DiI Labeling Research Articles

Evaluation of the effect of lecithin and nanolecithin in repairing membrane damage, maintaining membrane integrity, and improving human sperm function in the freezing-thawing process.

Our study aimed to evaluate the effects of lecithin nanoparticles on sperm quality during cryopreservation. In phase one, sperm-freezing media were prepared with lecithin concentrations (0.5%, 1%, and 2%) and lecithin nanoparticles of various sizes (50-100, 100-200, and ≥ 200nm). Post-thaw, sperm motility, viability, mitochondrial membrane potential (MMP), lipid peroxidation (measured by malondialdehyde, MDA), and DNA fragmentation were evaluated. In phase two, the acrosomal reaction was assessed in the best and worst-performing groups from phase one. DiI labeling detected interactions between lecithin nanoparticles and the sperm membrane. Field emission scanning electron microscopy (FESEM) examined the sperm membrane's surface structure and lecithin binding sites. Atomic force microscopy (AFM) assessed height differences in the sperm surface layer in the best-performing group from phase one. The group treated with 1% lecithin nanoparticles (50-100nm) showed significantly increased viability post-thaw compared to other groups, with reduced DNA fragmentation and MDA levels. While motility significantly decreased in all groups compared to before freezing levels, lower concentrations, and smaller particle sizes yielded better results. MMP also significantly decreased across all groups with no significant differences. The acrosomal reaction significantly decreased with 1% lecithin nanoparticles (50-100nm) compared to the 2% (≥ 200nm) group. DiI-labeled nanoparticles and FESEM revealed that lecithin nanoparticles primarily bound to and infiltrated the sperm membrane, particularly in the head and postacrosomal regions. Lecithin nanoparticles effectively bind to the sperm membrane, protecting it during the freeze-thaw process and improving sperm viability.

Cognitive integrity in Non-Demented Individuals with Alzheimer's Neuropathology is associated with preservation and remodeling of dendritic spines.

Individuals referred to as Non-Demented with Alzheimer's Neuropathology (NDAN) exhibit cognitive resilience despite presenting Alzheimer's disease (AD) histopathological signs. Investigating the mechanisms behind this resilience may unveil crucial insights into AD resistance. DiI labeling technique was used to analyze dendritic spine morphology in control (CTRL), AD, and NDAN post mortem frontal cortex, particularly focusing on spine types near and far from amyloid beta (Aβ) plaques. NDAN subjects displayed a higher spine density in regions distant from Aβ plaques versus AD patients. In distal areas from the plaques, NDAN individuals exhibited more immature spines, while AD patients had a prevalence of mature spines. Additionally, our examination of levels of Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1), a protein associated with synaptic plasticity and AD, showed significantly lower expression in AD versus NDAN and CTRL. These results suggest that NDAN individuals undergo synaptic remodeling, potentially facilitated by Pin1, serving as a compensatory mechanism to preserve cognitive function despite AD pathology. Spine density is reduced near Aβ plaques compared to the distal area in CTRL, AD, and NDAN dendrites. NDAN shows higher spine density than AD in areas far from Aβ plaques. Far from Aβ plaques, NDAN has a higher density of immature spines, AD a higher density of mature spines. AD individuals show significantly lower levels of Pin1 compared to NDAN and CTRL.

Comparison of tumor growth assessment using GFP fluorescence and DiI labeling in a zebrafish xenograft model

ABSTRACT DiI is a lipophilic fluorescent dye frequently used to label and trace cells in cell cultures and xenograft models. However, DiI can also transfer from labeled to unlabeled cells, including host organism cells, and label dead cells obscuring interpretation of the results. These limitations of DiI labeling in xenograft models have not been thoroughly investigated. Here we labeled green fluorescent protein (GFP)-expressing MDA-MB-231 cells with DiI to directly compare tumor growth assessment in zebrafish xenografts using the DiI labeling and GFP fluorescence. Our results indicate that the DiI based assessment significantly overestimated tumor growth in zebrafish xenograft models compared to the GFP fluorescence based assessment. The imaging of DiI labeled GFP-expressing MDA-MB-231 cell cultures indicated that the DiI labeling of the membrane is uneven. Analysis of the DiI labeled GFP-expressing MDA-MB-231 cell cultures with flow cytometry indicated that the DiI labeling varied over time while the GFP fluorescence remained unchanged, suggesting that the GFP fluorescence is a more reliable signal for monitoring tumor progression than the DiI labeling. Taken together, our results demonstrate limitations of using DiI labeling for xenograft models and emphasize the need for validating the results based on DiI labeling with other orthogonal methods, such as the ones utilizing genetically encoded fluorophores.

Combined DiI and Antibody Labeling Reveals Complex Dysgenesis of Hippocampal Dendritic Spines in a Mouse Model of Fragile X Syndrome.

Structural, functional, and molecular alterations in excitatory spines are a common hallmark of many neurodevelopmental disorders including intellectual disability and autism. Here, we describe an optimized methodology, based on combined use of DiI and immunofluorescence, for rapid and sensitive characterization of the structure and composition of spines in native brain tissue. We successfully demonstrate the applicability of this approach by examining the properties of hippocampal spines in juvenile Fmr1 KO mice, a mouse model of Fragile X Syndrome. We find that mutant mice display pervasive dysgenesis of spines evidenced by an overabundance of both abnormally elongated thin spines and cup-shaped spines, in combination with reduced density of mushroom spines. We further find that mushroom spines expressing the actin-binding protein Synaptopodin-a marker for spine apparatus-are more prevalent in mutant mice. Previous work identified spines with Synaptopodin/spine apparatus as the locus of mGluR-LTD, which is abnormally elevated in Fmr1 KO mice. Altogether, our data suggest this enhancement may be linked to the preponderance of this subset of spines in the mutant. Overall, these findings demonstrate the sensitivity and versatility of the optimized methodology by uncovering a novel facet of spine dysgenesis in Fmr1 KO mice.

Experimental study on co-culture of DiI labeled rat bone marrow mesenchymal stem cells and polycaprolactone film in vitro to make a cell patch.

In stem cell therapy, due to the lack of an effective carrier, a large number of transplanted stem cells are lost and die. Therefore, finding a suitable carrier has become a further direction of stem cell therapy. In research on the co-culture of polycaprolactone (PCL) with 1,1'-Dioctadecyl-3,3,3',3'- tetramethylindocarbocyanine perchlorate (DiI) labeled bone marrow mesenchymal stem cells (BMSCs), we observe the effect of materials on the growth and proliferation of DiI labeled stem cells, and the effect of DiI labeling on patch preparation, so as to find a kind of biomaterial suitable for the growth and proliferation of BMSCs, and find a suitable cell carrier for stem cell therapy of myocardial infarction and in vivo tracing. Clean grade Sprague Dawley rats were selected as experimental objects, BMSCs were isolated and cultured, and the surface markers were identified by flow cytometry. After the BMSCs were cultured for 3 passages, the BMSCs were stained with DiI dye, and the BMSCs DiI and PCL biomaterial film were co-cultured. After 24 hours, the cell growth was observed under fluorescence microscope, and fixed for scanning under electron microscope. The cell proliferation was detected by CCK-8 at 1, 4, 7, 10 days of culture. The measurement data conforming to normal distribution are expressed in the form of mean ± standard deviation (X¯±s). One way ANOVA was used for comparison among groups, LSD analysis was used for pairwise comparison. The difference was statistically significant (P <0.05). BMSCs were strongly positive for CD90, CD44H, but negative for CD11b/c, CD45. Under fluorescence microscope, BMSCs DiI showed red light, fusiform or polygonal. Under the scanning electron microscope, the cell patch formed by co-culture of PCL film and DiI-BMSCs had a large number of cells on the surface and normal cell state. CCK-8 assay showed that the OD value on the first day was 0.330 ± 0.025; The OD value was 0.620 ± 0.012 on the 4th day, 1.033 ± 0.144 on the 7th day and 1.223 ± 0.133 on the 10th day. There was significant difference among the time points (P <0.05). The cell patch made of PCL film and DiI labeled BMSCs can survive and proliferate on the surface, so it can be used as a scaffold material for stem cell therapy in vivo.

A Novel ex vivo Method for Investigating Vascularization of Transplanted Islets

Revascularization of transplanted pancreatic islets is critical for survival and treatment of type 1 diabetes. Questions concerning how islets influence local microvascular networks and how networks form connections with islets remain understudied and motivate the need for new models that mimic the complexity of real tissue. Recently, our laboratory established the rat mesentery culture model as a tool to investigate cell dynamics involved in microvascular growth. An advantage is the ability to observe blood vessels, lymphatics, and immune cells. The objective of this study was to establish the rat mesentery tissue culture model as a useful tool to investigate islet tissue integration. DiI-labeled islets were seeded onto adult rat mesentery tissues and cultured for up to 3 days. Live lectin labeling enabled time-lapse observation of vessel growth. During culture, DiI-positive islets remained intact. Radial lectin-positive capillary sprouts with DiI labeling were observed to form from islets and connect to host networks. Lectin-positive vessels from host networks were also seen growing toward islets. PECAM and NG2 labeling confirmed that vessels sprouting from islets contained endothelial cells and pericytes. Our results introduce the rat mesentery culture model as a platform for investigating dynamics associated with the initial revascularization of transplanted islets.

Double-layered two-directional somatopleural cell migration during chicken body wall development revealed with local fluorescent tissue labeling.

The thoracic ventral body wall consists of the rib, the sternum, the intercostal muscles, and the connective tissues surrounding them. The ribs and the intercostal muscles are derived from the somite. The connective tissues are derived from the somatic layer of the lateral plate mesoderm, somatopleure. The lateral growth of the somatopleure forms the primary ventral body wall. The migration of somitic cells into the somatopleure generates the secondary body wall. As the migrating behavior of the somatopleural cells during secondary body wall formation is still unclear, we investigate here the migratory behavior of the somatopleural cells in the thorax during chicken ventral body wall development by labeling the thoracic somatopleural cells one-somite-wide by DiI labeling or gene transfection of the enhanced green fluorescent protein and observe their distribution assisted by the tissue-clearing technique FRUIT. Our labeling experiments revealed the rostral migration of the somatopleural cells into a deep part of the thoracic body wall in embryonic day 6.5 chickens. For embryonic day 8.5 chickens, these deep-migrating somatopleural cells were found around the sternal ribs. Thus, we identified the double-layered two-directional migrating pathways of the somatopleural cells: the rostral migration of the deep somatopleural cells and the lateral migration of the superficial somatopleural cells. Our findings imply that the rostral migration of deep somatopleural cells and the lateral migration of superficial ones might be associated with the developing sternal ribs and the innervation of the thoracic cutaneous nerves, respectively.

Perineuronal Nets in the Dorsomedial Striatum Contribute to Behavioral Dysfunction in Mouse Models of Excessive Repetitive Behavior

BackgroundExcessive repetitive behavior is a debilitating symptom of several neuropsychiatric disorders. Parvalbumin-positive inhibitory interneurons in the dorsal striatum have been linked to repetitive behavior, and a sizable portion of these cells are surrounded by perineuronal nets (PNNs), specialized extracellular matrix structures. Although PNNs have been associated with plasticity and neuropsychiatric disease, no previous studies have investigated their involvement in excessive repetitive behavior. MethodsWe used histochemistry and confocal imaging to investigate PNNs surrounding parvalbumin-positive cells in the dorsal striatum of 4 mouse models of excessive repetitive behavior (BTBR, Cntnap2, Shank3, prenatal valproate treatment). We then investigated one of these models, the BTBR mouse, in detail, with DiI labeling, in vivo and in vitro recordings, and behavioral analyses. We next degraded PNNs in the dorsomedial striatum (DMS) using the enzyme chondroitinase ABC and assessed dendritic spine density, electrophysiology, and repetitive behavior. ResultsWe found a greater percentage of parvalbumin-positive interneurons with PNNs in the DMS of all 4 mouse models of excessive repetitive behavior compared with control mice. In BTBR mice, we found fewer dendritic spines on medium spiny neurons (targets of parvalbumin-positive interneurons) and differences in neuronal oscillations as well as inhibitory postsynaptic potentials compared with control mice. Reduction of DMS PNNs in BTBR mice altered dendritic spine density and inhibitory responses and normalized repetitive behavior. ConclusionsThese findings suggest that cellular abnormalities in the DMS are associated with maladaptive repetitive behaviors and that manipulating PNNs can restore normal levels of repetitive behavior while altering DMS dendritic spines and inhibitory signaling.

Modeling Joint Pain on a Chip: integrating sensory neurons in the microJoint to model osteoarthritis

The development of safe and effective methods for the treatment of osteoarthritis(OA)-associated pain is a critical need. Recently, our team successfully modeled OA-like pathology in an in vitro multi-component joint-on-a-chip (microJoint), which integrates engineered osteochondral complex, synovium, adipose tissue and given their crucial role in joint pain, macrophages. In the present project, we introduce the sensory neurons into the established microJoint to generate the neu-microJoint, enabling the dynamic interplay between the peripheral nervous system and joint tissues. By recording activity in sensory neurons, joint integrity as well as therapeutic efficacy can be monitored in real time. The neu-microJoint is a hybrid PDMS/3D printed system where microchannels establish a diffusion barrier but enable sensory neuron innervation of joint tissues. Neural activity is assessed with micro electrode arrays (MEAs) and fura2-based calcium imaging. With the use of L4 and L5 dorsal root ganglion (DRG) neurons recovered from organ donors, the neu-microJoint can be fully humanized. DRG neurons cultured for 4 days were screened with increasing concentrations of KCl (10, 30, and 50mM) to detect spiking in the MEAs. There was little to no spontaneous activity under baseline conditions, but activity was detected in response to 30mM KCl. Rodent and human DRG neurons plated in the neu-microJoint cultured between 10 – 28 days showed substantial axonal growth through the microchannels as visualized through DiI or fura2 labeling. Moreover, electrical stimulation applied to the neurites evoked an increase in calcium in the corresponding soma. Conditioned media from the OA-modeled microJoint applied to the neurons caused an increase in calcium in a subpopulation of neurons. These data suggest the potential to apply an in vitro microphysiological system to study OA pain. These results also indicate that a subset of neurons, now undergoing specific characterization, is potentially responsible for OA pain modeled in the neu-microJoint. F32 1F32NS110148-01A1, UG3TR003090.

MACS: Rapid Aqueous Clearing System for 3D Mapping of Intact Organs.

Tissue optical clearing techniques have provided important tools for large‐volume imaging. Aqueous‐based clearing methods are known for good fluorescence preservation and scalable size maintenance, but are limited by long incubation time, insufficient clearing performance, or requirements for specialized devices. Additionally, few clearing methods are compatible with widely used lipophilic dyes while maintaining high clearing performance. Here, to address these issues, m‐xylylenediamine (MXDA) is firstly introduced into tissue clearing and used to develop a rapid, highly efficient aqueous clearing method with robust lipophilic dyes compatibility, termed MXDA‐based Aqueous Clearing System (MACS). MACS can render whole adult brains highly transparent within 2.5 days and is also applicable for other intact organs. Meanwhile, MACS possesses ideal compatibility with multiple probes, especially for lipophilic dyes. MACS achieves 3D imaging of the intact neural structures labeled by various techniques. Combining MACS with DiI labeling, MACS allows reconstruction of the detailed vascular structures of various organs and generates 3D pathology of glomeruli tufts in healthy and diabetic kidneys. Therefore, MACS provides a useful method for 3D mapping of intact tissues and is expected to facilitate morphological, physiological, and pathological studies of various organs.

Acute slice preparation for electrophysiology increases spine numbers equivalently in the male and female juvenile hippocampus: a DiI labeling study.

Hippocampal slices are widely used for in vitro electrophysiological experiments to study underlying mechanisms for synaptic transmission and plasticity, and there is a growing appreciation for sex differences in synaptic plasticity. To date, several studies have shown that the process of making slices from male animals can induce synaptogenesis in cornu ammonis area 1 (CA1) pyramidal cells, but there is a paucity of data for females and other brain regions. In the current study we use microcrystals of the lipophilic carbocyanine dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate) to stain individual neurons in the CA1 and dentate gyrus (DG) hippocampal subfields of postnatal day 21 male and female rats. We show that the preparation of sections for electrophysiology produces significant increases in spines in sections obtained from females, similar to that observed in males. We also show that the procedures used for in vitro electrophysiology also result in significant spine increases in the DG and CA1 subfields. These results demonstrate the utility of this refined DiI procedure for staining neuronal dendrites and spines. They also show, for the first time, that in vitro electrophysiology slice preparations enhance spine numbers on hippocampal cells equivalently in both juvenile females and males.NEW & NOTEWORTHY This study introduces a new DiI technique that elucidates differences in spine numbers in juvenile female and male hippocampus, and shows that slice preparations for hippocampal electrophysiology in vitro may mask these differences.

Abstract 3766: Embryonal metastasis assay, an in vivo test of circulating tumorspheres to detect metastatic predisposition

Abstract INTRODUCTION One of the unsolved problems of cancer is its progression into systemic, metastatic disease. Malignant cells are released into circulation very early (circulating tumor cells or CTCs), but distant metastases are disseminated only by cancer stem cells (CSCs) that survive circulation. We investigated a unique feature of CSCs, formation of cell clusters (spheroids or tumorspheres) in vitro. We found that it also takes place in vivo, cancer tumorspheres circulate in blood and we showed that they carry cancer stem cells and strongly correlate with metastatic disease. In this project we developed a filtration method to collect live circulating cancer stem cell in tumorspheres and searched for a method to establish a prognostic assay to predict their metastatic potential. METHODS The conventional approaches use knockout mice to assay metastasis (NOD-SCID mice, etc.), but he high cost, the complex procedure and the lengthy assay (5 weeks) ruled them out as a routine clinical assay. Our goal was to establish an in vivo translational assay to detect metastatic predisposition early, with a chance to prevent systemic progression. We found that the rapidly progressing environment in the chicken embryo offers critical advantages to test the metastatic potential of a patient’s cancer. Easy access to chorioallantois circulation and to the embryo, the lack of immune response, the rapid turnaround time (5 days) and the technical simplicity rendered the new embryonal metastatic assay a promising and cost effective translational method. We used tumorspheres of both highly metastatic and non-metastatic cell lines. For image analysis a highly metastatic cell line (MDA-MB-436) was labeled by Green Fluorescent Protein. To model the technique of labeling real life patient spheroids (DiI membrane dye and protein tracing by CFDA-SA), we used the highly metastatic MDA-MB-231 and the non metastatic MCF7 and T47D cell lines. For quantitative analysis of embryonal metastasis we used methods applied in microchimerism detection. Embryonal DNA/RNA was extracted 5-9 days after cancer spheroid implantation and used qPCR to detect human sequences. RESULTS In the embryonal metastatic assays, fluorescent and confocal imaging detected metastatic cells in the embryo. DiI and CFDA-SA labeling of native tumorspheres indicated a level of toxicity and needed to be optimized. Molecular detection of human microchimerism in the embryos was extremely sensitive and generated quantitative metastatic data. CONCLUSIONS The results indicate that the embryonal metastasis assay using molecular microchimerism detection has the potential for a sensitive and quantitative method to assess the metastatic predisposition of a cancer patient. The method needs only an incubator, DNA extraction and a thermocycler, easily available in most hospitals and offers cost effective, relevant in vivo prognostic data within days. Citation Format: Peter Geck, James Kubilus, Andrew Makarovskiy, Viktoria Denes. Embryonal metastasis assay, an in vivo test of circulating tumorspheres to detect metastatic predisposition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3766.

Adipose-derived stem cell sheets accelerate bone healing in rat femoral defects.

In the present study, we investigated whether both adipose-derived stem cell (ADSC) and osteogenic-induced ADSC sheets could promote bone healing in a rat distal femoral metaphysis bone defect model. A through-hole defect of 1 mm diameter was drilled into each distal femur of 12 week old rats. Forty-five rats were randomly assigned to three groups: (1) control group; (2) ADSC sheet group; or (3) osteogenic-induced ADSC sheet group. We evaluated each group by analysis of computerized tomography scans every week after the surgery, histological analysis, and DiI labeling (a method of membrane staining for post implant cell tracing). Radiological and histological evaluations showed that a part of the hole persisted in the control group at four weeks after surgery, whereas the hole was restored almost completely by new bone formation in both sheet groups. The mean value of bone density (in Houndsfield units) for the bone defect area was significantly higher in both sheet groups than that in the control group (p = 0.05) at four weeks postoperative. A large number of osteocalcin positive osteoblasts were observed at the area of bone defect, especially in the osteogenic-induced ADCS sheet group. DiI labeling in the newly formed bone showed that each sheet had differentiated into bone tissue at four weeks after surgery. The ADSC and the osteogenic-induced ADSC sheets promoted significantly quicker bone healing in the bone defect. Moreover, the osteogenic-induced ADSC sheet may be more advantageous for bone healing than the ADSC sheet because of the higher number of osteocalcin positive osteoblasts via the transplantation.

Staurosporine Induces the Generation of Polyploid Giant Cancer Cells in Non-Small-Cell Lung Carcinoma A549 Cells

Cultivation of A549 non-small-cell lung carcinoma (NSCLC) cells in the presence of staurosporine (SSP) leads to a reduction or a lack of proliferation in a concentration-dependent manner. This inhibition of proliferation is accompanied by the generation of polyploid giant cancer cells (PGCCs) that are characterized by cell flattening, increased cell size, polyploidy, and polynucleation as determined by crystal violet staining, BrdU and DiI labelling, and flow cytometry as well as video time-lapse analysis. Continuous SSP treatment of A549 cells can preserve PGCCs for at least two months in a resting state. Upon removal of SSP, A549 PGCCs restart to divide and exhibit a proliferation pattern and cellular morphology indistinguishable from cells where PGCCs originally derived from. Thus, SSP-treated A549 cells represent a simple and reliable experimental model for the reversible generation of PGCCs and their subsequent experimental analysis.

From the primitive streak to the somitic mesoderm: labeling the early stages of chick embryos using EGFP transfection.

Mesoderm is derived from the primitive streak. The rostral region of the primitive streak forms the somitic mesoderm. We have previously shown the developmental origin of each level of the somitic mesoderm using DiI fluorescence labeling of the primitive streak. We found that the more caudal segments were derived from the primitive streak during the later developmental stages. DiI labeled several pairs of somites and showed the distinct rostral boundary; however, the fluorescence gradually disappeared in the caudal region. This finding can be explained in two ways: the primitive streak at a specific developmental stage is primordial of only a certain number of pairs of somites, or the DiI fluorescent dye was gradually diluted within the primitive streak by cell division. Here, we traced the development of the primitive streak cells using enhanced green fluorescent protein (EGFP) transfection. We confirmed that, the later the EGFP transfection stage, the more caudal the somites labeled. Different from DiI labeling, EGFP transfection performed at any developmental stage labeled the entire somitic mesoderm from the anterior boundary to the tail bud in 4.5-day-old embryos. Furthermore, the secondary neural tube was also labeled, suggesting that not only the somite precursor cells but also the axial stem cells were labeled.

Post mortem single-cell labeling with DiI and immunoelectron microscopy unveil the fine structure of kisspeptin neurons in humans

Kisspeptin (KP) synthesizing neurons of the hypothalamic infundibular region are critically involved in the central regulation of fertility; these cells regulate pulsatile gonadotropin-releasing hormone (GnRH) secretion and mediate sex steroid feedback signals to GnRH neurons. Fine structural analysis of the human KP system is complicated by the use of post mortem tissues. To gain better insight into the neuroanatomy of the somato-dendritic cellular compartment, we introduced the diolistic labeling of immunohistochemically identified KP neurons using a gene gun loaded with the lipophilic dye, DiI. Confocal microscopic studies of primary dendrites in 100-µm-thick tissue sections established that 79.3% of KP cells were bipolar, 14.1% were tripolar, and 6.6% were unipolar. Primary dendrites branched sparsely, contained numerous appendages (9.1 ± 1.1spines/100µm dendrite), and received rich innervation from GABAergic, glutamatergic, and KP-containing terminals. KP neuron synaptology was analyzed with immunoelectron microscopy on perfusion-fixed specimens. KP axons established frequent contacts and classical synapses on unlabeled, and on KP-immunoreactive somata, dendrites, and spines. Synapses were asymmetric and the presynaptic structures contained round and regular synaptic vesicles, in addition to dense-core granules. Although immunofluorescent studies failed to detect vesicular glutamate transporter isoforms in KP axons, ultrastructural characteristics of synaptic terminals suggested use of glutamatergic, in addition to peptidergic, neurotransmission. In summary, immunofluorescent and DiI labeling of KP neurons in thick hypothalamic sections and immunoelectron microscopic studies of KP-immunoreactive neurons in brains perfusion-fixed shortly post mortem allowed us to identify previously unexplored fine structural features of KP neurons in the mediobasal hypothalamus of humans.

Fluorescent Labeling and 2-Photon Imaging of Mouse Tooth Pulp Nociceptors

Retrograde fluorescent labeling of dental primary afferent neurons (DPANs) has been described in rats through crystalline fluorescent DiI, while in the mouse, this technique was achieved with only Fluoro-Gold, a neurotoxic fluorescent dye with membrane penetration characteristics superior to the carbocyanine dyes. We reevaluated this technique in the rat with the aim to transfer it to the mouse because comprehensive physiologic studies require access to the mouse as a model organism. Using conventional immunohistochemistry, we assessed in rats and mice the speed of axonal dye transport from the application site to the trigeminal ganglion, the numbers of stained DPANs, and the fluorescence intensity via 1) conventional crystalline DiI and 2) a novel DiI formulation with improved penetration properties and staining efficiency. A 3-dimensional reconstruction of an entire trigeminal ganglion with 2-photon laser scanning fluorescence microscopy permitted visualization of DPANs in all 3 divisions of the trigeminal nerve. We quantified DPANs in mice expressing the farnesylated enhanced green fluorescent protein (EGFPf) from the transient receptor potential cation channel subfamily M member 8 (TRPM8EGFPf/+) locus in the 3 branches. We also evaluated the viability of the labeled DPANs in dissociated trigeminal ganglion cultures using calcium microfluorometry, and we assessed the sensitivity to capsaicin, an agonist of the TRPV1 receptor. Reproducible DiI labeling of DPANs in the mouse is an important tool 1) to investigate the molecular and functional specialization of DPANs within the trigeminal nociceptive system and 2) to recognize exclusive molecular characteristics that differentiate nociception in the trigeminal system from that in the somatic system. A versatile tool to enhance our understanding of the molecular composition and characteristics of DPANs will be essential for the development of mechanism-based therapeutic approaches for dentine hypersensitivity and inflammatory tooth pain.

Lineage tracing of epithelial cells in developing teeth reveals two strategies for building signaling centers

An important event in organogenesis is the formation of signaling centers, which are clusters of growth factor-secreting cells. In the case of tooth development, sequentially formed signaling centers known as the initiation knot (IK) and the enamel knot (EK) regulate morphogenesis. However, despite the importance of signaling centers, their origin, as well as the fate of the cells composing them, remain open questions. Here, using lineage tracing of distinct epithelial populations, we found that the EK of the mouse incisor is derived de novo from a group of SRY-box 2 (Sox2)-expressing cells in the posterior half of the tooth germ. Specifically, EK progenitors are located in the posterior ventral basal layer, as demonstrated by DiI labeling of cells. Lineage tracing the formed EK with ShhCreER , which encodes an inducible Cre recombinase under the control of the Sonic hedgehog promoter, at subsequent developmental stages showed that, once formed, some EK cells in the incisor give rise to differentiated cells, whereas in the molar, EK cells give rise to the buccal secondary EK. This work thus establishes the developmental origin as well as the fate of the EK and reveals two strategies for the emergence of serially formed signaling centers: one through de novo establishment and the other by incorporation of progeny from previously formed signaling centers.

The role of miRNA-200a in the early stage of the mandibular development

BackgroundMicroRNAs are non-coding small RNA molecules that regulate various cellular functions. In this study, we analyzed the function of microRNA-200a (miR-200a) during the development of primary and secondary cartilages in the mandible during the embryonic stage. Materials and methodsMandibular processes were excised from ICR mouse embryos. Meckel’s cartilage and the complex of condylar and angular cartilages were examined as primary and secondary cartilages, respectively. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) and in situ hybridization were performed to examine the expression of miR-200a during normal mandibular development. Transfection of a miR-200a-mimic and -inhibitor were performed to analyze the effect of miR-200a on mandibular cartilage formation. Prior to transfection, DiI labeling was performed to detect the transfection sites and examine the effect of miR-200a on Meckel’s and condylar cartilage formation. Nine days after transfection, Alcian blue staining and quantification were performed to analyze the formation of the cartilage. ResultsThe expression levels of miR-200a gradually increased from embryonic day 9–14 in mandibular processes. However, the expression levels of miR-200a in Meckel’s cartilage were nearly identical from embryonic day 12–16. The positive hybridization signals were observed in Meckel’s cartilage and mesenchymal condensation of mandibular condylar cartilage. Alcian blue analysis showed a significant decrease in the complex of condylar and angular cartilage formation in the lateral-anterior-miR-200a-mimic transfected samples compared to miR-200a-inhibitor-transfected and control samples. ConclusionmiR-200a negatively regulates the formation of condylar cartilage in early mandibular development.

Monitoring Notch Signaling-Associated Activation of Stem Cell Niches within Injured Dental Pulp.

Dental pulp stem/progenitor cells guarantee tooth homeostasis, repair and regeneration throughout life. The decision between renewal and differentiation of these cells is influenced by physical and molecular interactions with stromal cells and extracellular matrix molecules forming the specialized microenvironment of dental pulp stem cell niches. Here we study the activation of putative pulp niches after tooth injury through the upregulation of Notch signaling pathway. Notch1, Notch2, and Notch3 molecules were used as markers of dental pulp stem/progenitor cells. Upon dental injury, Notch1 and Notch3 are detected in cells related to vascular structures suggesting a role of these proteins in the activation of specific pulpal perivascular niches. In contrast, a population of Notch2-positive cells that are actively proliferative is observed in the apical part of the pulp. Kinetics of these cells is followed up with a lipophilic DiI labeling, showing that apical pulp cells migrate toward the injury site where dynamic regenerative/repair events occur. The knowledge of the activation and regulation of dental pulp stem/progenitor cells within their niches in pathologic conditions may be helpful for the realization of innovative dental treatments in the near future.