Mount Immunofluorescence Staining Research Articles

Ontogenesis of the Mouse Ocular Surface Lymphatic Vascular Network.

Ocular lymphatic vessels play major physiological role in eye homeostasis and their dysfunction can contribute to the progression of several eye diseases. In this study, we characterized their spatiotemporal development and the cellular mechanisms occurring during their ontogenesis in the mouse eye. Whole mount immunofluorescent staining and imaging by standard or lightsheet fluorescence microscopy were performed on late embryonic and early postnatal eye mouse samples. We observed that the ocular surface lymphatic vascular network develops at the early postnatal stages (between P0 and P5) from two nascent trunks arising at the nasal side on both sides of the nictitating membrane. These nascent vessels further branch and encircle the whole eye surface by sprouting lymphangiogenesis. In addition, we got evidence for the existence of a transient lymphvasculogenesis process generating lymphatic vessel fragments that will mostly formed the corneolimbal lymphatic vasculature which further connect to the conjunctival lymphatic network. Our results also support that CD206-positive macrophages can transdifferentiate and then integrate into the lymphatic neovessels. Several complementary cellular processes participate in the development of the lymphatic ocular surface vasculature. This knowledge paves the way for the design of new therapeutic strategies to interfere with ocular lymphatic vessel formation when needed.

ErbB3 Control of Pmp22 Regulates Intestinal Barrier Function

Background: ErbB3 is a member of the ErbB/EGFR family of receptor tyrosine kinases and is the constitutively-expressed neuregulin (NRG) receptor in the intestinal epithelium. Pathways downstream of ErbB3 have been implicated in regulating tight junctions in other systems, but a role for ErbB3 in barrier function in the intestines has not been described. Here we tested the hypothesis that ErbB3 is required for maintenance of normal barrier function in the intestine. Methods: We generated Villin-Cre;ErbB3 flox/flox mice (ErbB3-IEKO) with ErbB3 deletion in the intestinal epithelium. All comparisons are to ErbB3 flox/flox littermate controls. Mice were given a FITC-dextran 4 kDa (FD4) gavage to assess intestinal permeability. Isolated epithelial ileal samples were collected for RT-qPCR, immunohistochemistry, and bulk RNA-seq analysis. Enteroids were generated and treated with the ErbB3 ligand NRG-1β as well as PD153035 and CP-724714 to inhibit EGFR and ErbB2, respectively. Enteroids were also fixed and stained for whole mount immunofluorescence staining. Caco-2BBe cells were grown on Transwell filters, treated with NRG-1β, and assessed for transepithelial electrical resistance (TEER) and FD4 flux. Results: ErbB3-IEKO mice displayed increased intestinal barrier permeability to FD4 compared to controls (p < 0.01). RNA-seq analysis showed a decrease in the expression of the putative tight junction protein Pmp22 in ErbB3-IEKO mice, while other tight junctional components were unchanged. RT-qPCR analysis of ileal samples showed an 80% Pmp22 downregulation (p < 0.01) with ErbB3 loss compared to controls, with similar results in ErbB3-deficient enteroids. Immunohistochemical analysis confirmed near complete loss of PMP22 in ErbB3-IEKO intestinal epithelium. In vitro, treatment with NRG-1β induced Pmp22 expression (p < 0.001) in control, but not ErbB3-deficient, enteroids. Treatment with PD153035 blocked NRG-1β-driven expression of Pmp22 while treatment with CP-724714 did not, suggesting that EGFR is a critical ErbB3 heterodimer partner in regulating Pmp22. Whole mount staining of enteroids confirmed co-localization of PMP22 with the tight junction protein ZO-1 at the apical junctions. Furthermore, treatment of Caco-2BBe monolayers with NRG-1β increased TEER and reduced FD4 flux, demonstrating active regulation of permeability. Conclusions: We demonstrate that loss of ErbB3 in the intestinal epithelium leads to reduced expression of the tight junction protein PMP22 and impaired barrier function in mice. Co-localization of PMP22 with ZO-1 suggests a role for PMP22 in forming the barrier and may represent a mechanism for regulation of intestinal permeability through the ErbB3-PMP22 axis. Furthermore, increased TEER in Caco-2BBe monolayers following NRG-1β treatment suggests induction of PMP22 may represent a means to enhance barrier function and a potential target for future therapeutic interventions targeting the barrier. Supported by NIH awards R01DK095004 and F31DK131856 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MP27-12 FUNCTIONAL ERECTOGENIC NERVE MAPPING OF THE ENTIRE RAT PROSTATE WITH OPTICAL CLEARING

You have accessJournal of UrologyCME1 Apr 2023MP27-12 FUNCTIONAL ERECTOGENIC NERVE MAPPING OF THE ENTIRE RAT PROSTATE WITH OPTICAL CLEARING Selman Unal, Biljana Musicki, Ahmet Hoke, and Arthur Burnett Selman UnalSelman Unal More articles by this author , Biljana MusickiBiljana Musicki More articles by this author , Ahmet HokeAhmet Hoke More articles by this author , and Arthur BurnettArthur Burnett More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003255.12AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Erectile dysfunction is an important complication of radical prostatectomy (RP) due to cavernous nerve (CN) injury, which is in part a result of the unknown exact distribution of the CN branches (CNBs) on the prostate. We hypothesized that preserving functional pro-erectile CNBs promotes the success of retaining erectile function after RP. The aim of this study was to determine the functional erectogenic nerve distribution surrounding the entire prostate using a rat model. METHODS: Submaximal electrical stimulation of the CN was performed in male rats to induce penile erection by stimulating nNOS phosphorylation on Ser-1412 (functional nNOS). For localization of the P-nNOS (Ser-1412)-containing and nNOS-containing nerves, whole mount immunofluorescence staining was performed on the prostate, using ß-III-tubulin, a non-specific nerve marker, as control. Furthermore, the staining was combined with optical clearing with benzylalcohol/benzylbenzoate (BABB), which removes lipids and minimizes light scattering, allowing imaging of the tissue in situ. Because the BABB clearing technique has not been used previously for the CN or the whole prostate, we first modified it by optimizing stringency of lipid removal. RESULTS: We achieved optimum staining signal and prostate transparency by modification of BABB clearing technique to include shortening dehydration to prevent fluorescence quenching, followed by incubation with tert-butanol and BABB (1:2) at decreased acidity for 1.5 hour (Figure 1a). Microscopic examination localized nNOS and P-nNOS (Ser-1412)-containing cell bodies in the major pelvic ganglia, CN, and CNBs on the prostate. All nerves stained with nNOS/P-nNOS were also stained with ß-III-tubulin (Figure 1b, c). While there was no background noise in ß-III-tubulin staining, a minor signal of the nNOS and P-nNOS (Ser-1412) staining was observed in other tissues such as vascular smooth muscles. CONCLUSIONS: This study presents the first mapping of the pro-erectile nerves, in contrast to all nerves, surrounding the anatomically preserved whole rat prostate. This approach allows a dimensional shift from 2D to 3D histology of the whole intact prostate, providing further information about the distribution of pro-erectile CNBs in penile erection. Source of Funding: This study was supported by the Patrick C. Walsh Prostate Cancer Research Grant. Selman Unal was supported by The Scientific and Technological Research Council of Turkey (TÜBİTAK) for this study © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e366 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Selman Unal More articles by this author Biljana Musicki More articles by this author Ahmet Hoke More articles by this author Arthur Burnett More articles by this author Expand All Advertisement PDF downloadLoading ...

Effect of Dual Endothelin Receptor Antagonist on a Retinal Degeneration Animal Model by Regulating Choroidal Microvascular Morphology.

Objective Clinical studies have found that increasing levels of plasma endothelin-1 (ET-1) might inhibit choroidal blood flow (BF) and promote choroidal vasoconstriction. This study was designed to investigate ET-1 levels and its effect on choroidal microvascular morphology in a retinitis pigmentosa (RP) animal model. Methods Mice with retinal degeneration (rd10) were intragastrically administered bosentan, a dual endothelin receptor antagonist. We detected plasma ET-1 levels using an enzyme-linked immunosorbent assay (ELISA) kit at P14, P21, and P28 and evaluated ET-1 expression in RPE/choroid/sclera complexes using western blot and whole mount immunofluorescence staining at P28. Retinal thickness was measured using hematoxylin and eosin (H&E) staining at P28. At the same time, we also estimated choroidal microvascular densities using vascular luminal casting with a scanning electron microscope (SEM). Results Plasma ET-1 levels were increased significantly in rd10 mice at P21 (65.48 ± 24.83 pg/ml) and P28 (85.89 ± 20.23 pg/ml) compared with C57BL/6J mice at P21 (33.52 ± 16.33 pg/ml) and P28 (42.38 ± 17.53 pg/ml); the expression of ET-1 was also upregulated in RPE/choroid/sclera complexes at P28. Bosentan inhibited ET-1 expression in plasma (P < 0.05) and RPE/choroid/sclera complexes at P28 in rd10 mice. Choroidal microvascular densities were decreased in rd10 mice, and bosentan could weaken these changes. Conclusion Plasma and local ET-1 was elevated in an animal model of RP, suggesting that it likely participates in the pathological progression of retinal degeneration and may thus provide a new intervention target. ET-1 blockade might exert its protective effect by elevating choroidal microvascular density via inhibition of ET-1.

The interaction of lead exposure and CCM3 defect plays an important role in regulating angiogenesis through eNOS/NO pathway

In this study, we aimed to explore the role of nitric oxide (NO) in regulating angiogenesis in cerebral cavernous malformations 3 gene (CCM3)-deficient mice exposed to lead during vascular development; further, we aimed to identify and study the potential mechanism involved as well. Angiogenesis was detected by whole mount immunofluorescent staining of retinal vessels in WT and CCM3+/− mice. Brain microvascular endothelial cells (BMECs) isolated from WT and CCM3+/− mice, primary HUVECs, and immortalized HUVECs (imHUVECs) (CCM3+/+ and CCM3-/-) were used and treated with lead acetate (PbAc). RT-PCR and Western blotting were used to detect the mRNA and protein expression of iNOS, eNOS, and VEGF genes. The results showed that both lead exposure and CCM3 gene deficiency adversely affected endothelial cell function, causing abnormal angiogenesis and vascular remodeling. The mRNA expression of eNOS and iNOS was significantly different in WT and CCM3+/− BMECs (0.04 ± 0.001 vs. 0.016 ± 0.002; 0.26 ± 0.002 vs. 0.306 ± 0.002, respectively), and the expression of eNOS and iNOS in imHUVECs (CCM3+/+ and CCM3-/-) also increased after PbAc exposure. In conclusion, CCM3 gene-deficient mice were more susceptible to abnormal vascular development after low-level lead exposure, probably due to the release of NO.

Participation of EML6 in the regulation of oocyte meiotic progression in mice.

The generation of a high-quality egg for reproduction requires faithful segregation of chromosome during oocyte meiosis. Here, we report that echinoderm microtubule-associated protein like 6 (EML6) is highly expressed in oocytes, and responsible for accurate segregation of homologous chromosomes in mice. Quantitative real-time RT-PCR and immunohistochemistry analyses revealed that EML6 was predominantly expressed by oocytes in the ovaries. Whole mount immunofluorescent staining showed that EML6 was colocalized with spindle microtubules in oocytes at various stages after meiotic resumption. This specialized localization was disrupted by nocodazole, the microtubule destabilizer, while enhanced by Taxol, a microtubule stabilizing reagent. In vivo knockdown of Eml6 expression by the specific siRNA resulted in chromosome misalignment and alteration in spindle dimension at both metaphase Ⅰ and Ⅱ stages, as well as the increased aneuploidy in the mature oocytes. Thus, these data suggest that EML family proteins participate in the control of oocyte meiotic division.

Vascular endothelial growth factor pathway promotes osseointegration and CD31hiEMCNhi endothelium expansion in a mouse tibial implant model: an animal study.

It is increasingly appreciated that coordinated regulation of angiogenesis and osteogenesis is needed for bone formation. How this regulation is achieved during peri-implant bone healing, such as osseointegration, is largely unclear. This study examined the relationship between angiogenesis and osteogenesis in a unique model of osseointegration of a mouse tibial implant by pharmacologically blocking the vascular endothelial growth factor (VEGF) pathway. An implant was inserted into the right tibia of 16-week-old female C57BL/6 mice (n = 38). Mice received anti-VEGF receptor-1 (VEGFR-1) antibody (25 mg/kg) and VEGF receptor-2 (VEGFR-2) antibody (25 mg/kg; n = 19) or an isotype control antibody (n = 19). Flow cytometric (n = 4/group) and immunofluorescent (n = 3/group) analyses were performed at two weeks post-implantation to detect the distribution and density of CD31hiEMCNhi endothelium. RNA sequencing analysis was performed using sorted CD31hiEMCNhi endothelial cells (n = 2/group). Osteoblast lineage cells expressing osterix (OSX) and osteopontin (OPN) were also detected with immunofluorescence. Mechanical pull-out testing (n = 12/group) was used at four weeks post-implantation to determine the strength of the bone-implant interface. After pull-out testing, the tissue attached to the implant surface was harvested. Whole mount immunofluorescent staining of OSX and OPN was performed to determine the amount of osteoblast lineage cells. Flow cytometry revealed that anti-VEGFR treatment decreased CD31hiEMCNhi vascular endothelium in the peri-implant bone versus controls at two weeks post-implantation. This was confirmed by the decrease of CD31 and endomucin (EMCN) double-positive cells detected with immunofluorescence. In addition, treated mice had more OPN-positive cells in both peri-implant bone and tissue on the implant surface at two weeks and four weeks, respectively. More OSX-positive cells were present in peri-implant bone at two weeks. More importantly, anti-VEGFR treatment decreased the maximum load of pull-out testing compared with the control. VEGF pathway controls the coupling of angiogenesis and osteogenesis in orthopaedic implant osseointegration by affecting the formation of CD31hiEMCNhi endothelium. Cite this article: Bone Joint J 2019;101-B(7 Supple C):108-114.

Neuroanatomical Localization of Galanin in Zebrafish Telencephalon and Anticonvulsant Effect of Galanin Overexpression.

Galanin is a neuropeptide widely expressed in the nervous system, but it is also present in non-neuronal locations. In the brain, galanin may function as an inhibitory neurotransmitter. Several studies have shown that galanin is involved in seizure regulation and can modulate epileptic activity in the brain. The overall goal of the study was to establish zebrafish as a model to study the antiepileptic effect of galanin. The goal of this study was achieved by (1) determining neuroanatomical localization of galanin in zebrafish lateral pallium, which is considered to be the zebrafish homologue of the mammalian hippocampus, the brain region essential for initiation of seizures, and (2) testing the anticonvulsant effect of galanin overexpression. Whole mount immunofluorescence staining and pentylenotetrazole (PTZ)-seizure model in larval zebrafish using automated analysis of motor function and qPCR were used in the study. Immunohistochemical staining of zebrafish larvae revealed numerous galanin-IR fibers innervating the subpallium, but only scarce fibers reaching the dorsal parts of telencephalon, including lateral pallium. In three-month old zebrafish, galanin-IR innervation of the telencephalon was similar; however, many more galanin-IR fibers reached the dorsal telencephalon, but in the lateral pallium only scarce galanin-IR fibers were visible. qRT-PCR revealed, as expected, a strong increase in the expression of galanin in the Tg(hsp70l:galn) line after heat shock; however, also without heat shock, the galanin expression was several-fold higher than in the control animals. Galanin overexpression resulted in downregulation of c-fos after PTZ treatment. Behavioral analysis showed that galanin overexpression inhibited locomotor activity in PTZ-treated and control larvae. The obtained results show that galanin overexpression reduced the incidence of seizure-like behavior episodes and their intensity but had no significant effect on their duration. The findings indicate that in addition to antiepileptic action, galanin modulates arousal behavior and demonstrates a sedative effect. The current study showed that galanin overexpression correlated with a potent anticonvulsant effect in the zebrafish PTZ-seizure model.

TLT-1-CONTROLS EARLY THROMBUS FORMATION AND STABILITY BY FACILITATING AIIBB3 OUTSIDE-IN SIGNALING IN MICE.

Platelets regulate inflammation as well as hemostasis. Inflammatory insults often induce hemostatic function through mechanisms that are not always understood. The triggering receptor expressed in myeloid cells (TREM)-like transcript 1 (TLT-1) is an abundantly expressed platelet receptor and its deletion leads to hemorrhage and edema after lipopolysaccharide and TNF-α treatment. To define a role for TLT-1 in immune derived bleeding we used a CXCL-2 mediated local inflammatory reaction in the vessels of the cremaster muscle of treml1 -/- and wild type mice. Our whole mount immunofluorescent staining of the cremaster muscle demonstrated a 50% reduction in clot size and increased extravasation of plasma molecules in treml1 -/- mice compared to wild type. We demonstrate that the decreased clotting in treml1 -/- mice is associated with a 2X reduction in integrin β3 phosphorylation on residue Y773 after platelet activation, which is consistent with treml1 -/- mice displaying reduced outside-in signaling and smaller thrombi. We further substantiate TLT-1's role in the regulation of immune derived bleeding using the reverse arthus reaction and demonstrate TLT-1's role in thrombosis using the thromboplastin initiated and collagen/epinephrine models of pulmonary embolism. Thus, the data presented here demonstrate that TLT-1 regulates early clot formation though the stabilization of αIIbβ3 outside-in signaling.

Enhancement of bradykinin-induced relaxation by focal brain ischemia in the rat middle cerebral artery: Receptor expression upregulation and activation of multiple pathways.

Focal brain ischemia markedly affects cerebrovascular reactivity. So far, these changes have mainly been related to alterations in the level of smooth muscle cell function while alterations of the endothelial lining have not yet been studied in detail. We have, therefore, investigated the effects of ischemia/reperfusion injury on bradykinin (BK)-induced relaxation since BK is an important mediator of tissue inflammation and affects vascular function in an endothelium-dependent manner. Focal brain ischemia was induced in rats by endovascular filament occlusion (2h) of the middle cerebral artery (MCA). After 22h reperfusion, both MCAs were harvested and the response to BK studied in organ bath experiments. Expression of the BK receptor subtypes 1 and 2 (B1, B2) was determined by real-time semi-quantitative RT-qPCR methodology, and whole mount immunofluorescence staining was performed to show the B2 receptor protein expression. In control animals, BK did not induce significant vasomotor effects despite a functionally intact endothelium and robust expression of B2 mRNA. After ischemia/reperfusion injury, BK induced a concentration-related sustained relaxation in all arteries studied, more pronounced in the ipsilateral than in the contralateral MCA. The B2 mRNA was significantly upregulated and the B1 mRNA displayed de novo expression, again more pronounced ipsi- than contralaterally. Endothelial cells displaying B2 receptor immunofluorescence were observed scattered or clustered in previously occluded MCAs. Relaxation to BK was mediated by B2 receptor activation, abolished after endothelium denudation, and largely diminished by blocking nitric oxide (NO) release or soluble guanylyl cyclase activity. Relaxation to BK was partially inhibited by charybdotoxin (ChTx), but not apamin or iberiotoxin suggesting activation of an endothelium-dependent hyperpolarization pathway. When the NO-cGMP pathway was blocked, BK induced a transient relaxation which was suppressed by ChTx. After ischemia/reperfusion injury BK elicits endothelium-dependent relaxation which was not detectable in control MCAs. This gain of function is mediated by B2 receptor activation and involves the release of NO and activation of an endothelium-dependent hyperpolarization. It goes along with increased B2 mRNA and protein expression, leaving the functional role of the de novo B1 receptor expression still open.

Interaction of Phospho‐Moesin and CD44 in Pericytes Attenuated the Maturation of Neovessles in AGE‐induced Angiogenesis

BackgroundWe have previously demonstrated that advanced glycation endproducts (AGEs) promoted angiogenesis by inducing ERM phosphorylation, especially, moesin phosphorylation in human umbilical vein endothelial cells (HUVECs). This study was to investigate the effect of moesin phosphorylation on angiogenesis and neovessel maturation in aortic ring and retina in AGE‐treated mice. This study also focus on the impact of interaction between phospho‐moesin and CD44 on AGE‐induced retinal microvascular pericyte (RMP) migration and neovessel maturation.MethodsAGE‐treated mouse model was developed in WT mice, in receptor for AGEs (RAGE)‐knockout (RAGE(−/−)) mice and in WT postnatal mice by intraperitoneal injection of AGEs. retinal vessels were observed by whole mount immunofluorescence staining for isolectin B4 (IB4). Mouse aorta rings were embedded in 3D collagen and the sprouted vascular endothelial cells was identified by immunofluorescent staining IB4. Pericytes were identified with NG2 or α‐SMA in retina and aortic ring. Primary RMPs were isolated, purified from weanling rats, and identified by cellular markers α‐SMA, PDGFR‐β, NG2 and desmin using immunofluorescence microscopy. Migration of RMPs was induced in different condition and detected with transwell. The phosphorylation of moesin was measured using western blotting. Colocalization and interaction between CD44, phospho‐moesin and F‐actin was detected by confocal laser scanning microscopy and coimmunoprecipitation.ResultsThe number of neovessels in retina and the phosphorylation of moesin were increased in WT mice after AGE treatment for 1, 2, and 6 months, and the increase of retinal neovessels was more obvious with time extension. There were no significant alteration in RAGE(−/−) mice after AGE treatment for six months. The phosphorylation of moesin and the number of neovessels in retina were increased in WT postnatal mice after AGE treatment for 7 d. Both in vivo and in vitro AGE treatment could enhanced the phosphorylation of moesin and promoted neovessel sprouting in mouse aortic ring, while there were not significant changes in RAGE(−/−) mouse aortic ring. The pericyte cover rate was reduced in neovessels in AGE‐treated mouse aortic ring, as well as in retinal microvessels in AGE‐treated mice. AGE‐BSA increased moesin phosphorylation in RMPs, which was in consistent with RMP migration in a dose‐ and time‐dependent manner. AGE‐BSA promoted CD44 and phospho‐moesin interaction, as well as the F‐actin stress fiber reassembly. The inhibition of ROCK by specific inhibitor Y27632 decreased AGE‐induced moesin phosphorylation and subsequently attenuated pericyte migration, suppressed the formation of CD44‐phospho‐ERM complex and redistribution of F‐actin. The inhibiting mutant of phosphorylation of Thr 558 in moesin suppressed AGE‐induced migration and F‐actin rearrangement, while the activating mutant of moesin phosphorylation of Thr 558 promoted cell migration and F‐actin alteration.ConclusionsAGEs promoted angiogenesis in mice by inducing moesin phosphorylation. The interaction of phospho‐moesin and CD44 participates in the pathogenesis of AGE‐induced RMP migration through ROCK activation and might hamper the maturation of neovessels.Support or Funding InformationSponsored by General Program from Natural Science Foundation of China (81370226, 81170297, and 80971201)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts

Organ Culture and Whole Mount Immunofluorescence Staining of Mouse Wolffian Ducts.

Tubal morphogenesis is a fundamental requirement for the development of most mammalian organs, including the male reproductive system. The epididymis, an integral part of the male reproductive tract, is responsible for sperm storage, maturation, and transport. The adult epididymis is a highly coiled tube that develops from a simple and straight embryonic precursor known as Wolffian duct (WD). Proper coiling of the epididymis is essential for male fertility, as sperm in the testis are unable to fertilize an oocyte. However, the mechanism responsible for epididymal development and coiling remains unclear, partially due to the lack of whole organ culture and imaging methods. In this study, we describe an in vitro culture system and whole mount immunofluorescence protocol to better visualize the process of WD coiling and development, which may also be applied to study other tubular organs.

Abstract 15945: Cdc42 is Required for Lymphatic Branching, Maturation and Valve Formation During Development

Cdc42 is a Ras-related GTPase that plays an important role in regulation of actin cytoskeletal architecture. Blocking the ability of Cdc42 to activate its effectors has been shown to inhibit a range of cellular functions including cell polarization, migration, proliferation and differentiation. Consistent with its critical roles in vitro, the inactivation of Cdc42 in mice resulted in embryonic lethality before E6.5. The early embryonic lethal phenotype of Cdc42-null mice has made it not useful for studies on the interesting questions of the roles and mechanisms of Cdc42 in the vascular development. To overcome this problem, we have generated an endothelial cell (EC) specific Cdc42 knockout mouse line by crossing Cdc42/flox mice with vascular endothelial cadherin Cre mice. Our results have demonstrated that the deletion of Cdc42 in ECs resulted in embryonic lethality with severe edema. Whole mount immunofluorescence staining showed mesentery collecting lymphatic vessel maturation and valve formation defects. Interestingly, lymphangiogenesis in the intestine wall was totally disrupted in Cdc42 EC knockout embryos. Moreover, we analyzed the role of Cdc42 in lymphatic vessel formation in the skin. We found that Cdc42 is required for tip cells filopodia formation and the deletion of Cdc42 in endothelial cells impaired lymphatic vessel sprouting, branching and the mutant lymphatic vessels displayed blunt-ended, bulbous lymphatic. We also noted that the size of lymphatic lumen was significantly increased. Taken together, our data suggested that Cdc42 plays an essential role in lymphatic branching, maturation and valve formation during development.

Comprehensive evaluation of peripheral nerve regeneration in the acute healing phase using tissue clearing and optical microscopy in a rodent model.

Peripheral nerve injury (PNI), a common injury in both the civilian and military arenas, is usually associated with high healthcare costs and with patients enduring slow recovery times, diminished quality of life, and potential long-term disability. Patients with PNI typically undergo complex interventions but the factors that govern optimal response are not fully characterized. A fundamental understanding of the cellular and tissue-level events in the immediate postoperative period is essential for improving treatment and optimizing repair. Here, we demonstrate a comprehensive imaging approach to evaluate peripheral nerve axonal regeneration in a rodent PNI model using a tissue clearing method to improve depth penetration while preserving neural architecture. Sciatic nerve transaction and end-to-end repair were performed in both wild type and thy-1 GFP rats. The nerves were harvested at time points after repair before undergoing whole mount immunofluorescence staining and tissue clearing. By increasing the optic depth penetration, tissue clearing allowed the visualization and evaluation of Wallerian degeneration and nerve regrowth throughout entire sciatic nerves with subcellular resolution. The tissue clearing protocol did not affect immunofluorescence labeling and no observable decrease in the fluorescence signal was observed. Large-area, high-resolution tissue volumes could be quantified to provide structural and connectivity information not available from current gold-standard approaches for evaluating axonal regeneration following PNI. The results are suggestive of observed behavioral recovery in vivo after neurorrhaphy, providing a method of evaluating axonal regeneration following repair that can serve as an adjunct to current standard outcomes measurements. This study demonstrates that tissue clearing following whole mount immunofluorescence staining enables the complete visualization and quantitative evaluation of axons throughout nerves in a PNI model. The methods developed in this study could advance PNI research allowing both researchers and clinicians to further understand the individual events of axonal degeneration and regeneration on a multifaceted level.

Characterization of human dental pulp cells-derived spheroids in serum-free medium: Stem cells in the core

Spheroid models have led to an increased understanding of differentiation, tissue organization and homeostasis. In the present study, we have observed that under a serum-free medium, human dental pulp cells (DPCs) spontaneously formed spheroids, and could survive over 15 weeks. To characterize these spheroids, we investigated their dynamics, microenvironment, cell distribution, molecular profiles, and neuronal/osteogenic potential. Cell tracking assay showed that cells inside the spheroids have very slow cycling. Although the spheroids had hypoxia microenvironments, there were not any massive cell die-offs even after long-term cultivation. Whole mount immunofluorescence staining and histological analysis showed a distribution of stem cells in the central/intermediate zones of spheroids. qRT-PCR analysis demonstrated that the expression of stemness markers NANOG, TP63, and CD44 in the spheroids were much higher than within the monolayer cultures. Gene expression levels of neural markers CDH2, NFM, TUBB3, and CD24 in the spheroids were much higher than the monolayer DPCs and increased in a culture time-dependent manner. Without any neural induction, spheroid-derived cells spontaneously converted into neuron-like cells with positive staining of neural markers HuC/D and P75 under the serum-free medium for about 2 weeks. When the spheroids were transferred into osteogenic medium, they rapidly differentiated into osteo/odontogenic cells, especially the central original cells. Compared to the monolayer DPCs, mineralization in spheroids were significantly increased. This spheroid model offers a study tool to explore the molecular bases of stem cell homeostasis and tissue organization, and can be wildly used for nerve tissue and bone regeneration.

The Effects of Age and Cx3cr1 Deficiency on Retinal Microglia in theIns2AkitaDiabetic Mouse

Diabetic retinopathy (DR) is a major cause of visual impairment in developed countries. While DR has been described classically as a microvascular disease, recent evidence suggests that changes to retinal microglia are an early feature of retinopathy. In our study, we assessed changes in microglial distribution and morphology in vivo and ex vivo in a mouse model of non-proliferative DR, and further examined effects of age and the absence of the functional chemokine receptor Cx(3)cr1 on the progression of these changes. To isolate the effects of the three variables: diabetic status, age, and role of Cx(3)cr1, the Ins2(Akita) mouse was crossed with Cx(3)cr1-eGFP reporter mice. Eyes were assessed clinically in vivo at 10, 20, 30, and 46 weeks of age, and the retinal structure and arrangement of GFP(+) microglia was examined ex vivo using whole mount immunofluorescence staining and confocal microscopy. clinical examination of the fundus, vasculature, or GFP(+) microglial distribution did not reveal any macroscopic changes related to diabetic status: however, ex vivo microscopic analysis revealed alterations in microglial network organization, and evidence of cell shape changes regarded classically as signs of activation, in Ins2(Akita) mice from 10 weeks of age. These changes were exacerbated in older diabetic mice whose microglia lacked Cx(3)cr1 (Ins2(Akita) Cx(3)cr1(gfp/gfp) mice). Diabetic status and Cx(3)cr1 deficiency led to accumulations of Iba-1(+) hyalocytes (vitreal macrophages) and subretinal macrophages. These data showed that changes to murine retinal microglia occur in response to systemic diabetic status in the absence of overt retinopathy and inflammation. These changes are exaggerated in mice lacking Cx(3)cr1, suggesting fractalkine- Cx(3)cr1 interactions may have a role in early neuronal changes in preproliferative DR.

The Impact of Ovarian Stimulation on the Expression of Candidate Reprogramming Genes in Mouse Preimplantation Embryos

Ovarian stimulation with gonadotrophins is an integral part of assisted reproductive technologies in human subfertility/infertility treatment. Recent findings have associated ovarian stimulation with the increased incidence of imprinting disorders in humans as well as defects in genome-wide methylation reprogramming and, in particular, imprinting in mice. Here, we present the first study that determined the impact of ovarian stimulation on the expression of developmentally important reprogramming genes (Apex1, Lig1, Lig3, Mbd2, Mbd3, Mbd4, and Polb) in single early mouse morula embryos (16-cell stage). Using absolute quantification of mRNA by quantitative real-time PCR, we observed an association of ovarian stimulation with a downregulation of mRNAs encoding the base excision repair proteins APEX1 and POLB as well as the 5-methyl-CpG-binding domain protein MBD3 in individual morula embryos. Whole mount immunofluorescence staining of early and late morula embryos with an antibody against APEX1 revealed individual embryos with lower protein expression levels after ovarian stimulation and a correlation of mRNA expression with protein abundance. Our data argue for a negative impact of ovarian stimulation during female gametogenesis and/or early embryo development affecting the expression of candidate reprogramming factors.

Decreased Hypoxia-Induced Neovascularization in Angiopoietin-2 Heterozygous Knockout Mouse through Reduced MMP Activity

Background/Aims: Proliferative diabetic retinopathy is characterized by the formation of retinal neovascularization. Angiopoietin-2 (Ang-2) and matrix metalloproteinase (MMP) play a critical role in angiogenesis. However, the precise location and function of Ang-2 during formation of retinal neovascularizations driven by hypoxia in relation to MMP activity have not been elucidated. In this study, we investigated the response of Ang-2 heterozygous knockout retinas (Ang2^+/- mouse) to hypoxia and its link to MMP activity in an oxygen-induced retinopathy (OIR) model. Methods: Pre-retinal neovascularizations were quantitated in vertical sections. Intra-retinal angiogenesis was assessed by whole mount immunofluorescence staining of retinas. MMP activity was examined in retinal protein lysate and whole mount retinal in situ zymography. Results: Ang2^+/- retinas subjected to the OIR model showed 33 % reduced neovascularization and 271 % increased avascular zones at postnatal day 17. In the OIR model, Ang-2 was modestly expressed in pre-retinal neovascularizations and venules, but strongly in arterioles and capillary sprouts. MMPs were activated in close association to where Ang-2 is expressed. MMP activity was substantially decreased in Ang2^+/- retinas. Conclusions: Our present data suggest the spatially concomitant expression of Ang2 and MMPs, and that Ang2 modulates hypoxia-induced neovascularization by regulating MMP activity.

M cell development occurs preferentially in distal small intestinal Peyer's patches in mice lacking B cells (μMT), CCR6, or TRANCE/RANKL

Peyer's patches (PP) are inductive sites for generation of mucosal IgA antibody responses in the small intestine (SI). M cells are specialized epithelial cells present in the follicle‐associated epithelium (FAE) of PP that acquire small particulate antigens and convey them to immune cells in the subepithelial dome. The exact molecular signals driving differentiation of FAE enterocytes into M cells have not yet been defined. In wild type (WT) mice, the extent of M cell development is equal in PP throughout the SI. B cell deficient mice (μMT) were previously shown to have smaller PP with fewer follicles and a reduced density of M cells. Whole mount immunofluorescence staining of PP from μMT mice with Ulex europaeus lectin (UEA‐1) labeled with rhodamine reveals a consistent proximal to distal gradient in the extent of M cell development with PP follicles from the proximal third of the SI containing 6.0% of the M cells in WT PP and follicles from the distal third containing 79.0% of the WT level. A proximal to distal gradient was also observed in 2 other mutant mice (CCR6 and TRANCE/RANKL knockout mice) with impaired M cell development. In TRANCE deficient mice no M cells are observed in PP from the proximal third of the SI and the number of M cells in the middle and distal third rises to just 1.9% and 12.3% of WT levels. The existence of a proximal to distal gradient in M cell development in PP from these mutant mice demonstrates that M cell development is a complex process shaped by molecular cues that differ regionally along the SI. The greater reduction in M cells in the proximal PP of the models studied may reflect the later initiation of PP organogenesis and/or the increased density of the enteric microflora in the distal SI.Supported by the NIH (DK‐64730, DK‐64399).