RESUMEN
OBJECTIVEHigh invasiveness of malignant gliomas frequently causes early local recurrence of the tumor, resulting in extremely poor outcome. To control such recurrence, novel therapies targeted toward infiltrating glioma cells around the tumor border are required. Here, the authors investigated the antitumor activity of sonodynamic therapy (SDT) combined with a sonosensitizer, 5-aminolevulinic acid (5-ALA), on malignant gliomas to explore the possibility for clinical use of 5-ALA-mediated SDT (5-ALA-SDT).METHODSIn vitro cytotoxicity of 5-ALA-SDT was evaluated in U87 and U251 glioma cells and in U251Oct-3/4 glioma stemlike cells. Treatment-related apoptosis was analyzed using flow cytometry and TUNEL staining. Intracellular reactive oxygen species (ROS) were measured and the role of ROS in treatment-related cytotoxicity was examined by analysis of the effect of pretreatment with the radical scavenger edaravone. Effects of 5-ALA-SDT with high-intensity focused ultrasound (HIFU) on tumor growth, survival of glioma-transplanted mice, and histological features of the mouse brains were investigated.RESULTSThe 5-ALA-SDT inhibited cell growth and changed cell morphology, inducing cell shrinkage, vacuolization, and swelling. Flow cytometric analysis and TUNEL staining indicated that 5-ALA-SDT induced apoptotic cell death in all gliomas. The 5-ALA-SDT generated significantly higher ROS than in the control group, and inhibition of ROS generation by edaravone completely eliminated the cytotoxic effects of 5-ALA-SDT. In the in vivo study, 5-ALA-SDT with HIFU greatly prolonged survival of the tumor-bearing mice compared with that of the control group (p < 0.05). Histologically, 5-ALA-SDT produced mainly necrosis of the tumor tissue in the focus area and induced apoptosis of the tumor cells in the perifocus area around the target of the HIFU-irradiated field. The proliferative activity of the entire tumor was markedly decreased. Normal brain tissues around the ultrasonic irradiation field of HIFU remained intact.CONCLUSIONSThe 5-ALA-SDT was cytotoxic toward malignant gliomas. Generation of ROS by the SDT was thought to promote apoptosis of glioma cells. The 5-ALA-SDT with HIFU induced tumor necrosis in the focus area and apoptosis in the perifocus area of the HIFU-irradiated field, whereas the surrounding brain tissue remained normal, resulting in longer survival of the HIFU-treated mice compared with that of untreated mice. These results suggest that 5-ALA-SDT with HIFU may present a less invasive and tumor-specific therapy, not only for a tumor mass but also for infiltrating tumor cells in malignant gliomas.
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Ácido Aminolevulínico/uso terapéutico , Apoptosis/efectos de los fármacos , Neoplasias Encefálicas/terapia , Tratamiento con Ondas de Choque Extracorpóreas/métodos , Glioma/terapia , Animales , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Glioma/tratamiento farmacológico , Glioma/patología , Ratones , Resultado del TratamientoRESUMEN
OBJECTIVE Drug clearance may be a limiting factor in the clinical application of convection-enhanced delivery (CED). Peptide-based nanofibers (NFPs) have a high aspect ratio, and NFPs loaded with drugs could potentially maintain effective drug concentrations for an extended period sufficient for cancer therapy. The objective of this study was to assess the volume of distribution (Vd) and clearance of variable lengths of NFPs when administered using CED. METHODS NFPs composed of multiple methoxypolyethylene glycol (mPEG)-conjugated constructs (mPEG2000-KLDLKLDLKLDL-K( FITC)-CONH2, for which FITC is fluorescein isothiocyanate) were assembled in an aqueous buffer. The NFPs were approximately 5 nm in width and were formulated into different lengths: 100 nm (NFP-100), 400 nm (NFP-400), and 1000 nm (NFP-1000). The NFP surface was covalently conjugated with multiple Cy5.5 fluorophores as the optical reporters to track the post-CED distribution. Forty-two 6- to 8-week-old Ntv-a;p53fl/fl mice underwent CED to the striatum. Animals were killed immediately, 24 hours or 72 hours after CED. The brains were extracted and sectioned for assessing NFP Vd to volume of infusion (Vi) ratio, and clearance using fluorescence microscopy. RESULTS CED of NFPs was well tolerated by all the animals. The average Vd/Vi ratios for NFP-100, NFP-400, NFP-1000, and unconjugated positive control (free Cy5.5) were 1.87, 2.47, 1.07, and 3.0, respectively, which were statistically different (p = 0.003). The percentages remaining of the original infusion volume at 24 hours for NFP-100, -400, and -1000 were 40%, 90%, and 74%, respectively. The percentages remaining at 72 hours for NFP-100, -400, and -1000 were 15%, 30%, and 46%, respectively. Unconjugated Cy5.5 was not detected at 24 or 72 hours after CED. CONCLUSIONS CED of NFPs is feasible with Vd/Vi ratios and clearance rates comparable to other nanocarriers. Of the 3 NFPs, NFP-400 appears to provide the best distribution and slowest clearance after 24 hours. NFP provides a dynamic theranostic platform, with the potential to deliver clinically efficacious drug payload to brain tumor after CED.
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Encéfalo/metabolismo , Nanofibras , Péptidos/metabolismo , Péptidos/farmacocinética , Animales , Portadores de Fármacos , Ratones , Distribución TisularRESUMEN
OBJECTIVE Over the last decade, a number of authors have investigated the utility of different biological and synthetic matrices as alternatives to conventional nerve grafts. However, the autologous nerve graft remains the gold standard, even though it often involves using a pure sensory nerve to reconstruct a mixed or even a pure motor nerve. Furthermore, limited donor sites often necessitate a significant mismatch of needed nerve tissue, especially for large proximal nerve defects such as brachial plexus lesions. Here, the authors present a new technique that overcomes these problems: the fascicular shift procedure (FSP). A fascicular group of the nerve distal to the injury is harvested in a sufficient length to bridge the nerve defect. METHODS The method of fascicular shifting was tested at the sciatic nerve in 45 Lewis rats. In the experimental group, a 15-mm nerve defect was created and reconstructed with a fascicular group that was harvested directly distal to the gap. This group was compared with 1 negative control group (defect without reconstruction) and 3 positive control groups (sensory, motor, and mixed graft). After 12 weeks of nerve regeneration, outcome was evaluated using retrograde labeling, histomorphometric analysis, and muscle force analysis. RESULTS All reconstructed groups showed successful regeneration with various levels of function. The negative control group showed minimal force measurements that were of no functional value. The fascicular shift provided sufficient guidance to overcome nerve defects, had higher (p < 0.1) motor neuron counts (1958.75 ± 657.21) than the sensory graft (1263.50 ± 538.90), and was equal to motor grafts (1490.43 ± 794.80) and mixed grafts (1720.00 ± 866.421). This tendency of improved motor regeneration was confirmed in all analyses. The mixed graft group was compared with the experimental group to investigate the influence of the potential damage induced by the fascicular shift distal to the repair site. However, none of the analyses revealed an impairment of nerve regeneration for both the tibial and common peroneal index muscles. CONCLUSIONS This study demonstrates that harvesting a transplant from the nerve segment distal to the injury site offers a mixed graft without causing additional donor-site morbidity. These grafts perform statistically better than a standard sensory graft in terms of motor recovery. The fascicular shift presents a novel method to reconstruct large proximal nerve defects, making it immensely attractive in brachial plexus reconstruction.
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Neuronas Motoras/fisiología , Músculo Esquelético/fisiopatología , Regeneración Nerviosa/fisiología , Enfermedades del Sistema Nervioso Periférico/cirugía , Nervio Ciático/cirugía , Animales , Modelos Animales de Enfermedad , Masculino , Procedimientos Neuroquirúrgicos/métodos , Ratas , Trasplante Autólogo/métodosRESUMEN
OBJECTIVE Diffuse astrocytomas (DAs) have a high recurrence rate due to diffuse infiltration into the brain and spinal cord. Micro RNAs (miRNAs) are small noncoding RNAs that regulate gene expression by binding to complementary sequences of target messenger RNA (mRNA). It has been reported that miRNA-22 (miR-22) is involved in the invasion of some cancer cell lines. The aim of this study was to identify the biological effects of miR-22 in regard to the invasion of human DAs. METHODS The authors evaluated whether the level of miR-22 is elevated in human spinal DAs by using miRNA chips. Next, the role of miR-22 in 1321N1 human astrocytoma cells was investigated. Finally, to elucidate whether miR-22 promotes invasion by astrocytoma cells in vivo, the authors transplanted miR-22 overexpressed astrocytoma cells into mouse thoracic spinal cord. RESULTS The miR-22 significantly upregulated the invasion capacity of 1321N1 cells. Computational in silico analysis predicted that tissue inhibitor of matrix metalloproteinase-2 (TIMP2) is a target gene of miR-22. This was confirmed by quantitative reverse transcription polymerase chain reaction and Western blotting, which showed that miR-22 inhibited TIMP2 mRNA and protein expression, respectively. Luciferase reporter assays demonstrated that miR-22 directly bound the 3'-untranslated regions of TIMP2. The authors further showed that miR-22 promoted invasiveness in 1321N1 astrocytoma cells when transplanted into mouse spinal cord. CONCLUSIONS These data suggest that miR-22 acts to regulate invasion of 1321N1 astrocytoma cells by targeting TIMP2 expression. Additional studies with more cases and cell lines are required to elucidate the findings of this study for a novel treatment target for spinal DAs.
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Astrocitoma/metabolismo , Movimiento Celular/fisiología , MicroARNs/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Proliferación Celular/fisiología , Femenino , Humanos , Masculino , Metaloproteinasa 2 de la Matriz/metabolismo , Persona de Mediana Edad , Invasividad Neoplásica/patologíaRESUMEN
OBJECTIVE The aim of this study was to evaluate whether combined gene therapy with vascular endothelial growth factor (VEGF) plus apelin during indirect vasoreconstructive surgery enhances brain angiogenesis in a chronic cerebral hypoperfusion model in rats. METHODS A chronic cerebral hypoperfusion model induced by the permanent ligation of bilateral common carotid arteries (CCAs; a procedure herein referred to as "CCA occlusion" [CCAO]) in rats was employed in this study. Seven days after the CCAO procedure, the authors performed encephalo-myo-synangiosis (EMS) and injected plasmid(s) into each rat's temporal muscle. Rats were divided into 4 groups based on which plasmid was received (i.e., LacZ group, VEGF group, apelin group, and VEGF+apelin group). Protein levels in the cortex and attached muscle were assessed with enzyme-linked immunosorbent assay (ELISA) on Day 7 after EMS, while immunofluorescent analysis of cortical vessels was performed on Day 14 after EMS. RESULTS The total number of blood vessels in the cortex on Day 14 after EMS was significantly larger in the VEGF group and the VEGF+apelin group than in the LacZ group (p < 0.05, respectively). Larger vessels appeared in the VEGF+apelin group than in the other groups (p < 0.05, respectively). Apelin protein on Day 7 after EMS was not detected in the cortex for any of the groups. In the attached muscle, apelin protein was detected only in the apelin group and the VEGF+apelin group. Immunofluorescent analysis revealed that apelin and its receptor, APJ, were expressed on endothelial cells (ECs) 7 days after the CCAO. CONCLUSIONS Combined gene therapy (VEGF plus apelin) during EMS in a chronic cerebral hypoperfusion model can enhance angiogenesis in rats. This treatment has the potential to be a feasible option in a clinical setting for patients with moyamoya disease.
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Apelina/uso terapéutico , Isquemia Encefálica/terapia , Terapia Genética , Enfermedad de Moyamoya/terapia , Factor A de Crecimiento Endotelial Vascular/uso terapéutico , Animales , Enfermedad Crónica , Terapia Combinada , Modelos Animales de Enfermedad , Masculino , Ratas , Ratas WistarRESUMEN
OBJECTIVE Shunt obstruction by cells and/or tissue is the most common cause of shunt failure. Ventricular catheter obstruction alone accounts for more than 50% of shunt failures in pediatric patients. The authors sought to systematically collect explanted ventricular catheters from the Seattle Children's Hospital with a focus on elucidating the cellular mechanisms underlying obstruction. METHODS In the operating room, explanted hardware was placed in 4% paraformaldehyde. Weekly, samples were transferred to buffer solution and stored at 4°C. After consent was obtained for their use, catheters were labeled using cell-specific markers for astrocytes (glial fibrillary acidic protein), microglia (ionized calcium-binding adapter molecule 1), and choroid plexus (transthyretin) in conjunction with a nuclear stain (Hoechst). Catheters were mounted in custom polycarbonate imaging chambers. Three-dimensional, multispectral, spinning-disk confocal microscopy was used to image catheter cerebrospinal fluid-intake holes (10× objective, 499.2-µm-thick z-stack, 2.4-µm step size, Olympus IX81 inverted microscope with motorized stage and charge-coupled device camera). Values are reported as the mean ± standard error of the mean and were compared using a 2-tailed Mann-Whitney U-test. Significance was defined at p < 0.05. RESULTS Thirty-six ventricular catheters have been imaged to date, resulting in the following observations: 1) Astrocytes and microglia are the dominant cell types bound directly to catheter surfaces; 2) cellular binding to catheters is ubiquitous even if no grossly visible tissue is apparent; and 3) immunohistochemical techniques are of limited utility when a catheter has been exposed to Bugbee wire electrocautery. Statistical analysis of 24 catheters was performed, after excluding 7 catheters exposed to Bugbee wire cautery, 3 that were poorly fixed, and 2 that demonstrated pronounced autofluorescence. This analysis revealed that catheters with a microglia-dominant cellular response tended to be implanted for shorter durations (24.7 ± 6.7 days) than those with an astrocyte-dominant response (1183 ± 642 days; p = 0.027). CONCLUSIONS Ventricular catheter occlusion remains a significant source of shunt morbidity in the pediatric population, and given their ability to intimately associate with catheter surfaces, astrocytes and microglia appear to be critical to this pathophysiology. Microglia tend to be the dominant cell type on catheters implanted for less than 2 months, while astrocytes tend to be the most prevalent cell type on catheters implanted for longer time courses and are noted to serve as an interface for the secondary attachment of ependymal cells and choroid plexus.
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Astrocitos , Derivaciones del Líquido Cefalorraquídeo/efectos adversos , Falla de Equipo , Hidrocefalia/cirugía , Macrófagos , Complicaciones Posoperatorias/etiología , Niño , Preescolar , Comprensión , Diseño de Equipo/tendencias , Femenino , Humanos , Hidrocefalia/diagnóstico , Lactante , Masculino , Complicaciones Posoperatorias/diagnóstico , Estudios RetrospectivosRESUMEN
Diagnosis and management of peripheral nerve injury is complicated by the inability to assess microstructural features of injured nerve fibers via clinical examination and electrophysiology. Diffusion tensor imaging (DTI) has been shown to accurately detect nerve injury and regeneration in crush models of peripheral nerve injury, but no prior studies have been conducted on nerve transection, a surgical emergency that can lead to permanent weakness or paralysis. Acute sciatic nerve injuries were performed microsurgically to produce multiple grades of nerve transection in rats that were harvested 1 hour after surgery. High-resolution diffusion tensor images from ex vivo sciatic nerves were obtained using diffusion-weighted spin-echo acquisitions at 4.7 T. Fractional anisotropy was significantly reduced at the injury sites of transected rats compared with sham rats. Additionally, minor eigenvalues and radial diffusivity were profoundly elevated at all injury sites and were negatively correlated to the degree of injury. Diffusion tensor tractography showed discontinuities at all injury sites and significantly reduced continuous tract counts. These findings demonstrate that high-resolution DTI is a promising tool for acute diagnosis and grading of traumatic peripheral nerve injuries.
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Imagen de Difusión Tensora , Traumatismos de los Nervios Periféricos/diagnóstico , Enfermedad Aguda , Animales , Anisotropía , Modelos Animales de Enfermedad , Femenino , Humanos , Extremidad Inferior/patología , Masculino , Curva ROC , Ratas Sprague-Dawley , Neuropatía Ciática/diagnóstico , Sensibilidad y Especificidad , Estadística como AsuntoRESUMEN
OBJECT: Cell therapy is a promising candidate among biological or technological innovations sought to augment microsurgical techniques in peripheral nerve repair. This report describes long-term functional regenerative effects of cell therapy in the rat injury model with a focus on sensory recovery. METHODS: Schwann cells were derived from isogenic nerve or skin precursor cells and injected into the transected and immediately repaired sciatic nerve distal to the injury site. Sensory recovery was assessed at weeks 4, 7, and 10. Axonal regeneration was assessed at Week 11. RESULTS: By Week 10, thermal sensitivity in cell therapy groups returned to a level indistinguishable from the baseline (p > 0.05). Immunohistochemistry at 11 weeks after injury showed improved regeneration of NF+ and IB4+ axons. CONCLUSIONS: The results of this study show that cell therapy significantly improves thermal sensation and the number of regenerated sensory neurons at 11 weeks after injury. These findings contribute to the view of skin-derived stem cells as a reliable source of Schwann cells with therapeutic potential for functional recovery in damaged peripheral nerve.