RESUMO
We report on an innovative, fabric-based conformable, and easily fabricated electroceutical wound dressing that inhibits bacterial biofilm infections and shows significant promise for healing chronic wounds. Cyclic voltammetry demonstrates the ability of the electroceutical to produce reactive oxygen species, primarily HOCl that is responsible for bacterial inhibition. In vitro investigation with the lawn biofilm grown on a soft tissue mimic assay shows the efficacy of the dressing against both gram-positive and gram-negative bacteria in the biofilm form. In vivo, the printed electroceutical dressing was utilized as an intervention treatment for a canine subject with a non-healing wound due to a year-long persistent polymicrobial infection. The clinical case study with the canine subject exhibited the applicability in a clinical setting with the results showing infection inhibition within 11 days of initial treatment. This printed electroceutical dressing was integrated with a Bluetooth® enabled circuit allowing remote monitoring of the current flow within the wound bed. The potential to monitor wounds remotely in real-time with a Bluetooth® enabled circuit proposes a new physical biomarker for management of infected, chronic wounds.
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We present a method for designing and optimizing an in-house designed electromagnetic probe for distinguishing morphological differences in biological tissues. The probe comprises concentric multi-wound coils, the inner being the primary coil and the outer being the detector coil. A time-varying voltage is imposed on the primary coil, resulting in an induced current in the detector coil. For highly conductive samples, eddy currents are induced in the sample and inductively couple with the electromagnetic probe. However, in weakly conducting samples, the primary coupling mechanism is found to be capacitive though there can be a non-negligible inductive component. Both the mutual inductive coupling and the capacitive coupling between the sample and the probe are detected as a change in the induced voltage of the detector coil using lock-in detection. The induced voltage in the detector coil is influenced more by the morphological structure of the specimen rather than by changes in electrical conductivity within different regions of the sample. The instrument response of the lock-in amplifier is also examined with simulated input voltage signals to relate its output to specific changes in inductive and capacitive coupling, in order to relate sample characteristics to a single voltage output. A circuit element model is used to interpret the experimental measurements. It is found that the sensitivity of the measurement for a given set of probe characteristics (resistances, inductances, and capacitances) can be optimized by adding a small amount of capacitance in the external circuit in parallel with the detector coil. Illustrative measurements are presented on animal (porcine and bovine) tissue and on human liver tissue containing a metastatic tumor to demonstrate the capabilities of the probe and measurement method in distinguishing different tissue types despite having similar electrical conductivities. Since biological tissues are multi-scale, heterogeneous materials comprising regions of differing conductivity, permittivity, and morphological structure, the electromagnetic method presented here has the potential to examine structural variations in tissue undergoing physical changes due to healing or disease.
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Técnicas Citológicas/instrumentação , Campos Eletromagnéticos , Animais , Bovinos , Desenho de Equipamento , HumanosRESUMO
Pulmonary arterial hypertension (PH) and chronic kidney disease (CKD) both profoundly impact patient outcomes, whether as primary disease states or as co-morbid conditions. PH is a common co-morbidity in CKD and vice versa. A growing body of literature describes the epidemiology of PH secondary to chronic kidney disease and end-stage renal disease (ESRD) (WHO group 5 PH). But, there are only limited data on the epidemiology of kidney disease in group 1 PH (pulmonary arterial hypertension [PAH]). The purpose of this review is to summarize the current data on epidemiology and discuss potential disease mechanisms and management implications of kidney dysfunction in PAH. Kidney dysfunction, determined by serum creatinine or estimated glomerular filtration rate, is a frequent co-morbidity in PAH and impaired kidney function is a strong and independent predictor of mortality. Potential mechanisms of PAH affecting the kidneys are increased venous congestion, decreased cardiac output, and neurohormonal activation. On a molecular level, increased TGF-ß signaling and increased levels of circulating cytokines could have the potential to worsen kidney function. Nephrotoxicity does not seem to be a common side effect of PAH-targeted therapy. Treatment implications for kidney disease in PAH include glycemic control, lifestyle modification, and potentially Renin-Angiotensin-Aldosterone System (RAAS) blockade.
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Hematopoietic stem cell transplantation (HSCT) is often used in the treatment of hematologic disorders. Although it can be curative, the pre-transplant conditioning regimen can be associated with neurotoxicity. In this prospective study, we examined white matter (WM) integrity with diffusion tensor imaging (DTI) and neuropsychological functioning before and one year after HSCT in twenty-two patients with hematologic disorders and ten healthy controls evaluated at similar intervals. Eighteen patients received conditioning treatment with high-dose (HD) chemotherapy, and four had full dose total body irradiation (fTBI) and HD chemotherapy prior to undergoing an allogeneic or autologous HSCT. The results showed a significant decrease in mean diffusivity (MD) and axial diffusivity (AD) in diffuse WM regions one year after HSCT (p-corrected <0.05) in the patient group compared to healthy controls. At baseline, patients treated with allogeneic HSCT had higher MD and AD in the left hemisphere WM than autologous HSCT patients (p-corrected <0.05). One year post-transplant, patients treated with allogeneic HSCT had lower fractional anisotropy (FA) and higher radial diffusivity (RD) in the right hemisphere and left frontal WM compared to patients treated with autologous HSCT (p-corrected <0.05).There were modest but significant correlations between MD values and cognitive test scores, and these were greatest for timed tests and in projection tracts. Patients showed a trend toward a decline in working memory, and had lower cognitive test scores than healthy controls at the one-year assessment. The findings suggest a relatively diffuse pattern of alterations in WM integrity in adult survivors of HSCT.
Assuntos
Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Substância Branca/patologia , Adulto , Células-Tronco Adultas/fisiologia , Células-Tronco Adultas/transplante , Idoso , Anisotropia , Encéfalo/patologia , Cognição/fisiologia , Transtornos Cognitivos/fisiopatologia , Imagem de Tensor de Difusão/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Substância Branca/anatomia & histologiaRESUMO
Point-wise ex vivo electrical impedance spectroscopy measurements were conducted on excised hepatic tissue from human patients with metastatic colorectal cancer using a linear four-electrode impedance probe. This study of 132 measurements from 10 colorectal cancer patients, the largest to date, reports that the equivalent electrical conductivity for tumor tissue is significantly higher than normal tissue (p < 0.01), ranging from 2-5 times greater over the measured frequency range of 100 Hz-1 MHz. Difference in tissue electrical permittivity is also found to be statistically significant across most frequencies. Furthermore, the complex impedance is also reported for both normal and tumor tissue. Consistent with trends for tissue electrical conductivity, normal tissue has a significantly higher impedance than tumor tissue (p < 0.01), as well as a higher net capacitive phase shift (33° for normal liver tissue in contrast to 10° for tumor tissue).
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Neoplasias Colorretais/secundário , Fígado/fisiopatologia , Fígado/cirurgia , Adulto , Idoso , Impedância Elétrica , Feminino , Humanos , Técnicas In Vitro , Masculino , Pessoa de Meia-Idade , Fotografação/instrumentação , Reprodutibilidade dos TestesRESUMO
BACKGROUND: Matrix metalloproteinase-9 (MMP-9) secreted by corneal epithelial cells has a role in the remodelling of extracellular matrix and migration of epithelial cells. Elevated levels of MMP-9 activity in the ocular surface may be involved in the pathogenesis of corneal diseases. N-acetylcysteine (NAC) has been used to treat corneal diseases, including recurrent epithelial erosions. In this study, its effects on the MMP-9 secretion and human corneal epithelial (HCE) cell migration were evaluated in vitro. METHODS: Confluent HCE cell cultures were treated with 0-20 mM NAC, and tested for MMP-9 secretion and epithelial cell migration by gelatin zymography and scratch wound assay, respectively. Comparisons between different treatment groups were made using analysis of variance, followed by multiple pairwise comparisons. RESULTS: Twenty mM NAC inhibited the secretion of MMP-9 significantly. Cell migration, assessed after 24 h of wounding, showed a highly significant dose-dependent inhibitory effect. CONCLUSIONS: This study shows that NAC reduces MMP-9 production by HCE cells and inhibits cell migration in vitro. This information helps to elucidate the mechanisms by which NAC may be beneficial therapeutically and suggests that NAC may be useful for managing corneal erosions and related conditions.
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Acetilcisteína/farmacologia , Movimento Celular/efeitos dos fármacos , Epitélio Corneano/efeitos dos fármacos , Sequestradores de Radicais Livres/farmacologia , Metaloproteinase 9 da Matriz/metabolismo , Células 3T3 , Animais , Células Cultivadas , Técnicas de Cocultura , Relação Dose-Resposta a Droga , Epitélio Corneano/citologia , Epitélio Corneano/enzimologia , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Camundongos , Doadores de Tecidos , CicatrizaçãoRESUMO
Mutations in bone morphogenetic protein receptor type 2 (BMPR2) cause familial pulmonary arterial hypertension (FPAH), but the penetrance is reduced and females are significantly overrepresented. In addition, gene expression data implicating the oestrogen-metabolising enzyme CYP1B1 suggests a detrimental role of oestrogens or oestrogen metabolites. We examined genetic and metabolic markers of altered oestrogen metabolism in subjects with a BMPR2 mutation. Genotypes for CYP1B1 Asn453Ser (N453S) were determined for 140 BMPR2 mutation carriers (86 females and 54 males). Nested from those subjects, a case-control study of urinary oestrogen metabolite levels (2-hydroxyoestrogen (2-OHE) and 16alpha-hydroxyoestrone (16alpha-OHE(1))) was conducted in females (five affected mutation carriers versus six unaffected mutation carriers). Among females, there was four-fold higher penetrance among subjects homozygous for the wild-type genotype (N/N) than those with N/S or S/S genotypes (p = 0.005). Consistent with this finding, the 2-OHE/16alpha-OHE(1) ratio was 2.3-fold lower in affected mutation carriers compared to unaffected mutation carriers (p = 0.006). Our findings suggest that variations in oestrogens and oestrogen metabolism modify FPAH risk. Further investigation of the role of oestrogens in this disease with profound sex bias may yield new insights and, perhaps, therapeutic interventions.
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Receptores de Proteínas Morfogenéticas Ósseas Tipo II/genética , Estrogênios/metabolismo , Hipertensão/diagnóstico , Hipertensão/epidemiologia , Artéria Pulmonar/fisiopatologia , Adulto , Idoso , Receptores de Proteínas Morfogenéticas Ósseas Tipo II/metabolismo , Estudos de Casos e Controles , Estudos de Coortes , Feminino , Genótipo , Heterozigoto , Humanos , Hipertensão/metabolismo , Masculino , Pessoa de Meia-Idade , Mutação , Polimorfismo Genético , Fatores SexuaisRESUMO
OBJECTIVE: To examine the neural basis of cognitive complaints in healthy older adults in the absence of memory impairment and to determine whether there are medial temporal lobe (MTL) gray matter (GM) changes as reported in Alzheimer disease (AD) and amnestic mild cognitive impairment (MCI). METHODS: Participants were 40 euthymic individuals with cognitive complaints (CCs) who had normal neuropsychological test performance. The authors compared their structural brain MRI scans to those of 40 patients with amnestic MCI and 40 healthy controls (HCs) using voxel-based morphometry and hippocampal volume analysis. RESULTS: The CC and MCI groups showed similar patterns of decreased GM relative to the HC group on whole brain analysis, with differences evident in the MTL, frontotemporal, and other neocortical regions. The degree of GM loss was associated with extent of both memory complaints and performance deficits. Manually segmented hippocampal volumes, adjusted for age and intracranial volume, were significantly reduced only in the MCI group, with the CC group showing an intermediate level. CONCLUSIONS: Cognitive complaints in older adults may indicate underlying neurodegenerative changes even when unaccompanied by deficits on formal testing. The cognitive complaint group may represent a pre-mild cognitive impairment stage and may provide an earlier therapeutic opportunity than mild cognitive impairment. MRI analysis approaches incorporating signal intensity may have greater sensitivity in early preclinical stages than volumetric methods.
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Envelhecimento/psicologia , Hipocampo/patologia , Transtornos da Memória/patologia , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/patologia , Análise de Variância , Atrofia , Mapeamento Encefálico , Feminino , Humanos , Imageamento por Ressonância Magnética/métodos , Masculino , Transtornos da Memória/psicologia , Testes Neuropsicológicos , Aprendizagem Verbal/fisiologiaRESUMO
AIM: Leukaemia inhibitory factor (LIF) is a pleotrophic cytokine expressed in a variety of cell types, and have shown to regulate stem cell proliferation, vascular genesis, inflammation, and immunity in various locations. Expression of LIF and its role in the cornea have not been studied previously. In this study, we examined the expression of LIF in the cornea. MATERIALS AND METHOD: Immunohistochemistry was performed using polyclonal LIF antibodies, and Avidin-Biotin ABC complex on cultured human corneal epithelium corneal fibroblasts and wild-type murine corneal epithelium. RESULTS: LIF was detected in the cytoplasm of murine corneal epithelium, cultured human corneal epithelium, and fibroblasts. The expression of LIF was mainly cytoplasmic. CONCLUSION: LIF is expressed in the corneal epithelium and fibroblasts. It may have an important role in the maintenance of homeostasis of the corneal epithelium and cornea stroma. Further studies are necessary to elucidate the role of LIF in the cornea.
Assuntos
Córnea/química , Interleucina-6/análise , Animais , Células Cultivadas , Citoplasma/química , Células Epiteliais/química , Epitélio Corneano/química , Fibroblastos/química , Humanos , Técnicas Imunoenzimáticas , Fator Inibidor de Leucemia , CamundongosRESUMO
Mosaic beta-galactosidase reporter staining patterns in the adult adrenal cortex of 21-OH/LacZ transgenic mice were compared to those observed in mouse chimeras and X-inactivation mosaics, which are known to have a lineage basis. This revealed similar patterns of blue and white radial stripes in all three experimental groups. Each blue stripe may contain one or more blue coherent clones of cells but this was taken into account by correcting the observed stripe numbers for the effects of different proportions of LacZ-positive (blue) and LacZ-negative (unstained) cells between adrenals. The corrected stripe numbers were similar in all three experimental groups, which supports the hypothesis that the stripes in the adrenals of 21-OH/LacZ transgenic mice are formed in a similar way to those in chimeras and X-inactivation mosaics (i.e., they have a lineage basis). This suggests that the 21-OH/LacZ transgenic mouse is likely to be a valid model for studying steroidogenic cell lineage in the adrenal cortex, thereby providing additional support for the centripetal migration hypothesis of adrenocortical cytogenesis.
Assuntos
Córtex Suprarrenal/citologia , Óperon Lac , Esteroide 21-Hidroxilase/genética , Córtex Suprarrenal/enzimologia , Animais , Linhagem Celular , Quimera , Inativação Gênica , Genes Reporter , Camundongos , Camundongos Transgênicos , Modelos Animais , Modelos Biológicos , Mosaicismo , Coloração e Rotulagem , Cromossomo X , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Mouse embryos homozygous for a null allele of Gpi1 which encodes the glycolytic enzyme glucose phosphate isomerase fail to complete gastrulation and die at about embryonic day 7.5, but mutant cells can survive in fetal chimaeras in which they are mixed with wild-type cells. An adult female mouse chimaera, composed of wild-type cells and homozygous Gpi1(-/-) null mutant cells, was produced to test whether the presence of wild-type cells in the ovary allowed mutant oocytes to survive and function. This mouse produced 28 offspring, eight of which were derived from homozygous Gpi1(-/-) null oocytes. DNA in situ hybridization also showed that some Gpi1(-/-) follicle cells were able to survive in chimaeric ovarian follicles. It is likely that the survival of mutant follicle cells and fully functional mutant oocytes was mediated by the presence of wild-type cells that could provide metabolic intermediates and so bypass the block in the glycolytic pathway. Wild-type cumulus cells probably supported the growing GPI-deficient oocytes via metabolic co-operation, by passing ATP and other glycolytic products through gap junctions. It was concluded that female mouse germ cells and ovarian follicle cells do not need an intact endogenous glycolytic pathway if they can obtain appropriate metabolites from an exogenous source.
Assuntos
Glucose-6-Fosfato Isomerase/genética , Glicólise/genética , Oócitos/fisiologia , Anemia Hemolítica Congênita não Esferocítica , Animais , Comunicação Celular/genética , Sobrevivência Celular/genética , Quimera , Feminino , Camundongos , Camundongos Mutantes , Mutação , Oócitos/enzimologia , Folículo Ovariano/fisiologiaRESUMO
We describe lens defects in heterozygous small eye mice, and autonomous deficiencies of Pax6(+/-) cells in the developing lens of Pax6(+/+) <--> Pax6(+/-) chimeras. Two separate defects of the lens were identified by analyzing the distribution of heterozygous cells in chimeras: Pax6(+/-) cells are less readily incorporated into the lens placode than wild type, and those that are incorporated into the lens are not maintained efficiently in the proliferating lens epithelium. The lens of chimeric eyes is, therefore, predominantly wild type from embryonic day 16.5 onwards, whereas heterozygous cells contribute normally to all other eye tissues. Eye size and defects of the iris and cornea are corrected in fetal and adult chimeras with up to 80% mutant cells. Therefore, these aspects of the phenotype may be secondary consequences of primary defects in the lens, which has clinical relevance for the human aniridia (PAX6(+/-)) phenotype.
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Segmento Anterior do Olho/anormalidades , Anormalidades do Olho/genética , Proteínas do Olho/fisiologia , Proteínas de Homeodomínio/fisiologia , Cristalino/anormalidades , Animais , Segmento Anterior do Olho/embriologia , Linhagem da Célula , Quimera , Modelos Animais de Doenças , Células Epiteliais/patologia , Proteínas do Olho/genética , Heterozigoto , Proteínas de Homeodomínio/genética , Cristalino/embriologia , Camundongos , Camundongos Mutantes , Morfogênese/genética , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Proteínas Repressoras , Seleção GenéticaRESUMO
Mouse chimaeras made by aggregating two 8-cell stage embryos undergo size regulation shortly after implantation. Thus chimaeric pups are approximately normal size at birth despite their origin from two complete embryos. Chimaeras of some strain combinations are genotypically unbalanced such that cells of one strain almost always predominate. For example, the BALB/c inbred strain often makes a low contribution to chimaeras. This genotypic imbalance in the composition could arise by selection against BALB/c cells. Selection may be particularly acute at the time of size regulation. To investigate if the mechanism(s) responsible for size regulation could cause the low contribution of BALB/c cells, we compared the composition of an unbalanced series of chimaeras, produced by aggregating two complete 8-cell stage embryos, with a similar series of chimaeras made by aggregating two half 8-cell stage embryos. In each case the unbalanced strain combination was BALB/c<-->[(C57BL x CBA/Ca)F1 x TGB] and parallel studies were undertaken with a genotypically balanced strain combination. For each chimaera, the composition of the fetus, placenta and extraembryonic membranes were determined at E12.5. When two half embryos were aggregated the BALB/c strain still made a poor contribution to all the tissues of the mid-gestation conceptus. This implies that this strain combination remained unbalanced even when size regulation was absent or minimal. Therefore, size regulation did not play a major role in reducing the contribution of BALB/c cells and producing the phenotypic imbalance in the chimaeras.
Assuntos
Quimera/embriologia , Quimera/genética , Animais , Animais Recém-Nascidos , Blastocisto/citologia , Constituição Corporal/genética , Contagem de Células , Feminino , Genótipo , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Fenótipo , GravidezAssuntos
Preparo de Canal Radicular/métodos , Tratamento do Canal Radicular/métodos , Cavidade Pulpar/anatomia & histologia , Cavidade Pulpar/diagnóstico por imagem , Restauração Dentária Permanente , Humanos , Radiografia , Materiais Restauradores do Canal Radicular/química , Irrigantes do Canal Radicular/uso terapêutico , Obturação do Canal Radicular/métodos , Preparo de Canal Radicular/classificação , Preparo de Canal Radicular/instrumentação , Propriedades de Superfície , Ápice Dentário/anatomia & histologia , Ápice Dentário/diagnóstico por imagem , Resultado do TratamentoRESUMO
Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid<-->8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid<-->8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell<-->8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid<-->8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.
Assuntos
Blastocisto/citologia , Quimera , Trofoblastos/citologia , Fatores Etários , Animais , Agregação Celular , Tamanho Celular , Ectoderma/citologia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBARESUMO
In a previous study of mouse tetraploid<-->diploid chimaeric blastocysts, tetraploid cells were found to be more abundant in the trophectoderm than the inner cell mass (ICM) and more abundant in the mural trophectoderm than the polar trophectoderm. This non-random allocation of tetraploid cells to different regions of the chimaeric blastocyst may contribute to the restricted tissue distribution seen in post-implantation stage tetraploid<-->diploid chimaeras. However, the tetraploid and diploid embryos that were aggregated together differed in several respects: the tetraploid embryos had fewer cells and these cells were bigger and differed in ploidy. Each of these factors might underlie a non-random allocation of tetraploid cells to the chimaeric blastocyst. A combination of micromanipulation and electrofusion was used to produce two series of chimaeras that distinguished between the effects of cell size and ploidy on the allocation of cells to different tissues in chimaeric blastocysts. When aggregated cells differed in cell size but not ploidy, the derivatives of the larger cell contributed significantly more to the mural trophectoderm and polar trophectoderm than the ICM. When aggregated cells differed in ploidy but not cell size, the tetraploid cells contributed significantly more to the mural trophectoderm than the ICM. In both experiments the contributions to the polar trophectoderm tended to be intermediate between those of the mural trophectoderm and ICM. These experiments show that both the larger size and increased ploidy of tetraploid cells could have contributed to the non-random cell distribution that was observed in a previous study of tetraploid<-->diploid chimaeric blastocysts.
Assuntos
Blastocisto/citologia , Quimera/genética , Ploidias , Animais , Blastocisto/ultraestrutura , Tamanho Celular , Cruzamentos Genéticos , Diploide , Feminino , Globinas/genética , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Poliploidia , TransgenesRESUMO
Studies of tetraploid<-->diploid (4n<-->2n) mouse chimaeras have demonstrated unequal contributions of 4n cells to different tissues of the midgestation conceptus. Such a pattern has also been reported in chimaeras as early as E3.5d, which show an enhanced contribution of 4n cells to the mural trophectoderm (Everett & West, 1996). In this study, sectioned 4n<-->2n and 2n<-->2n control chimaeric blastocysts were digitised and reconstructed in 3 dimensions (3-D). The 3-D images revealed only limited mixing of cells from the 2 contributing embryos of individual blastocysts in both chimaera groups. Consequently, the distribution pattern of the 2 cell types was dependent on the spatial relationship between the orientation of the blastocyst and the boundary between the 2 clusters of cells. The distribution patterns observed were not strikingly different for 4n<-->2n and 2n<-->2n chimaeras, each showing some transgenic positive cell contribution in all 3 identifiable developmental lineages. It was notable, however, that in all 4n<-->2n blastocysts at least some 4n cells were located adjacent to the blastocyst cavity. Such a consistent pattern was not evident in 2n<-->2n chimaeras. This study has demonstrated the value of 3-D reconstructions for the analysis of spatial relationships of 2 cell populations in chimaeric mouse blastocysts.
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Blastocisto/ultraestrutura , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Ploidias , Animais , Linhagem da Célula , Quimera , Diploide , Idade Gestacional , Camundongos , PoliploidiaRESUMO
Chimaeric mice were made by aggregating Pax6(-/-) and wild-type mouse embryos, in order to study the interaction between the optic vesicle and the prospective lens epithelium during early stages of eye development. Histological analysis of the distribution of homozygous mutant cells in the chimaeras showed that the cell-autonomous removal of Pax6(-/-) cells from the lens, shown previously at E12.5, is nearly complete by E9.5. Most mutant cells are eliminated from an area of facial epithelium wider than, but including, the developing lens placode. This result suggests a role for Pax6 in maintaining a region of the facial epithelium that has the tissue competence to undergo lens differentiation. Segregation of wild-type and Pax6(-/-) cells occurs in the optic vesicle at E9.5 and is most likely a result of different adhesive properties of wild-type and mutant cells. Also, proximo-distal specification of the optic vesicle (as assayed by the elimination of Pax6(-/-) cells distally), is disrupted in the presence of a high proportion of mutant cells. This suggests that Pax6 operates during the establishment of patterning along the proximo-distal axis of the vesicle. Examination of chimaeras with a high proportion of mutant cells showed that Pax6 is required in the optic vesicle for maintenance of contact with the overlying lens epithelium. This may explain why Pax6(-/-) optic vesicles are inefficient at inducing a lens placode. Contact is preferentially maintained when the lens epithelium is also wild-type. Together, these results demonstrate requirements for functional Pax6 in both the optic vesicle and surface epithelia in order to mediate the interactions between the two tissues during the earliest stages of eye development.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Olho/embriologia , Proteínas de Homeodomínio , Morfogênese/fisiologia , Animais , Quimera , Cruzamentos Genéticos , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Epitélio/embriologia , Olho/citologia , Proteínas do Olho/genética , Proteínas do Olho/fisiologia , Face/embriologia , Feminino , Genótipo , Heterozigoto , Cristalino/citologia , Cristalino/embriologia , Masculino , Camundongos , Camundongos Knockout , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados , Epitélio Pigmentado Ocular/embriologia , Proteínas RepressorasRESUMO
Previously we found that male mice carrying either of two attenuated herpes simplex virus thymidine kinase reporter transgenes displayed low level ectopic expression of the reporter gene in the testis and, although fertile, exhibited reduced fecundity. In contrast to males of later generations, many of the founder males failed to transmit the transgene to their progeny. This led to the suggestion that these fertile non-transmitting males are mosaic, with the sperm developing from the non-transgenic lineage outperforming those from the heterozygous transgenic lineage. Here we present the results of artificial insemination (AI) and in vitro fertilization (IVF) experiments designed to test this hypothesis. Albino CF(1) hybrid females were inseminated with mixtures of equal numbers of sperm from heterozygous transgenic (HT) males (equivalent to C57BL/6 x CBAF(2)) and CF(1) males. Similar mixed inseminations were carried out in parallel with sperm from non-transgenic (NT) siblings of the HT mice and 13-day fetuses were scored by eye color to determine their paternity. The pooled data from five experiments gave ratios of CF(1) to HT and CF(1) to NT offspring of 8.13 and 0.22 respectively, implying a calculated HT to NT ratio of 0.027. This indicates that, in competition with each other, the NT sperm would be almost 40 times more successful in fertilization than the HT sperm. Smaller differences were observed between HT and NT when AI was performed with unmixed sperm, consistent with the fertility of HT non-founder males. However, in five IVF experiments carried out with unmixed sperm, 142/212 oocytes exposed to NT sperm were activated and divided, while only 8/226 oocytes treated with HT sperm reached the two-cell stage. This confirms that HT sperm are defective and indicates that the IVF method employed amplified these deficiencies, which may have only a small effect upon natural reproduction when the HT sperm are not in competition with normal sperm.