RESUMEN
Parkinson's disease (PD) is a disabling neurodegenerative disease that manifests with resting tremor, bradykinesia, rigidity and postural instability. Since the discovery of microRNAs (miRNAs) in 1993, miRNAs have been shown to be important biological molecules involved in diverse processes to maintain normal cellular functions. Over the past decade, many studies have reported dysregulation of miRNA expressions in PD. Here, we identified 15 miRNAs from 34 reported screening studies that demonstrated dysregulation in the brain and/or neuronal models, cerebrospinal fluid (CSF) and blood. Specific miRNAs-of-interest that have been implicated in PD pathogenesis include miR-30, miR-29, let-7, miR-485 and miR-26. However, there are several challenges and limitations in drawing definitive conclusions due to the small sample size in clinical studies, varied laboratory techniques and methodologies and their incomplete penetrance of the blood-brain barrier. Developing an optimal delivery system and unravelling druggable targets of miRNAs in both experimental and human models and clinical validation of the results may pave way for novel therapeutics in PD.
Asunto(s)
Regulación de la Expresión Génica , MicroARNs/genética , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Animales , HumanosRESUMEN
Pharmaceutically active compounds require different modes of drug delivery systems to accomplish therapeutic activity without loss of its activity and lead to exhibit no adverse effects. Originating from ancient days, pulmonary mode of drug delivery is gaining much importance compared to other modes of drug delivery systems with respect to specific diseases. Pulmonary drug delivery is a non-invasive route for local and systemic therapies together with more patient convenience, compliance and is a needleless system. In this review, we addressed the vaccine delivery via non- or minimally invasive routes. Polymeric nanoparticles are preferred for use in the pulmonary delivery devices owing to a prolonged retention in lungs. Small site for absorption, mucociliary clearance, short residence time and low bioavailability are some of the limitations in pulmonary drug delivery have been resolved by generating micro- and nano-sized aerosol particles. We have classified the breathable medicine on the basis of available devices for inhalation and also prominent diseases treated through pulmonary mode of drug delivery. Owing to increasing toxicity of pharmacological drugs, the use of natural medicines has been rapidly gaining importance recently. The review article describes breathability of medicines or the pulmonary mode of drug delivery system and their drug release profile, absorption, distribution and efficacy to cure asthma and diabetes.
Asunto(s)
Administración por Inhalación , Sistemas de Liberación de Medicamentos , Nanomedicina , Nanopartículas/administración & dosificación , Humanos , Pulmón/metabolismo , Pulmón/fisiología , Extractos Vegetales/administración & dosificación , Polvos , Vacunas/administración & dosificaciónRESUMEN
BACKGROUND/AIM: Microglia, the resident macrophages in the central nervous system, secrete various proinflammatory cytokines and undergo proliferation upon activation in various neurodegenerative diseases. Activation of microglia has been implicated in exacerbation of various neurodegenerative diseases. Recently, it has been proposed that mesenchymal stem cells (MSC) have immunosuppressive properties and the potential to moderate inflammation. This study aimed to elucidate the effects of MSC-conditioned medium (MSC-CM) in modulating microglial activation by analyzing microglial proinflammatory and anti-inflammatory factors [interleukin (IL)-6, tumor necrosis factor (TNF)-α, inducible nitric oxide synthase (iNOS) and IL-10], signaling pathway molecules [NFκB, c-Jun N-terminal kinase (JNK) and MKP-1) and NO production. METHODS: Immortalized murine microglia cell line, BV2 microglia and primary microglia isolated from C57BL/6 mouse pup brains were used in this study. Mouse MSC were isolated from the male C57BL/6 mouse tibia and fibula. The effects of MSC-CM on the expression of inflammatory cytokines and signaling molecules in microglia were elucidated using RT-PCR, immunofluorescence analysis and Western blot analysis. NO production in microglia was assessed using a Griess kit. RESULTS: MSC-CM significantly reduced the mRNA and protein expression levels of proinflammatory cytokines (IL-6 and TNF-α) in microglia activated by lipopolysaccharide (LPS). In addition, MSC-CM significantly reduced the protein expression of NFκB, JNK and c-Jun, but increased the expression levels of IL-10 and MKP-1 in activated BV2 microglia. NO production and iNOS expression by BV2 microglia in MSC-CM were increased. CONCLUSIONS: Overall, our findings suggest that MSC immunomodulate microglial activities through paracrine effects.
Asunto(s)
Citocinas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Microglía/metabolismo , Óxido Nítrico/metabolismo , Comunicación Paracrina/fisiología , Animales , Corteza Cerebral , Medios de Cultivo Condicionados , Masculino , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Maternal diabetes alters gene expression leading to neural tube defects (NTDs) in the developing brain. The mechanistic pathways that deregulate the gene expression remain unknown. It is hypothesized that exposure of neural stem cells (NSCs) to high glucose/hyperglycemia results in activation of epigenetic mechanisms which alter gene expression and cell fate during brain development. METHODS AND FINDINGS: NSCs were isolated from normal pregnancy and streptozotocin induced-diabetic pregnancy and cultured in physiological glucose. In order to examine hyperglycemia induced epigenetic changes in NSCs, chromatin reorganization, global histone status at lysine 9 residue of histone H3 (acetylation and trimethylation) and global DNA methylation were examined and found to be altered by hyperglycemia. In NSCs, hyperglycemia increased the expression of Dcx (Doublecortin) and Pafah1b1 (Platelet activating factor acetyl hydrolase, isoform 1b, subunit 1) proteins concomitant with decreased expression of four microRNAs (mmu-miR-200a, mmu-miR-200b, mmu-miR-466a-3p and mmu-miR-466 d-3p) predicted to target these genes. Knockdown of specific microRNAs in NSCs resulted in increased expression of Dcx and Pafah1b1 proteins confirming target prediction and altered NSC fate by increasing the expression of neuronal and glial lineage markers. CONCLUSION/INTERPRETATION: This study revealed that hyperglycemia alters the epigenetic mechanisms in NSCs, resulting in altered expression of some development control genes which may form the basis for the NTDs. Since epigenetic changes are reversible, they may be valuable therapeutic targets in order to improve fetal outcomes in diabetic pregnancy.
Asunto(s)
Células Madre Embrionarias/metabolismo , Epigénesis Genética/genética , Hiperglucemia/genética , 1-Alquil-2-acetilglicerofosfocolina Esterasa/genética , Animales , Células Cultivadas , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Embrión de Mamíferos , Células Madre Embrionarias/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Femenino , Glucosa/farmacología , Histonas/metabolismo , Ratones , MicroARNs/genética , Proteínas Asociadas a Microtúbulos/genética , Neuropéptidos/genética , EmbarazoRESUMEN
Congenital heart defects (CHD) are one of the most common defects in offspring of diabetic mothers. There is a clear association between maternal diabetes and CHD; however the underlying molecular mechanism remains unknown. We hypothesized that maternal diabetes affects with the expression of early developmental genes that regulate the essential developmental processes of the heart, thereby resulting in the pathogenesis of CHD. We analyzed genome-wide expression profiling in the developing heart of embryos from diabetic and control mice by using the oligonucleotide microarray. Microarray analysis revealed that a total of 878 genes exhibited more than 1.5 fold changes in expression level in the hearts of experimental embryos in either E13.5 or E15.5 compared with their respective controls. Expression pattern of genes that is differentially expressed in the developing heart was further examined by the real-time reverse transcriptase-polymerase chain reaction. Several genes involved in a number of molecular signaling pathways such as apoptosis, proliferation, migration and differentiation in the developing heart were differentially expressed in embryos of diabetic pregnancy. It is concluded that altered expression of several genes involved in heart development may contribute to CHD in offspring of diabetic mothers.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Corazón/embriología , Embarazo en Diabéticas , Transcriptoma , Animales , Análisis por Conglomerados , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/embriología , Diabetes Mellitus Experimental/genética , Femenino , Masculino , Ratones , Análisis por Micromatrices , Miocardio/metabolismo , Embarazo , Embarazo en Diabéticas/genética , Efectos Tardíos de la Exposición Prenatal/genética , Estreptozocina , Estudios de Validación como AsuntoRESUMEN
Oxidative stress induced by maternal diabetes plays an important role in the development of cardiac malformations. Zinc (Zn) supplementation of animals and humans has been shown to ameliorate oxidative stress induced by diabetic cardiomyopathy. However, the role of Zn in the prevention of oxidative stress induced by diabetic cardiac embryopathy remains unknown. We analyzed the preventive role of Zn in diabetic cardiac embryopathy by both in vivo and in vitro studies. In vivo study revealed a significant decrease in lipid peroxidation, superoxide ions, and oxidized glutathione and an increase in reduced glutathione, nitric oxide, and superoxide dismutase in the developing heart at embryonic days (E) 13.5 and 15.5 in the Zn-supplemented diabetic group when compared to the diabetic group. In addition, significantly down-regulated protein and mRNA expression of metallothionein (MT) in the developing heart of embryos from diabetic group was rescued by Zn supplement. Further, the nuclear microscopy results showed that trace elements such as phosphorus, calcium, and Zn levels were significantly increased (P<0.001), whereas the iron level was significantly decreased (P<0.05) in the developing heart of embryos from the Zn-supplemented diabetic group. In vitro study showed a significant increase in cellular apoptosis and the generation of reactive oxygen species (ROS) in H9c2 (rat embryonic cardiomyoblast) cells exposed to high glucose concentrations. Supplementation with Zn significantly decreased apoptosis and reduced the levels of ROS. In summary, oxidative stress induced by maternal diabetes could play a role in the development and progression of cardiac embryopathy, and Zn supplementation could be a potential therapy for diabetic cardiac embryopathy.
Asunto(s)
Apoptosis/efectos de los fármacos , Diabetes Mellitus Experimental/fisiopatología , Suplementos Dietéticos , Embrión de Mamíferos/efectos de los fármacos , Cardiopatías Congénitas/prevención & control , Estrés Oxidativo , Zinc/administración & dosificación , Animales , Glucemia/metabolismo , Western Blotting , Células Cultivadas , Complicaciones de la Diabetes/etiología , Complicaciones de la Diabetes/patología , Complicaciones de la Diabetes/prevención & control , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Femenino , Glutatión/genética , Glutatión/metabolismo , Cardiopatías Congénitas/etiología , Cardiopatías Congénitas/patología , Técnicas para Inmunoenzimas , Peroxidación de Lípido/efectos de los fármacos , Metalotioneína/genética , Metalotioneína/metabolismo , Ratones , Miocitos Cardíacos/citología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Microscopía Nuclear , ARN Mensajero/genética , Ratas , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismoRESUMEN
While OX42(+) microglia/macrophages have been considered as a scavenger in the brain, NG2(+) cells are generally considered as oligodendrocyte progenitor cells or function-unknown glial cells. Recent evidence showed that under some pathological conditions, certain cells have become positive for both anti-NG2 and anti-OX42 antibodies. Our results suggested that some OX42(+) microglia or macrophages were induced to express NG2 proteins 3 and 5 days later after focal injection of lipopolysaccharide into the brain cortex of Sprague-Dawley rats. In consideration of the induction of NG2 expression may associate with gaining or losing functions of microglia/macrophages, we further showed that, while OX42(+) or ED1(+) microglia/macrophages presented active phagocytic function, NG2(+) /OX42(+) cells failed to engulf latex beads. The induced expression of NG2 protein may possibly indicate the functional diversity of activated microglia/macrophages in the brain.
Asunto(s)
Antígenos/metabolismo , Encéfalo/inmunología , Lipopolisacáridos/farmacología , Microglía/inmunología , Monocitos/inmunología , Fagocitosis/fisiología , Proteoglicanos/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Masculino , Microglía/efectos de los fármacos , Microglía/metabolismo , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Fagocitosis/efectos de los fármacos , Ratas , Ratas Sprague-DawleyRESUMEN
Diabetes mellitus rightly regarded as a silent-epidemic is continually on the rise and estimated to have a global prevalence of 6.4 % as of 2010. Diabetes during pregnancy is a well known risk factor for congenital anomalies in various organ systems that contribute to neonatal mortality, including cardiovascular, gastrointestinal, genitourinary and neurological systems, among which the neural tube defects are frequently reported. Over the last two to three decades, several groups around the world have focussed on identifying the molecular cues and cellular changes resulting in altered gene expression and the morphological defects and in diabetic pregnancy. In recent years, the focus has gradually shifted to looking at pre-programmed changes and activation of epigenetic mechanisms that cause altered gene expression. While several theories such as oxidative stress, hypoxia, and apoptosis triggered due to hyperglycemic conditions have been proposed and proven for being the cause for these defects, the exact mechanism or the link between how high glucose can alter gene expression/transcriptome and activate epigenetic mechanisms is largely unknown. Although preconceptual control of diabetes, (i.e., managing glucose levels during pregnancy), and in utero therapies has been proposed as an effective solution for managing diabetes during pregnancy, the impact that a fluctuating glycemic index can have on foetal development has not been evaluated in detail. A tight glycemic control started before pregnancy has shown to reduce the incidence of congenital abnormalities in diabetic mothers. On the other hand, a tight glycemic control after organogenesis and embryogenesis have begun may prove insufficient to prevent or reverse the onset of congenital defects. The importance of determining the extent to which glycemic levels in diabetic mothers should be regulated is critical as foetal hypoglycemia has also been shown to be teratogenic. Finally, the major question remaining is if this whole issue is negligible and not worthy of investigation as the efficient management of diabetes during pregnancy is well in place in many countries.
Asunto(s)
Anatomía/historia , Animales , Educación Médica/historia , Historia del Siglo XX , Humanos , SingapurRESUMEN
Immunomodulatory effects of transplanted mesenchymal stem cells (MSCs) in the treatment of Parkinson's disease were studied in the MPTP-induced mouse model. MPTP treatment induced a significant loss of dopaminergic neurons, decreased expressions of claudin 1, claudin 5 and occludin in the substantia nigra compacta (SNc), and functional damage of the blood brain barrier (BBB). Our study further discovered that infiltration of MBLs into the brain to bind with microglia was detected in the SNc of MPTP-treated mice, suggesting that the BBB compromise and MBL infiltration might be involved in the pathogenesis of MPTP-induced PD. In addition, MPTP treatment also increased the expression of mannose-binding lectins (MBLs) in the liver tissue. Intravenous transplantation of MSCs into MPTP-treated mice led to recovery of BBB integrity, suppression of MBL infiltration at SNc and MBL expression in the liver, suppression of microglial activation and prevention of dopaminergic neuron death. No transplanted MSCs were observed to differentiate into dopaminergic neurons, while the MSCs migrated into the SNc and released TGF-beta1 there. Therefore, intravenous transplantation of MSCs which protect dopaminergic neurons from MPTP toxicity may be engaged in anyone or a combination of these mechanisms: repair of the BBB, reduction of MBL in the brain, inhibition of microglial cytotoxicity, and direct protection of dopaminergic neurons.
Asunto(s)
Barrera Hematoencefálica/fisiopatología , Encefalitis/fisiopatología , Trasplante de Células Madre Mesenquimatosas/métodos , Neuronas/patología , Trastornos Parkinsonianos/cirugía , Sustancia Negra/fisiopatología , Animales , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/patología , Muerte Celular/fisiología , Células Cultivadas , Modelos Animales de Enfermedad , Dopamina/metabolismo , Encefalitis/inducido químicamente , Gliosis/metabolismo , Gliosis/fisiopatología , Gliosis/prevención & control , Infusiones Intravenosas , Hígado/metabolismo , Hígado/fisiopatología , Masculino , Lectinas de Unión a Manosa/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/fisiopatología , Degeneración Nerviosa/prevención & control , Neuronas/metabolismo , Trastornos Parkinsonianos/metabolismo , Trastornos Parkinsonianos/fisiopatología , Sustancia Negra/metabolismo , Resultado del TratamientoRESUMEN
The aim of this study was to investigate the role of nitric oxide (NO), and the expression of endothelial nitric oxide synthase (eNOS) and vascular endothelial growth factor (VEGF) genes in developing hearts at embryonic day 13.5 of embryos from diabetic mice. The protein and mRNA expression levels of eNOS and VEGF were significantly altered in the developing hearts of embryos from diabetic mice. The NO level was significantly decreased, whereas the VEGF concentration was significantly increased in the developing hearts of the embryos from diabetic mice. In vitro study showed a significant reduction in eNOS expression and cell proliferation in cardiac myoblast cells exposed to high glucose concentrations. Further, high glucose induced apoptosis in myoblast cells. Ultrastructural changes characteristics of apoptosis, including cell blebbing, aggregation of ribosomes and vacuoles in the cytoplasm were also evident in myoblast cells exposed to high glucose. It is suggested that hyperglycemia alters the expression of eNOS and VEGF genes that are involved in the regulation of cell growth and vasculogenesis, thereby contributing to the cardiac malformations seen in embryos from diabetic mice.
Asunto(s)
Diabetes Mellitus Experimental/genética , Desarrollo Embrionario/genética , Cardiopatías Congénitas/genética , Corazón/embriología , Óxido Nítrico Sintasa/genética , Factor A de Crecimiento Endotelial Vascular/genética , Animales , Apoptosis , Proliferación Celular , Células Cultivadas , Femenino , Glucosa/farmacología , Cardiopatías Congénitas/patología , Cardiopatías Congénitas/ultraestructura , Ratones , Mioblastos Cardíacos/citología , Mioblastos Cardíacos/efectos de los fármacos , Mioblastos Cardíacos/metabolismo , Miocardio/enzimología , Miocardio/metabolismo , Miocardio/patología , ARN Mensajero/metabolismo , RatasRESUMEN
BACKGROUND: Congenital heart defects are frequently observed in infants of diabetic mothers, but the molecular basis of the defects remains obscure. Thus, the present study was performed to gain some insights into the molecular pathogenesis of maternal diabetes-induced congenital heart defects in mice. METHODS AND RESULTS: We analyzed the morphological changes, the expression pattern of some genes, the proliferation index and apoptosis in developing heart of embryos at E13.5 from streptozotocin-induced diabetic mice. Morphological analysis has shown the persistent truncus arteriosus combined with a ventricular septal defect in embryos of diabetic mice. Several other defects including defective endocardial cushion (EC) and aberrant myofibrillogenesis have also been found. Cardiac neural crest defects in experimental embryos were analyzed and validated by the protein expression of NCAM and PGP 9.5. In addition, the protein expression of Bmp4, Msx1 and Pax3 involved in the development of cardiac neural crest was found to be reduced in the defective hearts. The mRNA expression of Bmp4, Msx1 and Pax3 was significantly down-regulated (p < 0.001) in the hearts of experimental embryos. Further, the proliferation index was significantly decreased (p < 0.05), whereas the apoptotic cells were significantly increased (p < 0.001) in the EC and the ventricular myocardium of the experimental embryos. CONCLUSION: It is suggested that the down-regulation of genes involved in development of cardiac neural crest could contribute to the pathogenesis of maternal diabetes-induced congenital heart defects.
Asunto(s)
Diabetes Mellitus Experimental/complicaciones , Regulación del Desarrollo de la Expresión Génica , Defectos del Tabique Interventricular/genética , Corazón/embriología , Miocitos Cardíacos/química , Cresta Neural/química , Tronco Arterial Persistente/genética , Animales , Apoptosis , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/análisis , Proteínas Morfogenéticas Óseas/genética , Proliferación Celular , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Regulación hacia Abajo , Embrión de Mamíferos/química , Femenino , Defectos del Tabique Interventricular/embriología , Defectos del Tabique Interventricular/metabolismo , Defectos del Tabique Interventricular/patología , Factor de Transcripción MSX1/análisis , Factor de Transcripción MSX1/genética , Ratones , Miocitos Cardíacos/ultraestructura , Moléculas de Adhesión de Célula Nerviosa/análisis , Moléculas de Adhesión de Célula Nerviosa/genética , Cresta Neural/embriología , Cresta Neural/patología , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/análisis , Factores de Transcripción Paired Box/genética , Embarazo , ARN Mensajero/análisis , Tronco Arterial Persistente/embriología , Tronco Arterial Persistente/metabolismo , Tronco Arterial Persistente/patología , Ubiquitina Tiolesterasa/análisis , Ubiquitina Tiolesterasa/genéticaRESUMEN
Opposing functions of activated microglia, namely neuroprotection or neurotrophy versus neurodestruction or neurotoxicity, have been observed in a number of experimental models of neurotrauma and neurodegenerative diseases. However, the mechanism(s) involved in the determination of which function activated microglia execute under a given set of conditions still remains to be elucidated. Our current in vitro study has revealed that a neuroprotective/neurotrophic or a neurodestructive/neurotoxic microglial function may be configured by the equilibrium among various microglial factors released into the microenvironment. When NSC-34 neurons were treated with lower concentrations of lipopolysaccharide-stimulated BV-2 microglial conditioned medium (LPS-BVCM), viability of the NSC-34 neurons increased, outgrowth of neuronal processes was promoted, and the formation of 2,5-hexanedione-induced aggregates was prevented. However, when NSC-34 neurons were treated with higher concentrations of the same LPS-BVCM, neuronal viability was reduced, apoptosis was induced and outgrowth of neuronal processes was prevented. Measurement of the cytokines tumor necrotic factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), and IL-6 in the LPS-BVCM has shown that the upregulation in expression for each cytokine varied both temporally and quantitatively. It is postulated that an alteration in the concentration of the LPS-BVCM might significantly affect the functional balance of microglial factors in the microenvironment with a resultant different microglial function.
Asunto(s)
Microglía/química , Microglía/fisiología , Análisis de Varianza , Animales , Anexina A5/metabolismo , Apoptosis/efectos de los fármacos , Aumento de la Célula , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular , Supervivencia Celular/efectos de los fármacos , Medios de Cultivo Condicionados/farmacología , Citocinas/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Ensayo de Inmunoadsorción Enzimática/métodos , Glicoles/farmacología , Hibridomas , Lipopolisacáridos/farmacología , Ratones , Microglía/efectos de los fármacos , Neuritas/efectos de los fármacos , Neuritas/fisiología , Factores de TiempoRESUMEN
Autologous bone marrow mesenchymal stem cell (MSC) transplantation has great potential in cell therapy used for the treatment of neurodegenerative disorders. Since many genetic deficiencies have been reported in pathogenesis of the diseases, genetic backgrounds of donor stem cells should be concerned. In this study, effects of neurofilament light subunit (NFL) gene deficiency on proliferation and neuronal differentiation of MSCs were studied in vitro. Lower proliferation rate was observed in NFL-/- MSCs. When exposed to retinoic acid (RA), both NFL-/- and normal MSCs could express several markers of neuronal lineage, such as Nestin, MAP-2, NeuN, O4 and GFAP. However, the NFL expression at mRNA and protein levels was observed only in normal MSCs but absent in NFL-/- MSCs. Significant reductions in amount of neurofilament heavy subunit (NFH) protein and number of neuron-like cells were detected in differentiated NFL-/- MSCs. Interestingly, NFH positive protein accumulations were observed in the neuron-like cells derived from NFL-/- MSCs. These accumulations were perinuclear and morphologically similar to protein aggregations in motoneurons of the spinal cord in NFL-/- mice. The results suggest that NFL gene deficiency could retard MSCs proliferation and neuronal generation, even though the capability of neuronal lineage differentiation of MSCs may not be deterred. Moreover, the NFL-/- MSCs differentiated neuron-like cells carried on the genetic and pathologic deficiency, suggesting that the genetic quality of donor cells must not only be tested, but also modified before transplantation. This also points towards the possibility of creating a stem cell-derived cell model for pathogenesis study.
Asunto(s)
Diferenciación Celular/genética , Cuerpos de Inclusión/metabolismo , Trasplante de Células Madre Mesenquimatosas/normas , Células Madre Mesenquimatosas/metabolismo , Proteínas de Neurofilamentos/genética , Neuronas/metabolismo , Animales , Biomarcadores/análisis , Biomarcadores/metabolismo , Linaje de la Célula/genética , Proliferación Celular , Células Cultivadas , Femenino , Genotipo , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/patología , Masculino , Trasplante de Células Madre Mesenquimatosas/efectos adversos , Células Madre Mesenquimatosas/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/metabolismo , Neuronas/patología , Tretinoina/farmacologíaRESUMEN
The pathogenesis of neurodegenerative diseases prior to the onset of symptoms is generally not clear. The present study has employed a mouse model with a lack of the low-molecular-weight neurofilament subunit (NFL-/-), in which formation of protein aggregates occurs in neurons, to investigate glial cellular reactions in the lumbar cord segments of NFL-/- mice at ages from 1 to 6 months. Age-matched C57BL/6 mice serve as the control. Apparent neurofilament positive aggregates in the cytoplasm of motoneurons have been observed in NFL-/- mice. However, there were no noticeable changes in microglial numbers and GFAP staining of astrocytes. Unexpectedly, a downregulation in expression of complement receptor type 3 alpha subunit (CD11b) was detected in the spinal cord of NFL-/- mice, while there was no obvious difference between NFL-/- and C57BL/6 mice in the CD11b staining intensity of macrophages from livers and spleens. In addition, retardation in morphological transformation from activated to amoeboid microglia in response to sciatic nerve injury, differential expressions of some cytokines in the lumbar cord segments and induction of Iba-1 (ionized calcium-binding adaptor molecule-1) expression in microglia were observed in NFL-/- mice. Our results suggest not only the existence of an inhibitory niche for CD11b expression in microglia in the lumbar cord segments of NFL-/- mice but also differential microglial reactions between earlier and later stages of neuropathogenesis. Although the real cause for such inhibition is still unknown, this effect might play a particular role in the survival of the abnormal protein aggregate-bearing motoneurons in the early development stage of neurodegeneration in the NFL-/- mice.
Asunto(s)
Antígeno CD11b/metabolismo , Regulación hacia Abajo/fisiología , Microglía/metabolismo , Neuronas Motoras/fisiología , Proteínas de Neurofilamentos/deficiencia , Médula Espinal/citología , Factores de Edad , Animales , Animales Recién Nacidos , Antígeno CD11b/genética , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/ultraestructura , Microscopía Electrónica de Transmisión/métodos , Neuronas Motoras/ultraestructura , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Neuropatía Ciática/metabolismo , Neuropatía Ciática/patologíaRESUMEN
After our studies on ganglion cell degeneration in the glaucomatous retina, the current work further confirmed the reduction of amacrine cells in the retina after the onset of glaucoma. Present study also tried to understand the possible mechanisms underlying neuronal degeneration in the glaucomatous retina. Changes of expressions in immediate early genes (IEGs), glutamate receptors (GluRs), calcium-binding proteins (CaBPs), 8-hydroxy-deoxyguanosine (8-OH-dG) and nitric oxide synthase (NOS), as well as apoptotic-related factors including caspase 3, bax, and bcl-2 were examined. IEGs such as c-fos and c-jun were induced in the retina of the glaucomatous rat as early as 2 hr after the onset of glaucoma and lasted up to 2 weeks. Expressions of GluRs and CaBPs (i.e., parvalbumin and calbindin D-28k) were observed to be increased in the retinal ganglion cell layer (GCL) and inner nuclear layer (INL) at 3 days and 1 week after the onset of glaucoma. The increase occurred well before and during the phase where significant neuronal death was observed in the GCL and INL of the glaucomatous retinae. Induction of 8-OH-dG was present in both the GCL and INL of the glaucomatous retina at 3 days after the onset of glaucoma before significant neuronal death was observed. Furthermore, confocal microscopy study showed the complete colocalization of immunohistochemical expression of caspase 3 with glial fibrillary acidic protein (GFAP), but not with neuronal nuclei (NeuN). It indicates that astrocytes and Müller cells are involved in the pathological processes of neuronal death. The relationship between the linked factors and neuronal degeneration is also discussed.
Asunto(s)
Glaucoma/fisiopatología , Degeneración Nerviosa/fisiopatología , Neuronas/patología , Retina/fisiopatología , Degeneración Retiniana/fisiopatología , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Apoptosis/fisiología , Astrocitos/patología , Proteínas de Unión al Calcio/metabolismo , Caspasa 3 , Caspasas/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Modelos Animales de Enfermedad , Genes Inmediatos-Precoces/fisiología , Glaucoma/complicaciones , Proteína Ácida Fibrilar de la Glía/metabolismo , Inmunohistoquímica , Masculino , Degeneración Nerviosa/etiología , Óxido Nítrico Sintasa/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Receptores de Glutamato/metabolismo , Retina/patología , Degeneración Retiniana/etiología , Regulación hacia Arriba/fisiologíaRESUMEN
The aim of this study was to investigate the expression of cytokines, tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta (IL-1beta), interleukin-6 (IL-6) and transforming growth factor-beta 1 (TGF-beta1) and chemokines, fractalkine, monocyte chemoattractant protein 1 (MCP-1) and stromal cell-derived factor 1 (SDF-1) in the dorsal motor nucleus of the vagus nerve (DMV) after right vagotomy. Results showed that the immunoreactivities of IL-1beta, IL-6, TGF-beta1, fractalkine and MCP-1 were upregulated in the DMV at 14 days and the upregulation persisted at least until 28 days following right vagotomy. Quantification analysis revealed significant increases in the number of their immunopositive cells in the right DMV at 14 and 28 days after right vagotomy. Moreover, the upregulation of TNF-alpha immunoreactivity and significantly increased number of TNF-alpha-immunopositive cells were observed in the injured DMV at 7 and 14 days, and the increase in SDF-1-immunopositive cells at 14 days, after right vagotomy. Real time RT-PCR analysis showed the significant increase in the mRNA expression of IL-1beta, fractalkine and MCP-1 at 7 days, and the upregulation of TNF-alpha mRNA expression at 1 day after vagotomy. However, the peak increase in TGF-beta1 mRNA expression was observed at 1 day and the significant increase persisted at least until 14 days following right vagotomy. Double immunofluorescence analysis showed co-localization of lectin, a marker for microglia with CX3CR1 but not with IL-1beta at 14 days following right vagotomy. This study suggests that cytokines and chemokines involved in neuroprotection and neurodestruction could be activated in the axotomized DMV. However, it warrants further investigation to understand the neurodestructive and neuroprotective mechanisms that determine the fate of the vagal motoneurons after vagotomy.
Asunto(s)
Quimiocinas/metabolismo , Citocinas/metabolismo , Regulación de la Expresión Génica/fisiología , Bulbo Raquídeo/metabolismo , Animales , Recuento de Células/métodos , Quimiocinas/genética , Citocinas/genética , Lateralidad Funcional/fisiología , Inmunohistoquímica/métodos , Lectinas/metabolismo , Masculino , Naftalenos , Oxepinas , ARN Mensajero/biosíntesis , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Factores de Tiempo , Vagotomía/métodosRESUMEN
We have examined the activation of glial cells and the upregulation of phosphorylated extracellular signaling-regulated kinase (ERK)-1 and -2 in upper thoracic segments of the spinal cord in rats following acute cardiac injury (ACI). ACI was established by intramyocardial injection of formalin and confirmed by hematoxylin and eosin (H&E) and terminal transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining. Following ACI, the astrocytes (determined by glial fibrillary acidic protein (GFAP) immunoreactivity (-IR)) and microglia (determined by OX-42-IR) were activated within the thoracic spinal cord. Phosphorylated (phospho-) ERK-IR was also activated in response to ACI. The upregulation of phospho-ERK was observed at 1h and became very obvious at 6h following ACI. The upregulated phospho-ERK was evidently expressed in the superficial and deep dorsal horn of the thoracic spinal cord. The activated ERK was also expressed in the intermediolateral nucleus (IML), nucleus intercalatus (IC) and the long processes projecting to the central canal, regions closely associated with autonomic outflow. Thus, the present study suggested that ACI could induce the activation of spinal ERK, which might link the nociceptive processing with the spinal sympathetic reflexes in myocardial injury in clinics.
Asunto(s)
Cardiopatías/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Neuroglía/fisiología , Médula Espinal/metabolismo , Médula Espinal/patología , Enfermedad Aguda , Animales , Activación Enzimática , Líquido Extracelular/metabolismo , Formaldehído , Cardiopatías/inducido químicamente , Inmunohistoquímica , Masculino , Proteína Quinasa 3 Activada por Mitógenos , Dolor/inducido químicamente , Dolor/metabolismo , Fosforilación , Ratas , Ratas Wistar , Tórax , Regulación hacia ArribaRESUMEN
Mesenchymal stem cells (MSCs), cultured ex vivo, recently were shown to be able to migrate into sites of brain injuries when transplanted systemically or locally, suggesting that MSCs possess migratory capacity. However, the mechanisms underlying the migration of these cells remain unclear. In this study, we examined the role of some chemokines and their receptors in the trafficking of rat MSCs (rMSCs) in a rat model of left hypoglossal nerve injury. rMSCs transplanted into the lateral ventricles of the rat brain migrated to the avulsed hypoglossal nucleus, where the expression of chemokines, stromal-cell-derived factor 1 (SDF-1), and fractalkine was observed to be increased. This increase temporally paralleled the migration of rMSCs into the avulsed nucleus at 1 and 2 weeks after operation. It has been found that rMSCs express CXCR4 and CX3CR1, the respective receptors for SDF-1 and fractalkine, and other chemokine receptors, CCR2 and CCR5. Furthermore, in vitro analysis revealed that recombinant human SDF-1 alpha (rhSDF-1alpha) and recombinant rat fractalkine (rrfractalkine) induced the migration of rMSCs in a G-protein-dependent manner. Intracerebral injection of rhSDF-1alpha has also been shown to stimulate the homing of transplanted rMSCs to the site of injection in the brain. These data suggest that the interactions of fractalkine-CX3CR1 and SDF-1-CXCR4 could partially mediate the trafficking of transplanted rMSCs. This study provides an important insight into the understanding of the mechanisms governing the trafficking of transplanted rMSCs and also significantly expands the potential role of MSCs in cell therapy for brain injuries and diseases.
Asunto(s)
Movimiento Celular/fisiología , Quimiocinas CXC/farmacología , Traumatismos del Nervio Hipogloso , Células Madre Mesenquimatosas/citología , Receptores de Quimiocina/metabolismo , Animales , Encéfalo/patología , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Quimiocina CX3CL1 , Quimiocina CXCL12 , Quimiocinas CX3C/farmacología , Proteínas de Unión al GTP/metabolismo , Nervio Hipogloso/citología , Nervio Hipogloso/metabolismo , Proteínas de la Membrana/farmacología , Células Madre Mesenquimatosas/metabolismo , Ratas , Ratas Wistar , Receptores CXCR4/metabolismoRESUMEN
We have studied the expression of chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1 at the mRNA and protein levels in adult neural progenitor cells (NPCs) in neurosphere cultures using RT-PCR and immunocytochemistry methods. NPCs were isolated from the subventricular zone of adult rat brain and propagated in vitro as neurospheres. The neurospheres showed immunoactivity of nestin, an intermediate filament marker for NPCs. NPCs in the neurosphere cultures differentiated into NeuN-, GFAP-, or GalC-positive cells in vitro. Using cultured cortical microglial cells as positive control, we demonstrated the mRNA expression of CXCR4, CCR2, CCR5, and CX3CR1 in neurospheres by RT-PCR. Double immunofluorescent staining further confirmed the co-localization of nestin with either CXCR4, CCR2, CCR5, or CX3CR1 on neurospheres. These results suggest that adult NPCs in the neurosphere cultures express chemokine receptors CXCR4, CCR2, CCR5, and CX3CR1.