Tissue-Targeted Transcriptomics Reveals SEMA3D Control of Hypoglossal Nerve Projection to Mouse Tongue Primordia.

The mouse hypoglossal nerve originates in the occipital motor nuclei at embryonic day (E)10.5 and projects a long distance, reaching the vicinity of the tongue primordia, the lateral lingual swellings, at E11.5. However, the details of how the hypoglossal nerve correctly projects to the primordia are poorly understood. To investigate the molecular basis of hypoglossal nerve elongation, we used a novel transcriptomic approach using the ROKU method. The ROKU algorithm identified 3825 genes specific for lateral lingual swellings at E11.5, of which 34 genes were predicted to be involved in axon guidance. Ingenuity Pathway Analysis-assisted enrichment revealed activation of the semaphorin signaling pathway during tongue development, and quantitative PCR showed that the expressions of Sema3d and Nrp1 in this pathway peaked at E11.5. Immunohistochemistry detected NRP1 in the hypoglossal nerve and SEMA3D as tiny granules in the extracellular space beneath the epithelium of the tongue primordia and in lateral and anterior regions of the mandibular arch. Fewer SEMA3D granules were localized around hypoglossal nerve axons and in the space where they elongated. In developing tongue primordia, tissue-specific regulation of SEMA3D might control the route of hypoglossal nerve projection via its repulsive effect on NRP1.

The relationship between Meckel's cartilage resorption and incisor tooth germ in mice.

Our understanding of the initiation and cellular mechanisms underlying endochondral resorption of Meckel's cartilage (MC) remains limited. Several studies have shown that the resorption site of MC and the mandibular incisor tooth germ are located close to each other. However, whether incisor tooth germ development is involved in MC resorption remains unclear. In this study, we aimed to elucidate the spatio-temporal interaction between the initiation site of MC resorption and the development of incisor tooth germs in an embryonic mouse model. To this effect, we developed a histology-based three-dimensional (3D) reconstruction technique using paraffin-embedded serial sections of various tissues in the jaw. The serial sections were cut in the frontal section and the tissue constituents (e.g., MC, incisor, and mineralized mandible) were studied using conventional and enzyme-based histochemistry. The outline of each component was marked on the frontal sectional images and 3D structures were constructed. To assess the vascular architecture at the site of MC resorption, immunohistochemical staining using anti-laminin, anti-factor VIII, and anti-VEGF antibodies was performed. MC resorption was first observed on the lateral incisor-facing side of the cartilage rods at sites anterior to the mental foramen on E16.0. The 3D analysis suggested that: (a) the posterior region of the clastic cartilage resorption corresponds to the cervical loop of the incisor; (b) the cervical portion of the tooth germ inflates probably due to temporal cellular congestion prior to differentiation into matrix-producing cells; (c) the incisor tooth germ tissue is present in close proximity to MC even in mouse with continuously growing tooth and determines the disappearance of MC as the tooth development.

Nuclear factor 1 X-type-associated regulation of myogenesis in developing mouse tongue.

OBJECTIVES: The tongue contains skeletal myofibers that differ from those in the trunk, limbs, and other orofacial muscles. However, the molecular basis of myogenic differentiation in the tongue muscles remains unclear. In this study, we conducted comprehensive gene expression profiling of the developing murine tongue. METHODS: Tongue primordia were dissected from mouse embryos at embryonic day (E)10.5-E18.5, while myogenic markers were detected via microarray analysis and quantitative polymerase chain reaction (PCR). In addition to common myogenic regulatory factors such as Myf5, MyoD, myogenin, and Mrf4, we focused on Nfix, which acts as a unique molecular switch triggering the shift from embryonic to fetal myoblast lineage during limb myogenesis. Nfix inhibition was performed using a specific antisense oligonucleotide in the organ culture of tongue primordia. RESULTS: Microarray and ingenuity pathway analyses confirmed the significant upregulation of myogenic signaling molecules, including Nfix, associated with the differentiation of myoblasts from myogenic progenitor cells during E10.5-E11.5. Quantitative PCR confirmed that Nfix expression started at E10.5 and peaked at E14.5. Fetal myoblast-specific genes, such as Mck and Myh8, were upregulated after E14.5, whereas embryonic myoblast-specific genes, such as Myh3 and Myh7, were downregulated. When Nfix was inhibited in the organ culture of tongue primordia, subtle morphological differences were noted in the tongue. Such an observation was only noted in the cultures of E10.5-derived tongue primordia. CONCLUSIONS: These results reveal the contribution of Nfix to tongue myogenesis. Nfix expression during early tongue development may play a vital role in tongue muscle development.

Comparison of the incidence of depression before and after endovascular treatment in patients with lower limb peripheral artery disease.

Depression is a chronic illness that affects mood, physical health, and overall vitality and quality of life. Depression has been associated with an increased risk of all-cause and cardiovascular mortality among patients with peripheral arterial disease (PAD). Therefore, this study aimed to compare the incidence of depression before and after endovascular treatment in patients with lower limb PAD. This is an important clinical issue considering the worldwide increase in PAD with the aging population and the known negative impact of depression on recovery. This was a retrospective sub-analysis of data from the Tokyo Peripheral Vascular Intervention Study using the TOMA-CODE registry. The presence and extent of depressive symptoms were evaluated using the patient health questionnaire (PHQ-9), with a depressive tendency score of ≥ 5. The PHQ-9 score was evaluated before endovascular treatment (EVT) and at 4 (± 1) weeks after EVT. The study population consisted of 87 patients who completed the PHQ-9 before EVT, with 76 completing the post-EVT PHQ-9. Of these 76, 19 had a pre-EVT score ≥ 5. Overall, there was no difference in the pre- and post-EVT scores (P = 0.091). There was no significant change in the 19 patients with a pre-EVT score ≥ 5 (mean 9.2 ± 4.4); however, there was a tendency to improve in the pre- to post-EVT score (mean, 6.9 ± 5.2; P = 0.059). Diabetes was a significant negative factor for pre- to post-EVT score improvement (P = 0.023). Overall, symptoms of depression showed the tendency to improve at 30 days post-EVT. However, diabetes was associated with lower improvement in symptoms.

[The case of an octogenarian who died of acute myocardial infarction complicated with acute pulmonary embolism from a right ventricular thrombus].

An 86-year-old female on dialysis experienced a decrease in blood pressure and worsening of her respiratory condition during dialysis, for which she visited our emergency unit. She was admitted to our Department of Cardiology with a diagnosis of acute myocardial infarction complicated with heart failure because of anterior wall of left ventricular dysfunction, positive troponin T levels and negative T wave on a precordial lead electrocardiogram. On the same day, she underwent coronary angiography and stenting at left anterior descending artery #7 with 99% stenosis. She also showed an elevated D-dimer level on admission, and contrast-enhanced computed tomography (CT) was performed the day after admission, considering the likelihood of respiratory failure due to pulmonary thromboembolism. However, the findings were negative. On the 4th day of hospitalization, she showed marked hypoxemia. Her D-dimer level was further elevated, and when she underwent enhanced CT again, there was no evidence of deep vein thrombosis, but thrombus in the pulmonary artery and apex of right ventricle was noted. She was therefore diagnosed with acute pulmonary embolism due to thrombosis from the right ventricle rather than from a deep vein. She rapidly received anticoagulant therapy and non-invasive positive pressure ventilation therapy for respiratory failure, but she entered cardiopulmonary arrest and quickly died. She was suspected to have been complicated with a right ventricular infarction and an acute anterior wall myocardial infarction, resulting in a large thrombus along the apex of the right ventricle. This case of both myocardial infarction and pulmonary embolism is very rare, and we report it here with consideration.

CXCL12 promotes CCR7 ligand-mediated breast cancer cell invasion and migration toward lymphatic vessels.

Chemokines are a family of cytokines that mediate leukocyte trafficking and are involved in tumor cell migration, growth, and progression. Although there is emerging evidence that multiple chemokines are expressed in tumor tissues and that each chemokine induces receptor-mediated signaling, their collaboration to regulate tumor invasion and lymph node metastasis has not been fully elucidated. In this study, we examined the effect of CXCL12 on the CCR7-dependent signaling in MDA-MB-231 human breast cancer cells to determine the role of CXCL12 and CCR7 ligand chemokines in breast cancer metastasis to lymph nodes. CXCL12 enhanced the CCR7-dependent in vitro chemotaxis and cell invasion into collagen gels at suboptimal concentrations of CCL21. CXCL12 promoted CCR7 homodimer formation, ligand binding, CCR7 accumulation into membrane ruffles, and cell response at lower concentrations of CCL19. Immunohistochemistry of MDA-MB-231-derived xenograft tumors revealed that CXCL12 is primarily located in the pericellular matrix surrounding tumor cells, whereas the CCR7 ligand, CCL21, mainly associates with LYVE-1+ intratumoral and peritumoral lymphatic vessels. In the three-dimensional tumor invasion model with lymph networks, CXCL12 stimulation facilitates breast cancer cell migration to CCL21-reconstituted lymphatic networks. These results indicate that CXCL12/CXCR4 signaling promotes breast cancer cell migration and invasion toward CCR7 ligand-expressing intratumoral lymphatic vessels and supports CCR7 signaling associated with lymph node metastasis.

Skin Wound Healing of the Adult Newt, Cynops pyrrhogaster: A Unique Re-Epithelialization and Scarless Model.

In surgical and cosmetic studies, scarless regeneration is an ideal method to heal skin wounds. To study the technologies that enable scarless skin wound healing in medicine, animal models are useful. However, four-limbed vertebrates, including humans, generally lose their competency of scarless regeneration as they transit to their terrestrial life-stages through metamorphosis, hatching or birth. Therefore, animals that serve as a model for postnatal humans must be an exception to this rule, such as the newt. Here, we evaluated the adult newt in detail for the first time. Using a Japanese fire-bellied newt, Cynops pyrrhogaster, we excised the full-thickness skin at various locations on the body, and surveyed their re-epithelialization, granulation or dermal fibrosis, and recovery of texture and appendages as well as color (hue, tone and pattern) for more than two years. We found that the skin of adult newts eventually regenerated exceptionally well through unique processes of re-epithelialization and the absence of fibrotic scar formation, except for the dorsal-lateral to ventral skin whose unique color patterns never recovered. Color pattern is species-specific. Consequently, the adult C. pyrrhogaster provides an ideal model system for studies aimed at perfect skin wound healing and regeneration in postnatal humans.

Utility of Saline-Induced Resting Full-Cycle Ratio Compared with Resting Full-Cycle Ratio and Fractional Flow Reserve.

BACKGROUND: The saline-induced distal coronary pressure/aortic pressure ratio predicted fractional flow reserve (FFR). The resting full-cycle ratio (RFR) represents the maximal relative pressure difference in a cardiac cycle. Therefore, the present study aimed to compare the results of saline-induced RFR (sRFR) with FFR. METHODS: Seventy consecutive lesions with only moderate stenosis were included. The FFR, RFR, and sRFR values were compared. The sRFR was assessed using an intracoronary bolus infusion of saline (2 mL/s) for five heartbeats. The FFR was obtained after an intravenous injection of papaverine. RESULTS: Overall, the FFR, sRFR, and RFR values were 0.78 ± 0.12, 0.79 ± 0.13, and 0.83 ± 0.14, respectively. With regard to anatomical morphology were 40, 18, and 12 cases of focal, diffuse, and tandem lesion. There was a significant correlation between the sRFR and FFR (R = 0.96, p < 0.01). There were also significant correlations between the sRFR and FFR in the left coronary and right coronary artery (R = 0.95, p < 0.01 and R = 0.98, p < 0.01). Furthermore, significant correlations between sRFR and FFR were observed in not only focal but also in nonfocal lesion including tandem and diffuse lesions (R = 0.93, p < 0.01 and R = 0.97, p < 0.01). A close agreement on FFR and sRFR was shown using the Bland-Altman analysis (95% CI of agreement: -0.08-0.07). In the receiver operating characteristic curve analysis, the cutoff value of sRFR to predict an FFR of 0.80 was 0.81 (area under curve, 0.97; sensitivity 90.6%; and specificity 98.2%). CONCLUSION: The sRFR can accurately and safely predict the FFR and might be effective for diagnosing ischemia.

The Determinants and Outcomes of Myocardial Injury After Transcatheter Aortic-Valve Implantation: SAPIEN 3 Study.

BACKGROUND: The effect of myocardial injury (MI) post-transcatheter aortic valve implantation (TAVI) on clinical outcomes is controversial. This study aimed to evaluate the effect of MI severity on clinical outcome and left ventricle function 30 days post-TAVI and determine MI post-TAVI predictors. METHODS: Overall, 138 consecutive patients who underwent successful transfemoral TAVI using SAPIEN3 and diagnosed using echocardiography and computed tomography were analyzed. High-sensitivity cardiac troponin T (TnT) was evaluated at baseline, immediately, and at 24, 48, and 72 h post-TAVI. Echocardiography findings and N-terminal pro-B-type natriuretic peptide (Nt-pro BNP) levels were evaluated 30 days post-TAVI. RESULTS: Mean age and STS score were 84.4 ±â€¯3.5 years and 6.4 ±â€¯3.2%, respectively. All cases showed severe aortic valve stenosis. Peri-procedural MI was observed in 48 of 100 patients (48.0%). Patients were grouped into MI (n = 48) and non-MI (n = 52), without significant difference in characteristics. Pre-balloon aortic valvuloplasty rate and total pacing time were significantly higher in MI vs non-MI. Total rapid pacing time (TRPT) was an independent predictor for MI (OR 1.06; 95% CI 1.01-1.16; p = 0.04). Echocardiography and Nt-pro BNP changes 30 days post-TAVI were similar between groups. CONCLUSION: Peri-procedural MI, assessed by TnT changes, was observed in 48% of patients. The MI was not associated with overt cardiac dysfunction, and the recovery of left ventricular function and Nt-pro BNP level occurred similarly by 30 day post-TAVI between both groups. In multivariate analysis, TRPT was associated with MI after SAPIEN3 implantation. TRIAL REGISTRATION NUMBER: UMIN000036669.

The effect of the debulking by excimer laser coronary angioplasty on long-term outcome compared with drug-coating balloon: insights from optical frequency domain imaging analysis.

This study evaluated the 1-year efficacy of excimer laser coronary angioplasty (ELCA) before drug-coated balloon (DCB) dilatation for the treatment of in-stent restenosis (ISR). Forty consecutive patients with ISR were treated by DCB with or without the use of ELCA (ELCA plus DCB, N = 20; DCB alone, N = 20). Debulking efficiency (DE) value was defined as the neointima area on optical frequency domain imaging (OFDI) debulked by ELCA. The patients in the ELCA plus DCB group were divided into two groups (greater DE (GDE), N = 10; smaller DE (SDE), N = 10) based on the median value of DE. Thereafter, the ISR segment was prepared with a scoring balloon, followed by DCB. At follow-up, binary restenosis and target lesion revascularization (TLR) were evaluated. There were no significant differences in baseline characteristics such as age, comorbidity, and ISR type. Overall, the incidence of neoatherosclerosis in the ISR segment was 17.5%. Post-PCI, acute gain of minimum lumen diameter on quantitative coronary angiography and of minimum lumen area on OFDI was numerically higher in the GDE than in the SDE and the DCB alone group. At follow-up, the occurrences of binary restenosis and TLR in the ELCA plus DCB group were 20.0% and 10.0%; these values in the DCB alone group were 20.0% and 20.0%, respectively. Two patients from the SDE and none from the GDE developed TLR. DCB alone treatment was inferior to ELCA plus DCB treatment. However, greater ELCA debulking might be required to obtain optimal outcomes.

Dynamic microstructural changes in alveolar bone in ligature-induced experimental periodontitis.

Periodontitis is an inflammatory disease that causes bone resorption. This study used a ligature-induced experimental periodontitis model to observe the kinetic process of microstructural changes in alveolar bone and introduced star volume analysis as a new methodology to assess microstructural changes. Thirty Wistar rats were used. To induce experimental periodontitis, ligatures were placed around the maxillary first molar. Rats were euthanized on days 0, 1, 3, 7, 14, and 28 after ligature placement. In addition to using hematoxylin and eosin staining, tartrate-resistant acid phosphatase (TRAP)/alkaline phosphatase (ALP) double staining, and micro-computed tomography were performed to analyze bone remodeling. From day 0 to day 7 (initiation phase), the model showed predominant inflammation with increased numbers of TRAP-positive cells, while ALP expression decreased. In contrast, from day 14 to day 28 (resolution phase), inflammatory cells and TRAP-positive cells decreased, whereas ALP expression recovered to levels comparable to that on day 0. Regarding microstructure parameters, in the initiation phase, bone volume fraction, bone mineral density, trabecular thickness, and star volume of the trabeculae decreased significantly, whereas trabecular separation and star volume of the marrow space increased significantly, indicating bone resorption. In the resolution phase, microstructure parameters normalized, indicated bone formation. We confirmed dynamic alveolar bone remodeling in ligature-induced periodontitis in rats. Furthermore, we assessed the potential for using star volume analysis as a sensitive new tool to clarify microstructural changes to alveolar bone in this model.

The comparison of early healing 1-month after PCI among CoCr-everolimus-eluting stent (EES), biodegradable polymer (BP)-EES and BP-sirolimus-eluting stent: Insights from OFDI and coronary angioscopy.

BACKGROUND: Third-generation stents with abluminal biodegradable polymer (BP) might facilitate early healing. Therefore, we compared early healing between second-generation and third-generation stents using coronary angioscopy (CAS) and optical frequency domain imaging [OFDI]. METHODS: We prospectively enrolled 30 consecutive patients with stent implantation for acute coronary syndrome (cobalt­chromium [CoCr] everolimus-eluting stent [EES] [n = 10], BP-EES [n = 10], and BP-sirolimus eluting stent [SES] [n = 10]). All patients underwent CAS and OFDI 1 month after initial percutaneous coronary intervention. On OFDI, the stent coverage (SC), thrombus, and peri-strut low intensity area (PLIA) were assessed. CAS findings were recorded for the grade of SC, grade of yellow color (YC), and grade of the thrombus (TG). RESULTS: On OFDI, the incidences of any thrombus at the 1-month follow-up were 70%, 80%, and 80% in the CoCr-EES, BP-EES, and BP-SES groups, respectively. The percentage of coverage was comparable among the groups (CoCr-EES 79.8 vs. BP-EES 79.9 vs. BP-SES 80.1%, P = 0.96). However, the number of struts with PLIA was numerically higher in the BP-SES group than in the CoCr-EES and BP-EES groups (46.4 ±â€¯25.1 vs. 21.6 ±â€¯13.2 vs. 22.0 ±â€¯7.2%, P = 0.08). In the CoCr-EES, BP-EES, and BP-SES groups, mean grades of SC were 1.25 ±â€¯0.5, 1.25 ±â€¯0.5, and 0.85 ±â€¯0.70 (P = 0.60); mean grades of YC were 0.75 ±â€¯0.5, 0.80 ±â€¯0.45, and 0.88 ±â€¯0.37 (P = 0.65), and mean grades of TG were 1.00 ±â€¯1.00, 1.20 ±â€¯0.83, and 0.88 ±â€¯0.64 (P = 0.75), respectively. CONCLUSION: Third-generation stents are not inferior to second-generation stents regarding stent coverage. However, PLIA on OFDI was often observed with BP-SESs, indicating involvement of the fibrin component.

Effects of theaflavins on tissue inflammation and bone resorption on experimental periodontitis in rats.

BACKGROUND AND OBJECTIVE: Theaflavins (TFs), the major polyphenol in black tea, have the ability to reduce inflammation and bone resorption. The aim of this study was to evaluate the effects of TFs on experimental periodontitis in rats. MATERIAL AND METHODS: Thirty rats were divided into five groups: Control (glycerol application without ligation), Ligature (glycerol application with ligation), TF1 (1 mg/mL TF application with ligation), TF10 (10 mg/mL TF application with ligation), and TF100 (100 mg/mL TF application with ligation). To induce experimental periodontitis, ligatures were placed around maxillary first molars bilaterally. After ligature placement, 100 µL glycerol or TFs were topically applied to the rats daily, and rats were euthanized 7 days after ligature placement. Micro-computed tomography was used to measure bone resorption in the left side of the maxilla, and quantitative polymerase chain reaction was used to measure the expression of interleukin (IL)-6, growth-regulated gene product/cytokine-induced neutrophil chemoattractant (Gro/Cinc-1, rat equivalent of IL-8), matrix metalloproteinase-9 (Mmp-9), receptor activator of nuclear factor-kappa Β ligand (Rankl), osteoprotegerin (Opg), and the Rankl/Opg ratio in gingival tissue. With tissue from the right side of the maxilla, hematoxylin and eosin staining was used for histological analysis, immunohistochemical staining for leukocyte common antigen (CD45) was used to assess inflammation, and tartrate-resistant acid phosphatase (TRAP) staining was used to observe the number of osteoclasts. RESULTS: The TF10 and TF100 groups, but not the TF1 group, had significant inhibition of alveolar bone loss, reduction in inflammatory cell infiltration in the periodontium, and significantly reduced numbers of CD45-positive cells and TRAP-positive osteoclasts compared with the Ligature group. Correspondingly, the TF10 and TF100 groups had significantly downregulated gene expression of IL-6, Gro/Cinc-1(IL-8), Mmp-9, and Rankl, but not of Opg. Consequently, Rankl/Opg expression was significantly increased in the Ligation group but was attenuated in the TF10 and TF100 groups. CONCLUSION: The results of this study suggest that topical application of TFs may reduce inflammation and bone resorption in experimental periodontitis. Therefore, TFs have therapeutic potential in the treatment of periodontal disease.

Migration of lymphatic endothelial cells and lymphatic vascular development in the craniofacial region of embryonic mice.

Lymphatic development in mice is initiated in the trunk at embryonic day (E) 9.5. This study aimed to examine the origin of craniofacial lymphatic endothelial cells (LECs) and the developmental process of lymphatic vessels in the mouse craniofacial region. Serial sections from ICR mouse embryos at E9.5-E14.5 were immunolabeled with LEC and venous endothelial cell (VEC) markers. These markers included prospero homeobox protein 1 (Prox1), vascular endothelial growth factor receptor 3 (Vegfr3), lymphatic vessel endothelial hyaluronan receptor 1 (Lyve1), and C-C motif chemokine 2 (Ccl21) for LEC, and COUP transcription factor 2 (CoupTF2) and endomucin (Emcn) for VEC. LECs were monitored as an index in Prox1/Vegfr3 double-positive cells using three-dimensional analysis because LECs express Prox1 and Vegfr3 ab initio during lymphatic vascular development. LECs appeared in VECs of the lateral walls of cardinal veins (CVs) at E9.5. These LECs were dichotomized into LEC populations that formed lymph sacs close to CVs and were scattered in the surrounding CVs. The scattered LECs formed cellular streams and extended from the trunk to the mandibular arches at E10.5 - E11.5. In the mandibular arches, individual LECs aggregated, and formed lymph sacs and tubular lymphatic vessels at E11.5-E14.5. Expression of the LEC marker proteins Lyve1 and Ccl21 in LECs changed during craniofacial lymphatic vascular development. Collectively, these findings suggest that craniofacial LECs originate from CVs of the trunk and migrate into the mandibular arches. Additionally, we found that craniofacial lymphatic vessels are formed according to morphogenesis of individual LECs that migrate from CVs.

The utility of total lipid core burden index/maximal lipid core burden index ratio within the culprit plaque to predict filter-no reflow: insight from near-infrared spectroscopy with intravascular ultrasound.

Filter-no reflow (FNR) is a phenomenon wherein flow improves after the retrieve of distal protection. Near-infrared spectroscopy with intravascular ultrasound (NIRS-IVUS) enables lipid detection. We evaluated the predictors of FNR during PCI using NIRS-IVUS. Thirty-two patients who underwent PCI using the Filtrap® for acute coronary syndrome (ACS) were enrolled. The culprit plaque (CP) was observed using NIRS-IVUS. Total lipid-core burden index (T-LCBI) and maximal LCBI over any 4-mm segment (max-LCBI4mm) within CP were evaluated. T-LCBI/max-LCBI4mm ratio within CP was calculated as an index of the extent of longitudinal lipid expansion. The attenuation grade (AG) and remodeling index (RI) in CP were analyzed. AG was scored based on the extent of attenuation occupying the number of quadrants. The patients were divided into FNR group (N = 8) and no-FNR group (N = 24). AG was significantly higher in FNR group than in no-FNR group (1.6 ± 0.6 vs. 0.9 ± 0.42, p = 0.01). RI in FNR group tended to be greater than in no-FNR group. T-LCBI/max-LCBI4mm ratio within the culprit plaque was significantly higher in FNR group than in no-FNR group (0.50 ± 0.10 vs. 0.33 ± 0.13, p < 0.01). In multivariate logistic regression analysis, AG > 1.04 (odds ratio [OR] 18.4, 95% confidence interval [CI] 1.5-215.7, p = 0.02) and T-LCBI/max-LCBI4mm ratio > 0.42 (OR 14.4, 95% CI 1.2-176.8, p = 0.03) were independent predictors for the occurrence of FNR. The use of T-LCBI/max-LCBI4mm ratio within CP might be an effective marker to predict FNR during PCI in patients with ACS.

Embryonic tongue morphogenesis in an organ culture model of mouse mandibular arches: blocking Sonic hedgehog signaling leads to microglossia.

Mouse tongue development is initiated with the formation of lateral lingual swellings just before fusion between the mediodorsal surfaces of the mandibular arches at around embryonic day 11.0. Here, we investigated the role of Sonic hedgehog (Shh) signaling in embryonic mouse tongue morphogenesis. For this, we used an organ culture model of the mandibular arches from mouse embryos at embryonic day 10.5. When the Shh signaling inhibitor jervine was added to the culture medium for 24-96 h, the formation of lateral lingual swellings and subsequent epithelial invagination into the mesenchyme were impaired markedly, leading to a hypoplastic tongue with an incomplete oral sulcus. Notably, jervine treatment reduced the proliferation of non-myogenic mesenchymal cells at the onset of forming the lateral lingual swellings, whereas it did not affect the proliferation and differentiation of a myogenic cell lineage, which created a cell community at the central circumferential region of the lateral lingual swellings as seen in vivo and in control cultures lacking the inhibitor. Thus, epithelium-derived Shh signaling stimulates the proliferation of non-myogenic mesenchymal cells essential for forming lateral lingual swellings and contributes to epithelial invagination into the mesenchyme during early tongue development.

Three-Dimensional Visualization of Developing Neurovascular Architecture in the Craniofacial Region of Embryonic Mice.

Recent studies have highlighted the mechanism of vascular and axonal guidance to ensure proper morphogenesis and organogenesis. We aimed to perform global mapping of developing neurovascular networks during craniofacial development of embryonic mice. To this end, we developed histology-based three-dimensional (3D) reconstructions using paraffin-embedded serial sections obtained from mouse embryos. All serial sections were dual-immunolabeled with Pecam1 and Pgp9.5/Gap43 cocktail antibodies. All immunolabeled serial sections were digitized with virtual microscopy to acquire high spatial resolution images. The 3D reconstructs warranted superior positional accuracy to trace the long-range connectivity of blood vessels and individual cranial nerve axons. It was feasible to depict simultaneously the details of angiogenic sprouting and axon terminal arborization and to assess quantitatively the locoregional proximity between blood vessels and cranial nerve axons. Notably, 3D views of the craniofacial region revealed the following: Branchial arch arteries and blood capillary plexi were formed without accompanying nerves at embryonic day (E) 9.5. Cranial nerve axons began to grow into the branchial arches, developing a labyrinth of small blood vessels at E10.5. Vascular remodeling occurred, and axon terminals of the maxillary, mandibular, chorda tympani, and hypoglossal nerve axons had arborized around the lateral lingual swellings at E11.5. The diverged patterning of trigeminal nerves and the arterial branches from the carotid artery became congruent at E11.5. The overall results support the advantage of dual-immunolabeling and 3D reconstruction technology to document the architecture and wiring of the developing neurovascular networks in mouse embryos.

Heterogeneous tumor stromal microenvironments of oral squamous cell carcinoma cells in tongue and nodal metastatic lesions in a xenograft mouse model.

BACKGROUND: Oral squamous cell carcinoma exhibits a poor prognosis, caused by aggressive progression and early-stage metastasis to cervical lymph nodes. Here, we developed a xenograft mouse model to explore the heterogeneity of the tumor microenvironment that may govern local invasion and nodal metastasis of tumor cells. METHODS: We transplanted five oral carcinoma cell lines into the tongues of nude mice and determined tongue tumor growth and micrometastatic dissemination by serially sectioning the tongue and lymph node lesions in combination with immunohistochemistry and computer-assisted image analysis. Our morphometric analysis enabled a quantitative assessment of blood and lymphatic endothelial densities in the intratumoral and host stromal regions. RESULTS: All cell lines tested were tumorigenic in mouse tongue. The metastatic lesion-derived carcinoma cell lines (OSC19, OSC20, and HSC2) yielded a 100% nodal metastasis rate, whereas the primary tumor-derived cell lines (KOSC2 and HO-1-u-1) showed <40% metastatic potential. Immunohistochemistry showed that the individual cell lines gave rise to heterogeneous tumor architecture and phenotypes and that their micrometastatic lesions assimilated the immunophenotypic properties of the corresponding tongue tumors. Notably, OSC19 and OSC20 cells shared similar aggressive tumorigenicity in both the tongue and lymph node environments but displayed markedly diverse immunophenotypes and gene expression profiles. CONCLUSIONS: Our model facilitated comparing the tumor microenvironments in tongue and lymph node lesions. The results support that tumorigenicity and tumor architecture in the host tongue environment depend on the origin and properties of the carcinoma cell lines and that metastatic progression may take place through heterogeneous tumor-host interactions.

Molecular contribution to cleft palate production in cleft lip mice.

Cleft palate following cleft lip may include a developmental disorder during palatogenesis. CL/Fr mice fetuses, which develop cleft lip and palate spontaneously, have less capability for in vivo cell proliferation in palatal mesenchyme compared with CL/Fr normal fetuses. In order to know the changes of signaling molecules contributing to cleft palate morphogenesis following cleft lip, the mRNA expression profiles were compared in palatal shelves oriented vertically (before elevation) in CL/Fr fetuses with or without cleft lip. The changes in mRNA profile of cleft palate morphogenesis were presented in a microarray analysis, and genes were restricted to lists contributing to cleft palate development in CL/Fr fetuses with cleft lip. Four candidate genes (Ywhab, Nek2, Tacc1 and Frk) were linked in a gene network that associates with cell proliferation (cell cycle, MAPK, Wnt and Tgf beta pathways). Quantitative real-time RT-PCR highlighted the candidate genes that significantly changed in CL/Fr fetuses with cleft lip (Ywhab, Nek2 and Tacc1). The results of these molecular contributions will provide useful information for a better understanding of palatogenesis in cleft palate following cleft lip. Our data indicated the genetic contribution to cleft palate morphogenesis following cleft lip.

Three-dimensional reconstruction of oral tongue squamous cell carcinoma at invasion front.

We conducted three-dimensional (3D) reconstruction of oral tongue squamous cell carcinoma (OTSCC) using serial histological sections to visualize the architecture of invasive tumors. Fourteen OTSCC cases were collected from archival paraffin-embedded specimens. Based on a pathodiagnostic survey of whole cancer lesions, a core tissue specimen (3 mm in diameter) was dissected out from the deep invasion front using a paraffin tissue microarray. Serial sections (4 µ m thick) were double immunostained with pan-cytokeratin and Ki67 antibodies and digitized images were acquired using virtual microscopy. For 3D reconstruction, image registration and RGB color segmentation were automated using ImageJ software to avoid operator-dependent subjective errors. Based on the 3D tumor architecture, we classified the mode of invasion into four types: pushing and bulky architecture; trabecular architecture; diffuse spreading; and special forms. Direct visualization and quantitative assessment of the parenchymal-stromal border provide a new dimension in our understanding of OTSCC architecture. These 3D morphometric analyses also ascertained that cell invasion (individually and collectively) occurs at the deep invasive front of the OTSCC. These results demonstrate the advantages of histology-based 3D reconstruction for evaluating tumor architecture and its potential for a wide range of applications.