0099-2399/92/1805-0205/$03.00/0 JOURNAL OF ENDODONTICS Copyright © 1992 by The American Association of Endodontists
Printed in U.S.A. VOL. 18, NO. 5, MAY 1992
SCIENTIFIC ARTICLES Concentrations of Leukotriene B4 in Symptomatic and Asymptomatic Periapical Lesions Mahmoud Torabinejad, DMD, MSD, Elisabetta Cotti, DDS, MS, and Timothy Jung, MD, PhD
Twelve periapical lesions from symptomatic and asymptomatic patients (six each) were obtained and immediately frozen in liquid nitrogen. Six pulps from unerupted third molars as well as chronically inflamed gingival tissues were also obtained, frozen, and used as negative and positive controls, respectively. The concentration of leukotriene (LT) B4 was determined by reverse phase high pressure liquid chromatography. Representative samples from each group were fixed in formalin, sectioned, and stained with hematoxylin and eosin. Extremely low levels of LTB4 were detected in the uninflamed pulpal samples in comparison to those found in chronically inflamed gingival tissues and periradicular lesions. A significant statistical difference was noted between concentrations of LTB4 in periapical lesions of symptomatic patients~and those found in asymptomatic patients and samples of chronically inflamed gingival tissues (p < 0.05). In addition, a positive correlation was found between the presence of symptoms, the concentration of LTB4, and presence of polymorphonuclear leukocytes in symptomatic periapical lesions. The results show presence of high concentrations of LTB4 in symptomatic human periapical lesions.
I IMEMBRANEPHOSPHOLIPIDS I
Y~OOX C yG~AFIACHIDACID@ ONIC ~
J THROMSOXANE TxA2 TxB2
.EUKOTRIENES! LIPOXINS LTA4 LTD4 LPA LTB4 LTE4 LPB LTC4 LTF4
FIG 1. Pathways of formation of various arachidonic acid metabolites.
clooxygenase pathway of arachidonic acid have been reported in inflamed pulps and periapical tissues of human and experimental animals (1-5). The 5-1ipoxygenase metabolites of arachidonic acid such as LTB4, and cysteinyl containing leukotrienes are proinflammatory (6). LTB4 causes chemotaxis of polymorphonuclear (PMN) leukocytes both in vitro and in vivo and promotes adhesion of PMN leukocytes to endothelium (7). In addition, it stimulates aggregation, degranulation, and generation of superoxide in PMN leukocytes. Furthermore, LTB4 has been implicated in infiltration of PMN leukocytes in psoriasis and rheumatoid arthritis and immune complex induced pleurisies (7). Despite the presence of numerous reports on the role of lipoxygenase metabolites of arachidonic acid in inflammation there are only a few reports in the dental literature focusing on the role of these substances in the pathogenesis of oral pathosis (8-11). The role of immunological reactions and most nonspecific mediators of inflammation such as complement components, lysosomal enzymes, and some cytokines has been reported in the literature (12). Histological examination of acute apical abscess shows a predominance of PMN leukocytes in these lesions (13). Among other mediators such as C3a and prostaglandin E2 (5), LTB4 has also been implicated in the influx of these cells with inflammatory regions. A search of the literature showed an absence of published research on presence or absence of LTB4 in human periapical lesions. The
As a consequence of pathological changes in the dental pulp, bacteria and their products, as well as denatured host tissues, can accumulate in the root canal system. Egress of these irritants into the periapical tissues results in the activation of nonspecific and specific host resistance factors in this region. Upon cellular damage and release of various phospholipases a large amount of arachidonic acid is released. The enzymatic oxidation of this substance results in the formation of many biologically active metabolites such as prostaglandins and thromboxanes via the cyclooxygenase pathway and leukotrienes (LT) and lipoxins through the lipoxygenase pathway (Fig. 1). Elevated levels of various metabolites from the cy-
205
206
Torabinejad et al.
purpose of this study was to determine the concentration of endogenous LTB4 in symptomatic and asymptomatic human periapical lesions. MATERIALS AND METHODS Collection of Tissue Samples Four groups of tissue samples were included in this study. The experimental tissue specimens were periapical lesions removed surgically from patients with and without symptoms. Pulpal tissue samples from unerupted third molars and inflamed gingival tissue specimens were used as negative and positive controls, respectively. A total of 24 tissue samples were taken from 24 different patients. The samples were obtained from four groups (six each) of patients. The grouping of periapical lesions was done according to the criteria of Torabinejad and Walton (13). A periapical lesion was defined as symptomatic when it was associated with a radiolucency and clinical signs of pain and/ or swelling. A lesion was considered asymptomatic when the patient had a periapical radiolucency and had no pain and/ or swelling. Periapical lesions were obtained from patients who had had previous root canal therapy. The indications for surgical intervention were based on the recommendations by Hovland (14). The size of the radiolucencies varied from 5 × 5 mm 2 to 13 × 9 mm% with an average size of 8 x 7.6 mm 2. The duration of presence of most asymptomatic periapical lesions was unknown. The symptomatic lesions had become symptomatic 1 to 3 days before their surgical removal. Patients who were on antibiotics or anti-inflammatory medications were excluded from the study. Group 1 consisted of periapical lesions from patients with pain and/or swelling and signs and symptoms of acute apical abscess (13) (experimental group). Group 2 was comprised of periapical lesions taken from asymptomatic patients who had radiographic and clinical signs of chronic apical periodontitis (13) (experimental group). Group 3 consisted of uninflamed pulpal tissues taken from unerupted third molars. These teeth had no history of pulpal or periradicular pathosis (negative control). Group 4 consisted of inflamed gingival tissues taken from patients with chronic periodontitis who were scheduled for periodontal surgery (positive control). The periodontal assessment of these patients was done by measuring periodontal pocket depths, soft tissue recession, and the degree of mobility of each tooth. The gingival tissue samples came from areas with an average Russell index score of 6 (moderate periodontitis) (15). The surgical procedures were performed under local anesthetic, using lidocaine with 1:100,000 epinephrine. After reflecting a full-thickness flap and removal of cortical bone, each periapical lesion was completely curetted out with a large spoon excavator. The pulps from unerupted third molars were obtained by making deep grooves on the buccal and lingual surface of each tooth with a fissure bur in a high-speed handpiece under water spray and splitting it in halves. The gingival tissue samples were obtained from patients who needed gingivectomies as a part of their periodontal therapy. After removal, depending on the size of the lesion, half or the whole tissue sample was snap-frozen in liquid nitrogen and stored at -70"C.
Journal of Endodontics
Histopathological Examination of Tissue Samples Half of the tissue samples from 16 large specimens were randomly placed in 10% formalin for histopathological examination: five from group 1, four from group 2, three from group 3, and four from group 4. Tissue samples were embedded in paraplast, sectioned at 6 um, and were stained with hematoxylin and eosin. Representative samples from each group were evaluated histologically by an independent oral pathologist under a light microscope. The sections were examined for the types (acute, chronic, and subacute) of inflammatory cells and the degree of inflammation (1, no inflammation; 2, mild inflammation (inflammatory cells more than two cell diameters apart); 3, moderate (inflammation cells one cell diameter apart); and 4, severe (inflammatory cells were less than one cell diameter apart).
Extraction of Leukotriene B4 The concentrations of LTB4 were determined by reverse phase high pressure liquid chromatography (HPLC). The methods for experimental tissues and appropriate standards as well as controls were performed according to procedures described by Jung et al. (16). After thawing and determining the wet weight of each tissue specimen, 1.0 ml of 100% ethanol was added to the sample and it was then homogenized in an Ultra-Turrax homogenizer. Then, 1.0 ml of HPLC grade water was added to each homogenized sample; the samples were centrifuged for 15 min at 3000 rpm in a Beckman T J-6 centrifuge. The supernatant was recovered and concentrated to 1 ml under nitrogen gas. The ethanolic sample was then dissolved in 19 ml of HPLC grade water and eluted through a C-18 column, Bond Elute/Vas Elute system (Analytical International, Harbor City, CA). The arachidonic acid metabolites retained in the cartilage were eluted with 10 ml of methanol. The methanolic eluent was dried and reconstituted with 1 ml of 50% ethanol. The sample (50 ul) was injected into the reverse phase-HPLC. The eluting solvent for lipoxygenase products was 70% methanol and 30% water (containing 0.1% acetic acid and 0.1% trifluoroacetic acid). Flow rate was 1.5 ml/min. The wavelength was set at 280 nm for the first 5.5 min, from 5.5 to 14.0 min at 235 nm, and from 14.0 to 22.0 min at 280 nm. The column was a DuPont, Reversed-Phase, Zorbax ODS column (4.6 mm x 25 cm) with an ODS guard column (4.6 mm x 5 cm). The HPLC system was a Shimadzu binary gradient LC-6A system with a C-R6A data processor and SPD-6A variable wavelength UV detector. The absolute calibration curve method was used for quantitative calculation, using the following equation: Content = (FliAi + F2i/Wspl) x 100 where Ai is the area (height); Fli is the response factor for slope; F2i is the response factor for constant term; and Wspl is the weight of sample. To determine the statistical difference in the concentrations of LTB4 among the groups, a Kruskal-Wallis one-way analysis of variance was used.
Leukotriene in Periapex
Vol. 18, No. 5, May 1992
207
TABLE 1. HistopathoIogical evaluation of representative samples showing degree (D) and type (T) of inflammation Group 1: symptomatic periapical lesions
Group 2: asymptomatic periapical lesions
Group 3: uninflamed pulpal tissue samples Group 4: inflamed gingival tissue samples
1 2 3 4 5 1 2 3 4 1 2
D4 D3 D3 D4 D4 D3 D4 D4 D4 ---
T3 T3 T1 T3 T3 T2 T2 T2 T2 ---
3
--
--
1 2 3 4
D3 D3 D4 D4
T2 T2 T2 T2
FIG 2. A section of a periapical lesion from a symptomatic patient showing an edematous granulation tissue infiltrated mainly by PMN leukocytes and some lymphocytes, plasma cells, and macrophages (hematoxylin and eosin; original magnification xl00).
RESULTS Evaluation of the results from the histopathological examination of the representative samples showed that the four groups were homogeneous (Table 1). Samples from group 1 showed the presence of moderate to severe acute and subacute inflammation (Fig. 2). Examination of the sections in group 2 showed the presence of moderate to severe chronic infiltrate (Fig. 3). Representative samples in group 3 consisted ofpulpal connective tissues. Group 4 showed areas of moderate to severe chronic inflammation (Fig. 4). The means and standard deviations of concentrations of LTB4 for each group were calculated in nanograms per milligram of wet tissue weight and are shown in Fig. 5 The Knaskal-Wallis one-way analysis of variance showed the presence of a significant difference in concentration of LTB4 between the four groups of tissues tested in this experiment (p = 0.001). Comparison between groups showed significant difference between the endogenous levels of LTB4 in the positive control (group 4) and the negative control (group 3, p < 0.05). In addition, significant differences were found in the levels of LTB4 in group 1 when compared with groups 2 and 3 (p
Printed in U.S.A. VOL. 18, NO. 5, MAY 1992
SCIENTIFIC ARTICLES Concentrations of Leukotriene B4 in Symptomatic and Asymptomatic Periapical Lesions Mahmoud Torabinejad, DMD, MSD, Elisabetta Cotti, DDS, MS, and Timothy Jung, MD, PhD
Twelve periapical lesions from symptomatic and asymptomatic patients (six each) were obtained and immediately frozen in liquid nitrogen. Six pulps from unerupted third molars as well as chronically inflamed gingival tissues were also obtained, frozen, and used as negative and positive controls, respectively. The concentration of leukotriene (LT) B4 was determined by reverse phase high pressure liquid chromatography. Representative samples from each group were fixed in formalin, sectioned, and stained with hematoxylin and eosin. Extremely low levels of LTB4 were detected in the uninflamed pulpal samples in comparison to those found in chronically inflamed gingival tissues and periradicular lesions. A significant statistical difference was noted between concentrations of LTB4 in periapical lesions of symptomatic patients~and those found in asymptomatic patients and samples of chronically inflamed gingival tissues (p < 0.05). In addition, a positive correlation was found between the presence of symptoms, the concentration of LTB4, and presence of polymorphonuclear leukocytes in symptomatic periapical lesions. The results show presence of high concentrations of LTB4 in symptomatic human periapical lesions.
I IMEMBRANEPHOSPHOLIPIDS I
Y~OOX C yG~AFIACHIDACID@ ONIC ~
J THROMSOXANE TxA2 TxB2
.EUKOTRIENES! LIPOXINS LTA4 LTD4 LPA LTB4 LTE4 LPB LTC4 LTF4
FIG 1. Pathways of formation of various arachidonic acid metabolites.
clooxygenase pathway of arachidonic acid have been reported in inflamed pulps and periapical tissues of human and experimental animals (1-5). The 5-1ipoxygenase metabolites of arachidonic acid such as LTB4, and cysteinyl containing leukotrienes are proinflammatory (6). LTB4 causes chemotaxis of polymorphonuclear (PMN) leukocytes both in vitro and in vivo and promotes adhesion of PMN leukocytes to endothelium (7). In addition, it stimulates aggregation, degranulation, and generation of superoxide in PMN leukocytes. Furthermore, LTB4 has been implicated in infiltration of PMN leukocytes in psoriasis and rheumatoid arthritis and immune complex induced pleurisies (7). Despite the presence of numerous reports on the role of lipoxygenase metabolites of arachidonic acid in inflammation there are only a few reports in the dental literature focusing on the role of these substances in the pathogenesis of oral pathosis (8-11). The role of immunological reactions and most nonspecific mediators of inflammation such as complement components, lysosomal enzymes, and some cytokines has been reported in the literature (12). Histological examination of acute apical abscess shows a predominance of PMN leukocytes in these lesions (13). Among other mediators such as C3a and prostaglandin E2 (5), LTB4 has also been implicated in the influx of these cells with inflammatory regions. A search of the literature showed an absence of published research on presence or absence of LTB4 in human periapical lesions. The
As a consequence of pathological changes in the dental pulp, bacteria and their products, as well as denatured host tissues, can accumulate in the root canal system. Egress of these irritants into the periapical tissues results in the activation of nonspecific and specific host resistance factors in this region. Upon cellular damage and release of various phospholipases a large amount of arachidonic acid is released. The enzymatic oxidation of this substance results in the formation of many biologically active metabolites such as prostaglandins and thromboxanes via the cyclooxygenase pathway and leukotrienes (LT) and lipoxins through the lipoxygenase pathway (Fig. 1). Elevated levels of various metabolites from the cy-
205
206
Torabinejad et al.
purpose of this study was to determine the concentration of endogenous LTB4 in symptomatic and asymptomatic human periapical lesions. MATERIALS AND METHODS Collection of Tissue Samples Four groups of tissue samples were included in this study. The experimental tissue specimens were periapical lesions removed surgically from patients with and without symptoms. Pulpal tissue samples from unerupted third molars and inflamed gingival tissue specimens were used as negative and positive controls, respectively. A total of 24 tissue samples were taken from 24 different patients. The samples were obtained from four groups (six each) of patients. The grouping of periapical lesions was done according to the criteria of Torabinejad and Walton (13). A periapical lesion was defined as symptomatic when it was associated with a radiolucency and clinical signs of pain and/ or swelling. A lesion was considered asymptomatic when the patient had a periapical radiolucency and had no pain and/ or swelling. Periapical lesions were obtained from patients who had had previous root canal therapy. The indications for surgical intervention were based on the recommendations by Hovland (14). The size of the radiolucencies varied from 5 × 5 mm 2 to 13 × 9 mm% with an average size of 8 x 7.6 mm 2. The duration of presence of most asymptomatic periapical lesions was unknown. The symptomatic lesions had become symptomatic 1 to 3 days before their surgical removal. Patients who were on antibiotics or anti-inflammatory medications were excluded from the study. Group 1 consisted of periapical lesions from patients with pain and/or swelling and signs and symptoms of acute apical abscess (13) (experimental group). Group 2 was comprised of periapical lesions taken from asymptomatic patients who had radiographic and clinical signs of chronic apical periodontitis (13) (experimental group). Group 3 consisted of uninflamed pulpal tissues taken from unerupted third molars. These teeth had no history of pulpal or periradicular pathosis (negative control). Group 4 consisted of inflamed gingival tissues taken from patients with chronic periodontitis who were scheduled for periodontal surgery (positive control). The periodontal assessment of these patients was done by measuring periodontal pocket depths, soft tissue recession, and the degree of mobility of each tooth. The gingival tissue samples came from areas with an average Russell index score of 6 (moderate periodontitis) (15). The surgical procedures were performed under local anesthetic, using lidocaine with 1:100,000 epinephrine. After reflecting a full-thickness flap and removal of cortical bone, each periapical lesion was completely curetted out with a large spoon excavator. The pulps from unerupted third molars were obtained by making deep grooves on the buccal and lingual surface of each tooth with a fissure bur in a high-speed handpiece under water spray and splitting it in halves. The gingival tissue samples were obtained from patients who needed gingivectomies as a part of their periodontal therapy. After removal, depending on the size of the lesion, half or the whole tissue sample was snap-frozen in liquid nitrogen and stored at -70"C.
Journal of Endodontics
Histopathological Examination of Tissue Samples Half of the tissue samples from 16 large specimens were randomly placed in 10% formalin for histopathological examination: five from group 1, four from group 2, three from group 3, and four from group 4. Tissue samples were embedded in paraplast, sectioned at 6 um, and were stained with hematoxylin and eosin. Representative samples from each group were evaluated histologically by an independent oral pathologist under a light microscope. The sections were examined for the types (acute, chronic, and subacute) of inflammatory cells and the degree of inflammation (1, no inflammation; 2, mild inflammation (inflammatory cells more than two cell diameters apart); 3, moderate (inflammation cells one cell diameter apart); and 4, severe (inflammatory cells were less than one cell diameter apart).
Extraction of Leukotriene B4 The concentrations of LTB4 were determined by reverse phase high pressure liquid chromatography (HPLC). The methods for experimental tissues and appropriate standards as well as controls were performed according to procedures described by Jung et al. (16). After thawing and determining the wet weight of each tissue specimen, 1.0 ml of 100% ethanol was added to the sample and it was then homogenized in an Ultra-Turrax homogenizer. Then, 1.0 ml of HPLC grade water was added to each homogenized sample; the samples were centrifuged for 15 min at 3000 rpm in a Beckman T J-6 centrifuge. The supernatant was recovered and concentrated to 1 ml under nitrogen gas. The ethanolic sample was then dissolved in 19 ml of HPLC grade water and eluted through a C-18 column, Bond Elute/Vas Elute system (Analytical International, Harbor City, CA). The arachidonic acid metabolites retained in the cartilage were eluted with 10 ml of methanol. The methanolic eluent was dried and reconstituted with 1 ml of 50% ethanol. The sample (50 ul) was injected into the reverse phase-HPLC. The eluting solvent for lipoxygenase products was 70% methanol and 30% water (containing 0.1% acetic acid and 0.1% trifluoroacetic acid). Flow rate was 1.5 ml/min. The wavelength was set at 280 nm for the first 5.5 min, from 5.5 to 14.0 min at 235 nm, and from 14.0 to 22.0 min at 280 nm. The column was a DuPont, Reversed-Phase, Zorbax ODS column (4.6 mm x 25 cm) with an ODS guard column (4.6 mm x 5 cm). The HPLC system was a Shimadzu binary gradient LC-6A system with a C-R6A data processor and SPD-6A variable wavelength UV detector. The absolute calibration curve method was used for quantitative calculation, using the following equation: Content = (FliAi + F2i/Wspl) x 100 where Ai is the area (height); Fli is the response factor for slope; F2i is the response factor for constant term; and Wspl is the weight of sample. To determine the statistical difference in the concentrations of LTB4 among the groups, a Kruskal-Wallis one-way analysis of variance was used.
Leukotriene in Periapex
Vol. 18, No. 5, May 1992
207
TABLE 1. HistopathoIogical evaluation of representative samples showing degree (D) and type (T) of inflammation Group 1: symptomatic periapical lesions
Group 2: asymptomatic periapical lesions
Group 3: uninflamed pulpal tissue samples Group 4: inflamed gingival tissue samples
1 2 3 4 5 1 2 3 4 1 2
D4 D3 D3 D4 D4 D3 D4 D4 D4 ---
T3 T3 T1 T3 T3 T2 T2 T2 T2 ---
3
--
--
1 2 3 4
D3 D3 D4 D4
T2 T2 T2 T2
FIG 2. A section of a periapical lesion from a symptomatic patient showing an edematous granulation tissue infiltrated mainly by PMN leukocytes and some lymphocytes, plasma cells, and macrophages (hematoxylin and eosin; original magnification xl00).
RESULTS Evaluation of the results from the histopathological examination of the representative samples showed that the four groups were homogeneous (Table 1). Samples from group 1 showed the presence of moderate to severe acute and subacute inflammation (Fig. 2). Examination of the sections in group 2 showed the presence of moderate to severe chronic infiltrate (Fig. 3). Representative samples in group 3 consisted ofpulpal connective tissues. Group 4 showed areas of moderate to severe chronic inflammation (Fig. 4). The means and standard deviations of concentrations of LTB4 for each group were calculated in nanograms per milligram of wet tissue weight and are shown in Fig. 5 The Knaskal-Wallis one-way analysis of variance showed the presence of a significant difference in concentration of LTB4 between the four groups of tissues tested in this experiment (p = 0.001). Comparison between groups showed significant difference between the endogenous levels of LTB4 in the positive control (group 4) and the negative control (group 3, p < 0.05). In addition, significant differences were found in the levels of LTB4 in group 1 when compared with groups 2 and 3 (p
