age - related changes in human salivary glands have been described by some studies in humans 














  and animals 


 . the literature regarding aging alterations in human sublingual glands is limited by the number of reports , and some phenomena are still unclear . despite the different methodologies used in the investigations of age - related microscopic changes in the salivary glands , the consensus is that there is a decrease in the volume of parenchyma that is accompanied by an increase in fat and connective tissue 














 . this aging process also includes acinar atrophy and an increase in the number of duct - like structures ( dls ) 














  ( figure 1 ) . the term  duct - like structures is commonly used because there is no evidence that the structures are true ducts . for many researchers , the appearance of dls with age represents the final process of acinar atrophy , and the making the distinction between a duct and a dls can be very difficult 














 . furthermore , the dls do not represent a specific phenomenon of senescence , and they also appear in atrophic glands 


  and glandular neoplasms 


 . figure 1a : normal gland  26 years old ( group i ) . preserved acini and absence of duct - like structures ( dls ) are observed ; b : altered gland  presence of dls , absence of acini and substitution of the glandular parenchyma by connective tissue  68 years old ( group ii ) ( hematoxylin and eosin 100x ) 
  some authors also believe that , during the aging process , atrophic acini can resemble ducts , and the distinguishing between them can be very difficult or even impossible 














 , demonstrating that the morphology of aged salivary glands is not completely established . regarding immunohistochemical studies related to the salivary glands , most of them only aim to characterize their epithelial and/or myoepithelial components 








  or to study pathological processes such as neoplasias 


 . it is evident that the literature regarding age - related changes in salivary glands is not abundant . in humans , studies are scarce and the sublingual gland is rarely contemplated . the increase in the number of dls allows speculation that a mitotic process is occurring within the cells or even a transition of an acinus to a duct 


  because the acini disappear while the dls increase in number . the aim of this work is to evaluate the phenotype and the cell proliferation index of dls in human sublingual glands during the aging process . this study was approved by the human research ethics committee of the bauru school of dentistry at the university of so paulo ( usp ) ( process n 59/2010 ) and also by the so paulo death verification service ( usp ) , where the glands were obtained . the methods and the exclusion criteria were the same as those used by azevedo , et al . individuals with the following systemic disorders or base diseases were excluded from the study : lymphoma , leukemia , mucoviscidosis , rheumatic diseases , sjgren s syndrome , obesity , cachexia , diabetes mellitus , alcoholic cirrhosis , collagen diseases , history of head or neck cancer - related surgery , history of cytotoxic drug administration , previous radiotherapy of the head or neck during the last 3 months and macroscopic autolysis . data regarding systemic involvement and cause of death were obtained from the family and the autopsy reports . the main causes of death included pulmonary edema , bronchopneumonia , acute myocardial infarction , cerebral vascular accident , ischemic heart disease , cerebral edema and congestive heart failure . the interval between the time of death and the autopsy ranged from 6:05 to 92:55 h , with a mean time of 16:46 h. the cadaver glands were completely removed along with a portion of the overlying mucosa of the mouth floor . the glands from the cadavers were divided into two groups : group i ( control ; 0 - 30 years old ) and group ii ( 60 - 90 years old ) . the paraffin - embedded material was cut into 3 - 5 m - thick sequential slices . for the study of the cellular phenotype of the dls , antibodies against cytokeratin 19 ( ck 19 ; clone rck108 ; isotype igg1/mouse ; monoclonal ; dako , dako inc , carpinteria , ca , usa , cod#m0888 ; dilution 1:100 ) and the s-100 protein ( s-100 ; isotype igg / rabbit ; polyclonal ; dako , dako inc , carpinteria , ca , usa , cod#z0311 ; dilution 1:2000 ) were used . the anti - ck 19 antibody was used to stain the cells of the ductal system 






  and anti s-100 to stain the serous acinar cells 


 . the histochemical markers periodic acid - schiff ( pas ) and alcian blue ( ph 2.5 ) were used to stain the mucous acinar cells . for the analysis of the cell proliferation capacity of the dls , the ki-67 ( ki-67 ; clone mib-1 ; isotype igg1/mouse ; monoclonal ; dako , dako inc , carpinteria , ca , usa , cod#m720 ; dilution 1:400 ) antibody was used 




 . the immunohistochemical study was performed using the polymeric horseradish peroxidase ( hrp)-linker antibody conjugate technique . the material was incubated in a humidity chamber at 4c for 18 hours . considering the large s-100 protein family , the polyclonal rabbit anti - s-100 was used , which reacts strongly with all family members , including human s100a and b sub - units . the specimens were counterstained with harris hematoxylin , and the slides were mounted using adhesive resin ( entellan , merck , darmstadt , hessen , germany ) and coverslips . for all immunohistochemical markers , both a positive and a negative control were prepared according to the manufacturers recommendations . a digital image - capture computer system ( axiocam mrc , zeiss , jena , thuringia , germany ) and a binocular microscope ( axioskop 2 plus , zeiss , jena , thuringia , germany ) with a 40x objective were used . the images were analyzed using the software supplied with the microscope ( axiovision 4.5 , zeiss , jena , thuringia , germany ) . each microscopic field was previously standardized in this program to avoid differences in the expression intensity of the antibodies . in each captured microscopic field , the dls were counted to establish their percentage in the staining profile . the most representative areas of the event ( spots ) , identified using a lower magnification , were selected . when the dls were diffusely distributed and spread out , 20 spots were randomly selected . in group i , all microscopic fields containing dls were captured and in group ii , 20 fields containing dls were randomly captured ( figure 2 ) . true ducts or acini that were easily recognized were excluded from the count . 
 figure 2schematic representation of the selection of spots . a : due to the small quantity of duct - like structures ( dls ) in group i , all microscopic fields containing dls were captured ; b : a spot containing three dls ; c : 20 fields containing dls were randomly captured in group ii ; d : diffuse distribution of dls in group ii 
  for the localization and the counting of the dls , slides stained with hematoxylin - eosin ( h.e . ) were used . we considered dls to be those with shrunken seromucous cells , granule depletion and widened lumens that were adjacent to ductal epithelial cells and resembled intralobular non - striated ducts , which is consistent with the characterization used in our previous study 
 . we excluded true excretory ducts , which exhibited a dilated lumen lined by cuboidal cells with central nuclei and an eosinophilic cytoplasm 
 . a section of each gland was analyzed with each studied molecule , totaling 60 slides for each staining ( 30 per group ) . therefore , a total of 360 slides were analyzed [ 60 for each marker : h.e . , pas , alcian blue ( ph 2.5 ) , ck 19 , s-100 and ki-67 ] . a semi - quantitative scoring system was applied to the immunohistochemical and histochemical analysis [ adapted from soini , et al . both analyses resulted in a final score that represented no expression , weak expression or strong expression . in each microscopic field , the percentage of positive ( with partial or total staining ) dls was calculated for each marker . scores ranging from 0 to 4 were attributed to those percentage values : 0 ) absence of positive dls ; 1 ) < 25% of positive dls ; 2 ) 26 - 50% of positive dls ; 3 ) 51 - 75% of positive dls ; 4 ) > 75% of positive dls . then , for the dls positive samples , the partial or total covering and the intensity of the immunostaining in the positive cells were calculated following the same method applied previously . finally , the sum of the values attributed to the quantity , covering and intensity of the immunostaining resulted in a final score where 3 - 7 represented weak to moderate expression and 8 - 12 was a strong expression of the antibody . basically , the pas and alcian blue staining in the dls followed the same scoring system , except for the intensity values , which did not exist for these stains . the cellular proliferative activity in the dls , revealed by ki-67 staining , was calculated as the percentage of positive cells to establish a cellular proliferation index in the same previously captured fields . the comparison between groups and between markers was accomplished by the student s t - test or the mann - whitney test . the correlation among the staining groups the exclusion criteria were the same as those used by azevedo , et al . individuals with the following systemic disorders or base diseases were excluded from the study : lymphoma , leukemia , mucoviscidosis , rheumatic diseases , sjgren s syndrome , obesity , cachexia , diabetes mellitus , alcoholic cirrhosis , collagen diseases , history of head or neck cancer - related surgery , history of cytotoxic drug administration , previous radiotherapy of the head or neck during the last 3 months and macroscopic autolysis . data regarding systemic involvement and cause of death were obtained from the family and the autopsy reports . the main causes of death included pulmonary edema , bronchopneumonia , acute myocardial infarction , cerebral vascular accident , ischemic heart disease , cerebral edema and congestive heart failure . the interval between the time of death and the autopsy ranged from 6:05 to 92:55 h , with a mean time of 16:46 h. the cadaver glands were completely removed along with a portion of the overlying mucosa of the mouth floor . the glands from the cadavers were divided into two groups : group i ( control ; 0 - 30 years old ) and group ii ( 60 - 90 years old ) . the paraffin - embedded material was cut into 3 - 5 m - thick sequential slices . for the study of the cellular phenotype of the dls , antibodies against cytokeratin 19 ( ck 19 ; clone rck108 ; isotype igg1/mouse ; monoclonal ; dako , dako inc , carpinteria , ca , usa , cod#m0888 ; dilution 1:100 ) and the s-100 protein ( s-100 ; isotype igg / rabbit ; polyclonal ; dako , dako inc , carpinteria , ca , usa , cod#z0311 ; dilution 1:2000 ) were used . the anti - ck 19 antibody was used to stain the cells of the ductal system 






  and anti s-100 to stain the serous acinar cells 


 . the histochemical markers periodic acid - schiff ( pas ) and alcian blue ( ph 2.5 ) were used to stain the mucous acinar cells . for the analysis of the cell proliferation capacity of the dls , the ki-67 ( ki-67 ; clone mib-1 ; isotype igg1/mouse ; monoclonal ; dako , dako inc , carpinteria , ca , usa , cod#m720 ; dilution 1:400 ) antibody was used 




 . the immunohistochemical study was performed using the polymeric horseradish peroxidase ( hrp)-linker antibody conjugate technique . the material was incubated in a humidity chamber at 4c for 18 hours . considering the large s-100 protein family , the polyclonal rabbit anti - s-100 was used , which reacts strongly with all family members , including human s100a and b sub - units . the specimens were counterstained with harris hematoxylin , and the slides were mounted using adhesive resin ( entellan , merck , darmstadt , hessen , germany ) and coverslips . for all immunohistochemical markers , both a positive and a negative control were prepared according to the manufacturers recommendations . a digital image - capture computer system ( axiocam mrc , zeiss , jena , thuringia , germany ) and a binocular microscope ( axioskop 2 plus , zeiss , jena , thuringia , germany ) with a 40x objective were used . the images were analyzed using the software supplied with the microscope ( axiovision 4.5 , zeiss , jena , thuringia , germany ) . each microscopic field was previously standardized in this program to avoid differences in the expression intensity of the antibodies . in each captured microscopic field the most representative areas of the event ( spots ) , identified using a lower magnification , were selected . when the dls were diffusely distributed and spread out , 20 spots were randomly selected . in group i , all microscopic fields containing dls were captured and in group ii , 20 fields containing dls were randomly captured ( figure 2 ) . a : due to the small quantity of duct - like structures ( dls ) in group i , all microscopic fields containing dls were captured ; b : a spot containing three dls ; c : 20 fields containing dls were randomly captured in group ii ; d : diffuse distribution of dls in group ii 
  for the localization and the counting of the dls , slides stained with hematoxylin - eosin ( h.e . ) were used . we considered dls to be those with shrunken seromucous cells , granule depletion and widened lumens that were adjacent to ductal epithelial cells and resembled intralobular non - striated ducts , which is consistent with the characterization used in our previous study 
 . we excluded true excretory ducts , which exhibited a dilated lumen lined by cuboidal cells with central nuclei and an eosinophilic cytoplasm 
 . a section of each gland was analyzed with each studied molecule , totaling 60 slides for each staining ( 30 per group ) . therefore , a total of 360 slides were analyzed [ 60 for each marker : h.e . , pas , alcian blue ( ph 2.5 ) , ck 19 , s-100 and ki-67 ] . a semi - quantitative scoring system was applied to the immunohistochemical and histochemical analysis [ adapted from soini , et al . both analyses resulted in a final score that represented no expression , weak expression or strong expression . in each microscopic field , the percentage of positive ( with partial or total staining ) dls was calculated for each marker . scores ranging from 0 to 4 were attributed to those percentage values : 0 ) absence of positive dls ; 1 ) < 25% of positive dls ; 2 ) 26 - 50% of positive dls ; 3 ) 51 - 75% of positive dls ; 4 ) > 75% of positive dls . then , for the dls positive samples , the partial or total covering and the intensity of the immunostaining in the positive cells were calculated following the same method applied previously . finally , the sum of the values attributed to the quantity , covering and intensity of the immunostaining resulted in a final score where 3 - 7 represented weak to moderate expression and 8 - 12 was a strong expression of the antibody . basically , the pas and alcian blue staining in the dls followed the same scoring system , except for the intensity values , which did not exist for these stains . the cellular proliferative activity in the dls , revealed by ki-67 staining , was calculated as the percentage of positive cells to establish a cellular proliferation index in the same previously captured fields . the comparison between groups and between markers was accomplished by the student s t - test or the mann - whitney test . the correlation among the staining groups the kappa coefficients ( k ) for the s-100 , ck 19 , pas and alcian blue variables were 87.50% , 78.13% , 96.88% , and 98.88% , respectively . this variation could be observed among different glands or even among different lobes of the same gland . overall , the glands of young people were intact , with serous and mucous acini well delineated and absent or few and sparse dls ( figure 1a ) . in the glands of the elderly , certain sections had preserved lobes while others had characteristics inherent to senility , which is indicated by the intense replacement of the glandular parenchyma by fibrous and fatty tissue and many dls ( figure 1b ) . in group i , from the 30 slides analyzed , only 12 had dls , whereas in group ii , only 2 slides did not show these structures . when comparing both groups , only the immunostaining for ck 19 showed a statistically significant difference between them ( p=0.033 ) , with stronger expression in the elderly group . although the expression for s-100 was also stronger in group ii , the difference was not significant ( p=1.000 ) ( table 1 ) . table 1comparison between groups regarding histochemical and immunohistochemical staininggroups  iiip  ( n=12)(n=28 )  median1 quartile3 quartilemedian1 quartile3 quartile  s-1000.0000.0006.0001.2860.0003.8751.000ck-199.0006.00011.62510.8409.08312.0000.033*pas0.0000.0000.0000.0000.0000.4580.536aa0.0000.0001.8750.0000.0000.9170.907mann - whitney test ( p<0.05 ) . alcian blue serous acini ( identified by s-100 expression ) were present in the dls with a lower frequency and a weaker intensity than the ck 19 expression levels . in contrast , the expression of ck 19 was strong in the dls and very similar to the immunostaining in the ducts . the serous acini exhibited strong immunostaining for s-100 and moderate , weak or absent expression of ck 19 . the ducts were invariably negative for s-100 and positive for ck 19 ( figure 3 ) . figure 3a : duct - like structures ( dls ) with weak or absent s-100 expression . a strong expression of s100 seen in the nerve cells ( short arrow ) can be regarded as the positive control . serous cells with strong expression ( arrow ) ( group ii ) ; b : strong expression of ck 19 in some cells in the dls . serous cells with weak expression ( group ii ) ; c : positive s-100 expression in serous acinar cells ( arrows ) and negative in ducts and dls ( short arrows ) ( group i ) ; d : strong ck 19 expression in ducts and dls ( arrows ) and weak or moderate expression in serous acinar cells ( group ii ) 
  regarding group i , the immunostaining of the dls for ck 19 was stronger than for s-100 , with a statistically significant difference observed ( p=0.004 ) ( table 2 ) . table 2comparison between s-100 and ck 19 immunostaining of duct - like structures in groups i and iigroup i 
 s-100ck 19  meansdsemmeansdsemp2.5833.3700.9737.6254.2161.2170.004 * 

 group ii 

 s-100 

 ck 19 

 median 

 1 
  quartile 

 3 
  quartile 

 median 

 1 
 quartile 

 3 
  quartile 


 1.2860.0003.87510.8409.08312.000<0.001 * 
 student s t - test ( p<0.05 ) . ii , the same circumstance was observed and this difference was even more significant ( p<0.001 ) ( table 2 ) . this condition was also seen in the descriptive microscopic analysis because in the senile glands , the dls strongly expressed ck 19 ( figure 4a ) , showing a ductal pattern . in some sections , we also observed the presence of areas with a mixture of acinar and ductal cells ( figure 4b ) . figure 4a : similar ck 19 expression between ducts ( * ) and duct - like structures ( group ii ) . the ductal cells are stained with the anti- ck 19 ( group ii ) . 
  when the pearson correlation coefficient was applied , the correlation between ck 19 and s-100 was absent ( =-0.163 ; p=0.315 ) . regarding the pas and alcian blue staining for mucous acini , both markers worked similarly , where the staining was evident in the mucous acini and quite rare in the dls ( figure 5a ) . when there was staining , it was restricted to a small portion of the structure and was classified as partial ( figure 5b ) . these markers also showed , in some cases , the presence of mucin within the ducts or dls ( figure 5c ) . there was no statistically significant difference between pas and alcian blue ( p=0.270 ) . figure 5a : mucous acini stain for periodic acid - schiff ( pas ) and alcian blue . the isolated duct - like structures ( dls ) is negative for both markers ( arrows )  group i ( 21 years old ) ; b : partial staining of a dls for pas and alcian blue ( arrows ) . group ii ( 76 years old ) ; c : negative dls for pas and alcian blue . presence of mucus in the lumen of a dls ( arrows ) . group ii ( 76 years old ) 
  in both groups , there was no immunostaining for the ki-67 antibody in any dls ( figure 6 ) . the expression of various basal cells of the epithelium of the oral mucosa worked as a positive controlfor the antibody used . figure 6absence of nuclei expression of ki-67 in the duct - like structures ( group ii ) this variation could be observed among different glands or even among different lobes of the same gland . overall , the glands of young people were intact , with serous and mucous acini well delineated and absent or few and sparse dls ( figure 1a ) . in the glands of the elderly , certain sections had preserved lobes while others had characteristics inherent to senility , which is indicated by the intense replacement of the glandular parenchyma by fibrous and fatty tissue and many dls ( figure 1b ) . in group i , from the 30 slides analyzed , only 12 had dls , whereas in group ii , only 2 slides did not show these structures . when comparing both groups , only the immunostaining for ck 19 showed a statistically significant difference between them ( p=0.033 ) , with stronger expression in the elderly group . although the expression for s-100 was also stronger in group ii , the difference was not significant ( p=1.000 ) ( table 1 ) . table 1comparison between groups regarding histochemical and immunohistochemical staininggroups  iiip  ( n=12)(n=28 )  median1 quartile3 quartilemedian1 quartile3 quartile  s-1000.0000.0006.0001.2860.0003.8751.000ck-199.0006.00011.62510.8409.08312.0000.033*pas0.0000.0000.0000.0000.0000.4580.536aa0.0000.0001.8750.0000.0000.9170.907mann - whitney test ( p<0.05 ) . alcian blue serous acini ( identified by s-100 expression ) were present in the dls with a lower frequency and a weaker intensity than the ck 19 expression levels . in contrast , the expression of ck 19 was strong in the dls and very similar to the immunostaining in the ducts . the serous acini exhibited strong immunostaining for s-100 and moderate , weak or absent expression of ck 19 . the ducts were invariably negative for s-100 and positive for ck 19 ( figure 3 ) . figure 3a : duct - like structures ( dls ) with weak or absent s-100 expression . a strong expression of s100 seen in the nerve cells ( short arrow ) can be regarded as the positive control . serous cells with strong expression ( arrow ) ( group ii ) ; b : strong expression of ck 19 in some cells in the dls . serous cells with weak expression ( group ii ) ; c : positive s-100 expression in serous acinar cells ( arrows ) and negative in ducts and dls ( short arrows ) ( group i ) ; d : strong ck 19 expression in ducts and dls ( arrows ) and weak or moderate expression in serous acinar cells ( group ii ) 
  regarding group i , the immunostaining of the dls for ck 19 was stronger than for s-100 , with a statistically significant difference observed ( p=0.004 ) ( table 2 ) . table 2comparison between s-100 and ck 19 immunostaining of duct - like structures in groups i and iigroup i 
 s-100ck 19  meansdsemmeansdsemp2.5833.3700.9737.6254.2161.2170.004 * 

 group ii 

 s-100 

 ck 19 

 median 

 1 
  quartile 

 3 
  quartile 

 median 

 1 
 quartile 

 3 
  quartile 


 1.2860.0003.87510.8409.08312.000<0.001 * 
 student s t - test ( p<0.05 ) . ii , the same circumstance was observed and this difference was even more significant ( p<0.001 ) ( table 2 ) . this condition was also seen in the descriptive microscopic analysis because in the senile glands , the dls strongly expressed ck 19 ( figure 4a ) , showing a ductal pattern . in some sections , we also observed the presence of areas with a mixture of acinar and ductal cells ( figure 4b ) . figure 4a : similar ck 19 expression between ducts ( * ) and duct - like structures ( group ii ) . b : mixed structure with ductal and acinar cells . the ductal cells are stained with the anti- ck 19 ( group ii ) . 
  when the pearson correlation coefficient was applied , the correlation between ck 19 and s-100 was absent ( =-0.163 ; p=0.315 ) . regarding the pas and alcian blue staining for mucous acini , both markers worked similarly , where the staining was evident in the mucous acini and quite rare in the dls ( figure 5a ) . when there was staining , it was restricted to a small portion of the structure and was classified as partial ( figure 5b ) . these markers also showed , in some cases , the presence of mucin within the ducts or dls ( figure 5c ) . there was no statistically significant difference between pas and alcian blue ( p=0.270 ) . figure 5a : mucous acini stain for periodic acid - schiff ( pas ) and alcian blue . the isolated duct - like structures ( dls ) is negative for both markers ( arrows )  group i ( 21 years old ) ; b : partial staining of a dls for pas and alcian blue ( arrows ) . group ii ( 76 years old ) ; c : negative dls for pas and alcian blue . presence of mucus in the lumen of a dls ( arrows ) . in both groups , there was no immunostaining for the ki-67 antibody in any dls ( figure 6 ) . the expression of various basal cells of the epithelium of the oral mucosa worked as a positive controlfor the antibody used . figure 6absence of nuclei expression of ki-67 in the duct - like structures ( group ii ) the sublingual glands used in this work are the same as those used by azevedo , et al . ( 2005).in regards to the gender of the cadavers and the side of the glands , previous studies have already demonstrated that there are no differences in these variables 




 . we consider our samples to be very rich and consistent because the sublingual glands were removed from the cadavers after a short post - mortem period . moreover , the sublingual glands are rarely addressed in previous studies , where they are typically derived from rats 




 . only the works of the present line of research use human sublingual glands 






 . because dls are not an exclusive phenomenon of senescence , group i was used in this study as a control to observe whether there were differences in the staining pattern of dls in this group in comparison to group ii . when a gland from group i exhibited dls , the number was very low and often limited to the extremity of the lobe . we believe that the presence of dls in the glands of young individuals may be due to some injury , i.e. , small traumas to the mouth floor during feeding and microbstructions of the peripheral ductal system . moreover , these structures have been shown in studies that induce glandular atrophy through the duct obstruction 


  or in pathological processes such as neoplasms 


 . therefore , despite the presence of dls upon cellular senescence , they are not exclusive in this age group , which justifies why we opted to include group i in the present study . in regards to normal salivary glands , several studies demonstrate that ck 19 is strongly expressed in all parts of the ductal system and shows weak response to serous and mucous acinar cells 


 . for geiger , et al . ( 2002 ) , ck 19 is expressed in ductal cells but completely absent in acinar cells . regarding the s-100 protein , studies in normal salivary glands and tumors showed strong expression in serous acinar cells and weak expression in the ductal segments 


 . in this study , ck 19 was strongly expressed in true ducts and most of the dls . the ck 19 expression in the serous acini was weaker or absent and was not detected in the mucous acini . however , the s-100 protein was expressed in serous acinar cells and absent in true ducts and mucous acinar cells . the difference in the immunostaining of ck 19 in the dls between groups was significant ( p=0.033 ) ( table 1 ) . the expression of this protein was higher in the elderly , indicating the possibility that , over time , the dls begin to express the molecules of the ductal system more strongly . in both groups , there was a statistically significant difference between ck 19 and s-100 ( tables 4 and 5 ) with more intense ck 19 staining , demonstrating a ductal pattern of the dls . it is possible that the dls increase their ductal pattern with age while they simultaneously decrease the expression of s-100 . regarding the cell proliferation activity , we did not find any cells positive for ki-67 in the dls of both groups ( figure 6 ) . the effectiveness of the antibody used was proven by the presence of nuclear staining in various basal cells of the oral mucosa epithelium in the same sections . ( 2002 ) who found a proliferation index from 0% in acinar and ductal cells from young and senile human labial glands . ( 2002 ) , the staining for pcna in ductal cells is high in the early stages of glandular atrophy and subsequently decreases . instead , the dls are commonly observed in the later stages of atrophy , suggesting that both mitosis ( early stages ) and the transition from acinar to ductal cells ( later stages ) contribute to the increase in the number of ducts in the atrophic sublingual glands . these findings may explain the absence of immunostaining for ki-67 in the dls , demonstrating that they represent a late stage of atrophy ( or aging ) where mitosis is no longer observed . however , even in the dls of young glands , there was no evidence of cell proliferation , which is difficult to explain unless the authors used another marker ( pcna ) . based on our results , we do not support the hypothesis that mitosis explains the increase in the number of dls . ( 2002 ) that the dls represent the final process of acinar atrophy , we can infer that , in this investigation , s-100 was expressed in the dls due to the presence of serous acinar cells that remained from the atrophy process . unlike serous cells , there was rarely any trace of mucous cells in the dls ( figure 5b ) . therefore , we hypothesized that , if these structures are actually products of an acinar regression , this acinus is mixed , where mucous cells degenerate while the serous portion is more resistant and occasionally remains active . this finding corroborates other studies in humans 




 , in which the serous cells showed greater resistance to autolysis compared to the mucous cells . frequently , the areas of mixed serous and ductal cells were visualized as illustrated in figure 4b . ( 2002 ) demonstrated that evidence of a mixed phenotype suggests the transition and differentiation of cells through metaplasia . this transition from one acinus to a duct may be a result of epithelial metaplasia , which is defined as the transition from a reactive differentiated epithelium to another type of differentiated epithelium characterized by an impaired function or an alteration that is not physiologically normal in that place 
 . salivary glands exhibit a variety of metaplastic changes 
 and this could explain the cellular phenotypic changes resulting from aging . for mintz and mooradian 
  ( 1987 ) , the sublingual glands underwent squamous metaplasia of the ductal epithelium with age . according to ihrler , et al . ( 2002 ) , the majority of salivary gland metaplasias develop from basal cells of striated and excretory ducts that have an enormous capacity for pluridirectional morphogenetic differentiation . for us , our results strongly suggest metaplasia to explain this age - related change in human salivary glands . we believe in a dynamic process that arises in the acinus and culminates in a complete transformation of a duct . because the acinar cells originate from the primitive ductal cells , the embryological phenomenon maybe the way that the reverse process can occur . extrapolating this data to the clinic , we understand that the acini fail to work as secretory units and start to conduct the saliva . at this moment this could explain the decrease in the function of the saliva with age . according to dayan , et al . ( 2000 ) , the decrease in acini volume may explain the decrease in secretion by the salivary glands with age . according to scott 
  ( 1977 ) , the junction of all age - related microscopic changes may explain the decrease in glandular function . however , despite proof that acini decrease with age , certain studies support that saliva gland functions remain stable in healthy elderly patients 


 . ( 2014 ) demonstrated that symptoms of oral dryness ( xerostomia ) are not associated with age - related degenerative changes in human labial salivary glands . the authors suggest that the autonomic innervation of labial salivary glands may play a role in the occurrence of xerostomia and that the integrity of labial salivary gland acini is related to the parenchymal autonomic innervation 
 . one hypothesis is that the secretion reserve capacity of the glands can be affected by external factors such as radiation or drug use , but not by senescence 
 . because there is a difficulty in collecting saliva coming from the sublingual gland and lacked studies with direct tests of salivary flow , we can not firmly conclude if there is a decrease in salivary flow of the sublingual gland with age . although the clinical impact of the age - related changes in the salivary glands is still unclear , we think this work represents a contribution to pathologists . because these structures are commonly found in mouth floor , pre - prosthetic surgery specimens and in neoplasias of the major and minor salivary glands , these findings may help explain the phenomenon of the appearance of multiple dls when elaborating microscopic pathology reports . the present study demonstrated that duct - like structures present a ductal phenotypic profile and do not present cell proliferation activity . they may represent a regressive process arising from acini or represent the result of metaplasia .