Facial nerve dissection in parotid surgery: a microscopic investigation study.

In parotid surgery, it is crucial to identify and preserve the facial nerve, which runs through the parotid gland. The purpose of this study was to histologically clarify two clinical questions: whether "superficial" and "deep" lobes exist anatomically and what are the structures surrounding facial nerve. Parotid gland tissues were obtained from dissection of donated cadavers. The gland was cut perpendicular to the facial nerve plane at 5 mm intervals, and the pieces were embedded in paraffin, thinly sliced, and stained. The morphology of the nerve was observed at each site, and the relationships between the thickness of the perineural tissue (defined as the tissue between the groups of nerve fasciculi and the glandular parenchyma), nerve diameter, and distance from the proximal end of the nerve were examined. In addition, the dissection layer was examined histologically in isolated parotid tissues. The interlobular connective tissue was spread like a mesh within the parotid gland and subdivided the glandular parenchyma. The facial nerve was located in the interlobular connective tissue, and its course was not restricted to the boundary plane between the superficial and deep lobes. The thickness of the perineural tissue decreased with increasing distance from the proximal end of the nerve. The dissection layer was clarified that located in the perineural tissue. The perineural tissue is thinner in more distal regions, which may make dissection more difficult there. No particular anatomical structure appears to separate the superficial and deep lobes.

Visualization of basement membranes by a nidogen-based fluorescent reporter in mice.

Basement membranes (BMs) are thin, sheet-like extracellular structures that cover the basal side of epithelial and endothelial tissues and provide structural and functional support to adjacent cell layers. The molecular structure of BMs is a fine meshwork that incorporates specialized extracellular matrix proteins. Recently, live visualization of BMs in invertebrates demonstrated that their structure is flexible and dynamically rearranged during cell differentiation and organogenesis. However, the BM dynamics in mammalian tissues remain to be elucidated. We developed a mammalian BM imaging probe based on nidogen-1, a major BM-specific protein. Recombinant human nidogen-1 fused with an enhanced green fluorescent protein (Nid1-EGFP) retains its ability to bind to other BM proteins, such as laminin, type IV collagen, and perlecan, in a solid-phase binding assay. When added to the culture medium of embryoid bodies derived from mouse ES cells, recombinant Nid1-EGFP accumulated in the BM zone of embryoid bodies, and BMs were visualized in vitro. For in vivo BM imaging, a knock-in reporter mouse line expressing human nidogen-1 fused to the red fluorescent protein mCherry (R26-CAG-Nid1-mCherry) was generated. R26-CAG-Nid1-mCherry showed fluorescently labeled BMs in early embryos and adult tissues, such as the epidermis, intestine, and skeletal muscles, whereas BM fluorescence was unclear in several other tissues, such as the lung and heart. In the retina, Nid1-mCherry fluorescence visualized the BMs of vascular endothelium and pericytes. In the developing retina, Nid1-mCherry fluorescence labeled the BM of the major central vessels; however, the BM fluorescence were hardly observed in the peripheral growing tips of the vascular network, despite the presence of endothelial BM. Time-lapse observation of the retinal vascular BM after photobleaching revealed gradual recovery of Nid1-mCherry fluorescence, suggesting the turnover of BM components in developing retinal blood vessels. To the best of our knowledge, this is the first demonstration of in vivo BM imaging using a genetically engineered mammalian model. Although R26-CAG-Nid1-mCherry has some limitations as an in vivo BM imaging model, it has potential applications in the study of BM dynamics during mammalian embryogenesis, tissue regeneration, and pathogenesis.

Changes in the Incidence of Infantile Spinal Muscular Atrophy in Shikoku, Japan between 2011 and 2020.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder. Al-though there was no cure for SMA, newly developed therapeutic drugs (nusinersen, onasemnogene abeparvovec, and risdiplam) have been proven effective for the improvement of motor function and prevention of respiratory insufficiency of infants with SMA. Nusinersen was introduced in Japan in 2017 and onasemnogene abeparvovec in 2020. We hypothesized that the introduction of these drugs might influence the incidence of SMA (more precisely, increase the diagnosis rate of SMA) in Japan. To test this hypothesis, we conducted a second epidemiological study of infantile SMA using questionnaires in Shikoku, Japan between October 2021 and February 2022. The incidence of infantile SMA during the period 2016-2020 was 7.08 (95% confidence interval [CI] 2.45-11.71) per 100,000 live births. According to our previous epidemiological study, the incidence of infantile SMA during 2011-2015 was 2.70 (95% CI 0.05-5.35) per 100,000 live births. The increased incidence of infantile SMA suggests that the widespread news in Japan regarding the introduction of therapeutic agents, nusinersen and onasemnogene abeparvovec, raised clinicians' awareness about SMA, leading to increased and earlier diagnosis of SMA in Shikoku.

Cyclosporine for the treatment of multisystem inflammatory syndrome in children with coronary artery aneurysms.

Multisystem inflammatory syndrome in children (MIS-C) is a newly described syndrome related to the COVID-19, resembling other known aetiologies, including Kawasaki disease. Cardiovascular involvement is common; left ventricle dysfunction and coronary artery aneurysm (CAA) are also observed. Many treatment guidelines recommend using intravenous immunoglobulin (IVIG) alone or with glucocorticoids as the first-line therapy. Biological agents, such as anakinra, are recommended for refractory cases, but the evidence is still accumulating. Moreover, the use of other treatment agents can be beneficial, especially when anakinra is unavailable. Here, we report the case of a 9-year-old girl who presented with MIS-C with CAAs. She received cyclosporine because two rounds of IVIG treatment were ineffective and the use of anakinra is not approved in Japan. Her cytokine profile showed that cyclosporine prevented exacerbation. The case highlights that cyclosporine therapy can be an option for the treatment of refractory MIS-C with CAA.

Severe pediatric asthma with a poor response to omalizumab: a report of three cases and three-dimensional bronchial wall analysis.

Omalizumab is used for the treatment of persistent severe allergic asthma in adults and children. However, some patients remain symptomatic even after omalizumab treatment. In bronchial asthma, chronic inflammation of the bronchial wall causes thickening of the airway wall, resulting from irreversible airway remodeling. Progression of airway remodeling causes airflow obstruction, leading to treatment resistance. We report three Japanese children with severe asthma who had a poor response to omalizumab treatment. They had a long period of inadequate management of asthma before initiating omalizumab. Even after omalizumab treatment, their symptoms persisted, and the parameters of spirometry tests did not improve. We hypothesized that omalizumab was less effective in these patients because airway wall remodeling had already progressed. We retrospectively evaluated the bronchial wall thickness using a three-dimensional bronchial wall analysis with chest computed tomography. The bronchial wall thickness was increased in these cases compared with six responders. Progressed airway wall thickness caused by airway remodeling may be associated with a poor response to omalizumab in children with severe asthma.

Morphology of cervical periradicular fibrous sheath and nerve roots in relation to postoperative C5 palsy.

BACKGROUND CONTEXT: C5 palsy is a major complication of cervical spine surgery, however, its exact pathogenesis remains unclear. Some studies have shown that the superficial layer of the posterior longitudinal ligament extends laterally and forms the periradicular fibrous sheath (PFS), and envelopes the nerve roots. However, the anatomical relationship between the PFS and nerve root at each cervical level has not been fully revealed. PURPOSE: To examine the difference of the PFS that covers the nerve root at each cervical level, and to consider its potential in the onset of postoperative C5 palsy. STUDY DESIGN: Anatomical study of cervical dissection of 13 embalmed cadavers. METHODS: Thirteen human formalin-fixed cadavers were dissected from posterior approach, and were observed their cervical nerves bilaterally from C3 to C8 (the total number of nerves was 156). The bare area length (BAL), which is the distance between the medial posterior edge of the PFS and the bifurcation of the nerve and dura mater, was measured by using electronic calipers. Thus, BAL is the uncovered area of the nerve root by the PFS. We examined whether BAL significantly varied at each cervical level. RESULTS: We confirmed the PFS macro- and/or microanatomically in all cadavers. The average BAL gradually increased craniocaudally, and there was a significant step between that of C5 and C6 level. CONCLUSION: The average BAL of the C5 root was significantly shorter than that of C6, C7, and C8, suggesting that C5 root was more tightly anchored. This could be one reason for C5 palsy, making C5 nerve root vulnerable to the traction caused by the postoperative spinal cord shift. CLINICAL SIGNIFICANCE: This study provides clinicians an additional understanding of the anatomical factor of C5 palsy. Consideration of the anchoring effect of the PFS for nerve roots, release of the PFS could be a preventive procedure for C5 palsy.

Growth recovery lines in children: A comparison between psychosocial short stature and other pathological causes of short stature.

BACKGROUND: Psychosocial short stature (PSS) is a rare disorder associated with emotional deprivation. Growth recovery lines (GRLs), the radiodense bands in metaphyseal bones, are indicators of stress. OBJECTIVE: To evaluate the efficacy of using GRLs in the distal radius to identify PSS. PARTICIPANTS AND SETTING: This retrospective cohort study included children 15-138 months of age with short stature whose hands and wrists were radiographed between 2011 and 2020 at Matsuyama Red Cross Hospital in Japan. METHODS: PSS was determined if a child with short stature had been reported to be abused or neglected. Other pathological short statures were diagnosed per the established criteria. GRLs, height velocity before and after specific treatment, insulin-like growth factor 1, and the difference between chronological and skeletal age were assessed. RESULTS: The PSS and other short stature groups comprised of 7 and 11 children, respectively. The body mass index of the PSS group was smaller than that of other short stature group (-1.15 standard deviation [SD] vs. 0.57 SD, P = 0.003). The PSS group had significantly more GRLs than the other group (5.3 vs. 0.5, P = 0.011). Height velocity before treatment in the PSS group was significantly lower (-5.46 SD vs. -1.86 SD, P = 0.005), with no significant differences in other variables. The specificity for PSS was >90% when children with short stature had at least three GRLs in both distal radii. CONCLUSIONS: Abuse or neglect should be considered in children with short stature having multiple GRLs in the distal radius.

Lactoferrin-like Immunoreactivity in Distinct Neuronal Populations in the Mouse Central Nervous System.

Lactoferrin (Lf) is an iron-binding glycoprotein mainly found in exocrine secretions and the secondary granules of neutrophils. In the central nervous system (CNS), expression of the Lf protein has been reported in the lesions of some neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis, as well as in the aged brain. Lf is primarily considered an iron chelator, protecting cells from potentially toxic iron or iron-requiring microorganisms. Other biological functions of Lf include immunomodulation and transcriptional regulation. However, the roles of Lf in the CNS have yet to be fully clarified. In this study, we raised an antiserum against mouse Lf and investigated the immunohistochemical localization of Lf-like immunoreactivity (Lf-LI) throughout the CNS of adult mice. Lf-LI was found in some neuronal populations throughout the CNS. Intense labeling was found in neurons in the olfactory systems, hypothalamic nuclei, entorhinal cortex, and a variety of brainstem nuclei. This study provides detailed information on the Lf-LI distribution in the CNS, and the findings should promote further understanding of both the physiological and pathological significance of Lf in the CNS.

Paediatric case of group A streptococcal pharyngitis, arthritis and osteomyelitis associated with dental neglect.

Group A streptococcus (GAS) causes a wide variety of infections in the paediatric population, ranging from pharyngitis to rare but severe invasive diseases, such as bacterial arthritis and osteomyelitis. Dental neglect is a type of child neglect in which caregivers fail to provide adequate care and treatment for dental diseases. This results in poor oral hygiene and can lead to complications including sepsis. We report the case of a 4-year-old boy, suffering from child neglect, presenting with GAS pharyngitis and subsequent bacterial arthritis in the right ankle, osteomyelitis in the right talus and abscess in the right calcaneus. He was first treated with penicillin, which was changed to clindamycin because of a suspected drug-induced rash. He was discharged after 6 weeks of intravenous therapy when symptoms had resolved and inflammatory markers were within the normal range. The case highlights that dental neglect may present a risk for subsequent invasive infections.

The therapeutic potential of exosomal miR-22 for cervical cancer radiotherapy.

Cervical cancer is the fourth-most prevalent malignancy in women. For advanced cervical cancer, radiotherapy is a major treatment. Micro RNAs (miRNAs) are small, noncoding RNAs that negatively regulate the target gene expression posttranscriptionally. miR-22 is frequently downregulated in various cancers including cervical cancer, and is associated with a poor prognosis in cervical cancer. Exosomes are small endosomally secreted vesicles that carry components such as proteins, messenger RNA (mRNA), DNA and miRNA. We investigated whether or not exosomes can efficiently deliver miR-22 to recipient cervical cancer cells and affect the gene expression in the cells, as well as assessed the role of exosomal miR-22 in radiosensitivity. Exosomes containing high levels of miR-22 were extracted by ultracentrifugation and then characterized by Western blotting, a nanoparticle tracking analysis and electron microscopy. The high presence of miR-22 in the exosome was confirmed by real-time polymerase chain reaction. After the administration of the collected exosomal miR-22 to SKG-II and C4-I cervical cancer cells, the level of miR-22 in the cells was significantly increased, indicating the absorption of the exosomal miR-22. When miR-22 encapsulated in exosomes was administered to the SKG-II cells, the level of c-Myc binding protein (MYCBP) and human telomerase reverse transcriptase (hTERT) was significantly decreased in correlation with increased radiosensitivity determined by a clonogenic assay. Taken together, these results suggest that the administration of exosomal miR-22 may be a novel drug delivery system for cervical cancer radiotherapy.

Survey of patients with spinal muscular atrophy on the island of Shikoku, Japan.

BACKGROUND: Spinal muscular atrophy (SMA) is an inherited neuromuscular disorder associated with spinal motor neuron loss and characterized by generalized muscle weakness. Only a few reports exist on SMA epidemiology in Japan. Additionally, nusinersen recently became available as a treatment for this condition. We estimated the prevalence of each type of SMA on Shikoku, Japan's fourth-largest major island. METHODS: We sent a questionnaire to all 131 hospitals in Shikoku that have pediatrics or neurology departments from March to September 2019, asking whether each hospital had SMA patients at that time. If so, we sent a second questionnaire to obtain more detailed information on the clinical data and treatment of each patient. RESULTS: A total of 117 hospitals (89.3%) responded to our first questionnaire, and 21 SMA patients were reported, 16 of whom had homozygous deletion of SMN1. Of the 21, nine had SMA type 1, five were type 2, five were type 3, one was type 4, and one was unidentified. The estimated prevalence for all instances of SMA and 5q-SMA was 0.56 and 0.43 per 100,000 people, respectively. Thirteen patients had received nusinersen therapy. Its outcomes varied from no obvious effects and being unable to sit to being able to sit independently. CONCLUSION: Our data showed the prevalence of SMA types 2 and 3 was relatively low on Shikoku compared with previous reports from other countries, suggesting delayed diagnosis may affect the results. Remaining motor function may be one predicting factor. Greater awareness of SMA among clinicians and patients seems necessary for more accurate epidemiological studies.

Soluble Vegfr3 gene therapy suppresses multi-organ metastasis in a mouse mammary cancer model.

Accumulating evidence on the association of VEGF-C with lymphangiogenesis and lymph node metastasis implicates lymphatic vessels as a potential target in anti-cancer therapy. To evaluate whether blocking VEGF-C and VEGFR-3 signaling can inhibit multi-organ metastases, a mouse metastatic mammary cancer model was subjected to gene therapy using a soluble VEGFR-3 expression vector (psVEGFR-3). We showed that psVEGFR-3 significantly diminished cell growth in vitro with or without added VEGF-C, and significantly reduced primary tumor growth and tumor metastases to wide-spectrum organs in vivo. Although apoptotic cell death and angiogenesis levels did not differ between the control and psVEGFR-3 groups, cell proliferation and lymphangiogenesis in the mammary tumors were significantly decreased in the psVEGFR-3 group. Furthermore, lymphatic vessel invasion was significantly inhibited in this group. Real-time RT-PCR analysis revealed significantly high expression of the Vegfr3 gene due to gene therapy, and the transcriptional levels of Pcna and Lyve1 tended to decrease in the psVEGFR-3 group. Immunofluorescence staining indicated that phospho-tyrosine expression was considerably lower in tumor cells of psVEGFR-3-treated mammary carcinomas than those of control tumors. Double immunofluorescence staining indicated that phospho-tyrosine+ /LYVE-1+ (a lymphatic vessel marker) tended to decrease in psVEGFR-3-treated mammary carcinomas compared with control mice, indicating a decline in the activity of the VEGF-C/VEGFR-3 axis. These findings showed that a blockade of VEGF-C/VEGFR-3 signaling caused by sVEGFR-3 sequestered VEGF-C and prevented the side-effects of anti-angiogenesis and suppressed overall metastases, suggesting their high clinical significance.

Urethane-induced Mammary Carcinogenesis Susceptibility in Transgenic Mice Expressing a Dominant-negative TGF-ß Type II Receptor.

BACKGROUND/AIM: Transforming growth factor-ß (TGF-ß) plays dual suppressive and oncogenic roles in mammary carcinogenesis. MATERIALS AND METHODS: To analyze whether TGF-ß exerts suppressive or oncogenic actions on mammary carcinogenesis, transgenic mice overexpressing a dominant-negative mutant type II TGF-ß receptor (TßRII-DNR) driven by the mouse mammary tumor virus (MMTV) promoter were treated with a low dose of urethane, a carcinogen present in fermented food products and alcoholic beverages. RESULTS: Lobular proliferative lesions, showing high ß-casein expression, developed in the mammary glands of TßRII-DNR+/+ mice aged >61 weeks. Compared with wild-type mice, TßRII-DNR+/+ mice administered with urethane showed significant increases in dysplastic hyperplasias and adenocarcinomas of the mammary glands. CONCLUSION: The functional decline of TGF-ß signaling in mammary glands led to a high susceptibility to urethane-induced mammary carcinogenesis. TGF-ß signaling may act as a tumor suppressor during mammary tumor development.

Serum exosomal microRNA-34a as a potential biomarker in epithelial ovarian cancer.

BACKGROUND: Ovarian cancer (OC) is a leading cause of cancer-related death in women, and thus an accurate diagnosis of the predisposition and its early detection is necessary. The aims of this study were to determine whether serum exosomal microRNA-34a (miR-34a) in ovarian cancer could be used as a potential biomarker. METHODS: Exosomes from OC patients' serum were collected, and exosomal miRNAs were extracted. The relative expression of miR-34a was calculated from 58 OC samples by quantitative real-time polymerase chain reaction. RESULTS: Serum exosomal miR-34a levels were significantly increased in early-stage OC patients compared with advanced-stage patients. Its levels were significantly lower in patients with lymph node metastasis than in those with no lymph node metastasis. Furthermore, its levels in the recurrence group were significantly lower than those in the recurrence-free group. CONCLUSIONS: Serum exosomal miR-34a could be a potential biomarker for improving the diagnostic efficiency of OC.

Hemophagocytic lymphohistiocytosis complicating invasive pneumococcal disease: a pediatric case report.

BACKGROUND: Hemophagocytic lymphohistiocytosis (HLH) is an infrequent but life-threatening disease due to excessive immune activation. Secondary HLH can be triggered by infections, autoimmune diseases, and malignant diseases. Streptococcus pneumoniae is a pathogenic bacterium responsible for invasive pneumococcal disease (IPD) such as meningitis and bacteremia. Although the pneumococcal conjugate vaccine (PCV) has led to reductions in IPD incidence, cases of IPD caused by serotypes not included in PCV are increasing. There are few reports of secondary HLH caused by IPD in previously healthy children. We herein report a rare case of a previously healthy boy with secondary HLH complicating IPD of serotype 23A, which is not included in the pneumococcal 13-valent conjugate vaccine (PCV-13). CASE PRESENTATION: An 11-month-old boy who had received three doses of PCV-13 was hospitalized with prolonged fever, bilateral otitis media, neutropenia and elevated C-reactive protein (CRP) levels. Blood culture on admission revealed S. pneumoniae, leading to a diagnosis of IPD. HLH was diagnosed based on a prolonged fever, neutropenia, anemia, hepatosplenomegaly, hemophagocytosis in the bone marrow, and elevated serum levels of triglycerides, ferritin, and soluble interleukin-2 receptor. He received broad-spectrum antibiotics and intravenous immunoglobulins for IPD and high-dose steroid pulse therapy and cyclosporine A for HLH; thereafter, his fever resolved, and laboratory findings improved. The serotype of the isolated S. pneumoniae was 23A, which is not included in PCV-13. CONCLUSIONS: It is important to consider secondary HLH as a complication of IPD cases with febrile cytopenia or hepatosplenomegaly, and appropriate treatment for HLH should be started without delay.

Interleukin-24 is a novel diagnostic biomarker for the severity of acute kidney injury.

There is a clinical need for sensitive acute kidney injury (AKI) biomarkers that enable early therapeutic interventions and prediction of disease prognosis. In this study, we monitored interleukin (IL)-24 expressed in kidneys with severe AKI that progresses to atrophic kidney in a mouse model of ischemia-reperfusion injury (IRI). Therefore, we evaluated IL-24 as a potential biomarker not only for early diagnosis of AKI, but also for predicting progression to chronic kidney disease (CKD). Serum IL-24 was detected earlier than the elevation of serum creatinine levels and urinary IL-24 was detected as early as neutrophil gelatinase associated lipocalin (NGAL) in severe AKI (60 min of IRI). In addition, serum and urine IL-24 levels tended to increase in relation to ischemia duration. In such kidneys, vascular smooth muscle cells expressed IL-24 in response to the injury in the renal tubular epithelial cell and its target was the renal tubular epithelial cell itself. IL-24 may play a pivotal role in the communication between tubular epithelial cells and vascular smooth muscle cells and, in conclusion, IL-24 can be used as a sensitive biomarker for AKI.

Dickkopf3 (Dkk3) is required for maintaining the integrity of secretory vesicles in the mouse adrenal medulla.

REIC (reduced expression in immortalized cells) has been identified as a gene whose expression was reduced in immortalized cultured cells. The REIC gene is identical to Dickkopf-3 (Dkk3), which encodes a secreted glycoprotein belonging to the Dkk family. Previously, we showed that Dkk3 protein is present in the mouse adrenal medulla. However, its role in this tissue has not been elucidated. To explore it, we performed electron microscopic (EM) studies and RNA-sequencing (RNA-seq) analysis on Dkk3-null adrenal glands. EM studies showed that the number of dense core secretory vesicles were significantly reduced and empty vesicles were increased in the medulla endocrine cells. Quantitative PCR (qPCR) analysis showed relative expression levels of chromogranin A (Chga) and neuropeptide Y (Npy) were slightly but significantly reduced in the Dkk3-null adrenal glands. From the result of RNA-seq analysis as a parallel study, we selected three of the downregulated genes, uncoupled protein-1 (Ucp1), growth arrest and DNA-damage-inducible 45 gamma (Gadd45g), and Junb with regard to the estimated expression levels. In situ hybridization confirmed that these genes were regionally expressed in the adrenal gland. However, expression levels of these three genes were not consistent as revealed by qPCR. Thus, Dkk3 maintains the integrity of secreting vesicles in mouse adrenal medulla by regulating the expression of Chga and Npy.