RESUMO
BACKGROUND: Sensory systems evolved intricate designs to accurately encode perplexing environments. However, this encoding task may become particularly challenging for animals harboring a small number of sensory neurons. Here, we studied how the compact resource-limited chemosensory system of Caenorhabditis elegans uniquely encodes a range of chemical stimuli. RESULTS: We find that each stimulus is encoded using a small and unique subset of neurons, where only a portion of the encoding neurons sense the stimulus directly, and the rest are recruited via inter-neuronal communication. Furthermore, while most neurons show stereotypical response dynamics, some neurons exhibit versatile dynamics that are either stimulus specific or network-activity dependent. Notably, it is the collective dynamics of all responding neurons which provides valuable information that ultimately enhances stimulus identification, particularly when required to discriminate between closely related stimuli. CONCLUSIONS: Together, these findings demonstrate how a compact and resource-limited chemosensory system can efficiently encode and discriminate a diverse range of chemical stimuli.
Assuntos
Caenorhabditis elegans , Células Quimiorreceptoras , Animais , Caenorhabditis elegans/fisiologia , Células Quimiorreceptoras/fisiologia , Células Receptoras Sensoriais/fisiologiaRESUMO
We demonstrate that a deep neural network can be trained to virtually refocus a two-dimensional fluorescence image onto user-defined three-dimensional (3D) surfaces within the sample. Using this method, termed Deep-Z, we imaged the neuronal activity of a Caenorhabditis elegans worm in 3D using a time sequence of fluorescence images acquired at a single focal plane, digitally increasing the depth-of-field by 20-fold without any axial scanning, additional hardware or a trade-off of imaging resolution and speed. Furthermore, we demonstrate that this approach can correct for sample drift, tilt and other aberrations, all digitally performed after the acquisition of a single fluorescence image. This framework also cross-connects different imaging modalities to each other, enabling 3D refocusing of a single wide-field fluorescence image to match confocal microscopy images acquired at different sample planes. Deep-Z has the potential to improve volumetric imaging speed while reducing challenges relating to sample drift, aberration and defocusing that are associated with standard 3D fluorescence microscopy.
Assuntos
Aprendizado Profundo , Microscopia de Fluorescência/métodos , Animais , Caenorhabditis elegans/ultraestrutura , Microscopia Confocal , Neurônios/ultraestruturaRESUMO
We hypothesized that in head and neck squamous cell carcinoma (HNSCC), the neurotrophin brain-derived neurotrophic factor (BDNF) and its high affinity receptor TrkB regulate tumor cell survival, invasion, and therapy resistance. We used in situ hybridization for BDNF and immunohistochemistry (IHC) for TrkB in 131 HNSCC samples. Brain-derived neurotrophic factor was highly expressed in normal mucosa in HNSCC tissue and in cell lines, whereas only 42.74% of HNSCC tissue was TrkBâº. One fourth of HNSCC cases was human papilloma virus (HPV)- positive, but the TrkB IHC frequency was not different in HPV-positive (HPVâº) and negative cases. The UPCI-SCC090 cells expressed constitutive levels of TrkB. Transforming-growth-factor-ß1 (1 ng/mL TGF-ß1) induced TrkB in a subpopulation of SCC-25 cells. A single 10-µg/mL mitomycin C treatment in UPCI-SCC090 cells induced apoptosis and BDNF did not rescue them. The SCC-25 cells were resistant to the MMC treatment, and their growth decreased after TGF-ß1 treatment, but was restored by BDNF if it followed TGF-ß1. Taken together, BDNF might be ineffective in HPV⺠HNSCC patients. In HPV- HNSCC patients, tumor cells did not die after chemotherapeutic challenge and BDNF with TGF-ß1 could improve tumor cell survival and contribute to worse patient prognosis.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Glicoproteínas de Membrana/metabolismo , Infecções por Papillomavirus/metabolismo , Receptor trkB/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Idoso , Fator Neurotrófico Derivado do Encéfalo/genética , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/genética , Neoplasias de Cabeça e Pescoço/virologia , Humanos , Masculino , Glicoproteínas de Membrana/genética , Pessoa de Meia-Idade , Mitomicina/farmacologia , Infecções por Papillomavirus/genética , Receptor trkB/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço/virologia , Fator de Crescimento Transformador beta1/farmacologiaRESUMO
Photodynamic therapy (PDT) is suggested to have an impact on the treatment of early stage head and neck cancers (HNSCC). We investigated the effect of PDT with methylene blue (MB) and a diode laser (660 nm) as the laser source on HNSCC cell lines as an in vitro model of surface oral squamous cell carcinoma. Cell-cultures were exposed to 160 µM MB for 4 min and to laser light for 8 min. Viability was proven via cell viability assay and clonogenic survival via clone counting assay. The combination of MB and diode laser evidenced high efficient loss of cell viability by 5% of the control, while treatment with the same concentration of MB for 4 min alone showed a viability of 46% of the control. In both SCC-25 and Detroit 562 HNSCC cells, MB combined with the laser allowed a significant abrogation of clonogenic growth (p < 0.01), especially in the case of Detroit 562 cells less than 1% of the suspension plated cells were able to grow tumor cell nests. Multiresistant (Detroit 562) HNSCC cells expressing cancer stem cell markers are sensitive to MB/red laser combined PDT.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Azul de Metileno/farmacologia , Neoplasias Bucais/tratamento farmacológico , Fármacos Fotossensibilizantes/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Humanos , FotoquimioterapiaRESUMO
Fibroblasts play a central role in tumor invasion, recurrence, and metastasis in head and neck squamous cell carcinoma. The aim of this study was to investigate the influence of tumor cell self-produced factors and paracrine fibroblast-secreted factors in comparison to indirect co-culture on cancer cell survival, growth, migration, and epithelial-mesenchymal transition using the cell lines SCC-25 and human gingival fibroblasts. Thereby, we particularly focused on the participation of the fibroblast-secreted transforming growth factor beta-1.Tumor cell self-produced factors were sufficient to ensure tumor cell survival and basic cell growth, but fibroblast-secreted paracrine factors significantly increased cell proliferation, migration, and epithelial-mesenchymal transition-related phenotype changes in tumor cells. Transforming growth factor beta-1 generated individually migrating disseminating tumor cell groups or single cells separated from the tumor cell nest, which were characterized by reduced E-cadherin expression. At the same time, transforming growth factor beta-1 inhibited tumor cell proliferation under serum-starved conditions. Neutralizing transforming growth factor beta antibody reduced the cell migration support of fibroblast-conditioned medium. Transforming growth factor beta-1 as a single factor was sufficient for generation of disseminating tumor cells from epithelial tumor cell nests, while other fibroblast paracrine factors supported tumor nest outgrowth. Different fibroblast-released factors might support tumor cell proliferation and invasion, as two separate effects.
Assuntos
Carcinoma de Células Escamosas/patologia , Transição Epitelial-Mesenquimal/fisiologia , Fibroblastos/metabolismo , Neoplasias de Cabeça e Pescoço/patologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Comunicação Parácrina/fisiologia , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Sobrevivência Celular/fisiologia , Técnicas de Cocultura , Fibroblastos/patologia , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Carcinoma de Células Escamosas de Cabeça e Pescoço , Microambiente Tumoral/fisiologiaRESUMO
Co-culture of periodontal ligament fibroblasts (PDLs) and SCC-25 oral squamous carcinoma cells (OSCC) results in conversion of PDLs into carcinoma-associated fibroblasts (CAFs) and induces epithelial-to mesenchymal transition (EMT) of OSCC tumor cells. We hypothesized that Curcumin targets this dynamic mutual interaction between CAFs and tumor cells. Normal and 2 µM Curcumin-treated co-culture were performed for 4 days, followed by analysis of tumor cell invasivity, mRNA/protein expression of EMT-markers and mediators, activity measure of matrix metalloproteinase 9 (MMP-9), and western blot analysis of signal transduction in tumor cells and fibroblasts. In Curcumin-treated co-culture, in tumor cells, the levels of nuclear factor κB (NFκBα) and early response kinase (ERK)-decreased, in fibroblasts, integrin αv protein synthesis decreased compared to corresponding cells in normal co-culture. The signal modulatory changes induced by Curcumin caused decreased release of EMT-mediators in CAFs and reversal of EMT in tumor cells, which was associated with decreased invasion. These data confirm the palliative potential of Curcumin in clinical application.
Assuntos
Carcinoma de Células Escamosas/patologia , Comunicação Celular/efeitos dos fármacos , Curcumina/farmacologia , Fibroblastos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Biomarcadores Tumorais/metabolismo , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Técnicas de Cocultura/métodos , Transição Epitelial-Mesenquimal , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Integrina alfaV/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Neoplasias Bucais/metabolismo , Neoplasias Bucais/patologia , NF-kappa B/metabolismo , Invasividade Neoplásica/patologia , Ligamento Periodontal/citologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de SinaisRESUMO
A major goal in neuroscience is to elucidate the principles by which memories are stored in a neural network. Here, we have systematically studied how four types of associative memories (short- and long-term memories, each as positive and negative associations) are encoded within the compact neural network of Caenorhabditis elegans worms. Interestingly, sensory neurons were primarily involved in coding short-term, but not long-term, memories, and individual sensory neurons could be assigned to coding either the conditioned stimulus or the experience valence (or both). Moreover, when considering the collective activity of the sensory neurons, the specific training experiences could be decoded. Interneurons integrated the modulated sensory inputs and a simple linear combination model identified the experience-specific modulated communication routes. The widely distributed memory suggests that integrated network plasticity, rather than changes to individual neurons, underlies the fine behavioral plasticity. This comprehensive study reveals basic memory-coding principles and highlights the central roles of sensory neurons in memory formation.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/fisiologia , Interneurônios , Proteínas de Caenorhabditis elegans/fisiologia , Células Receptoras Sensoriais/fisiologia , Redes Neurais de ComputaçãoRESUMO
OBJECTIVE: To evaluate the pathophysiologic changes in the inner ear during the course of severe cerebral malaria in an established animal model, C57 BL/6J mice. METHODS: This study aims to examine the hearing threshold, the histological changes and ICAM-1 expression in the murine cochlea. RESULTS: Four of seven mice showed an expected hearing loss of 20 dB or more. The light microscopy of the inner ear did not show any morphologic alterations. The immunohistochemical analysis for ICAM-1 showed intensive staining in the stria vascularis of sick animals and hardly any reaction in healthy controls. CONCLUSION: The up-regulation of ICAM-1 in the stria vascularis - generating the endocochlear potential - suggests its involvement in plasmodial infection.
Assuntos
Perda Auditiva/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Malária Cerebral/metabolismo , Estria Vascular/metabolismo , Animais , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Modelos Animais , Plasmodium bergheiRESUMO
C. elegans worms exhibit a natural chemotaxis towards food cues. This provides a potential platform to study the interactions between stimulus valence and innate behavioral preferences. Here we perform a comprehensive set of choice assays to measure worms' relative preference towards various attractants. Surprisingly, we find that when facing a combination of choices, worms' preferences do not always follow value-based hierarchy. In fact, the innate chemotaxis behavior in worms robustly violates key rationality paradigms of transitivity, independence of irrelevant alternatives and regularity. These violations arise due to asymmetric modulatory effects between the presented options. Functional analysis of the entire chemosensory system at a single-neuron resolution, coupled with analyses of mutants, defective in individual neurons, reveals that these asymmetric effects originate in specific sensory neurons.
Assuntos
Comportamento Animal/fisiologia , Caenorhabditis elegans/fisiologia , Quimiotaxia/fisiologia , Células Receptoras Sensoriais/fisiologia , Animais , Sinais (Psicologia) , Tomada de Decisões/fisiologia , Modelos BiológicosRESUMO
We would like to make our readers aware of the publication by Cohen et al., which reports irrational behaviour in C. elegans olfactory preference[1] . These complementary studies establish C. elegans as a model system to explore the neural mechanisms of decision making.
RESUMO
Organisms' capacity to anticipate future conditions is key for survival. Associative memories are instrumental for learning from past experiences, yet little is known about the processes that follow memory retrieval and their potential advantage in preparing for impending developments. Here, using C. elegans nematodes, we demonstrate that odor-evoked retrieval of aversive memories induces rapid and protective stress responses, which increase animal survival prospects when facing imminent adversities. The underlying mechanism relies on two sensory neurons: one is necessary during the learning period, and the other is necessary and sufficient for memory retrieval. Downstream of memory reactivation, serotonin secreted from two head neurons mediates the systemic stress response. Thus, evoking stressful memories, stored within individual sensory neurons, allows animals to anticipate upcoming dire conditions and provides a head start to initiate rapid and protective responses that ultimately increase animal fitness.
Assuntos
Caenorhabditis elegans/fisiologia , Memória , Odorantes , Células Receptoras Sensoriais/fisiologia , Adaptação Fisiológica , AnimaisRESUMO
OBJECTIVE: EMT (epithelial to mesenchymal transition) contributes to tumor progression and metastasis. We aimed to investigate the effects of EMT on CDDP resistance in HNSCC (head and neck squamous cell carcinoma)-cells. METHODS: EMT was induced using conditioned medium from a tumor cell/fibroblast co-culture. HNSCC cells were alternatively treated with TGF-ß1. The response to CDDP was evaluated with viability and clonogenic assays. RESULTS: Treatment of SCC-25/ Detroit 562 cells with conditioned medium increased viability of the tumor cells. Moreover, it doubled the IC50 of CDDP of SCC-25 cells from 6.2 µM to 13.1 µM (p < 0.001). The IC50 of CDDP of Detroit 562 cells was increased following treatment with conditioned medium from 13.1 µM to 26.8 µM (p < 0.01). Colony forming ability after treatment with 5 or 10 µM CDDP was significantly higher in HNSCC cells treated with co-culture conditioned medium than in controls (p < 0.05). Treatment with TGF-ß1 had no effect on the IC50 of CDDP (p > 0.1). CONCLUSIONS: Cell free medium from a co-culture was able to induce EMT in HNSCC cells. Co-culture treated HNSCC cells revealed increased viability and were less sensitive to CDDP treatment. TGF-ß1 also induced a mesenchymal phenotype, but did not alter resistance to CDDP in HNSCC cells.
Assuntos
Carcinoma de Células Escamosas/patologia , Cisplatino/farmacologia , Meios de Cultivo Condicionados/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Fibroblastos/patologia , Gengiva/patologia , Neoplasias de Cabeça e Pescoço/patologia , Antineoplásicos/farmacologia , Apoptose , Western Blotting , Carcinoma de Células Escamosas/tratamento farmacológico , Proliferação de Células , Técnicas de Cocultura , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Gengiva/efeitos dos fármacos , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Humanos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Crescimento Transformador beta1/farmacologia , Células Tumorais CultivadasRESUMO
AIM: Systemic pharmacotherapies have limitation due to blood-labyrinth barrier, so local delivery via the round window membrane opens a path for effective treatment. Multifunctional nanoparticle (NP)-mediated cell specific drug delivery may enhance efficacy and reduce side effects. Different NPs with ligands to target TrkB receptor were tested. Distribution, uptake mechanisms, trafficking, and bioefficacy of drug release of rolipram loaded NPs were evaluated. METHODS: We tested lipid based nanocapsules (LNCs), Quantum Dot, silica NPs with surface modification by peptides mimicking TrkB or TrkB activating antibodies. Bioefficacy of drug release was tested with rolipram loaded LNCs to prevent cisplatin-induced apoptosis. We established different cell culture models with SH-SY-5Y and inner ear derived cell lines and used neonatal and adult mouse explants. Uptake and trafficking was evaluated with FACS and confocal as well as transmission electron microscopy. RESULTS: Plain NPs show some selectivity in uptake related to the in vitro system properties, carrier material, and NP size. Some peptide ligands provide enhanced targeted uptake to neuronal cells but failed to show this in cell cultures. Agonistic antibodies linked to silica NPs showed TrkB activation and enhanced binding to inner ear derived cells. Rolipram loaded LNCs proved as effective carriers to prevent cisplatin-induced apoptosis. DISCUSSION: Most NPs with targeting ligands showed limited effects to enhance uptake. NP aggregation and unspecific binding may change uptake mechanisms and impair endocytosis by an overload of NPs. This may affect survival signaling. NPs with antibodies activate survival signaling and show effective binding to TrkB positive cells but needs further optimization for specific internalization. Bioefficiacy of rolipram release confirms LNCs as encouraging vectors for drug delivery of lipophilic agents to the inner ear with ideal release characteristics independent of endocytosis.
RESUMO
Sprouty (Spry) proteins are negative feedback inhibitors of receptor tyrosine kinase signaling. Downregulation of Spry2 has been demonstrated to promote elongative axon growth of cultured peripheral and central neurons. Here, we analyzed Spry2 global knockout mice with respect to axon outgrowth in vitro and peripheral axon regeneration in vivo. Neurons dissociated from adult Spry2 deficient sensory ganglia revealed stronger extracellular signal-regulated kinase activation and enhanced axon outgrowth. Prominent axon elongation was observed in heterozygous Spry2(+/-) neuron cultures, whereas homozygous Spry2(-/-) neurons predominantly exhibited a branching phenotype. Following sciatic nerve crush, Spry2(+/-) mice recovered faster in motor but not sensory testing paradigms (Spry2(-/-) mice did not tolerate anesthesia required for nerve surgery). We attribute the improvement in the rotarod test to higher numbers of myelinated fibers in the regenerating sciatic nerve, higher densities of motor endplates in hind limb muscles and increased levels of GAP-43 mRNA, a downstream target of extracellular regulated kinase signaling. Conversely, homozygous Spry2(-/-) mice revealed enhanced mechanosensory function (von Frey's test) that was accompanied by an increased innervation of the epidermis, elevated numbers of nonmyelinated axons and more IB4-positive neurons in dorsal root ganglia. The present results corroborate the functional significance of receptor tyrosine kinase signaling inhibitors for axon outgrowth during development and nerve regeneration and propose Spry2 as a novel potential target for pharmacological inhibition to accelerate long-distance axon regeneration in injured peripheral nerves.
Assuntos
Axônios/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Proteínas de Membrana/deficiência , Regeneração Nervosa/genética , Neurônios/metabolismo , Animais , Proteína GAP-43/metabolismo , Heterozigoto , Homozigoto , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos BALB C , Camundongos Knockout , Atividade Motora/fisiologia , Regeneração Nervosa/fisiologia , Proteínas Serina-Treonina Quinases , Recuperação de Função Fisiológica/fisiologia , Nervo Isquiático/lesõesRESUMO
BACKGROUND: Due to their biochemical versatility, nanoparticles (NPs) have become one of the most important future carriers for drugs and genes. NP-mediated delivery could enable an effective pharmacotherapy to the inner ear and combat hearing loss. AIMS: This study investigates the endocytic trafficking of silica NPs within HEI-OC1 cells, a cell line derived from the inner ear. MATERIALS & METHODS: To investigate the interaction between 50-, 70- and 100-nm silica NPs and the cells, the authors employed a set of commonly available methods involving light and electron microscopy, and sample processing methods, which preserve the native cell shape and the fragile endocytic structures. RESULTS: The authors observed that 50-nm NPs were the most efficiently internalized. They also identified macropinocytosis as the dominant mechanism of uptake, showed localization of NPs in the early endosome and observed that silica NPs were delayed during trafficking to the lysosomes, where these NPs stayed confined, showing no endosomal escape. CONCLUSION: These silica NPs mostly rely on macropinocytosis for internalization. A successful use of silica NPs as vectors would involve smaller NPs and an endosomal escape strategy. Original submitted 21 December 2011; Revised submitted 23 May 2012; Published online 14 August 2012.
Assuntos
Nanopartículas/química , Órgão Espiral/citologia , Dióxido de Silício/química , Animais , Transporte Biológico/fisiologia , Linhagem Celular , Camundongos , Células NIH 3T3 , Nanopartículas/ultraestruturaRESUMO
The highly compartmentalized anatomy of the ear aggravates drug delivery, which is used to combat hearing-related diseases. Novel nanosized drug vehicles are thought to overcome the limitations of classic approaches. In this article, we summarize the nanotechnology-based efforts involving nano-objects, such as liposomes, polymersomes, lipidic nanocapsules and poly(lactic-co-glycolic acid) nanoparticles, as well as nanocoatings of implants to provide an efficient means for drug transfer in the ear. Modern strategies do not only enhance drug delivery efficiency, in the inner ear these vector systems also aim for specific uptake into hair cells and spiral ganglion neurons. These novel peptide-mediated strategies for specific delivery are reviewed in this article. Finally, the biosafety of these vector systems is still an outstanding issue, since long-term application to the ear has not yet been assessed.
Assuntos
Sistemas de Liberação de Medicamentos/métodos , Orelha , Nanomedicina/métodos , Animais , Cóclea/metabolismo , HumanosRESUMO
Hearing loss is frequent in intensive care patients and can be due to several causes. However, sepsis has not been examined as a possible cause. The aim of this study is to assess the influence of experimental sepsis on hearing thresholds and to evaluate pathological changes in the cochlea. The cecal ligation puncture technique was used to induce sepsis in 18 mice. Results were compared with those from 13 sham-operated and 13 untreated control mice. The hearing thresholds of the animals were evaluated with auditory evoked brainstem responses prior to the induction of sepsis and again at the peak of the disease. Immediately after the second measurement, the mice were sacrificed and the inner ears harvested and prepared for further evaluation. The cochleae were examined with light microscopy, electron microscopy and immunohistochemistry for Bax, cleaved caspase-3 and Bcl-2. The mice with sepsis showed a significant hearing loss but not the control groups. Induction of apoptosis could be shown in the supporting cells of the organ of Corti. Furthermore, excitotoxicity could be shown at the basal pole of the inner hair cells. In this murine model, sepsis leads to significant hearing impairment. The physiological alteration could be linked to apoptosis in the supporting cells of the organ of Corti and to a disturbance of the synapses of the inner hair cells.
Assuntos
Apoptose/efeitos dos fármacos , Ácido Glutâmico/toxicidade , Perda Auditiva/complicações , Perda Auditiva/patologia , Neurotoxinas/toxicidade , Sepse/complicações , Sepse/patologia , Animais , Temperatura Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Caspase 3/metabolismo , Cóclea/enzimologia , Cóclea/patologia , Cóclea/fisiopatologia , Cóclea/ultraestrutura , Potenciais Evocados Auditivos do Tronco Encefálico , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Ciliadas Auditivas Internas/patologia , Células Ciliadas Auditivas Internas/ultraestrutura , Perda Auditiva/fisiopatologia , Imuno-Histoquímica , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Punções , Sepse/fisiopatologia , Proteína X Associada a bcl-2/metabolismoRESUMO
Numerous studies on nanocarriers use fluorescent dye labeling to investigate their biodistribution or cellular trafficking. However, when the fluorescence dye is not grafted to the nanocarrier, the question of the stability of the labeling arises. How can it be validated that the fluorescence observed during an experiment corresponds to the nanocarriers, and not to the free dye released from the nanocarriers? Studying the integrity of the labeling is challenging. Therefore, an innovative approach to confirm the labeling stability was developed, based on the transfer of a fluorescent dye from its hosting nanocarrier to a lipophilic compartment. Lipid nanocapsules (LNC) and triglyceride oil were used as models. The protocol involved mixing of LNC suspension and oil, and then separation by centrifugation. The quality of the separation was controlled by light scattering, using the derived count rate tool. Dye transfer from loaded LNCs to the lipophilic compartment or from a lipophilic compartment containing dye to non-loaded LNC was investigated by varying the nature of the dye and the oil, the oil volume and the LNC dilution. Tensiometry was used to define the dye location in the nanocarrier. Results showed that when dyes such as Nile Red and Coumarin-6 are located in oily core, the transfer occurred in a partition-dependent manner. In contrast, when the dye was entrapped in the surfactant shell of LNCs such as lipophilic indocarbocyanines (i.e. DiO, DiI and DiD), no transfer was observed. Dye diffusion was also observed in cell culture, with Nile Red inside lipid bodies of HEI-OC1 cells, without uptake of LNCs. In contrast, DiO-loaded LNCs had to be internalized to observe fluorescence inside the cells, providing a further confirmation of the absence of transfer in this case, and the stability of fluorescence labeling of the LNCs.
Assuntos
Portadores de Fármacos/administração & dosagem , Corantes Fluorescentes/administração & dosagem , Nanocápsulas/administração & dosagem , Animais , Linhagem Celular , Portadores de Fármacos/química , Corantes Fluorescentes/química , Lipídeos/química , Camundongos , Nanocápsulas/química , Imagem ÓpticaRESUMO
Carbon dots were synthesized by a simple and green strategy for selective and sensitive Cu(2+) ion detection using both down and upconversion fluorescence. These fluorescent nanosensors show low cytotoxicity and are applied for intracellular sensing and imaging of Cu(2+) in biological systems.