Histopathological Evaluation of Combined Impacts of the Synthetic Progestin Levonorgestrel and Temperature on the Female Zebrafish Maturation Using a Semi-quantitative Grading Analysis-Is it Enough?

Pharmaceuticals contamination (e.g., synthetic progestins), and global climate change, represent two of the most stressful factors affecting aquatic species. To our knowledge, there is huge gap of data regarding the combined effects of both stressors on vertebrates' reproduction. Thus, it is crucial to implement rapid screenings of measurable histopathological alterations in fish gonads. For that, we propose: (1) an evaluation of the combined effects of progestin (levonorgestrel) and temperature on maturation of zebrafish female gonads, using a semi-quantitative method (i.e., grading) and (2) testing the robustness of the grading analysis comparatively to a quantitative method (i.e., stereology). Grading analysis showed a decrease on maturation stage of ovaries exposed to both stressors. Although grading is less robust than stereological analysis, it is recommended for a preliminary approach, since it gives a correct idea on trends and it is fast and cost-effective. For a detailed histological assessment, we recommend a stereological study.

Experimental sepsis induces sustained inflammation and acetylcholinesterase activity impairment in the hypothalamus.

Sepsis is one of the leading causes of mortality in intensive care units besides causing profound alterations in the brain. One of the structures notably affected during sepsis is the hypothalamus, resulting in important physiopathological consequences. Recently, we provided evidence that the presence of neuroinflammation, oxidative stress, and apoptosis in the hypothalamus of septic rats, is accompanied by impairment of arginine vasopressin (AVP) secretion. We had also demonstrated that sepsis survivor animals present attenuated AVP secretion after osmotic challenge, suggesting a persistent inflammation in the hypothalamus. However, the long-term course of inflammation in the hypothalamus remains unclear. Thus, we induced sepsis by cecal ligation and puncture (CLP) in Wistar rats and, five days after sepsis induction, the hypothalamus of each animal was collected for analysis. Nonmanipulated animals (naive) were used as controls. We found that CLP-induced morphological alterations in microglial cells are accompanied by an increase in Iba-1 immunoreactivity. Moreover, we observed enhanced expression of NF-κB and CREB transcription factors, which are well known to modulate the immune response. Additionally, we found that phosphorylation of GSK3α/ß (a kinase upstream to the CREB signaling pathway) was increased, as well as COX-2, iNOS, and IL-6 that are canonic inflammatory proteins. Thus, our results indicated the presence of sustained activation of resident glial cells that may result in neuroinflammation and cholinergic neurotransmission disruptions in the hypothalamus.

Alterations in hypothalamic synaptophysin and death markers may be associated with vasopressin impairment in sepsis survivor rats.

The impairment in arginine vasopressin (AVP) secretion during sepsis is described in clinical and experimental studies and has been associated with oxidative stress, apoptosis, and diminished activation of hypothalamic neurons. Few studies have, however, assessed these abnormalities in sepsis survivors. Here we performed two sets of experiments on Wistar rats that had been subjected to sepsis by cecal ligation and puncture (CLP) or nonmanipulated (naive) as control. In the first set, tissues and blood were collected from survivor rats 10 days after CLP to quantify hypothalamic Bcl-2, cleaved caspase- 3 and synaptophysin content, and bacterial load. In the second set, survivor rats were submitted to an acute osmotic stimulus (hypertonic saline), and after 30 minutes the water intake and AVP secretion were analyzed. The sepsis-surviving rats did not show bacterial load in tissues, but their hypothalamic synaptophysin and Bcl-2 levels were decreased, and the cleaved caspase- 3 level was increased when compared with the control group. However, AVP secretion was significantly attenuated in the CLP survivor animals submitted to an acute osmotic stimulus. These results suggest that the persistent AVP impairment in sepsis survivor animals may be due to a hypothalamic dysfunction associated with a synaptic deficit and decreased anti-apoptotic protein expression. This article is protected by copyright. All rights reserved.

Interactive effects of increased temperature, pCO2 and the synthetic progestin levonorgestrel on the fitness and breeding of the amphipod Gammarus locusta.

Given the lack of knowledge regarding climate change-chemical exposure interactions, it is vital to evaluate how these two drivers jointly impact aquatic species. Thus, for the first time, we aimed at investigating the combined effects of increased temperature, pCO2 and the synthetic progestin levonorgestrel on survival, growth, consumption rate and reproduction of the amphipod Gammarus locusta. For that, a full factorial design manipulating temperature [ambient temperature and warming (+4 °C)], pCO2 [normocapnia and hypercapnia (Δ pH 0.5 units)] and the progestin levonorgestrel (LNG: L1 - 10 ngLL-1 and L2 - 1000 ngLL-1, control - no progestin and solvent control - vehicle ethanol (0.01%)) was implemented for 21 days. G. locusta was strongly negatively affected by warming, experiencing higher mortality rates (50-80%) than in any other treatments. Instead, growth rates were significantly affected by interactions of LNG with temperature and pCO2. It was observed, in the short-term (7d) that under ambient temperature (18 °C) and hypercapnic conditions (pH 7.6), the LNG presence promoted the amphipod's growth, while in the medium-term (21d) this response was not observed. Relative consumption rates (RCRs), during the first week were higher than in the third week. Furthermore, in the first week, RCRs were negatively affected by higher temperature while in the third week, RCRs were negatively affected by acidification. Furthermore, it was observed a negative effect of higher temperature and acidification on G. locusta fecundity, contrarily to LNG. Concluding, the impact of increased temperature and pCO2 was clearly more adverse for the species than exposure to the synthetic progestin, however, some interactions between the progestin and the climate factors were observed. Thus, in a future scenario of global change, the presence of LNG (and other progestins alike) may modulate to a certain level the effects of climate drivers (and vice-versa) on the gammarids fitness and reproduction.

Warming modulates the effects of the endocrine disruptor progestin levonorgestrel on the zebrafish fitness, ovary maturation kinetics and reproduction success.

Interactive effects between multiple stressors, namely climate drivers (e.g., temperature) and chemical pollution (e.g., endocrine disruptors) are poorly studied. Here, it was for the first time evaluated the combinatory effects of temperature and a synthetic progestin, levonorgestrel (LNG), on the fitness and reproductive-related endpoints of zebrafish (Danio rerio). A multi-factorial design was implemented by manipulating both temperature [setting as baseline an ambient temperature of 27 °C, against warming (+3 °C)] and LNG levels (10 ngL-1 and 1000 ngL-1). Groups of males and females were exposed sub-acutely, for 21-days. Increased temperature caused an overall decrease in the females' gonadosomatic index (GSI), during the pre-reproduction phase, LNG did not affect GSI. In addition, fecundity (number of ovulated eggs) was negatively affected by both temperature and LNG, being the effect of the latter more intense. Fish exposed to the highest LNG concentration (at both temperatures) did not reproduce, but also in those exposed to the lowest dose of progestin at a higher temperature, a complete reproductive failure occurred. These results reflect what was observed in the stereological analysis of the ovary maturation stages prior to reproduction. Accordingly, the higher the LNG concentration, the lower the degree of maturation of the ovary. This was exacerbated by the higher temperature. As to embryonated eggs, they hatched significantly faster at higher temperatures, but exposure to 10 ngL-1 of LNG (at 27 °C) reduced significantly the hatching rate, comparing to control. Further, the recrudescence of the ovary 48 h after spawning seems to be not affected by both stressors. Our data suggest that in a future scenario of global warming and synthetic hormones exposure, the reproduction of fish species, such as the zebrafish, can be endangered, which can put at risk their success, and consequently affect the structure and functioning of associated aquatic ecosystems.

Differential Role of Neurohypophysial Hormones in Hypotension and Nitric Oxide Production During Endotoxaemia.

Besides their well-established endocrine roles, vasopressin and oxytocin are also important regulators of immune function, participating in a complex neuroendocrine-immune network. In the present study, we investigated whether and how vasopressin and oxytocin could modulate lipopolysaccharide (LPS)-induced nitric oxide (NO) production in a well-established model of experimental endotoxaemia. Male Wistar rats were previously treated i.v. with vasopressin V1 or oxytocin receptor antagonists and then received either an i.v. LPS injection to induce endotoxaemia or a saline imjection as a control. The animals were divided into two groups: in the first group, blood was collected at 2, 4 and 6 h after LPS injection; in the second group, mean arterial blood pressure (MABP) and heart rate (HR) were recorded over 6 h. Plasma vasopressin and oxytocin values were higher in LPS- compared to saline-injected animals at 2 and 4 h but returned to basal levels at 6 h. NO levels exhibited an opposite pattern, showing a progressive increase over the entire period. The previous administration of a vasopressin V1 receptor antagonist significantly reduced NO plasma concentrations at 2 and 4 h but not at 6 h. By contrast, oxytocin receptor agonist pre-treatment had no effect on the NO plasma concentration. In relation to MABP, previous treatment with vasopressin V1 receptor antagonist reversed the LPS-induced hypotension at 4 h, although this was not the case for oxytocin antagonist-treated animals. None of the antagonists affected HR. Our findings indicate that vasopressin (but not oxytocin) has effects on NO production during endotoxaemia in rats, although they do not lend support to the proposed anti-inflammatory actions of vasopressin during endotoxaemia.

The impact of compression therapy with Unna's boot on the functional status of VLU patients.

OBJECTIVE: To assess disability in patients with venous leg ulcers treated with compression therapy with Unna's boot. METHOD: A descriptive analytic case control study was conducted from June 2010 to May 2011 in an outpatient wound care clinic in interior Brazil. Fifty patients of both sexes, aged 18 years or above, who had had a venous leg ulcer for more than 1 year and a Doppler ankle-brachial index of 0.8-1.0 were selected for the study. Patients were treated with wound dressings and Unna's boot. Disability was assessed using the 20-item Stanford Health Assessment Disability Scale (HAQ-20). Statistical analysis was performed using the Student's t-test, the Kruskal-Wallis test and the chi-square test of independence, all at a significance level of 0.05 (p < 0.05). RESULTS: The mean overall HAQ score at inclusion (baseline) was 2.98, indicating impaired functional capacity. After 8 and 12 months of compression treatment with Unna's boot, the mean overall HAQ scores were 1.35 and 1.0, respectively, indicating good functional capacity. CONCLUSION: Patients with venous leg ulcer reported severe difficulty or serious disability in their daily functioning at baseline; after 8 months of treatment with Unna's boot, these patients were able to perform activities of daily living.

Acinetobacter baumannii infections in a tertiary care hospital in Mexico over the past 13 years.

BACKGROUND: Acinetobacter baumannii has evolved from an opportunistic pathogen into a common and persistent nosocomial bacterium capable of causing severe infections during endemic and epidemic periods. METHODS: The study period extended from January 1999 to December 2011 and involved patients hospitalized at the Hospital Civil de Guadalajara, Fray Antonio Alcalde, Jalisco, Mexico. From each patient, a single isolate was obtained, and a total of 3,680 unique isolates were collected. Susceptibility tests were performed according to the guidelines of the Clinical and Laboratory Standards Institute. RESULTS: A. baumannii has disseminated throughout the Hospital Civil de Guadalajara, Fray Antonio Alcalde, since 1999. A. baumannii isolates obtained from patients treated in the adult intensive care unit represent the majority of the isolates that have been collected. In addition, A. baumannii was isolated from the adult neurosurgical ward and the adult internal medicine ward, and these isolates were frequently obtained from secretions. A persistent decrease in the susceptibility of A. baumannii isolates to meropenem (92% in 1999 to 12% in 2011), imipenem and amikacin has been observed. CONCLUSIONS: A. baumannii became an endemic nosocomial pathogen during the study period at the Hospital Civil de Guadalajara, Fray Antonio Alcalde, and has exhibited a persistent decrease in susceptibility to all categories of antimicrobial agents over the past 13 years.

Bacterial intensity and localization in primary molars with caries disease.

AIM: The aim was to assess the characteristics and outcomes of infections affecting the structures of carious primary molars. MATERIALS AND METHODS: Forty primary molars were used and classified according to the following clinical situation: With profound caries lesion, with bone loss at the furcation region, with perforation of the pulp chamber floor, and residual roots. The teeth were demineralized, cut, and stained with both haematoxylin-eosin and Brown and Brenn staining techniques. Assessment was performed using optical microscopy. RESULTS: Statistical analysis of the data by means of the Chi-square test suggests that there was a significant relationship (P<0.001) between the intensity and localization of infection and the level of destruction of dental structures. A significant difference was also observed in the intensity and localization of infection between the groups regarding crown, furca, and root (P<0.001). CONCLUSION: More intense and profound the infection, more severe is the dental destruction. The groups of residual roots showed the most severe bacterial infection compared to other groups.

Effects of ethinylestradiol and of an environmentally relevant mixture of xenoestrogens on steroidogenic gene expression and specific transcription factors in zebrafish.

In natural environments fish are exposed to endocrine disrupting compounds (EDCs) present at low concentrations and with different modes of actions. Here, adult zebrafish of both sexes were exposed for 21 days to an estrogenic mixture (Mix) of eleven EDCs previously quantified in Douro River estuary (Portugal) and to 100 ng/L 17α-ethinylestradiol (EE2) as positive control. Vitellogenin mRNA and HSI in males confirmed both exposure regimes as physiologically active. Potential candidates for estrogenic disturbance of steroidogenesis were identified (StAR, 17ß-HSD1, cyp19a1), but Mix only affected cyp19a1 in females. Significant differences in the response of FSHß, cypa19a2, 20ß-HSD were observed between EE2 and Mix. Mtf-1 and tfap2c transcription factor binding sites were discovered in the putative promoter regions and corresponding transcription factors were found to be differentially expressed in response to Mix and EE2. The results suggest that "non-classical effects" of estrogenic EDC in fish are mediated via transcription factors.

Celecoxib treatment does not alter recruitment and activation of osteoclasts in the initial phase of experimental tooth movement.

In a previous study, we reported that the short-term treatment with celecoxib, a non-steroidal anti-inflammatory drug (NSAID) attenuates the activation of brain structures related to nociception and does not interfere with orthodontic incisor separation in rats. The conclusion was that celecoxib could possibly be prescribed for pain in orthodontic patients. However, we did not analyze the effects of this drug in periodontium. The aim of this follow-up study was to analyze effects of celecoxib treatment on recruitment and activation of osteoclasts and alveolar bone resorption after inserting an activated orthodontic appliance between the incisors in our rat model. Twenty rats (400-420 g) were pretreated through oral gavage with celecoxib (50 mg/kg) or vehicle (carboxymethylcellulose 0.4%). After 30 min, they received an activated (30 g) orthodontic appliance, set not to cause any palate disjunction. In sham animals, the appliance was immediately removed after introduction. All animals received ground food and, every 12 h, celecoxib or vehicle. After 48 h, they were anesthetized and transcardiacally perfused through the aorta with 4% formaldehyde. Subsequently, maxillae were removed, post-fixed and processed for histomorphometry or immunohistochemical analyses. As expected, incisor distalization induced an inflammatory response with certain histological changes, including an increase in the number of active osteoclasts at the compression side in group treated with vehicle (appliance: 32.2 ± 2.49 vs sham: 4.8 ± 1.79, P<0.05) and celecoxib (appliance: 31.0 ± 1.45 vs sham: 4.6 ± 1.82, P<0.05). The treatment with celecoxib did not modify substantially the histological alterations and the number of active osteoclasts after activation of orthodontic appliance. Moreover, we did not see any difference between the groups with respect to percentage of bone resorption area. Taken together with our previous results we conclude that short-term treatment with celecoxib can indeed be a therapeutic alternative for pain relieve during orthodontic procedures.

Central NO-cGMP pathway in thermoregulation and survival rate during polymicrobial sepsis.

Sepsis induces production of inflammatory mediators such as nitric oxide (NO) and causes physiological alterations, including changes in body temperature (Tb). We evaluated the involvement of the central NO-cGMP pathway in thermoregulation during sepsis induced by cecal ligation and puncture (CLP), and analyzed its effect on survival rate. Male Wistar rats with a Tb probe inserted in their abdomen were intracerebroventricularly injected with 1 microL NG-nitro-L-arginine methyl ester (L-NAME, 250 microg), a nonselective NO synthase (NOS) inhibitor; or aminoguanidine (250 microg), an inducible NOS inhibitor; or 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (ODQ, 0.25 microg), a guanylate cyclase inhibitor. Thirty minutes after injection, sepsis was induced by cecal ligation and puncture (CLP), or the rats were sham operated. The animals were divided into 2 groups for determination of Tb for 24 h and assessment of survival during 3 days. The drop in Tb seen in the CLP group was attenuated by pretreatment with the NOS inhibitors (p < 0.05) and blocked with ODQ. CLP rats pretreated with either of the inhibitors showed higher survival rates than vehicle injected groups (p < 0.05), and were even higher in the ODQ pretreated group. Our results showed that the effect of NOS inhibition on the hypothermic response to CLP is consistent with the role of nitrergic pathways in thermoregulation.

Effects of short-term acetaminophen and celecoxib treatment on orthodontic tooth movement and neuronal activation in rat.

Non-steroidal anti-inflammatory drugs (NSAIDs) have been used for pain relief in orthodontics, but clinical studies reported that they may reduce tooth movement (TM). By other side, TM seems to activate brain structures related to nociception, but the effects of NSAIDs in this activation have not been studied yet. We analyzed the effect of short-term treatment with acetaminophen or celecoxib in the separation of rat upper incisors, as well as in neuronal activation of the spinal trigeminal nucleus, following tooth movement. Thirty rats (400-420 g) were pretreated through oral gavage (1 ml/dose) with acetaminophen (200mg/kg), celecoxib (50mg/kg) or vehicle (carboxymethylcellulose 0.4%). After 30 min, they received an activated (30 g) orthodontic appliance for TM. In controls, this appliance was immediately removed after its introduction. Rats received ground food, and every 12h, one of the drugs or vehicle. After 48 h, they were anesthetized, maxilla was radiographed, and were perfused with 4% paraformaldehyde. Brains were further processed for Fos immunohistochemistry. TM induced incisor distalization (p<0.05) and neuronal activation of the spinal trigeminal nucleus. Treatment with both drugs did not affect tooth movement, but reduced c-fos expression in the caudalis subnucleus. No changes in c-fos expression were seen in the oralis and interpolaris subnuclei. We conclude that neither celecoxib nor acetaminophen seems to affect tooth movement, when used for 2 days, but both drugs are able to reduce the activation of brain structures related to nociception. Short-term treatment with celecoxib, thus, may be a therapeutic alternative to acetaminophen when the latter is contraindicated.

Blocking central leukotrienes synthesis affects vasopressin release during sepsis.

Recent studies revealed that vasopressinergic neurons have a high content of cys-leukotriene C(4) (LTC(4)) synthase, a critical enzyme in cys-leukotriene synthesis that may play a role in regulating vasopressin secretion. This study investigates the role of this enzyme in arginine vasopressin (AVP) release during experimentally induced sepsis. Male Wistar rats received an i.c.v. injection of 3-[1-(p-chlorobenzyl)-5-(isopropyl)-3-tert-butylthioindol-2-yl]-2, 2-dimethylpropanoic acid (MK-886) (1.0 microg/kg), a leukotrienes (LTs) synthesis inhibitor, or vehicle, 1 h before cecal ligation and puncture (CLP) or sham operation. In one group of animals the survival rate was monitored for 3 days. In another group, the animals were decapitated at 0, 4, 6, 18 and 24 h after CLP or sham operation, and blood was collected for hematocrit, serum sodium and nitrate, plasma osmolality, protein and AVP determination. A third group was used for blood pressure measurements. The neurohypophysis was removed for quantification of AVP content, and the hypothalamus was dissected for LTC(4) synthase analysis by Western blot. Mortality after CLP was reduced by the central administration of MK-886. The increase in plasma AVP levels and hypothalamus LTC(4) synthase content in the initial phase of sepsis was blocked, whereas the decrease in neurohypophyseal AVP content was partially reversed. Also the blood pressure drop was abolished in this phase. The increase of serum nitric oxide and hematocrit was reduced, and the decrease in plasma protein and osmolality was not affected by the LTs blocker. In the final phase of sepsis, the plasma AVP level and the hypothalamic LTC(4) synthase content were at basal levels. The central administration of MK-886 increased the hypothalamic LTC(4) synthase content but did not alter the plasma and neurohypophysis AVP levels observed, or the blood pressure during this phase. These results suggest that the central LTs are involved in the vasopressin release observed during sepsis.

Rapid maxillary expansion causes neuronal activation in brain structures of rats.

A correlation between pain sensation and neuronal c-fos expression has been analyzed following experimental rapid maxillar expansion (RME). Adult male Wistar rats were anaesthetized and divided into three groups: animals that received an orthodontic apparatus, which was immediately removed after the insertion (control), animals that received an inactivated orthodontic apparatus (without force), and animals that received an orthodontic apparatus previously activated (140 g force). After 6, 24, 48, or 72 h, the animals were re-anaesthetized, and perfused with 4% paraformaldehyde. The brains were removed, fixed, and sections containing brain structures related to nociception were processed for Fos protein immunohistochemistry (IHC). The insertion of the orthodontic apparatus with 140 g was able to cause RME that could be seen by radiography. The IHC results showed that the number of activated neurons in the different nuclei changed according to the duration of appliance insertion and followed a temporal pattern similar to that of sensations described in clinics. The animals that received the orthodontic apparatus without force did not show RME but a smaller c-fos expression in the same brain structures. In conclusion, we demonstrate that orthodontic force used for palate disjunction activates brain structures that are related to nociception, and that this activation is related to the pain sensation described during orthodontic treatment.

Efficacy of intracoronal bleaching techniques with different light activation sources.

AIM: To evaluate ex vivo the efficacy of 35% hydrogen peroxide for intracoronal bleaching when activated by LEDs, halogen lamp or by the walking bleach technique. METHODOLOGY: Forty extracted human maxillary central incisors had their crowns resected 1 mm below the amelo-cemental junction and were submitted to artificial staining in centrifuged rat haemolysed blood. A 2-mm thick glass ionomer cervical plug was placed inside the canal, at the level of the amelo-cemental junction. Samples were divided randomly into five groups: group I received 35% hydrogen peroxide gel activated by LEDs. Group II received 35% hydrogen peroxide gel activated by a halogen lamp-based light curing unit. Group III received 35% hydrogen peroxide gel followed by the walking bleach technique. Group IV was neither artificially stained nor bleached (positive control) and group V was stained, but not bleached (negative control). The shade of the teeth was assessed visually by three independent and calibrated evaluators, before and after bleaching. The results were analysed using Kruskal-Wallis one-way analysis of variance and Dunn's post-test. RESULTS: No statistical differences regarding sample shades were found amongst groups for the tested internal bleaching techniques (P > 0.05). CONCLUSIONS: Hydrogen peroxide for intracoronal bleaching when activated either by LEDs, halogen lamp or by the walking bleach technique presented similar efficacy.

Lamotrigine pharmacokinetic evaluation in epileptic patients submitted to VEEG monitoring.

OBJECTIVE: The aim of the present study was to evaluate the pharmacokinetic profile of lamotrigine (LTG) in epileptic patients submitted to video-electroencephalography (VEEG) monitoring and, in addition, to investigate the influence of concomitant antiepileptic drugs (AEDs) on the kinetics of LTG. METHODS: The analysis assumed a one-compartment open model with first-order absorption and elimination. The kinetic estimates obtained in this population were validated by using the Prediction-Error approach. The influence of medication was also assessed by the calculation of the LTG concentration-to-dose ratio. Patients (n=135) were divided into four groups according to the co-medication: Group 1, patients taking LTG with enzyme-inducer agents; Group 2, patients receiving LTG with valproic acid; Group 3, patients receiving both inducers and inhibitors of LTG metabolism; Group 4, patients under AEDs not known to alter LTG metabolism. RESULTS: The obtained estimates for clearance (CL) (L/h/kg) [0.075+/-0.029 (Group 1), 0.014+/-0.005 (Group 2), 0.025+/-0.008 (Group 3) and 0.044+/-0.011 (Group 4)] appear to be the most appropriate set to be implemented in clinical practice as prior information, as demonstrated by the accuracy and precision of the measurements. In addition, the influence of co-medication on the LTG profile was further confirmed by the basal LTG concentration-to-dose ratio. CONCLUSION: The results of the present investigation may contribute to achieving the goal of optimizing patients' clinical outcomes by managing their medication regimen through measured drug concentrations. Patients submitted to VEEG monitoring may benefit from this study, as the results may be used to provide better drug management in this medical setting.

The effect of mineral trioxide aggregate on the apexification and periapical healing of teeth with incomplete root formation.

AIM: To evaluate the influence of mineral trioxide aggregate (MTA) on apexification and periapical healing of teeth in dogs with incomplete root formation and previously contaminated canals and to verify the necessity of employing calcium hydroxide paste before using MTA. METHODOLOGY: Twenty premolars from two 6-month old dogs were used. After access to the root canals and complete removal of the pulp, the canal systems remained exposed to the oral environment for 2 weeks. Canal preparation was then carried out using Hedström files, under irrigation with 1% sodium hypochlorite, 1 mm short of the radiographic apex. After drying, the canals of two premolars in each dog were left empty (control group). The other eight teeth in each animal were divided into two experimental groups. The apical thirds of the canals of group 1 were filled with MTA. In the teeth of group 2, the canals were dressed with a calcium hydroxide-propylene glycol paste. After 1 week, the paste was removed and the apical third was filled with MTA. All teeth were restored with reinforced zinc oxide cement (IRM) and amalgam. The animals were killed 5 months later, and blocks of the teeth and surrounding tissues were submitted to histological processing. The sections were studied to evaluate seven parameters: formation of an apical calcified tissue barrier, level of barrier formation, inflammatory reaction, bone and root resorption, MTA extrusion, and microorganisms. Results of experimental groups were analysed by Wilcoxon's nonparametric tests and by the test of proportions. The critical value of statistical significance was 5%. RESULTS: Significant differences (P < 0.05) were found in relation to the position of barrier formation and MTA extrusion. The barrier was formed in the interior of the canal in 69.2% of roots from MTA group only. In group 2, it was formed beyond the limits of the canal walls in 75% of the roots. MTA extrusion occurred mainly in roots from group 2. There was similarity between the groups for the other parameters. CONCLUSIONS: Mineral trioxide aggregate used after root canal preparation favoured the occurrence of the apexification and periapical healing. The initial use of calcium hydroxide paste was not necessary for apexification to occur, and has shown to be strongly related to the extrusion of MTA and formation of barriers beyond the limits of the root canal walls.

Ex vivo evaluation of the capacity of the Tri Auto ZX to locate the apical foramen during root canal retreatment.

AIM: To evaluate ex vivo the capacity of the Tri Auto ZX to locate the apical foramen during root canal retreatment. METHODOLOGY: The root canals of 62 maxillary and mandibular canines were prepared to a length 1 mm short of the apical foramen, to an apical size 35 using 1% sodium hypochlorite as an irrigant. Once prepared, the length of each tooth was measured directly using a size 15 K-Flexofile introduced in the canal until the tip was visible at the apical foramen. After the file was removed, its length was recorded to a precision of 0.01 mm using a calliper. These direct lengths (DL) became the 'gold standard' for comparison with the electronic lengths (EL) derived from the Tri Auto ZX. After direct measurement, the tooth was measured electronically (EL1) and the canals were filled using lateral condensation of gutta-percha cones and sealer. Seven days later the root filling was removed using solvent, Gates-Glidden burs, and K-files, and new electronic lengths determined (EL2). The electronic lengths (EL1 and EL2) were compared with the DL, and the differences were analysed statistically using the proportions test and Student's t-test. RESULTS: At a tolerance limit of +/-0.5 mm, EL1 coincided with the DL in 76% of cases. Lengths obtained in the presence of remnant of filling material (EL2) coincided in 81% of cases. The proportions test used to compare these percentages showed no statistically significant difference between EL1 and EL2 (P > 0.05). The Student's t-test revealed a statistically significant difference (P < 0.05) between the means of the differences between DL/EL1 (-0.36 mm) and DL/EL2 (-0.04 mm). CONCLUSION: The Tri Auto ZX was accurate to +/-0.5 mm in more than 80% of teeth following removal of root fillings.