The Role Of Metabotropic Glutamate Receptor 5 In Traumatic Brain Injury And Its Mechanism

Traumatic brain injury（TBI）is a common and often life-threatening condition in clinic.About 30–35%of patients with moderate or severe TBI die within the first 30 days after admitted to the hospital,while the survivors would suffer from motor and neuropsychological malfunctions,losing the ability to carry out normal work and even daily living activities.Accordingly,this trauma will lead a heavy spirit and economic burden to both family and society.To elucidate its mechanism and find out efficient strategy for the treatment of TBI is an urgent need.Besides the uncontrollable and irreversible physical injury leading to damage of brain tissues,inflammation is a critical pathological process to aggravate the progress of TBI.However,the triggers and participants of inflammation after TBI include both native inflammatory cells（such as macrophages and microglial cells）in the central nervous system（CNS）as well as infiltrated peripheral blood immune cells.Neutrophils are the fir st peripheral immune cells transmigrating across the blood-brain barrier（BBB）and infiltrating into brain after TBI,which is an important contributor to inflammatory injury of brain during the acute phase.Several experimental results indicated that eliminating neutrophils were beneficial during the acute phase of brain injuries.Therefore,it is a potential strategy to halt the brain inflammation via blocking neutrophils’infiltration during the acute phase.Metabotropic glutamate receptor 5（mGluR5）is a group I metabotropic glutamate receptor which is considered to exert an important effect on inflammatory regulation after TBI.However,the data are inconsistent,since both activation and inactivation of mGluR5have been reported to play an inflammatory protective role.For example,activation of mGluR5 reduces the neuronal apoptosis under the condition of oxygen-glucose deprivation and inhibits microglial associated inflammation.Furthermore,Loane and Wang et al.reported that activation of mGluR5,（RS）-2-chloro-5-hydroxyphenylglycine（CHPG）injected intracerebroventricularly,inhibits caspase-dependent apoptosis of neurons and suppresses inflammatory cytokine release from microglias.However,inactivation of mGluR5 also alleviates neuronal injury in vitro and suppresses astrocytes releasing proinflammatory cytokines or chemokines.Most of these studies mainly focused on the central functions of mGluR5 on the brain native cells participating in inflammation which including astrocytes,microglias and neurons,but ignored the role of mGluR5 in modulating peripheral infiltrated immune cells（such as neutrophils）.We speculated that it is a potential reason for the contradictory effects of mGluR5 agonists or antagonists observed in TBI as the previous studies described,which needs to be elucidated.Accordingly,in the present study,we focused on the effect of mGluR5 on peripheral inflammatory cell infiltration and associated brain damages in the acute phase of TBI by using mGluR5 global knockout（KO）mice,primary isolated murine neutrophils and brain microvascular endothelial cells（BMECs）,associated cell lines and in vitro BBB model.These researches are expected to give an insight of mGluR5 in TBI and provide some novel clues for TBI therapy.Methods1.An automatic brain impact machine was used to make the moderate TBI of mice.Briefly,after anesthetizing mice with an intraperitoneal injection of 1.5%sodium pentobarbital and fixed in the stereotaxic frame,the scalp was incised and a dental drill was used to perform a craniotomy with a trephine bit（?=3 mm）on the leftmotor cortex（anterior-posterior 2 mm,medial-lateral 2 mm from bregma）.Then the cortex surface at the center of the craniotomy was targeted vertically by a metal tip（?=2.5 mm）.When coarse adjustment and fine adjustment were finished,the machine induced the TBI by retracting the tip upward by 2 cm,and then a down stroke（velocity:1.5 m/s,deformation depth:2mm,duration:120 ms）.The wound was sutured immediately and the mice were returned to the animal facility.2.The Longa scale,a modified neurological severity score（mNSS）and foot-fault test were used to assess neurological deficits of each animal before sacrifice by two observers blinded to the treatment.Motor,sensory,reflex,and balance tests were included.A higher score or more foot faults represented a more severe injury.The number of foot faults for the right hindlimb was recorded over 50 steps while mice crossed a narrow wooden beam（6mm wide and 120 mm in length）.All mice were trained to perform those tests for 3 days before TBI.3.Mice were anesthetized 24 h after injury and killed transcardially with saline,followed by 4%paraformaldehyde.Brains were immediately removed and post-fixed in4%paraformaldehyde for 24 h.Paraffin-embedded sections（10μm thick）were stained with hematoxylin and eosin（HE）and observed under a microscope.4.Immunofluorescence analyses were carried out as follows.After quenching of endogenous peroxidase activity and blocking of nonspecific binding,sections were allowed to react with the primary antibodies（rat anti-CD15,rabbit anti-CD177,or rabbit anti-vWF with goat anti-claudin-5）at 4°C overnight.Sections were then washed,incubated with Alexa Fluor 488-or Alexa Fluor 555-conjugated secondary antibodies for 1 h,observed under a fluorescence microscope,and photographed with nuclei stained with DAPI.Murine peripheral blood were stained with specific marker Gr-1 conjugated Alexa 488 diluted with PBS containing 2%BSA for 45 min at 4°C,and they were observed and calculated under the fluorescence microscope.5.The Evans blue assay was carried out as below.Mice were anesthetized,and 2%Evans blue dye（4 ml/kg）was injected intravenously and allowed to circulate for 2 h.T hen mice were perfused transcardially with physiological saline until no blue color was evident in effluent.The brain was removed and sliced into 2 mm thick sections.Extravasated Evans blue was visualized by fluorescence microscope in fixed,frozen cryostat sections.6.Neutrophils were isolated from femur and fibula of mice as follows.Murine bone marrow was dispersed in HBSS(without Ca²⁺and Mg²⁺)with 0.5%fetal calf serum（FCS）（HBSS/FCS）,straining through a 70-μm cell strainer,and was centrifuged（1000 g,30 min,room temperature）over 2-layer（0/62.5%）Percoll gradient in HBSS.Mature EGFP-labelled neutrophils were obtained from the lower part of 62.5%Percoll（purity,80–90%by cytospin）,and contaminating red blood cells were removed by ammonium chloride lysis.Finally,cells were washed twice in HBSS/FCS.The attained neutrophils were diluted in RPMI-1640 media with 10%（v/v）fetal bovine serum（FBS）to a final concentration of 10⁶/ml and cultured at 37?°C in an atmosphere of 5%（v/v）CO₂ for indicated time.Brain microvascular endothelial cells were isolated from mouse brains as below.In brief,brains were removed from mice and stored in Dulbecco’s modified Eagle’s medium（DMEM）on ice.Meninges were carefully removed from forebrains,and forebrains were mashed with forceps and minced into small pieces of approximately 1 mm3,then digested with 2 mg/ml collagenase/dispase in DMEM for 45 min in 37°C（occasionally shaking）.Cold DMEM was added to the homogenate and centrifuged at 1000 g,4°C for 8 min.The pellet was resuspended in 25%bovine serum albumin（BSA）and centrifuged at 1000 g,4°C for 20 min.Then the resulting suspension containing vascular component was filtered through a 59μm nylon mesh.BMECs cultures were washed and maintained in the Endothelial Cell Medium（ECM）supplemented with 100 mg/ml heparin and 4 mg/ml puromycin.After 24 h,the culture medium was replaced and puromycin was removed from ECM.The isolated cells were identified with vWF by immunofluorescence,the endothelial cell marker,to make sure the purity of cells.When the cultures reached 80%confluency,the purified BMECs were used to construct in vitro BBB models.Murine brain endothelial cells（bEnd.3）,recognized to present brain endothelium like properties,were obtained from American Type Culture Collection（ATCC）.This cell model is an appropriate choice to study blood-brain barrier function.The cells were cultured in DMEM supplemented with 10%（v/v）FBS and antibiotics（100 U/ml penicillin and 100μg/ml streptomycin）.7.The isolated bone marrow’s neutrophils were stained with specific marker Gr-1conjugated Alexa 488 diluted with PBS containing 2%BSA for 45 min at 4°C.Flow cytometry data of bone marrow’s neutrophils were acquired with a FACSCanto II and analyzed with FlowJo software.8.Total RNA of neutrophils isolated from bone marrows of WT or mGluR5 KO mice were prepared by using TRIzol Reagent.Standard cDNA synthesis,probe labeling,hybridization,and scanning were performed using the standard protocols of CapitalBio Technology.Expression profilings were carried out by using Affymetrix Mouse Genome430 2.0 arrays.A total of 4 samples were assayed,including 2 samples from WT mice and 2samples from mGluR5 KO mice.Microarray visualization of GO pathways analysis and data filtering of chemokine related genes were accomplished by the specific manager of CapitalBio Technology.9.Total RNA from tissues or cultured cells was isolated using TRIzol Reagent.2μg of template RNA was used to synthesize cDNA using a reverse transcription kit.Quantitative PCR was performed using an ABI 7500 thermocycler employing SYBR Premix Ex TaqTMⅡKit.The interest mRNA expression levels were normalized to that of endogenous control by the 2^-ΔΔCt method.10.Mechanical scratches were performed to mimic traumatic injury on brain microvascular endothelial cells.In brief,traumatic injury was performed on cultured brain endothelial cells（bEnd.3）by using a 10μl pipette tip.We made 4 crosswise lines in the cell culture holes symmetrically.And the injury lines were produced on the confluent surface of endothelial layer with equal distances between the scratches,and detached cells were removed by washing with DMEM.11.Blood-brain barrier models were performed as described previously with modifications.Briefly,BMECs（5×10⁵/cm²）were seeded on the upper side of the collagen-coated polyester membrane of transwell inserts（polyester membranes,3μm pore size,?=6.5 mm）placed in the holes of the 24-well culture plates.The BBB models were maintained in ECM with media replacement occurring every other day until the monolayers reached confluency.Transmigration assays were carried out as follows.After mechanical scratches（2symmetrical crosswise lines）were performed on the monolayers of BMECs,EGFP-labelled neutrophils（1×10⁶ cells per well）were added to the upper inserts of the transwell system of in vitro BBB models.Over a 6-h period at 37°C,the number of transmigrated cells from the lower chambers were observed and calculated by fluorescence microscope.12.To selectively knock out mGluR5 on bone marrow cells,bone marrow transplantation was carried out as below.Briefly,female mice,aged 8–10 weeks,to be used as the recipient mice were irradiated twice with 8 Gy separated by 3 h.Bone marrow donor male mice 12 weeks of age were killed and the bone marrow cells were achieved at 2×10⁷cells per milliliter.Approcimately 300μL suspended cells were injected into recipient mice through caudal vein.The chimeras were used in the following experiments after 4 weeks.Results1.To explore the important role of mGluR5 in TBI,we evaluated the differences between eight to ten-week-old WT and mGluR5 KO mice’s behavioral recovery from CCI-induced TBI.The brain water content,breath rate and weight showed no difference between WT and KO groups before trauma.Longa score and foot-fault test were carried out to assess motor and balance function,while motor,sensory,reflex,and balance tests were covered in modified neurological severity score.We used Longa scale first,but did not find any significant differences between WT and KO groups on Day 1 or Days 3 after TBI.The modified neurological severity score were nearly the same on Day 1 post-TBI,but on Days3,mGluR5 KO mice displayed significantly lower modified neurological severity score than WT.The differences of foot-fault test were again absent on Day 1,but hindlimb faults were fewer in KO group than WT on Days 3 after TBI.Unlike the previously reported no behavioral difference between the mGluR5 antagonist-treated and untreated injured groups before 14 days after TBI,these results demonstrate that neurological dysfunction was less severe in the mGluR5 KO mice in the acute phase of TBI.2.In order to further understand the significance of mGluR5 in protection against TBI,we investigated the leukocyte recruitment following CCI-induced TBI.Once the peripheral leukocytes infiltrated the brain parenchyma,devastating overinflammation may cause serious secondary injury.We found out that mGluR5 KO mice had markedly fewer infiltrated immunocytes near the site of injury in brain sections 24 h after TBI.Microglias and all blood-born leukocytes,including macrophages,monocytes,neutrophils,and T cells are CD15 positive,while CD177 is a specific marker for neutrophils.CD15 positive cells,especially CD177 positive neutrophils,were more in WT mice’s brain slices than KO near the site of injury by immunofluorescence.For immunocytes secretes many sorts of inflammatory factors after TBI,we discovered that the mRNA expressions of IL-1βand TNF-αof brain tissues were significantly higher in WT mice than KO ones,but IL-6 had no significant difference.These data suggest that less neutrophils’infiltration and fewer inflammatory factor expressions were detected in brain tissues of the mGluR5 KO mice after TBI.3.To investigate how the leukocytes infiltrated the brain parenchyma,we detected the integrity of BBB after TBI.Since plasma albumin passes the BBB after TBI and Evans blue stain binds to serum albumin immediately after injection,Evans blue stain accumulation is a reliable way to estimate BBB permeability.At 24 h after TBI,Evans blue assay revealed apparently less leakage of albumin in brain slices of mGluR5 KO mice compared with WT.Next,the expression of claudin-5,the tight junction protein that helps build the BBB,was examined by fluorescence microscope.KO mice had higher and more continuous claudin-5expression than WT in brain sections,indicating that knockout of mGluR5 protects against the decrease of the tight junction protein claudin-5.These results show knockout of mGluR5 reduced the increase of BBB permeability induced by TBI.4.After the examination of the defensive construction against infiltration,we analyzed the factor about enhancement of neutrophil recruitment and infiltration after TBI.The transmigration of inflammatory cells from microvessels to brain parenchyma is usually the result of oriented movement of chemotaxis.So we investigated the gene expression of the main brain microvascular endothelial chemokines that attract neutrophils in brain tissues.The receptor of CXCL1 and CXCL2 is CXCR2 on neutrophils,and CCL2,CCL4 and CCL5 pair with CCR1.At 24 h after CCI-induced TBI,both the WT and mGluR5 KO groups had markedly higher expression of CXCL1,CXCL2,CCL2,CCL4 and CCL5 of brain tissues than the sham groups.And all the five chemokines were significantly lower in mGluR5 KO TBI group than WT TBI group.These findings suggest that the mRNA expressions of chemokines in brain tissues were downregulated by knockout of mGluR5after TBI.5.To decipher whether neutrophils themselves alter before infiltration under traumatic brain injury condition,microarray was performed on bone marrow-derived neutrophils from WT and KO mice separately.The pathway analysis indicated that several pathways changed apparently when mGluR5 was knocked out,especially cytokine and chemokine related activities.Because the chemokines and chemokine receptors were important for the function of neutrophils’infiltration,we selected chemokine and chemokine receptor related genes to show that these genes decreased significantly in KO groups,reiterating that mGluR5 plays an important role in chemotaxis of neutrophils.To investigate whether the number of neutrophils changes before infiltration,we isolated the peripheral blood and counted the Gr-1（murine neutrophil specific marker）positive cells by immunofluorescence.Then we calculated the Gr-1 positive neutrophils in bone marrow from femur and fibula of mice by flow cytometry.There is both no significant difference between WT and KO mice.To some extent,we conclude that knockout of mGluR5 did not change the number of neutrophils in mouse blood or bone marrow.6.Back to the BBB,we focused on the role of the main component BMECs.BEnd.3,a murine brain endothelial cell line,was used to confirm the observation in vivo.We developed the mechanical scratch cell model that mimics TBI-induced traumatic brain microvascular endothelial injury in vitro.Cultures were pre-treated with the mGluR5selective antagonist MPEP to inactivate mGluR5 post injury.Then we examined the same mRNA expressions of the chemokines in mouse TBI models described above.The expression levels of CXCL1,CXCL2,CCL2,CCL4 and CCL5 were detected in scratch-injured bEnd.3 cells and were found to increase 24 h after stretch injury when compared with uninjured cells.It clearly showed that MPEP significantly suppressed the scratch-induced expression levels of CXCL1,CXCL2,CCL2,CCL4 and CCL5 24 h post-injury compared to untreated injured cells.From these results,we conclude that the mRNA expressions of chemokines in BMECs were downregulated by mGluR5 inactivation in vitro.7.Holding neutrophils and BMECs together,we further examined the impact of mGluR5 on neutrophils’transmigration through BBB.Primary murine brain microvascular endothelial cells were isolated to build the in vitro BBB models.When the monolayers reached confluency and the TEER were above 150Ω·cm²,the in vitro BBB models were ready for use.Then EGFP-labelled neutrophils were extracted from bone marrows of EGFP-transgenic mice.After pretreatment with mGluR5 antagonist MPEP and mechanical scratches were performed on the monolayers of primary BMECs on transwells,EGFP-labelled neutrophils were added to the upper inserts of in vitro BBB models.MPEP significantly reduced the number of transmigrated cells between the scratch group and the scratch pretreated with MPEP group.These findings indicate that mGluR5 inactivation suppresses neutrophils’transmigration through BBB in vitro.8.PKC pathway,the main downstream of mGluR5,was examined in mechanical scratch models of bEnd.3 cells（a BMEC cell line）in vitro.The mRNA expressions of CXCL1,CXCL2,CCL2,CCL4 and CCL5 of bEnd.3 cells were detected by qRT-PCR at 24h post injury.There were no siginificant differences among the PKC inhibitor GF109203X pretreated injured group,the GF109203X and MPEP pretreated injured group and the MPEP pretreated injured group,whose mRNA expressions of chemokines were all significantly lower than the untreated injured group.However,there was no significant difference between GF109203X pretreated and untreated injured groups,indicating that other pathway was involved in the downregulation of CCL5 by MPEP.Taken together,PKC pathway was involved in the regulation of mGluR5 on neutrophils’trafficking.9.The mRNA expression of mGluR5 on neutrophils from bone marrow before trauma and on Day 1 and Days 3,7,14 post-TBI was detected.The expression of mGluR5 on neutrophils was significantly induced after TBI within 24 h,followed by the declination to the nearly same on Days 14.10.In order to confirm that mGluR5 expressed in peripheral is important for neutrophil’s infiltration in TBI,we constructed the chimeras to exert the TBI model.We found that selective mGluR5 KO group performed better at modified neurological severity score compared with WT transplantation group on Day 1 and Days 3 after TBI.On Days 3,selective mGluR5 KO group displayed significantly lower Longa score and fewer hindlimb faults as whole mGluR5 KO group did.While on Days 7 and 14 these two groups showed no statistical difference.These data supported that selective blocking mGluR5 in peripheral was beneficial to alleviate the outcomes of TBI in acute phase.ConclusionsIn this study,we investigated the role of global knockout of mGluR5 in a mouse TBI model and demonstrate that inhibition of neutrophil infiltration are involved in the mGluR5deficiency-mediated protection against inflammation and brain damage post TBI.Here we for the first time address the important regulatory effect of mGluR5 on peripheral immune cells to affect the progress brain inflammation and the outcome of TBI and elucidate its associated mechanisms.Previously,to some extent,the role of mGluR5 in TBI have been reported.However,most of these studies treated animal models with mGluR5 agonists or antagonists via intra-cerebroventricular injetction.This treatment way predominantly affected mGluR5 in the brain,neglecting the peripheral effects exerted by mGluR5.Therefore,although these data provide a vital clue about mGluR5 function in TBI,they might not give people a whole and clear picture of mGluR5 activity in this injury,which limited the application of mGluR5-targeted therapy on TBI.In this study,we observed the role of mGluR5 in the acute phase of TBI via using mGluR5 global knockout mice and demonstrated that global knockout of mGluR5 protects against brain damage and attenuates neurological dysfunction in the acute phase of TBI.It has been reported that cell surface and intracellular mGluR5has different effects.Global knockout of mGluR5 inactivates both cell surface and intracellular mGluR5,while using mGluR5 antagonist generally regulates surface mGluR5.Accordingly,our results to some extent provide more credible data to understand the whole role of mGluR5 might play during TBI.Further,we found that much less neutrophil infiltration and inflammation was detected in the brain tissue of mGluR5 KO mice post-TBI,which is consistent with the protection of mGluR5 KO described above and supports the reports that the infiltrated neutrophils were the main contributor to brain inflammatory injury during the acute stage after TBI.On one hand,our results showed that mGluR5 deficiency dramatically reduced the BBB permeability accompanied with the upregulation of claudin-5 expression.These data are consistent with the evidence that mGluR5 on brain endothelial cells could modulate the permeability through phosphorylated vasodilator-stimulated phosphoprotein in hypoxic mouse model.On the other hand,we presented that the chemokine and chemokine receptor expressions of mGluR5 KO neutrophils and BMECs were significantly decreased when compared to that of WT mice.However,mGluR5 KO did not affect the number of neutrophils in blood or bone marrow.This confirmed that mGluR5 deficiency suppressed neutrophil infiltration rather than neutrophil generation.Since brain endothelial cells play important role of chemotaxis of leukocytes,we presented that blocking mGluR5 could inhibit chemokines secreted by brain endothelial cells to alleviate the infiltration of neutrophils.Meanwhile,this is possibly the first time to confirm that mGluR5 deficiency impairs chemokine related genes of neutrophils.The detailed mechamisms about how mGluR5 affect neutrophils’function need further investigations.Several researches have pointed out that mGluRs not only exert the essential effect in the CNS but also play a role in peripheral tissues,like gastrointestinal system,immune system and endothelial functions.For instance,mGluRs modulate gastrointestinal vagovagal reflexes,trafficking and activation of immune cells,permeability of endothelial cells and angiogenesis in tumor.Despite the fact that mGluR5s are relatively highly expressed in the CNS compared with peripheral tissues,some researchers demonstrated that mGluR5 was developmentally regulated.The expression of mGluR5 of peripheral immune cells including neutrophils,T cells,and dendritic cells could be induced and significantly regulates the activities of these cells in cases of pathological condition.Meanwhile,we confirmed that mGluR5 expression of neutrophils could be induced under TBI condition within 24 h,indicating mGluR5 plays an important role in functions during this process.The mGluR5 deficiency experiments also addressed the importance of mGluR5 peripheral effect on the CNS inflammation and brain damage.The function of mGluR5,especially in peripheral immune cells,needs to be re-examined and evaluated.Since in vitro BBB model was established by monolayers of rCMECs for the first time,numerous models built with cultured cells have been widely used in the structural and functional organization of BBB,the study of BBB permeability of drugs,and the process of inflammatory cells crossing the barrier.To mimic the neutrophil trafficking and infiltration after TBI,we constructed in vitro scratched BBB model.For the polyester membrane of transwell inserts is unelastic,we chose scratch instead of stretch.Using this model,we demonstrated that using mGluR5 specific antagonist could inhibit neutrophils’transmigration as mGluR5 KO animal did through suppressing CXCL1,CXCL2,CCL2,CCL4 and CCL5 release.During this process,the classical PKC pathway was crucial for the modulation of chemokines exerted by mGluR5.MGluR5 couples to G_q/G₁₁ and activates phospholipase Cβ,resulting in the hydrolysis of phosphotinositides and generation of inositol 1,4,5-trisphosphate and diacylglycerol.This classical pathway leads to the calcium mobilization and the activation of PKC.Being consistent with our results,Schmidt et al.found that the stimulation of mGluR5 is tightly connected with the activation of PLC and PKCγin accumbens nucleus shell.Chen et al.presented that mGluR5 modulates neuronal injury in PKC-dependent activation of MEK/ERK pathway.Last but not least,the comparisons between chimeras emphasized the significance of mGluR5 in peripheral.Although some issues,such as how mGluR5 influences the expression of chemokines in neutrophils,when is the suitable time window to block peripehral mGluR5,and why mGluR5 exerts opposite effects in the peripheral and the CNS,need be further addressed,our results show a new aspect of mGluR5 function in inflammation and brain damage after TBI,which might provide some potential novel strategies for TBI therapy.