periodontal health can be described as  a dynamic state where the activity of proinflammatory / antimicrobial cytokines to control infection is optimally balanced by anti - inflammatory mechanisms to prevent unwarranted inflammation  . in subjects susceptible to periodontal disease ( pd ) , an imbalance of the inflammatory response results in excessive production of proinflammatory cytokines and the subsequent loss of periodontal attachment . on the other hand ,
furthermore , the release of tissue regenerating factors may contribute to periodontal regeneration by regulating the function of periodontal ligament cells , endothelial cells , and cementoblasts . in this
setting , neurotrophin brain - derived neurotrophic factor ( bdnf ) has been reported to enhance periodontal tissue regeneration [ 4 , 5 ] .
bdnf is a member of the neurotrophin family which is expressed by vascular endothelium and osteoblastic , immune , and neuronal cells .
bdnf is reported to be involved in the joint inflammatory process and its production is increased in response to proinflammatory cytokines . although a role for bdnf in periodontal regeneration has been proposed , no information is available concerning bdnf and periodontal disease .
the aim of this study was to measure the levels of bdnf in periodontal tissues from patients with chronic periodontitis .
the presence of polymorphisms rs6265 and rs4923463 of the bdnf gene and its correlation with inflammatory and clinical parameters were also assessed .
twenty - eight patients with cp , treated at the periodontal clinic , school of dentistry , at universidade federal de minas gerais ( ufmg , brazil ) , were enrolled in this study .
patients in this study met the following inclusion criteria : previous history of cp , diagnosed according to previously described criteria : ( 1 ) exhibiting more than one tooth with probing depth higher than 5  mm , ( 2 ) exhibiting more than two sites with clinical attachment loss deeper than 6  mm , and ( 3 ) exhibiting lesions distributed in more than two teeth in each quadrant .
patients who met the following criteria were excluded : ( 1 ) having a history of smoking , ( 2 ) use of antibiotic , ( 3 ) usage of anti - inflammatory and/or immunosuppressive medications during the 6 preceding months , and ( 4 ) a history of any systemic diseases ( i.e. , immunologic and autoimmune disorders , diabetes mellitus ) .
the control group ( hc ) comprised 29 age and gender matched periodontally healthy patients enrolled for third molar removal surgery .
periodontal examination was performed in both groups of patients , cp and hc , at the initial visit to determine probing depth ( pd ) , clinical attachment loss ( cal ) , and bleeding on probing ( bop ) .
measurements were performed full - mouth at 6 sites per tooth ( mesiobuccal , midbuccal , distobuccal , mesiolingual , midlingual , and distolingual ) .
all the measurements were performed by the same examiner . at the time of the examination a peripheral blood sample
periodontal tissue samples from periodontal pockets or healthy oral mucosa extracted during surgery of impacted third molars were fixed in 10% buffered formalin , embedded in paraffin wax , and cut longitudinally ( 3  m ) .
the sections were deparaffinized , rehydrated , and stained with h&e for evaluation of the inflammatory infiltrate .
inflammatory cells were counted in four fields in two independent sections , using a light microscope ( axioskop 40 zeiss ; carl zeiss , gottingen , germany ) at 400x magnification .
the concentrations of the il-17a , bdnf , il-10 , and tnf- and the chemokine cxcl10 were measured in periodontal tissues by enzyme - linked immunosorbent assay ( elisa ) using commercially available kits ( r&d systems , minneapolis , mn , usa ) .
the lower limit of detection for each cytokine was 15  pg / ml , 3.9  pg / ml , 5.5  pg / ml , 20  pg / ml , and 4.5  pg / ml , respectively , for il-17a , il-10 , tnf- , bdnf , and cxcl10 . the data were determined using a standard curve prepared for each assay and expressed as picograms of cytokine / chemokine per 100  mg of tissue .
periodontal tissue samples were also used for determination of myeloperoxidase ( mpo ) activity , a neutrophil enzyme marker , as described earlier .
the mpo activity in homogenized periodontal tissues was evaluated by enzymatic reaction and absorbance was measured at 450  nm .
the mpo content was expressed as relative units calculated from standard curves based on mpo activities from 5% casein peritoneal - induced neutrophils assayed in parallel .
total genomic dna was extracted from blood samples using qiaamp dna blood mini kit ( qiagen , valencia , ca , usa ) according to manufacturer 's instructions .
quality , integrity , and quantity of dna were analyzed by nanodrop spectrophotometer ( thermo scientific , wilmington , de , usa ) .
all amplifications were carried out in an abi 7900h thermal cycler ( applied biosystems , foster city , ca , usa ) using taqman genotyping master mix and following manufacturer 's recommended amplification conditions .
chi - square test analysis was used to test for deviation of genotype frequencies from hardy - weinberg equilibrium .
the levels of cytokines in periodontal tissues and the frequency of gene polymorphisms were compared by the student 's t - test and chi - square tests .
the sample included in the current study was composed by age and gender matched groups .
the clinical features pd , cal , and bop were significantly higher in the cp than in the hc group ( p < 0.0001 ) ( table 1 ) .
the levels of il-17a , cxcl10 , il-10 , tnf- , and bdnf in periodontal tissues were greater in cp patients than in controls ( figure 1 ) .
moreover , the mpo activity and the inflammatory infiltrate in the periodontal tissues , characterized by polymorphonuclear and mononuclear leukocytes , were significantly higher in the cp than in the hc group ( figure 2 ) .
the bdnf and il-10 levels in periodontal tissues were negatively correlated ( r = 0.691 , p = 0.002 ) , whereas no correlation between bdnf and il-17a , tnf- , cxcl10 , or clinical parameters was observed ( pd , cal , and bop ) . following the clinical investigation , the frequencies of polymorphisms ( bdnf ) were assessed in blood samples of hc and cp subjects ( table 2 ) . the frequency of these genotypes agreed with the hardy - weinberg equilibrium ( p > 0.05 ) .
the distribution of the bdnf polymorphisms was similar between the groups ( table 2 ) .
we also investigated whether some of these polymorphisms were associated with worse clinical periodontal parameters .
as shown in table 3 , no differences in clinical parameters were found when comparing the genotypes .
the levels of bdnf and the inflammatory mediators cxcl10 and tnf- were increased in gg genotype of bdnf rs6265 polymorphism ( figures 3(a ) , 3(b ) , and 3(c ) ) , but mpo levels did not alter significantly ( figure 3(d ) ) . in bdnf
rs4923463 polymorphism the levels of bdnf and mpo did not differ , but the levels of cxcl10 and tnf- were higher in patients with aa genotype ( figures 3(e)3(h ) ) .
a wide range of nonneural cells in peripheral tissues or in the immune system expresses neurotrophins and their receptors .
thus , the mitogenic and immune regulatory functions of neurotrophins have been discussed [ 6 , 1012 ] .
the neurotrophin bdnf is reported to be involved in inflammatory reactions , and its production is increased in response to proinflammatory cytokines .
the present study is the first to demonstrate that bdnf levels were increased in periodontal tissues from chronic periodontitis compared to healthy subjects . in agreement with our findings
, bdnf was found in high levels in the plasma of patients with osteoarthritis   and in patients with rheumatoid arthritis .
while some authors   reported that bdnf levels were significantly correlated with self - reported pain , others   did not find association between bdnf and clinical parameters of arthritis .
several studies analyzed bdnf rs6265 polymorphisms in psychiatric disorders [ 1618 ] . to date , few studies examined the rs4923463 polymorphism [ 16 , 1820 ] .
while one study found correlation between snp rs4923463 and attention - deficit / hyperactivity disorder , schizophrenia , and risk of suicide in bipolar disorder , another study did not find any correlation between schizophrenia and rs4923463 polymorphism .
previously , two studies have evaluated the effects of bdnf polymorphisms in bone [ 21 , 22 ] , but there are no available studies in periodontal disease . in the present study we did not find differences in bdnf genotype distribution between patients with cp and controls .
nevertheless , we found that subjects with gg ( rs6265 ) genotype expressed higher levels of bdnf in periodontal tissues , in agreement with a previous report showing that bdnf - m66 variant alters intracellular trafficking and impairs bdnf secretion . on the other hand , rs6265 polymorphism
interestingly , the snp rs6265 was reported as a phossnp , which means this snp regulates protein phosphorylation .
the snp rs6265 affects substrate - kinase interaction between bdnf protein and chek2 kinase and regulates bdnf phosphorylation at site t62 .
subjects aa genotype carriers exhibited lower bone mineral density compared to g carriers . specifically , bdnf - v66 ( major allele g at rs6265 ) transfection significantly increases expression of osteoblast specific markers ( opn , bmp2 , and alp ) and promotes osteoblast differentiation and maturation in cell culture .
an association of rs6265 with bone metabolism was also suggested in the largest meta - analysis involving 32,961 individuals of european and east asian ancestry .
they found that homozygous minor allele a carriers ( aa ) have significantly decreased bmd compared to major allele g carriers ( ga and gg ) .
it has been reported that bdnf is able to induce an increase in il-10 expression . however , a negative correlation between the production of bdnf and il-10 was observed in samples from patients with periodontitis .
il-10 can inhibit the release of proinflammatory cytokines from monocytes / macrophages and can therefore inhibit the lipopolysaccharide- and ifn--induced secretion of inflammatory cytokines ( e.g. , tnf- , il-1 , il-6 , cxcl8 , and others ) . in periodontal disease , il-10 is thought to be associated with lower disease severity .
previous studies demonstrated that bdnf induces periodontal tissue regeneration by activation of cementoblasts differentiation , vascular endothelial cell migration , and also has a positive effect on bone remodeling [ 5 , 28 ] .
this data together suggested that bdnf has a role in bone remodeling and any change in this neurotrophin levels could have an impact in bone repair .
finally , we observe that cp subjects with gg ( rs6265 ) and aa ( rs4923463 ) genotypes demonstrated increased levels of tnf- and cxcl10 .
cxcl10 has several roles , such as chemoattraction of macrophages , t cells , nk cells , and dendritic cells .
in addition , previous studies showed that exposure to bdnf substantially and synergistically enhanced tnf- levels in vitro , and tnf- preconditioning increased proliferation , mobilization , and osteogenic differentiation in vitro .
we can hypothesize that the concomitant increase of bdnf , tnf- , and cxcl10 in patients with the gg genotype may be an attempt of the host to induce periodontal healing .
so , maybe if these patients were examined after periodontal treatment , they could display higher and better levels of tissue regeneration compared to patient who do not exhibit the gg genotype .
in conclusion , bdnf seems to be related to periodontal pathogenesis and also involved in tissue repair .
the results obtained here provide a benchmark for future studies with a large cohort of patients to help strengthen and understand the influence of neurotrophins in periodontal disease .
 brain - derived neurotrophic factor ( bdnf ) is a member of the neurotrophic factor family . outside the nervous system 
 , bdnf has been shown to be expressed in various nonneural tissues , such as periodontal ligament , dental pulp , and odontoblasts . 
 although a role for bdnf in periodontal regeneration has been suggested , a function for bdnf in periodontal disease has not yet been studied . 
 the aim of this study was to analyze the bdnf levels in periodontal tissues of patients with chronic periodontitis ( cp ) and periodontally healthy controls ( hc ) . 
 all subjects were genotyped for the rs4923463 and rs6265 bdnf polymorphisms . 
 periodontal tissues were collected for elisa , myeloperoxidase ( mpo ) , and microscopic analysis from 28 cp patients and 29 hc subjects . 
 bdnf levels were increased in cp patients compared to hc subjects . 
 a negative correlation was observed when analyzing concentration of bdnf and il-10 in inflamed periodontium . 
 no differences in frequencies of bdnf genotypes between cp and hc subjects were observed . 
 however , bdnf genotype gg was associated with increased levels of bdnf , tnf- , and cxcl10 in cp patients . in conclusion 
 , bdnf seems to be associated with periodontal disease process , but the specific role of bdnf still needs to be clarified . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion