Analysis of arsenic-modulated expression of hypothalamic estrogen receptor, thyroid receptor, and peroxisome proliferator-activated receptor gamma mRNA and simultaneous mitochondrial morphology and respiration rates in the mouse.

Arsenic has been identified as an environmental toxicant acting through various mechanisms, including the disruption of endocrine pathways. The present study assessed the ability of a single intraperitoneal injection of arsenic, to modify the mRNA expression levels of estrogen- and thyroid hormone receptors (ERα,ß; TRα,ß) and peroxisome proliferator-activated receptor gamma (PPARγ) in hypothalamic tissue homogenates of prepubertal mice in vivo. Mitochondrial respiration (MRR) was also measured, and the corresponding mitochondrial ultrastructure was analyzed. Results show that ERα,ß, and TRα expression was significantly increased by arsenic, in all concentrations examined. In contrast, TRß and PPARγ remained unaffected after arsenic injection. Arsenic-induced dose-dependent changes in state 4 mitochondrial respiration (St4). Mitochondrial morphology was affected by arsenic in that the 5 mg dose increased the size but decreased the number of mitochondria in agouti-related protein- (AgRP), while increasing the size without affecting the number of mitochondria in pro-opiomelanocortin (POMC) neurons. Arsenic also increased the size of the mitochondrial matrix per host mitochondrion. Complex analysis of dose-dependent response patterns between receptor mRNA, mitochondrial morphology, and mitochondrial respiration in the neuroendocrine hypothalamus suggests that instant arsenic effects on receptor mRNAs may not be directly reflected in St3-4 values, however, mitochondrial dynamics is affected, which predicts more pronounced effects in hypothalamus-regulated homeostatic processes after long-term arsenic exposure.

Determination of Bile Acids in Canine Biological Samples: Diagnostic Significance.

The comprehensive examination of bile acids is of paramount importance across various fields of health sciences, influencing physiology, microbiology, internal medicine, and pharmacology. While enzymatic reaction-based photometric methods remain fundamental for total BA measurements, there is a burgeoning demand for more sophisticated techniques such as liquid chromatography-tandem mass spectrometry (LC-MS/MS) for comprehensive BA profiling. This evolution reflects a need for nuanced diagnostic assessments in clinical practice. In canines, a BA assessment involves considering factors, such as food composition, transit times, and breed-specific variations. Multiple matrices, including blood, feces, urine, liver tissue, and gallbladder bile, offer insights into BA profiles, yet interpretations remain complex, particularly in fecal analysis due to sampling challenges and breed-specific differences. Despite ongoing efforts, a consensus regarding optimal matrices and diagnostic thresholds remains elusive, highlighting the need for further research. Emphasizing the scarcity of systematic animal studies and underscoring the importance of ap-propriate sampling methodologies, our review advocates for targeted investigations into BA alterations in canine pathology, promising insights into pathomechanisms, early disease detection, and therapeutic avenues.

Functional Aspects of Hypothalamic Asymmetry.

Anatomically, the brain is a symmetric structure. However, growing evidence suggests that certain higher brain functions are regulated by only one of the otherwise duplicated (and symmetric) brain halves. Hemispheric specialization correlates with phylogeny supporting intellectual evolution by providing an ergonomic way of brain processing. The more complex the task, the higher are the benefits of the functional lateralization (all higher functions show some degree of lateralized task sharing). Functional asymmetry has been broadly studied in several brain areas with mirrored halves, such as the telencephalon, hippocampus, etc. Despite its paired structure, the hypothalamus has been generally considered as a functionally unpaired unit, nonetheless the regulation of a vast number of strongly interrelated homeostatic processes are attributed to this relatively small brain region. In this review, we collected all available knowledge supporting the hypothesis that a functional lateralization of the hypothalamus exists. We collected and discussed findings from previous studies that have demonstrated lateralized hypothalamic control of the reproductive functions and energy expenditure. Also, sporadic data claims the existence of a partial functional asymmetry in the regulation of the circadian rhythm, body temperature and circulatory functions. This hitherto neglected data highlights the likely high-level ergonomics provided by such functional asymmetry.

Metabolic Lateralization in the Hypothalamus of Male Rats Related to Reproductive and Satiety States.

The hypothalamus is the main regulatory center of many homeostatic processes, such as reproduction, food intake, and sleep-wake behavior. Recent findings show that there is a strongly interdependent side-linked localization of hypothalamic functions between the left and right hemispheres. The goal of the present study was to trace functional asymmetry of the hypothalamus related to the regulation of food intake and reproduction, in male rodents. Subjects were examined through measurements of mitochondrial metabolism ex vivo. Impact of gonadectomy and scheduled feeding was tested on the modulation of hypothalamic metabolic asymmetry. Results show that in male rats, functional lateralization of the hypothalamus can be attributed to the satiety state rather than to reproductive control. Fasting caused left-sided metabolic dominance, while satiety was linked to the right hemisphere; trends and direction in sided dominance gradually followed the changes in satiety state. Our findings revealed satiety state-dependent metabolic differences between the two hypothalamic hemispheres. It is therefore concluded that, at least in male rats, the hypothalamic hemispheres control the satiety state-related functions in an asymmetric manner.

Endocrine Disruptors Induced Distinct Expression of Thyroid and Estrogen Receptors in Rat versus Mouse Primary Cerebellar Cell Cultures.

The endocrine system of animals consists of fine-tuned self-regulating mechanisms that maintain the hormonal and neuronal milieu during tissue development. This complex system can be influenced by endocrine disruptors (ED)-substances that can alter the hormonal regulation even in small concentrations. By now, thousands of substances-either synthesized by the plastic, cosmetic, agricultural, or medical industry or occurring naturally in plants or in polluted groundwater-can act as EDs. Their identification and testing has been a hard-to-solve problem; Recent indications that the ED effects may be species-specific just further complicated the determination of biological ED effects. Here we compare the effects of bisphenol-A, zearalenone, and arsenic (well-known EDs) exerted on mouse and rat neural cell cultures by measuring the differences of the ED-affected neural estrogen- and thyroid receptors. EDs alters the receptor expression in a species-like manner detectable in the magnitude as well as in the nature of biological responses. It is concluded that the interspecies differences (or species specificity) in ED effects should be considered in the future testing of ED effects.

Comparative Analysis of Zearalenone Effects on Thyroid Receptor Alpha (TRα) and Beta (TRß) Expression in Rat Primary Cerebellar Cell Cultures.

Thyroid receptors play an important role in postnatal brain development. Zearalenone (ZEN), a major mycotoxin of Fusarium fungi, is well known to cause serious health problems in animals and humans through various mechanisms, including the physiological pathways of thyroid hormone (TH). In the present study, we aimed to investigate the expression of thyroid receptors α (TRα) and ß (TRß) in primary cerebellar neurons in the presence or absence of glia and following ZEN treatment, using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Primary cerebellar granule cells were treated with low doses of ZEN (0.1 nM) in combination with physiologically relevant concentrations of l-thyroxine (T4), 3,3',5-triiodo-l-thyronine (T3) and 17ß-estradiol (E2). Expression levels of TRα and TRß at mRNA and protein levels were slightly modified by ZEN administered alone; however, along with thyroid and steroid hormones, modelling the physiological conditions, expression levels of TRs varied highly depending on the given treatment. Gene expression levels were also highly modulated by the presence or absence of glial cells, with mostly contrasting effects. Our results demonstrate divergent transcriptional and translational mechanisms involved in the expression of TRs implied by ZEN and hormonal milieu, as well as culturing conditions.

Utilization and acceptance of virtual patients in veterinary basic sciences - the vetVIP-project.

Context: In medical and veterinary medical education the use of problem-based and cased-based learning has steadily increased over time. At veterinary faculties, this development has mainly been evident in the clinical phase of the veterinary education. Therefore, a consortium of teachers of biochemistry and physiology together with technical and didactical experts launched the EU-funded project "vetVIP", to create and implement veterinary virtual patients and problems for basic science instruction. In this study the implementation and utilization of virtual patients occurred at the veterinary faculties in Budapest, Hannover and Lublin. Methods: This report describes the investigation of the utilization and acceptance of students studying veterinary basic sciences using optional online learning material concurrently to regular biochemistry and physiology didactic instruction. The reaction of students towards this offer of clinical case-based learning in basic sciences was analysed using quantitative and qualitative data. Quantitative data were collected automatically within the chosen software-system CASUS as user-log-files. Responses regarding the quality of the virtual patients were obtained using an online questionnaire. Furthermore, subjective evaluation by authors was performed using a focus group discussion and an online questionnaire. Results: Implementation as well as usage and acceptance varied between the three participating locations. High approval was documented in Hannover and Lublin based upon the high proportion of voluntary students (>70%) using optional virtual patients. However, in Budapest the participation rate was below 1%. Due to utilization, students seem to prefer virtual patients and problems created in their native language and developed at their own university. In addition, the statement that assessment drives learning was supported by the observation that peak utilization was just prior to summative examinations. Conclusion: Veterinary virtual patients in basic sciences can be introduced and used for the presentation of integrative clinical case scenarios. Student post-course comments also supported the conclusion that overall the virtual cases increased their motivation for learning veterinary basic sciences.

Bisphenol A influences oestrogen- and thyroid hormone-regulated thyroid hormone receptor expression in rat cerebellar cell culture.

Thyroid hormones (THs) and oestrogens are crucial in the regulation of cerebellar development. TH receptors (TRs) mediate these hormone effects and are regulated by both hormone families. We reported earlier that THs and oestradiol (E2) determine TR levels in cerebellar cell culture. Here we demonstrate the effects of low concentrations (10-10 M) of the endocrine disruptor (ED) bisphenol A (BPA) on the hormonal (THs, E2) regulation of TRα,ß in rat cerebellar cell culture. Primary cerebellar cell cultures, glia-containing and glia-destroyed, were treated with BPA or a combination of BPA and E2 and/or THs. Oestrogen receptor and TH receptor mRNA and protein levels were determined by real-time qPCR and Western blot techniques. The results show that BPA alone decreases, while BPA in combination with THs and/or E2 increases TR mRNA expression. In contrast, BPA alone increased receptor protein expressions, but did not further increase them in combination with THs and/or E2. The modulatory effects of BPA were mediated by the glia; however, the degree of changes also depended on the specific hormone ligand used. The results signify the importance of the regulatory mechanisms interposed between transcription and translation and raise the possibility that BPA could act to influence nuclear hormone receptor levels independently of ligand-receptor interaction.

Preparation of purified perikaryal and synaptosomal mitochondrial fractions from relatively small hypothalamic brain samples.

In order to measure the activity of neuronal mitochondria, a representative proof of neuronal processes, physiologically relevant mitochondrial samples need to be gained as simply as possible. Existing methods are, however, either for tissue samples of large size and/or homogenous microstructures only, or are not tested for mitochondrial function measurements. In the present article we describe a gradient fractionation method to isolate viable and well-coupled mitochondria from relatively heterogeneous histological microstructures such as the hypothalamus. With this new method, we are able to isolate a sufficient amount of functional mitochondria for determination of respiratory activity, in a short period of time, using affordable equipment. •Verified by electron microscopy, our method separates highly enriched and well-preserved perikaryal and synaptosomal mitochondria. Both fractions contain minimal cell debris and no myelin. Respiratory measurements (carried out by Clark-type electrode) confirmed undisturbed mitochondrial function providing well-evaluable records. The demonstrated protocol yields highly viable mitochondrial subfractions within 3 h from small brain areas for high-precision examinations. Using this procedure, brain regions with relatively heterogeneous histological microstructure (hypothalamus) can also be efficiently sampled.•Up to our present knowledge, our method is the shortest available procedure with the lowest sample size to gain debris-free, fully-viable mitochondria.

Comparison of Individual and Combined Effects of Four Endocrine Disruptors on Estrogen Receptor Beta Transcription in Cerebellar Cell Culture: The Modulatory Role of Estradiol and Triiodo-Thyronine.

BACKGROUND: Humans and animals are continuously exposed to a number of environmental substances that act as endocrine disruptors (EDs). While a growing body of evidence is available to prove their adverse health effects, very little is known about the consequences of simultaneous exposure to a combination of such chemicals; METHODS: Here, we used an in vitro model to demonstrate how exposure to bisphenol A, zearalenone, arsenic, and 4-methylbenzylidene camphor, alone or in combination, affect estrogen receptor ß (ERß) mRNA expression in primary cerebellar cell cultures. Additionally, we also show the modulatory role of intrinsic biological factors, such as estradiol (E2), triiodo-thyronine (T3), and glial cells, as potential effect modulators; RESULTS: RESULTS show a wide diversity in ED effects on ERß mRNA expression, and that the magnitude of these ED effects highly depends on the presence or absence of E2, T3, and glial cells; CONCLUSION: The observed potency of the EDs to influence ERß mRNA expression, and the modulatory role of E2, T3, and the glia suggests that environmental ED effects may be masked as long as the hormonal milieu is physiological, but may tend to turn additive or superadditive in case of hormone deficiency.

Estrogen- and Satiety State-Dependent Metabolic Lateralization in the Hypothalamus of Female Rats.

Hypothalamus is the highest center and the main crossroad of numerous homeostatic regulatory pathways including reproduction and energy metabolism. Previous reports indicate that some of these functions may be driven by the synchronized but distinct functioning of the left and right hypothalamic sides. However, the nature of interplay between the hemispheres with regard to distinct hypothalamic functions is still unclear. Here we investigated the metabolic asymmetry between the left and right hypothalamic sides of ovariectomized female rats by measuring mitochondrial respiration rates, a parameter that reflects the intensity of cell and tissue metabolism. Ovariectomized (saline injected) and ovariectomized+estrogen injected animals were fed ad libitum or fasted to determine 1) the contribution of estrogen to metabolic asymmetry of hypothalamus; and 2) whether the hypothalamic asymmetry is modulated by the satiety state. Results show that estrogen-priming significantly increased both the proportion of animals with detected hypothalamic lateralization and the degree of metabolic difference between the hypothalamic sides causing a right-sided dominance during state 3 mitochondrial respiration (St3) in ad libitum fed animals. After 24 hours of fasting, lateralization in St3 values was clearly maintained; however, instead of the observed right-sided dominance that was detected in ad libitum fed animals here appeared in form of either right- or left-sidedness. In conclusion, our results revealed estrogen- and satiety state-dependent metabolic differences between the two hypothalamic hemispheres in female rats showing that the hypothalamic hemispheres drive the reproductive and satiety state related functions in an asymmetric manner.

Hypothalamic sidedness in mitochondrial metabolism: new perspectives.

Morphofunctional changes in hypothalamic neurons are highly energy dependent and rely on mitochondrial metabolism. Therefore, mitochondrial adenosine triphosphate production plays a permissive role in hypothalamic regulatory events. Here, we demonstrated that in the female rat hypothalamus, mitochondrial metabolism and tissue oxygenation show an asymmetric lateralization during the estrous cycle. This asymmetry was not detected in males. The observed sidedness suggests that estrous cycle-linked hypothalamic functions in females are based on hemispheric distinction. The novel concept of hypothalamic asymmetry necessitates the revision of hypothalamic neural circuits, synaptic reorganization, and the role of hypothalamic sides in the regulation of integrated homeostatic functions.

Ligand-induced changes in Oestrogen and thyroid hormone receptor expression in the developing rat cerebellum: A comparative quantitative PCR and Western blot study.

Oestrogen (E2) and thyroid hormones (THs) are key regulators of cerebellar development. Recent reports implicate a complex mechanism through which E2 and THs influence the expression levels of each other's receptors (ERs and TRs) to precisely mediate developmental signals and modulate signal strength. We examined the modulating effects of E2 and THs on the expression levels of their receptor mRNAs and proteins in cultured cerebellar cells obtained from 7-day-old rat pups. Cerebellar granule cell cultures were treated with either E2, THs or a combination of these hormones, and resulting receptor expression levels were determined by quantitative PCR and Western blot techniques. The results were compared to non-treated controls and to samples obtained from 14-day-old in situ cerebella. Additionally, we determined the glial effects on the regulation of ER-TR expression levels. The results show that (i) ER and TR expression depends on the combined presence of E2 and THs; (ii) glial cells mediate the hormonal regulation of neuronal ER-TR expression and (iii) loss of tissue integrity results in characteristic changes in ER-TR expression levels. These observations suggest that both E2 and THs, in adequate amounts, are required for the precise orchestration of cerebellar development and that alterations in the ratio of E2/THs may influence signalling mechanisms involved in neurodevelopment. Comparison of data from in vitro and in situ samples revealed a shift in receptor expression levels after loss of tissue integrity, suggesting that such adjusting/regenerative mechanisms may function after cerebellar tissue injury as well.

Determining the apical preparation size in premolars: part III.

OBJECTIVE: The objective of this study was to investigate cross sections of the apical part of premolar root canals and determine recommendations for their optimal apical preparation size. METHODOLOGY: A total of 140 root canals from extracted human premolars (86 maxillary, 54 mandibular) were selected. After preflaring, the electronic working length (WL) was measured to identify where the apical preparation size (APS) should be established. Subsequently, nontapered, noncutting special instruments (SI) were used to reach the WL. The diameter of the largest SI width that reached the WL was defined as d(SI). The roots were apically sectioned at 2 levels, and the diameter of a potential rotary instrument was determined, allowing complete circumferential cutting of the root canal wall for each section. The estimation of APS was then related to d(SI). RESULTS: Apically enlarging upper premolars with 2 canals and lower premolars to an individual APS of d(SI) +0.3 mm (6 file sizes) resulted in a 71% to 75% complete preparation of the root canal walls, whereas an enlarged preparation of d(SI) +0.4 mm (8 file sizes) reached 82% to 96%. In upper premolars with a single canal, an APS of d(SI) +0.3 mm completely processed the root canal walls in only 37%, whereas an APS of d(SI) +0.4 mm reached 63%. CONCLUSIONS: The APS in 2-canal upper premolars and mandibular premolars should be at least 6 sizes larger than the first apically binding file, whereas in upper premolars with a central canal, the APS should be enlarged to 8 sizes larger. Normally, this implies shaping premolars with 1 canal to #60 to 70 and with 2 canals to #40 to 50.

Effects of energy restriction on thyroid hormone metabolism in chickens.

Energy restriction induces changes in thyroid hormone economy in the form of a complex adaptation mechanism, in order to conserve energy storage and protein reserves. In the present work, thyroid hormone serum concentrations, hepatic deiodinase enzyme activities and hepatic deiodinase mRNA expression were examined after feed restriction and fasting. We demonstrate that during energy restriction, T 3 concentration is lowered due to a decreased T 4 activation and increased T 3 inactivation. We show that hepatic type-I deiodinase (D1) is not affected by energy restriction, however, hepatic D2 is decreased on both transcriptional and enzyme activity levels. Furthermore, hepatic D3 is increased after feed restriction in the liver. We also show that the hypothalamic feedback is not involved in the changes in serum T 3 and T 4 concentrations. Our data indicate that D2 enzyme contributes to the special hormone-exporting role of the chicken liver and this enzyme can be modulated by feed restriction.

NTPDases in the neuroendocrine hypothalamus: possible energy regulators of the positive gonadotrophin feedback.

BACKGROUND: Brain-derived ectonucleoside triphosphate diphosphohydrolases (NTPDases) have been known as plasma membrane-incorporated enzymes with their ATP-hydrolyzing domain outside of the cell. As such, these enzymes are thought to regulate purinergic intercellular signaling by hydrolyzing ATP to ADP-AMP, thus regulating the availability of specific ligands for various P2X and P2Y purinergic receptors. The role of NTPDases in the central nervous system is little understood. The two major reasons are the insufficient knowledge of the precise localization of these enzymes in neural structures, and the lack of specific inhibitors for the various NTPDases. To fill these gaps, we recently studied the presence of neuron-specific NTPDase3 in the mitochondria of hypothalamic excitatory neurons by morphological and functional methods. Results from those studies suggested that intramitochondrial regulation of ATP levels may play a permissive role in the neural regulation of physiological functions by tuning the level of ATP-carried energy that is needed for neuronal functions, such as neurotransmission and/or intracellular signaling. PRESENTATION OF THE HYPOTHESIS: In the lack of highly specific inhibitors, the determination of the precise function and role of NTPDases is hardly feasable. Yet, here we attempt to find an approach to investigate a possible role for hypothalamic NTPDase3 in the initiation of the midcycle luteinizing hormone (LH) surge, as such a biological role was implied by our recent findings. Here we hypothesize that NTPDase-activity in neurons of the AN may play a permissive role in the regulation of the estrogen-induced pituitary LH-surge. TESTING THE HYPOTHESIS: We propose to test our hypothesis on ovariectomized rats, by stereotaxically injecting 17beta-estradiol and/or an NTPDase-inhibitor into the arcuate nucleus and determine the consequential levels of blood LH, mitochondrial respiration rates from arcuate nucleus synaptosomal preparations, NTPDase3-expression from arcuate nucleus tissue samples, all compared to sham and intact controls. IMPLICATIONS OF THE HYPOTHESIS: Results from these studies may lead to the conclusion that estrogen may modulate the activity of mitochondrial, synapse-linked NTPDase3, and may show a correlation between mitochondrial NTPDase3-activity and the regulation of LH-release by estrogen.

Glycogenic induction of thyroid hormone conversion and leptin system activation in the liver of postpartum dairy cows.

In the regulation of energy metabolism, the liver plays an important role in the reinforcement of energy production. In periparturient cows the energy homeostasis turns into a negative energy balance that may shift the physiological regulation of energy balance towards pathological processes. Propylene glycol (PG), as a complementary source of energy used in the nutrition of dairy cows, alters systemic thyroid hormone economy; however, the exact mechanism through which highly glycogenic feed supplements impact liver metabolism is little known. Previous studies showed that only leptin receptors are expressed in the liver of cows, and now we report that leptin mRNA is expressed in the liver of cows as well. The present results show that the mRNA of leptin and its receptors are differentially modulated by the increased energy content of the feed consumed. Simultaneous changes in hepatic type I deiodinase activity suggest that hepatic modulation of the leptin system by PG supplementation may be mediated by an increased local thyroxine-triiodothyronine conversion. Since PG supplementation with simultaneous T4-T3 turnover and increased hepatic leptin- and short-form leptin receptor mRNA were not associated with a significant change in hepatic total lipid levels, it is suggested that the leptin system, directly or indirectly modulated by thyroid hormones, may represent a local defence mechanism to prevent fatty liver formation.

Ecto-nucleoside triphosphate diphosphohydrolase 3 in the ventral and lateral hypothalamic area of female rats: morphological characterization and functional implications.

BACKGROUND: Based on its distribution in the brain, ecto-nucleoside triphosphate diphosphohydrolase 3 (NTPDase3) may play a role in the hypothalamic regulation of homeostatic systems, including feeding, sleep-wake behavior and reproduction. To further characterize the morphological attributes of NTPDase3-immunoreactive (IR) hypothalamic structures in the rat brain, here we investigated: 1.) The cellular and subcellular localization of NTPDase3; 2.) The effects of 17beta-estradiol on the expression level of hypothalamic NTPDase3; and 3.) The effects of NTPDase inhibition in hypothalamic synaptosomal preparations. METHODS: Combined light- and electron microscopic analyses were carried out to characterize the cellular and subcellular localization of NTPDase3-immunoreactivity. The effects of estrogen on hypothalamic NTPDase3 expression was studied by western blot technique. Finally, the effects of NTPDase inhibition on mitochondrial respiration were investigated using a Clark-type oxygen electrode. RESULTS: Combined light- and electron microscopic analysis of immunostained hypothalamic slices revealed that NTPDase3-IR is linked to ribosomes and mitochondria, is predominantly present in excitatory axon terminals and in distinct segments of the perikaryal plasma membrane. Immunohistochemical labeling of NTPDase3 and glutamic acid decarboxylase (GAD) indicated that gamma-amino-butyric-acid- (GABA) ergic hypothalamic neurons do not express NTPDase3, further suggesting that in the hypothalamus, NTPDase3 is predominantly present in excitatory neurons. We also investigated whether estrogen influences the expression level of NTPDase3 in the ventrobasal and lateral hypothalamus. A single subcutaneous injection of estrogen differentially increased NTPDase3 expression in the medial and lateral parts of the hypothalamus, indicating that this enzyme likely plays region-specific roles in estrogen-dependent hypothalamic regulatory mechanisms. Determination of mitochondrial respiration rates with and without the inhibition of NTPDases confirmed the presence of NTPDases, including NTPDase3 in neuronal mitochondria and showed that blockade of mitochondrial NTPDase functions decreases state 3 mitochondrial respiration rate and total mitochondrial respiratory capacity. CONCLUSION: Altogether, these results suggest the possibility that NTPDases, among them NTPDase3, may play an estrogen-dependent modulatory role in the regulation of intracellular availability of ATP needed for excitatory neuronal functions including neurotransmission.

A clinical method to determine the optimal apical preparation size. Part I.

OBJECTIVE: To establish a new approach for determination of the optimal apical preparation size (APS). STUDY DESIGN: After preflaring 212 root canals from 80 extracted molars, electronic working length (WL) was measured to establish where the apical preparation size (APS) should be determined. Subsequently, nontapered, noncutting special instruments were used to reach WL. The diameter of the largest special instrument (SI) width that had to overcome some resistance to advance to WL was defined as d(SI). After sectioning the roots apically, the diameter of a potential rotary instrument was determined for each section, allowing a complete cutting of the root canal wall. The estimation of APS was related to d(SI). RESULTS: Theoretically, individual APS of d(SI) + 0.60 mm yielded circumferential apical preparations in 98% of the cases. When shaping palatal/distal root canals of molars to d(SI) + 0.40 mm, and mesiobuccal/mesiolingual/distobuccal root canals of molars to d(SI) + 0.30 mm, complete preparations of root canal walls resulted in 78% (95% CI, 69%-87%) and 72% (95% CI, 65%-80%) of canals, respectively. CONCLUSION: The described approach allowed evaluation of the APS for most root canals. Root canals should be shaped to larger sizes than normally recommended.

Extended apical enlargement with hand files versus rotary NiTi files. Part II.

OBJECTIVE: The objective of this study was to compare 2 preparation techniques performed under simulated clinical conditions with extended apical enlargement following determination of the optimal apical preparation size (APS). STUDY DESIGN: After preflaring 240 root canals, APS was evaluated as outlined in Part I. The apical portion was shaped to APS either with rotary NiTi Lightspeed instruments (LS) or NiTi hand instruments (HA) using the balanced force technique in a phantom head. After sectioning the apical area at 3 levels, every cross section was analyzed microscopically for circumferential removal of canal wall dentine. Loss of working length, instrument separation, and perforation were additionally recorded. RESULTS: In 70% (LS) and 69% (HA) of the root canals, 2 of 3 levels demonstrated that the root canal dentin was cut circumferentially. Neither loss of working length nor perforation occurred in both groups. Four LS instruments separated. CONCLUSIONS: APS frequently results in a nearly complete apical preparation regardless of the preparation techniques. In a few cases apical enlargement to APS does not achieve complete cutting of the canal walls. There was a rather slight risk of serious procedural errors.