Low blood glucose and fatigue accumulation at peak hours of occupational trauma in secondary industry workers.

Objectives. The incidence of occupational traumatic injuries caused by human error has been reported to occur at 11:00 and 8-9 h after commencing work. Impaired attention is closely related to the incidence of these accidents. Therefore, this study aimed to clarify the changes in blood glucose, fatigue and stress response hormone levels over time among workers in a secondary industry. Methods. The blood glucose and subjective fatigue levels of 26 male secondary-industry workers were measured on workdays. In addition, the cortisol and dehydroepiandrosterone levels in saliva were measured on one workday and one holiday. Results. Blood glucose levels at 11:00 and 17:30 on the workday were significantly lower than those at 09:30. Moreover, hypoglycemia was observed in some participants. A significant increase in subjective fatigue levels was observed during the workday. However, no significant differences in salivary cortisol levels were observed between the workday and the holiday at any time point. Conclusions. Blood glucose levels decreased and subjective fatigue levels increased at the time points that occupational accidents were reported to occur most frequently during work. These factors may contribute to human errors due to impaired attentional function.

Wearing high-heeled shoes on a side slope makes standing balance unstable.

INTRODUCTION: Many women wearing high-heeled footwear are at high risk of falls. Past studies have examined the balance on level ground or balance during walking. We measured the standing balance on the ground and side slopes for 18 healthy women. METHOD: Body sway was evaluated based on the center of pressure (COP) while participants stood on level ground on a side slope. The total locus length as well as rectangular and outer peripheral areas were then measured using a Zebris system. Measurements were compared under bare feet, normal shoe, and high-heeled shoe conditions. RESULTS: On level ground, there were no significant differences among the three conditions. On the side slope, the total locus length (TLL), rectangular area (RA) and outer peripheral areas (OPA) were significantly greater for the high-heeled shoes than for the bare feet and normal shoes. Standing on the side slope caused larger body sway than on the level ground, along with a higher risk of falling. DISCUSSION: In TLL, OPA, and RA, the COP moved outside substantially when participants stood on a slope in high heels than in shoes. High heels were highly unstable for standing on a slope since the ankle joint of one leg is in plantar flexion, the foot is pronated, and the other side is plantarflexed at the ankle with pronation of the foot. CONCLUSION: High-heeled shoes significantly alter a person's balance when standing on a side slope, suggesting a high risk of falling.

Stroke impairment, balance, and cognitive status on admission predict walking independence up to 90 days post-stroke but their contributions change over time.

This study aimed to identify predictors of gait independence in three successive patient cohorts that received inpatient rehabilitation for at least 30 days, 60 days, or 90 days post-stroke. A total of 26 independent variables were collected within 3 days of stroke onset, including basic information (age, sex, stroke type), sensorimotor function (Stroke Impairment Assessment Set), gait function, balance function, and cognitive function. The dependent variable was walking independence (without assistance from another person) at 30, 60, or 90 days post-stroke. A decision tree was developed for predicting gait independence at each assessment time point. The predictors of walking independence differed among the cohorts that received inpatient rehabilitation for at least 30, 60, and 90 days post-stroke. Specifically, the Short Form Berg Balance Scale score was in the higher layer and was a strong predictor of gait at all time points. The cognitive Functional Independence Measure progressed to the higher layer at later time points. The lower extremity motor function was an additional predictor in the 30-day cohort. For later cohorts, the predictive value of balance and cognitive function declined whereas the contribution of the paralyzed grip strength and trunk function increased. These results suggest that sensorimotor and cognitive function within 3 days of stroke can predict walking independence between 1 and 3 months post-stroke; however, the prognostic value of predictors varies among the patients who receive inpatient rehabilitation for shorter versus longer time.

Comparison of flatfeet and normal feet using data of the gait cycle, contact area, and foot pressure.

Although the foot is involved in load-bearing and shock absorption, foot pressure (FP), ground contact area (CA), and gait cycle (GC) in flatfeet (FF) have not been examined in detail. We aimed to analyze the influence of FF on FP, CA, and GC. We included 20 and 21 women with FF and normal feet (NF), respectively. A Footscan plantar pressure plate (RsScan International, Belgium) was used to analyze FP, CA, and GC. FP was applied to the unit area of 10 compartments. GC analysis was performed using phase-time measurements by dividing the GC into four phases. In the analysis, FP and CA were compared between the FF and NF groups. A comparison of GC was similarly performed between the two groups. The data provided in this article will be useful when designing studies on the effect of foot shape on FP, CA, and GC during gait.

Relationship between Perceived Leg Length Discrepancy at One Month and Preoperative Hip Abductor Muscle Elasticity in Patients after Total Hip Arthroplasty.

OBJECTIVE: Preoperative factors related to perceived leg length discrepancy (PLLD) after total hip arthroplasty (THA) are not well studied. This study aimed to examine the preoperative factors, including hip abductor modulus, related to PLLD one month after THA. METHODS: The study included 73 patients diagnosed with osteoarthritis secondary to developmental dysplasia of the hip and a posterior approach to surgery. Multiple logistic regression analysis was performed using the presence or absence of PLLD as the dependent variable and preoperative hip abductor's modulus of elasticity, pain, hip abduction range of motion, hip abductor muscle strength and pelvic obliquity as the independent variable. Additionally, receiver operating characteristic curves were used for the extracted variables for calculating the cutoffs, sensitivity, specificity and area under the curve (AUC) to determine the presence or absence of PLLD. The significance level was set at p<0.05. RESULTS: The hip abductor modulus (odds ratio=1.13; 95% confidence interval=1.06-1.21; p<0.001) was selected as a preoperative factor. The cutoff value to determine the presence or absence of a PLLD was 16.32 kPa. The sensitivity and specificity were 81.8% and 72.5%, respectively, and the AUC was 0.8137. CONCLUSION: The hip abductor muscle elastic modulus affected PLLD one month after THA. If the preoperative hip abductor elastic modulus is higher than the cutoff value, it may affect the appearance of PLLD at one month postoperatively.

Multiple roles of afadin in the ultrastructural morphogenesis of mouse hippocampal mossy fiber synapses.

A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses.

Performance of an improperly sized and stretched-out loose-fitting powered air-purifying respirator: Manikin-based study.

The objective of this study was to investigate the protection level offered by a Powered Air-Purifying Respirator (PAPR) equipped with an improperly sized or stretched-out loose-fitting facepiece using constant and cyclic flow conditions. Improperly sized PAPR facepieces of two models as well as a stretched-out facepiece were tested. These facepieces were examined in two versions: with and without exhaust holes. Loose-fitting facepieces (size "large") were donned on a small manikin headform and challenged with sodium chloride (NaCl) aerosol particles in an exposure chamber. Four cyclic flows with mean inspiratory flows (MIFs) of 30, 55, 85, and 135 L/min were applied using an electromechanical Breathing Recording and Simulation System (BRSS). The manikin Fit Factor (mFF) was determined as the ratio of aerosol concentrations outside (Cout) to inside (Cin) of the facepiece, measured with a P-Trak condensation particle counter (CPC). Results showed that the mFF decreased exponentially with increasing MIF. The mFF values of the stretched-out facepiece were significantly lower than those obtained for the undamaged ones. Facepiece type and MIF were found to significantly affect the performance of the loose-fitting PAPR. The effect of the exhaust holes was less pronounced and depended on the facepiece type. It was concluded that an improperly sized facepiece might potentially offer relatively low protection (mFF < 250) at high to strenuous workloads. The testing was also performed at a constant inhalation flow to explore the mechanism of the particle-facepiece interaction. Results obtained with cyclic flow pattern were consistent with the data generated when testing the loose-fitting PAPR under constant flow conditions. The time-weighted average values of mFF calculated from the measurements conducted under the constant flow regime were capable of predicting the protection under cyclic flow regime. The findings suggest that program administrators need to equip employees with properly sized facepieces and remove stretched-out ones from workplace. Manufacturers should emphasize the importance of proper sizing with their user instructions.

Real-time histological imaging of kidneys stained with food dyes using multiphoton microscopy.

We have developed a real-time imaging technique for diagnosis of kidney diseases which is composed of two steps, staining renal cells safely with food dyes and optical sectioning of living renal tissue to obtain histological images by multiphoton microscopy (MPM). Here, we demonstrated that the MPM imaging with food dyes, including erythrosine and indigo carmine, could be used as fluorescent agents to visualize renal functions and structures such as glomerular bloodstreams, glomerular filtration, and morphology of glomeruli and renal tubules. We also showed that the kidneys of IgA nephropathy model-mice stained with the food dyes presented histopathological characteristics different from those observed in normal kidneys. The use of the food dyes enhances the quality of tissue images obtained by MPM and offers the potential to contribute to a clinical real-time diagnosis of kidney diseases.

Nectin-1 spots regulate the branching of olfactory mitral cell dendrites.

Olfactory mitral cells extend lateral secondary dendrites that contact the lateral secondary and apical primary dendrites of other mitral cells in the external plexiform layer (EPL) of the olfactory bulb. The lateral dendrites further contact granule cell dendrites, forming dendrodendritic reciprocal synapses in the EPL. These dendritic structures are critical for odor information processing, but it remains unknown how they are formed. We recently showed that the immunoglobulin-like cell adhesion molecule nectin-1 constitutes a novel adhesion apparatus at the contacts between mitral cell lateral dendrites, between mitral cell lateral and apical dendrites, and between mitral cell lateral dendrites and granule cell dendritic spine necks in the deep sub-lamina of the EPL of the developing mouse olfactory bulb and named them nectin-1 spots. We investigated here the role of the nectin-1 spots in the formation of dendritic structures in the EPL of the mouse olfactory bulb. We showed that in cultured nectin-1-knockout mitral cells, the number of branching points of mitral cell dendrites was reduced compared to that in the control cells. In the deep sub-lamina of the EPL in the nectin-1-knockout olfactory bulb, the number of branching points of mitral cell lateral dendrites and the number of dendrodendritic reciprocal synapses were reduced compared to those in the control olfactory bulb. These results indicate that the nectin-1 spots regulate the branching of mitral cell dendrites in the deep sub-lamina of the EPL and suggest that the nectin-1 spots are required for odor information processing in the olfactory bulb.

Nectin-1 spots as a novel adhesion apparatus that tethers mitral cell lateral dendrites in a dendritic meshwork structure of the developing mouse olfactory bulb.

Mitral cells project lateral dendrites that contact the lateral and primary dendrites of other mitral cells and granule cell dendrites in the external plexiform layer (EPL) of the olfactory bulb. These dendritic structures are critical for odor information processing, but it remains unknown how they are formed. In immunofluorescence microscopy, the immunofluorescence signal for the cell adhesion molecule nectin-1 was concentrated on mitral cell lateral dendrites in the EPL of the developing mouse olfactory bulb. In electron microscopy, the immunogold particles for nectin-1 were symmetrically localized on the plasma membranes at the contacts between mitral cell lateral dendrites, which showed bilateral darkening without dense cytoskeletal undercoats characteristic of puncta adherentia junctions. We named the contacts where the immunogold particles for nectin-1 were symmetrically accumulated "nectin-1 spots." The nectin-1 spots were 0.21 µm in length on average and the distance between the plasma membranes was 20.8 nm on average. In 3D reconstruction of serial sections, clusters of the nectin-1 spots formed a disc-like structure. In the mitral cell lateral dendrites of nectin-1-knockout mice, the immunogold particles for nectin-1 were undetectable and the plasma membrane darkening was electron-microscopically normalized, but the plasma membranes were partly separated from each other. The nectin-1 spots were further identified between mitral cell lateral and primary dendrites and between mitral cell lateral dendrites and granule cell dendritic spine necks. These results indicate that the nectin-1 spots constitute a novel adhesion apparatus that tethers mitral cell dendrites in a dendritic meshwork structure of the developing mouse olfactory bulb.

Breathing simulator of workers for respirator performance test.

Breathing machines are widely used to evaluate respirator performance but they are capable of generating only limited air flow patterns, such as, sine, triangular and square waves. In order to evaluate the respirator performance in practical use, it is desirable to test the respirator using the actual breathing patterns of wearers. However, it has been a difficult task for a breathing machine to generate such complicated flow patterns, since the human respiratory volume changes depending on the human activities and workload. In this study, we have developed an electromechanical breathing simulator and a respiration sampling device to record and reproduce worker's respiration. It is capable of generating various flow patterns by inputting breathing pattern signals recorded by a computer, as well as the fixed air flow patterns. The device is equipped with a self-control program to compensate the difference in inhalation and exhalation volume and the measurement errors on the breathing flow rate. The system was successfully applied to record the breathing patterns of workers engaging in welding and reproduced the breathing patterns.

Glutamate acts as a key signal linking glucose metabolism to incretin/cAMP action to amplify insulin secretion.

Incretins, hormones released by the gut after meal ingestion, are essential for maintaining systemic glucose homeostasis by stimulating insulin secretion. The effect of incretins on insulin secretion occurs only at elevated glucose concentrations and is mediated by cAMP signaling, but the mechanism linking glucose metabolism and cAMP action in insulin secretion is unknown. We show here, using a metabolomics-based approach, that cytosolic glutamate derived from the malate-aspartate shuttle upon glucose stimulation underlies the stimulatory effect of incretins and that glutamate uptake into insulin granules mediated by cAMP/PKA signaling amplifies insulin release. Glutamate production is diminished in an incretin-unresponsive, insulin-secreting ß cell line and pancreatic islets of animal models of human diabetes and obesity. Conversely, a membrane-permeable glutamate precursor restores amplification of insulin secretion in these models. Thus, cytosolic glutamate represents the elusive link between glucose metabolism and cAMP action in incretin-induced insulin secretion.

Sevoflurane in combination with propofol, not thiopental, induces a more robust neuroapoptosis than sevoflurane alone in the neonatal mouse brain.

PURPOSE: Sevoflurane is the most widely used volatile anesthetic of general anesthesia. In children and neonates, it is commonly used alone or in combination with thiopental or propofol. A few recent studies reported that sevoflurane induced neuronal death in the developing rodent brain. We measured the neurotoxicity of these anesthetics at clinical doses, alone and in combination, in the developing mouse brain. METHODS: Seven-day-old C57BL/6 mice were randomly assigned to 6 treatment groups. Three groups were exposed to 3% sevoflurane for 6 h after injection of saline, thiopental (5 mg/kg), or propofol (10 mg/kg), whereas three groups were exposed to room air for 6 h after injection of equal doses of saline, thiopental, or propofol. Apoptosis in the hippocampal CA1 region (CA1) and retrosplenial cortex (RC) was assessed using caspase-3 immunostaining. RESULTS: Sevoflurane alone caused significantly higher apoptosis in the CA1 compared with saline plus air (P = 0.04). Sevoflurane in combination with propofol resulted in significantly greater numbers of apoptotic neurons than sevoflurane alone in both the CA1 and the RC (P = 0.04). However, there was no significant difference in apoptotic neuron density in both the regions between the groups treated with sevoflurane alone and in combination with thiopental (P = 0.683). CONCLUSION: Sevoflurane alone can induce neuronal apoptosis, and this effect is enhanced by propofol. Thiopental did not exacerbate the neurotoxicity of sevoflurane. There is the possibility that the combination of sevoflurane and propofol is a more harmful anesthetic technique than sevoflurane alone in pediatric patients.

Laboratory evaluation of the particle size effect on the performance of an elastomeric half-mask respirator against ultrafine combustion particles.

OBJECTIVES: This study quantified the particle size effect on the performance of elastomeric half-mask respirators, which are widely used by firefighters and first responders exposed to combustion aerosols. METHODS: One type of elastomeric half-mask respirator equipped with two P-100 filters was donned on a breathing manikin while challenged with three combustion aerosols (originated by burning wood, paper, and plastic). Testing was conducted with respirators that were fully sealed, partially sealed (nose area only), or unsealed to the face of a breathing manikin to simulate different faceseal leakages. Three cyclic flows with mean inspiratory flow (MIF) rates of 30, 85, and 135 L/min were tested for each combination of sealing condition and combustion material. Additional testing was performed with plastic combustion particles at other cyclic and constant flows. Particle penetration was determined by measuring particle number concentrations inside and outside the respirator with size ranges from 20 to 200 nm. RESULTS: Breathing flow rate, particle size, and combustion material all had significant effects on the performance of the respirator. For the partially sealed and unsealed respirators, the penetration through the faceseal leakage reached maximum at particle sizes >100 nm when challenged with plastic aerosol, whereas no clear peaks were observed for wood and paper aerosols. The particles aerosolized by burning plastic penetrated more readily into the unsealed half-mask than those aerosolized by the combustion of wood and paper. The difference may be attributed to the fact that plastic combustion particles differ from wood and paper particles by physical characteristics such as charge, shape, and density. For the partially sealed respirator, the highest penetration values were obtained at MIF = 85 L/min. The unsealed respirator had approximately 10-fold greater penetration than the one partially sealed around the bridge of the nose, which indicates that the nose area was the primary leak site.

Tiam1 interaction with the PAR complex promotes talin-mediated Rac1 activation during polarized cell migration.

Migrating cells acquire front-rear polarity with a leading edge and a trailing tail for directional movement. The Rac exchange factor Tiam1 participates in polarized cell migration with the PAR complex of PAR3, PAR6, and atypical protein kinase C. However, it remains largely unknown how Tiam1 is regulated and contributes to the establishment of polarity in migrating cells. We show here that Tiam1 interacts directly with talin, which binds and activates integrins to mediate their signaling. Tiam1 accumulated at adhesions in a manner dependent on talin and the PAR complex. The interactions of talin with Tiam1 and the PAR complex were required for adhesion-induced Rac1 activation, cell spreading, and migration toward integrin substrates. Furthermore, Tiam1 acted with talin to regulate adhesion turnover. Thus, we propose that Tiam1, with the PAR complex, binds to integrins through talin and, together with the PAR complex, thereby regulates Rac1 activity and adhesion turnover for polarized migration.

Distinct distribution and localization of Rho-kinase in mouse epithelial, muscle and neural tissues.

The small GTP-binding protein Rho plays a crucial role in a wide variety of cellular functions through various effector proteins. Rho-kinase is a key effector protein of Rho, which is composed of two isoforms, ROCK1 and ROCK2. To clarify the site of action of ROCK1 and ROCK2, we performed immunofluorescence and immunoelectron microscopic analyses using isoform-specific antibodies in mouse tissues. In the large and small intestines, ROCK1 immunoreactivity was predominantly identified in epithelial cells, and ROCK2 immunoreactivity was negligible. In these epithelial cells, ROCK1 immunoreactivity was distributed on plasma membranes, while ROCK1 immunogold signals were localized at cell-cell contacts and cell adhesion sites, especially at the adherens junctions at the ultrastructural level. In the bladder epithelium, however, ROCK1 and ROCK2 signals were identified at intermediate filaments, and ROCK2 signals were also observed in nuclei. In the three types of muscular cells-smooth, cardiac, and skeletal muscle cells-ROCK1 and ROCK2 also showed differential distribution. ROCK1 signals were localized at actin filaments, plasma membranes, and vesicles near plasma membranes in smooth muscle cells; at the lysosomes in skeletal muscle cells; and were undetectable in cardiac muscle cells. ROCK2 signals were localized at actin filaments and centrosomes in smooth muscle cells, at intercalated discs in cardiac muscle cells, and at Z-discs and sarcoplasmic reticulum in skeletal muscle cells. In the brain, ROCK1 immunoreactivity was distributed in glia, whereas ROCK2 immunoreactivity was observed in neurons. These results indicate that the two isoforms of Rho-kinase distribute differentially to accomplish their specific functions.

Intravital dual-colored visualization of colorectal liver metastasis in living mice using two photon laser scanning microscopy.

A major challenge of cancer biology is to visualize the dynamics of the metastatic process in secondary organs at high optical resolution in vivo real-time. Here, we presented intravital, dual-colored imaging of liver metastasis formation from a single cancer cell to metastatic colonies in the living liver of living mice using two photon laser scanning microscopy (TPLSM). Red fluorescent protein expressing murine (SL4) or human (HT29) colorectal cancer cell lines were inoculated to the spleen of green fluorescent protein expressing mice. Intravital TPLSM was performed by exteriorizing and fixing the liver lobe of living mice. This was repeated several times for the long-term imaging of the same mouse. Viable cancer cells in the living liver of living mice were visualized intravitally at a magnification of over 600×. Single cancer cells were arrested within hepatic sinusoids 2 h after injection. Platelet aggregation surrounding a cancer cell was observed, indicating a phenomenon of tumor-cell induced platelet aggregation. Cancer cells were extravasated from hepatic sinusoids to the space of Disse. Protrusions of Kupffer cells surrounding a cancer cell were observed, indicating that Kupffer cells appear to phagocytose cancer cells. SL4 cells formed liver metastatic colonies with extensive stromal reaction. Liver metastases by HT29 cells were observed as a cluster of micrometastatic nodules. High-resolution, dual-colored, real-time visualization of cancer metastasis using intravital TLPSM can help to understand spatiotemporal tumor-host interactions during metastatic processes in the living organs of living animals.

Behavioral alterations associated with targeted disruption of exons 2 and 3 of the Disc1 gene in the mouse.

Disrupted-In-Schizophrenia 1 (DISC1) is a promising candidate gene for susceptibility to psychiatric disorders, including schizophrenia. DISC1 appears to be involved in neurogenesis, neuronal migration, axon/dendrite formation and synapse formation; during these processes, DISC1 acts as a scaffold protein by interacting with various partners. However, the lack of Disc1 knockout mice and a well-characterized antibody to DISC1 has made it difficult to determine the exact role of DISC1 in vivo. In this study, we generated mice lacking exons 2 and 3 of the Disc1 gene and prepared specific antibodies to the N- and C-termini of DISC1. The Disc1 mutant mice are viable and fertile, and no gross phenotypes, such as disorganization of the brain's cytoarchitecture, were observed. Western blot analysis revealed that the DISC1-specific antibodies recognize a protein with an apparent molecular mass of ~100 kDa in brain extracts from wild-type mice but not in brain extracts from DISC1 mutant mice. Immunochemical studies demonstrated that DISC1 is mainly localized to the vicinity of the Golgi apparatus in hippocampal neurons and astrocytes. A deficiency of full-length Disc1 induced a threshold shift in the induction of long-term potentiation in the dentate gyrus. The Disc1 mutant mice displayed abnormal emotional behavior as assessed by the elevated plus-maze and cliff-avoidance tests, thereby suggesting that a deficiency of full-length DISC1 may result in lower anxiety and/or higher impulsivity. Based on these results, we suggest that full-length Disc1-deficient mice and DISC1-specific antibodies are powerful tools for dissecting the pathophysiological functions of DISC1.

Intravital three-dimensional dynamic pathology of experimental colitis in living mice using two-photon laser scanning microscopy.

BACKGROUND: Intravital three-dimensional (3D) visualization of treatment efficacy in experimental colitis in living mice using two-photon laser scanning microscopy (TPLSM) has not been described. METHODS: Colitis was induced with dextran sulfate sodium (DSS) in green fluorescent protein (GFP) transgenic mice. The 3D tomographic image of DSS-induced colitis with or without prednisolone was obtained intravitally using TPLSM. A serosal-approaching method was developed, by which we could observe all layers of the cecum from serosa to luminal mucosa without opening and everting the cecum. The dynamic pathology and treatment efficacy were assessed in the same mouse on several occasions. RESULTS: The time-lapse 3D tomographic movie of DSS-induced colitis was obtained in living mice at a magnification of greater than ×600, which demonstrated irregularity of crypts, disappearance of crypts, inflammatory cell infiltrates in the lamina propria, and abscess formation at the bottom of crypts. Intravital TPLSM in the same mice demonstrated fewer infiltrating leukocytes and crypt abscesses on day 14 in the steroid group compared with the nonsteroid group. CONCLUSIONS: Intravital 3D tomographic visualization of experimental colitis using TPLSM in combination with the serosal-approaching method can provide dynamic pathology at a high magnification, which may be useful in evaluating treatment efficacy in the same living mice.

Intravital imaging of DSS-induced cecal mucosal damage in GFP-transgenic mice using two-photon microscopy.

BACKGROUND: Two-photon laser-scanning microscopy (TPLSM) is a powerful diagnostic tool for real-time, high-resolution structural imaging. However, obtaining high-quality in vivo TPLSM images of intra-abdominal organs remains technically challenging. MATERIALS AND METHODS: An organ-stabilizing system was applied to high-quality TPLSM imaging. Real-time imaging of visceral organs, such as the liver, spleen, kidney and intestine, of transgenic green fluorescent protein (GFP) mice was performed in vivo using TPLSM. The bacterial translocation model using dextran sodium sulfate (DSS)-induced colitis was also investigated in prepared GFP mice following simple surgery. This allowed the capture of morphological real images using in vivo TPLSM. Immunohistochemical analysis of ZO-1 was performed to support the morphological findings of TPLSM. RESULTS AND CONCLUSIONS: We established an organ-stabilizing system to evaluate the real-time imaging of visceral organs in actin-GFP transgenic mice using in vivo TPLSM. DSS-induced colitis showed irregularity of crypt architecture, disappearance of crypts, inflammatory cell infiltration and increased rolling of white blood cells along the vasculature. In addition, the intercellular distance of mucosal cells in the crypt and vascular endothelial cells in the intestinal wall was increased in the intestinal mucosa during DSS colitis. In DSS colitis, there was remarkable loss of mucosal and vascular endothelial ZO-1 expression, as could be seen by a decrease in ZO-1 staining. In conclusion, our observations suggested the possibility that our TPLSM imaging system can be used to clarify the pathophysiological changes in various diseases using longitudinal studies of microscopic changes in the same animal over long periods of time.