Fibroblast-like synoviocytes from fluid and synovial membrane from primary osteoarthritis demonstrate similar production of interleukin 6, and metalloproteinases 1 and 3.

OBJECTIVES: The objective of the study was to compare the production of metalloproteinases (MMP)-1, -3 and interleukin (IL)-6 by fibroblast-like synoviocytes (FLS) derived from synovial fluid (FD-FLS), and FLS derived from synovial tissue (TD-FLS) of patients with primary osteoarthritis (OA). The more accessible FD-FLS could facilitate the study of the role of these cells in OA pathophysiology. METHODS: MMP-1, MMP-3, and IL-6 levels were measured in the supernatant culture at baseline and 22 hours after stimulation with TNF-α and IL-1 ß. RESULTS: There was no difference at baseline between MMP-1, MMP-3 and IL-6 production by FD-FLS and TDFLS. Analogous to baseline, stimulation of FD-FLS and TD-FLS with IL-1ß and TNF-α did not result in difference on MMP-3 and IL-6 production. However, TD-FLS produced more MMP-1 than FD-FLS after stimulation with IL-1ß (p=0.01). Additionally, there was a positive correlation for production of MMP-1, MMP-3 and IL-6 between FD-FLS and TD-FLS (r=0.40 and p<0.0008; r=0.66 and p<0.0001; r=0.76 and p<0.0001, respectively). Supporting this statistical significant positive correlation, the Bland-Altman plotting, showed a homogeneous distribution of the values and low mean disagreement rates between all results of FD-FLS and TD-FLS (23.1%, 56.8% and 48.1%, respectively). CONCLUSIONS: Our data demonstrated functional similarity between FD-FLS and TD-FLS and support the use of a more accessible source of FLS for the study of the pathogenesis of joint destruction and therapeutic targets in primary OA.

The use of smartphones in radiographic diagnosis: accuracy on the detection of marginal gaps.

OBJECTIVE: To compare the accuracy of computer monitor and smartphone screen for radiographic diagnosis of marginal gap. MATERIALS AND METHODS: Forty teeth with mesial-occlusal-distal inlays (each tooth with a perfect fit and a 0.4-mm marginal gap restoration) were imaged with a phosphor plate system. Original digital radiographs were exported and analyzed with two different methods: computer monitor and smartphone screen; for the last method, images were shared with WhatsApp. Three examiners assessed all radiographs (n = 160) for the presence of marginal gap by using a dichotomous scale (yes/no). Diagnostic performance of each examiner and viewing method was evaluated by means of sensitivity (Se), specificity (Sp), and overall accuracy (Ac). Difference between the frequencies of gap detection of each method was analyzed using the McNemar test. Intra- and inter-examiner agreements were calculated using kappa statistics. RESULTS: Intra- and inter-examiner agreements were ≥ 0.80 for both methods. Similar diagnostic performance was found for computer monitor (Se = 0.87-1; Sp = 0.8-0.97; Ac = 0.84-0.99) and smartphone (Se = 0.77-1; Sp = 0.87-1; Ac = 0.88-0.95) viewing methods. No statistically significant differences in the frequency of gap detection were observed between the methods (P > 0.05). CONCLUSION: Diagnostic accuracy of smartphone screens was similar to that of computer monitor for marginal gap detection. CLINICAL RELEVANCE: Smartphones are becoming a common daily tool. In this sense, it might be an important new aid in Dentistry, including radiographic evaluation, which could benefit patients and dentists.

Analysis of the genetic ancestry of patients with oral clefts from South American admixed populations.

Increased susceptibility to cleft lip, with or without cleft palate (CL±P) has been observed in South America, as related to Amerindian ancestry, using epidemiological data, uniparental markers, and blood groups. In this study, it was evaluated whether this increased risk remains when Amerindian ancestry is estimated using autosomal markers and considered in the predictive model. Ancestry was estimated through genotyping 62 insertion and deletion (INDEL) markers in sample sets of patients with CL±P, patients with cleft palate (CP), and controls, from Patagonia in southern Argentina and Belém in northern Brazil. The Amerindian ancestry in patients from Patagonia with CL±P was greater than in controls although it did not reach statistical significance. The European ancestry in patients with CL±P from Belém and in patients with CP from Belém and Patagonia was higher than in controls and statistically significant for patients with CP who were from Belém. This high contribution of European genetic ancestry among patients with CP who were from Belém has not been previously observed in American populations. Our results do not corroborate the currently accepted risks for CL±P and CP estimated by epidemiological studies in the North American populations and probably reflect the higher admixture found in South American ethnic groups when compared with the same ethnic groups from the North American populations.

Uniparental ancestry markers in Chilean populations.

The presence of Native Americans, Europeans, and Africans has led to the development of a multi-ethnic, admixed population in Chile. This study aimed to contribute to the characterization of the uniparental genetic structure of three Chilean regions. Newborns from seven hospitals in Independencia, Providencia, Santiago, Curicó, Cauquenes, Valdívia, and Puerto Montt communes, belonging to the Chilean regions of Santiago, Maule, and Los Lagos, were studied. The presence of Native American mitochondrial DNA (mtDNA) haplogroups and two markers present in the non-recombinant region of the Y chromosome, DYS199 and DYS287, indicative of Native American and African ancestry, respectively, was determined. A high Native American matrilineal contribution and a low Native American and African patrilineal contributions were found in all three studied regions. As previously found in Chilean admixed populations, the Native American matrilineal contribution was lower in Santiago than in the other studied regions. However, there was an unexpectedly higher contribution of Native American ancestry in one of the studied communes in Santiago, probably due to the high rate of immigration from other regions of the country. The population genetic sub-structure we detected in Santiago using few uniparental markers requires further confirmation, owing to possible stratification for autosomal and X-chromosome markers.

Joint-specific rheumatoid arthritis fibroblast-like synoviocyte regulation identified by integration of chromatin access and transcriptional activity.

The mechanisms responsible for the distribution and severity of joint involvement in rheumatoid arthritis (RA) are not known. To explore whether site-specific fibroblast-like synoviocyte (FLS) biology might be associated with location-specific synovitis and explain the predilection for hand (wrist/metacarpal phalangeal joints) involvement in RA, we generated transcriptomic and chromatin accessibility data from FLS to identify the transcription factors and pathways. Networks were constructed by integration of chromatin accessibility and gene expression data. Analysis revealed joint-specific patterns of FLS phenotype, with proliferative, migratory, proinflammatory, and matrix-degrading characteristics observed in resting FLS derived from the hand joints compared with hip or knee. TNF stimulation amplified these differences, with greater enrichment of proinflammatory and proliferative genes in hand FLS compared with hip and knee FLS. Hand FLS also had the greatest expression of markers associated with an "activated" state relative to the "resting" state, with the greatest cytokine and MMP expression in TNF-stimulated hand FLS. Predicted differences in proliferation and migration were biologically validated with hand FLS exhibiting greater migration and cell growth than hip or knee FLS. Distinctive joint-specific FLS biology associated with a more aggressive inflammatory response might contribute to the distribution and severity of joint involvement in RA.

Joint-specific rheumatoid arthritis fibroblast-like synoviocyte regulation identified by integration of chromatin access and transcriptional activity.

The mechanisms responsible for the distribution and severity of joint involvement in rheumatoid arthritis (RA) are not known. To explore whether site-specific FLS biology might be associated with location-specific synovitis and explain the predilection for hand (wrist/metacarpal phalangeal joints) involvement in RA, we generated transcriptomic and chromatin accessibility data from FLS to identify the transcription factors (TFs) and pathways. Networks were constructed by integration of chromatin accessibility and gene expression data. Analysis revealed joint-specific patterns of FLS phenotype, with proliferative, migratory, proinflammatory, and matrix-degrading characteristics observed in resting FLS derived from the hand joints compared with hip or knee. TNF-stimulation amplified these differences, with greater enrichment of proinflammatory and proliferative genes in hand FLS compared with hip and knee FLS. Hand FLS also had the greatest expression of markers associated with an 'activated' state relative to the 'resting' state, with the greatest cytokine and MMP expression in TNF-stimulated hand FLS. Predicted differences in proliferation and migration were biologically validated with hand FLS exhibiting greater migration and cell growth than hip or knee FLS. Distinctive joint-specific FLS biology associated with a more aggressive inflammatory response might contribute to the distribution and severity of joint involvement in RA.

Dysregulated NUB1 and Neddylation Enhances Rheumatoid Arthritis Fibroblast-Like Synoviocyte Inflammatory Responses.

OBJECTIVE: Fibroblast-like synoviocytes (FLS) contribute to the pathogenesis of rheumatoid arthritis (RA), in part due to activation of the proinflammatory transcription factor NF-κB. Neddylation is modulated by the negative regulator of ubiquitin-like protein (NUB) 1. We determined whether NUB1 and neddylation are aberrant in the models with RA FLS, thereby contributing to their aggressive phenotype. METHODS: Models with RA or osteoarthritis (OA) FLS were obtained from arthroplasty synovia. Real-time quantitative polymerase chain reaction and Western blot analysis assessed gene and protein expression, respectively. NUB1 was overexpressed using an expression vector. NF-κB activation was assessed by stimulating FLS with interleukin (IL)-1ß. Neddylation inhibitor (MLN4924) and proteasome inhibitor were used in migration and gene expression assays. MLN4924 was used in the model with K/BxN serum-transfer arthritis. RESULTS: Enhanced H3K27ac and H3K27me3 peaks were observed in the NUB1 promoter in the OA FLS compared with the RA FLS. NUB1 was constitutively expressed by FLS, but induction by IL-1ß was significantly greater in the OA FLS. The ratio of neddylated cullin (CUL) 1 to nonneddylated CUL1 was lower in the OA FLS than the RA FLS. NUB1 overexpression decreased NF-κB nuclear translocation and IL-6 messenger RNA (mRNA) in IL-1ß-stimulated the RA FLS. MLN4924 decreased CUL1 neddylation, NF-κB nuclear translocation, and IL-6 mRNA in IL-1ß-stimulated the RA FLS. MLN4924 significantly decreased arthritis severity in the model with K/BxN serum-transfer arthritis. CONCLUSION: CUL1 neddylation and NUB1 induction is dysregulated in the models with RA, which increases FLS activation. Inhibition of neddylation is an effective therapy in an animal model of arthritis. These data suggest that the neddylation system contributes to the pathogenesis of RA and that regulation of neddylation could be a novel therapeutic approach.

Joint-specific regulation of homeobox D10 expression in rheumatoid arthritis fibroblast-like synoviocytes.

Rheumatoid arthritis (RA) is a systemic immune-mediated disease characterized by joint inflammation and destruction. The disease typically affects small joints in the hands and feet, later progressing to involve larger joints such as the knees, shoulders, and hips. While the reasons for these joint-specific differences are unclear, distinct epigenetic patterns associated with joint location have been reported. In this study, we evaluated the unique epigenetic landscapes of fibroblast-like synoviocytes (FLS) from hip and knee synovium in RA patients, focusing on the expression and regulation of Homeobox (HOX) transcription factors. These highly conserved genes play a critical role in embryonic development and are known to maintain distinct expression patterns in various adult tissues. We found that several HOX genes, especially HOXD10, were differentially expressed in knee FLS compared with hip FLS. Epigenetic differences in chromatin accessibility and histone marks were observed in HOXD10 promoter between knee and hip FLS. Histone modification, particularly histone acetylation, was identified as an important regulator of HOXD10 expression. To understand the mechanism of differential HOXD10 expression, we inhibited histone deacetylases (HDACs) with small molecules and siRNA. We found that HDAC1 blockade or deficiency normalized the joint-specific HOXD10 expression patterns. These observations suggest that epigenetic differences, specifically histone acetylation related to increased HDAC1 expression, play a crucial role in joint-specific HOXD10 expression. Understanding these mechanisms could provide insights into the regional aspects of RA and potentially lead to therapeutic strategies targeting specific patterns of joint involvement during the course of disease.

Side effects of different head and neck radiotherapy doses on wistar rat's behavior.

Radiotherapy (RT) is a common treatment for head and neck tumors. However, it causes several physical and behavioral side effects, and no study has assessed the emotional effects in rats. Therefore, the present study evaluated the influence of head and neck RT on the behavior and body weight gain in Wistar rats. Fifty-four male Wistar rats were allocated into six groups (n = 9) according to the irradiation dose, which was applied at the first day of the experiment: RT-7.5 (single dose of 7.5 Gy); RT-10 (single dose of 10 Gy); RT-15 (single dose of 15 Gy); RT-30 (single dose of 30 Gy); Control (without RT). The animals were irradiated in the region of the right face, and behavioral tests and weighing were performed on days one, seven, and 28. The open field and Y-maze tests were undertaken to analyze the animal's behavior. The dose of 30 Gy was lethal when applied to the head and neck region. The irradiated animals had less weight gain when compared to the control ones, but there was no statistical difference. In the open field and Y-maze tests, lower mobility of animals in the RT groups was observed both on day seven and at the end of the experiment (day 28) when compared to the control rats (p < 0.05). It was possible to conclude that the different doses of radiation induced depressive behavior in the animals, and that the weight gain tended to be lower in the irradiated groups, however, without statistical difference.

Laser Capture Microscopy RNA Sequencing for Topological Mapping of Synovial Pathology During Rheumatoid Arthritis.

OBJECTIVE: Rheumatoid arthritis (RA) is an autoimmune disease in which the joint lining or synovium becomes highly inflamed and majorly contributes to disease progression. Understanding pathogenic processes in RA synovium is critical for identifying therapeutic targets. We performed laser capture microscopy (LCM) followed by RNA sequencing (LCM-RNAseq) to study regional transcriptomes throughout RA synovium. METHODS: Synovial lining, sublining, and vessel samples were captured by LCM from seven patients with RA and seven patients with osteoarthritis (OA). RNAseq was performed on RNA extracted from captured tissue. Principal component analysis was performed on the sample set by disease state. Differential expression analysis was performed between disease states based on log2 fold change and q value parameters. Pathway analysis was performed using the Reactome Pathway Database on differentially expressed genes among disease states. Significantly enriched pathways in each synovial region were selected based on the false discovery rate. RESULTS: RA and OA transcriptomes were distinguishable by principal component analysis. Pairwise comparisons of synovial lining, sublining, and vessel samples between RA and OA revealed substantial differences in transcriptional patterns throughout the synovium. Hierarchical clustering of pathways based on significance revealed a pattern of association between biologic function and synovial topology. Analysis of pathways uniquely enriched in each region revealed distinct phenotypic abnormalities. As examples, RA lining samples were marked by anomalous immune cell signaling, RA sublining samples were marked by aberrant cell cycle, and RA vessel samples were marked by alterations in heme scavenging. CONCLUSION: LCM-RNAseq confirms reported transcriptional differences between the RA synovium and the OA synovium and provides evidence supporting a relationship between synovial topology and molecular anomalies in RA.

Morphofunctional analysis of fibroblast-like synoviocytes in human rheumatoid arthritis and mouse collagen-induced arthritis.

BACKGROUND: Fibroblast-like synoviocytes (FLS) play a prominent role in rheumatoid synovitis and degradation of the extracellular matrix through the production of inflammatory cytokines and metalloproteinases (MMPs). Since animal models are frequently used for elucidating the disease mechanism and therapeutic development, it is relevant to study the ultrastructural characteristics and functional responses in human and mouse FLS. The objective of the study was to analyze ultrastructural characteristics, Interleukin-6 (IL-6) and Metalloproteinase-3 (MMP-3) production and the activation of intracellular pathways in Fibroblast like synoviocytes (FLS) cultures obtained from patients with rheumatoid arthritis (RA) and from mice with collagen-induced arthritis (CIA). METHODS: FLSs were obtained from RA patients (RA-FLSs) (n = 8) and mice with CIA (CIA-FLSs) (n = 4). Morphology was assessed by transmission and scanning electron microscopy. IL-6 and MMP-3 production was measured by ELISA, and activation of intracellular signaling pathways (NF-κB and MAPK: p-ERK1/2, p-P38 and p-JNK) was measured by Western blotting in cultures of RA-FLSs and CIA-FLSs stimulated with tumor necrosis factor-alpha (TNF-α) and IL-1ß. RESULTS: RA-FLS and CIA-FLS cultures exhibited rich cytoplasm, rough endoplasmic reticula and prominent and well-developed Golgi complexes. Transmission electron microscopy demonstrated the presence of lamellar bodies, which are cytoplasmic structures related to surfactant production, in FLSs from both sources. Increased levels of pinocytosis and numbers of pinocytotic vesicles were observed in RA-FLSs (p < 0.05). Basal production of MMP-3 and IL-6 was present in RA-FLSs and CIA-FLSs. Regarding the production of MMP-3 and IL-6 and the activation of signaling pathways, the present study demonstrated a lower response to IL-1ß by CIA-FLSs than by RA-FLSs. CONCLUSION: This study provides a comprehensive understanding of the biology of RA-FLS and CIA-FLS. The differences and similarities in ultrastructural morphology and important inflammatory cytokines shown, contribute to future in vitro studies using RA-FLS and CIA-FLS, in addition, they indicate that the adoption of CIA-FLS for studies should take careful and be well designed, since they do not completely resemble human diseases.

Systems-biology analysis of rheumatoid arthritis fibroblast-like synoviocytes implicates cell line-specific transcription factor function.

Rheumatoid arthritis (RA) is an immune-mediated disease affecting diarthrodial joints that remains an unmet medical need despite improved therapy. This limitation likely reflects the diversity of pathogenic pathways in RA, with individual patients demonstrating variable responses to targeted therapies. Better understanding of RA pathogenesis would be aided by a more complete characterization of the disease. To tackle this challenge, we develop and apply a systems biology approach to identify important transcription factors (TFs) in individual RA fibroblast-like synoviocyte (FLS) cell lines by integrating transcriptomic and epigenomic information. Based on the relative importance of the identified TFs, we stratify the RA FLS cell lines into two subtypes with distinct phenotypes and predicted active pathways. We biologically validate these predictions for the top subtype-specific TF RARα and demonstrate differential regulation of TGFß signaling in the two subtypes. This study characterizes clusters of RA cell lines with distinctive TF biology by integrating transcriptomic and epigenomic data, which could pave the way towards a greater understanding of disease heterogeneity.

Performance of High Mobility Protein Group 1 and Interleukin-6 as Predictors of Outcomes Resulting from Variceal Bleeding in Patients with Advanced Chronic Liver Disease.

Variceal bleeding is a serious complication in cirrhotic patients and is related to increased expression of inflammatory mediators that accentuate circulatory dysfunction. The study aims to evaluate the performance of high mobility protein group 1 (HMG1) and interleukin-6 (IL-6) as predictors of acute kidney injury (AKI), infection and death in these patients. Fifty patients who were diagnosed with advanced chronic liver disease with variceal bleeding were included. The mean age was 52.8 ± 10.8 years, and 33 (66%) were male. Twenty-one (42%) patients were classified as Child-Pugh C, 21 (42%) Child-Pugh B and 8 (16%) Child-Pugh A. The mean HMG1 serum level was 2872.36 pg/mL ± 2491.94, and the median IL-6 serum level was 47.26 pg/mL (0-1102.4). In AKI, the serum level of HMG1 that performed best on the ROC curve was 3317.9 pg/mL. The IL-6 serum level was not associated with AKI. HMG1 and IL-6 cut-off values that better predicted infection were 3317.9 pg/mL and 72.9 pg/mL, and for mortality, the values were 2668 pg/mL and 84.5 pg/mL, respectively. In multivariate analysis, the variables that were associated with AKI and infection outcomes were model for end-stage liver disease and HMG1. Infections were related to the risk of death. Clinical and laboratory variables related to the outcomes were identified. Serum levels of HMG1 were associated with AKI and infection and had good performance in the ROC curve. IL-6 levels were not maintained in logistic regression outcomes but had good performance in infection and death outcomes. Such data will be useful for comparisons and possible future validations.

Low-dose Multidetector Computed Tomographic and Cone-beam Computed Tomographic Protocols for Volumetric Measurement of Simulated Periapical Lesions.

INTRODUCTION: The purpose of this study was to evaluate the accuracy of low-dose multidetector computed tomographic (LD-MDCT) imaging for the volumetric measurement of simulated periapical lesions. METHODS: Eighteen monoradicular teeth were introduced in bone blocks, and periapical lesions were simulated at the periapical region of each tooth. All teeth were imaged using 4 acquisition protocols: large (dentoalveolar) field of view (FOV) cone-beam computed tomographic (CBCT) imaging (120 kV, 5 mA, and 0.2-mm voxel), small (dental) FOV CBCT imaging (90 kV, 10 mA, and 0.2-mm voxel), standard multidetector computed tomographic imaging (120 kV, 50 mA, and 0.62-mm voxel), and LD-MDCT imaging (120 kV, 10 mA, and 0.62-mm voxel). Tomographic images were evaluated by a single trained and calibrated examiner (intraclass correlation coefficient = 0.991) using ITK-SNAP segmentation software (University of Pennsylvania, Philadelphia, PA). The gold standard was obtained by the impressions of the lesions with regular fluid addition silicone and individual weighing using a precision analytical scale. Data were evaluated by the repeated measures analysis of variance test; the significance level was defined as P < .05. RESULTS: No statistical differences (P > .05) were found among the groups regardless of the device, milliamperage, FOV, or voxel size. CONCLUSIONS: LD-MDCT shows performance comparable with other standard reference methods for measuring the volume of periapical lesions and can be a useful and safe protocol in clinical situations in which CBCT imaging is not available, such as in cases of patients admitted to hospitals.

High-fat diet effect on periapical lesions and hepatic enzymatic antioxidant in rats.

AIMS: To evaluate the effects of a high-fat diet (HFD) on the progression of apical periodontitis (AP), local inflammation, systemic antioxidant status, and blood lipid profile in rats. MAIN METHODS: Sixteen male Wistar rats were fed a standard diet (SD) or a HFD. At the sixth experimental week, the pulp chambers of the mandibular first molars were exposed to develop AP. A glucose tolerance test was performed the week before euthanasia. At the tenth experimental week, the animals were euthanized and the livers were collected to estimate catalase (CAT) and reduced glutathione (GSH) levels. Blood was acquired for biochemical analysis. The size of AP was estimated from radiographs and described as AP size-to-body weight ratio; inflammatory grade of AP was determined by histological analysis. KEY FINDINGS: At the end of the experimental period, the rats fed the HFD had 30% less weight (P < 0.0001) and higher blood glucose levels after 30 min of sucrose intake (P < 0.05) than those fed the SD. Animals from the HFD group had lower levels of CAT (P < 0.01), but the same was not observed in the GSH levels. Plasma insulin and total cholesterol were not affected by the diet. The rats fed the HFD presented greater AP than those fed the SD (P < 0.05). However, the local inflammatory infiltrate was similar in both groups. SIGNIFICANCE: The alterations promoted by the consumption of a HFD were not only observed systemically, but also locally, producing greater AP in rats than a SD.

Apical periodontitis induces changes on oxidative stress parameters and increases Na+/K+-ATPase activity in adult rats.

OBJECTIVES: Endodontic infection can cause systemic alterations. The involvement of oxidative stress (OS) and transmembrane enzymes compose the pathogenesis of various systemic diseases. However, the relation among apical periodontitis (AP), OS parameters, and Na+/K+-ATPase (NKA) pump was not reported in the literature. This study evaluated the AP influence on OS parameters and NKA activity in adult rats. METHODS: Adult male Wistar rats (sixteen weeks old) were randomly assigned to two experimental groups: control (CT group; n = 8) and AP (AP group; n = 9), which was induced in the first right mandibular molar tooth. After 21 days of AP induction, mandibles were dissected for radiographic analysis. In addition, the heart, liver, pancreas, and kidney were collected for analysis of endogenous OS parameters and NKA activity. Data were analyzed by Student's T-test. Values of p < 0.05 were considered statistically significant. RESULTS: AP presence increased reactive species (RS) generation only in the heart, while the other analyzed organs did not have this parameter modified. Heart and pancreas had a decreased endogenous antioxidant system (catalase activity and vitamin C levels), liver and kidney had an increased one. AP increased NKA activity in the heart, liver, and pancreas, but not in the kidney. CONCLUSION: The modulation of both endogenous antioxidant defense system and NKA activity in vital organs suggested that alterations in the antioxidant status and cellular electrochemical gradient may be involved in the AP pathophysiology.

Detectability of Middle Mesial Canal in Mandibular Molar After Troughing Using Ultrasonics and Magnification: An Ex Vivo Study.

The aim of this study was to evaluate the frequency of the identification of middle mesial (MM) canals in extracted permanent first and second mandibular molars before and after executing a troughing technique with high magnification. Sample consisted of 105 mandibular molars. After gaining access to the pulp chamber and cleaning the chamber floor, root canals were detected in three different stages. The initial location was performed under direct viewing without magnification. In the second stage, a dental operating microscope was employed at 12 magnification. If the MM canal was not observed, a standardized troughing technique was executed with the use of an ultrasonic tip between the mesiobuccal and mesiolingual canals under magnification. Statistical differences in the frequency of MM canals before and after troughing were determined using McNemar's test, with the significance level set at 5%. No significant increase in the identification of the MM canal was found when comparing observations before (9.52%) and after (12.38%) the troughing technique under high magnification. However, viewing under magnification and the execution of troughing significantly increased the location of the MM canal (12.38%) when compared to viewing without magnification (3.81%) (p<0.01). The troughing technique with the aid of a dental operating microscope significantly improved the identification of MM canals compared to viewing without magnification. Troughing is a safe, minimally invasive procedure that benefits the treatment of mandibular molars.