Satoshi Shimo

Conductive resins improve charging and resolution of acquired images in electron microscopic volume imaging

Recent advances in serial block-face imaging using scanning electron microscopy (SEM) have enabled the rapid and efficient acquisition of 3-dimensional (3D) ultrastructural information from a large volume of biological specimens including brain tissues. However, volume imaging under SEM is often hampered by sample charging, and typically requires specific sample preparation to reduce charging and increase image contrast. In the present study, we introduced carbon-based conductive resins for 3D analyses of subcellular ultrastructures, using serial block-face SEM (SBF-SEM) to image samples. Conductive resins were produced by adding the carbon black filler, Ketjen black, to resins commonly used for electron microscopic observations of biological specimens. Carbon black mostly localized around tissues and did not penetrate cells, whereas the conductive resins significantly reduced the charging of samples during SBF-SEM imaging. When serial images were acquired, embedding into the conductive resins improved the resolution of images by facilitating the successful cutting of samples in SBF-SEM. These results suggest that improving the conductivities of resins with a carbon black filler is a simple and useful option for reducing charging and enhancing the resolution of images obtained for volume imaging with SEM.

Effect of streptozotocin-induced diabetes on motoneurons and muscle spindles in rats

This study examined the alterations in the number and size of motoneurons innervating the medial gastrocnemius (MG) and biceps femoris (BF) motor nuclei in diabetic rats (12 or 22 weeks after injection of streptozotocin) and age-matched controls using retrograde labeling technique. Additionally, morphological alterations of muscle spindles in BF and MG muscles were tested. Significantly fewer labeled MG motoneurons were found in 12- and 22-week diabetic rats as compared with age-matched control animals. In contrast, the number of BF motoneurons was preserved in each group. Compared to control animals, the ratio of larger motoneurons of MG and BF muscle were decreased at 12 weeks, and smaller MG motoneurons were drastically decreased at 22 weeks. Moreover, MG muscle spindle showed reduction of its number and increase of intrafusal muscle fibers; however, BF muscle spindles showed little or no difference from control animals. We conclude that there is an early loss of alpha motoneurons for both MG and BF muscles followed by a later loss of gamma motoneurons in MG muscle in diabetic animals. Moreover, loss of gamma motoneuron might induce atrophy of MG muscle spindles.

Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets.

Validation of the shoulder36 for the activities of daily living with shoulder disorders

[Purpose] The Shoulder36 (V 1. 3) is a regional questionnaire in Japan that has not been validated as a functional evaluation of the shoulder via a thorough comparison with other questionnaires (e.g., QuickDASH). The purpose of this study was to test the reliability, validity, and responsiveness of the Shoulder36 (V 1. 3). [Subjects and Methods] A series of 46 patients with upper extremity disorders completed the Shoulder36 (V 1. 3) and the QuickDASH Japanese version (QuickDASH-JSSH). The reliability of the Shoulder36 was assessed for consistency and validity. The correlation coefficients between the Shoulder36 (V 1. 3) and the QuickDASH-JSSH were obtained. [Results] The total of the Cronbach’s alpha coefficients for the Shoulder36 (V 1. 3) was 0.98. The intraclass correlation coefficients for the six domains of the Shoulder36 (V 1. 3) were similarly high, ranging from 0.81 to 0.94. The correlations between the six domains of the Shoulder36 (V 1. 3) and the three domains of the QuickDASH subscales ranged from −0.43 to −0.78. [Conclusion] The Shoulder36 (V 1. 3) was able to evaluate the relationship between activities of daily living and shoulder joint function with the same degree of accuracy but in more detail than QuickDASH-JSSH. Therefore, it should prove to be a valuable asset in physiotherapy plans and have multiple research applications.

Early rehabilitation affects functional outcomes and activities of daily living after arthroscopic rotator cuff repair: a case report.

[Purpose] The effect of early rehabilitation protocols after arthroscopic rotator cuff repair is currently unknown. We examined short-term effects of early rehabilitation on functional outcomes and activities of daily living after arthroscopic rotator cuff repair. [Subject and Methods] An 82-year-old male fell during a walk, resulting in a supraspinatus tear. Arthroscopic rotator cuff repair was performed using a single-row technique. He wore an abduction brace for 6 weeks after surgery. [Results] From day 1 after surgery, passive range of motion exercises, including forward flexion and internal and external rotation were performed twice per day. Starting at 6 weeks after surgery, active range of motion exercises and muscle strengthening exercises were introduced gradually. At 6 weeks after surgery, his active forward flexion was 150°, UCLA shoulder rating scale score was 34 points, and Quick Disabilities of the Arm, Shoulder, and Hand questionnaire disability/symptom score was 36 points. At 20 weeks after surgery, his active forward flexion was 120°, UCLA shoulder rating scale score was 34 points, and Quick Disabilities of the Arm, Shoulder, and Hand questionnaire disability/symptom score was 0 points. [Conclusion] These protocols are recommended to physical therapists during rehabilitation for arthroscopic rotator cuff repair to support rapid reintegration into activities of daily living.

Effect of streptozotocin-induced diabetes on motor representations in the motor cortex and corticospinal tract in rats

Motor disorders in patients with diabetes are associated with diabetic peripheral neuropathy, which can lead to symptoms such as lower extremity weakness. However, it is unclear whether central motor system disorders can disrupt motor function in patients with diabetes. In a streptozotocin-induced rat model of type 1 diabetes, we used intracortical microstimulation to evaluate motor representations in the motor cortex, recorded antidromic motor cortex responses to spinal cord stimulation to evaluate the function of corticospinal tract (CST) axons, and used retrograde labeling to evaluate morphological alterations of CST neurons. The diabetic rats exhibited size reductions in the hindlimb area at 4 weeks and in trunk and forelimb areas after 13 weeks, with the hindlimb and trunk area reductions being the most severe. Other areas were unaffected. Additionally, we observed reduced antidromic responses in CST neurons with axons projecting to lumbar spinal segments (CST-L) but not in those with axons projecting to cervical segments (CST-C). This was consistent with the observation that retrograde-labeled CST-L neurons were decreased in number following tracer injection into the spinal cord in diabetic animals but that CST-C neurons were preserved. These results show that diabetes disrupts the CST system components controlling hindlimb and trunk movement. This disruption may contribute to lower extremity weakness in patients.

Immunohistochemical Detection of Soluble Immunoglobulins in Small Intestines

We performed immunohistochemical or ultrastructural analyses of living mouse small intestines prepared by “in vivo cryotechnique” (IVCT). Living morphological states of small intestinal tissues, including flowing erythrocytes and opening blood vessels, were observed on paraffin-embedded sections prepared with IVCT. IgA was immunolocalized in many plasma cells of the lamina propria mucosa, intestinal matrices, and also in epithelial cells of the intestinal villi and crypts. Both IgG1 and IgM immunoreactivities were mainly detected in blood vessels, whereas only IgG1 was also immunolocalized in interstitial matrices of mucous membranes. Confocal laser scanning micrographs of double-fluorescence immunostaining for IgA immunoreactivity are detected in the cytoplasm of epithelial cells as well as plasma cells in the lamina propria mucosa. On the other hand, by electron microscopy, intracellular ultrastructures of epithelial cells were well preserved in tissue areas 5–10 μm away from the cryogen-contact surface tissues. Apical microvilli of epithelial cells contained dynamically waving actin filaments. Furthermore, highly electron-dense organelles, such as mitochondria, in addition to endoplasmic reticulum and ribosomes, were well preserved under the widely organized terminal web. Additionally, Epon-embedded thick sections were treated with sodium ethoxide, followed by antigen retrieval, and immunostained for various proteins, such as IgA, Igκ, IgG1, IgM, J-chain, and albumin. IgA immunoreactivity was detected as a tiny dot-like pattern in the cytoplasm of some epithelial cells and plasma cells localized in the lamina propria. The J-chain and Igκ immunoreactivities were also detected in the same local areas as those of IgA. Thus, IVCT was useful for the preservation of soluble serum proteins and ultrastructural analyses of dynamically changing epithelial cells of living mouse small intestines.