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Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Lipid-mediated nucleic acid delivery is an alternative to viral vector-mediated gene delivery and has the following advantages. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, since transit across the nuclear membrane is not necessary, and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Delivery of RNA to target organ(s) has previously been challenging due to RNA's rapid degradation in biological systems, but cationic lipids complexed with RNA, as well as lipid nanoparticles (LNPs), have allowed for delivery and expression of the complexed RNA both in vitro and in vivo. This review will focus on the non-viral lipid-mediated delivery of RNAs, including mRNA, siRNA, shRNA, and microRNA, to the central nervous system (CNS), an organ with at least two unique challenges. The CNS contains a large number of slowly dividing or non-dividing cell types and is protected by the blood brain barrier (BBB). In non-dividing cells, RNA-lipid complexes demonstrated increased transfection efficiency relative to DNA transfection. The efficiency, timing of the onset, and duration of expression after transfection may determine which nucleic acid is best for which proposed therapy. Expression can be seen as soon as 1 h after RNA delivery, but duration of expression has been limited to 5-7 h. In contrast, transfection with a DNA lipoplex demonstrates protein expression within 5 h and lasts as long as several weeks after transfection.
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BACKGROUND: Perioperative seizures may affect 1% to 50% of patients undergoing craniotomy and adversely impact outcomes. However, data on intraoperative seizures are limited. This retrospective case-control study investigated the incidence and risk factors for intraoperative seizures during elective supratentorial craniotomy involving evoked potential monitoring. MATERIALS AND METHODS: Patients aged 18 years or above undergoing elective supratentorial craniotomy with evoked potential monitoring who experienced intraoperative seizures at our institution between December 2008 and March 2014 were compared with a control group generated using a random number generator. Six controls were used for each case from among the patients who underwent elective supratentorial craniotomy during the same calendar year. Multivariate analysis was conducted using logistic regression to identify the risk factors for intraoperative seizures. RESULTS: Among the 1916 patients who met the inclusion criteria, 45 (2.3%) had intraoperative seizures. The majority of seizures occurred during burr-hole placement or craniotomy, before lesion manipulation. Timing of seizures relative to motor evoked potential runs and stimulus intensity was variable. Significant risk factors for intraoperative seizures were seizure history (odds ratio [OR], 2.18; 95% confidence interval [CI], 1.07-4.46; P=0.03), diagnosis of brain tumor (OR, 2.41; 95% CI, 1.16-4.19; P=0.02), and temporal craniotomy (OR, 5.18; 95% CI, 2.03-13.25; P=0.001). Intraoperative prophylactic use of phenytoin/fosphenytoin and levetiracetam was protective against seizure (phenytoin/fosphenytoin: OR, 0.12; 95% CI, 0.04-0.35; P<0.001 and levetiracetam: OR, 0.40; 95% CI, 0.17-0.94; P=0.04). Phenytoin/fosphenytoin was more protective than levetiracetam (OR, 0.31; 95% CI, 0.10-0.99; P=0.048). CONCLUSIONS: The overall incidence of intraoperative seizures was 2.3%. Independent risk factors for intraoperative seizures were seizure history, diagnosis of intracranial tumor, and temporal craniotomy. Intraoperative prophylactic anticonvulsant use was protective.
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Craneotomía/efectos adversos , Complicaciones Intraoperatorias/epidemiología , Convulsiones/epidemiología , Adulto , Anciano , Anticonvulsivantes/uso terapéutico , Neoplasias Encefálicas/complicaciones , Estudios de Casos y Controles , Potenciales Evocados Motores , Femenino , Humanos , Incidencia , Complicaciones Intraoperatorias/prevención & control , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Riesgo , Convulsiones/prevención & control , Adulto JovenRESUMEN
We examined the effect of early intensive care unit (ICU) adherence to 2007 Brain Trauma Foundation Guideline indicators after traumatic brain injury (TBI) on inpatient mortality at a level 1 trauma center in India (Jay Prakash Narayan Apex Trauma Center [JPNATC]) and Harborview Medical Center (HMC) in U.S. among adults older than 18 years with severe TBI. At each site, ICU Guideline adherence in first 72 hours for 17 indicators was determined and expressed as a percentage. Outcomes were in-hospital mortality and Glasgow Outcome Scale (GOS) scores at 3, 6, and 12 months after discharge. JPNATC and HMC Guideline adherence rates were 74.9% [11.0] and 71.6 % (SD ±10.4), and overall in-hospital mortality was 24% and 27%, respectively. At JPNATC, less than 65% ICU Guideline adherence was associated with higher inpatient mortality (adjusted relative risk [aRR], 1.92; 95% confidence interval [CI], 1.11-3.33) and an increase in ICU Guideline adherence rate by 1% was associated with a 3% lower in-hospital mortality (aRR, 0.97; 95% CI, 0.95-0.99). Among patients discharged with a GOS score of 2-4 at JPNATC, 67% improved at 12 months (R(2) = 0.991; P < 0.01; 99% follow-up rate) compared with discharge, but 35%, 25%, and 14% of patients discharged with a GOS score of 3-5 deteriorated at 3, 6, and 12 months to a lower GOS at home. Achieving early ICU adherence to guideline indicators was feasible and associated with significantly lower in-hospital mortality at JPNATC. Although the intracranial pressure (ICP) monitoring rates varied, in-hospitals deaths were similar between the two institutions. Although long-term outcomes generally improved, patients discharged with favorable GOS score often deteriorated at home.
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Lesiones Traumáticas del Encéfalo/terapia , Adhesión a Directriz , Adulto , Lesiones Traumáticas del Encéfalo/mortalidad , Lesiones Traumáticas del Encéfalo/fisiopatología , Femenino , Escala de Consecuencias de Glasgow , Mortalidad Hospitalaria , Humanos , India , Unidades de Cuidados Intensivos , Presión Intracraneal , Masculino , Alta del Paciente , Guías de Práctica Clínica como Asunto , Estudios Prospectivos , Estudios Retrospectivos , Riesgo , Centros Traumatológicos , Resultado del Tratamiento , WashingtónRESUMEN
Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery. Lipid-mediated delivery of DNA or mRNA is usually more rapid than viral-mediated delivery, offers a larger payload, and has a nearly zero risk of incorporation. Lipid-mediated delivery of DNA or RNA is therefore preferable to viral DNA delivery in those clinical applications that do not require long-term expression for chronic conditions. Delivery of RNA may be preferable to non-viral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary and onset of expression with RNA is therefore even faster than with DNA, although both are faster than most viral vectors. Here, we describe techniques for cationic lipid-mediated delivery of nucleic acids encoding reporter genes in a variety of cell lines. We describe optimized formulations and transfection procedures that we previously assessed by bioluminescence and flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in non-dividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. Duration of expression in eukaryotic cells after mRNA transcript delivery depends on multiple factors, including transcript stability, protein turnover, and cell type. Delivery of DNA results in onset of expression within 5 h after transfection, a peak in expression 24-48 h after transfection, and a return to baseline that can be as long as several weeks after transfection. In vitro results are consistent with our in vivo delivery results, techniques for which are described as well. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression and greater efficiency, particularly in non-dividing cells, while the longer duration and the higher mean levels of expression per cell that are ultimately obtained following DNA delivery confirm a continuing role for DNA gene delivery in clinical applications that require longer term transient gene expression.
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ADN/administración & dosificación , ARN Mensajero/administración & dosificación , Transfección/métodos , Animales , Células CHO , Sistema Nervioso Central/metabolismo , Cricetulus , Lípidos/química , Ratones , Células 3T3 NIHRESUMEN
Appropriate gene delivery systems are essential for successful gene therapy in clinical medicine. Cationic lipid-mediated delivery is an alternative to viral vector-mediated gene delivery where transient gene expression is desirable. However, cationic lipid-mediated delivery of DNA to post-mitotic cells is often of low efficiency, due to the difficulty of DNA translocation to the nucleus. Rapid lipid-mediated delivery of RNA is preferable to nonviral DNA delivery in some clinical applications, because transit across the nuclear membrane is not necessary. Here we describe techniques for cationic lipid-mediated delivery of RNA encoding reporter genes in a variety of in vitro cell lines and in vivo. We describe optimized formulations and transfection procedures that we have previously assessed by flow cytometry. RNA transfection demonstrates increased efficiency relative to DNA transfection in nondividing cells. Delivery of mRNA results in onset of expression within 1 h after transfection and a peak in expression 5-7 h after transfection. These results are consistent with our in vivo delivery results, techniques for which are shown as well. Longer duration and the higher mean levels of expression per cell that are ultimately obtained following DNA delivery confirm a continuing role for DNA gene delivery in clinical applications that require long term transient gene expression. RNA delivery is suitable for short-term transient gene expression due to its rapid onset, short duration of expression, and greater efficiency, particularly in nondividing cells.
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Expresión Génica , Lípidos/química , ARN Mensajero , Transfección/métodos , ADN/química , ADN/genética , Biosíntesis de Proteínas , ARN Mensajero/síntesis química , ARN Mensajero/química , ARN Mensajero/genética , Factores de TiempoRESUMEN
AIM: To report our institutional experience with intraoperative jugular venous oximetry during pediatric intracranial surgery to guide anesthetic care. BACKGROUND: The utility of intraoperative jugular venous oximetry in adults undergoing intracranial surgery is well known. However, there is a little information on its' application in children during intracranial surgery. METHODS: After IRB approval, we examined patient, equipment, placement, and sampling characteristics for jugular bulb catheters in children aged <18 years who were monitored with jugular oximetry during elective intracranial surgery between 2006 and 2010. We also determined the prevalence of intraoperative cerebral desaturation (SjvO(2) < 55%), its causes, and the interventions based on jugular oximetry values. RESULTS: Data from 19 children (10 males and nine females), aged 12 ± 1 years (range 7-17) who underwent craniotomy for arteriovenous malformation (AVM) resection (68%), tumor removal (21%), or aneurysm clipping (11%), were reviewed. We analyzed 88 coincident SjvO(2), PaCO(2), and mean arterial pressure data points. Eleven (58%) patients experienced at least one episode of cerebral desaturation. There were 25 (28%) episodes of cerebral desaturation, six of which we attributed to relative hypotension, four to hypocarbia, and 15 to a combination of both. There were no intraoperative or immediate postoperative (first 24 h) complications because of jugular oximetry. CONCLUSION: Findings from this series indicate that (i) intraoperative jugular venous oximetry in children is feasible in experienced hands, (ii) cerebral desaturation detected by jugular oximetry is common during pediatric intracranial procedures, and (iii) monitoring jugular venous saturation can impact anesthetic interventions to optimize cerebral physiology.
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Circulación Cerebrovascular/fisiología , Venas Yugulares/fisiopatología , Monitoreo Intraoperatorio/métodos , Procedimientos Neuroquirúrgicos , Oximetría/métodos , Adolescente , Niño , Estudios de Factibilidad , Femenino , Humanos , Masculino , Oximetría/instrumentación , Estudios RetrospectivosRESUMEN
Intrathecal delivery of gene therapeutics is a route of administration that overcomes several of the limitations that plague current immunosuppressive treatments for autoimmune diseases of the central nervous system (CNS). Here we report intrathecal delivery of small amounts (3 µg) of plasmid DNA that codes for an immunomodulatory fusion protein, OX40-TRAIL, composed of OX40, a tumor necrosis factor receptor, and tumor necrosis factor related apoptosis inducing ligand (TRAIL). This DNA was delivered in a formulated nucleic acid-lipid complex (lipoplexes) with an asymmetric two-chain cationic lipid myristoyl (14:0) and lauroyl (12:1) rosenthal inhibitor-substituted compound (MLRI) formed from the tetraalkylammonium glycerol-based compound N-(1-(2,3-dioleoyloxy)-propyl-N-1-(2-hydroxy)ethyl)-N,N-dimethyl ammonium iodide. Delivery and expression in the CNS of OX40-TRAIL in the mouse prior to onset of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, decreased the severity of clinical disease. We believe this preclinical demonstration of rapid, widespread, and biologically therapeutic nonviral gene delivery to the CNS is important in further development of clinical lipid-based therapeutics for CNS disorders.
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ADN/química , Encefalomielitis Autoinmune Experimental/terapia , Técnicas de Transferencia de Gen , Terapia Genética , Lípidos/química , Animales , Sistema Nervioso Central/metabolismo , Cisterna Magna , ADN/administración & dosificación , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Femenino , Expresión Génica , Genes Reporteros , Inyecciones Espinales , Lauratos/química , Lípidos/administración & dosificación , Ratones , Ratones Endogámicos C57BL , Esclerosis Múltiple/metabolismo , Esclerosis Múltiple/terapia , Miristatos/química , Plásmidos/administración & dosificación , Plásmidos/química , Receptores OX40/genética , Receptores OX40/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Estearatos/administración & dosificación , Estearatos/química , Ligando Inductor de Apoptosis Relacionado con TNF/genética , Ligando Inductor de Apoptosis Relacionado con TNF/metabolismoRESUMEN
The heat shock proteins (HSPs) are members of highly conserved families of molecular chaperones that have multiple roles in vivo. We discuss the HSPs in general, and Hsp70 and Hsp27 in particular, and their rapid induction by severe stress in the context of tissue and organ expression in physiology and disease. We describe the current state of knowledge of the relationship and interactions between extra- and intracellular HSPs and describe mechanisms and significance of extracellular expression of HSPs. We focus on the role of the heat shock proteins as biomarkers of central nervous system (CNS) ischemia and other severe stressors and discuss recent and novel technologies for rapid measurement of proteins in vivo and ex vivo. The HSPs are compared to other proposed small molecule biomarkers for detection of CNS injury and to other methods of detecting brain and spinal cord ischemia in real time. While other biomarkers may be of use in prognosis and in design of appropriate therapies, none appears to be as rapid as the HSPs; therefore, no other measurement appears to be of use in the immediate detection of ongoing severe ischemia with the intention to immediately intervene to reduce the severity or risk of permanent damage.
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Aneurisma de la Aorta Torácica/diagnóstico , Isquemia Encefálica/diagnóstico , Proteínas de Choque Térmico , Isquemia de la Médula Espinal/diagnóstico , Biomarcadores/análisis , Proteínas de Choque Térmico/sangre , Proteínas de Choque Térmico/líquido cefalorraquídeo , Proteínas de Choque Térmico/química , Humanos , Factores de TiempoRESUMEN
We, and others, have observed that the structure of cationic lipids appears to have a significant effect on the transfection efficacy of optimized nucleic acid/cationic lipid complexes (lipoplexes) used for in vitro and in vivo gene delivery and expression. Although there are many in vitro comparisons of lipid reagents for gene delivery, few comparisons have been made in vivo. We previously reported the effects of changes in hydrophobic domain chain length and chain asymmetry, changes in headgroup composition, and counterion exchange. We have observed in our own work over many years the apparent superiority of asymmetric versus symmetric hydrocarbon domains for otherwise similar lipids. In this investigation we use in vivo whole animal brain imaging to evaluate the contribution of symmetric versus asymmetric hydrophobic domains on what we previously determined to be optimal chain lengths for in vitro transfections. We specifically investigated several glycerol-based lipids; however, the rare reports of asymmetric non-glycerol-based lipids also support our observations. We found that asymmetric, two-chain cationic lipids of 14 to 18 carbons perform significantly better in vivo, as analyzed by whole animal imaging, than the paired symmetric lipids.
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Transfección , Animales , Encéfalo/metabolismo , Terapia Genética , Vectores Genéticos , Interacciones Hidrofóbicas e Hidrofílicas , Lípidos/química , Mediciones Luminiscentes , Espectroscopía de Resonancia Magnética , Ratones , Ácidos Nucleicos/químicaRESUMEN
The balance of redox is pivotal for normal function and integrity of tissues. Ischemic insults occur as results of a variety of conditions, leading to an accumulation of reactive oxygen species (ROS) and an imbalanced redox status in the tissues. The oxidant stress may activate signaling mechanisms provoking more toxic events, and eventually cause tissue damage. Therefore, treatments with antioxidants, free radical scavengers and their mimetics, as well as gene transfer approaches to overexpress antioxidant genes represent potential therapeutic options to correct the redox imbalance. Among them, antioxidant gene transfer may enhance the production of antioxidant scavengers, and has been employed to experimentally prevent or treat ischemic injury in cardiovascular, pulmonary, hepatic, intestinal, central nervous or other systems in animal models. With improvements in vector systems and delivery approaches, innovative antioxidant gene therapy has conferred better outcomes for myocardial infarction, reduced restenosis after coronary angioplasty, improved the quality and function of liver grafts, as well as outcome of intestinal and cerebral ischemic attacks. However, it is crucial to be mindful that like other therapeutic armentarium, the efficacy of antioxidant gene transfer requires extensive preclinical investigation before it can be used in patients, and that it may have unanticipated short- or long-term adverse effects. Thus, it is critical to balance between the therapeutic benefits and potential risks, to develop disease-specific antioxidant gene transfer strategies, to deliver the therapy with an optimal time window and in a safe manner. This review attempts to provide the rationale, the most effective approaches and the potential hurdles of available antioxidant gene transfer approaches for ischemic injury in various organs, as well as the possible directions of future preclinical and clinical investigations of this highly promising therapeutic modality.
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Antioxidantes/administración & dosificación , Técnicas de Transferencia de Gen , Isquemia/enzimología , Isquemia/genética , Animales , Técnicas de Transferencia de Gen/tendencias , Terapia Genética/métodos , Terapia Genética/tendencias , Humanos , Isquemia/terapia , Estrés Oxidativo/efectos de los fármacos , Estrés Oxidativo/genética , Especies Reactivas de Oxígeno/metabolismoRESUMEN
We previously showed that a vector:lipid delivery system, comprised of a plasmid DNA vector and cationic lipid (lipoplex), when injected into the cerebrospinal fluid (CSF) of rats can deliver reporter genes in vivo efficiently and with widespread expression to the Central Nervous System (CNS). To further characterize this delivery system, we now present experiments that demonstrate the in vivo time-to-peak expression of the reporter gene, firefly luciferase. We infused a formulated lipoplex containing the lipid MLRI [dissymmetric myristoyl (14:0) and lauroyl (12:1) rosenthal inhibitor-substituted compound formed from the tetraalkylammonium glycerol-based DORI] and pNDluc, a luciferase vector, into CSF in the cisterna magna (CM) of the rat. Luciferase activity was followed over time by bioluminescence imaging after injection of luciferin. Our results show that luciferase activity in the CNS of rats is widespread, peaks 72 hours after injection into CM and can be detected in vivo for at least 7-10 days after peak expression. We further show that in contrast to injection into CSF, enzyme activity is not widely distributed after injection of the vector into brain parenchyma, emphasizing the importance of CSF delivery to achieve widespread vector distribution. Finally, we confirm the distribution of firefly luciferase in brain by immunohistochemical staining from an animal that was euthanized at the peak of enzyme expression.
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Sistema Nervioso Central/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular , Cisterna Magna , Portadores de Fármacos , Técnicas de Transferencia de Gen , Genes Reporteros , Lípidos , Luciferasas de Luciérnaga/biosíntesis , Luciferasas de Luciérnaga/líquido cefalorraquídeo , Luciferasas de Luciérnaga/genética , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Attachment of targeted ligands to nonviral DNA or RNA delivery systems is a promising strategy that seeks to overcome the poor target selectivity generally observed in systemic delivery applications. Several methods have been developed for the conjugation of ligands to lipids or polymers, however, direct conjugation of ligands onto lipid- or polymer-nucleic acid complexes is not as straightforward. Here, we examine an oximation approach to directly label a lipoplex formulation. Specifically, we report the synthesis of a cationic diketo lipid DMDK, and its use as a convenient ligation tool for attachment of aminooxy-functionalized reagents after its complexation with DNA. We demonstrate the feasibility of direct lipoplex labeling by attaching an aminooxy-functionalized fluorescent probe onto pre-formed plasmid DNA-DMDK lipoplexes (luciferase, GFP). The results reveal that DMDK protects DNA from degradation on exposure to either DNase or human cerebral spinal fluid, and that simple mixing of DMDK lipoplexes with the aminooxy probe labels the complexes without sacrificing transfection efficiency. The biocompatibility and selectivity of this method, as well as the ease of bioconjugation, make this labeling approach ideal for biological applications.
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ADN/química , Portadores de Fármacos/síntesis química , Ácidos Grasos Insaturados/síntesis química , Lípidos/química , Oximas/química , Animales , Células CHO , Líquido Cefalorraquídeo/química , Líquido Cefalorraquídeo/enzimología , Cricetinae , Cricetulus , Desoxirribonucleasas/química , Portadores de Fármacos/química , Portadores de Fármacos/farmacología , Ácidos Grasos Insaturados/química , Colorantes Fluorescentes/química , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/química , Proteínas Fluorescentes Verdes/genética , Humanos , Ligandos , Luciferasas/química , Estructura Molecular , Oximas/síntesis química , Oximas/farmacología , Estereoisomerismo , Transfección/métodosRESUMEN
An understanding of the time course and correlation with injury of heat shock proteins (HSPs) released during brain and/or spinal cord cellular stress (ischemia) is critical in understanding the role of the HSPs in cellular survival, and may provide a clinically useful biomarker of severe cellular stress. We have analyzed the levels of HSPs in the cerebrospinal fluid (CSF) from patients who are undergoing thoracic aneurysm repair. Blood and CSF samples were collected at regular intervals, and CSF was analyzed by enzyme-linked immunosorbent assay for HSP70 and HSP27. These results were correlated with intraoperative somatosensory-evoked potentials measurements and postoperative paralysis. We find that the levels of these proteins in many patients are elevated and that the degree of elevation correlates with the risk of permanent paralysis. We hypothesize that sequential measurement intraoperatively of the levels of the heat shock proteins HSP70 and HSP27 in the CSF can predict those patients who are at greatest risk for paralysis during thoracic aneurysm surgery and will allow us to develop means of preventing or attenuating this severe and often fatal complication.
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Aneurisma de la Aorta Torácica/líquido cefalorraquídeo , Aneurisma de la Aorta Torácica/cirugía , Proteínas de Choque Térmico HSP27/líquido cefalorraquídeo , Proteínas HSP70 de Choque Térmico/líquido cefalorraquídeo , Parálisis/etiología , Complicaciones Posoperatorias/etiología , Cicatrización de Heridas , Anciano , Aneurisma de la Aorta Torácica/complicaciones , Procedimientos Quirúrgicos Cardiovasculares/efectos adversos , Intervalos de Confianza , Demografía , Femenino , Proteínas de Choque Térmico , Humanos , Masculino , Persona de Mediana Edad , Chaperonas Moleculares , Análisis Multivariante , Parálisis/líquido cefalorraquídeo , Parálisis/complicaciones , Complicaciones Posoperatorias/líquido cefalorraquídeo , Análisis de RegresiónRESUMEN
Inhaled anesthetics produce many effects and bind to a large number of brain proteins, but it is not yet clear if this is accompanied by widespread changes in gene expression of the biological targets. Such changes in expression might implicate functionally important targets from the large pool of binding targets. Both rats and isolated primary cortical neurons were exposed to anesthetics and DNA oligonucleotide microarrays were used to detect and quantify transcriptional changes in neural tissue. Using analysis of variance with multiple testing correction, multiple exposures of rats to 0.8 MAC (minimum alveolar concentration) halothane only produced significant changes in a few metabolic genes. No significant in-vivo gene transcriptional response to 0.8 MAC isoflurane was detected. The use of primary cortical neurons allowed exposure to 3 MAC anesthetics without evidence of toxicity. Isoflurane altered several genes involved with neurotransmitter transport, signaling and cellular structure, whereas halothane produced few detectable changes in these cultured cells. Selected genes were confirmed by quantitative reverse transcription-polymerase chain reaction. Although indicating only a small degree of transcriptional regulation, these data implicate several plausible targets, including synaptic vesicle handling, that might contribute to drug action. In addition, the data show different gene expression profiles for the two inhaled anesthetics, suggesting unique pharmacological targets and mechanisms in each case.
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Anestésicos por Inhalación/farmacología , Encéfalo/metabolismo , ADN/metabolismo , Perfilación de la Expresión Génica/métodos , Halotano/farmacología , Isoflurano/farmacología , Farmacogenética/métodos , Animales , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Oligonucleótidos/química , Ratas , Ratas Sprague-DawleyRESUMEN
Heat shock protein (Hsp)70 can suppress both necrosis and apoptosis induced by various injuries in vivo and in vitro. However, the relative importance of different functions and binding partners of Hsp70 in ischemic protection is unknown. To explore this question, we tested the ability of Hsp70-K71E, an adenosine triphosphate (ATP)ase-deficient point mutant, and Hsp70-381-640, a deletion mutant lacking the ATPase domain and encoding the carboxyl-terminal portion, to protect against ischemia-like injury in vivo and in vitro. Heat shock protein 70-wild type (-WT), -K71E, -381-640, and control vector plasmid LXSN were expressed in primary murine astrocyte cultures. Astrocytes overexpressing Hsp70-WT, -K71E, or -381-640 were all significantly protected from 4 h combined oxygen-glucose deprivation and 24 h reperfusion when assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay or propidium iodide staining and cell counting (P < 0.05). Brains of rats were transfected with plasmids encoding Hsp70-WT, -K71E, -381-640, or LXSN 24 h before 2 h middle cerebral artery occlusion followed by 24 h reperfusion. Animals that overexpressed either of the mutant proteins or Hsp70-WT had significantly better neurological scores and smaller infarcts than control animals. Protection by both mutants was associated with reduced protein aggregation, as assessed by ubiquitin immunohistochemistry and reduced nuclear translocation of apoptosis-inducing factor. The results show that the carboxyl-terminal portion of Hsp70 is sufficient for neuroprotection. This indicates that neither the ability to fold denatured proteins nor interactions with cochaperones or other proteins that bind the amino-terminal half of Hsp70 are essential to ischemic protection.
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Isquemia Encefálica/prevención & control , Terapia Genética/métodos , Proteínas HSP70 de Choque Térmico/uso terapéutico , Animales , Animales Recién Nacidos , Apoptosis/efectos de los fármacos , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Isquemia Encefálica/fisiopatología , Muerte Celular/efectos de los fármacos , Células Cultivadas , Regulación de la Expresión Génica , Glucosa/farmacología , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Ratones , Neuronas/metabolismo , Oxígeno/farmacología , Mutación Puntual , Estructura Terciaria de Proteína/genética , Ubiquitina/antagonistas & inhibidores , Ubiquitina/metabolismoRESUMEN
Fluid percussion brain injury (FPI) impairs pial artery dilation to activators of the ATP-sensitive (K(ATP)) and calcium-activated (K(Ca)) K(+) channels. This study investigated the role of heat shock protein (HSP) in the modulation of K(+) channel-induced pial artery dilation after FPI in newborn pigs equipped with a closed cranial window. Under nonbrain injury conditions, topical coadministration of exogenous HSP-27 (1 mug/ml) blunted dilation to cromakalim, CGRP, and NS-1619 (10(-8) and 10(-6) M; cromakalim and CGRP are K(ATP) agonists and NS-1619 is a K(Ca) agonist). In contrast, coadministration of exogenous HSP-70 (1 mug/ml) potentiated dilation to cromakalim, CGRP, and NS-1619. FPI increased the cerebrospinal fluid (CSF) concentration of HSP-27 from 0.051 +/- 0.012 to 0.113 +/- 0.035 ng/ml but decreased the CSF concentration of HSP-70 from 50.42 +/- 8.96 to 30.9 +/- 9.9 ng/ml at 1 h postinsult. Pretreatment with topical exogenous HSP-70 (1 mug/ml) before FPI fully blocked injury-induced impairment of cromakalim and CGRP dilation and partially blocked injury-induced impairment of dilation to NS-1619. These data indicate that HSP-27 and HSP-70 contribute to modulation of K(+) channel-induced pial artery dilation. These data suggest that HSP-70 is an endogenous protectant of which its actions may be unmasked and/or potentiated with exogenous administration before brain injury.
Asunto(s)
Lesiones Encefálicas/fisiopatología , Canales de Calcio/fisiología , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas de Choque Térmico/fisiología , Canales de Potasio/fisiología , Vasodilatación/fisiología , Animales , Animales Recién Nacidos , Bencimidazoles/farmacología , Presión Sanguínea , Lesiones Encefálicas/tratamiento farmacológico , Péptido Relacionado con Gen de Calcitonina/farmacología , Circulación Cerebrovascular/fisiología , Cromakalim/farmacología , Femenino , Proteínas HSP70 de Choque Térmico/farmacología , Proteínas de Choque Térmico/farmacología , Masculino , Piamadre/irrigación sanguínea , Porcinos , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaRESUMEN
Clinical applications of gene therapy require advances in gene delivery systems. Although numerous clinical trials are already underway, the ultimate success of gene therapies will depend on gene transfer vectors that facilitate the expression of a specific gene at therapeutic levels in the desired cell populations without eliciting cytotoxicity. In clinical applications for which transient expression is desirable, mRNA delivery is of particular interest. We have shown cationic lipid-mediated mRNA delivery to be feasible, efficient, and reproducible in vitro. mRNA delivery to the cerebrospinal fluid (CSF) in vivo would provide a means of vector distribution throughout the central nervous system (CNS). This study examined the functional integrity and protection from degradation of mRNA/cationic complexes (lipoplexes) in human cerebrospinal fluid (hCSF) in vitro and expression of these lipoplexes in vivo. Results obtained from gel electrophoresis indicate that cationic lipids protect mRNA transcripts from RNases in hCSF for at least 4 hr. This is in contrast to the total disappearance of nonlipid-complexed mRNA in less than 5 min. We confirmed the importance of RNase activity by incubating mRNA transcripts encoding luciferase or green fluorescent protein (GFP) in hCSF to which RNase inhibitors had been added. After incubation, these solutions were used to transfect Chinese hamster ovary (CHO) cells in vitro. Next, assays for both GFP and luciferase were used to demonstrate functional integrity and translation of the mRNA transcripts. Finally, we delivered in vitro transcribed mRNA vectors encoding for Hsp70 and luciferase to the lateral ventricle of the rat in a series of preliminary in vivo experiments. Initial immunohistochemistry analysis demonstrates that the distribution, uptake, and expression of reporter sequences using lipid-mediated mRNA vector delivery is extensive, as we earlier reported using similar methods with DNA vectors but that the expression may be less intense. Expression was noted in coronal sections throughout the rat brain, confirming the potential for lipid-mediated mRNA delivery to the CNS. These findings confirm that complexing mRNA with cationic lipid before exposure to CSF confers protection against RNase activity, facilitating distribution, cellular uptake, and expression of mRNA delivered into the CNS.
Asunto(s)
Sistema Nervioso Central/metabolismo , Técnicas de Transferencia de Gen , Metabolismo de los Lípidos , ARN Mensajero/metabolismo , Animales , Células CHO , Cricetinae , Genes Reporteros , Humanos , Lípidos/líquido cefalorraquídeo , ARN Mensajero/líquido cefalorraquídeo , ARN Mensajero/aislamiento & purificación , Ratas , Transfección/métodosRESUMEN
Low levels of transfection efficacy and lipid-associated cytotoxicity have complicated the use of cationic lipids to facilitate transfer of exogenous DNA to eukaryotic cells. To address these issues, we synthesized a panel of six tetraester polyamines that were designed to minimize cytotoxicity by using pentaerythritol to link the hydrophobic and the DNA-binding domains. We conducted this study to probe the effects of structural modifications around pentaerythritol as a linker on transfection activity and cell viability. We compared polyamines against commercial lipid reagents using luciferase and green fluorescent protein transfection assays in both CHO and NIH3T3 cells. Measurement of transfection activity and cytotoxicity using flow cytometry showed that the more active polyamine analogs exhibited activities comparable to LipofectAMINE PLUS and TransFast. Flow cytometry analyses revealed that all the pentaerythritol-based polyamines were uniformly nontoxic, whereas transfection activity was dependent on headgroup and sidechain composition. These results demonstrate that pentaerythritol is a useful core material for the development of active, nontoxic transfection agents.