RESUMO
Metal dyshomeostasis is associated with neurodegenerative disorders, cancers and vascular disease. We report the effects of age (range: 3 to 18 months) on regional copper, iron and zinc levels in the brain of the C57BL/6 mouse, a widely used inbred strain with a permissive background allowing maximal expression of mutations in models that recapitulate these disorders. We present formulae that can be used to determine regional brain metal concentrations in the C57BL/6 mouse at any age in the range of three to eighteen months of life. Copper levels in the C57BL/6 mouse adult brain were highest in the striatum and cerebellum and increased with age, excepting the cortex and hippocampus. Regional iron levels increased linearly with age in all brain regions, while regional zinc concentrations became more homogeneous with age. Knockdown of the copper transporter Ctr1 reduced brain copper, but not iron or zinc, concentrations in a regionally-dependent manner. These findings demonstrate biometals in the brain change with age in a regionally-dependent manner. These data and associated formulae have implications for improving design and interpretation of a wide variety of studies in the C57BL/6 mouse.
Assuntos
Cobre , Zinco , Camundongos , Animais , Zinco/metabolismo , Ferro/metabolismo , Camundongos Endogâmicos C57BL , Encéfalo/metabolismoRESUMO
Alpha-Synuclein (α-Syn) is expressed in the central nervous system and the nervous system of the gut (enteric nervous system, ENS), and is well known to be the major constituent of Lewy bodies which are the hallmark of Parkinson's disease. Gastrointestinal disorders frequently manifest several years before motor deficits develop in Parkinson's patients. Despite extensive research on pathological rodent models, the physiological role of α-Syn in the normal ENS is unclear hampering analysis of its neuropathology. We compared the ENS in colons of α-Syn-knockout (α-Syn KO) and wild-type mice using immunohistochemistry and calcium-imaging of responses to synaptic input. We found that α-Syn is predominantly expressed in cholinergic varicosities, which contain vesicular acetylcholine transporter. α-Syn KO mice had higher enteric neuron density and a larger proportion of cholinergic neurons, notably those containing calretinin, demonstrating a role for α-Syn in regulating development of these neurons. Moreover, α-Syn deletion enhanced the amplitude of synaptically activated [Ca2+]i transients that are primarily mediated by acetylcholine activating nicotinic receptors suggesting that α-Syn modulates the availability of acetylcholine in enteric nerve terminals.
Assuntos
Neurônios Colinérgicos/fisiologia , Colo/inervação , Sistema Nervoso Entérico/crescimento & desenvolvimento , alfa-Sinucleína/fisiologia , Animais , Cálcio/metabolismo , Contagem de Células/estatística & dados numéricos , Neurônios Colinérgicos/metabolismo , Colo/fisiologia , Sistema Nervoso Entérico/metabolismo , Feminino , Masculino , Camundongos , Camundongos Knockout , alfa-Sinucleína/biossíntese , alfa-Sinucleína/genéticaRESUMO
OBJECTIVE: This study sought to assess the potential efficacy of a novel class of metal chaperone on the outcomes in an animal model of a controlled cortical impact. This work was predicated on previous observations that this class of compound has exhibited neuroprotective potential in other models of aging and neurodegeneration. RESEARCH DESIGN: The study employed a controlled cortical impact traumatic brain injury in three month old mice with subsequent behavioral and cellular assessments to determine therapeutic efficacy. METHODS: Cognitive (Y-maze) and motor assessments (Rotarod and Open Field) were employed to determine behavioral end points. Histological-based methods were utilized to assess neuronal integrity, astrocytosis, and lesion volume. OUTCOMES: We demonstrate here that acute post-injury treatment with PBT2 (Prana Biotechnology) is sufficient to maintain neuronal integrity (evidenced by decreased lesion area and increased numbers of neurons; decreased astrocytosis was also present) and to normalize performance in cognitive testing (Y-maze). These effects occurred within days and were maintained for the entire duration of the study (26 days post-injury). These data support the further interrogation of the utility of metal chaperones for the treatment and/or prevention of the neuroanatomical, biochemical, and behavioral deficits that occur following brain injuries of different etiologies.
Assuntos
Lesões Encefálicas Traumáticas/tratamento farmacológico , Quelantes/uso terapêutico , Fármacos Neuroprotetores/uso terapêutico , Animais , Astrócitos/patologia , Encéfalo/patologia , Lesões Encefálicas Traumáticas/patologia , Lesões Encefálicas Traumáticas/psicologia , Cognição , Locomoção , Masculino , Aprendizagem em Labirinto , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/patologia , Desempenho Psicomotor/efeitos dos fármacos , Zinco/metabolismoRESUMO
Prion diseases are phenotypically diverse, transmissible, neurodegenerative disorders affecting both animals and humans. Misfolding of the normal prion protein (PrPC) into disease-associated conformers (PrPSc) is considered the critical etiological event underpinning prion diseases, with such misfolded isoforms linked to both disease transmission and neurotoxicity. Although important advances in our understanding of prion biology and pathogenesis have occurred over the last 3-4 decades, many fundamental questions remain to be resolved, including consensus regarding the principal pathways subserving neuronal dysfunction, as well as detailed biophysical characterization of PrPSc species transmitting disease and/or directly associated with neurotoxicity. In vivo and in vitro models have been, and remain, critical to furthering our understanding across many aspects of prion disease patho-biology. Prion animal models are arguably the most authentic in vivo models of neurodegeneration that exist and have provided valuable and multifarious insights into pathogenesis; however, they are expensive and time-consuming, and it can be problematic to clearly discern evidence of direct PrPSc neurotoxicity in the overall context of pathogenesis. In vitro models, in contrast, generally offer greater tractability and appear more suited to assessments of direct acute neurotoxicity but have until recently been relatively simplistic, and overall there remains a relative paucity of validated, biologically relevant models with heightened reliability as far as translational insights, contributing to difficulties in redressing our knowledge gaps in prion disease pathogenesis. In this review, we provide an overview of the spectrum and methodological diversity of in vivo and in vitro models of prion acute toxicity, as well as the pathogenic insights gained from these studies.
Assuntos
Síndromes Neurotóxicas/metabolismo , Proteínas PrPSc/metabolismo , Doenças Priônicas/metabolismo , Príons/metabolismo , Animais , Humanos , Modelos Biológicos , Neurônios/metabolismoRESUMO
Lithium is a first-line therapy for bipolar affective disorder. However, various adverse effects, including a Parkinson-like hand tremor, often limit its use. The understanding of the neurobiological basis of these side effects is still very limited. Nigral iron elevation is also a feature of Parkinsonian degeneration that may be related to soluble tau reduction. We found that magnetic resonance imaging T2 relaxation time changes in subjects commenced on lithium therapy were consistent with iron elevation. In mice, lithium treatment lowers brain tau levels and increases nigral and cortical iron elevation that is closely associated with neurodegeneration, cognitive loss and parkinsonian features. In neuronal cultures lithium attenuates iron efflux by lowering tau protein that traffics amyloid precursor protein to facilitate iron efflux. Thus, tau- and amyloid protein precursor-knockout mice were protected against lithium-induced iron elevation and neurotoxicity. These findings challenge the appropriateness of lithium as a potential treatment for disorders where brain iron is elevated (for example, Alzheimer's disease), and may explain lithium-associated motor symptoms in susceptible patients.
Assuntos
Lítio/efeitos adversos , Lítio/metabolismo , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Encéfalo/metabolismo , Humanos , Ferro/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Transtornos Parkinsonianos/metabolismo , Proteínas tau/antagonistas & inibidoresRESUMO
Patients with Parkinson's disease often experience non-motor symptoms including constipation, which manifest prior to the onset of debilitating motor signs. Understanding the causes of these non-motor deficits and developing disease modifying therapeutic strategies has the potential to prevent disease progression. Specific neuronal subpopulations were reduced within the myenteric plexus of mice 21 days after intoxication by the intraperitoneal administration of MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) and was associated with a reduction in stool frequency, indicative of intestinal dysfunction. Oral administration of the divalent copper complex, Cu(II)(atsm), which has been shown to be neuroprotective and restore motor performance to MPTP lesioned mice, improved stool frequency and was correlated with restoration of neuronal subpopulations in the myenteric plexus of MPTP lesioned mice. Restoration of intestinal function was associated with reduced enteric glial cell reactivity and reduction of markers of inflammation. Therapeutics that have been shown to be neuroprotective in the central nervous system, such as Cu(II)(atsm), therefore also provide symptom relief and are disease modifying in the intestinal tract, suggesting that there is a common cause of Parkinson's disease pathogenesis in the enteric nervous system and central nervous system.
Assuntos
Constipação Intestinal/tratamento farmacológico , Defecação/efeitos dos fármacos , Intoxicação por MPTP/tratamento farmacológico , Plexo Mientérico/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Compostos Organometálicos/farmacologia , Tiossemicarbazonas/farmacologia , Administração Oral , Animais , Constipação Intestinal/complicações , Constipação Intestinal/metabolismo , Constipação Intestinal/fisiopatologia , Complexos de Coordenação , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/fisiopatologia , Defecação/fisiologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Injeções Intraperitoneais , Intoxicação por MPTP/complicações , Intoxicação por MPTP/metabolismo , Intoxicação por MPTP/fisiopatologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Atividade Motora/efeitos dos fármacos , Plexo Mientérico/metabolismo , Plexo Mientérico/fisiopatologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Neuroglia/patologia , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Substância Negra/fisiopatologiaRESUMO
Aberrant posttranslational modifications (PTMs) of proteins, namely phosphorylation, induce abnormalities in the biological properties of recipient proteins, underlying neurological diseases including Parkinson's disease (PD). Genome-wide studies link genes encoding α-synuclein (α-Syn) and Tau as two of the most important in the genesis of PD. Although several kinases are known to phosphorylate α-Syn and Tau, we focused our analysis on GSK-3ß because of its accepted role in phosphorylating Tau and to increasing evidence supporting a strong biophysical relationship between α-Syn and Tau in PD. Therefore, we investigated transgenic mice, which express a point mutant (S9A) of human GSK-3ß. GSK-3ß-S9A is capable of activation through endogenous natural signaling events, yet is unable to become inactivated through phosphorylation at serine-9. We used behavioral, biochemical, and in vitro analysis to assess the contributions of GSK-3ß to both α-Syn and Tau phosphorylation. Behavioral studies revealed progressive age-dependent impairment of motor function, accompanied by loss of tyrosine hydroxylase-positive (TH+ DA-neurons) neurons and dopamine production in the oldest age group. Magnetic resonance imaging revealed deterioration of the substantia nigra in aged mice, a characteristic feature of PD patients. At the molecular level, kinase-active p-GSK-3ß-Y216 was seen at all ages throughout the brain, yet elevated levels of p-α-Syn-S129 and p-Tau (S396/404) were found to increase with age exclusively in TH+ DA-neurons of the midbrain. p-GSK-3ß-Y216 colocalized with p-Tau and p-α-Syn-S129. In vitro kinase assays showed that recombinant human GSK-3ß directly phosphorylated α-Syn at a single site, Ser129, in addition to its known ability to phosphorylate Tau. Moreover, α-Syn and Tau together cooperated with one another to increase the magnitude or rate of phosphorylation of the other by GSK-3ß. Together, these data establish a novel upstream role for GSK-3ß as one of several kinases associated with PTMs of key proteins known to be causal in PD.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/fisiopatologia , alfa-Sinucleína/metabolismo , Proteínas tau/metabolismo , Animais , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Camundongos , Camundongos Transgênicos , Transtornos Parkinsonianos/genética , Transtornos Parkinsonianos/patologia , alfa-Sinucleína/genética , Proteínas tau/genéticaRESUMO
The catecholamines dopamine (DA), norepinephrine (NE) and epinephrine (E) are neurotransmitters and hormones that mediate stress responses in tissues and plasma. The expression of ß-amyloid precursor protein (APP) is responsive to stress and is high in tissues rich in catecholamines. We recently reported that APP is a ferroxidase, subsuming, in neurons and other cells, the iron-export activity that ceruloplasmin mediates in glia. Here we report that, like ceruloplasmin, APP also oxidizes synthetic amines and catecholamines catalytically (K(m) NE=0.27 mM), through a site encompassing its ferroxidase motif and selectively inhibited by zinc. Accordingly, APP knockout mice have significantly higher levels of DA, NE and E in brain, plasma and select tissues. Consistent with this, these animals have increased resting heart rate and systolic blood pressure as well as suppressed prolactin and lymphocyte levels. These findings support a role for APP in extracellular catecholaminergic clearance.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Catecolaminas/metabolismo , Monoaminoxidase/metabolismo , Precursor de Proteína beta-Amiloide/deficiência , Animais , Pressão Sanguínea/efeitos dos fármacos , Pressão Sanguínea/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Dopamina/toxicidade , Embrião de Mamíferos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Frequência Cardíaca/genética , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução/efeitos dos fármacosRESUMO
By the time a patient first presents with symptoms of Parkinson's disease at the clinic, a significant proportion (50-70%) of the cells in the substantia nigra (SN) has already been destroyed. This degeneration progresses until, within a few years, most of the cells have died. Except for rare cases of familial PD, the initial trigger for cell loss is unknown. However, we do have some clues as to why the damage, once initiated, progresses unabated. It would represent a major advance in therapy to arrest cell loss at the stage when the patient first presents at the clinic. Current therapies for Parkinson's disease focus on relieving the motor symptoms of the disease, these unfortunately lose their effectiveness as the neurodegeneration and symptoms progress. Many experimental approaches are currently being investigated attempting to alter the progression of the disease. These range from replacement of the lost neurons to neuroprotective therapies; each of these will be briefly discussed in this review. The main thrust of this review is to explore the interactions between dopamine, alpha synuclein and redox-active metals. There is abundant evidence suggesting that destruction of SN cells occurs as a result of a self-propagating series of reactions involving dopamine, alpha synuclein and redox-active metals. A potent reducing agent, the neurotransmitter dopamine has a central role in this scheme, acting through redox metallo-chemistry to catalyze the formation of toxic oligomers of alpha-synuclein and neurotoxic metabolites including 6-hydroxydopamine. It has been hypothesized that these feed the cycle of neurodegeneration by generating further oxidative stress. The goal of dissecting and understanding the observed pathological changes is to identify therapeutic targets to mitigate the progression of this debilitating disease.
RESUMO
Electrospinning has been employed extensively in tissue engineering to generate nanofibrous scaffolds from either natural or synthetic biodegradable polymers to simulate the cellular microenvironment. Electrospinning rapidly produces fibers of the nanolength scale and the process offers many opportunities to tailor the physical, chemical, and biological properties of a material for specific applications and cellular environments. There is growing evidence that nanofibers amplify certain biological responses such as contact guidance and differentiation, however this has not been fully exploited in tissue engineering. This review addresses the cellular interactions with electrospun scaffolds, with particular focus on neural, bone, cartilage, and vascular tissue regeneration. Some aspects of scaffold design, including architectural properties, surface functionalization and materials selection are also addressed.
Assuntos
Materiais Biocompatíveis/química , Fenômenos Fisiológicos Celulares , Técnicas Eletroquímicas/métodos , Nanoestruturas/química , Engenharia Tecidual/métodos , Animais , Técnicas de Cultura de Células/métodos , Humanos , Nanotecnologia/métodos , Medicina Regenerativa/métodos , Alicerces TeciduaisRESUMO
In this study, thermoresponsive xyloglucan hydrogel scaffolds were investigated as candidates for neural tissue engineering of the spinal cord. The hydrogels were optimized to provide similar mechanical properties to that of native spinal cord, although also being functionalized through the immobilization of poly-D-lysine to promote neurone adhesion and neurite outgrowth. Under 2D and 3D culture conditions, xyloglucan scaffolds supported the differentiation of primary cortical neurones. Furthermore, functionalization provided a means of controlling and optimizing the cell diameter, number, migration and the neurite density, and the direction of growth. The interaction of neural stem cells (NSCs) was also investigated on the xyloglucan scaffolds in vitro. The survival of the NSCs and the axonal extensions on the scaffolds were similar to that of the primary cortical neurones. These findings suggest that xyloglucan-based materials are suitable for providing a neurotrophic milieu.
Assuntos
Hidrogel de Polietilenoglicol-Dimetacrilato/química , Células-Tronco Multipotentes/fisiologia , Regeneração Nervosa/fisiologia , Neuritos/fisiologia , Neurônios/citologia , Traumatismos da Medula Espinal , Alicerces Teciduais , Compostos de Anilina/química , Animais , Compostos Azo/química , Materiais Biocompatíveis/química , Materiais Biocompatíveis/metabolismo , Diferenciação Celular , Células Cultivadas , Glucanos/química , Glucanos/metabolismo , Hidrogel de Polietilenoglicol-Dimetacrilato/metabolismo , Teste de Materiais , Camundongos , Camundongos Endogâmicos C57BL , Estrutura Molecular , Células-Tronco Multipotentes/citologia , Polilisina/química , Polímeros/química , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/terapia , Temperatura , Engenharia Tecidual/métodos , Xilanos/química , Xilanos/metabolismoRESUMO
Leucine-rich repeat-containing G-protein-coupled receptor 8 (LGR8; also classified as relaxin family peptide 2 receptor; RXFP2) has been identified as a cognate receptor for the peptide hormone, insulin-like peptide 3 (INSL3) and INSL3-LGR8 signaling plays an essential role in testis descent and germ cell development in human and rodents. Lgr8 mRNA has been detected in human tissues including testis, kidney and brain, but its regional and cellular distribution in these tissues in human or other species is largely unknown. In an initial step to elucidate the physiological function of a putative INSL3-LGR8 system in rat brain, the localization of Lgr8 mRNA was investigated using in situ hybridization histochemistry, revealing a discrete distribution in forebrain, with expression highly enriched in the thalamus. High densities were detected in the parafascicular nucleus (Pf), the dorsolateral, ventrolateral and posterior thalamic nuclei, and in the medial habenula. Lgr8 transcripts were also detected in frontal and motor cortices. The comparative distribution of LGR8 (receptor protein) was examined by autoradiography of [125I]-human INSL3 binding sites, with high densities detected in the thalamus, especially in Pf, and in the entire striatum--the caudate putamen (CPmicro), islands of Calleja, olfactory tubercle, nucleus accumbens--with lower levels in distinct layers of cerebral cortex. Notably, these areas also receive dopaminergic projections. These findings demonstrate the existence of LGR8 in neuronal soma in the thalamus and axons/terminals in thalamic target areas such as the striatum and frontal cortex. LGR8 was also detected throughout the medial habenula-fasciculus retroflexus-interpeduncular nucleus pathway, further indicating that the receptor is transported from mRNA-expressing soma to remote axonal/terminal sites. These findings suggest the existence of a broadly distributed LGR8 signaling system in the rat involved in sensorimotor, limbic and cognitive functions. Further studies are now required to elucidate the precise function of LGR8, under normal and pathological conditions, as importantly, several of the equivalent receptor-positive areas in human brain are part of the pathology of neurodegenerative conditions including Parkinson's disease.
Assuntos
Encéfalo/metabolismo , Neurônios/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Tálamo/citologia , Animais , Gânglios da Base/metabolismo , Encéfalo/anatomia & histologia , Vias Eferentes/fisiologia , Humanos , Insulina/metabolismo , Masculino , Ligação Proteica/fisiologia , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genéticaRESUMO
The pathogenesis of Parkinson's disease (PD) involves ongoing apoptotic loss of dopaminergic neurons in the substantia nigra pars compacta. Local delivery of the trophic factors can rescue dopaminergic neurons and halt the progression of PD. In this study we show that fetal E11 striatum-derived neurospheres and E14.5 ventral mesencephalon (VM) -derived neurospheres (NS E11 and NSvm, respectively) are a source of factors that rescue dopaminergic neurons. First, long-term expanded NS E11 and NSvm rescued primary dopaminergic neurons from serum-deprivation induced apoptosis and promoted survival of dopaminergic neurons for 14 days in vitro and this effect was due to soluble contact-independent factor/s. Second, green fluorescent protein-expressing NS E11 and NSvm grafted into the midbrain of mice with unilateral 6-hydroxydopamine-induced Parkinsonism resulted in partial rescue of the nigro-striatal system and improvement of the hypo-dopaminergic behavioral deficit. Reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that intact NS E11 and NSvm expressed fibroblast growth factor-2, brain-derived neurotrophic factor (BDNF), pleiotrophin, neurotrophin-3, but not glial cell line-derived neurotrophic factor (GDNF). GDNF expression was also undetectable in vivo in grafted NS E11 and NSvm suggesting that NS-derived factor/s other than GDNF mediated the rescue of nigral dopaminergic neurons. Identification of NS-derived soluble factor(s) may lead to development of novel neuroprotective therapies for PD. An unexpected observation of the present study was the detection of the ectopic host-derived tyrosine hydroxylase (TH) -expressing cells in sham-grafted mice and NS E11- and NSvm -grafted mice. We speculate that injury-derived signals (such as inflammatory cytokines that are commonly released during transplantation) induce TH expression in susceptible cells.
Assuntos
Transplante de Células/fisiologia , Dopamina/fisiologia , Mesencéfalo/fisiologia , Neostriado/fisiologia , Neurônios/fisiologia , Neurônios/transplante , Substância Negra/fisiologia , Anfetamina/toxicidade , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Estimulantes do Sistema Nervoso Central/toxicidade , Técnicas de Cocultura , Meios de Cultivo Condicionados , Meios de Cultura Livres de Soro , Feminino , Hidroxidopaminas/toxicidade , Imuno-Histoquímica , Mesencéfalo/citologia , Camundongos , Camundongos Endogâmicos C57BL , Neostriado/citologia , Gravidez , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Comportamento Estereotipado/efeitos dos fármacos , Substância Negra/citologia , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
The interaction of murine embryonic cortical neurons on randomly orientated electrospun scaffolds of poly(L-lactide) (P(L)LA) and poly(lactide-co-glycolide) (PLGA) is investigated in this study. The scaffolds were surface treated with different concentrations of KOH to partially hydrolyze the surface and therefore change the surface tension. Hydrophilicity did not significantly influence the number of primary and secondary branches; however, it had a considerable effect on neurite extension. For scaffolds with surface tensions of 40-47 dyn cm(-1) there was a significantly greater overall neurite length for both the primary and secondary branches compared with more hydrophilic scaffolds. Another major finding of this work was that the interfibre distance influenced how the neurites extended. When the interfibre distance was greater than approximately 15 microm the neurites followed the fibres and avoided regions of very high fibre density. At interfibre distances less than approximately 15 microm, the neurites traversed between the fibres. Therefore, this study provided little evidence that contact guidance was the dominating cue in directing neurite extension, instead inferring that chemical cues, possibly from adjacent neurons had induced directional change.
Assuntos
Córtex Cerebral/embriologia , Regeneração Tecidual Guiada/métodos , Ácido Láctico/química , Nanoestruturas/química , Neurônios/citologia , Neurônios/fisiologia , Ácido Poliglicólico/química , Polímeros/química , Engenharia Tecidual/métodos , Animais , Materiais Biocompatíveis/química , Técnicas de Cultura de Células/métodos , Crescimento Celular , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Regeneração Tecidual Guiada/instrumentação , Camundongos , Camundongos Endogâmicos C57BL , Nanoestruturas/ultraestrutura , Tamanho da Partícula , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Propriedades de SuperfícieRESUMO
Foetal mouse cortical cells were cultured on 2D films and within 3D thermally responsive chitosan/glycerophosphate salt (GP) hydrogels. The biocompatibility of chitosan/GP 2D films was assessed in terms of cell number and neurites per cell. Osmolarity of the hydrogel was a critical factor in promoting cell survival with isotonic GP concentrations providing optimal conditions. To improve cell adhesion and neurite outgrowth, poly-D-lysine (PDL) was immobilised onto chitosan via azidoaniline photocoupling. Increase in PDL concentrations did not alter cell survival in 2D cultures but neurite outgrowth was significantly inhibited. Neurons exhibited a star-like morphology typical of 2D culture systems. The effects of PDL attachment on cell number, cell morphology and neurite outgrowth were more distinct in 3D culture conditions. Neurones exhibited larger cell bodies and sent out single neurites within the macroporous gel. Immobilised PDL improved cell survival up to an optimum concentration of 0.1%, however, further increases resulted in drops in cell number and neurite outgrowth. This was attributed to a higher cell interaction with PDL within a 3D hydrogel compared to the corresponding 2D surface. The results show that thermally responsive chitosan/GP hydrogels provide a suitable 3D scaffolding environment for neural tissue engineering.
Assuntos
Materiais Biocompatíveis , Quitosana/química , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Neurônios/metabolismo , Polilisina/química , Engenharia Tecidual/métodos , Animais , Técnicas de Cultura de Células/métodos , Sobrevivência Celular , Glicerofosfatos/farmacologia , Temperatura Alta , Hidrogéis , Camundongos , Neuritos/metabolismoRESUMO
Chitosan is a well-known biomaterial that, with the addition of glycerophosphate salt (GP), gels at physiological temperatures and therefore is useful for tissue engineering purposes. This study examines the procedure of injecting chitosan/ GP to the brain in order to form a gel track. The gel system and surgical technique were successful in this endeavour; however, on examining the inflammatory response to the material it was found that the chitosan/GP was wholly engulfed by macrophages within 7 days. This was determined by staining for both the gel and the macrophages, an important technique for localising injected material. The chitosan/GP-containing macrophages formed a neat tract at the lesion site, but after 45 days no chitosan/GP was found. It was concluded that, although chitosan/GP is present after implantation, it is not available for direct scaffolding in the brain.
Assuntos
Materiais Biocompatíveis/efeitos adversos , Quitosana/efeitos adversos , Glicerofosfatos/efeitos adversos , Inflamação/patologia , Animais , Materiais Biocompatíveis/química , Quitosana/administração & dosagem , Quitosana/química , Géis/metabolismo , Glicerofosfatos/administração & dosagem , Glicerofosfatos/química , Imuno-Histoquímica , Inflamação/induzido quimicamente , Injeções Intraventriculares , Ativação de Macrófagos , Macrófagos/metabolismo , Masculino , Microscopia Eletrônica de Varredura , Estrutura Molecular , Peso Molecular , Ratos , Ratos Wistar , Fatores de Tempo , Engenharia Tecidual/métodosRESUMO
The aim of this study was to investigate the interaction of mouse embryonic cortical neurons on P(L)LA and PLGA substrates, which were partially hydrolysed using potassium hydroxide (KOH). The chemical and topographical properties of the surfaces were characterized, and it was discovered that there was a decrease in the hydrophilicity for the P(L)LA with increasing concentration of KOH. This was due to chemical modifications to the surfaces of the substrates. Alternatively for the PLGA substrate, only the 0.1 M KOH treated sample had a significantly different hydrophilicity highlighting that surface erosion resulted at higher concentrations. The morphology of the neurons grown on the two substrates were compared to poly(D)lysine (positive control). The neurons formed colonies on all of the substrates, but were dramatically reduced in size in the case of the 0.1 M KOH treated substrates. This finding was attributed to the increases in cell spreading and the size of the cells, as they were larger, more elongated and bipolar like those on the positive control. However, there was a significant decrease in the total number of live cells per unit area. Therefore, on these materials when there was increased cellular spreading there was significantly higher cell death. Furthermore, unlike the 0, 0.2, and 0.4 M KOH treated substrates, there was an absence of large bundles of axons that extended between colonies on the 0.1 M sample, instead exhibiting short axons that grew in free space.
Assuntos
Córtex Cerebral/embriologia , Neurônios/fisiologia , Animais , Materiais Biocompatíveis , Técnicas de Cultura de Células , Divisão Celular , Membrana Celular/fisiologia , Membrana Celular/ultraestrutura , Concentração de Íons de Hidrogênio , Hidróxidos , Ácido Láctico , Camundongos , Microscopia de Força Atômica , Microscopia Eletrônica de Varredura , Neurônios/citologia , Poliésteres , Ácido Poliglicólico , Copolímero de Ácido Poliláctico e Ácido Poliglicólico , Polímeros , Compostos de Potássio , Propriedades de SuperfícieRESUMO
Galactose modified xyloglucan is a thermally reversible hydrogel that is increasingly used in the biomedical field due to the ease of altering the gelation time and temperature by modifying the galactose removal ratio. However there is little information concerning the morphology and rheological properties of the hydrogel under physiological conditions. Differential scanning microcalorimetry (DSmicroC) showed the thermal gelation process to occur over a broad temperature range (5-50 degrees C). The rheological properties of the hydrogels were investigated as a function of concentration, temperature and ionic strength. The final elastic moduli of the hydrogels increased with increases in concentration. Isothermal rheology suggests that the gelation occurred in two distinct stages, which was influenced by the solution media. Scanning electron microscopy (SEM) was used to characterize the morphology of the xyloglucan which were thermally gelled at 37 degrees C. The resultant morphology was strongly dependent on the concentration of the hydrogel. Strong hydrogels were only obtained at 3 wt.% at 37 degrees C, and the morphology characterized by an open 3-dimensional network, comprised of thin membranes. It is proposed that the first stage of the isothermal gelation is the formation and growth of the thin membranes, followed by the formation of a three dimensional network.
Assuntos
Glucanos/química , Hidrogéis/química , Xilanos/química , Varredura Diferencial de Calorimetria , Glucanos/ultraestrutura , Estrutura Molecular , Reologia , Temperatura , Xilanos/ultraestruturaRESUMO
Neuronal nicotinic acetylcholine receptors (nAChRs) at presynaptic sites can modulate dopaminergic synaptic transmission by regulating dopamine (DA) release and uptake. Dopaminergic transmission in nigrostriatal and mesolimbic pathways is vital for the coordination of movement and is associated with learning and behavioral reinforcement. We reported recently that the D2 DA receptor plays a central role in regulating the arbor size of substantia nigra dopaminergic neurons. Given the known effects of nAChRs on dopaminergic neurotransmission, we assessed the ability of the alpha4 nAChR subunit to regulate arbor size of dopaminergic neurons by comparing responses of wild-type and alpha4 nAChR subunit knockout [alpha4(-/-)] mice to long-term exposure to cocaine, amphetamine, nicotine, and haloperidol, and after substantia nigra neurotoxic lesioning. We found that dopaminergic neurons in adult drug-naive alpha4(-/-) mice had significantly larger terminal arbors, and despite normal short-term behavioral responses to drugs acting on pre- and postsynaptic D2 DA receptors, they were unable to modulate their terminal arbor in response to pharmacological manipulation or after lesioning. In addition, although synaptosome DA uptake studies showed that the interaction of the D2 DA receptor and the dopamine transporter (DAT) was preserved in alpha4(-/-) mice, DAT function was found to be impaired. These findings suggest that the alpha4 subunit of the nAChR is an independent regulator of terminal arbor size of nigrostriatal dopaminergic neurons and that reduced functionality of presynaptic DAT may contribute to this effect by impairing DA uptake.
Assuntos
Corpo Estriado/citologia , Dopamina/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurônios/citologia , Receptores Nicotínicos/fisiologia , Substância Negra/citologia , Animais , Comportamento Animal/efeitos dos fármacos , Contagem de Células , Cocaína/análogos & derivados , Cocaína/metabolismo , Camundongos , Proteínas do Tecido Nervoso/análise , Oxidopamina , Receptores Nicotínicos/análise , Transmissão Sináptica , Sinaptossomos/metabolismoRESUMO
It is well established that mammalian skeletal muscles exhibit a considerable degree of plasticity and one of the main determining factors of this plasticity is the activity pattern and duration of motoneurone discharge. Lesions to the right substantia nigra pars compacta (SNpc) of six adult rats were made to determine whether altered output from the SNpc ultimately leads to a change in the expression of proteins in contralateral skeletal muscles. After 4 months, altered motor performance was identified by the administration of amphetamine. After 7 months, 30-70% of dopaminergic cells in the SNpc had been destroyed. The protein content of muscles was then quantified from densitometric scans of gels, and expressed as a % of the amount of actin (the protein used as a reference in this study). The lesion affected the expression of different protein isoforms in the fast- and slow-twitch muscles. In slow-twitch soleus muscles, the lesion decreased the proportion of alpha-tropomyosin and increased the proportion of beta-tropomyosin. In the fast-twitch extensor digitorum longus muscles, the lesion increased the proportion of the fast isoform of troponin-T1f, and decreased the proportions of the two isoforms of myosin light chain. This study establishes a connection between the chronic effects of a lesion to the SNpc, with a loss of dopaminergic neurones, impaired motor performance, and altered expression of proteins in skeletal muscle. The implication of these results is that the altered motor function observed in Parkinson's disease may be associated with alterations to the expression of skeletal muscle proteins.