RESUMO
Swept-source optical coherence tomography (SS-OCT) shows potential for the in vivo quantitative evaluation of micro-structural enamel surface phenomena occurring during early erosive demineralization. This randomized controlled single-blind cross-over clinical study aimed to evaluate the use of SS-OCT for detecting optical changes in the enamel of 30 healthy volunteers subjected to orange juice rinsing (erosive challenge) in comparison to mineral water rinsing (control), according to wiped and non-wiped enamel surface states. Participants were randomly allocated to 60 min of orange juice rinsing (pH 3.8) followed by 60 min of water rinsing (pH 6.7) and vice versa, with a 2-week wash-out period. In addition, the labial surfaces of the right or left maxillary incisors were wiped prior to SS-OCT imaging. An automated ImageJ algorithm was designed to analyse the back-scattered OCT signal intensity (D) after orange juice rinsing compared to after water rinsing. D was quantified as the OCT signal scattering from the 33 µm sub-surface enamel, normalised by the total OCT signal intensity entering the enamel. The back-scattered OCT signal intensity increased by 3.1% (95% CI 1.1-5.1%) in the wiped incisors and by 3.5% (95% CI 1.5-5.5%) in the unwiped incisors (p < 0.0001). Wiping reduced the back-scattered OCT signal intensity by 1.7% (95% CI -3.2 to -0.3%; p = 0.02) in comparison to the unwiped enamel surfaces for both rinsing solutions (p = 0.2). SS-OCT detected OCT signal changes in the superficial sub-surface enamel of maxillary central incisor teeth of healthy volunteers after orange juice rinsing.
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Esmalte Dentário/diagnóstico por imagem , Esmalte Dentário/patologia , Tomografia de Coerência Óptica/métodos , Desmineralização do Dente/diagnóstico por imagem , Erosão Dentária/diagnóstico por imagem , Adulto , Estudos Cross-Over , Feminino , Humanos , Masculino , Método Simples-Cego , Adulto JovemRESUMO
The regulation of Ubiquitin (Ub) conjugates generated by the complex network of proteins that promote the mammalian DNA double-strand break (DSB) response is not fully understood. We show here that the Ub protease POH1/rpn11/PSMD14 resident in the 19S proteasome regulatory particle is required for processing poly-Ub formed in the DSB response. Proteasome activity is required to restrict tudor domain-dependent 53BP1 accumulation at sites of DNA damage. This occurs both through antagonism of RNF8/RNF168-mediated lysine 63-linked poly-Ub and through the promotion of JMJD2A retention on chromatin. Consistent with this role POH1 acts in opposition to RNF8/RNF168 to modulate end-joining DNA repair. Additionally, POH1 acts independently of 53BP1 in homologous recombination repair to promote RAD51 loading. Accordingly, POH1-deficient cells are sensitive to DNA damaging agents. These data demonstrate that proteasomal POH1 is a key de-ubiquitinating enzyme that regulates ubiquitin conjugates generated in response to damage and that several aspects of the DSB response are regulated by the proteasome.
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Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Transativadores/metabolismo , Linhagem Celular , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Histona Desmetilases com o Domínio Jumonji/metabolismo , Lisina/metabolismo , Poliubiquitina/metabolismo , Rad51 Recombinase/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Apoptin, the VP3 protein from chicken anaemia virus (CAV), induces tumour cell-specific cell death and represents a potential future anti-cancer therapeutic. In tumour but not in normal cells, Apoptin is phosphorylated and translocates to the nucleus, enabling its cytotoxic activity. Recently, the ß isozyme of protein kinase C (PKCß) was shown to phosphorylate Apoptin in multiple myeloma cell lines. However, the exact mechanism and nature of interaction between PKCß and Apoptin remain unclear. Here we investigated the physical and functional link between PKCß and CAV-Apoptin as well as with the recently identified Apoptin homologue derived from human Gyrovirus (HGyV). In contrast to HCT116 colorectal cancer cells the normal colon mucosa cell lines expressed low levels of PKCßI and showed reduced Apoptin activation, as evident by cytoplasmic localisation, decreased phosphorylation and lack of cytotoxic activity. Co-immunoprecipitation and proximity ligation assay studies identified binding of both CAV- and HGyV-Apoptin to PKCßI in HCT116 cells. Using Apoptin deletion constructs the N-terminal domain of Apoptin was found to be required for interacting with PKCßI. FRET-based PKC activity reporter assays by fluorescence lifetime imaging microscopy showed that expression of Apoptin in cancer cells but not in normal cells triggers a significant increase in PKC activity. Collectively, the results demonstrate a novel cancer specific interplay between Apoptin and PKCßI. Direct interaction between the two proteins leads to Apoptin-induced activation of PKC and consequently activated PKCßI mediates phosphorylation of Apoptin to promote its tumour-specific nuclear translocation and cytotoxic function.
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Proteínas do Capsídeo/metabolismo , Proteína Quinase C beta/metabolismo , Núcleo Celular/metabolismo , Células HCT116 , Humanos , FosforilaçãoRESUMO
Breast cancer heterogeneity demands that prognostic models must be biologically driven and recent clinical evidence indicates that future prognostic signatures need evaluation in the context of early compared with late metastatic risk prediction. In pre-clinical studies, we and others have shown that various protein-protein interactions, pertaining to the actin microfilament-associated proteins, ezrin and cofilin, mediate breast cancer cell migration, a prerequisite for cancer metastasis. Moreover, as a direct substrate for protein kinase Cα, ezrin has been shown to be a determinant of cancer metastasis for a variety of tumour types, besides breast cancer; and has been described as a pivotal regulator of metastasis by linking the plasma membrane to the actin cytoskeleton. In the present article, we demonstrate that our tissue imaging-derived parameters that pertain to or are a consequence of the PKC-ezrin interaction can be used for breast cancer prognostication, with inter-cohort reproducibility. The application of fluorescence lifetime imaging microscopy (FLIM) in formalin-fixed paraffin-embedded patient samples to probe protein proximity within the typically <10 nm range to address the oncological challenge of tumour heterogeneity, is discussed.
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Neoplasias da Mama/patologia , Proteína Quinase C-alfa/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Neoplasias da Mama/enzimologia , Neoplasias da Mama/metabolismo , Proteínas do Citoesqueleto/metabolismo , Feminino , Transferência Ressonante de Energia de Fluorescência , Humanos , Metástase Neoplásica , Fosforilação , Frações Subcelulares/metabolismo , Especificidade por Substrato , Resultado do TratamentoRESUMO
A 1 MHz frame rate complementary metal-oxide semiconductor (CMOS) camera was used in combination with an image intensifier for wide-field time-correlated single-photon counting (TCSPC) imaging. The system combines an ultrafast frame rate with single-photon sensitivity and was employed on a fluorescence microscope to image decays of ruthenium compound Ru(dpp) with lifetimes from around 1 to 5 µs. A submicrowatt excitation power over the whole field of view is sufficient for this approach, and compatibility with live-cell imaging was demonstrated by imaging europium-containing beads with a lifetime of 570 µs in living HeLa cells. A standard two-photon excitation scanning fluorescence lifetime imaging (FLIM) system was used to independently verify the lifetime for the europium beads. This approach brings together advantageous features for time-resolved live-cell imaging such as low excitation intensity, single-photon sensitivity, ultrafast camera frame rates, and short acquisition times.
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Microscopia/métodos , Fótons , Európio/química , Európio/metabolismo , Glicerol/química , Células HeLa , Humanos , Medições Luminescentes , Compostos Organometálicos/química , Fenantrolinas/química , Semicondutores , Fatores de Tempo , Água/químicaRESUMO
A novel, fast, and easy mechano-chemistry-based (dry milling) method has been developed to exfoliate graphene with hydrophobic drugs generating few-layer graphene mesosheets (< 10 nm in thickness and â¼1 µm in width). The electronic properties of the graphitic structure were partially preserved after the milling treatment compared with graphene oxide prepared by Hummers' method. Several characterization techniques such as thermogravimetric analysis, Raman spectroscopy, atomic force microscopy, electron microscopy, and molecular dynamics simulation were used to characterize this material. The drug-exfoliated mesosheets were pharmacologically inactive, offering a new approach for making water-soluble few-layer graphene mesosheets upon dry milling with hydrophobic drugs, mainly used as exfoliating agents.
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Anfotericina B/farmacologia , Grafite/química , Água/química , Antibacterianos/farmacologia , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Microscopia de Força Atômica , Simulação de Dinâmica Molecular , Solubilidade , Propriedades de SuperfícieRESUMO
We have developed an interferometric implementation of coherent anti-Stokes Raman scattering which enables broadband coherent Raman spectroscopy free from the nonresonant background, with a signal strength proportional to concentration. Spectra encode mode symmetry information into the amplitude response, which can be directly compared to polarized spontaneous Raman spectra. The method requires only passive polarization optics and is suitable for a wide range of laser linewidths and pulse durations. The method's application to Raman spectral imaging is demonstrated.
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Interferometria/métodos , Modelos Teóricos , Análise Espectral Raman/métodosRESUMO
OBJECTIVES: To investigate the potential mineralising effects of calcium silicate-based dentine replacement material (Biodentine™) in comparison with glass-ionomer cement (GIC) (Fuji IX™) on different human dentine substrates using a multimodal non-invasive optical assessment. METHODS: Cements were applied on artificially demineralised or naturally carious dentine and stored for 4 weeks in phosphate-rich media +/- tetracycline used for mineralisation labelling. Interfacial dentine was examined from the same sample and location before and after aging using two-photon fluorescence microscopy, fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) imaging. Additionally, Raman spectroscopy was used to detect changes in the mineral content of dentine. RESULTS: Significant changes in the fluorescence intensity and lifetime were detected in partially demineralised dentine and caries-affected dentine underneath both tested cements, after storage (p < 0.001). This was associated with a significant increase in the mineral content as indicated by the increased intensity of the phosphate Raman peak located at 959 cm-1 (p < 0.0001). Caries-infected dentine showed significant fluorescence changes under Biodentine™ after storage (p < 0.001), but not under GIC (p = 0.44). Tetracycline binding induced a reduction in the fluorescence lifetime with comparable increase in the fluorescence intensity in both cements' groups within the affected dentine (p < 0.001). Significance Two-photon fluorescence microscopy can be used efficiently for non-destructive in-vitro dentine caries characterisation providing a technique for studying the same dentine-cement interface over time and detect changes. Biodentine™ demonstrated comparable remineralising potential to GIC, in addition to inducing remineralisation of caries-infected dentine. This may suggest using Biodentine™ as part of minimally invasive operative dentistry (MID) in caries management.
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Cárie Dentária , Análise Espectral Raman , Resinas Acrílicas , Compostos de Cálcio , Dentina/química , Cimentos de Ionômeros de Vidro/química , Cimentos de Ionômeros de Vidro/farmacologia , Humanos , Microscopia de Fluorescência , Minerais , Fosfatos , Silicatos , Dióxido de Silício , Tetraciclinas/análiseRESUMO
Development of remote imaging for diagnostic purposes has progressed dramatically since endoscopy began in the 1960's. The recent advent of a clinically licensed intensity-based fluorescence micro-endoscopic instrument has offered the prospect of real-time cellular resolution imaging. However, interrogating protein-protein interactions deep inside living tissue requires precise fluorescence lifetime measurements to derive the Förster resonance energy transfer between two tagged fluorescent markers. We developed a new instrument combining remote fiber endoscopic cellular-resolution imaging with TCSPC-FLIM technology to interrogate and discriminate mixed fluorochrome labeled beads and expressible GFP/TagRFP tags within live cells. Endoscopic-FLIM (e-FLIM) data was validated by comparison with data acquired via conventional FLIM and e-FLIM was found to be accurate for both bright bead and dim live cell samples. The fiber based micro-endoscope allowed remote imaging of 4 microm and 10 microm beads within a thick Matrigel matrix with confident fluorophore discrimination using lifetime information. More importantly, this new technique enabled us to reliably measure protein-protein interactions in live cells embedded in a 3D matrix, as demonstrated by the dimerization of the fluorescent protein-tagged membrane receptor CXCR4. This cell-based application successfully demonstrated the suitability and great potential of this new technique for in vivo pre-clinical biomedical and possibly human clinical applications.
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Endoscópios , Tecnologia de Fibra Óptica/instrumentação , Transferência Ressonante de Energia de Fluorescência/instrumentação , Aumento da Imagem/instrumentação , Mapeamento de Interação de Proteínas/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Miniaturização , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
This study aimed to investigate the null hypothesis that there is no relationship between the microhardness of carious dentine and its native autofluorescence (AF). Six extracted, carious molars were sectioned through natural lesions in the mesio-distal longitudinal plane. The Knoop microhardness (Knoop hardness number, KHN) of the cut surfaces of each sample was recorded at regular intervals through sound and carious dentine. Confocal fibre-optic micro-endoscopic (CFOME) examination of the carious dentine and the sound dentine was carried out at the same intervals using the Cellvizio system (600 microm wide, flat-end probe) with an excitation wavelength of 488 nm. The blindly collected numerical data were analysed using the original microhardness KHN. The data analysis indicated that the autofluorescence signals increased significantly when the microhardness of dentine dropped below 25 KHN. Therefore, the null hypothesis was disproved, and it was concluded from this investigation that the autofluorescent signal intensity recorded using CFOME could produce an objective and reproducible correlation to the microhardness of carious dentine. Confocal fibre-optic micro-endoscopic examination could have clinical potential as a technology to help delineate the carious dentine that might be excavated in a clinical procedure in vivo.
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Testes de Atividade de Cárie Dentária/métodos , Cárie Dentária/patologia , Dentina/patologia , Tecnologia de Fibra Óptica , Fluorescência , Dureza , Humanos , Microscopia Confocal , Dente Serotino/patologiaRESUMO
BACKGROUND: The aim of this longitudinal, one-year cohort study was to explore the hypothesis that fluorescence sampling of the root canal space prior to obturation could predict the outcome of root canal treatment (RCT). METHODS: Sixty-five teeth underwent primary RCT and were followed up clinically and radiographically. The outcome was determined radiographically with periapical radiographs (PR) and cone beam computed tomography (CBCT) scans. RESULTS: Success at 12 months was predictable based on the fluorescence score. When the fluorescence score (defined as the percentage of signal over total signal including background) was lower than 67, there was a 4.5 times (Odds ratio (OR) = 0.028; 95% confidence interval (CI): 0.003, 0.291, p = 0.001) greater chance of success (90% overall). When the readings were above this threshold, the success rate was 20%. CONCLUSION: A chairside sampling method is able to predict the outcome of RCT, through the use of paper point sampling and fluorescence staining. This has reduced the prevalence of persistent infections by guiding the optimum time for obturation. ClinicalTrials.gov trial NCT03660163.
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Lymphatic vasculature is crucial for metastasis in triple-negative breast cancer (TNBC); however, cellular and molecular drivers controlling lymphovascular metastasis are poorly understood. We define a macrophage-dependent signaling cascade that facilitates metastasis through lymphovascular remodeling. TNBC cells instigate mRNA changes in macrophages, resulting in ß4 integrin-dependent adhesion to the lymphovasculature. ß4 integrin retains macrophages proximal to lymphatic endothelial cells (LECs), where release of TGF-ß1 drives LEC contraction via RhoA activation. Macrophages promote gross architectural changes to lymphovasculature by increasing dilation, hyperpermeability, and disorganization. TGF-ß1 drives ß4 integrin clustering at the macrophage plasma membrane, further promoting macrophage adhesion and demonstrating the dual functionality of TGF-ß1 signaling in this context. ß4 integrin-expressing macrophages were identified in human breast tumors, and a combination of vascular-remodeling macrophage gene signature and TGF-ß signaling scores correlates with metastasis. We postulate that future clinical strategies for patients with TNBC should target crosstalk between ß4 integrin and TGF-ß1.
Assuntos
Integrina beta4/metabolismo , Vasos Linfáticos/citologia , Vasos Linfáticos/patologia , Macrófagos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Animais , Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Células Endoteliais/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Células HEK293 , Humanos , Integrina beta4/genética , Metástase Linfática , Vasos Linfáticos/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Transdução de Sinais/genética , Fator de Crescimento Transformador beta1/genética , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , CalininaRESUMO
OBJECTIVE: To test the null hypothesis that chitosan application has no impact on the remineralisation of artificial incipient enamel white spot lesions (WSLs). METHODS: 66 artificial enamel WSLs were assigned to 6 experimental groups (n=11): (1) bioactive glass slurry, (2) bioactive glass containing polyacrylic acid (BG+PAA) slurry, (3) chitosan pre-treated WSLs with BG slurry (CS-BG), (4) chitosan pre-treated WSLs with BG+PAA slurry (CS-BG+PAA), (5) remineralisation solution (RS) and (6) de-ionised water (negative control, NC). Surface and cross-sectional Raman intensity mapping (960cm-1) were performed on 5 samples/group to assess mineral content. Raman spectroscopy and attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) were used to identify the type of newly formed minerals. Surface and cross-sectional Knoop microhardness were implemented to evaluate the mechanical properties after remineralisation. Surface morphologies and Ca/P ratio were observed using scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy (EDX). Data were statistically analysed using one-way ANOVA with Tukey's test. RESULTS: BG+PAA, CS-BG, RS presented significantly higher mineral regain compared to NC on lesion surfaces, while CS-BG+PAA had higher subsurface mineral content. Newly mineralised crystals consist of type-B hydroxycarbonate apatite. CS-BG+PAA showed the greatest hardness recovery, followed by CS-BG, both significantly higher than other groups. SEM observations showed altered surface morphologies in all experimental groups except NC post-treatment. EDX suggested a higher content of carbon, oxygen and silicon in the precipitations in CS-BG+PAA group. There was no significant difference between each group in terms of Ca/P ratio. SIGNIFICANCE: The null hypothesis was rejected. Chitosan pre-treatment enhanced WSL remineralisation with either BG only or with BG-PAA complexes. A further investigation using dynamic remineralisation/demineralisation system is required with regards to clinical application.
Assuntos
Quitosana/farmacologia , Desmineralização do Dente/tratamento farmacológico , Remineralização Dentária/métodos , Resinas Acrílicas/química , Cerâmica/química , Esmalte Dentário/química , Esmalte Dentário/efeitos dos fármacos , Dureza , Teste de Materiais , Microscopia Eletrônica de Varredura , Espectrometria por Raios X , Espectroscopia de Infravermelho com Transformada de Fourier , Análise Espectral Raman , Propriedades de SuperfícieRESUMO
Modifiable hydrogels have revealed tremendous insight into how physical characteristics of cells' 3D environment drive stem cell lineage specification. However, in native tissues, cells do not passively receive signals from their niche. Instead they actively probe and modify their pericellular space to suit their needs, yet the dynamics of cells' reciprocal interactions with their pericellular environment when encapsulated within hydrogels remains relatively unexplored. Here, we show that human bone marrow stromal cells (hMSC) encapsulated within hyaluronic acid-based hydrogels modify their surroundings by synthesizing, secreting and arranging proteins pericellularly or by degrading the hydrogel. hMSC's interactions with this local environment have a role in regulating hMSC fate, with a secreted proteinaceous pericellular matrix associated with adipogenesis, and degradation with osteogenesis. Our observations suggest that hMSC participate in a bi-directional interplay between the properties of their 3D milieu and their own secreted pericellular matrix, and that this combination of interactions drives fate.
Assuntos
Comunicação Celular , Linhagem da Célula , Junções Célula-Matriz/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Amidas/farmacologia , Comunicação Celular/efeitos dos fármacos , Linhagem da Célula/efeitos dos fármacos , Junções Célula-Matriz/efeitos dos fármacos , Humanos , Hidrogel de Polietilenoglicol-Dimetacrilato , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Paclitaxel/farmacologia , Piridinas/farmacologia , Células-Tronco/efeitos dos fármacosRESUMO
In the original version of this Article the dataset identifier in the Data Availability statement was incorrect. The correct dataset identifier is PXD009500. This has been corrected in the HTML and PDF versions of this Article.
RESUMO
The original version of this Article contained an error in the author affiliations. The affiliation of Marjan Enayati with 'Ludwig Boltzmann Cluster for Cardiovascular Research at the Center for Biomedical Research, Medical University of Vienna, Austria' was inadvertently omitted. This has now been corrected in both the PDF and HTML versions of the Article.
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Fluorescence lifetime imaging (FLIM) represents a key optical technique for imaging proteins and protein interaction in vivo. We review the principles and recent advances in the application of the technique, instrumentation and molecular probe development.
Assuntos
Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Animais , Anisotropia , Fluorescência , Transferência Ressonante de Energia de Fluorescência , Humanos , Microscopia de Fluorescência por Excitação Multifotônica , Sondas MolecularesRESUMO
Molar incisor hypomineralization (MIH) affects the permanent incisors and molars, whose undermineralized matrix is evidenced by lesions ranging from white to yellow/brown opacities to crumbling enamel lesions incapable of withstanding normal occlusal forces and function. Diagnosing the condition involves clinical and radiographic examination of these teeth, with known limitations in determining the depth extent of the enamel defects in particular. Optical coherence tomography (OCT) is an emerging hard and soft tissue imaging technique, which was investigated as a new potential diagnostic method in dentistry. A comparison between the diagnostic potential of the conventional methods and OCT was conducted. Compared to conventional imaging methods, OCT gave more information on the structure of the enamel defects as well as the depth extent of the defects into the enamel structure. Different types of enamel defects were compared, each type presenting a unique identifiable pattern when imaged using OCT. Additionally, advanced methods of OCT image analysis including backscattered light intensity profile analysis and enface reconstruction were performed. Both methods confirmed the potential of OCT in enamel defects diagnosis. In conclusion, OCT imaging enabled the identification of the type of enamel defect and the determination of the extent of the enamel defects in MIH with the advantage of being a radiation free diagnostic technique.
Assuntos
Hipoplasia do Esmalte Dentário/diagnóstico por imagem , Esmalte Dentário/diagnóstico por imagem , Interpretação de Imagem Assistida por Computador/métodos , Tomografia de Coerência Óptica/métodos , Esmalte Dentário/patologia , Hipoplasia do Esmalte Dentário/patologia , Humanos , Dente/diagnóstico por imagem , Dente/patologiaRESUMO
Earlier studies proved the success of using chemically functionalised multi-walled carbon nanotubes (f-MWNTs) as nanocarriers to the brain. Little insight into the kinetics of brain distribution of f-MWNTs in vivo has been reported. This study employed a wide range of qualitative and quantitative techniques with the aim of shedding the light on f-MWNT's brain distribution following intravenous injection. γ-Scintigraphy quantified the uptake of studied radiolabelled f-MWNT in the whole brain parenchyma and capillaries while 3D-single photon emission computed tomography/computed tomography imaging and autoradiography illustrated spatial distribution within various brain regions. Raman and multiphoton luminescence together with transmission electron microscopy confirmed the presence of intact f-MWNT in mouse brain, in a label-free manner. The results evidenced the presence of f-MWNT in mice brain parenchyma, in addition to brain endothelium. Such information on the rate and extent of regional and cellular brain distribution is needed before further implementation into neurological therapeutics can be made.