Spectroscopic studies for identifying the chemical states of the periostracum of the Corbicula species in Lake Biwa.

Corbicula clam shells consist of thin periostracum and calcareous layers made of calcium carbonate (CaCO3). Depending on habitat conditions, the shell exhibits various colorations, such as yellow, brown, and black. The chemical state of the periostracum of the Corbicula species in Lake Biwa was studied by X-ray absorption fine structure (XAFS) and Raman scattering spectroscopies. Fe K-edge X-ray absorption near edge structure (XANES) revealed that the Fe3+ intensity increases as the color of the shell changes from yellow to black. Raman spectra suggested that quinone-based polymers cover the yellow shell, and the black shell is further covered by dihydroxyphenylalanine (DOPA) rings of amino acid derivatives. From Fe K-edge extended X-ray absorption fine structure (EXAFS), it was found that Fe3+ in the periostracum was surrounded by five to six oxygen atoms with an average Fe-O ligand distance of 2.0 Å. Accordingly, a tris-DOPA-Fe3+ complex is formed, which is responsible for the periostracum's black color.

Microscopic image-based classification of adipocyte differentiation by machine learning.

Adipocyte differentiation is a sequential process involving increased expression of peroxisome proliferator-activated receptor gamma (PPARγ), adipocyte-specific gene expression, and accumulation of lipid droplets in the cytoplasm. Expression of the transcription factors involved is usually detected using canonical biochemical or biomolecular procedures such as Western blotting or qPCR of pooled cell lysates. While this provides a useful average index for adipogenesis for some populations, the precise stage of adipogenesis cannot be distinguished at the single-cell level, because the heterogenous nature of differentiation among cells limits the utility of averaged data. We have created a classifier to sort cells, and used it to determine the stage of adipocyte differentiation at the single-cell level. We used a machine learning method with microscopic images of cell stained for PPARγ and lipid droplets as input data. Our results show that the classifier can successfully determine the precise stage of differentiation. Stage classification and subsequent model fitting using the sequential reaction model revealed the action of pioglitazone and rosiglitazone to be promotion of transition from the stage of increased PPARγ expression to the next stage. This indicates that these drugs are PPARγ agonists, and that our classifier and model can accurately estimate drug action points and would be suitable for evaluating the stage/state of individual cells during differentiation or disease progression. The incorporation of both biochemical and morphological information derived from immunofluorescence image of cells and so overcomes limitations of current models.

Discovery of Brain-Penetrant Glucosylceramide Synthase Inhibitors with a Novel Pharmacophore.

Inhibition of glucosylceramide synthase (GCS) is a major therapeutic strategy for Gaucher's disease and has been suggested as a potential target for treating Parkinson's disease. Herein, we report the discovery of novel brain-penetrant GCS inhibitors. Assessment of the structure-activity relationship revealed a unique pharmacophore in this series. The lipophilic ortho-substituent of aromatic ring A and the appropriate directionality of aromatic ring B were key for potency. Optimization of the absorption, distribution, metabolism, elimination, toxicity (ADMETox) profile resulted in the discovery of T-036, a potent GCS inhibitor in vivo. Pharmacophore-based scaffold hopping was performed to mitigate safety concerns associated with T-036. The ring opening of T-036 resulted in another potent GCS inhibitor with a lower toxicological risk, T-690, which reduced glucosylceramide in a dose-dependent manner in the plasma and cortex of mice. Finally, we discuss the structural aspects of the compounds that impart a unique inhibition mode and lower the cardiovascular risk.

Discovery of Pyrazolo[1,5-a]pyrazin-4-ones as Potent and Brain Penetrant GluN2A-Selective Positive Allosteric Modulators Reducing AMPA Receptor Binding Activity.

N-Methyl-d-aspartate receptors (NMDARs) are members of the ionotropic glutamate receptor family and play a crucial role in learning and memory by regulating synaptic plasticity. Activation of NMDARs containing GluN2A, one of the NMDAR subunits, has recently attracted attention as a promising therapeutic approach for neuropsychiatric diseases such as schizophrenia, depression, and epilepsy. In the present study, we developed potent and brain-penetrable GluN2A-selective positive allosteric modulators. Lead compound 2b was generated by scaffold hopping of hit compound 1, identified from the internal alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-focused compound library through a high-throughput screening campaign. Subsequent optimization of the lead compound, including a structure-based drug design approach, resulted in the identification of a potent GluN2A PAM (R)-9, which possessed high selectivity against both subtypes of AMPAR and NMDAR. Furthermore, (R)-9 significantly enhanced long-term potentiation in the rat hippocampus 24 h after oral administration, indicating that this molecule is a potentially useful in vivo pharmacological tool for treating psychiatric diseases.

Design and Synthesis of Novel Spiro Derivatives as Potent and Reversible Monoacylglycerol Lipase (MAGL) Inhibitors: Bioisosteric Transformation from 3-Oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl Moiety.

The therapeutic potential of monoacylglycerol lipase (MAGL) inhibitors in central nervous system-related diseases has attracted attention worldwide. However, the availability of reversible-type inhibitor is still limited to clarify the pharmacological effect. Herein, we report the discovery of novel spiro chemical series as potent and reversible MAGL inhibitors with a different binding mode to MAGL using Arg57 and His121. Starting from hit compound 1 and its co-crystal structure with MAGL, structure-based drug discovery (SBDD) approach enabled us to generate various spiro scaffolds like 2a (azetidine-lactam), 2b (cyclobutane-lactam), and 2d (cyclobutane-carbamate) as novel bioisosteres of 3-oxo-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl moiety in 1 with higher lipophilic ligand efficiency (LLE). Optimization of the left hand side afforded 4f as a promising reversible MAGL inhibitor, which showed potent in vitro MAGL inhibitory activity (IC50 6.2 nM), good oral absorption, blood-brain barrier penetration, and significant pharmacodynamic changes (2-arachidonoylglycerol increase and arachidonic acid decrease) at 0.3-10 mg/kg, po. in mice.

Pilocarpine induces the residual secretion of salivary fluid in perfused submandibular glands of rats.

Pilocarpine is an M3 muscarinic agonist that is widely used for the treatment of xerostomia caused by various diseases and medical conditions. Pilocarpine induced the secretion of salivary fluid in perfused submandibular glands of rats. The secretion of salivary fluid observed after removal of pilocarpine was referred to as residual fluid secretion. The volume of fluid and time of the residual secretion depended on the dose of pilocarpine. Such a residual effect of pilocarpine was observed on fluid secretion via the paracellular pathway and oxygen consumption. When a muscarinic antagonist was added to the perfusate immediately after cessation of pilocarpine, residual secretion of salivary fluid did not occur. These observations indicate that the residual secretion of salivary fluid is a characteristics of the interaction of pilocarpine with muscarinic receptors.

Stress Characterization of the Interface Between Thermal Oxide and the 4H-SiC Epitaxial Layer Using Near-Field Optical Raman Microscopy.

Stresses induced in the silicon carbide (SiC) epitaxial layer near the interface between thermal silicon oxide and 4H-SiC epitaxial substrate were measured using a near-field optical Raman microscope equipped with a hollow pyramid probe (aperture size: approximately 250 nm). The E2 phonon was observed to undergo a 0.17 cm-1 redshift owing to reduction in oxide-layer thickness from 300 nm to 0 nm; this result was compared against that obtained using a standard Raman microprobe sans the pyramidal probe. The result indicates that the epitaxial layer near the SiO2-4H-SiC interface was maintained under a constant tensile stress of the order of 50 MPa. This agrees well with the result obtained using the finite element method (FEM). Based on results obtained using the said Raman microprobe and Fourier transform infrared (FT-IR) measurements, use of an inhomogeneity formation model at the SiO2-4H-SiC interface has been proposed in this study.

LLO-mediated Cell Resealing System for Analyzing Intracellular Activity of Membrane-impermeable Biopharmaceuticals of Mid-sized Molecular Weight.

Cell-based assays have become increasingly important in the preclinical studies for biopharmaceutical products such as specialty peptides, which are of interest owing to their high substrate specificity. However, many of the latter are membrane impermeable and must be physically introduced into cells to evaluate their intracellular activities. We previously developed a "cell-resealing technique" that exploited the temperature-dependent pore-forming activity of the streptococcal toxin, streptolysin O (SLO), that enabled us to introduce various molecules into cells for evaluation of their intracellular activities. In this study, we report a new cell resealing method, the listeriolysin O (LLO)-mediated resealing method, to deliver mid-sized, membrane-impermeable biopharmaceuticals into cells. We found that LLO-type resealing required no exogenous cytosol to repair the injured cell membrane and allowed the specific entry of mid-sized molecules into cells. We use this method to introduce either a membrane-impermeable, small compound (8-OH-cAMP) or specialty peptide (Akt-in), and demonstrated PKA activation or Akt inhibition, respectively. Collectively, the LLO-type resealing method is a user-friendly and reproducible intracellular delivery system for mid-sized membrane-impermeable molecules into cells and for evaluating their intracellular activities.

TRPV4 heats up ANO1-dependent exocrine gland fluid secretion.

Several ion channels and transporters regulate fluid secretion in salivary and lacrimal glands. In salivary glands, the major anion channel involved in fluid secretion is the calcium-activated chloride channel anoctamin 1 (ANO1). Several members of the transient receptor potential (TRP) channel superfamily regulate ANO1 activity. Here, we report a functional interaction between thermosensitive TRP vanilloid (TRPV)4 and ANO1 in acinar cells isolated from mouse salivary and lacrimal glands. TRPV4 activation induced chloride currents and shrinkage of acinar cells by increasing intracellular calcium concentrations. The chloride currents evoked by a TRPV4-specific activator (GSK1016790A) were identified as ANO1-mediated currents. Moreover, TRPV4 activation by an inositol 1,4,5-trisphosphate (IP3)-dependent mechanism was found to contribute to the muscarinic pathway of fluid secretion. Muscarinic stimulation of saliva and tear secretion was down-regulated in both TRPV4-deficient mice and in acinar cells treated with a TRPV4-specific antagonist (HC-067047). Furthermore, the temperature dependence of muscarinic salivation was shown to depend mainly on TRPV4. Our results suggest that TRPV4 interacts with IP3 receptors and ANO1 to regulate the muscarinic pathway that mediates salivation and lacrimation.-Derouiche, S., Takayama, Y., Murakami, M., Tominaga, M. TRPV4 heats up ANO1-dependent exocrine gland fluid secretion.

A portable infrared photoplethysmograph: heartbeat of Mytilus galloprovincialis analyzed by MRI and application to Bathymodiolus septemdierum.

Infrared photoplethysmogram (IR-PPG) and magnetic resonance image (MRI) of the Mytilus galloprovincialis heart were obtained simultaneously. Heart rate was varied by changing temperature, aerial exposure and hypoxia. Higher heart rates (35-20 beat min-1) were usually observed at 20°C under the aerobic condition, and typical IR-PPG represented a single peak (peak v). The upward and downward slopes of the peak v corresponded to the filling and contracting of the ventricle, respectively. A double-peak IR-PPG was observed in a wide range of heart rates (5 to 35 beats min-1) under various conditions. The initial peak v corresponded to the filling of the ventricle, and the origin of the second peak (v') varied with the heart rate. A flat IR-PPG with a noise-level represented cardiac arrest. Although large movement of the shells and the foot caused slow waves or a baseline drift of the IR-PPG, the heart rate can be calculated from the v-v interval. Based on these results, we assembled a portable IR-PPG recording system, and measured the heartbeats of Bathymodiolus septemdierum (Mytilidae) for 24 h on a research vessel just after sampling from the deep sea, showing that IR-PPG is a noninvasive, economical, robust method that can be used in field experiments.

Mechanism involved in Danshen-induced fluid secretion in salivary glands.

AIM: Danshen's capability to induce salivary fluid secretion and its mechanisms were studied to determine if it could improve xerostomia. METHODS: Submandibular glands were isolated from male Wistar rats under systemic anesthesia with pentobarbital sodium. The artery was cannulated and vascularly perfused at a constant rate. The excretory duct was also cannulated and the secreted saliva was weighed in a cup on an electronic balance. The weight of the accumulated saliva was measured every 3 s and the salivary flow rate was calculated. In addition, the arterio-venous difference in the partial oxygen pressure was measured as an indicator of oxygen consumption. In order to assess the mechanism involved in Danshen-induced fluid secretion, either ouabain (an inhibitor of Na(+)/K(+) ATPase) or bumetanide (an inhibitor of NKCC1) was additionally applied during the Danshen stimulation. In order to examine the involvement of the main membrane receptors, atropine was added to block the M3 muscarinic receptors, or phentolamine was added to block the α1 adrenergic receptors. In order to examine the requirement for extracellular Ca(2+), Danshen was applied during the perfusion with nominal Ca(2+) free solution. RESULTS: Although Danshen induced salivary fluid secretion, 88.7 ± 12.8 µL/g-min, n = 9, (the highest value around 20 min from start of DS perfusion was significantly high vs 32.5 ± 5.3 µL/g-min by carbamylcholine, P = 0.00093 by t-test) in the submandibular glands, the time course of that secretion differed from that induced by carbamylcholine. There was a latency associated with the fluid secretion induced by Danshen, followed by a gradual increase in the secretion to its highest value, which was in turn followed by a slow decline to a near zero level. The application of either ouabain or bumetanide inhibited the fluid secretion by 85% or 93%, and suppressed the oxygen consumption by 49% or 66%, respectively. These results indicated that Danshen activates Na(+)/K(+) ATPase and NKCC1 to maintain Cl(-) release and K(+) release for fluid secretion. Neither atropine or phentolamine inhibited the fluid secretion induced by Danshen (263% ± 63% vs 309% ± 45%, 227% ± 63% vs 309% ± 45%, P = 0.899, 0.626 > 0.05 respectively, by ANOVA). Accordingly, Danshen does not bind with M3 or α1 receptors. These characteristics suggested that the mechanism involved in DS-induced salivary fluid secretion could be different from that induced by carbamylcholine. Carbamylcholine activates the M3 receptor to release inositol trisphosphate (IP3) and quickly releases Ca(2+) from the calcium stores. The elevation of [Ca(2+)]i induces chloride release and quick osmosis, resulting in an onset of fluid secretion. An increase in [Ca(2+)]i is essential for the activation of the luminal Cl(-) and basolateral K(+) channels. The nominal removal of extracellular Ca(2+) totally abolished the fluid secretion induced by Danshen (1.8 ± 0.8 µL/g-min vs 101.9 ± 17.2 µL/g-min, P = 0.00023 < 0.01, by t-test), suggesting the involvement of Ca(2+) in the activation of these channels. Therefore, IP3-store Ca(2+) release signalling may not be involved in the secretion induced by Danshen, but rather, there may be a distinct signalling process. CONCLUSION: The present findings suggest that Danshen can be used in the treatment of xerostomia, to avoid the systemic side effects associated with muscarinic drugs.

Magnetic resonance imaging analysis of water flow in the mantle cavity of live Mytilus galloprovincialis.

Water flow inside the shell of Mytilus galloprovincialis was measured by phase-contrast magnetic resonance imaging (MRI). In seawater without algal cells at 23 °C, water approached the mussel from the posterior-ventral side, and entered through the inhalant aperture at a velocity of 40-20 mm s(-1). The flow rate in the lower mantle cavity decreased to 10-20 mm s(-1), the water flowed in the anterior-dorsal direction and approached the demibranches at a velocity of 5-10 mm s(-1). After passing through the lamellae to the upper mantle cavity, the water stretched the interlamellar cavity, turned to the posterior-dorsal direction and accumulated in the epibranchial cavity. The water flows came together at the ventral side of the posterior adductor muscle. The velocity increased more to than 50 mm s(-1) in the exhalant siphon, and exhaled out in the posterior-dorsal direction. The anterior-posterior direction of the flow was imaged every 1.92 s by the inflow effect of T1-weighted MRI. The flow seemed to be constant, and no cyclic motion of the mantles or the gills was detected. Spontaneous closure of the shells caused a quick drop of the flow in the mantle cavity. In the opening process of the shells, water flow in the interlamellar cavities increased before the opening, followed by an increase of flows in the exhalant siphon and inhalant aperture with minimum delay, reaching a plateau within 1 min of the shells opening. This provides direct evidence that the lateral cilia drive water in the mussel M. galloprovincialis.

SUMO-modification and elimination of the active DNA demethylation enzyme TDG in cultured human cells.

Thymine DNA glycosylase (TDG) is a base excision repair enzyme that interacts with the small ubiquitin-related modifier (SUMO)-targeted ubiquitin E3 ligase RNF4 and functions in the active DNA demethylation pathway. Here we showed that both SUMOylated and non-modified forms of endogenous TDG fluctuated during the cell cycle and in response to drugs that perturbed cell cycle progression, including hydroxyurea and nocodazole. Additionally, we detected a SUMOylation-independent association between TDG and RNF4 in vitro as well as in vivo, and observed that both forms of TDG were efficiently degraded in RNF4-depleted cells when arrested at S phase. Our findings provide insights into the in vivo dynamics of TDG SUMOylation and further clarify the TDG-RNF4 interaction.

Testing the constant-volume hypothesis by magnetic resonance imaging of Mytilus galloprovincialis heart.

The constant-volume (CV) hypothesis was tested using the Mytilus galloprovincialis heart under two conditions. The volume of the ventricle, auricles and pericardium, and the flow in the heart and adjacent vessels were measured by magnetic resonance imaging. In synthetic seawater at 23°C (immersed condition), the end-diastolic volume (EDV), end-systolic volume (ESV) and stroke volume (SV) were 50%, 21% and 29% of the heart volume, respectively, and the auricle volume (VA) was maximized at end-systole. Assuming a constant volume of the heart, venous return to the auricles (IV) was constant, and out-flow from the pericardium to the kidney (IPK) was 2/3 of SV. During aerial exposure (emersed condition), EDV, ESV and SV decreased to 33%, 22% and 11%, respectively. VA was maximized at end-diastole and associated with the decrease of systolic IV to 1/2 of diastolic IV, while IPK remained at 80% of the immersed condition. Based on these results--in addition to two postulates of the CV hypothesis: (1) the total volume of the heart is always the same, and (2) ventricle contraction causes a decrease in pressure in the pericardium--we modified two postulates: (3) the low pericardial pressure maintains venous return from the anterior oblique vein to the auricle, and (4) the pressure difference between the auricle and the pericardium drives haemolymph filtration through the auricle walls. We also added a new postulate: (5) dilatation of the ventricle is associated with the haemolymph output to the kidney via the renopericardial canals.

Mn-citrate and Mn-HIDA: intermediate-affinity chelates for manganese-enhanced MRI.

In this study we investigated two manganese chelates in order to improve the image enhancement of manganese-enhanced MRI and decrease the toxicity of free manganese ions. Since both MnCl2 and a low-affinity chelate were associated with a slow continuous decrease of cardiac functions, we investigated intermediate-affinity chelates: manganese N-(2-hydroxyethyl)iminodiacetic acid (Mn-HIDA) and Mn-citrate. The T1 relaxivity values for Mn-citrate (4.4 m m⁻¹ s⁻¹) and Mn-HIDA (3.3 m m⁻¹ s⁻¹) in artificial cerebrospinal fluid (CSF) were almost constant in a concentration range from 0.5 to 5 m m at 37 °C and 4.7 T. In human plasma, the relaxivity values increased when the concentrations of these Mn chelates were decreased, suggesting the presence of free Mn²âº bound with serum albumin. Mn-HIDA and Mn-citrate demonstrated a tendency for better contractility when employed with an isolated perfused frog heart, compared with MnCl2. Only minimal changes were demonstrated after a venous infusion of 100 m m Mn-citrate or Mn-HIDA (8.3 µmol kg⁻¹ min⁻¹) in rats and a constant heart rate, arterial pressure and sympathetic nerve activity were maintained, even after breaking the blood-brain barrier (BBB). Mn-citrate and Mn-HIDA could not cross the intact BBB and appeared in the CSF, and then diffused into the brain parenchyma through the ependymal layer. The responses in the supraoptic nucleus induced by the hypertonic stimulation were detectable. Therefore, Mn-citrate and Mn-HIDA appear to be better choices for maintaining the vital conditions of experimental animals, and they may improve the reproducibility of manganese-enhanced MRI of the small nuclei in the hypothalamus and thalamus.

Involvement of AQP6 in the Mercury-sensitive osmotic lysis of rat parotid secretory granules.

In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73-80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg²âº, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg²âº (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5-2.0 µM Hg²âº, concentrations that activate AQP6. The Hg lysis was completely blocked by ß-mercaptoethanol which disrupts Hg²âº-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO(3) (-) > Br⁻ > I⁻ > Cl⁻ and was facilitated by acidic pH. The anion selectivity for NO(3) (-) and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg²âº-sensitive anion channel in rat parotid secretory granules.

The actin-specific reagent jasplakinolide induces apoptosis in primary rat parotid acinar cells.

Jasplakinolide is a reagent that stabilizes and polymerizes actin filaments and is a commonly used tool in cell biology. In primary rat parotid acinar cells, jasplakinolide partially inhibited the release of amylase induced by ß-adrenergic receptor activation, as previously reported. However, in confocal microscopic observation with fluorescence conjugated anti-actin antibody, the jasplakinolide-treated cells not only showed decreased fluorescence intensity and aggregation of cortical F-actin but also revealed events characteristic of apoptosis such as cell shrinkage, membrane blebbing and apoptotic body formation. Such characteristic events of apoptosis were confirmed by transmission electron microscopy. The occurrence of apoptosis in jasplakinolide-treated cells was further confirmed by biochemical analysis: a DNA ladder was detected by electrophoresis, and DNA fragmentation was revealed using ELISA with an antibody to single-stranded DNA. Moreover, the degradation of fodrin was detected in jasplakinolide-treated cells by Western blotting, and the K(+) release induced by the fluid secretagogue carbachol was impaired. Taken together, these results demonstrate that jasplakinolide induces apoptosis and suppresses the secretory functions of rat parotid acinar cells.

Magnetic resonance imaging of the temporomandibular joint in the rat compared with low-powered light microscopy.

OBJECTIVE: High magnetic field magnetic resonance imaging (MRI) was applied to the temporomandibular joint (TMJ) in the rat. The purpose of this study was the depiction of the internal structure of the TMJ, including the articular disc, articular cartilage, and the upper and lower joint cavities. We also proposed MRI settings and slices suitable for imaging the TMJ in the rat. METHODS: Temporomandibular joints from one female and eight male Sprague Dawley rats (5-8 weeks old) and four male Wistar-Hamamatsu rats (7-8 weeks old) were used. Using scout images, the horizontal plane was defined as being parallel to the body of the basisphenoid bone underneath the base of the brain. The coronal plane was defined as a slice vertical to the horizontal plane and vertical to the longitudinal fissure of the cerebrum. The sagittal plane was defined as a slice vertical to the horizontal plane and parallel to the longitudinal fissure of the cerebrum. RESULTS: T(1)-weighted MR images with a spatial resolution of 75 µm were obtained for 5 min. The temporal bone and mandibular condyle were depicted as lower signal intensity images and the articular disc was depicted as an intermediate signal intensity image. In accordance with Gd-DTPA-enhanced MR or T(2)-weighted MR images, the articular disc, articular cartilage, and the upper and lower joint cavities could be assigned clearly. CONCLUSION: These MRI findings closely agreed with those observed with haematoxylin-eosin staining under light microscopy, suggesting that MRI is a useful method for analyzing the complex structure of the TMJ in the rat.