chlorhexidine has been proposed in endodontic therapy in various concentrations as an irrigating solution , intracanal medication and in association with other substances , for cleansing of infected root canal systems ( barbosa , et al.1 1997 ; siqueira jr . and uzeda22 1997 ; estrela , et al.4 2001 ; schafer and bssmann20 2005 ) . this substance is a cationic detergent of the biguanide group , which is almost insoluble in water . for this reason , it is almost always prepared in salt form , chlorhexidine digluconate , which increases its solubility ( davies3 1973 ) . it is considered a wide spectrum antimicrobial substance because in addition to its efficacy against gram - positive bacteria , it also acts against gram - negative bacteria and has variable fungicidal action , depending on the specie . furthermore , it has a very important property called substantivity , that is , residual effect ( rlla , et al.18 1970 ; rosenthal , et al.19 2004 ) , which means that its presence is maintained in the blood and other bodily fluids . although the use of chlorhexidine has been widely diffused in many dental fields , its efficacy in microbial control has been proven ( heling , et al.8,9 1992 ; siqueira jr . and uzeda22 1997 ; dametto , et al.2 2005 ) and the tolerance of oral tissues to this substance has been shown ( le e schiott14 1970 ; yates , et al.24 1993 ) , few studies have demonstrated the behavior of conjunctive tissues in its presence ( fellippe , et al.5 1998 ) . when used as an intracanal medication , both in solution and in association with calcium hydroxide , chlorhexidine remains in intimate contact with the periapical tissues , usually for a prolonged period . moreover , longitudinal observation of these reactions seems to be a relevant factor for elucidation of the type of response that host tissues can present in the presence of this substance . thus , the purpose of this study was to evaluate the response of rat buccal submucosa tissue to the presence of distilled water , a mixture of calcium hydroxide ( p.a . ) and distilled water , a 2% chlorhexidine solution and a mixture of calcium hydroxide ( p.a . ) and 2% chlorhexidine solution . the experimental protocol was reviewed and approved by the ethics in human and animal research committee of the brazil 's lutheran university ( ulbra , canoas , rs ) . the animals were subjected to a light / dark cycle of 12 h , mean temperature of 22c , 40 - 60% humidity , with free access to solid chow and water ad libitum , thus closely preserving their habitual situation . the rats were randomly separated and identified both as individuals and in groups . for inoculation of the test materials , 120 cylinders measuring 5 mm in length and 1.3 mm internal diameter were obtained from polyethylene 19 g tubes ( venescalp , feira de santana , ba , brazil ) . atoxic polyurethane sponges cut in sizes of approximately 621 mm were used for placement of 2% chlorhexidine solution and distilled water . as much as 0.02 ml of liquid was injected into the center of the sponges using a 1-ml insulin syringe to avoide liquid overflow at the edge of the cylinder . to obtain the pastes , calcium hydroxide powder ( iodontosul - industrial odontolgica do sul ltda . , porto alegre , rs , brazil ) was divided in two portions of approximately 4.6 g , weighed in an analytical balance . concerning the quantity of liquid , whether using distilled water or 2% chlorhexidine , as much as 5.1 ml was stipulated , maintaining a proportion of 1 g of powder to 1.1 ml of liquid , resulting in a homogenous mixture with toothpaste consistency . the paste was injected into the cylinder using a 10 ml disposable syringe without needle . all surgical procedures were performed under general anesthesia by intramuscular administration of 0.1 ml of ketamine hydrochloride ( dopalen , agribrands . sade animal , paulnia , sp , brazil ) associated with 0.05 ml of xylazine hydrochloride ( rompun , bayer , sade animal , so paulo , sp , brazil ) per 100 g of rat body weight . for material insertion , the rat 's mouth was opened and a 3-mm - long incision was made at the region of the buccal gingival sulcus with a # 15 surgical scalpel ( med blade , medgoldman ind . manaus , am , brazil ) , after which a randomly chosen cylinder was inserted in the submucosal tissue of the region . after implantation of each of the cylinders , the incision was closed using resorbable , # 3 catgut sutures ( ethicon , johnson & johnson , so paulo , sp , brazil ) . following completion of each experimental time interval ( 7 , 15 and 30 postoperative days ) , 10 randomly chosen rats were anesthetized as described above and sacrificed by cervical dislocation , the pieces being immediately immersed in 10% formalin . prior to histological processing , the cylinders were carefully located , removed from the histological samples , dehydrated in a gradual alcohol series and embedded in paraffin . the blocks were cut at the central portion , where 5 standardized semi - serial 5-m - thick histological sections were obtained and stained with harris hematoxylin and eosin . five glass slides belonging to each histological block were examined with an optical microscope ( olympikus , tokyo , japan ) by a single senior examiner who was blinded to the tested substances . in this initial stage , the whole extension of the glass slides was examined at 100 , 200 and 400 magnification in order to visualize the tissue adjacent to at least one of the ends of the cylinder . this was established as an inclusion criterion to select the slide with the best quality staining and histological details ( figure 1 ) . regarding the region corresponding to the cylinder extremity once these selection criteria were established , a new phase began , in which the slides were analyzed qualitatively ( figueiredo , et al.6 2001 ) , observing the presence of neutrophils , lymphocytes , plasmocytes , eosinophils , macrophages , gigantocytes , condensation fibrosis and abscess . based on these findings , the slides were then classified according to the following scores : 0  absence ; 1  presence ; 2  infiltrate . for cell events , the following scores were considered : 0 - when inflammatory cells were absent or found only within the blood vessels ; 1 - when inflammatory cells were sparsely present or in very small groups ; and 2 - when inflammatory cells , in an area close to a tube extremity , were found in the entire field or were present in a large number , configuring great severity . condensation fibrosis received the following scores : 0 when absent ; 1 when a fine layer involving the study material was formed ; and 2 when a thick layer was formed . regarding abscess formation , the following scores were given : 0 - absent ; 1 - the abscess was limited to the proximity of the cylinder containing the material ; and 2 - the abscess reached more distant areas . after qualitative analysis and attribution of scores to the observed events , the slides were scored according to inflammation severity . a ranking scale was established , which ordered the slides in increasing degree of inflammatory reaction , ranging from absent to highly inflammatory . all analyzes were undertaken at 100 , 200 and 400 magnification in the region close to a cylinder extremity , chosen as described above . for comparison of the cell events observed using the tested materials at 7 , 15 and 30 days , the data obtained were submitted to kruskal - wallis nonparametric test at 5% level of significance . the analysis of the degree of inflammation denoted in the ranking scale was also performed with kruskal - wallis nonparametric test at 5% significance level . initially , the data were submitted to statistical analysis of the possible influence of the quadrants used in the rat mouth in relation to the inflammatory response ( kruskal - wallis ; 5% significance level ) . as no statistically significant difference was observed , all p values were over 0.05 , it was inferred that the factor quadrant exerted no influence on the results . for comparison of the cell events observed using the tested materials at 7 , 15 and 30 days , the data obtained were submitted to kruskal - wallis nonparametric test at 5% level of significance . the analysis of the degree of inflammation denoted in the ranking scale was also performed with kruskal - wallis nonparametric test at 5% significance level . initially , the data were submitted to statistical analysis of the possible influence of the quadrants used in the rat mouth in relation to the inflammatory response ( kruskal - wallis ; 5% significance level ) . as no statistically significant difference was observed , all p values were over 0.05 , it was inferred that the factor quadrant exerted no influence on the results . the results are summarized in table 1 , which present the means and standard deviations of the cell events for the tested materials and the time interval . no statistically significant differences were found in the analysis of cell events when comparing the tested materials ( figures 2 and 3 ) . on the other hand , statistically significant differences were found while analyzing the tissue response caused by a single material according to the time interval : the severity of the inflammatory reaction to all substances decreased along the duration of the experiment ( figure 4 ) . analyzing the results of the ranking scale , established according to the degree of general inflammation and with the cell events ordered gradually from those showing absence of inflammation to those of greatest inflammation , no significant differences were found regarding among the tested materials ( table 2 ) . however , when testing the influence of the time interval on the degree of inflammation , the inflammatory process clearly diminished as a result of a longer experimental time interval ( table 3 ) . the use of the submucosal region of rat buccal gingival sulcus was chosen due to the marked physiological similarity of this location with the periapical tooth region , rather than the dorsum of these animals , which is a commonly used area in several studies . figueiredo , et al.6 ( 2001 ) used the submucosal tissue of rabbits with success in a study that , aside from sample size , was similar to the present investigations in its methodological steps . the ease of execution of this technique , especially because it does not require trichotomy and tissue divulsion as necessary when using the dorsal region , should also be highlighted . the stipulation of time intervals of 7 , 15 and 30 days , contemplated the commonly used periods in endodontics regarding the use of calcium hydroxide as an intracanal medication , permitting the observation of histological responses during short- , medium- and long - term periods , and was in agreement with the protocol of other studies that analyzed tissue response ( holland , et al.10 1999 ; nelson filho , et al.15 1999 ; holland , et al.11 2001 ) . the criteria for histological evaluation with 0-to-1 scores and the choice for neutrophils , eosinophils , lymphocytes / plasmocytes and macrophages / giant cells , condensation fibrosis and abscess as markers of tissue reaction , were basically due to the direct relation of these elements with the inflammatory process ( figueiredo , et al.6 2001 ) . an expert examiner analyzed the inflammatory events because in addition to providing quantitative data using the above - mentioned scoring system , the examiner could present qualitative data on the inflammatory process , by interpreting tissue responses , such as the presence of abscess , condensation fibrosis and other events . peters , et al.17 ( 2002 ) suggested quantification of angiogenesis using a specific software to provide a faster and more practical . however , it is our understanding that such a count is only quantitative , providing no information regarding the quality of the inflammatory process and allowing no data interpretation . the use of a scoring system aimed to rank each slide according to the degree of inflammation present . having observed that the quadrant position did not interfere with the inflammatory response , the scoring system was designed by mixing all materials and time intervals . the analysis of data referring to the measurement of the inflammatory events was performed ( kruskal - wallis test ; a=5% ) at experimental periods of 7 , 15 and 30 days . however , some findings were interesting and should be addressed . in relation to the number of neutrophils , distilled water presented higher means than 2% chlorhexidine within a 7-day period . this is in agreement with the findings of knuuttila et al.13 ( 1978 ) who , using sponges impregnated with 0.01 to 0.2% chlorhexidine , observed that chlorhexidine demonstrated a certain influence on the diminished migration of leukocytes to the implantation sites when compared to saline over 12- , 24- and 36-hour periods . moreover , they reported that the greater the chlorhexidine concentration , the lower the number of inflammatory cells . using the same methodology , paunio , et al.16 ( 1978 ) , maintained the previously mentioned sponges for longer time intervals , between 3 and 23 days , obtaining results that indicated that chlorhexidine presented a clear delaying effect on the formation of granulation tissue . watts , et al.23 ( 1989 ) affirmed that chlorhexidine presented a mild chemotaxis over neutrophils at low concentrations ; however , at high concentrations , this substance are able to immobilize cells . these results are in agreement with those of a previous study21 , in which it was observed that , at low concentrations , chlorhexidine caused a slight increase in the movement of neutrophils , while at higher concentrations , this substance determined a reduction in this movement . perhaps for this reason , in the present study , a large number of neutrophils were verified in relation to the analysis of distilled water after the first time interval . kenney , et al.12 ( 1972 ) analyzed the effect of chlorhexidine on polymorphonuclear leukocytes and demonstrated that a concentration of 0.2% caused such a severe destruction of these cells that it was not possible to count them . goldschmidt , et al.7 ( 1977 ) concluded that chlorhexidine , even at low concentrations , such as 0.001 and 0.2% , was shown to be cytotoxic . another event observed during the course of the study was the formation of abscesses throughout the experimental periods . to clarify the influence of the materials on the origin of these abscesses , a statistical analysis was performed ( table 1 ) , which demonstrated that the materials did not influence abscess formation , since no differences were found between the tested materials . in general , the cell events of this study resembled one another , denoting a certain similarity in the quality of inflammation . regarding the analysis of the influence of time interval on the tested substances , in accordance with the present methodology and the obtained results , it can be affirmed that all materials presented a statistically significant reduction in the inflammatory reaction at longer time intervals , which led us to infer that perhaps the initial inflammatory response was strongly influence by the aggression suffered during the surgical process and not only by the substances under analysis . with regard to the scoring system , which positioned the inflammatory events according to the degree of inflammation presented , tables 2 and 3 show that no significant differences occurred in relation to the analysis of the substances . however , confirming previous results , significant differences were verified when analyzing the time factor . data should , however , be analyzed with caution because the mean position , also called the mean ranking by statisticians , is an " n " dependent measure , that is , it completely depends on the study sample number . thus , this position can not be compared to other studies that have a different sample size . despite this fact , it is still an excellent method for situating and demonstrating the mean position of a tested substance within a scale . the results presented hereby do not agree with those of fellippe , et al.5 ( 1998 ) , who found differences between the irritating potential of 0.12% chlorhexidine and saline for a 3- hour time interval . this different result may be attributed to methodological differences , especially because the present study used distilled water and longer time intervals . this study did not intend to demonstrate that chlorhexidine solution and the association of this substance with calcium hydroxide are as biocompatible as distilled water and or the calcium hydroxide / distilled water pastes . on the contrary , we agree on the need for future works that may clarify the processes that take place in the periapical tissues when chlorhexidine is used as an intracanal medication . based on the methodology used in this study and according to the obtained results , it may be concluded that : 1 . no differences in tissue response were verified regarding the use of 2% chlorhexidine solution , distilled water , the association of 2% chlorhexidine and calcium hydroxide and the association of distilled water and calcium hydroxide , at any of the experimental time intervals ; 2 . the severity of the inflammatory reaction caused by all substances decreased at the longer time intervals .
the aim of this study was to analyze the buccal tissue responses of wistar rats to 2% chlorhexidine solution , calcium hydroxide and the association of both products . for this purpose , 
 30 specimens were randomly implanted in the filtrum of the four upper and lower hemiarches with a polyethylene tube containing one of the following substances : 2% chlorhexidine solution , calcium hydroxide and 2% chlorhexidine solution ( test groups ) ; calcium hydroxide and distilled water and distilled water ( control groups ) . 
 ten rats each were distributed according to time interval of evaluation at 7 , 15 and 30 days . 
 the histological sections were stained with harris hematoxylin and eosin . 
 analysis was performed with an optical microscope at x100 , x200 and x400 magnifications by an expert examiner blinded to the materials . 
 the sections were classified by scores attributed to inflammatory events and by a ranking determined according to the severity of the inflammation . 
 the results of the inflammatory events and severity ranking were submitted to the kruskal - wallis test at a 0.05 level of significance . 
 no statistically significant difference occurred among the tested materials ; however , all materials showed a decreased of severity with respect to longer time intervals .