i , 
 sections of cervix were stained for collagen and smooth muscle using masson s trichrome and 
 determined by computerized image analysis . part ii determined the types and proportions of 
 cervical gags using alcian blue 8gx staining . healthy bitches were grouped according to the 
 stage of their estrous cycle , and bitches with pyometra were classified as open- or 
 closed - cervix . animals : twenty eight healthy cyclic bitches of various breeds were used 
 in the study . the average age was 2.1  0.8 years ( mean  sem , range 14 years ) . at the time 
 of ovariohysterectomy , 10 bitches were classified as in anestrus , 7 in estrus and 11 in 
 diestrus . another group of 28 bitches with pyometra ( open - cervix , n=18 ; closed - cervix , n=10 ) 
 and a mean age of 7.0  3.9 years ( range , 215 years ) were also included . the stages of the estrous cycle were initially sorted by the reproductive history , vaginal 
 cytology , ovarian morphology ( i.e. , number and size of follicles , corpora hemorrhagica and 
 corpora lutea ) , combined with serum progesterone concentrations and histology of 
 hematoxylin - eosin stained sections of the uterine part of the cervix ( table 1table 1.criteria for estrous cycle staging in healthy bitchesstage of estrous cyclemajority of vaginalepithelial cellsovarian structureprogesterone concentration 
 ( ng / ml ) ( mean  sd ) ( range)uterine histologyanestrusbasal cellsquiescent 0.5  0.4 ( 0.11.2)inactive phaseestruscornified cellslarge follicles ( > 0.5 cm ) and corpora 
 hemorrhagica5.1  2.9 ( 1.18.9)proliferative to early secretory stagediestrusnon - cornified cellscorpora lutea ( > 0.5 cm ) and/or corpora 
 hemorrhagica12.8  10.0 ( 1.337.8)secretory stage ) . a diagnosis of pyometra was based on clinical signs , hematology and an 
 ultrasound scan of the uterus . open- and closed - cervix pyometra were classified according to 
 the presence or absence of purulent vulvar discharge , respectively . collection and processing of samples : cervical tissue samples were 
 collected surgically and prepared as previously described . briefly , the cervix was longitudinally cut from the internal to external os , 
 fixed in 4% paraformaldehyde for 3648 hr and embedded in paraffin wax . samples were 
 sectioned at 4 m and then placed on coated slides 
 ( 3-aminopropyl - triethoxysilane , minimum 98% ; sigma - aldrich , taufkirchen , germany ) for 
 masson s trichrome or alcian blue staining . hormone analyses : blood samples were collected from the cephalic vein by 
 venipuncture ; serum was separated and stored at 20c . progesterone concentrations were 
 determined using a luminescence immunoassay ( immulite progesterone , diagnostic products , los 
 angeles , ca , u.s.a . ) . the intra - assay coefficients of variation were 3.9% at 0.1 
 ng / ml and 6.5% at 36.1 
 ng / ml . the inter - assay coefficients of variation were 
 3.8% at 0.1 ng / ml and 16.3% at 36.1 
 ng / ml . the minimal assay sensitivity was 0.04 
 ng / ml . staining with masson s trichrome and the determination of collagen and smooth 
 muscle areas : to determine the proportion of collagen to smooth muscle , tissue 
 sections were deparaffinized , rehydrated with graded alcohols , immersed in warm bouin s 
 solution ( sigma - aldrich , st . louis , mo , u.s.a . ) ( 5560c ) for 2 hr and washed in running tap 
 water for 2 min followed by distilled water for 3060 sec . thereafter , they were stained 
 with weigert hematoxylin ( merck , darmstadt , germany ) for 10 min and rinsed until only the 
 cell nuclei were stained . smooth muscle was stained red with biebrich scarlet - acid - fuschin 
 ( sigma - aldrich ) for 10 min , rinsed and immersed in phosphomolybdic phosphotungstic acid 
 ( sigma - aldrich ) for 15 min . subsequently , collagen was stained blue with aniline blue 
 ( sigma - aldrich ) for 10 min and rinsed in distilled water followed by immersion in 1% acetic 
 acid for 5 min . finally , the tissues were rehydrated with 95 and 100% ethanol , left to air 
 dry and mounted with mounting medium . color image analysis was performed under a light microscope at 40 magnification . a total 
 of 5 to 10 reading fields the blue ( collagen ) and red ( muscle ) stained area of the cervical tissue was measured using 
 a software program ( image - pro plus 6.0 , media cybernetics , inc . , proportion of collagen and muscle was calculated by the number of pixels of either blue 
 ( collagen ) or red ( muscle ) color using an automated detection system , producing a percentage 
 area for collagen and muscle in a tissue section . according to the lack of collagen in the 
 luminal epithelium , the proportion of collagen to smooth muscle was evaluated only in the 
 two tissue layers , e.g. stroma and muscle . the mean proportion of collagen and muscle and 
 standard errors of means were calculated from 5 to 10 readings per section . staining with alcian blue 8gx and the determination of gags : tissue gags 
 were determined by alcian blue staining as previously described [ 14 , 23 ] . briefly , tissue sections 
 were de - waxed , rehydrated and immersed in 0.025 m sodium acetate buffer , ph 5.8 , containing 
 0.05% alcian blue 8gx ( sigma , poole , u.k . ) with different concentrations of mgcl2 
 ( 0.05 m , 0.4 m and 0.8 m ) for 18 hr at room temperature . sections were washed three times in 
 the corresponding buffer containing mgcl2 , dehydrated and mounted in dpx . alcian blue stains different types of gags at different concentrations of mgcl2 
 . at low concentrations ( 0.05 m 
 mgcl2 ) , all gags stain ; at 0.4 m mgcl2 , all the sulfated gags 
 ( chondroitin sulfate , cs ; dermatan sulfate , ds ; heparan sulfate , hs ; and keratan sulfate , 
 ks ) stain and at 0.8 m mgcl2 only highly sulfated gags ( ks and hs ) stain . three cervical cell layers , i.e. luminal epithelium , stroma and muscle , were separately 
 analyzed for the intensity of staining with alcian blue using a vickers m85a scanning and 
 integrating microdensitometer . ten readings of 
 each cell layer at each concentration of mgcl2 were taken at 550 nm . absolute 
 alcian blue staining intensity was expressed as the mean integrated extinction ( mie  100 ) 
 per unit area . the calculation of mie of all gags in each layer was performed as previously 
 described . in brief , the percentage of sulfated 
 gags ( cs , ds , ks and hs ) was subtracted from the percentage of total gags ( taken as 100% ) to 
 determine the percentage of non - sulfated gags ( i.e. hyaluronan ; ha ) . similarly , percentage 
 of highly sulfated gags ( ks and hs ) was subtracted from that for all the sulfated gags to 
 determine the percentages of cs and ds and ks and hs . results are expressed as ( i ) total 
 gags , ( ii ) percentage of ha , ( iii ) percentage of total sulfated gags , ( iv ) percentage of cs 
 plus ds and ( v ) percentage of ks plus hs . statistical analysis : data on the proportions of smooth muscle and 
 collagen and the percentages of gags were analyzed to compare the effect of the stage of the 
 estrous cycle by an analysis of variance using spss version 13.0 ( spss inc . , the t - test was for animals with open - cervix or closed - cervix 
 pyometra comparing the proportions of smooth muscle and collagen and the percentages of gags 
 between these two conditions . proportion of collagen and smooth muscle : examples of the blue - stained 
 collagen and red - stained smooth muscle in the cervix of a cyclic bitch and a bitch with 
 pyometra are shown in fig . 1fig . 1.massons trichrome staining for blue - stained collagen and red - counterstained smooth 
 muscle in the cervix of healthy bitches at estrus ( a ) , diestrus ( b ) , anestrus ( c ) and 
 in bitches with open - cervix ( d ) and closed - cervix pyometra ( e ) . bar=100 
 m .. as the technique used in this study determined the relative area of collagen to 
 smooth muscle , the area of collagen was in an inverse relationship with the area of smooth 
 muscle . overall , the relative area occupied by collagen was lower ( p0.05 ) 
 during anestrus compared to estrus , but did not differ between anestrus and diestrus 
 ( p>0.05 , table 
 2table 2.proportion ( % ) of collagen in relative to smooth muscle ( mean  sem ) in the 
 cervices of bitches during different stages of the estrous cycle and with open- and 
 closed - cervix pyometrastatus of bitchesn% collagenhealthyanestrus 1047.03  2.50 estrus 757.87  2.39 diestrus1150.89  2.14 pyometraopen - cervix 1839.57  1.67 closed - cervix 826.49  2.23 values with different superscripts within healthy bitches ( a and b ) and bitches with 
 pyometra ( x and y ) differed significantly ( p0.05 ) . ) . a higher proportion of collagen proportion was found in the cervical tissue 
 of bitches with open- compared to closed - cervix pyometra ( p0.05 , table 2 ) . masson s trichrome staining for blue - stained collagen and red - counterstained smooth 
 muscle in the cervix of healthy bitches at estrus ( a ) , diestrus ( b ) , anestrus ( c ) and 
 in bitches with open - cervix ( d ) and closed - cervix pyometra ( e ) . values with different superscripts within healthy bitches ( a and b ) and bitches with 
 pyometra ( x and y ) differed significantly ( p0.05 ) . glycosaminoglycans : the pattern of staining with alcian blue at 3 
 concentrations of mgcl2 for 3 groups of gags ; non - sulfated gags ( mainly ha ) , 
 sulfated gags ( cs plus ds ) and highly sulfated gags ( ks plus hs ) are shown in fig . 2fig . 2.alcian blue staining in body of the cervical tissue ( a  c ) of an anestrous bitch . differences in staining intensity among the 3 varying concentrations of 
 mgcl2 can be noted ; total gags at 0.05 mgcl2 ( a ) , total 
 sulfated gags at 0.4 mgcl2 ( b ) and highly sulfated gags at 0.8 
 mgcl2 ( c ) . cervical cell layers are labeled : luminal epithelium ( le ) , 
 stroma ( s ) and smooth muscle ( m ) . bar=100 m .. regardless of gag type , the gags were primarily confined to the smooth muscle layers 
 ( tables 3 and 4table 3.intensity of glycosaminoglycan ( gag ) staining ( mie  sem ) in different tissue 
 layers of the bitch cervix during different stages of the estrous cyclestage of estrous cycleluminal epithelium stroma muscle total gagsanestrus 10.58  1.096.79  0.5616.50  2.23estrus 15.84  2.097.13  0.5019.31  1.83diestrus 14.06  1.736.30  0.6317.53  1.67hyaluronan ( ha)anestrus 7.31  1.303.23  0.5410.27  2.29estrus 12.80  2.303.24  0.6712.15  2.19diestrus 10.68  1.613.09  0.5911.89  1.55total sulfated gagsanestrus 3.26  0.273.56  0.286.23  0.26estrus 3.04  0.313.88  0.337.15  0.94diestrus 3.38  0.453.21  0.415.64  0.56chondroitin sulfate and dermatan sulfate 
 ( cs and ds)anestrus 2.02  0.242.06  0.144.52  0.22estrus 2.30  0.312.43  0.275.56  0.83diestrus 2.53  0.391.85  0.333.80  0.41keratan sulfate and heparan sulfate ( ks 
 and hs)anestrus 1.24  0.141.53  0.261.71  0.22estrus 0.74  0.0.81.45  0.151.59  0.18diestrus 0.84  0.091.35 0.211.77  0.23within columns , values with different superscripts ( a and b ) differed significantly 
 ( p0.05 ) . ) . the total gags in the cervix occupied about 46.8% of the muscle layer , 23.4% 
 of the luminal epithelium and 28% of the stroma . hyaluronan was the predominant gag not only 
 in the cervix of healthy bitches , irrespective of the stage of estrous cycle , but also in 
 bitches with pyometra ( tables 3 and 4 ) . in the luminal epithelium and muscle layers , ha 
 was the predominant gag ( 69.0% of total gags ) . alcian blue staining in body of the cervical tissue ( a  c ) of an anestrous bitch . differences in staining intensity among the 3 varying concentrations of 
 mgcl2 can be noted ; total gags at 0.05 mgcl2 ( a ) , total 
 sulfated gags at 0.4 mgcl2 ( b ) and highly sulfated gags at 0.8 
 mgcl2 ( c ) . cervical cell layers are labeled : luminal epithelium ( le ) , 
 stroma ( s ) and smooth muscle ( m ) . bar=100 m . within columns , values with different superscripts ( a and b ) differed significantly 
 ( p0.05 ) . in the luminal epithelium of healthy bitches , ha and ks plus hs were both significantly 
 ( p0.05 ) higher in estrus than anestrus ( table 3 ) . in the muscle layer , however , no significant differences 
 were observed among the stages of the estrous cycle in the stroma of healthy bitches ( table 3 ) . in bitches with pyometra , all types of gags in the luminal epithelium and muscle layers 
 were not different between open- and closed - cervix pyometra . however , in stroma , the ks plus 
 hs were higher ( p0.05 ) in cervices from closed- compared to open - cervix 
 pyometra ( table 4table 4.intensity of glycosaminoglycan ( gag ) staining ( mie  sem ) in different tissue 
 layers of the cervices of bitches with open- and closed - cervix pyometrastatus of pyometraluminal epitheliumstromamuscle total gagsopen12.97  1.138.31  0.5720.92  1.58closed12.07  1.658.30 1.1119.54  2.50hyaluronan ( ha ) open9.73  1.164.21  0.5314.85  1.62 closed8.48  1.724.01  1.1213.37  2.4total sulfated gagsopen3.23  0.194.09  0.186.07  0.25closed3.58  0.274.29  0.366.17  0.50chondroitin sulfate and dermatan 
 sulfate ( cs and ds)open2.34  0.182.90  0.174.40  0.21closed2.45  0.212.38  0.274.04  0.61keratan sulfate and heparan sulfate ( ks 
 and hs)open0.89  0.121.18  0.101.66  0.17closed1.13  0.171.90  0.282.12  0.19within columns , values with different superscripts ( a and b ) differed significantly 
 ( p0.05 ) . ) . within columns , values with different superscripts ( a and b ) differed significantly 
 ( p0.05 ) . the results of this study demonstrated that proportion of collagen to smooth muscle and gag 
 components in the cervix of healthy intact bitches differed according to the stage of the 
 estrous cycle , i.e. estrus , diestrus and anestrus , suggesting an influence of sex steroids 
 on the ecm of the canine cervix . in bitches with pyometra , the proportion of collagen was 
 higher in those with open - cervix pyometra , but the gags did not differ between open- and 
 closed - cervix pyometra , except for the highly sulfated gags , ks plus hs , in stroma where it 
 was higher in cervical tissues from bitches with closed - cervix pyometra . these findings 
 suggest that mechanisms regulating cervical patency in pyometra have a greater impact on 
 collagen remodeling rather than on the patterns of gags . in line with a recent study on the sheep cervix , the increase in cervical area occupied by 
 collagen during estrus observed in the present study appeared to be associated with high 
 estradiol concentrations . it has been suggested 
 that the ovarian steroid hormones have a role in the synthesis and remodeling of collagen in 
 the uterus of the rabbit , the uterine cervix of 
 women   and the cervix of the ewe   and the lower urinary tract of the dog . moreover , the estradiol - induced disruption of 
 collagen bundles has been demonstrated in the cervix of pregnant   and estrus   ewes . our 
 results were also in line with the previous report , demonstrating the decreased collagen 
 concentration and increased collagenase activity in relation to cervical relaxation in the 
 bitches with pyometra . the disruption of 
 collagen bundles in this study could be characterized by the increased interfibrillary 
 distance of collagen fibrils likely due to increased water content in the cervical tissue 
 [ 2 , 6 ] . in 
 this study , the proportions of collagen and smooth muscle differed among estrus , diestrus 
 and anestrus , suggesting an influence of hormonal status on collagen remodeling . however , 
 the involvement of collagen in cervical patency ( open versus closed ) in the dog remained an 
 interesting subject for further investigation . in this respect , it would be interesting to 
 compare samples of cervix taken from estrous bitches when the cervix is open , approximately 
 2 days before the lh surge , or closed , 3 days before end of the estrus , including cervical tissue from pregnant and 
 peripartum bitches . the bitches affected with open - cervix pyometra had increased proportion of collagen area in 
 the cervix , in other words , a decreased proportion of smooth muscle , suggesting disruption 
 of collagen leading to cervical relaxation ( opening ) . this phenomenon associated with an 
 inflammatory reaction in the uterus and also in the cervix as evidenced by the higher degree 
 of neutrophil infiltration in cervical tissues of bitches with open - cervix pyometra . the loosening of collagen in the cervix is 
 characterized by a decrease in gag - protein interactions probably induced by collagenolytic 
 enzymes , such as the matrix metalloproteinases ( mmps ) from cervical neutrophils . although the exact underlying mechanism ( s ) that 
 alter cervical patency in bitches with pyometra is still unclear , neutrophils that induce 
 collagenolytic enzymes and other cytokines are likely participants . to our knowledge , the 
 gags in the canine cervix have not been reported previously . this study revealed variation 
 in distribution of the various gags among the tissue layers ( luminal epithelium , stroma and 
 muscle ) of the cervix . the findings that ha , the predominant gag , was more abundant in the 
 muscle layers than in the luminal epithelium or the stroma were in agreement with findings 
 in the sheep cervix [ 8 , 14 ] . our results in the cyclic bitches are also in line with a previous 
 report for sheep showing that cervical ha was highest during estrus compared to other stages 
 of the cycle . hyaluronan has hydrophilic 
 properties which cause tissue edema , dissociation of collagen fibers and decreased 
 extensibility of cervical tissue , all of which assist cervical ripening / relaxation [ 9 , 34 ] . in the 
 cyclic bitches , increased ha in the luminal epithelium during estrus was likely related to 
 high estradiol concentrations . studies in human cervical tissues support this notion that 
 estradiol positively regulates ha synthesis in the cervix   while progesterone has the opposite ( inhibitory ) effect on ha synthesis . the local application of ha to the rabbit 
 cervix resulted in a higher number of neutrophils that produce mmps and thus regulate the 
 remodeling collagen and facilitate cervical ripening . in cyclic bitches , during estrus , ha was increased , whereas the number of 
 neutrophils was not . in pyometra bitches , there 
 was no difference in ha between open- and closed - cervix pyometra , although there was an 
 increased density of neutrophils in the bitches with open - cervix pyometra . taken together , these findings suggest different 
 regulatory roles of ha and/or neutrophils in regulating cervical ecm and cervical patency in 
 normal cyclic bitches and bitches with pyometra . similar to our findings , the 4 sulfated gags , cs , ds , ks and hs , are present in human 
 [ 3 , 16 , 20 , 34 ] and rat 
  cervical tissues . however , no cs is detected 
 in the sheep cervix , and thus , the cervical 
 distribution of gags may vary according to the species . because sulfated gags increase the 
 strength of electrostatic interactions between collagen fibers and the binding of ds to 
 collagen , a reduction in sulfated gags may 
 weaken the cross - links between collagen fibers . these observations support our findings of significantly lower concentrations of the highly 
 sulfated gags ( ks and hs ) in the cervical stroma of bitches with open - cervix pyometra . furthermore , hs regulated leukocytic infiltration , and in genetically - modified mice lacking hs , increased leukocyte recruitment 
 was reported in cardiac muscle . the lower 
 combined amount of hs and ks and increased neutrophil density observed in bitches with 
 open - cervix pyometra may act to re - arrange collagen , thus weakening it and causing cervical 
 relaxation . in conclusion , the results of this study have demonstrated that ( i ) the proportion of 
 collagen to smooth muscle altered depending on stages of the estrous cycle and suggest that 
 such changes are related to the milieu of ovarian hormones , ( ii ) a higher proportion of 
 collagen was found in bitches with open - cervix pyometra where the cervix was fully relax and 
 a lower proportion of collagen was found in cases of closed - cervix pyometra where the cervix 
 was tightly closed , indicating different structural function of cervical collagen in dogs 
 with a uterine pathology and ( iii ) the distribution of cervical gags varied among tissue 
 layers of the canine cervix , the stages of the estrous cycle and between open- closed - cervix 
 pyometra . ha , the predominant cervical gag , may have a role in remodeling collagen and in 
 cervical relaxation via either an increase in the water content of the tissue or by acting 
 in conjunction with tissue cytokines . it is interesting to note that our results suggest 
 that ha is not involved in the patency of the cervix in bitches with pyometra , but that the 
 highly sulfated gags ( ks and hs ) along with cytokines produced by local neutrophils seem to 
 be responsible .
abstractthe extracellular matrix of the cervix that comprises collagen , elastin , proteoglycans 
 and glycosaminoglycans ( gags ) is thought to have an essential role in cervical relaxation . 
 this study investigated the proportion of collagen and smooth muscle as well as the gags 
 in cervices obtained from healthy bitches at different stages of the estrous cycle and 
 bitches with open- and closed - cervix pyometra . 
 cervices were collected after 
 ovariohysterectomy . 
 the proportion of collagen to smooth muscle was determined using 
 masson s trichrome staining . 
 alcian blue staining was used to evaluate the relative 
 distribution of cervical gags . 
 the proportion of cervical collagen relative to smooth 
 muscle was higher at estrus compared to anestrus ( p0.05 ) . 
 it was also 
 higher ( p0.05 ) in bitches with open- compared to those with 
 closed - cervix pyometra . 
 overall , hyaluronan ( ha ) was the predominant gag in the canine 
 cervix . in the luminal epithelium , 
 the staining intensity for ha was stronger in estrus 
 than in anestrus ( p0.05 ) , but not in diestrus 
 ( p>0.05 ) . 
 on the contrary , the intensity for the combined keratan 
 sulfate ( ks ) and heparan sulfate ( hs ) was stronger in anestrus than in estrus and diestrus 
 ( p0.05 ) . in bitches with pyometra , 
 the staining intensity of the 
 stroma for ks and hs was weaker in open- compared to closed - cervix pyometra 
 ( p0.05 ) . 
 collectively , the different profiles of collagen and gag 
 suggest that the metabolism of both collagen and gags in the canine cervix is associated 
 with hormonal statuses during the estrous cycle and cervical patency of bitches with 
 pathological uterine conditions , such as pyometra .