Induced Rat Periapical Lesions Research Articles

Selenium ameliorates inflammation by decreasing autophagic flux and mitogen-activated protein kinase signalling on experimentally induced rat periapical lesions.

To reveal the molecular mechanisms that targets mitogen-activated protein kinase (MAPK) signalling and the autophagic flux and to investigate the possible effects of the systemic administration of selenium (Se) on experimentally induced rat periapical lesions. Thirty adult Sprague-Dawley rats were divided equally into negative control, positive control and Se groups. In the positive control and Se groups, the pulp chambers of their mandibular first molars were exposed to the oral environment to induce periapical lesions The Se group received daily intraperitoneal injections of Se at a dose of 0.1mg kg-1 . After 28 days, the amount of bone destruction; severity of inflammation; penetration of microorganisms along the root canal; collagen degradation in periodontal ligament; interleukin (IL)-6, hypoxia-inducible factor-1 (HIF-1), cyclooxygenase-2 (COX-2) and caspase-3 expression; autophagic flux; and p38 MAPK signalling were evaluated using radiographic, histopathological, Gram staining, picrosirius red stain, immunohistochemical, quantitative real-time polymerase chain (qRT-PCR) and Western blot methods, respectively. These data were analysed through the Kruskal-Wallis and Dunnett's tests (p < .05). The area of radiographic periapical bone loss, histopathological scores, the area of periapical bone loss and the scores for the bacteria localisation, the intensity of immunohistochemical staining for IL-6, HIF-1, COX-2 and caspase-3 in the Se group was significantly less than those of the positive control group (p < .01). The mRNA expression levels of Beclin-1, Atg3, Atg5, Atg7 and Atg16L1 were lower in the Se group than in the positive control group (p < .01). The protein expressions of Beclin-1, Atg5 and LC3-II, the phosphorylation ratio of the p38 MAPK and the ratios of LC3II/LC3I were significantly higher (p < .05) in the positive control and Se groups. On the contrary, the expression of the p62/SQSTM1 protein was significantly lower (p < .05) in the positive control and Se groups than in the negative control group. The induction of periapical lesions in rats increased autophagic flux and activated p38 MAPK signal transduction processes. Se suppressed the inflammatory process, reduced bone destruction and both the autophagic flux and p38 MAPK activation.

Expression of hypoxia-induced semaphorin 7A correlates with the severity of inflammation and osteoclastogenesis in experimentally induced periapical lesions

ObjectiveWhile hypoxia and inflammation are intimately linked, the effects of inflammatory hypoxia on the pathogenesis of periapical lesions remain largely unknown. The aim of this study was to examine hypoxia during the progression of experimentally induced rat periapical lesions, and to derive correlations between hypoxia-induced Semaphorin 7A (Sema7a) expression, severity of inflammation, and osteoclastogenesis in the lesions. DesignPeriapical lesions were developed after mandibular first molar pulp exposure in forty Sprague-Dawley rats. The animals were randomly divided into four groups and sacrificed at 0, 7, 14, and 28days after pulpal exposure. The bilateral mandibles containing the first molar were obtained and routinely prepared for histological, immunohistochemical, enzyme histochemical analyses and quantitative polymerase chain reaction detecting Sema7a mRNA expression. Data were analysed by one-way analysis of variance and the Pearson’s correlation and linear tendency test. ResultsPeriapical tissues become hypoxic during the development of experimentally induced periapical lesions, with steadily increasing numbers of HIF-1α-positive cells that positively correlate with the expression of Sema7a mRNA in the lesions. Furthermore, significant positive correlates were derived for the expression of Sema7a and the degree of inflammatory infiltration and osteoclast number, respectively. ConclusionsHypoxia-induced Sema7a participates in the pathogenesis of periapical lesions, providing a novel therapeutic target for the treatment of this inflammatory disease in the future.

Cathepsin K Inhibitor Regulates Inflammation and Bone Destruction in Experimentally Induced Rat Periapical Lesions

IntroductionCathepsin K is highly expressed in osteoclasts and plays an essential role in bone resorption. NC-2300 is an artificially designed cathepsin K inhibitor, and its application to experimentally induced arthritis induces down-regulation of bone destruction. In this study, we evaluated the effects of NC-2300 on inflammation and bone destruction in experimentally induced rat periapical lesions. MethodsThe dental pulps of lower first molars in rats were extirpated, and the pulp chambers were left open to the oral environment. NC-2300 and phosphate-buffered saline were administered orally twice a day in the experimental and control groups, respectively. Animals were sacrificed on day 21, and the mandibles were extracted. The left hemimandibles were used for micro–computed tomographic and histologic examination. For the right hemimandibles, RNA was extracted from the periapical tissues surrounding the root apices, and inflammatory mediator expression was examined by real-time polymerase chain reaction using complementary DNA converted from extracted RNA. ResultsThe size of the periapical lesion, number of tartrate-resistant acid phosphatase–positive osteoclasts and major histocompatibility complex class II molecule–expressing macrophages in the experimental group decreased significantly when compared with the control group. The expression of proinflammatory cytokines in the experimental group was significantly suppressed when compared with the control group. ConclusionsThese results suggest that the cathepsin K inhibitor may inhibit not only cathepsin K activity in osteoclasts but also inflammatory mediator synthesis relating to osteoclastogenesis, and these synergistic effects may be involved in the suppression of periapical lesion expansion.

Histochemical Localization of Dickkopf-1 in Induced Rat Periapical Lesions

IntroductionThe purpose of this study was to evaluate the expression of Dickkopf-1 (DKK-1), a secreted antagonist of the Wnt (wingless)/beta-catenin signaling pathway, during the development of periapical lesions in rats. MethodsPeriapical lesions were induced in Wistar rats by occlusal exposure of the pulp of their mandibular first molars. The animals were sacrificed randomly at 0, 7, 14, 21, and 28 day after pulpal exposure. Jaws containing the first molar were obtained and routinely prepared for histologic, immunohistochemical, and enzyme histochemical double immunofluorescence analyses. Data were analyzed using 1-way analysis of variance and Pearson correlation test. ResultsThe expansion of the area of periapical lesions was visible from days 7–21 and slowed down thereafter. A few DKK-1- and receptor activator of nuclear factor kappa B ligand (RANKL)-positive cells and osteoclasts were observed on day 7. All positive samples peaked in number on day 14. The expression levels of DKK-1 and RANKL and the number of osteoclasts decreased on days 21 and 28. DKK-1 expression was positively correlated with RANKL expression and osteoclast number from days 7–28. ConclusionsDKK-1 expression was up-regulated during periapical lesion development. DKK-1 may be associated with the inflammatory response and bone resorption in periapical lesions.

Kinetics of glycogen synthase kinase (GSK)3β and phosphorylated GSK3β (Ser 9) expression in experimentally induced periapical lesions

To investigate the kinetics of GSK3β and p-GSK3β (Ser 9) expression in experimentally induced rat periapical lesions and to explore their possible functions in the pathogenesis of periapical lesions. Periapical lesions were established in Wistar rats by occlusal pulp exposure in mandibular first molar teeth. The animals were killed on days 0, 7, 14, 21 and 28. Micro-computed tomographic, histological and enzyme histochemical analyses were performed to detect the progression of periapical lesions. Immunohistochemistry, double-dye immunofluorescence and Western blot were performed to determine the expression of GSK3β and p-GSK3β (Ser 9) in periapical tissues. From day 0 to day 28, the lesion volume and area gradually expanded, and the GSK3β-positive cells gradually ascended. A few p-GSK3β (Ser 9)-positive cells and osteoclasts appeared on day 7 and then climaxed on day 14. The numbers then simultaneously decreased from day 21 to day 28. Western blot analysis revealed that p-GSK3β (Ser 9) and GSK3β proteins were expressed at all time-points. The positive cells and protein expression ratio of p-GSK3β (Ser 9) against GSK3β increased from day 0 to day 14 and then decreased from day 14 to day 28. Finally, double-dye immunofluorescence assay revealed that p-GSK3β (Ser 9)-positive and RANKL-positive cells were co-localized around periapical lesions on days 14 and 28. GSK3β and p-GSK3β (Ser 9) can be observed and may be involved in alveolar bone resorption and inflammatory response in periapical lesions, as well as associated with periapical lesion pathogenesis.

Effect of Simvastatin on kinetics of Osteoprotegrin/receptor activator nuclear kappa B Ligand mRNA expression in periapical lesions

To evaluate the therapeutic effect of Simvastatin on apical periodontitis in rats by assessing the osteoblast production of receptor activator of nuclear factor-Kappa B Ligand (RANKL) and Osteoprotegerin (OPG) cytokines that are essential for bone resorption. Twenty-five healthy 8- to 10-week-old male rats were selected. Periapical lesions were induced by exposure of pulps through the occlusal surfaces of mandibular first molars. The pulps were extirpated and canals contaminated with saliva. The teeth were filled temporarily to make animal feeding feasible. The experimental group (n=12) was placed on 20mg(-1) kg(-1) day(-1) of Simvastatin for 10days (3days before and 7days after pulp exposure) and the control group (n=12) received a placebo. After 1, 2 and 4weeks, the animals were sacrificed and mRNA expression of OPG/RANKL was evaluated by real-time PCR. One rat was sacrificed without any interventions (negative control) to determine the baseline expression of the mediators involved. Data were analysed by one-way anova. Simvastatin significantly (P<0.05) enhanced the expression of OPG and reduced the mRNA expression of RANKL in the experimental group compared with the control group. Oral administration of Simvastatin attenuated RANKL expression and accelerated OPG expression in induced rat periapical lesions.

The Expression of Macrophage Migration Inhibitory Factor Is Correlated with Receptor Activator of Nuclear Factor Kappa B Ligand in Induced Rat Periapical Lesions

IntroductionMacrophage migration inhibitory factor (MIF) has been defined as a key cytokine in regulation of innate and adaptive immunity. The purpose of this study was to investigate the immunohistochemical localization of MIF and its relationship with receptor activator of nuclear factor kappa B ligand (RANKL) protein during the development of periapical lesions in rats. MethodsApical periodontitis was induced in Wistar rats by occlusal pulp exposure in mandibular first molar teeth. The animals were randomly killed at 0, 7, 14, 21, 28, and 35 days after pulp exposure. The jaws that contained the first molar were obtained and were prepared for histologic analysis, enzyme histochemistry, immunohistochemistry, and double immunofluorescence staining. ResultsFrom day 0 to day 35, the areas of periapical bone loss increased and seemed to be stabilized on day 35. A few MIF-positive and RANKL-positive cells and osteoclasts could be observed on day 7, and all climaxed on day 14. From day 21 to day 35, the expression of MIF and RANKL protein decreased, and fewer osteoclasts could be observed. ConclusionsThese findings showed that MIF might be associated with the differentiation of osteoclasts in the periapical lesions. MIF contributes to the pathogenesis of the periapical lesions through the induction of RANKL protein.

Kinetics of Th17‐related cytokine expression in experimentally induced rat periapical lesions

Th17-related cytokines are essential factors in various pathological states, including inflammatory bone destruction. This study investigated the contribution of Th17-related cytokines to the progress of experimentally induced rat periapical lesions. Periapical pathoses were induced by unsealed exposure of the pulp chamber of the lower first molars. A variety of immunocompetent cells, including CD68(+) macrophages, Ia antigen(+) cells and TCRαβ(+) T cells, were observed in the lesions. The expression levels of Th17-related cytokines, IL-17 and IL-23, and of pro-inflammatory cytokines, IL-1β and IL-6, were significantly increased at 14 days (expansion stage) compared with normal periapical tissues. The expression levels of Foxp3, a regulatory T cell (Treg)-related gene, and of IL-10, an anti-inflammatory cytokine, were higher at 28 days (chronic stage) than at 14 days. These findings suggest that Th17-related cytokines may be primary contributors to the initiation of periapical bone destruction, and that lesion expansion may be regulated by anti-inflammatory mediators.

Long‐term sequential receptor activator of NF‐κB ligand (RANKL) and osteoprotegrin (OPG) expression in lipopolysaccharide‐induced rat periapical lesions

Long-term sequential expression of receptor activator of NF-κB ligand (RANKL) and osteoprotegrin (OPG) in lipopolysaccharide (LPS)-induced rat periapical lesions has not been studied. Seventy-two 4-week-old Wistar rats were divided into eight experimental groups and one control group (eight animals in each). Lipopolysaccharide-induced periapical lesions were produced in rats by occlusal exposure of the pulp of their lower first molars in all experimental groups but not the control group. The extent of periapical destruction was measured by radiographic imaging. RANKL and OPG mRNA were measured in all tissue sections containing the periapical lesions as well as the control group every week from week 1 to week 8 by real-time quantitative reverse transcription polymerase chain reaction. RANKL and OPG protein were determined by immunohistochemistry. Osteoclasts were identified by enzyme histochemistry. The sequential changes in the mRNA and protein expression of RANKL and OPG were largely compatible with the occurrence of osteoclasts histologically and enzymes histochemically, as well as the mean areas of the periapical lesions radiographically during long-term observation of the LPS-induced rat periapical lesions. This study may be the first to demonstrate the long-term RANKL and OPG expression every week from week 1 to week 8 using LPS to produce periapical infection in a Wistar rat model. The long-term findings of high expressions of RANKL and OPG further extend the potential application of the Wistar rat model for future experimental trials using RANKL inhibitor to evaluate the treatment outcome for LPS-induced rat periapical lesions.

Simvastatin as a Novel Strategy To Alleviate Periapical Lesions

Hydroxymethylglutaryl-coenzyme A reductase inhibitors (statins) are widely used cholesterol-lowering agents that also possess anti-inflammatory activities. Cysteine-rich 61 (Cyr61) and CCL2 are potential osteolytic mediators in inflammatory bone diseases. The study assessed the effect of simvastatin on tumor necrosis factor α (TNF- α)--induced synthesis of Cyr61 and CCL2 in MG-63 human osteoblastic cells. The therapeutic effect of simvastatin on rat apical periodontitis was also examined. The synthesis of Cyr61 in MG-63 was assessed by Western analysis. Expression of CCL2 was examined by an enzyme-linked immunosorbent assay. The effect of simvastatin on induced rat periapical lesion was examined radiographically and immunohistochemically. Western blot showed that TNF-α stimulated Cyr61 synthesis in MG-63, whereas simvastatin attenuated this effect in a dose-dependent manner. Simvastatin also reduced the levels of TNF-α–induced CCL2, and exogenous Cyr61 restored the inhibitory effects. Radiography and histopathology revealed that the administration of simvastatin markedly diminished the severity of induced rat periapical lesions. The numbers of Cyr61-synthesizing osteoblasts and CD-68–positive macrophages were also decreased. Simvastatin suppresses the progression of apical periodontitis, possibly by diminishing Cyr61 expression in osteoblasts and, subsequently, macrophage chemotaxis into the lesions.

Immunohistochemical Localization of IL-17 in Induced Rat Periapical Lesions

Interleukin (IL)-17 is a member of a novel family of proinflammatory cytokines produced almost exclusively by a newly recognized subclass of activated T cells called “Th17” cells. From an endodontic perspective, IL-17 potently regulates cells of the innate immune system, serving as an important bridging molecule between the adaptive and innate immune systems. The purpose of this study was to investigate the immunohistochemical localization of IL-17 during the development of periapical lesions in rats. Periapical lesions developed within 28 days after mandibular first molar pulp exposure in Sprague-Dawley rats. The animals were randomly sacrificed at 0, 7, 14, 21, and 28 days after pulpal exposure. The jaws that contained the first molar were obtained and routinely prepared for histologic analysis, immunohistochemistry, and enzyme histochemistry. From day 0 to day 28, the number of IL-17–positive cells and neutrophils ascended and peaked on day 28. Osteoclast numbers substantially multiplied from day 0 to day 14 and then gradually decreased from day 14 to day 28. In addition, the osteoclast decrease contrasted with the increased number of IL-17–positive cells and neutrophils. These findings showed that IL-17 could be observed and might possibly be involved in the inflammatory response and bone resorption of periapical tissues as well as associated with periapical lesion pathogenesis.

Kinetics of RANKL, RANK and OPG expressions in experimentally induced rat periapical lesions

The object of this study was to elucidate the kinetics of receptor activator of NFkB ligand (RANKL), RANK, osteoprotegerin (OPG), and cytokine expressions in experimentally induced rat periapical lesions. The mRNA expressions of RANKL, RANK, OPG, and cytokines in experimentally induced rat periapical lesions were evaluated by real-time PCR. The lesions were induced in male Wistar rats (n = 48, 5 weeks of age) by unsealed pulp exposure of the lower first molars. Expression of RANKL was up-regulated at the beginning of lesion expansion, and expression ratio of RANKL against OPG, a competitor of RANKL, peaked at 2 and 3 weeks. Expression of inflammatory cytokines, such as TNF-alpha, IL-1alpha, and IL-1beta also increased at this stage, suggesting contribution of synergic effects of RANKL and proinflammatory cytokine signaling to lesion expansion. Most of RANKL+ cells were fibroblastic, but few of them were T cells. Expression of RANKL and proinflammatory cytokines was correlated with periapical lesion expansion.

Kinetic Study of Immunohistochemical Colocalization of Antigen-presenting Cells and Nerve Fibers in Rat Periapical Lesions

Immune and nervous systems play key roles in periapical pathosis; however, their spatial associations, which may be a prerequisite for paracrine interactions in the progression of periapical lesions, remain to be examined. In this study we examined the distribution and frequency of spatial associations between major histocompatibility complex class II molecule-expressing antigen-presenting cells (APCs) and protein gene product 9.5-immunoreactive nerve fibers in experimentally induced rat periapical lesions using double-immunofluorescence staining and confocal laser scanning microscopy. When active lesion expansion started, macrophage-like APCs frequently associated with nerve fibers around the apex. When the lesions were starting to stabilize, however, close associations between APCs with dendritic morphology and nerve fibers were found mostly in the periphery of lesions. CD86 + mature dendritic cells were also observed in this area. These findings suggest that functional interactions between APCs and nerve fibers may play some roles in the development of self-defense reactions in periapical lesions.

Immunolocalization of Macrophages and Transforming Growth Factor-β 1 in Induced Rat Periapical Lesions

Apical periodontitis was induced in Wistar rats by exposing the pulp chamber of right mandibular first molars to the oral environment. Animals were killed 0, 5, 10, 15, 20, 30, 60, and 80 days after lesion induction. Microradiographic and automated image analysis showed that the lesions expanded significantly in a time-dependent manner from day 0 to day 20 (0.039 mm2/day, p < 0.05, active phase) and stabilized thereafter (chronic phase). A linear regression test revealed a positive correlation between the numbers of ED-1 positive macrophage per microscopic high power field and the periapical lesion size during the active phase (r = 0.98, p < 0.01). Immunohistochemical studies showed that transforming growth factor-beta 1 positive macrophages distributed around the root apex and areas showing bone resorption during active lesion phase, whereas TGF-beta 1-positive osteoblasts were detected during the chronic stage (days 30, 60, and 80 after pulp exposure). Histologically TGF-beta 1 positive osteoblasts possessed a large, round nucleus as well as an abundant cytoplasm and located in close vicinity to areas exhibiting reparative bone formation. These results suggest that macrophages may play important role(s) in the initiation and development of periapical lesions and TGF-beta 1 may play dual roles in both bone resorption and deposition in induced rat periapical lesions.

Enhanced expression of activation-associated molecules on macrophages of heterogeneous populations in expanding periapical lesions in rat molars

Exudative macrophages are the most prevalent inflammatory cells during the entire pathogenetic process in experimentally induced rat periapical lesions. To clarify the significance of macrophages in the pathogenesis of periapical lesions, the way in which the phenotype of ED1 (a general marker for mononuclear phagocytes)-positive cells is modulated in actively expanding lesions was investigated, by immunoperoxidase staining with a panel of antibodies that recognize several activation-associated molecules on macrophages. Periapical lesions were induced experimentally by exposing the pulp in the lower first molars of Wistar rats. Active lesion expansion with morphological diversification of ED1-positive cells occurred between 14 and 28 days after the injury. Double immunoperoxidase staining revealed that ED1-positive cells coexpressing class II molecules of the major histocompatibility complex (MHC) molecules, inducible nitric oxide synthase (iNOS) and/or CD11a increased during the period of active lesion expansion. Increases of endothelial cells expressing intracellular adhesion molecule-1 and CD25 (interleukin-2 receptor)-expressing lymphocytes were also seen during the same period. Moreover, there existed two particular subpopulations of ED1+ cells in the established lesion at 28 days: (1) ED1++/class II MHC−/iNOS+ cells, located around the periapical abscess, and (2) ED1+/class II MHC+/iNOS− cells with slender or dendritic morphology, distributed predominantly in the outer portion of the lesion where T lymphocytes were abundant. The first cell type could be a macrophage with potent phagocytic and antimicrobial actions, and the second might possess sufficient antigen-presenting capacity to cause the activation of T lymphocytes. It was concluded that macrophages, when activated, may participate in triggering lesion expansion. Functionally distinct subpopulations of macrophages may occupy different sites within the lesion where they can most effectively exert their specific functions.

Immunohistochemical examination of the distribution of macrophages and CGRP-immunoreactive nerve fibers in induced rat periapical lesions.

To clarify the role of calcitonin gene-related peptide (CGRP) in the development of periapical lesions, we examined the distribution of CGRP-immunoreactive (IR) nerve fibers and macrophages, and the behavior of bone tissues in experimentally induced rat periapical lesions by immunohistochemical and quantitative methods. Although no extensive changes were observed at 7 days after pulp exposure, CGRP-IR nerve fibers increased in number until 28 days with a decrease thereafter. These neural changes were closely correlated with the alteration in number of macrophages except on day 7 when macrophages were significantly increased in number as compared with control rats. Tissue repair began to take place and a decrease in number of osteoclasts was observed when the density of CGRP-IR nerve fibers reached a peak. These results suggested that there might be a close relationship between macrophages and CGRP-IR nerve fibers and that CGRP-IR nerve fibers might participate in tissue repair in experimentally induced rat periapical lesions.

Pathogenesis of induced rat periapical lesions

Studies of the mechanisms of pathogenesis of periapical lesions were undertaken using a rat model of surgical pulp exposure. In this model, periapical lesions develop rapidly between days 0 and 15 (active phase) and more slowly thereafter (chronic phase). A Gram-negative anaerobic flora, similar to that seen in human beings, are quickly established. Lesions contain a mixed inflammatory cell infiltrate consisting of T cells, neutrophils, B cells, macrophages, and plasma cells. Helper T cells predominate during the active phase, whereas suppressor T cells are more frequent in the chronic phase. Extracts of periapical lesions contain bone-resorbing activity, the highest levels of which are present when lesions are actively expanding. Most bone-resorbing activity is mediated by the cytokine interleukin-1α, as determined by biochemical criteria and antibody neutralization studies. Prostaglandin 2 accounts for 10% to 15% of resorptive activity. Cells that express interleukin-1α were identified in pulp beginning on day 2 after exposure and in periapical tissue beginning on day 7, as determined by in situ hybridization and immunostaining. Macrophages, fibroblasts, neutrophils, and osteoclasts were positive for interleukin-1α mRNA and protein. Cells that express tumor necrosis factor α were also detected, whereas cells expressing interleukin-1β or tumor necrosis factor β were absent. Finally, periapical bone destruction was inhibited by 60% by treatment with interleukin-1 receptor antagonist. These studies establish a key role for interleukin-1α in the pathogenesis of periapical lesions in the rat model.

Immnohistochemical examination on the localization of macrophages and plasma cells in induced rat periapical lesions

To investigate the role of plasma cells and macrophages in the development of periapical lesions, we immunohistochemically examined the distribution of these inflammatory cells in experimental induced rat periapical lesions after pulpectomy. The number of ED1-positive mononuclear cells increased rapidly, reached a plateau which remained stable between days 10 and 60, and subsequently decreased. Immunoglobulin (Ig)-bearing plasma cells appeared after 60 days, and, of these, IgG-bearing plasma cells were predominant after 90 days. The radiographic and histopathological findings indicated the development of bone destruction at 10 days which continued until 60 days; tissue repair began to take place after 90 days. The results suggested that macrophages had a close relation to bone destruction and that plasma cells might participate in tissue repair rather than the development of periapical lesions.

T Helper and T Suppressor Cell Reversal During the Development of Induced Rat Periapical Lesions

T helper (TH) and T suppressor (TS) cells were enumerated in developing rat periapical lesions in order to identify the cell type associated with the active phase of lesion formation. Experimental pulp exposures were made in all molar teeth of 29 Sprague-Dawley rats, and teeth were left open to the oral environment. On days 15, 20, 30, and 90 after induction, inflammatory cells were isolated from periapical lesions, and T helper (W3/25+), T suppressor (OX8+), and total T cells (W3/13+) were enumerated by reactivity with monoclonal antibodies. During the early, most active phase of lesion development (day 15), TH cells outnumbered TS cells (TH/TS = 1.7). However, by day 20, when lesion expansion had slowed, the TH/TS ratio was reversed (0.9) and remained so for up to 90 days after induction (TH/TS = 0.7). Total T cell numbers did not change over the period of observation. TH cells thus predominated during the active phase of lesion development, whereas TS cells were associated with the chronic phase. This dynamic TH/TS reversal indicates that immune responses which occur within periapical lesions are highly regulated. In addition, the predominance of TH during the active phase of lesion development suggests that TH-mediated activities may be involved in the bone destruction and immune cell activation within periapical lesions.