RESUMEN
Complex interactions of the branching ureteric bud (UB) and surrounding mesenchymal cells during metanephric kidney development determine the final number of nephrons. Impaired nephron endowment predisposes to arterial hypertension and chronic kidney disease. In the kidney, extracellular matrix (ECM) proteins are usually regarded as acellular scaffolds or as the common histological end-point of chronic kidney diseases. Since only little is known about their physiological role in kidney development, we aimed for analyzing the expression and role of fibronectin. In mouse, fibronectin was expressed during all stages of kidney development with significant changes over time. At embryonic day (E) 12.5 and E13.5, fibronectin lined the UB epithelium, which became less pronounced at E16.5 and then switched to a glomerular expression in the postnatal and adult kidneys. Similar results were obtained in human kidneys. Deletion of fibronectin at E13.5 in cultured metanephric mouse kidneys resulted in reduced kidney sizes and impaired glomerulogenesis following reduced cell proliferation and branching of the UB epithelium. Fibronectin colocalized with alpha 8 integrin and fibronectin loss caused a reduction in alpha 8 integrin expression, release of glial-derived neurotrophic factor and expression of Wnt11, both of which are promoters of UB branching. In conclusion, the ECM protein fibronectin acts as a regulator of kidney development and is a determinant of the final nephron number.
Asunto(s)
Fibronectinas , Riñón , Animales , Fibronectinas/metabolismo , Fibronectinas/genética , Ratones , Humanos , Riñón/metabolismo , Riñón/embriología , Proteínas Wnt/metabolismo , Proteínas Wnt/genética , Factor Neurotrófico Derivado de la Línea Celular Glial/metabolismo , Factor Neurotrófico Derivado de la Línea Celular Glial/genética , Proliferación Celular , Integrinas/metabolismo , Integrinas/genética , Ratones Endogámicos C57BL , Matriz Extracelular/metabolismo , Cadenas alfa de IntegrinasRESUMEN
In oligodendrocytes of the vertebrate central nervous system a complex network of transcriptional regulators is required to ensure correct and timely myelination of neuronal axons. Here we identify Zfp276, the only mammalian ZAD-domain containing zinc finger protein, as a transcriptional regulator of oligodendrocyte differentiation and central myelination downstream of Sox10. In the central nervous system, Zfp276 is exclusively expressed in mature oligodendrocytes. Oligodendroglial deletion of Zfp276 led to strongly reduced expression of myelin genes in the early postnatal mouse spinal cord. Retroviral overexpression of Zfp276 in cultured oligodendrocyte precursor cells induced precocious expression of maturation markers and myelin genes, further supporting its role in oligodendroglial differentiation. On the molecular level, Zfp276 directly binds to and represses Sox10-dependent gene regulatory regions of immaturity factors and functionally interacts with the transcriptional repressor Zeb2 to enable fast transition of oligodendrocytes to the myelinating stage.
Asunto(s)
Oligodendroglía , Médula Espinal/citología , Factores de Transcripción , Animales , Diferenciación Celular , Ratones , Vaina de Mielina/fisiología , Neurogénesis , Oligodendroglía/citología , Oligodendroglía/metabolismo , Médula Espinal/metabolismo , Células Madre , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Formation of intraretinal capillaries and inner blood-retinal barrier during development requires norrin, a ligand of the canonical wingless/integrated (Wnt)/ß-catenin signaling pathway. Here we addressed the question whether retinal pigmented epithelium (RPE)-derived overexpression of norrin in transgenic mice rescues the vascular phenotype caused by norrin deficiency. To this end, we generated NdpKO/Rpe65-Norrin mice and analyzed the activation of ß-catenin signaling, the development of intraretinal capillaries, and the expression of blood-retinal barrier marker molecules. RPE-derived norrin induced retinal ß-catenin signaling but failed to rescue the vascular developmental defects and the breakdown of the blood-retinal barrier in norrin-deficient mice. Sites of ectopic norrin expression and the amounts of secreted transgenic protein are critical factors to enable the angiogenic properties of norrin.
Asunto(s)
Retina , beta Catenina , Ratones , Animales , Ratones Transgénicos , beta Catenina/genética , beta Catenina/metabolismo , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Epitelio , Proteínas del Ojo/fisiologíaRESUMEN
Schwann cells are the nerve ensheathing cells of the peripheral nervous system. Absence, loss and malfunction of Schwann cells or their myelin sheaths lead to peripheral neuropathies such as Charcot-Marie-Tooth disease in humans. During Schwann cell development and myelination chromatin is dramatically modified. However, impact and functional relevance of these modifications are poorly understood. Here, we analyzed histone H2B monoubiquitination as one such chromatin modification by conditionally deleting the Rnf40 subunit of the responsible E3 ligase in mice. Rnf40-deficient Schwann cells were arrested immediately before myelination or generated abnormally thin, unstable myelin, resulting in a peripheral neuropathy characterized by hypomyelination and progressive axonal degeneration. By combining sequencing techniques with functional studies we show that H2B monoubiquitination does not influence global gene expression patterns, but instead ensures selective high expression of myelin and lipid biosynthesis genes and proper repression of immaturity genes. This requires the specific recruitment of the Rnf40-containing E3 ligase by Egr2, the central transcriptional regulator of peripheral myelination, to its target genes. Our study identifies histone ubiquitination as essential for Schwann cell myelination and unravels new disease-relevant links between chromatin modifications and transcription factors in the underlying regulatory network.
Asunto(s)
Proteína 2 de la Respuesta de Crecimiento Precoz/fisiología , Neuropatía Hereditaria Motora y Sensorial/metabolismo , Histonas/metabolismo , Sistema Nervioso Periférico/metabolismo , Células de Schwann/metabolismo , Animales , Línea Celular Tumoral , Células HEK293 , Humanos , Ratones , Ratones Transgénicos , Sistema Nervioso Periférico/patología , Ratas , Células de Schwann/patología , Ubiquitina-Proteína Ligasas/genética , UbiquitinaciónRESUMEN
The cause of the elevated outflow resistance and consequent ocular hypertension characteristic of glaucoma is unknown. To investigate possible causes for this flow resistance, we used atomic force microscopy (AFM) with 10-µm spherical tips to probe the stiffness of the inner wall of Schlemm's canal as a function of distance from the tissue surface in normal and glaucomatous postmortem human eyes, and 1-µm spherical AFM tips to probe the region immediately below the tissue surface. To localize flow resistance, perfusion and imaging methods were used to characterize the pressure drop in the immediate vicinity of the inner wall using giant vacuoles that form in Schlemm's canal cells as micropressure sensors. Tissue stiffness increased with increasing AFM indentation depth. Tissues from glaucomatous eyes were stiffer compared with normal eyes, with greatly increased stiffness residing within â¼1 µm of the inner-wall surface. Giant vacuole size and density were similar in normal and glaucomatous eyes despite lower flow rate through the latter due to their higher flow resistance. This implied that the elevated flow resistance found in the glaucomatous eyes was localized to the same region as the increased tissue stiffness. Our findings implicate pathological changes to biophysical characteristics of Schlemm's canal endothelia and/or their immediate underlying extracellular matrix as cause for ocular hypertension in glaucoma.
RESUMEN
Transforming growth factor ß (TGFß) signaling has manifold functions such as regulation of cell growth, differentiation, migration, and apoptosis. Moreover, there is increasing evidence that it also acts in a neuroprotective manner. We recently showed that TGFß receptor type 2 (Tgfbr2) is upregulated in retinal neurons and Müller cells during retinal degeneration. In this study we investigated if this upregulation of TGFß signaling would have functional consequences in protecting retinal neurons. To this end, we analyzed the impact of TGFß signaling on photoreceptor viability using mice with cell type-specific deletion of Tgfbr2 in retinal neurons and Müller cells (Tgfbr2ΔOC) in combination with a genetic model of photoreceptor degeneration (VPP). We examined retinal morphology and the degree of photoreceptor degeneration, as well as alterations of the retinal transcriptome. In summary, retinal morphology was not altered due to TGFß signaling deficiency. In contrast, VPP-induced photoreceptor degeneration was drastically exacerbated in double mutant mice (Tgfbr2ΔOC; VPP) by induction of pro-apoptotic genes and dysregulation of the MAP kinase pathway. Therefore, TGFß signaling in retinal neurons and Müller cells exhibits a neuroprotective effect and might pose promising therapeutic options to attenuate photoreceptor degeneration in humans.
Asunto(s)
Degeneración Retiniana , Factor de Crecimiento Transformador beta , Animales , Modelos Animales de Enfermedad , Células Ependimogliales/metabolismo , Ratones , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptor Tipo II de Factor de Crecimiento Transformador beta/genética , Receptor Tipo II de Factor de Crecimiento Transformador beta/metabolismo , Retina/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo , Factor de Crecimiento Transformador beta/metabolismoRESUMEN
CCN2/CTGF is a matricellular protein that is known to enhance transforming growth factor-ß signaling and to induce a myofibroblast-like phenotype in a variety of cell types. Here, we investigated Ccn2/Ctgf promotor activity during development and in the adult mouse eye, using CTGFLacZ/+ mice in which the ß-galactosidase reporter gene LacZ had been inserted into the open reading frame of Ccn2/Ctgf. Promotor activity was assessed by staining for ß-galactosidase activity and by immunolabeling using antibodies against ß-galactosidase. Co-immunostaining using antibodies against glutamine synthetase, glial fibrillary acidic protein, choline acetyltransferase, and CD31 was applied to identify specific cell types. Ccn2/Ctgf promotor activity was intense in neural crest-derived cells differentiating to corneal stroma and endothelium, and to the stroma of choroid, iris, ciliary body, and the trabecular meshwork during development. In the adult eye, a persistent and very strong promotor activity was present in the trabecular meshwork outflow pathways. In addition, endothelial cells of Schlemm's canal, and of retinal and choroidal vessels, retinal astrocytes, Müller glia, and starburst amacrine cells were stained. Very strong promoter activity was seen in the astrocytes of the glial lamina at the optic nerve head. We conclude that CCN2/CTGF signaling is involved in the processes that govern neural crest morphogenesis during ocular development. In the adult eye, CCN2/CTGF likely plays an important role for the trabecular meshwork outflow pathways and the glial lamina of the optic nerve head.
Asunto(s)
Factor de Crecimiento del Tejido Conjuntivo/fisiología , Células Endoteliales , Retina , Animales , Células Endoteliales/citología , Células Endoteliales/metabolismo , Ratones , Regiones Promotoras Genéticas , Retina/citología , Retina/metabolismoRESUMEN
Differentiating oligodendrocytes generate myelin to ensure rapid saltatory conduction in the vertebrate central nervous system. Although oligodendroglial differentiation and myelination are accompanied by dramatic chromatin reorganizations, previously studied chromatin remodelers had only limited direct effects on the process. To study the functional significance of chromatin changes for myelination and identify relevant remodelers, we deleted Ep400, the central ATP-hydrolyzing subunit of the TIP60/EP400 complex, at defined times of mouse oligodendrocyte development. Whereas Ep400-deficient oligodendrocyte precursors develop normally, terminal differentiation and myelination are dramatically impaired. Mechanistically, Ep400 interacts with transcription factor Sox10, binds to regulatory regions of the Myrf gene and is required to induce this central transcriptional regulator of the myelination program. In addition to reduced and aberrant myelin formation, oligodendrocytes exhibit increased DNA damage and apoptosis so that numbers never reach wildtype levels during the short lifespan of Ep400-deficient mice. Ep400 deletion in already mature oligodendrocytes remains phenotypically inapparent arguing that Ep400 is dispensable for myelin maintenance. Given its essential function in myelin formation, modulation of Ep400 activity may be beneficial in conditions such as multiple sclerosis where this process is compromised.
Asunto(s)
ADN Helicasas/fisiología , Proteínas de Unión al ADN/fisiología , Vaina de Mielina/fisiología , Oligodendroglía/citología , Médula Espinal/citología , Animales , Encéfalo/citología , Encéfalo/embriología , Encéfalo/crecimiento & desarrollo , Encéfalo/metabolismo , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , ADN Helicasas/genética , ADN Helicasas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Redes Reguladoras de Genes , Células HEK293 , Humanos , Ratones Transgénicos , Vaina de Mielina/ultraestructura , Oligodendroglía/metabolismo , Ratas , Ratas Wistar , Médula Espinal/embriología , Médula Espinal/crecimiento & desarrollo , Médula Espinal/metabolismoRESUMEN
Hereditary retinal degenerations like retinitis pigmentosa (RP) are among the leading causes of blindness in younger patients. To enable in vivo investigation of cellular and molecular mechanisms responsible for photoreceptor cell death and to allow testing of therapeutic strategies that could prevent retinal degeneration, animal models have been created. In this study, we deeply characterized the transcriptional profile of mice carrying the transgene rhodopsin V20G/P23H/P27L (VPP), which is a model for autosomal dominant RP. We examined the degree of photoreceptor degeneration and studied the impact of the VPP transgene-induced retinal degeneration on the transcriptome level of the retina using next generation RNA sequencing (RNASeq) analyses followed by weighted correlation network analysis (WGCNA). We furthermore identified cellular subpopulations responsible for some of the observed dysregulations using in situ hybridizations, immunofluorescence staining, and 3D reconstruction. Using RNASeq analysis, we identified 9256 dysregulated genes and six significantly associated gene modules in the subsequently performed WGCNA. Gene ontology enrichment showed, among others, dysregulation of genes involved in TGF-ß regulated extracellular matrix organization, the (ocular) immune system/response, and cellular homeostasis. Moreover, heatmaps confirmed clustering of significantly dysregulated genes coding for components of the TGF-ß, G-protein activated, and VEGF signaling pathway. 3D reconstructions of immunostained/in situ hybridized sections revealed retinal neurons and Müller cells as the major cellular population expressing representative components of these signaling pathways. The predominant effect of VPP-induced photoreceptor degeneration pointed towards induction of neuroinflammation and the upregulation of neuroprotective pathways like TGF-ß, G-protein activated, and VEGF signaling. Thus, modulation of these processes and signaling pathways might represent new therapeutic options to delay the degeneration of photoreceptors in diseases like RP.
Asunto(s)
Perfilación de la Expresión Génica , Neuroprotección/genética , Retinitis Pigmentosa/genética , Transcripción Genética , Regulación hacia Arriba/genética , Animales , Quimiocina CCL2/metabolismo , Femenino , Proteínas de Unión al GTP/metabolismo , Redes Reguladoras de Genes , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Ratones , Ratones Transgénicos , Neuroglía/metabolismo , Degeneración Retiniana/complicaciones , Degeneración Retiniana/patología , Células Fotorreceptoras Retinianas Bastones/metabolismo , Células Fotorreceptoras Retinianas Bastones/patología , Rodopsina/genética , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Human SOX10 mutations lead to various diseases including Waardenburg syndrome, Hirschsprung disease, peripheral demyelinating neuropathy, central leukodystrophy, Kallmann syndrome and various combinations thereof. It has been postulated that PCWH as a combination of Waardenburg and Hirschsprung disease, peripheral neuropathy and central leukodystrophy is caused by heterozygous SOX10 mutations that result in the presence of a dominantly acting mutant SOX10 protein in the patient. One such protein with postulated dominant action is SOX10 Q377X. In this study, we generated a mouse model, in which the corresponding mutation was introduced into the Sox10 locus in such a way that Sox10 Q377X is constitutively expressed. Heterozygous mice carrying this mutation exhibited pigmentation and enteric nervous system defects similar to mice in which one Sox10 allele was deleted. However, despite presence of the mutant protein in Schwann cells and oligodendrocytes throughout development and in the adult, we found no phenotypic evidence for neurological defects in peripheral or central nervous systems. In the nervous system, the mutant Sox10 protein did not act in a dominant fashion but rather behaved like a hypomorph with very limited residual function. Our results question a strict genotype-phenotype correlation for SOX10 mutations and argue for the influence of additional factors including genetic background.
Asunto(s)
Factores de Transcripción SOXE/metabolismo , Alelos , Animales , Proteínas de Unión al ADN/genética , Enfermedades Desmielinizantes/genética , Modelos Animales de Enfermedad , Estudios de Asociación Genética , Heterocigoto , Proteínas del Grupo de Alta Movilidad/genética , Ratones , Ratones Endogámicos C3H , Mutación , Fenotipo , Factores de Transcripción SOXE/genética , Factores de Transcripción/genéticaRESUMEN
Purpose: To analyze whether activation of endogenous wingless (Wnt)/ß-catenin signaling in Müller cells is involved in protection of retinal ganglion cells (RGCs) following excitotoxic damage. Methods: Transgenic mice with a tamoxifen-dependent ß-catenin deficiency in Müller cells were injected with N-methyl-D-aspartate (NMDA) into the vitreous cavity of one eye to induce excitotoxic damage of the RGCs, while the contralateral eye received PBS only. Retinal damage was quantified by counting the total number of RGC axons in cross sections of optic nerves and measuring the thickness of the retinal layers on meridional sections. Then, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay was performed to identify apoptotic cells in retinas of both genotypes. Western blot analyses to assess the level of retinal ß-catenin and real-time RT-PCR to quantify the retinal expression of neuroprotective factors were performed. Results: Following NMDA injection of wild-type mice, a statistically significant increase in retinal ß-catenin protein levels was observed compared to PBS-injected controls, an effect that was blocked in mice with a Müller cell-specific ß-catenin deficiency. Furthermore, in mice with a ß-catenin deficiency in Müller cells, NMDA injection led to a statistically significant decrease in RGC axons as well as a substantial increase in TUNEL-positive cells in the RGC layer compared to the NMDA-treated controls. Moreover, in the retinas of the control mice a NMDA-mediated statistically significant induction of leukemia inhibitory factor (Lif) mRNA was detected, an effect that was substantially reduced in mice with a ß-catenin deficiency in Müller cells. Conclusions: Endogenous Wnt/ß-catenin signaling in Müller cells protects RGCs against excitotoxic damage, an effect that is most likely mediated via the induction of neuroprotective factors, such as Lif.
Asunto(s)
Células Ependimogliales/metabolismo , Nervio Óptico/metabolismo , Retina/metabolismo , Células Ganglionares de la Retina/metabolismo , Tamoxifeno/farmacología , Vía de Señalización Wnt/efectos de los fármacos , beta Catenina/metabolismo , Animales , Apoptosis/efectos de los fármacos , Axones/efectos de los fármacos , Axones/metabolismo , Células Ependimogliales/efectos de los fármacos , Etiquetado Corte-Fin in Situ , Factor Inhibidor de Leucemia/metabolismo , Ratones , Ratones Transgénicos , N-Metilaspartato/toxicidad , Nervio Óptico/efectos de los fármacos , Retina/efectos de los fármacos , Retina/patología , Células Ganglionares de la Retina/efectos de los fármacos , Vía de Señalización Wnt/genética , beta Catenina/deficienciaRESUMEN
Quantifying the number of axons in the optic nerve is of interest in many research questions. Here, we show that a stereological method allows simple, efficient, precise and unbiased determination of the total axon number in the murine optic nerve. Axons in semi-thin optic nerve cross sections from untreated eyes (nâ¯=â¯21) and eyes subjected to retinal damage by intravitreous NMDA injections (nâ¯=â¯32) or PBS controls (nâ¯=â¯5) were manually identified, counted and digitally labeled by hand. A stereological procedure was empirically tested with systematic combinations of different sampling methods (simple random sampling without replacement, systematic uniform random sampling, stratified random sampling) and sampling parameters. Extensive numerical Monte Carlo experiments were performed to evaluate their large-sample properties. Our results demonstrate reliable determination of total axon number and superior performance compared to other methods at a small fraction of the time required for a full manual count. We specify suitable sampling parameters for the adoption of an efficient stereological sampling scheme, give empirical estimates of the additionally introduced sampling variance to facilitate experimental planning, and offer AxonCounter, an easy-to-use plugin implementing these stereological methods for the multi-platform image processing application NIH ImageJ.
Asunto(s)
Recuento de Células/métodos , Técnicas Citológicas , Nervio Óptico/citología , Animales , Agonistas de Aminoácidos Excitadores/farmacología , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Ratones Endogámicos BALB C , N-Metilaspartato/farmacología , Nervio Óptico/efectos de los fármacosRESUMEN
Sequence variants in LOXL1 coding for the secreted enzyme lysyl oxidase homolog 1 (LOXL1) associate with pseudoexfoliation (PEX) syndrome, a condition that is characterized by the deposition of extracellular ï¬brillar PEX material in the anterior eye and other parts of the body. Since the specific role of LOXL1 in the pathogenesis of PEX is unclear, and an increase in its expression was reported for early stages of PEX syndrome, we generated and studied transgenic mice with ocular overexpression of its mouse ortholog Loxl1. The chicken ßB1-crystallin promoter was used to overexpress Loxl1 in the lenses of ßB1-crystallin-Loxl1 transgenic mice. Transgenic lenses contained high levels of the protein LOXL1 and its mRNA, which were both not detectable in lenses of wildtype littermates. In wildtype mice, immunoreactivity for LOXL1 was mainly seen extracellularly in region of the ciliary zonules. ßB1-crystallin-Loxl1 littermates showed an additional diffuse immunostaining in lens fibers and capsule, and in the inner limiting membrane and retina indicating secretion of soluble LOXL1 from transgenic lenses. In addition, lens fibers of transgenic animals contained multiple distinct spots of very intense LOXL1 immunoreactivity. By transmission electron microscopy, those spots correlated with electron-dense round or oval bodies of 20-50â¯nm in diameter which were localized in the rough endoplasmic reticulum and not seen in wildtype lenses. Immunogold electron microscopy confirmed that the electron-dense bodies contained LOXL1 indicating aggregation of insoluble LOXL1. Similar structures were seen in the extracellular lens capsule suggesting their secretion from lens fibers. Otherwise, no changes were seen between the eyes of ßB1-crystallin-Loxl1 mice and their wildtype littermates, neither by light microscopy and funduscopy of whole eyes, nor by scanning and quantitative transmission electron microscopy of ciliary epithelium and zonules. At one month of age, intraocular pressure was significantly higher in transgenic mice than in wildtype littermates. No differences in IOP were seen though at 2-5 months of age. We conclude that LOXL1 has a strong tendency to aggregate in the rER when expressed in vivo at high amounts. A similar scenario, involving intracellular aggregation of LOXL1 and secretion of LOXL1 aggregates into the extracellular space, may be involved in the early pathogenetic events in eyes of PEX patients.
Asunto(s)
Aminoácido Oxidorreductasas/genética , Cuerpo Ciliar/metabolismo , Síndrome de Exfoliación/metabolismo , Regulación de la Expresión Génica/fisiología , Cristalino/metabolismo , Agregado de Proteínas/fisiología , Aminoácido Oxidorreductasas/metabolismo , Animales , Western Blotting , Cuerpo Ciliar/ultraestructura , Síndrome de Exfoliación/etiología , Femenino , Inmunohistoquímica , Presión Intraocular , Cápsula del Cristalino/metabolismo , Cristalino/ultraestructura , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microscopía Electrónica de Transmisión , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Cadena B de beta-Cristalina/genéticaRESUMEN
BACKGROUND: The marginal mandibular branch (MMB) of the facial nerve provides lower lip symmetry apparent during human smile or crying and is mandatory for vocal phonation. In treating facial palsy patients, so far, little attention is directed at the MMB in facial reanimation surgery. However, isolated paralysis may occur congenital, in Bell's palsy or iatrogenic during surgery, prone to its anatomical course. A variety of therapies address symmetry with either weakening of the functional side or reconstruction of the paralyzed side. To further clarify the histoanatomic basis of facial reanimation procedures using nerve transfers, we conducted a human cadaver study examining macroanatomical and microanatomical features of the MMB including its axonal capacity. METHODS: Nerve biopsies of the MMB were available from 96 facial halves. Histological processing, digitalization, nerve morphometry investigation, and semiautomated axonal quantification were performed. Statistical analysis was conducted with P < 0.05 as level of significance. RESULTS: The main branch of 96 specimens contained an average of 3.72 fascicles 1 to 12, and the axonal capacity was 1603 ± 849 (398-5110, n = 85). Differences were found for sex (P = 0.018), not for facial sides (P = 0.687). Diameters were measured with 1130 ± 327 µm (643-2139, n = 79). A significant difference was noted between sexes (P = 0.029), not for facial sides (P = 0.512.) One millimeter in diameter corresponded to 1480 ± 630 axons (n = 71). A number of 900 axons was correlated with 0.97 mm (specificity, 90%; sensitivity, 72%). CONCLUSIONS: Our morphometric results for the MMB provide basic information for further investigations, among dealing with functional reconstructive procedures such as nerve transfers, nerve grafting for direct neurotization or babysitter procedures, and neurectomies to provide ideal power and authenticity.
Asunto(s)
Parálisis de Bell/cirugía , Nervio Facial/cirugía , Parálisis Facial/cirugía , Transferencia de Nervios/métodos , Procedimientos de Cirugía Plástica/métodos , Adulto , Axones/trasplante , Parálisis de Bell/patología , Biopsia con Aguja , Cadáver , Expresión Facial , Nervio Facial/anatomía & histología , Parálisis Facial/fisiopatología , Femenino , Humanos , Inmunohistoquímica , Masculino , Mandíbula/inervación , Recuperación de la Función , SonrisaRESUMEN
The facial nerve is responsible for any facial expression channeling human emotions. Facial paralysis causes asymmetry, lagophthalmus, oral incontinence, and social limitations. Facial dynamics may be re-established with cross-face-nerve-grafts (CFNG). Our aim was to reappraise the zygomaticobuccal branch system relevant for facial reanimation surgery with respect to anastomoses and crossings. Dissection was performed on 106 facial halves of 53 fresh frozen cadavers. Study endpoints were quantity and relative thickness of branches, correlation to "Zuker's point", interconnection patterns and crossings. Level I and level II branches were classified as relevant for CFNG. Anastomoses and fusion patterns were assessed in both levels. The zygomatic branch showed 2.98 ± 0.86 (range 2-5) twigs at level II and the buccal branch 3.45 ± 0.96 (range 2-5), respectively. In the zygomatic system a single dominant branch was present in 50%, two co-dominant branches in 9% and three in 1%. In 66% of cases a single dominant buccal twig, two co-dominant in 12.6%, and three in 1% of cases were detected. The most inferior zygomatic branch was the most dominant branch (P = 0.003). Using Zuker's point, a facial nerve branch was found within 5 mm in all facial halves. Fusions were detected in 80% of specimens. Two different types of fusion patterns could be identified. Undercrossing of branches was found in 24% at levels I and II. Our study describes facial nerve branch systems relevant for facial reanimation surgery in a three-dimensional relationship of branches to each other. Clin. Anat. 32:480-488, 2019. © 2019 Wiley Periodicals, Inc.
Asunto(s)
Nervio Facial/anatomía & histología , Anastomosis Quirúrgica , Mejilla/inervación , Nervio Facial/cirugía , Humanos , Valores de Referencia , Cigoma/inervaciónRESUMEN
In Schwann cells of the vertebrate peripheral nervous system, induction of myelination and myelin maintenance both depend on the HMG-domain-containing transcription factor Sox10. In oligodendrocytes of the central nervous system, Sox10 is also essential for the induction of myelination. Its role in late phases of myelination and myelin maintenance has not been studied so far. Here, we show that these processes are largely unaffected in mice that lack Sox10 in mature oligodendrocytes. As Sox10 is co-expressed with the related Sox8, we also analyzed oligodendrocytes and myelination in Sox8-deficient mice. Again, we could not detect any major abnormalities. Expression of many myelin genes was only modestly reduced in both mouse mutants. Dramatic reductions in expression levels and phenotypic disturbances became only apparent once Sox8 and Sox10 were both absent. This argues that Sox8 and Sox10 are jointly required for myelin maintenance and impact myelin gene expression. One direct target gene of both Sox proteins is the late myelin gene Mog. Our results point to at least partial functional redundancy between both related Sox proteins in mature oligodendrocytes and are the first report of a substantial function of Sox8 in the oligodendroglial lineage.
Asunto(s)
Vaina de Mielina/metabolismo , Oligodendroglía/metabolismo , Factores de Transcripción SOXE/biosíntesis , Células de Schwann/metabolismo , Animales , Linaje de la Célula , Expresión Génica , Células HEK293 , Células HeLa , Humanos , Ratones , Ratones Noqueados , Factores de Transcripción SOXE/genéticaRESUMEN
Dysfunction of the retinal pigmented epithelium (RPE) results in degeneration of photoreceptors and vision loss and is correlated with common blinding disorders in humans. Although many protein-coding genes are known to be expressed in RPE and are important for its development and maintenance, virtually nothing is known about the in vivo roles of non-coding transcripts. The expression patterns of microRNAs (miRNAs) have been analyzed in a variety of ocular tissues, and a few were implicated to play role in RPE based on studies in cell lines. Here, through RPE-specific conditional mutagenesis of Dicer1 or Dgcr8 in mice, the importance of miRNAs for RPE differentiation was uncovered. miRNAs were found to be dispensable for maintaining RPE fate and survival, and yet they are essential for the acquisition of important RPE properties such as the expression of genes involved in the visual cycle pathway, pigmentation and cell adhesion. Importantly, miRNAs of the RPE are required for maturation of adjacent photoreceptors, specifically for the morphogenesis of the outer segments. The alterations in the miRNA and mRNA profiles in the Dicer1-deficient RPE point to a key role of miR-204 in regulation of the RPE differentiation program in vivo and uncover the importance of additional novel RPE miRNAs. This study reveals the combined regulatory activity of miRNAs that is required for RPE differentiation and for the development of the adjacent neuroretina.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , MicroARNs/metabolismo , Retina/embriología , Epitelio Pigmentado de la Retina/citología , Animales , Adhesión Celular , Diferenciación Celular , Linaje de la Célula , Supervivencia Celular , ARN Helicasas DEAD-box/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Ratones , Ratones Transgénicos , Mutagénesis , Mutación , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Fotorreceptoras/metabolismo , Pigmentación , Retina/metabolismo , Rodopsina/metabolismo , Ribonucleasa III/metabolismo , TranscriptomaRESUMEN
The molecular pathogenesis of choroidal neovascularization (CNV), an angiogenic process that critically contributes to vision loss in age-related macular degeneration, is unclear. Herein, we analyzed the role of transforming growth factor (TGF)-ß signaling for CNV formation by generating a series of mutant mouse models with induced conditional deletion of TGF-ß signaling in the entire eye, the retinal pigment epithelium (RPE), or the vascular endothelium. Deletion of TGF-ß signaling in the eye caused CNV, irrespectively if it was ablated in newborn or 3-week-old mice. Areas of CNV showed photoreceptor degeneration, multilayered RPE, basal lamina deposits, and accumulations of monocytes/macrophages. The changes progressed, leading to marked structural and functional alterations of the retina. Although the specific deletion of TGF-ß signaling in the RPE caused no obvious changes, specific deletion in vascular endothelial cells caused CNV and a phenotype similar to that observed after the deletion in the entire eye. We conclude that impairment of TGF-ß signaling in the vascular endothelium of the eye is sufficient to trigger CNV formation. Our findings highlight the importance of TGF-ß signaling as a key player in the development of ocular neovascularization and indicate a fundamental role of TGF-ß signaling in the pathogenesis of age-related macular degeneration.
Asunto(s)
Neovascularización Coroidal/metabolismo , Degeneración Macular/patología , Epitelio Pigmentado de la Retina/patología , Transducción de Señal , Factor de Crecimiento Transformador beta/metabolismo , Animales , Coroides/patología , Neovascularización Coroidal/genética , Modelos Animales de Enfermedad , Ratones Noqueados , Retina/metabolismo , Factor de Crecimiento Transformador beta/genéticaRESUMEN
Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.
Asunto(s)
Técnicas de Cultivo de Célula , Separación Celular/métodos , Guías como Asunto , Malla Trabecular/citología , Factores de Edad , Animales , Biomarcadores/metabolismo , Consenso , Feto , Humanos , Donantes de Tejidos , Conservación de Tejido , Recolección de Tejidos y Órganos , Malla Trabecular/metabolismoRESUMEN
Olfactomedin 1 (OLFM1) is a secreted glycoprotein and member of the olfactomedin protein family, which is preferentially expressed in various areas throughout the central nervous system. To learn about the functional properties of OLFM1 in the eye, we investigated its localization in the mouse and pig eye. In addition, we analyzed the ocular phenotype of Olfm1 mutant mice in which 52 amino acids were deleted in the central part (M2 region) of OLFM1. OLFM1 was detected in cornea, sclera, retina, and optic nerve of both wild-type and Olfm1 mutant littermates. By immunohistochemistry and double labeling with the lectin peanut agglutinin, OLFM1 was found in the interphotoreceptor matrix (IPM) of mouse and pig retina where it was directly localized to the inner segments of photoreceptors. Western blotting confirmed the presence of the OLFM1 isoforms pancortin 1 (BMY) and pancortin 2 (BMZ) in the IPM. The retinal phenotype of Olfm1 mutant mice did not obviously differ from that of wild-type littermates. In addition, outer nuclear layer (ONL) and total retinal thickness were not different, and the same was true for the area of the optic nerve in cross sections. Functional changes were observed though by electroretinography, which showed significantly lower a- and b-wave amplitudes in Olfm1 mutant mice when compared to age-matched wild-type mice. When light damage experiments were performed as an experimental paradigm of photoreceptor apoptosis, significantly more TUNEL-positive cells were observed in Olfm1 mutant mice 30 h after light exposure. One week after light exposure, the ONL was significantly thinner in Olfm1 mutant mice than in wild-type littermates indicating increased photoreceptor loss. No differences were observed when rhodopsin turnover or ERK1/2 signaling was investigated. We conclude that OLFM1 is a newly identified IPM molecule that serves an important role for photoreceptor homeostasis, which is significantly compromised in the eyes of Olfm1 mutant mice.