RESUMEN
Retinal ganglion cells generate a pattern of action potentials to communicate visual information from the retina to cortical areas. Myelin, an insulating sheath, wraps axonal segments to facilitate signal propagation and when deficient, can impair visual function. Optic nerve development and initial myelination has largely been considered completed by the fifth postnatal week. However, the relationship between the extent of myelination and axonal signaling in the maturing optic nerve is not well characterized. Here, we examine the relationship between axon conduction and elements of myelination using extracellular nerve recordings, immunohistochemistry, western blot analysis, scanning electron microscopy, and simulations of nerve responses. Comparing compound action potentials from mice aged 4-12 weeks revealed five functional distinct axonal populations, an increase in the number of functional axons, and shifts toward fast-conducting axon populations at 5 and 8 weeks postnatal. At these ages, our analysis revealed increased myelin thickness, lower g-ratios and changes in the 14 kDa MBP isoform, while the density of axons and nodes of Ranvier remained constant. At 5 postnatal weeks, axon diameter increased, while at 8 weeks, increased expression of a mature sodium ion channel subtype, Nav 1.6, was observed at nodes of Ranvier. A simulation model of nerve conduction suggests that ion channel subtype, axon diameter, and myelin thickness are more likely to be key regulators of nerve function than g-ratio. Such refinement of axonal function and myelin rearrangement identified an extended period of maturation in the normal optic nerve that may facilitate the development of visual signaling patterns.
Asunto(s)
Vaina de Mielina , Nervio Óptico , Animales , Axones/fisiología , Ratones , Vaina de Mielina/fisiología , Conducción Nerviosa/fisiología , Células Ganglionares de la RetinaRESUMEN
INTRODUCTION: Here we study the impact of using either CO2 gas or cervical dislocation (CD) for euthanasia and using different techniques to enucleate the eye on preserving axonal density and morphology of the intraepithelial corneal nerves (ICNs). OBJECTIVES: To determine whether using CO2 gas or CD for euthanasia and enucleating by cutting or pulling eyes out impacts axon density and nerve terminal morphology in the mouse cornea. METHODS: Mice were euthanized by CO2 gas or CD; the impact of delaying fixation for 5 min post-euthanasia was also assessed. We tested two different techniques to enucleate the eyes: cutting the optic nerve by curved scissors or pulling the eye out. A minimum of 10 corneas from 5 male and female BALB/c mice were used for each variable. Axons and intraepithelial corneal nerve terminals (ICNTs) were visualized utilizing ßIII tubulin and L1CAM and quantified using confocal microscopy. RESULTS: The variations seen in axon density between individual mice are not gender- or euthanasia-dependent. A significant reduction in axon density and loss of ICNT morphology are observed in eyes enucleated by pulling the optic nerve out. Similar results are obtained in male and female mice. CONCLUSION: While the variations tested in euthanasia do not affect axon density in male and female mouse corneas, enucleation by proptosing and gently cutting out the eyes yields increased axon density and improved ICNT morphology compared to pulling eyes out and leaving the optic nerve attached.
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Axones , Córnea , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía ConfocalRESUMEN
The optic nerve represents one of the simplest regions of the CNS and has been useful in developing an understanding of glial development and myelination. While the visual system is frequently affected in demyelinating conditions, utilizing the optic nerve to model demyelination/remyelination studies has been difficult due to its accessibility, relatively small size, and dense nature that makes direct injections challenging. Taking advantage of the lack of oligodendrocytes and myelination in the mouse retina, we have developed a model in which the induction of apoptosis in mature oligodendrocytes allows for the selective, non-invasive generation of demyelinating lesions in optic nerve. Delivery of an inducer of oligodendrocyte apoptosis by intravitreous injection minimizes trauma to the optic nerve and allows for the assessment of oligodendrocyte death in the absence of injury related factors. Here we show that following induction of apoptosis, oligodendrocytes are lost within 3 days. The loss of oligodendrocytes is associated with limited microglial and astrocyte response, is patchy along the nerve, and results in localized myelin loss. Unlike in other regions of the murine CNS, where local demyelination stimulates activation of local oligodendrocyte precursors and remyelination, optic nerve demyelination induced by oligodendrocyte apoptosis fails to recover and results in persistent areas of myelin loss. Over time these chronic lesions change cellular composition and ultimately become devoid of GFAP+ astrocytes and OPCs. Why the optic nerve lesions fail to repair may reflect the lack of early immune responsiveness and provide a novel model of chronic demyelination.
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Apoptosis , Astrocitos/patología , Enfermedades Desmielinizantes/patología , Oligodendroglía/patología , Nervio Óptico/patología , Animales , Enfermedades Desmielinizantes/etiología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Regeneración Nerviosa , Células MadreRESUMEN
Multiple sclerosis (MS) is a CNS neurodegenerative autoimmune disease characterized by loss of oligodendrocytes and myelin in the brain and the spinal cord that results in localized functional deficits. Several risk factors have been associated with MS, however none fully explain the enhanced susceptibility seen in older individuals. Epidemiological data, based on geographical prevalence studies suggest that susceptibility is established early in life and frequently long before the diagnosis of disease raising the possibility that developmental events influence adult disease onset and progression. Here we test the hypothesis that selective loss of mature oligodendrocytes during postnatal development results in enhanced susceptibility to a demyelinating insult to the mature CNS. A transgenic mouse model was utilized to specifically induce apoptotic cell death in a subset of mature oligodendrocytes (MBP-iCP9) during the first 2 postnatal weeks followed by either a local LPC spinal cord injection or the induction of EAE in the adult animal. Immunostaining, immunoblotting, behavioral testing, and electron microscopy were utilized to examine the differences in the response between animals with developmental loss of oligodendrocytes and controls. We show that during development, oligodendrocyte apoptosis results in transient reductions in myelination and functional deficits that recover after 10-14 days. Compared to animals in which oligodendrocyte development was unperturbed, animals subjected to postnatal oligodendrocyte loss showed delayed recovery from an LPC lesion to the mature spinal cord. Unexpectedly, the induction and severity of MOG induced EAE was not significantly altered in animals following oligodendrocyte developmental loss even though there was a substantial increase in spinal cord tissue damage and CNS inflammation. It is unclear why the elevated glial responses seen in developmentally compromised animals were not reflected in enhanced functional deficits. These observations suggest that developmental loss of oligodendrocytes results in long lasting tissue changes that alter its response to subsequent insults and the capacity for repair in the adult.
RESUMEN
Multiple sclerosis (MS) is an autoimmune mediated neurodegenerative disease characterized by demyelination and oligodendrocyte (OL) loss in the central nervous system and accompanied by local inflammation and infiltration of peripheral immune cells. Although many risk factors and symptoms have been identified in MS, the pathology is complicated and the cause remains unknown. It is also unclear whether OL apoptosis precedes the inflammation or whether the local inflammation is the cause of OL death and demyelination. This review briefly discusses several models that have been developed to specifically ablate oligodendrocytes in an effort to separate the effects of demyelination from inflammation.
RESUMEN
Corneal epithelial basement membrane dystrophies and superficial injuries caused by scratches can lead to recurrent corneal erosion syndrome (RCES). Patients and animals with reduced corneal sensory nerve innervation can also develop recurrent erosions. Multiple wild-type mouse strains will spontaneously develop recurrent corneal erosions after single 1.5 mm debridement wounds. Here we show that this wound is accompanied by an increase in corneal epithelial cell proliferation after wound closure but without a commensurate increase in corneal epithelial thickness. We investigated whether excess corneal epithelial cell proliferation contributes to erosion formation. We found that topical application of Mitomycin C (MMC), a drug used clinically to improve healing after glaucoma and refractive surgery, reduces erosion frequency, enhances subbasal axon density to levels seen in unwounded corneas, and prevents excess epithelial cell proliferation after debridement wounding. These results suggest that topically applied MMC, which successfully reduces corneal haze and scarring after PRK, may also function to enhance subbasal nerve regeneration and epithelial adhesion when used to treat RCES.
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Córnea/efectos de los fármacos , Lesiones de la Cornea/tratamiento farmacológico , Mitomicina/farmacología , Regeneración Nerviosa/efectos de los fármacos , Análisis de Varianza , Animales , Axones/patología , Proliferación Celular/efectos de los fármacos , Córnea/patología , Lesiones de la Cornea/patología , Desbridamiento , Modelos Animales de Enfermedad , Células Epiteliales/efectos de los fármacos , Epitelio Corneal/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Cicatrización de Heridas/fisiologíaRESUMEN
BACKGROUND: We previously identified compound niches (CNs) at the limbal:corneal border of the mouse cornea that contain corneal epithelial progenitor cells, express Keratin 8 (K8), and goblet cell mucin Muc5AC. During re-epithelialization after 2.5 mm epithelial debridement wounds, CNs migrate onto the cornea and expand in number mimicking conjunctivalization. When CNs form during development and whether they express corneal epithelial progenitor cell enriched K14 was not known. RESULTS: To provide insight into corneal epithelial homeostasis, we quantify changes in expression of simple (K8, K18, K19) and stratified squamous epithelial keratins (K5, K12, K14, and K15) during postnatal development and in response to 2.5 mm wounds using quantitative polymerase chain reaction (Q-PCR), confocal imaging and immunoblots. K14 + CNs are present 7 days after birth. By 21 days, when the eyelids are open, K8, K19, and Muc5AC are also expressed in CNs. By 28 days after wounding, the corneal epithelium shows enhanced mRNA and protein expression for K14 and retains mRNA and protein for corneal epithelial specific K12. CONCLUSIONS: The keratin phenotype observed in corneal epithelial cells before eyelid opening is similar to that seen during wound healing. Data show K14 + corneal epithelial progenitor cells expand in number after 2.5 mm wounds.
Asunto(s)
Córnea/metabolismo , Lesiones de la Cornea/metabolismo , Epitelio Corneal/metabolismo , Cicatrización de Heridas/fisiología , Animales , Movimiento Celular/fisiología , Desbridamiento , Queratina-8/metabolismo , Ratones , Mucina 5AC/metabolismoRESUMEN
Although sensory reinnervation occurs after injury in the peripheral nervous system, poor reinnervation in the elderly and those with diabetes often leads to pathology. Here we quantify sub-basal axon density in the central and peripheral mouse cornea over time after three different types of injury. The mouse cornea is highly innervated with a dense array of sub-basal nerves that form a spiral called the vortex at the corneal center or apex; these nerves are readily detected within flat mounted corneas. After anesthesia, corneal epithelial cells were removed using either a dulled blade or a rotating burr within an area demarcated centrally with a 1.5 mm trephine. A third wound type, superficial trephination, involved demarcating the area with the 1.5 mm trephine but not removing cells. By 7 days after superficial trephination, sub-basal axon density returns to control levels; by 28 days the vortex reforms. Although axon density is similar to control 14 days after dulled blade and rotating burr wounding, defects in axon morphology at the corneal apex remain. After 14 days, axons retract from the center leaving the sub-basal axon density reduced by 37.2 and 36.8% at 28 days after dulled blade and rotating burr wounding, respectively, compared with control. Assessment of inflammation using flow cytometry shows that persistent inflammation is not a factor in the incomplete reinnervation. Expression of mRNAs encoding 22 regeneration-associated genes involved in axon targeting assessed by QPCR reveals that netrin-1 and ephrin signaling are altered after wounding. Subpopulations of corneal epithelial basal cells at the corneal apex stop expressing ki67 as early as 7 days after injury and by 14 and 28 days after wounding, many of these basal cells undergo apoptosis and die. Although sub-basal axons are restored to their normal density and morphology after superficial trephination, sub-basal axon recovery is partial after debridement wounds. The increase in corneal epithelial basal cell apoptosis at the apex observed at 14 days after corneal debridement may destabilize newly reinnervated sub-basal axons and lead to their retraction toward the periphery.
Asunto(s)
Axones , Lesiones de la Cornea/cirugía , Desbridamiento , Desnervación , Animales , Apoptosis , Lesiones de la Cornea/fisiopatología , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Factores de Crecimiento Nervioso/genética , Netrina-1 , ARN Mensajero/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
Significance: The binding of cytokines and growth factors to heparan sulfate (HS) chains on proteoglycans generates gradients that control development and regulate wound healing. Syndecan-1 (sdc1) is an integral membrane HS proteoglycan. Its structure allows it to bind with cytosolic, transmembrane, and extracellular matrix (ECM) proteins. It plays important roles in mediating key events during wound healing because it regulates a number of important processes, including cell adhesion, cell migration, endocytosis, exosome formation, and fibrosis. Recent Advances: Recent studies reveal that sdc1 regulates wound healing by altering integrin activation. Differences in integrin activation lead to cell-type-specific changes in the rate of cell migration and ECM assembly. Sdc1 also regulates endocytosis and the formation and release of exosomes. Critical Issues: Understanding how sdc1 facilitates wound healing and resolution will improve treatment options for elderly and diabetic patients with delayed wound healing. Studies showing that sdc1 function is altered in cancer are relevant to those interested in controlling fibrosis and scarring. Future Directions: The key to understanding the various functions ascribed to sdc1 is resolving how it interacts with its numerous binding partners. The role played by chondroitin sulfate glycosaminoglycan (GAG) chains on the ability of sdc1 to associate with its ligands needs further investigation. At wound sites heparanase can cleave the HS GAG chains of sdc1, alter its ability to bind cytokines, and induce shedding of the ectodomain. This review will discuss how the unique structure of sdc1 allows it to play key roles in cell signaling, ECM assembly, and wound healing.
RESUMEN
Amyloid-ß (Aß) is produced through the enzymatic cleavage of amyloid precursor protein (APP) by ß (Bace1) and γ-secretases. The accumulation and aggregation of Aß as amyloid plaques is the hallmark pathology of Alzheimer׳s disease and has been found in other neurological disorders, such as traumatic brain injury and multiple sclerosis. Although the role of Aß after injury is not well understood, several studies have reported a negative correlation between Aß formation and functional outcome. In this study we show that levels of APP, the enzymes cleaving APP (Bace1 and γ-secretase), and Aß are significantly increased from 1 to 3 days after impact spinal cord injury (SCI) in mice. To determine the role of Aß after SCI, we reduced or inhibited Aß in vivo through pharmacological (using DAPT) or genetic (Bace1 knockout mice) approaches. We found that these interventions significantly impaired functional recovery as evaluated by white matter sparing and behavioral testing. These data are consistent with a beneficial role for Aß after SCI.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Presenilina-1/metabolismo , Recuperación de la Función/fisiología , Traumatismos de la Médula Espinal/metabolismo , Médula Espinal/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Secretasas de la Proteína Precursora del Amiloide/genética , Animales , Ácido Aspártico Endopeptidasas/genética , Western Blotting , Inhibidores Enzimáticos/farmacología , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/fisiología , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Fibras Nerviosas Mielínicas/efectos de los fármacos , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/patología , Presenilina-1/antagonistas & inhibidores , Recuperación de la Función/efectos de los fármacos , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Factores de TiempoRESUMEN
PURPOSE: An in vivo mouse model reproducibly induces recurrent epithelial erosions in wild-type mice spontaneously 2 weeks after a single 1.5-mm corneal debridement wound made with a dulled blade. When 1.5-mm wounds are made by a rotating burr so that the corneal epithelial basement membrane is removed, corneas heal without developing erosions. Here, we characterize differences in cytokine deposition and changes in leukocytes between 0 and 6 hours after dulled-blade and rotating-burr wounding. METHODS: BALB/c mice were used to study 1.5-mm corneal wounds made using a dulled blade or a rotating burr. Mice were studied immediately after wounding (0 hour) and at 6 hours in vivo and in vitro in organ culture. Corneas, corneal extracts, and collagenase digests from naïve and wounded mice were used for three-dimensional (3D) confocal imaging, cytokine arrays, and flow cytometry. RESULTS: Confocal imaging showed CD45, a protein derived from leukocytes, accumulates at the wound edge by 3 and 6 hours after wounding in vivo but not in vitro with more CD45 accumulating after dulled-blade compared with rotating-burr wounds. Morphologic changes occurred in CD45+ leukocytes and higher levels for several cytokines were detected in the stromal wound bed within minutes following dulled-blade wounds. Flow cytometry showed significantly more monocytes (CD45+/CD11b+/Ly6C+) and γδT cells (CD45+/GL3+) recruited into the corneas of mice with dulled-blade wounds by 6 hours. CONCLUSIONS: Differences in cytokine-driven leukocyte responses are seen after dulled-blade debridement compared with rotating-burr injury.
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Lesiones de la Cornea , Citocinas/metabolismo , Lesiones Oculares/inmunología , Inmunidad Innata , Leucocitos/patología , Cicatrización de Heridas/inmunología , Animales , Membrana Basal/metabolismo , Membrana Basal/patología , Córnea/inmunología , Córnea/patología , Modelos Animales de Enfermedad , Epitelio Corneal/lesiones , Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Lesiones Oculares/metabolismo , Lesiones Oculares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Monocitos/patologíaRESUMEN
Corneal wound healing studies have a long history and rich literature that describes the data obtained over the past 70 years using many different species of animals and methods of injury. These studies have lead to reduced suffering and provided clues to treatments that are now helping patients live more productive lives. In spite of the progress made, further research is required since blindness and reduced quality of life due to corneal scarring still happens. The purpose of this review is to summarize what is known about different types of wound and animal models used to study corneal wound healing. The subject of corneal wound healing is broad and includes chemical and mechanical wound models. This review focuses on mechanical injury models involving debridement and keratectomy wounds to reflect the authors' expertise.
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Córnea/cirugía , Lesiones de la Cornea , Modelos Animales de Enfermedad , Cicatrización de Heridas/fisiología , Animales , Desbridamiento , Ratones , Técnicas de Cultivo de Órganos , ConejosRESUMEN
Inflammation has long been implicated in secondary tissue damage after spinal cord injury (SCI). Our previous studies of inflammatory gene expression in rats after SCI revealed two temporally correlated clusters: the first was expressed early after injury and the second was up-regulated later, with peak expression at 1-2 weeks and persistent up-regulation through 6 months. To further address the role of inflammation after SCI, we examined inflammatory genes in a second species, mice, through 28 days after SCI. Using anchor gene clustering analysis, we found similar expression patterns for both the acute and chronic gene clusters previously identified after rat SCI. The acute group returned to normal expression levels by 7 days post injury. The chronic group, which included C1qB, p22(phox) and galectin-3, showed peak expression at 7 days and remained up-regulated through 28 days. Immunohistochemistry and western blot analysis showed that the protein expression of these genes was consistent with the mRNA expression. Further exploration of the role of one of these genes, galectin-3, suggests that galectin-3 may contribute to secondary injury. In summary, our findings extend our prior gene profiling data by demonstrating the chronic expression of a cluster of microglial associated inflammatory genes after SCI in mice. Moreover, by demonstrating that inhibition of one such factor improves recovery, the findings suggest that such chronic up-regulation of inflammatory processes may contribute to secondary tissue damage after SCI, and that there may be a broader therapeutic window for neuroprotection than generally accepted.
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Galectina 3/fisiología , Regulación de la Expresión Génica , Mediadores de Inflamación/metabolismo , Traumatismos de la Médula Espinal/metabolismo , Animales , Animales Recién Nacidos , Células Cultivadas , Galectina 3/biosíntesis , Galectina 3/genética , Masculino , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Traumatismos de la Médula Espinal/genética , Traumatismos de la Médula Espinal/patologíaRESUMEN
Goblet cells are terminally differentiated cells secreting mucins and antibacterial peptides that play an important role in maintaining the health of the cornea. In corneal stem cell deficiency, the progenitor cells giving rise to goblet cells on the cornea are presumed to arise from differentiation of cells that migrate onto the cornea from the neighboring conjunctiva. This occurs in response to the inability of corneal epithelial progenitor cells at the limbus to maintain an intact corneal epithelium. This study characterizes clusters of cells we refer to as compound niches at the limbal:corneal border in the unwounded mouse. Compound niches are identified by high expression of simple epithelial keratin 8 (K8) and 19 (K19). They contain variable numbers of cells in one of several differentiation states: slow-cycling corneal progenitor cells, proliferating cells, nonproliferating cells, and postmitotic differentiated K12+Muc5ac+ goblet cells. Expression of K12 differentiates these goblet cells from those in the conjunctival epithelium and suggests that corneal epithelial progenitor cells give rise to both corneal epithelial and goblet cells. After wounds that remove corneal epithelial cells near the limbus, compound niches migrate from the limbal:corneal border onto the cornea where K8+ cells proliferate and goblet cells increase in number. By contrast, no migration of goblet cells from the bulbar conjunctiva onto the cornea is observed. This study is the first description of compound niches and corneal goblet cells and demonstration of a role for these cells in the pathology typically associated with corneal stem cell deficiency.
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Córnea/citología , Enfermedades de la Córnea/patología , Células Caliciformes/citología , Animales , Diferenciación Celular , Epitelio Corneal/patología , Humanos , Ratones , Ratones Endogámicos BALB C , Células Madre/citologíaRESUMEN
BACKGROUND: Traumatic spinal cord injury (SCI) induces secondary tissue damage that is associated with astrogliosis and inflammation. We previously reported that acute upregulation of a cluster of cell-cycle-related genes contributes to post-mitotic cell death and secondary damage after SCI. However, it remains unclear whether cell cycle activation continues more chronically and contributes to more delayed glial change. Here we examined expression of cell cycle-related proteins up to 4 months following SCI, as well as the effects of the selective cyclin-dependent kinase (CDKs) inhibitor CR8, on astrogliosis and microglial activation in a rat SCI contusion model. METHODS: Adult male rats were subjected to moderate spinal cord contusion injury at T8 using a well-characterized weight-drop model. Tissue from the lesion epicenter was obtained 4 weeks or 4 months post-injury, and processed for protein expression and lesion volume. Functional recovery was assessed over the 4 months after injury. RESULTS: Immunoblot analysis demonstrated a marked continued upregulation of cell cycle-related proteins - including cyclin D1 and E, CDK4, E2F5 and PCNA - for 4 months post-injury that were highly expressed by GFAP+ astrocytes and microglia, and co-localized with inflammatory-related proteins. CR8 administrated systemically 3 h post-injury and continued for 7 days limited the sustained elevation of cell cycle proteins and immunoreactivity of GFAP, Iba-1 and p22PHOX - a key component of NADPH oxidase - up to 4 months after SCI. CR8 treatment significantly reduced lesion volume, which typically progressed in untreated animals between 1 and 4 months after trauma. Functional recovery was also significantly improved by CR8 treatment after SCI from week 2 through week 16. CONCLUSIONS: These data demonstrate that cell cycle-related proteins are chronically upregulated after SCI and may contribute to astroglial scar formation, chronic inflammation and further tissue loss.
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Astrocitos/metabolismo , Proteínas de Ciclo Celular/biosíntesis , Cicatriz/metabolismo , Regulación de la Expresión Génica , Traumatismos de la Médula Espinal/metabolismo , Animales , Astrocitos/patología , Enfermedad Crónica , Cicatriz/patología , Inflamación/metabolismo , Inflamación/patología , Masculino , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal/patología , Factores de TiempoRESUMEN
Traumatic spinal cord injury (SCI) causes tissue loss and associated neurological dysfunction through mechanical damage and secondary biochemical and physiological responses. We have previously described the pathobiological role of cell cycle pathways following rat contusion SCI by examining the effects of early intrathecal cell cycle inhibitor treatment initiation or gene knockout on secondary injury. Here, we delineate changes in cell cycle pathway activation following SCI and examine the effects of delayed (24 h) systemic administration of flavopiridol, an inhibitor of major cyclin-dependent kinases (CDKs), on functional recovery and histopathology in a rat SCI contusion model. Immunoblot analysis demonstrated a marked upregulation of cell cycle-related proteins, including pRb, cyclin D1, CDK4, E2F1 and PCNA, at various time points following SCI, along with downregulation of the endogenous CDK inhibitor p27. Treatment with flavopiridol reduced induction of cell cycle proteins and increased p27 expression in the injured spinal cord. Functional recovery was significantly improved after SCI from day 7 through day 28. Treatment significantly reduced lesion volume and the number of Iba-1(+) microglia in the preserved tissue and increased the myelinated area of spared white matter as well as the number of CC1(+) oligodendrocytes. Furthermore, flavopiridol attenuated expression of Iba-1 and glactin-3, associated with microglial activation and astrocytic reactivity by reduction of GFAP, NG2, and CHL1 expression. Our current study supports the role of cell cycle activation in the pathophysiology of SCI and by using a clinically relevant treatment model, provides further support for the therapeutic potential of cell cycle inhibitors in the treatment of human SCI.
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Ciclo Celular , Flavonoides/farmacología , Piperidinas/farmacología , Traumatismos de la Médula Espinal/fisiopatología , Animales , Apoptosis , Proteínas de Unión al Calcio/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclina D1/metabolismo , Quinasa 4 Dependiente de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Factor de Transcripción E2F1/metabolismo , Flavonoides/administración & dosificación , Inmunohistoquímica , Locomoción , Masculino , Proteínas de Microfilamentos/metabolismo , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuronas/efectos de los fármacos , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Piperidinas/administración & dosificación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ratas , Ratas Sprague-Dawley , Médula Espinal/metabolismo , Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Factores de TiempoRESUMEN
The adult spinal cord contains a pool of endogenous glial precursor cells, which spontaneously respond to spinal cord injury (SCI) with increased proliferation. These include oligodendrocyte precursor cells that express the NG2 proteoglycan and can differentiate into mature oligodendrocytes. Thus, a potential approach for SCI treatment is to enhance the proliferation and differentiation of these cells to yield more functional mature glia and improve remyelination of surviving axons. We previously reported that soluble glial growth factor 2 (GGF2)- and basic fibroblast growth factor 2 (FGF2)-stimulated growth of NG2(+) cells purified from injured spinal cord in primary culture. This study examines the effects of systemic administration of GGF2 and/or FGF2 after standardized contusive SCI in vivo in both rat and mouse models. In Sprague-Dawley rats, 1 week of GGF2 administration, beginning 24 h after injury, enhanced NG2(+) cell proliferation, oligodendrogenesis, chronic white matter at the injury epicenter, and recovery of hind limb function. In 2',3'-cyclic-nucleotide 3'-phosphodiesterase-enhanced green fluorescent protein mice, GGF2 treatment resulted in increased oligodendrogenesis and improved functional recovery, as well as elevated expression of the stem cell transcription factor Sox2 by oligodendrocyte lineage cells. Although oligodendrocyte number was increased chronically after SCI in GGF2-treated mice, no evidence of increased white matter was detected. However, GGF2 treatment significantly increased levels of P0 protein-containing peripheral myelin, produced by Schwann cells that infiltrate the injured spinal cord. Our results suggest that GGF2 may have therapeutic potential for SCI by enhancing endogenous recovery processes in a clinically relevant time frame.
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Regeneración Nerviosa/efectos de los fármacos , Neurregulina-1/fisiología , Recuperación de la Función , Traumatismos de la Médula Espinal/tratamiento farmacológico , Regulación hacia Arriba , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Modelos Animales de Enfermedad , Femenino , Sustancias de Crecimiento/biosíntesis , Sustancias de Crecimiento/fisiología , Masculino , Ratones , Ratones Transgénicos , Fibras Nerviosas Mielínicas/patología , Fibras Nerviosas Mielínicas/fisiología , Regeneración Nerviosa/fisiología , Neurregulina-1/biosíntesis , Neurregulina-1/farmacología , Oligodendroglía/efectos de los fármacos , Oligodendroglía/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/farmacología , Recuperación de la Función/efectos de los fármacos , Recuperación de la Función/genética , Células de Schwann/efectos de los fármacos , Células de Schwann/fisiología , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
Recurrent corneal erosions are painful and put patients' vision at risk. Treatment typically begins with debridement of the area around the erosion site followed by more aggressive treatments. An in vivo mouse model has been developed that reproducibly induces recurrent epithelial erosions in wild-type mice spontaneously within two weeks after a single 1.5 mm corneal debridement wound created using a dulled-blade. This study was conducted to determine whether 1) inhibiting MMP9 function during healing after dulled-blade wounding impacts erosion development and 2) wounds made with a rotating-burr heal without erosions. Oral or topical inhibition of MMPs after dulled-blade wounding does not improve healing. Wounds made by rotating-burr heal with significantly fewer erosions than dulled-blade wounds. The localization of MMP9, ß4 integrin and basement membrane proteins (LN332 and type VII collagen), immune cell influx, and reinnervation of the corneal nerves were compared after both wound types. Rotating-burr wounds remove the anterior basement membrane centrally but not at the periphery near the wound margin, induce more apoptosis of corneal stromal cells, and damage more stromal nerve fibers. Despite the fact that rotating-burr wounds do more damage to the cornea, fewer immune cells are recruited and significantly more wounds resolve completely.
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Membrana Basal/patología , Córnea/patología , Enfermedades de la Córnea/patología , Cicatrización de Heridas , Administración Oftálmica , Administración Oral , Animales , Apoptosis , Membrana Basal/inmunología , Membrana Basal/metabolismo , Membrana Basal/cirugía , Moléculas de Adhesión Celular/metabolismo , Colágeno Tipo VII/metabolismo , Córnea/efectos de los fármacos , Córnea/inmunología , Córnea/inervación , Córnea/metabolismo , Córnea/cirugía , Enfermedades de la Córnea/tratamiento farmacológico , Enfermedades de la Córnea/etiología , Enfermedades de la Córnea/inmunología , Enfermedades de la Córnea/metabolismo , Desbridamiento/instrumentación , Modelos Animales de Enfermedad , Integrina beta4/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Endogámicos BALB C , Inhibidores de Proteasas/administración & dosificación , Factores de Tiempo , Cicatrización de Heridas/efectos de los fármacos , KalininaRESUMEN
Integrin α6ß4 is an integral membrane protein within hemidesmosomes and it mediates adhesion of epithelial cells to their underlying basement membrane. During wound healing, disassembly of hemidesmosomes must occur for sheet movement-mediated cell migration. The mechanisms of disassembly and reassembly of hemidesmosomes are not fully understood. The current study was initiated to understand the underlying cause of recurrent corneal erosions in the mouse. Here, we show that in vivo: (1) MMP9 levels are elevated and ß4 integrin is partially cleaved in epithelial cell extracts derived from debridement wounded corneas; (2) the ß4 ectodomain is missing from sites where erosions develop; and (3) ß4 cleavage can be reduced by inhibiting MMP activity. Although ß4, α3 and ß1 integrins were all cleaved by several MMPs, only MMP9 was elevated in cell extracts derived from corneas with erosions. Coimmunoprecipitation studies showed that ß4 integrin associates with MMP9, and protein clustering during immunoprecipitation induced proteolytic cleavage of the ß4 integrin extracellular domain, generating a 100 kDa ß4 integrin cytoplasmic domain fragment. Confocal imaging with three-dimensional reconstruction showed that MMP9 localizes at erosion sites in vivo where the ectodomain of ß4 integrin is reduced or absent. MMP activation experiments using cultured corneal and epidermal keratinocytes showed reduced levels of α6ß4 and ß1 integrins within 20 minutes of phorbol ester treatment. This report is the first to show that ß4 integrin associates with MMP9 and that its ectodomain is a target for cleavage by MMP9 in vivo under pathological conditions.
Asunto(s)
Epitelio Corneal/metabolismo , Epitelio Corneal/patología , Integrina beta4/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Animales , Células Cultivadas , Immunoblotting , Inmunoprecipitación , Técnicas In Vitro , Integrina beta4/genética , Queratinocitos/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Reacción en Cadena de la Polimerasa , Unión ProteicaRESUMEN
Injury to the spinal cord is known to result in inflammation. To date, the preponderance of research has focused on the acute neuroinflammatory response, which begins immediately and is believed to terminate within hours to (at most) days after the injury. However, recent studies have demonstrated that postinjury inflammation is not restricted to the first few hours or days after injury, but can last for months to years after a spinal cord injury (SCI). These chronic studies have revealed that increased numbers of inflammatory cells, such as microglia and macrophages, and inflammatory factors, including cytokines, chemokines, and enzyme products are found at markedly delayed times after injury. Here we review experimental work on a selection of the novel inflammatory factors observed chronically after SCI, including the nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) oxidase enzyme and galectin-3. We will discuss the role of these proteins in inflammation with regard to both detrimental and beneficial effects of neuroinflammation after injury. Finally, the potential of these proteins to serve as therapeutic targets will be considered, and a novel therapeutic approach (i.e., the agonist for metabotropic glutamate receptor 5 [mGluR5], [RS]-2-Chloro-5-hydroxyphenylglycine [CHPG]) will be discussed. This review will demonstrate the expression and activity profiles, roles in potentiation of injury, and therapy studies of these inflammatory factors suggest that not only are these chronically expressed factors viable targets for SCI treatment, but that the therapeutic window is broader than has previously been thought.