RESUMEN
Traumatic brain injury (TBI) can lead to chronic diseases, including neurodegenerative disorders and epilepsy. The hippocampus, one of the most affected brain region after TBI, plays a critical role in learning and memory and is one of the only two regions in the brain in which new neurons are generated throughout life from neural stem cells (NSC) in the dentate gyrus (DG). These cells migrate into the granular layer where they integrate into the hippocampus circuitry. While increased proliferation of NSC in the hippocampus is known to occur shortly after injury, reduced neuronal maturation and aberrant migration of progenitor cells in the hilus contribute to cognitive and neurological dysfunctions, including epilepsy. Here, we tested the ability of a novel, proprietary non-invasive nano-pulsed laser therapy (NPLT), that combines near-infrared laser light (808 nm) and laser-generated, low-energy optoacoustic waves, to mitigate TBI-driven impairments in neurogenesis and cognitive function in the rat fluid percussion injury model. We show that injured rats treated with NPLT performed significantly better in a hippocampus-dependent cognitive test than did sham rats. In the DG, NPLT significantly decreased TBI-dependent impaired maturation and aberrant migration of neural progenitors, while preventing TBI-induced upregulation of specific microRNAs (miRNAs) in NSC. NPLT did not significantly reduce TBI-induced microglia activation in the hippocampus. Our data strongly suggest that NPLT has the potential to be an effective therapeutic tool for the treatment of TBI-induced cognitive dysfunction and dysregulation of neurogenesis, and point to modulation of miRNAs as a possible mechanism mediating its neuroprotective effects.
Asunto(s)
Lesiones Traumáticas del Encéfalo/fisiopatología , Movimiento Celular/fisiología , Cognición/fisiología , Hipocampo/fisiopatología , Terapia por Láser , Células-Madre Neurales/fisiología , Animales , Masculino , Memoria a Corto Plazo/fisiología , Actividad Motora/fisiología , Neurogénesis/fisiología , Ratas , Ratas Sprague-Dawley , Recuperación de la Función/fisiologíaRESUMEN
There is growing evidence that type 2 diabetes or insulin resistance is linked to cognitive impairment. We recently confirmed altered lipid composition, down-regulation of insulin receptor expression and impaired basal synaptic transmission in the hippocampus of our transgenic murine model of adipocyte insulin resistance (AtENPP1-Tg). Here we evaluated whether the correction of adipose tissue dysfunction [via the subcutaneous transplantation of mesenchymal stem cells (MSC)] can improve the hippocampal synaptic transmission in AtENPP1-Tg mice versus their wildtype littermates. Animals were simply randomized to receive MSC, then weighed weekly for 12 weeks. At euthanasia, we assessed leptin in the collected serum and hippocampal synaptic high-frequency stimulation long-term potentiation (HFS-LTP) using brain slices. MSC transplantation normalized AtENPP1-Tg body and epididymal fat weights and was associated with increased leptin levels, a sign of adipocyte maturation. More importantly, transplantation restored the deficiency observed in AtENPP1-Tg HFS-LTP, the cellular readout of memory. Our results further corroborate the role of adipocyte maturation arrest in adipose tissue and highlight a role for the adipose tissue in modulating hippocampal cellular mechanisms. Further studies are warranted to explore the mechanisms for the MSC-induced improvement of hippocampal HFS-LTP.
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Tejido Adiposo/fisiopatología , Diabetes Mellitus Tipo 2/fisiopatología , Hipocampo/fisiopatología , Trasplante de Células Madre Mesenquimatosas , Tejido Adiposo/citología , Animales , Diabetes Mellitus Tipo 2/genética , Dieta Alta en Grasa , Ácidos Grasos no Esterificados , Humanos , Resistencia a la Insulina/genética , Leptina/sangre , Potenciación a Largo Plazo , Células Madre Mesenquimatosas , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/genética , Transmisión Sináptica/fisiologíaRESUMEN
Patients with traumatic brain injury (TBI) are frequently diagnosed with depression. Together, these two leading causes of death and disability significantly contribute to the global burden of healthcare costs. However, there are no drug treatments for TBI and antidepressants are considered off-label for depression in patients with TBI. In molecular profiling studies of rat hippocampus after experimental TBI, we found that TBI altered the expression of a subset of small, non-coding, microRNAs (miRNAs). One known neuroprotective compound (17ß-estradiol, E2), and two experimental neuroprotective compounds (JM6 and PMI-006), reversed the effects of TBI on miRNAs. Subsequent in silico analyses revealed that the injury-altered miRNAs were predicted to regulate genes involved in depression. Thus, we hypothesized that drug-induced miRNA profiles can be used to identify compounds with antidepressant properties. To confirm this hypothesis, we examined miRNA expression in hippocampi of injured rats treated with one of three known antidepressants (imipramine, fluoxetine and sertraline). Bioinformatic analyses revealed that TBI, potentially via its effects on multiple regulatory miRNAs, dysregulated transcriptional networks involved in neuroplasticity, neurogenesis, and circadian rhythms- networks known to adversely affect mood, cognition and memory. As did E2, JM6, and PMI-006, all three antidepressants reversed the effects of TBI on multiple injury-altered miRNAs. Furthermore, JM6 reduced TBI-induced inflammation in the hippocampus and depression-like behavior in the forced swim test; these are both properties of classic antidepressant drugs. Our results support the hypothesis that miRNA expression signatures can identify neuroprotective and antidepressant properties of novel compounds and that there is substantial overlap between neuroprotection and antidepressant properties.
Asunto(s)
Antidepresivos/farmacología , Lesiones Traumáticas del Encéfalo/tratamiento farmacológico , Depresión/tratamiento farmacológico , MicroARNs/genética , Animales , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/genética , Lesiones Traumáticas del Encéfalo/patología , Biología Computacional , Depresión/complicaciones , Depresión/genética , Depresión/patología , Modelos Animales de Enfermedad , Estradiol/farmacología , Fluoxetina/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/patología , Humanos , Imipramina/farmacología , Ratas , Sertralina/farmacología , Sulfonamidas/farmacología , Tiazoles/farmacologíaRESUMEN
BACKGROUND: Adult hippocampal neurogenesis plays an important role in synaptic plasticity and cogntive function. We reported that higher numbers of neural stem cells (NSC) in the hippocampus of cognitively-intact individuals with high Alzheimer's disease (AD) pathology (plaques and tangles) is associated with decreased synaptic amyloid beta oligomers (Aßο), an event linked to onset of dementia in AD. While these findings suggest a link between NSC and synaptic resistance to Aßο, the involved mechanism remains to be determined. With this goal in mind, here we investigated the ability of exosomes secreted from hippocampal NSC to promote synaptic resilience to Aßo. METHODS: Exosomes isolated from media of hippocampus NSC (NSC-exo) or mature hippocampal neuronal (MN-exo) cultures were delivered intracerebroventricularly (ICV) to mice before assessment of Aßο-induced suppression of hippocampal long-term potentiation (LTP) and memory deficits. Aßο binding to synapses was assessed in cultured hippocampal neurons and on synaptosomes isolated from hippocampal slices from wild type mice and from an inducible mouse model of NSC ablation (Nestin-δ-HSV-TK mice) treated with exosomes. Expression of CaMKII and of AMPA and NMDA glutamate receptor subunits in synaptosomes was measured by western blot. Small RNA Deep sequencing was performed to identify microRNAs enriched in NSC-exo as compared to MN-exo. Mimics of select miRNAs were injected ICV. RESULTS: NSC-exo, but not MN-exo, abolished Aßo-induced suppression of LTP and subsequent memory deficits. Furthermore, in hippocampal slices and cultured neurons, NSC-exo significantly decreased Aßo binding to the synapse. Similarly, transgenic ablation of endogenous NSC increased synaptic Aßo binding, which was reversed by exogenous NSC-exo. Phosphorylation of synaptic CaMKII was increased by NSC-exo, while AMPA and NMDA receptors were not affected. Lastly, we identified a set of miRNAs enriched in NSC-exo that, when injected ICV, protected the synapses from Aßo-binding and Aßo-induced LTP inhibition. CONCLUSIONS: These results identify a novel mechanism linking NSC-exo and synaptic susceptibility to Aßo that may underscore cognitive resilience of certain individuals with increased neurogenesis in spite of AD neuropathology and unmask a novel target for the development of a new treatment concept for AD centered on promoting synaptic resilience to toxic amyloid proteins.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Exosomas/metabolismo , Hipocampo/metabolismo , Plasticidad Neuronal/fisiología , Enfermedad de Alzheimer/metabolismo , Animales , Potenciación a Largo Plazo/fisiología , Ratones Endogámicos C57BL , Ratas , Sinapsis/metabolismoRESUMEN
Only a handful of published reports exist today that describe neurological complications following smoke inhalation injury. In this study, we characterize acute pathophysiological changes in the brain of sheep exposed to smoke inhalation, with- and without third-degree skin burn that models the injuries sustained by human victims of fire accidents. Blood-brain barrier integrity and hemorrhage were analyzed throughout the brain using specific histological stains: Hematoxylin & Eosin, Luxol fast blue, Periodic acid-Schiff (PAS), and Martius, Scarlet and Blue (MSB). Our data show that, following smoke inhalation injury, alone and in combination with third-degree skin burn, there was a significant increase in the number of congested and dilated blood vessels in the frontal cortex, basal ganglia, amygdala, hippocampus, pons, cerebellum, and pituitary gland as compared to sham-injured controls. Positive PAS staining confirmed damage to the basement membrane of congested and dilated blood vessels throughout the brain. Severe rupturing of blood vessels, microvascular hemorrhaging and bleeding throughout the brain was also observed in the injured groups. No significant changes in hemodynamics and PaO2 were observed. Our data demonstrate for the first time that acute smoke inhalation alone results in diffuse blood-brain barrier dysfunction and massive bleeding in the brain in the absence of hypoxia and changes in hemodynamics. These findings provide critical information and prompt further mechanistic and interventional studies necessary to develop effective and novel treatments aimed at alleviating CNS dysfunction in patients with smoke and burn injuries.
Asunto(s)
Barrera Hematoencefálica/fisiopatología , Quemaduras/fisiopatología , Piel/lesiones , Lesión por Inhalación de Humo/fisiopatología , Animales , Análisis de los Gases de la Sangre , Encéfalo/patología , Encéfalo/fisiopatología , Sistema Nervioso Central/patología , Femenino , Hemodinámica , Hemorragia/fisiopatología , Hipoxia , Pulmón/patología , Microcirculación , Oxígeno/metabolismo , Intercambio Gaseoso Pulmonar , Resucitación , OvinosRESUMEN
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RESUMEN
We have developed a novel, non-invasive nano-pulsed laser therapy (NPLT) system that combines the benefits of near-infrared laser light (808 nm) and ultrasound (optoacoustic) waves, which are generated with each short laser pulse within the tissue. We tested NPLT in a rat model of blast-induced neurotrauma (BINT) to determine whether transcranial application of NPLT provides neuroprotective effects. The laser pulses were applied on the intact rat head 1 h after injury using a specially developed fiber-optic system. Vestibulomotor function was assessed on post-injury days (PIDs) 1-3 on the beam balance and beam walking tasks. Cognitive function was assessed on PIDs 6-10 using a working memory Morris water maze (MWM) test. BDNF and caspase-3 messenger RNA (mRNA) expression was measured by quantitative real-time PCR (qRT-PCR) in laser-captured cortical neurons. Microglia activation and neuronal injury were assessed in brain sections by immunofluorescence using specific antibodies against CD68 and active caspase-3, respectively. In the vestibulomotor and cognitive (MWM) tests, NPLT-treated animals performed significantly better than the untreated blast group and similarly to sham animals. NPLT upregulated mRNA encoding BDNF and downregulated the pro-apoptotic protein caspase-3 in cortical neurons. Immunofluorescence demonstrated that NPLT inhibited microglia activation and reduced the number of cortical neurons expressing activated caspase-3. NPLT also increased expression of BDNF in the hippocampus and the number of proliferating progenitor cells in the dentate gyrus. Our data demonstrate a neuroprotective effect of NPLT and prompt further studies aimed to develop NPLT as a therapeutic intervention after traumatic brain injury (TBI).
Asunto(s)
Traumatismos por Explosión/complicaciones , Lesiones Traumáticas del Encéfalo/etiología , Terapia por Luz de Baja Intensidad/métodos , Ultrasonografía/métodos , Animales , Traumatismos por Explosión/fisiopatología , Lesiones Traumáticas del Encéfalo/fisiopatología , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Virally mediated RNA interference (RNAi) to knock down injury-induced genes could improve functional outcome after traumatic brain injury (TBI); however, little is known about the consequences of gene knockdown on downstream cell signaling pathways and how RNAi influences neurodegeneration and behavior. Here, we assessed the effects of adeno-associated virus (AAV) siRNA vectors that target two genes with opposing roles in TBI pathogenesis: the allegedly detrimental neuronal nitric oxide synthase (nNOS) and the potentially protective glutathione peroxidase 1 (GPx-1). In rat hippocampal progenitor cells, three siRNAs that target different regions of each gene (nNOS, GPx-1) effectively knocked down gene expression. However, in vivo, in our rat model of fluid percussion brain injury, the consequences of AAV-siRNA were variable. One nNOS siRNA vector significantly reduced the number of degenerating hippocampal neurons and showed a tendency to improve working memory. GPx-1 siRNA treatment did not alter TBI-induced neurodegeneration or working memory deficits. Nevertheless, microarray analysis of laser captured, virus-infected neurons showed that knockdown of nNOS or GPx-1 was specific and had broad effects on downstream genes. Since nNOS knockdown only modestly ameliorated TBI-induced working memory deficits, despite widespread genomic changes, manipulating expression levels of single genes may not be sufficient to alter functional outcome after TBI.
Asunto(s)
Lesiones Traumáticas del Encéfalo/genética , Dependovirus/genética , Glutatión Peroxidasa/genética , Trastornos de la Memoria/genética , Óxido Nítrico Sintasa de Tipo I/genética , Interferencia de ARN , Animales , Lesiones Traumáticas del Encéfalo/metabolismo , Lesiones Traumáticas del Encéfalo/fisiopatología , Dependovirus/metabolismo , Perfilación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Glutatión Peroxidasa/antagonistas & inhibidores , Glutatión Peroxidasa/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatología , Captura por Microdisección con Láser , Masculino , Aprendizaje por Laberinto , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/fisiopatología , Memoria a Corto Plazo/fisiología , Redes y Vías Metabólicas/genética , Análisis por Micromatrices , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/metabolismo , Neuronas/patología , Óxido Nítrico Sintasa de Tipo I/antagonistas & inhibidores , Óxido Nítrico Sintasa de Tipo I/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal , Glutatión Peroxidasa GPX1RESUMEN
The underlying molecular mechanisms of how dysregulated microRNAs (miRNAs) cause neurodegeneration after traumatic brain injury (TBI) remain elusive. Here we analyzed the biological roles of approximately 600 genes - we previously found these dysregulated in dying and surviving rat hippocampal neurons - that are targeted by ten TBI-altered miRNAs. Bioinformatic analysis suggests that neurodegeneration results from a global miRNA-mediated suppression of genes essential for maintaining proteostasis; many are hub genes - involved in RNA processing, cytoskeletal metabolism, intracellular trafficking, cell cycle progression, repair/maintenance, bioenergetics and cell-cell signaling - whose disrupted expression is linked to human disease. Notably, dysregulation of these essential genes would significantly impair synaptic function and functional brain connectivity. In surviving neurons, upregulated miRNA target genes are co-regulated members of prosurvival pathways associated with cellular regeneration, neural plasticity, and development. This study captures the diversity of miRNA-regulated genes that may be essential for cell repair and survival responses after TBI.
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Lesiones Traumáticas del Encéfalo/fisiopatología , Muerte Celular , Regulación de la Expresión Génica , Hipocampo/fisiopatología , Deficiencias en la Proteostasis/complicaciones , Animales , Lesiones Traumáticas del Encéfalo/complicaciones , Lesiones Traumáticas del Encéfalo/genética , Supervivencia Celular , Perfilación de la Expresión Génica , Masculino , Enfermedades Neurodegenerativas/etiología , Plasticidad Neuronal , Neuronas/fisiología , Deficiencias en la Proteostasis/etiología , RatasRESUMEN
According to current dogma, there is little or no ongoing neurogenesis in the fully developed adult enteric nervous system. This lack of neurogenesis leaves unanswered the question of how enteric neuronal populations are maintained in adult guts, given previous reports of ongoing neuronal death. Here, we confirm that despite ongoing neuronal cell loss because of apoptosis in the myenteric ganglia of the adult small intestine, total myenteric neuronal numbers remain constant. This observed neuronal homeostasis is maintained by new neurons formed in vivo from dividing precursor cells that are located within myenteric ganglia and express both Nestin and p75NTR, but not the pan-glial marker Sox10. Mutation of the phosphatase and tensin homolog gene in this pool of adult precursors leads to an increase in enteric neuronal number, resulting in ganglioneuromatosis, modeling the corresponding disorder in humans. Taken together, our results show significant turnover and neurogenesis of adult enteric neurons and provide a paradigm for understanding the enteric nervous system in health and disease.
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Apoptosis , Sistema Nervioso Entérico/metabolismo , Nestina/metabolismo , Neurogénesis , Receptores de Factor de Crecimiento Nervioso/metabolismo , Factores de Transcripción SOXE/metabolismo , Animales , Humanos , Ratones , Ratones Transgénicos , Nestina/genética , Receptores de Factor de Crecimiento Nervioso/genética , Factores de Transcripción SOXE/genéticaRESUMEN
Rare individuals remain cognitively intact despite the presence of neuropathology usually associated with fully symptomatic Alzheimer's disease (AD), which we refer to as Non-Demented with Alzheimer's disease Neuropathology (NDAN). Understanding the involved mechanism(s) of their cognitive resistance may reveal novel strategies to treat AD-related dementia. In the pursuit of this goal, we determined the number of hippocampal neural stem cells (NSCs) and investigated the expression of several miRNAs in NDAN and AD subjects. Laser-capture microdissection of autopsy human hippocampus DG and qRT-PCR miRNA analyses were combined with immunofluorescence in this study. The number of SOX2(+) NSCs in the DG was significantly increased in NDAN individuals as compared to AD subjects. Further, the prevalence of SOX2(+) NSCs was found to correlate with cognitive capacity. Neurogenesis-regulating miRNAs were decreased in NDAN individuals as compared to AD patients. An increased number of NSCs and new neurons in NDAN individuals is associated with a unique expression of regulating miRNAs and strongly support a role of neurogenesis in mediating, in part, the ability of these individuals to resist the pathological burden of AD.
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Enfermedad de Alzheimer/patología , Demencia/patología , Hipocampo/patología , Células-Madre Neurales/patología , Neurogénesis/genética , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/genética , Autopsia , Demencia/complicaciones , Demencia/genética , Progresión de la Enfermedad , Regulación de la Expresión Génica , Humanos , Captura por Microdisección con Láser , MicroARNs/genética , Factores de Transcripción SOXB1/metabolismoRESUMEN
INTRODUCTION: Stem cells have been evaluated as a potential therapeutic approach for several neurological disorders of the central and peripheral nervous system as well as for traumatic brain and spinal cord injury. Currently, the lack of a reliable and safe method to accurately and non-invasively locate the site of implantation and track the migration of stem cells in vivo hampers the development of stem cell therapy and its clinical application. In this report, we present data that demonstrate the feasibility of using the human sodium iodide symporter (hNIS) as a reporter gene for tracking neural stem cells (NSCs) after transplantation in the brain by using single-photon emission tomography/computed tomography (SPECT/CT) imaging. METHODS: NSCs were isolated from the hippocampus of adult rats (Hipp-NSCs) and transduced with a lentiviral vector containing the hNIS gene. Hipp-NSCs expressing the hNIS (NIS-Hipp-NSCs) were characterized in vitro and in vivo after transplantation in the rat brain and imaged by using technetium-99m ((99m)Tc) and a small rodent SPECT/CT apparatus. Comparisons were made between Hipp-NSCs and NIS-Hipp-NSCs, and statistical analysis was performed by using two-tailed Student's t test. RESULTS: Our results show that the expression of the hNIS allows the repeated visualization of NSCs in vivo in the brain by using SPECT/CT imaging and does not affect the ability of Hipp-NSCs to generate neuronal and glial cells in vitro and in vivo. CONCLUSIONS: These data support the use of the hNIS as a reporter gene for non-invasive imaging of NSCs in the brain. The repeated, non-invasive tracking of implanted cells will accelerate the development of effective stem cell therapies for traumatic brain injury and other types of central nervous system injury.
Asunto(s)
Encéfalo/patología , Diagnóstico por Imagen/métodos , Hipocampo/citología , Hipocampo/metabolismo , Animales , Western Blotting , Proliferación Celular/fisiología , Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Cultivadas , Masculino , Células-Madre Neurales/fisiología , Ratas , Ratas Sprague-Dawley , Tomografía Computarizada de Emisión de Fotón ÚnicoRESUMEN
Cognitive deficits in survivors of traumatic brain injury (TBI) are associated with irreversible neurodegeneration in brain regions such as the hippocampus. Comparative gene expression analysis of dying and surviving neurons could provide insight into potential therapeutic targets. We used two pathway-specific PCR arrays (RT2 Profiler Apoptosis and Neurotrophins & Receptors PCR arrays) to identify and validate TBI-induced gene expression in dying (Fluoro-Jade-positive) or surviving (Fluoro-Jade-negative) pyramidal neurons obtained by laser capture microdissection (LCM). In the Apoptosis PCR array, dying neurons showed significant increases in expression of genes associated with cell death, inflammation, and endoplasmic reticulum (ER) stress compared with adjacent, surviving neurons. Pro-survival genes with pleiotropic functions were also significantly increased in dying neurons compared to surviving neurons, suggesting that even irreversibly injured neurons are able to mount a protective response. In the Neurotrophins & Receptors PCR array, which consists of genes that are normally expected to be expressed in both groups of hippocampal neurons, only a few genes were expressed at significantly different levels between dying and surviving neurons. Immunohistochemical analysis of selected, differentially expressed proteins supported the gene expression data. This is the first demonstration of pathway-focused PCR array profiling of identified populations of dying and surviving neurons in the brain after TBI. Combining precise laser microdissection of identifiable cells with pathway-focused PCR array analysis is a practical, low-cost alternative to microarrays that provided insight into neuroprotective signals that could be therapeutically targeted to ameliorate TBI-induced neurodegeneration.
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Lesiones Encefálicas/genética , Lesiones Encefálicas/patología , Hipocampo/patología , Captura por Microdisección con Láser , Reacción en Cadena de la Polimerasa/métodos , Transducción de Señal/genética , Animales , Apoptosis/genética , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/terapia , Supervivencia Celular/genética , Perfilación de la Expresión Génica , Hipocampo/metabolismo , Masculino , Factores de Crecimiento Nervioso/genética , Neuronas/metabolismo , Neuronas/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Ratas , Ratas Sprague-DawleyRESUMEN
Substance P (SP) is commonly coexpressed with ACh in enteric motor neurons, and, according to the classical paradigm, both these neurotransmitters excite smooth muscle via parallel pathways. We hypothesized that, in addition, SP was responsible for maintaining the muscular responsiveness to ACh. We tested this hypothesis by using botulinum toxin (BoNT/A), a known blocker of vesicular release of neurotransmitters including ACh and neuropeptides. BoNT/A was injected into rat pyloric sphincter in different doses; as control we used boiled BoNT/A. At the desired time point, pylorus was dissected out and pyloric contractility was measured ex vivo in an organ bath and by measuring phosphorylation of myosin light chain 20 (MLC20). BoNT/A (10 IU) significantly reduced the response of pyloric muscle to exogenous ACh, an effect that was accompanied by reduced MLC20 phosphorylation in the muscle. Both effects were reversed by exogenous SP. CP-96345, a NK1 receptor antagonist, blocked the ability of exogenous SP to reverse the cholinergic hyporesponsiveness as well as the reduction in MLC20 phosphorylation induced by BoNT/A. In conclusion, we have identified a novel role for SP as a coneurotransmitter that appears to be important for the maintenance of muscular responsiveness to the principal excitatory neurotransmitter, ACh. These results also provide new insight into the effects of botulinum toxin on the enteric nervous system and gastrointestinal smooth muscle.
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Toxinas Botulínicas/farmacología , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Sustancia P/fisiología , Acetilcolina/farmacología , Animales , Compuestos de Bifenilo/farmacología , Estimulación Eléctrica , Técnicas In Vitro , Masculino , Músculo Liso/fisiología , Cadenas Ligeras de Miosina/metabolismo , Antro Pilórico/efectos de los fármacos , Antro Pilórico/fisiología , Píloro/efectos de los fármacos , Ratas , Receptores de Neuroquinina-1/efectos de los fármacos , Sustancia P/farmacologíaRESUMEN
Developing new pharmacotherapies for traumatic brain injury (TBI) requires elucidation of the neuroprotective mechanisms of many structurally and functionally diverse compounds. To test our hypothesis that diverse neuroprotective drugs similarly affect common gene targets after TBI, we compared the effects of two drugs, metyrapone (MT) and carbenoxolone (CB), which, though used clinically for noncognitive conditions, improved learning and memory in rats and humans. Although structurally different, both MT and CB inhibit a common molecular target, 11ß hydroxysteroid dehydrogenase type 1, which converts inactive cortisone to cortisol, thereby effectively reducing glucocorticoid levels. We examined injury-induced signaling pathways to determine how the effects of these two compounds correlate with pro-survival effects in surviving neurons of the injured rat hippocampus. We found that treatment of TBI rats with MT or CB acutely induced in hippocampal neurons transcriptional profiles that were remarkably similar (i.e., a coordinated attenuation of gene expression across multiple injury-induced cell signaling networks). We also found, to a lesser extent, a coordinated increase in cell survival signals. Analysis of injury-induced gene expression altered by MT and CB provided additional insight into the protective effects of each. Both drugs attenuated expression of genes in the apoptosis, death receptor and stress signaling pathways, as well as multiple genes in the oxidative phosphorylation pathway such as subunits of NADH dehydrogenase (Complex1), cytochrome c oxidase (Complex IV) and ATP synthase (Complex V). This suggests an overall inhibition of mitochondrial function. Complex 1 is the primary source of reactive oxygen species in the mitochondrial oxidative phosphorylation pathway, thus linking the protective effects of these drugs to a reduction in oxidative stress. The net effect of the drug-induced transcriptional changes observed here indicates that suppressing expression of potentially harmful genes, and also, surprisingly, reduced expression of pro-survival genes may be a hallmark of neuroprotective therapeutic effects.
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Lesiones Encefálicas/tratamiento farmacológico , Lesiones Encefálicas/genética , Carbenoxolona/uso terapéutico , Metirapona/uso terapéutico , Transducción de Señal/genética , Animales , Lesiones Encefálicas/complicaciones , Lesiones Encefálicas/patología , Carbenoxolona/farmacología , Muerte Celular/efectos de los fármacos , Muerte Celular/genética , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Regulación de la Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/patología , Humanos , Masculino , Metirapona/farmacología , Degeneración Nerviosa/complicaciones , Degeneración Nerviosa/tratamiento farmacológico , Degeneración Nerviosa/patología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Ratas , Ratas Sprague-Dawley , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genéticaRESUMEN
Circadian rhythm disturbances are frequently reported in patients recovering from traumatic brain injury (TBI). Since circadian clock output is mediated by some of the same molecular signaling cascades that regulate memory formation (cAMP/MAPK/CREB), cognitive problems reported by TBI survivors may be related to injury-induced dysregulation of the circadian clock. In laboratory animals, aberrant circadian rhythms in the hippocampus have been linked to cognitive and memory dysfunction. Here, we addressed the hypothesis that circadian rhythm disruption after TBI is mediated by changes in expression of clock genes in the suprachiasmatic nuclei (SCN) and hippocampus. After fluid-percussion TBI or sham surgery, male Sprague-Dawley rats were euthanized at 4 h intervals, over a 48 h period for tissue collection. Expression of circadian clock genes was measured using quantitative real-time PCR in the SCN and hippocampus obtained by laser capture and manual microdissection respectively. Immunofluorescence and Western blot analysis were used to correlate TBI-induced changes in circadian gene expression with changes in protein expression. In separate groups of rats, locomotor activity was monitored for 48 h. TBI altered circadian gene expression patterns in both the SCN and the hippocampus. Dysregulated expression of key circadian clock genes, such as Bmal1 and Cry1, was detected, suggesting perturbation of transcriptional-translational feedback loops that are central to circadian timing. In fact, disruption of circadian locomotor activity rhythms in injured animals occurred concurrently. These results provide an explanation for how TBI causes disruption of circadian rhythms as well as a rationale for the consideration of drugs with chronobiotic properties as part of a treatment strategy for TBI.
Asunto(s)
Lesiones Encefálicas/genética , Relojes Circadianos/genética , Regulación de la Expresión Génica , Núcleo Supraquiasmático/metabolismo , Factores de Transcripción ARNTL/genética , Factores de Transcripción ARNTL/metabolismo , Animales , Western Blotting , Lesiones Encefálicas/metabolismo , Lesiones Encefálicas/fisiopatología , Factor Neurotrófico Derivado del Encéfalo/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Relojes Circadianos/fisiología , Criptocromos/genética , Criptocromos/metabolismo , Hipocampo/metabolismo , Hipocampo/fisiopatología , Masculino , Actividad Motora/genética , Actividad Motora/fisiología , Proteínas Circadianas Period/genética , Proteínas Circadianas Period/metabolismo , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Núcleo Supraquiasmático/fisiopatologíaRESUMEN
BACKGROUND: Stem cell-based therapy has recently been explored for the treatment of disorders of the enteric nervous system (ENS). Pluripotent embryonic stem (ES) cells represent an attractive cell source; however, little or no information is currently available on how ES cells will respond to the gut environment. In this study, we investigated the ability of ES cells to respond to environmental cues derived from the ENS and related tissues, both in vitro and in vivo. METHODS: Neurospheres were generated from mouse ES cells (ES-NS) and co-cultured with organotypic preparations of gut tissue consisting of the longitudinal muscle layers with the adherent myenteric plexus (LM-MP). RESULTS: LM-MP co-culture led to a significant increase in the expression of pan-neuronal markers (ßIII-tubulin, PGP 9.5) as well as more specialized markers (peripherin, nNOS) in ES-NS, both at the transcriptional and protein level. The increased expression was not associated with increased proliferation, thus confirming a true neurogenic effect. LM-MP preparations exerted also a myogenic effect on ES-NS, although to a lesser extent. After transplantation in vivo into the mouse pylorus, grafted ES-NS failed to acquire a distinct phenotype al least 1 week following transplantation. CONCLUSIONS: This is the first study reporting that the gut explants can induce neuronal differentiation of ES cells in vitro and induce the expression of nNOS, a key molecule in gastrointestinal motility regulation. The inability of ES-NS to adopt a neuronal phenotype after transplantation in the gastrointestinal tract is suggestive of the presence of local inhibitory influences that prevent ES-NS differentiation in vivo.
Asunto(s)
Tratamiento Basado en Trasplante de Células y Tejidos/métodos , Células Madre Embrionarias/trasplante , Sistema Nervioso Entérico/embriología , Tracto Gastrointestinal/citología , Músculo Liso/citología , Plexo Mientérico/citología , Animales , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Técnicas de Cocultivo , Células Madre Embrionarias/citología , Tracto Gastrointestinal/metabolismo , Intestino Delgado/citología , Intestino Delgado/metabolismo , Ratones , Ratones Endogámicos , Músculo Liso/metabolismo , Óxido Nítrico Sintasa de Tipo I/metabolismo , Fenotipo , Píloro/citología , Píloro/metabolismo , Trasplante de Células Madre/métodosRESUMEN
Enteric neural stem cells (ENSCs) are a population of neural crest-derived multipotent stem cells present in postnatal gut that may play an important role in regeneration of the enteric nervous system. In most studies, these cells have been isolated from the layer of the gut containing the myenteric plexus. However, a recent report demonstrated that neurosphere-like bodies (NLBs) containing ENSCs could be isolated from mucosal biopsy specimens from children, suggesting that ENSCs are present in multiple layers of the gut. The aim of our study was to assess whether NLBs isolated from layers of gut containing either myenteric or submucosal plexus are equivalent. We divided the mouse small intestine into two layers, one containing myenteric plexus and the other submucosal plexus, and assessed for NLB formation. Differences in NLB density, proliferation, apoptosis, neural crest origin, and phenotype were investigated. NLBs isolated from the myenteric plexus layer were present at a higher density and demonstrated greater proliferation, lower apoptosis, and higher expression of nestin, p75, Sox10, and Ret than those from submucosal plexus. Additionally, they contained a higher percentage of neural crest-derived cells (99.4 ± 1.5 vs. 0.7 ± 1.19% of Wnt1-cre:tdTomato cells; P < 0.0001) and produced more neurons and glial cells than those from submucosal plexus. NLBs from the submucosal plexus layer expressed higher CD34 and produced more smooth muscle-like cells. NLBs from the myenteric plexus layer contain more neural crest-derived ENSCs while those from submucosal plexus appear more heterogeneous, likely containing a population of mesenchymal stem cells.
Asunto(s)
Intestino Delgado/citología , Células-Madre Neurales/citología , Animales , Diferenciación Celular , Supervivencia Celular , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos C57BLRESUMEN
Experimental evidence suggests that random, spontaneous (stochastic) fluctuations in gene expression have important biological consequences, including determination of cell fate and phenotypic variation within isogenic populations. We propose that fluctuations in gene expression represent a valuable tool to explore therapeutic strategies for patients who have suffered traumatic brain injury (TBI), for which there is no effective drug therapy. We have studied the effects of TBI on the hippocampus because TBI survivors commonly suffer cognitive problems that are associated with hippocampal damage. In our previous studies we separated dying and surviving hippocampal neurons by laser capture microdissection and observed unexplainable variations in post-TBI gene expression, even though dying and surviving neurons were adjacent and morphologically identical. We hypothesized that, in hippocampal neurons that subsequently are subjected to TBI, randomly increased pre-TBI expression of genes that are associated with neuroprotection predisposes neurons to survival; conversely, randomly decreased expression of these genes predisposes neurons to death. Thus, to identify genes that are associated with endogenous neuroprotection, we performed a comparative, high-resolution transcriptome analysis of dying and surviving hippocampal neurons in rats subjected to TBI. We found that surviving hippocampal neurons express a distinct molecular signature--increased expression of networks of genes that are associated with regeneration, cellular reprogramming, development, and synaptic plasticity. In dying neurons we found decreased expression of genes in those networks. Based on these data, we propose a hypothetical model in which hippocampal neuronal survival is determined by a rheostat that adds injury-induced genomic signals to expression of pro-survival genes, which pre-TBI varies randomly and spontaneously from neuron to neuron. We suggest that pharmacotherapeutic strategies that co-activate multiple survival signals and enhance self-repair mechanisms have the potential to shift the cell survival rheostat to favor survival and therefore improve functional outcome after TBI.
Asunto(s)
Lesiones Encefálicas/genética , Lesiones Encefálicas/patología , Regulación de la Expresión Génica , Animales , Lesiones Encefálicas/fisiopatología , Linaje de la Célula/genética , Proliferación Celular , Supervivencia Celular/genética , Reprogramación Celular/genética , Perfilación de la Expresión Génica , Hipocampo/patología , Homeostasis , Inmunohistoquímica , Plasticidad Neuronal/fisiología , Neuronas/metabolismo , Neuronas/patología , Fármacos Neuroprotectores/metabolismo , Ratas , Reacción en Cadena en Tiempo Real de la Polimerasa , Reproducibilidad de los Resultados , Coloración y Etiquetado , Procesos Estocásticos , Sinapsis/patología , TranscriptomaRESUMEN
Recent studies have explored the potential of central nervous system-derived neural stem cells (CNS-NSC) to repopulate the enteric nervous system. However, the exact phenotypic fate of gut-transplanted CNS-NSC has not been characterized. The aim of this study was to investigate the effect of the gut microenvironment on phenotypic fate of CNS-NSC in vitro. With the use of Transwell culture, differentiation of mouse embryonic CNS-NSC was studied when cocultured without direct contact with mouse intestinal longitudinal muscle-myenteric plexus preparations (LM-MP) compared with control noncocultured cells, in a differentiating medium. Differentiated cells were analyzed by immunocytochemistry and quantitative RT-PCR to assess the expression of specific markers and by whole cell patch-clamp studies for functional characterization of their phenotype. We found that LM-MP cocultured cells had a significant increase in the numbers of cells that were immune reactive against the panneuronal marker ß-tubulin, neurotransmitters neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and neuropeptide vasoactive intestinal peptide (VIP) and showed an increase in expression of these genes, compared with control cells. Whole cell patch-clamp analysis showed that coculture with LM-MP decreases cell excitability and reduces voltage-gated Na(+) currents but significantly enhances A-current and late afterhyperpolarization (AHP) and increases the expression of the four AHP-generating Ca(2+)-dependent K(+) channel genes (KCNN), compared with control cells. In a separate experiment, differentiation of LM-MP cocultured CNS-NSC produced a significant increase in the numbers of cells that were immune reactive against the neurotransmitters nNOS, ChAT, and the neuropeptide VIP compared with CNS-NSC differentiated similarly in the presence of neonatal brain tissue. Our results show that the gut microenvironment induces CNS-NSC to produce neurons that share some of the characteristics of classical enteric neurons, further supporting the therapeutic use of these cells for gastrointestinal disorders.