Whole body vibration accelerates the functional recovery of motor nerve components in sciatic nerve-crush injury model rats.

This study aimed to investigate the effect of whole body vibration (WBV) on the sensory and motor nerve components with sciatic nerve injury model rats. Surgery was performed on 21 female Wister rats (6-8 weeks) under intraperitoneal anesthesia. The nerve-crush injuries for the left sciatic nerve were inflicted using a Sugita aneurysm clip. The sciatic nerve model rats were randomly divided into two groups (n=9; control group, n=12; WBV group). The rats in the WBV group walked in the cage with a vibratory stimulus (frequency 50 Hz, 20 min/day, 5 times/wk), while those in the control group walked in the cage without any vibratory stimulus. We used heat stimulation-induced sensory threshold and lumbar magnetic stimulation-induced motor-evoked potentials (MEPs) to measure the sensory and motor nerve components, respectively. Further, morphological measurements, bilateral hind-limb dimension, bilateral gastrocnemius dimension, and weight were evaluated. Consequently, there were no significant differences in the sensory threshold at the injury side between the control and WBV groups. However, at 4 and 6 weeks postoperatively, MEPs latencies in the WBV group were significantly shorter than those in the control group. Furthermore, both sides of the hind-limb dimension at 6 weeks postoperatively, the left side of the gastrocnemius dimension, and both sides of the gastrocnemius weight significantly increased. In conclusion, WBV especially accelerates the functional recovery of motor nerve components in sciatic nerve-crush injury model rats.

CD34 is Expressed in Endothelial Cells in Embryonic Testes and is Additionally Expressed in Non-Endothelial Cells in Postnatal Mouse Testes.

CD34 is expressed in various cell types in various tissues/organs, and has been regarded as being expressed in progenitors in various differentiation pathways. On the other hand, morphological studies have reported the presence of a special type of interstitial cells, telocytes, which generally express CD34, and have extremely long moniliform prolongations in various tissues/organs in vertebrates. We have recently reported the successful reconstruction of testicular structures by 3-D re-aggregation culture of dissociated prepubertal mouse testicular cells, and the roles of CD34 + cells in the reconstruction. However, it was unknown whether CD34 is expressed in embryonic through adult testes, and if so, in what cell type it is expressed. In order to clarify the expression of CD34 and behavior of CD34 + cells during development of mouse testes, we performed immunohistochemical studies. The results show that CD34 is expressed in two cell types in testes; one is endothelial cells which co-express CD31, VE-cadherin, and integrin ß1, but barely express PDGFRα and integrin α4 and α9, throughout development, while the other one is non-endothelial cells in which CD34 expression is initiated after birth, and which co-express PDGFRα and integrin α4, α9, and ß1. The latter corresponds to telocytes. The present findings will lead to clarifying the roles of these two types of CD34 + cells in spermatogenesis.

Pulmonary Neuroendocrine Cells and Small Cell Lung Carcinoma: Immunohistochemical Study Focusing on Mechanisms of Neuroendocrine Differentiation.

Neuroendocrine (NE) differentiation has been histochemically detected in normal and cancer tissues and cells. Immunohistochemical analyses have provided a more detailed understanding of NE biology and pathology. Pulmonary NE cells are a rare lung epithelial type, and small cell carcinoma of the lung (SCLC) is a high-grade NE tumor. Pulmonary NE and SCLC cells share common mechanisms for NE differentiation. Neural or NE cell lineage-specific transcription factors, such as achaete-scute homologue 1 (Ascl1) and insulinoma-associated protein 1 (INSM1), are crucial for the development of pulmonary NE cells, and NE differentiation is influenced by the balance between Ascl1 and the suppressive neural transcription factor, hairy-enhancer of split 1, a representative target molecule of the Notch signaling pathway. In this review, we discuss the importance of Ascl1 and INSM1 in identifying pulmonary NE and SCLC cells and introduce Ascl1-related molecules detected by comparative RNA-sequence analyses. The molecular classification of SCLC based on the expression of lineage-specific transcription or co-transcription factors, including ASCL1, NEUROD1, POU2F3, and YAP1, was recently proposed. We attempted to characterize these 4 SCLC subtypes using integrated immunohistochemical studies, which will provide insights into the molecular characteristics of these subtypes and clarify the inter- and intratumor heterogeneities of SCLC.

VCAM1-α4ß1 integrin interaction mediates interstitial tissue reconstruction in 3-D re-aggregate culture of dissociated prepubertal mouse testicular cells.

Roles of interstitial tissue in morphogenesis of testicular structures remain less well understood. To analyze the roles of CD34+ cells in the reconstruction of interstitial tissue containing Leydig cells (LCs), and testicular structures, we used 3D-reaggregate culture of dissociated testicular cells from prepubertal mouse. After a week of culture, adult Leydig cells (ALCs) were preferentially incorporated within CD34+ cell-aggregates, but fetal LCs (FLCs) were not. Immunofluorescence studies showed that integrins α4, α9 and ß1, and VCAM1, one of the ligands for integrins α4ß1 and α9ß1, are expressed mainly in CD34+ cells and ALCs, but not in FLCs. Addition of function-blocking antibodies against each integrin and VCAM1 to the culture disturbed the reconstruction of testicular structures. Antibodies against α4 and ß1 integrins and VCAM1 robustly inhibited cell-to-cell adhesion between testicular cells and between CD34+ cells. Cell-adhesion assays indicated that CD34+ cells adhere to VCAM1 through the interaction with α4ß1 integrin. Live cell imaging showed that CD34+ cells adhered around ALC-aggregates. CD34+ cells on the dish moved toward the aggregates, extending filopodia, and entered into them, which was disturbed by VCAM1 antibody. These results indicate that VCAM1-α4ß1 integrin interaction plays pivotal roles in formation of testicular interstitial tissues in vitro and also in vivo.

Integrated Immunohistochemical Study on Small-Cell Carcinoma of the Lung Focusing on Transcription and Co-Transcription Factors.

Small-cell lung cancer (SCLC) is an aggressive malignant cancer that is classified into four subtypes based on the expression of the following key transcription and co-transcription factors: ASCL1, NEUROD1, YAP1, and POU2F3. The protein expression levels of these key molecules may be important for the formation of SCLC characteristics in a molecular subtype-specific manner. We expect that immunohistochemistry (IHC) of these molecules may facilitate the diagnosis of the specific SCLC molecular subtype and aid in the appropriate selection of individualized treatments. We attempted IHC of the four key factors and 26 candidate SCLC target molecules selected from the gene expression omnibus datasets of 47 SCLC samples, which were grouped based on positive or negative results for the four key molecules. We examined differences in the expression levels of the candidate targets and key molecules. ASCL1 showed the highest positive rate in SCLC samples, and significant differences were observed in the expression levels of some target molecules between the ASCL1-positive and ASCL1-negative groups. Furthermore, the four key molecules were coordinately and simultaneously expressed in SCLC cells. An IHC study of ASCL1-positive samples showed many candidate SCLC target molecules, and IHC could become an essential method for determining SCLC molecular subtypes.

Significance of achaete-scute complex homologue 1 (ASCL1) in pulmonary neuroendocrine carcinomas; RNA sequence analyses using small cell lung cancer cells and Ascl1-induced pulmonary neuroendocrine carcinoma cells.

ASCL1 is one of the master transcription factors of small cell lung carcinoma (SCLC). To investigate the significance of ASCL1 in pulmonary neuroendocrine carcinoma, we performed 2 comparative RNA-seq studies between H69 (ASCL1-positive, classical type SCLC) and H69AR (ASCL1-negative, variant type SCLC) and between ASCL1-transfected A549 adenocarcinoma cell lines (A549(ASCL1+) cell lines) and A549(control) cell lines. RNA-seq analyses revealed that 940 genes were significantly different between the H69 and H69AR cell lines, and 728 between the A549(ASCL1+) and A549(control) cell lines. In total, 120 common genes between these analyses were selected as candidate ASCL1-related genes, and included genes with various cellular functions, such as neural development, secretion, growth, and morphology. Their expression degrees in three classical and two variant SCLC cell lines, two A549(ASCL1+) and two A549(control) cell lines were subjected to quantitative PCR analyses. Since the candidate ASCL1-related genes were strongly expressed in the classical SCLC and A549(ASCL1+) cell lines and more weakly expressed in the variant SCLC and A549(control) cell lines, the ASCL1-related 7 molecules INSM1, ISL1, SYT4, KCTD16, SEZ6, MS4A8, and COBL were further selected. These molecules suggested diverse functions for A549(ASCL1+): INSM1 and ISL1 are transcription factors associated with neuroendocrine differentiation, while SYT4, KTCD16, and SEZ6 may be related to neurosecretory functions and MS4A8 and COBL to cell growth and morphology. An immunohistochemistry of these seven molecules was performed on lung carcinoma tissues and the xenotransplanted tumors of A549(ASCL1+), and they were preferentially and positively stained in ASCL1-postive tumor tissues.

Significance of Stat3 Signaling in Epithelial Cell Differentiation of Fetal Mouse Lungs.

To study the significance of signal transducer and activator of transcription (Stat) 3 in lung epithelial development of fetal mice, we examined fetal mouse lungs, focusing on the expression of Clara cell secretory protein (CCSP), Forkhead box protein J1 (Foxj1), calcitonin gene-related peptide (CGRP), phosphorylated Stat3 (Tyr705), and hairy/enhancer of split (Hes) 1, and observed cultured fetal lungs upon treatment with IL-6, a Stat3 activator, or cucurbitacin I, a Stat3 inhibitor. Moreover, the interaction of Stat3 signaling and Hes1 was studied using Hes1 gene-deficient mice. Phosphorylated Stat3 was detected in fetal lungs and, immunohistochemically, phosphorylated Stat3 was found to be co-localized in developing Clara cells, but not in ciliated cells. In the organ culture studies, upon treatment with IL-6, quantitative RT-PCR revealed that CCSP mRNA increased with increasing Stat3 phosphorylation, while cucurbitacin I decreased Hes1, CCSP, Foxj1 and CGRP mRNAs with decreasing Stat3 phosphorylation. In the lungs of Hes1 gene-deficient mice, Stat3 phosphorylation was not markedly different from wild-type mice, the expression of CCSP and CGRP was enhanced, and the treatment of IL-6 or cucurbitacin I induced similar effects on mouse lung epithelial differentiation regardless of Hes1 expression status. Stat3 signaling acts in fetal mouse lung development, and seems to regulate Clara cell differentiation positively. Hes1 could regulate Clara cell differentiation in a manner independent from Stat3 signaling.

Notch1 controls cell chemoresistance in small cell lung carcinoma cells.

BACKGROUND: Small cell lung carcinoma (SCLC) is characterized by a high rate of relapse and failure of chemotherapy because of the emergence of drug resistant cells. Notch signaling controls carcinogenesis in several human malignancies and could be involved in the resistance of cells to several chemotherapeutic agents. Herein, we analyzed the role of Notch1 signaling in the resistance of human SCLC cells to doxorubicin. METHODS: Small interfering ribonucleic acid technology was used to knock down (KD) Notch1 in H69AR and SBC-3 SCLC cells. We detected the effect of inhibiting Notch1 on the expression of drug resistant related molecules: multidrug resistance-associated protein (MRP-1) and anti-apoptotic factor B-cell lymphoma-2, as well as to cell adhesion molecule E-cadherin, which contributes to the adhesion of SCLC cells to the extracellular matrix and confers chemoresistance in a process known as cell adhesion-mediated drug resistance (CAM-DR). We also observed the effect of KD Notch1 on cell survival under high concentrations of doxorubicin treated media. RESULTS: H69AR and SBC-3 cells expressed Notch1 protein and grew as adherent aggregates, which confer resistance to high concentrations of doxorubicin. On inhibiting Notch1, we observed activation of the apoptotic pathway in cells, possibly resulting from the loss of CAM-DR and, thus, SBC-3 cells showed a loss of chemoresistant ability. However, in H69AR cells with KD Notch1, the expression of MRP-1 was increased and, thus, sustained the chemoresistant ability of cells. CONCLUSION: The Notch1 signaling pathway is involved in mediating the drug resistance phenotype of SCLC cells.

Bromodeoxyuridine (BrdU)-label-retaining cells in mouse terminal bronchioles.

Adult male mice were continuously treated with bromodeoxyuridine (BrdU) for 1, 2, or 4 weeks by an osmotic pump. To detect BrdU-label-retaining cells (LRCs), putative progenitor/stem cells, other animals were continuously treated with BrdU for 2 weeks, and were then kept without any treatments for 2, 6, or 18 months. The lungs were fixed with 4% paraformaldehyde, and were paraffin-embedded. We observed terminal bronchioles with BrdU immunostaining alone or with BrdU immunostaining accompanying immunostaining for Clara cell secretory protein (CCSP), forkhead box protein J1 (FoxJ1), or calcitonin gene-related peptide (CGRP). The average incidences of BrdU-incorporated cells in the terminal bronchioles after 1, 2, and 4 weeks of continuous BrdU infusion were 6.2%, 11.9%, and 23.1%, respectively. Most BrdU-incorporated cells in these periods were CCSP-immunoreactive (91.7%, 91.3%, and 88.2%, respectively), which means progenitor function of Clara cells. FoxJ1-immunoreactive BrdU-incorporated cells were fewer (5.4%, 3.0%, 2.7%, respectively). The average incidences of BrdU-LRCs in the terminal bronchioles after 2, 6, and 18 months were 7.2%, 4.3, and 2.7%, respectively. Most BrdU-LRCs were CCSP-immunoreactive (91.0%, 92.7%, and 89.6%, respectively), and FoxJ1-immunoreactive BrdU-LRCs were fewer (6.0%, 5.7%, and 2.1%, respectively). CGRP-positive BrdU-incorporated cells were occasional. CGRP-positive BrdU-LRCs were detected in 17.6% of neuroepithelial bodies (NEBs) at 2 months, but disappeared at 6 months. BrdU-positive stem cell candidates, which locate at the brochiolo-alveolar duct junction or cover NEB, were few throughout this study. In conclusion, in the lungs treated only with BrdU, CCSP-immunoreactive cells are important to maintain homeostasis in the terminal bronchiolar epithelium.