotherapy.