Histological Study and LYVE-1 Immunolocalization of Mouse Mesenteric Lymph Nodes with “In Vivo Cryotechnique”

The “in vivo cryotechnique” (IVCT) is a powerful tool to directly freeze living animal organs in order to maintain biological components in frozen tissues, reflecting their native states. In this study, mesenteric lymph nodes of living mice were directly frozen with IVCT, and we did morphological studies and immunohistochemical analyses on a hyaluronic acid receptor, LYVE-1. In lymph nodes, widely open lymphatic sinuses were observed, and many lymphocytes adhered to inner endothelial cells along subcapsular sinuses. The LYVE-1 was clearly immunolocalized at inner endothelial cells of subcapsular sinuses, as well as those of medullary sinuses. Conventional pre-embedding electron microscopy also showed LYVE-1 immunolocalization along both the apical and basal sides of cell membranes of inner endothelial cells. By triple-immunostaining for LYVE-1, smooth muscle actin, and type IV collagen, the LYVE-1 was immunolocalized only in the inner endothelial cells, but not in outer ones which were surrounded by collagen matrix and smooth muscle cells. Thus, the functional morphology of lymph nodes in vivo was demonstrated and LYVE-1 immunolocalization in inner endothelial cells of subcapsular sinuses suggests hyaluronic acid incorporation into lymph node parenchyma

Immunohistochemical Distribution of Serum Proteins in Living Mouse Heart with In Vivo Cryotechnique

In vivo cryotechnique (IVCT), which immediately cryofixes target organs in situ, was used to clarify the morphological features of beating heart tissue of living mice. IVCT was performed for diastolic heart tissue under the condition of monitoring with electrocardiogram (ECG). Other mouse hearts were prepared with conventional perfusion-fixation (PF-DH) or immersion-fixation followed by dehydration (IM-DH), and quick-freezing of resected heart tissues (FQF). Immunolocalizations of albumin, immunoglobulin G1 (IgG1), intravenously injected bovine serum albumin (BSA), and connexin 43 were examined after different intervals of BSA injection. In the case of IVCT, the exact stop time of beating mouse hearts was recorded by ECG, and open blood vessels with flowing erythrocytes were observed with less artificial tissue shrinkage than with conventional preparation methods. Both albumin and BSA were well preserved in intercalated discs and t-tubules of cardiomyocytes in addition to blood vessels and interstitial matrices. IgG1 was immunolocalized in interstitial matrices of heart tissues in addition to their blood vessels. At 4 hr after BSA injection, it was immunolocalized in the intercalated discs of cardiomyocytes and lost later at 8 hr. IVCT should prove to be more useful for the morphofunctional examination of dynamically changing heart tissue than conventional preparation methods

Immunoreactivity of Glutamate in Mouse Retina Inner Segment of Photoreceptors With In Vivo Cryotechnique

The purpose of this study was to clarify a previously controversial issue concerning glutamate (Glu) immunoreactivity (IR) in the inner segment (IS) of photoreceptors by using in vivo cryotechnique (IVCT) followed by freeze substitution (FS), which enabled us to analyze the cells and tissues reflecting living states. Eyeballs from anesthetized mice were directly frozen using IVCT. The frozen tissues were processed for FS fixation in acetone containing chemical fixatives, and embedded in paraffin. Deparaffinized sections were immunostained with an anti-Glu antibody. The strongest Glu-IR was obtained in the specimens prepared by FS with paraformaldehyde or a low concentration of glutaraldehyde, whereas no Glu-IR was obtained without the chemical fixatives. The Glu was immunolocalized in the IS, outer and inner plexiform and ganglion cell layers. Thus, the immunolocalization of Glu in the IS was clearly demonstrated using IVCT. (J Histochem Cytochem 57:883–888, 2009

Essential Function of Protein 4.1G in Targeting of Membrane Protein Palmitoylated 6 into Schmidt-Lanterman Incisures in Myelinated Nerves

Protein 4.1G is a membrane skeletal protein found in specific subcellular structures in myelinated Schwann cells and seminiferous tubules. Here, we show that in the mouse sciatic nerve, protein 4.1G colocalized at Schmidt-Lanterman incisures (SLI) and the paranodes with a member of the membrane-associated guanylate kinase (MAGUK) family, membrane protein palmitoylated 6 (MPP6). Coimmunoprecipitation experiments revealed that MPP6 was interacting with protein 4.1G. In contrast to wild-type nerves, in 4.1G knockout mice, MPP6 was found largely in the cytoplasm near Schwann cell nuclei, indicating an abnormal protein transport. Although the SLI remained in the 4.1G knockout sciatic nerves, as confirmed by E-cadherin immunostaining, their shape was altered in aged 4.1G knockout nerves compared to their shape in wild-type nerves. In the seminiferous tubules, MPP6 was localized similarly to protein 4.1G along cell membranes of the spermatogonium and early spermatocytes. However, in contrast to myelinated peripheral nerves, the specific localization of MPP6 in the seminiferous tubules was unaltered in the absence of protein 4.1G. These results indicate that 4.1G has a specific role in the targeting of MPP6 to the SLI and the assembly of these subcellular structures

Successful treatment of limited-stage small-cell lung cancer in the right mainstem bronchus by a combination of chemotherapy and argon plasma coagulation

The current standard-of-care treatment for patients with limited-stage small-cell lung cancer (SCLC) is concurrent chemoradiotherapy for local and systemic control. However, standard-of-care treatment strategies have not been established for those with limited-stage SCLC who have a history of thoracic radiotherapy due to concerns with complications associated with radiation overdose. A 37-year-old male developed an aspergilloma in the postoperative left thoracic space after he was treated with concurrent chemoradiotherapy for mediastinal type lung adenocarcionoma and subsequent left upper lobectomy for heterochronous dual adenocarcinoma. Fiberoptic bronchoscopy was performed to examine the status of the suspected bronchopleural fistula when a polypoid mass was observed in the right mainstem bronchus. A histological examination showed that the mass was SCLC at a clinical stage of cTisN0M0, stageIA, without local invasion. Since thoracic radiotherapy was not an option due to a previous history of thoracic irradiation, a combination treatment of carboplatin and etoposide was administered for 4 cycles and resulted in good partial response. In addition, argon plasma coagulation (APC) was performed as an alternative to curative radiotherapy on day 22 of the 4th cycle. The 5th cycle was administered 7 days after APC at which the anticancer therapy was completed. The patient remains disease-free 60 months after the completion of treatment, which suggests that this combination therapy may resolve very early-stage SCLC

Recent Progress on Genetically Modified Animal Models for Membrane Skeletal Proteins: The 4.1 and MPP Families

The protein 4.1 and membrane palmitoylated protein (MPP) families were originally found as components in the erythrocyte membrane skeletal protein complex, which helps maintain the stability of erythrocyte membranes by linking intramembranous proteins and meshwork structures composed of actin and spectrin under the membranes. Recently, it has been recognized that cells and tissues ubiquitously use this membrane skeletal system. Various intramembranous proteins, including adhesion molecules, ion channels, and receptors, have been shown to interact with the 4.1 and MPP families, regulating cellular and tissue dynamics by binding to intracellular signal transduction proteins. In this review, we focus on our previous studies regarding genetically modified animal models, especially on 4.1G, MPP6, and MPP2, to describe their functional roles in the peripheral nervous system, the central nervous system, the testis, and bone formation. As the membrane skeletal proteins are located at sites that receive signals from outside the cell and transduce signals inside the cell, it is necessary to elucidate their molecular interrelationships, which may broaden the understanding of cell and tissue functions