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1.
J Cancer Res Ther ; 16(5): 1125-1128, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33004758

RESUMO

Objective: The objective was to evaluate the feasibility and safety of computed tomography (CT)-guided percutaneous irreversible electroporation (IRE) in porcine kidneys. Materials and Methods: Under CT guidance, two monopole probes were used to precisely puncture through the renal parenchyma into the renal hilum in nine anesthetized adult Bama miniature pigs. After which, IRE ablation was performed. Biochemical and pathological examinations were carried out 2 h, 2, 7, and 14 days after the procedure. Results: All procedures were performed successfully without any serious complications such as bleeding, infection, or death. All pigs survived until the end of the study. Pathological examinations showed that cells in the ablation area were dead within 2 days after the procedure, whereas the vascular endothelium showed only slight damage. After 2 days, endothelialization ensued and regrowth of smooth muscle cells was observed after 14 days. Hemogram tests indicated a transient increase but gradually returned to baseline levels 14 days after the procedure. Conclusion: IRE was essentially safe, however further studies on tumor ablation using several different animal models are needed.


Assuntos
Eletroporação/normas , Rim/cirurgia , Cirurgia Assistida por Computador/métodos , Tomografia Computadorizada por Raios X/métodos , Técnicas de Ablação/métodos , Animais , Creatina Quinase Forma MB/sangue , Eletroporação/métodos , Estudos de Viabilidade , Hidroxibutirato Desidrogenase/sangue , Rim/metabolismo , Rim/patologia , L-Lactato Desidrogenase/sangue , Leucócitos/patologia , Modelos Animais , Suínos , Resultado do Tratamento
2.
J Vis Exp ; (164)2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33104060

RESUMO

Electroporation has established itself as a critical method for transferring specific genes into cells to understand their function. Here, we describe a single-cell electroporation technique that maximizes the efficiency (~80%) of in vitro gene transfection in excitatory and class-specific inhibitory neurons in mouse organotypic hippocampal slice culture. Using large glass electrodes, tetrodotoxin-containing artificial cerebrospinal fluid and mild electrical pulses, we delivered a gene of interest into cultured hippocampal CA1 pyramidal neurons and inhibitory interneurons. Moreover, electroporation could be carried out in cultured hippocampal slices up to 21 days in vitro with no reduction in transfection efficiency, allowing for the study of varying slice culture developmental stages. With interest growing in examining the molecular functions of genes across a diverse range of cell types, our method demonstrates a reliable and straightforward approach to in vitro gene transfection in mouse brain tissue that can be performed with existing electrophysiology equipment and techniques.


Assuntos
Eletroporação/métodos , Hipocampo/citologia , Inibição Neural/fisiologia , Neurônios/fisiologia , Análise de Célula Única , Técnicas de Cultura de Tecidos , Animais , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Células Piramidais/fisiologia , Fixação de Tecidos , Transfecção
3.
Sci Rep ; 10(1): 15145, 2020 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-32934254

RESUMO

Two species of parasitic fungi from the phylum Chytridiomycota (chytrids) are annihilating global amphibian populations. These chytrid species-Batrachochytrium dendrobatidis and B. salamandrivorans-have high rates of mortality and transmission. Upon establishing infection in amphibians, chytrids rapidly multiply within the skin and disrupt their hosts' vital homeostasis mechanisms. Current disease models suggest that chytrid fungi locate and infect their hosts during a motile, unicellular 'zoospore' life stage. Moreover, other chytrid species parasitize organisms from across the tree of life, making future epidemics in new hosts a likely possibility. Efforts to mitigate the damage and spread of chytrid disease have been stymied by the lack of knowledge about basic chytrid biology and tools with which to test molecular hypotheses about disease mechanisms. To overcome this bottleneck, we have developed high-efficiency delivery of molecular payloads into chytrid zoospores using electroporation. Our electroporation protocols result in payload delivery to between 75 and 97% of living cells of three species: B. dendrobatidis, B. salamandrivorans, and a non-pathogenic relative, Spizellomyces punctatus. This method lays the foundation for molecular genetic tools needed to establish ecological mitigation strategies and answer broader questions in evolutionary and cell biology.


Assuntos
Anfíbios/crescimento & desenvolvimento , Doenças dos Animais/epidemiologia , Quitridiomicetos/patogenicidade , Eletroporação/métodos , Micoses/veterinária , Esporos Fúngicos/isolamento & purificação , Anfíbios/microbiologia , Animais , Interações Hospedeiro-Patógeno , Micoses/microbiologia , Esporos Fúngicos/fisiologia
4.
Zhonghua Wai Ke Za Zhi ; 58(10): 787-792, 2020 Oct 01.
Artigo em Chinês | MEDLINE | ID: mdl-32993267

RESUMO

Objective: To examine the safety and clinical efficacy of ultrasound-guided irreversible electroporation (IRE) using the open surgery approach, after induction chemotherapy, in the treatment of locally advanced pancreatic cancer (LAPC) . Methods: The data of 64 LAPC patients who underwent ultrasound-guided IRE using the open surgery approach after induction chemotherapy at Department of Pancreatobiliary Surgery, Sun Yat-sen University Cancer Center from August 2015 to March 2019 were retrospectively analyzed. The study comprised of 30 males and 34 females, with median age of 58.5 years old (range: 34 to 87 years old) , were included in this study.The tumor was located in the pancreatic head and body/tail in 30 and 34 patients, respectively.The largest recorded tumor size was 6.1 cm (≤4.0 cm: n=35; >4.0 cm: n=29) .To create an electric field around the tumor, Two to six probes were parallelly inserted into each patient's tumor, based on the size of the tumor, at a distance of 2 cm apart through the transverse mesocolon in a caudal-to-cranial direction.According to the numerical sequence of patients undergoing ultrasound-guided IRE, the first 15 cases and following 49 patients were categorized as the primary and secondary treatment group, respectively.T text or χ(2) test was analyzed to the data between two groups.The study endpoints were overall survival (OS) and progression free survival (PFS) , which were investigated using Kaplan-Meier method, and their differences were compared using log-rank test. Results: The overall length of hospital stay was (8.9±2.7) days (range: 5 to 20 days) . Four patients were lost to follow-up.The study follow-up rate was 93.8%, with a median follow-up time of 29.3 months (range: 13.5 to 55.7 months) .The median OS and PFS of the entire cohort was 24.6 months (95% CI: 22.0 to 27.3 months) and 12.0 months (95%CI: 8.8 to 15.2 months) , respectively.One month after IRE, abdominal pain was significantly relieved in 95.3% of the patients (t=-28.55, P<0.01) .The rate of complications in the entire cohort was 20.3% and all were classified as grade B.Of them, pancreatic fistula, incisional infection, and upper gastrointestinal hemorrhage were observed in 7, 4, and 2 cases, respectively.The rate of complications for patients in the primary and secondary treatment groups were significantly different (10/15 vs. 6.1%) , respectively (χ(2)=26.01, P<0.01) .Further, two deaths were observed after IRE in the primary treatment group, while none was observed in the secondary treatment group. Conclusions: Ultrasound-guided IRE using the open surgery approach after induction chemotherapy is found to be safe and effective in treating patients with LAPC.However, these findings should be validated in prospective randomized trials before wide clinical application.


Assuntos
Antineoplásicos/administração & dosagem , Eletroporação , Neoplasias Pancreáticas , Ultrassonografia de Intervenção , Adulto , Idoso , Idoso de 80 Anos ou mais , Antineoplásicos/uso terapêutico , Terapia Combinada , Eletroporação/métodos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Estudos Retrospectivos
5.
Sci Rep ; 10(1): 12806, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732955

RESUMO

Analyzing gene function in a broad range of research organisms is crucial for understanding the biological functions of genes and their evolution. Recent studies have shown that short hairpin RNAs (shRNAs) can induce gene-specific knockdowns in two cnidarian species. We have developed a detailed, straightforward, and scalable method to deliver shRNAs into fertilized eggs of the hydrozoan cnidarian Hydractinia symbiolongicarpus via electroporation, yielding effective gene-targeted knockdowns that can last throughout embryogenesis. Our electroporation protocol allows for the transfection of shRNAs into hundreds of fertilized H. symbiolongicarpus eggs simultaneously with minimal embryo death and no long-term harmful consequences on the developing animals. We show RT-qPCR and detailed phenotypic evidence of our method successfully inducing effective knockdowns of an exogenous gene (eGFP) and an endogenous gene (Nanos2), as well as knockdown confirmation by RT-qPCR of two other endogenous genes. We also provide visual confirmation of successful shRNA transfection inside embryos through electroporation. Our detailed protocol for electroporation of shRNAs in H. symbiolongicarpus embryos constitutes an important experimental resource for the hydrozoan community while also serving as a successful model for the development of similar methods for interrogating gene function in other marine invertebrates.


Assuntos
Cnidários/embriologia , Cnidários/genética , Eletroporação/métodos , Desenvolvimento Embrionário/genética , Técnicas de Silenciamento de Genes/métodos , RNA Interferente Pequeno/genética , Animais , Embrião não Mamífero , Proteínas de Fluorescência Verde/genética , Proteínas de Ligação a RNA/genética , Transfecção
6.
Br J Radiol ; 93(1115): 20200465, 2020 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-32783618

RESUMO

Management of musculoskeletal (MSK) tumours has traditionally been delivered by surgeons and medical oncologists. However, in recent years, image-guided interventional oncology (IO) has significantly impacted the clinical management of MSK tumours. With the rapid evolution of relevant technologies and the expanding range of clinical indications, it is likely that the impact of IO will significantly grow and further evolve in the near future.In this narrative review, we describe well-established and new interventional technologies that are currently integrating into the IO armamentarium available to radiologists to treat MSK tumours and illustrate new emerging IO indications for treatment.


Assuntos
Neoplasias Ósseas/terapia , Oncologia/tendências , Neoplasias Musculares/terapia , Antineoplásicos/administração & dosagem , Dor do Câncer/terapia , Terapia Combinada/métodos , Criocirurgia/métodos , Eletroporação/métodos , Potenciais Evocados , Feminino , Fluoroscopia/métodos , Previsões , Fraturas Ósseas/terapia , Ablação por Ultrassom Focalizado de Alta Intensidade , Humanos , Doença Iatrogênica/prevenção & controle , Lipossomos/administração & dosagem , Imagem por Ressonância Magnética Intervencionista/métodos , Masculino , Micro-Ondas/uso terapêutico , Traumatismos dos Nervos Periféricos/etiologia , Traumatismos dos Nervos Periféricos/prevenção & controle , Ablação por Radiofrequência/métodos , Terapia por Radiofrequência/métodos , Radiologia Intervencionista/métodos , Radiologia Intervencionista/tendências , Neoplasias da Coluna Vertebral/terapia , Coluna Vertebral/cirurgia , Tomografia Computadorizada por Raios X/métodos , Ultrassonografia de Intervenção/métodos
7.
Sci Rep ; 10(1): 13332, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32770110

RESUMO

Although electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells. Here, we developed a square-wave pulsing protocol using OptiMEM-GlutaMAX for highly efficient transfection of murine embryonic fibroblasts (MEF) and induced pluripotency stem (iPS) cells using reporter genes as well as gRNA/Cas9-encoding plasmids. An electrotransfection efficiency of > 95% was achieved for both MEF and iPS cells using reporter-encoding plasmids. The protocol was efficient for plasmid sizes ranging from 6.2 to 13.5 kb. Inducing the error prone non-homologous end joining repair by gRNA/Cas9 plasmid transfection, a high rate of targeted gene knockouts of up to 98% was produced in transgenic cells carrying a single-copy of Venus reporter. Targeted deletions in the Venus transgene were efficiently (up to 67% deletion rate) performed by co-electroporation of two gRNA-encoding plasmids. We introduced a plasmid electrotransfection protocol which is straight-forward, cost-effective, and efficient for CRISPRing murine primary cells. This protocol is promising to make targeted genetic engineering using the CRISPR/Cas9 plasmid system.


Assuntos
Eletroporação/métodos , Fibroblastos/fisiologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Transfecção/métodos , Animais , Animais Geneticamente Modificados/genética , Sistemas CRISPR-Cas/genética , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Reparo do DNA por Junção de Extremidades/genética , Edição de Genes/métodos , Técnicas de Inativação de Genes/métodos , Genes Reporter/genética , Camundongos , Plasmídeos/genética , RNA Guia/genética , Transgenes/genética
8.
Surgery ; 168(4): 610-616, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32631655

RESUMO

BACKGROUND: Combining immune checkpoint blockade therapy with operative disruptive immunomodulation using irreversible electroporation may overcome the resistance to systemic therapy found in patients with locally advanced, unresectable pancreatic cancer. We describe the safety profile and efficacy of IRE with nivolumab. METHODS: In the preclinical phase of study, human pancreatic cell lines were cultured with interferon-γ (10 ng/mL) and murine models of pancreatic cancer were treated with irreversible electroporation and programmed death ligand-1 (PD-L1) expression was measured. In this phase 1b clinical trial (NCT03080974), surgical ablative irreversible electroporation was performed followed by nivolumab. The primary end point was dose-limiting toxicity. RESULTS: Human pancreatic cells express PD-L1 when cultured with interferon-γ: quantitative polymerase chain reaction MiaPaca (15.2 rel. fold ± 0.5; P < .01) and S20-13 (31.0 rel. fold ± 4.4; P < .01). Murine orthotopic tumors treated by irreversible electroporation had an increase in signal intensity score for the expression of PD-L1 in residual tumor (P < .01). Ten patients were included in the safety analysis with a 12-month median follow-up (interquartile range 6.0, 15.8). No dose-limiting toxicities occurred. Seven patients developed grade 3/4 treatment-related adverse events; none required a dose modification of nivolumab; nivolumab-related adverse events occurred in 1 patient. Mean time to progression was 6.3 months (confidence interval 3.5-10.0) with current median overall survival of 18.0 months (confidence interval 9.2-26.8). CONCLUSION: Irreversible electroporation induces expression of PD-L1 in vitro. Combination therapy with concurrent nivolumab is well tolerated. A multicenter, phase 2 adjuvant trial is underway using irreversible electroporation and nivolumab in patients with locally advanced pancreatic cancer.


Assuntos
Adenocarcinoma/terapia , Antineoplásicos Imunológicos/uso terapêutico , Eletroporação/métodos , Nivolumabe/uso terapêutico , Neoplasias Pancreáticas/terapia , Adenocarcinoma/imunologia , Adenocarcinoma/patologia , Adulto , Idoso , Animais , Antineoplásicos Imunológicos/efeitos adversos , Antígeno B7-H1/genética , Linhagem Celular Tumoral , Terapia Combinada , Feminino , Expressão Gênica , Humanos , Interferon gama/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Nivolumabe/efeitos adversos , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Receptor de Morte Celular Programada 1/genética , Subpopulações de Linfócitos T
9.
Sci Rep ; 10(1): 10883, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32616770

RESUMO

Irreversible electroporation (IRE) is a non-thermal ablation modality that has been shown to be safe and effective in its application to tumors that are close to risky areas. This study aims to assess the safety and efficacy of IRE for unresectable hilar cholangiocarcinoma. Nine patients from two medical centers in Asia received IRE treatment between June 2015 and July 2017. Before IRE treatment, percutaneous biliary decompressions had been performed on eight patients, and internal stenting had been performed on one patient. All patients tolerated the procedure well without high-grade complications. The ablated tumors had constant size without contrast enhancement for more than three months in eight patients and the level of CA19-9 decreased significantly in all patients. The percutaneous biliary drainage tube was removed from two patients with recanalization of the bile duct. The internal stent in one patient was removed without further stenting. The median overall survival period was 26 months, and the progression-free survival was 18 months. Bile ducts remained narrow in the majority (2/3) of the treated patients. Nevertheless, IRE ablation of unresectable hilar cholangiocarcinoma involving vital structures is a safe and feasible primary treatment for local tumor control and is effective in prolonging survival.


Assuntos
Técnicas de Ablação/métodos , Neoplasias dos Ductos Biliares/cirurgia , Eletroporação/métodos , Tumor de Klatskin/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias dos Ductos Biliares/tratamento farmacológico , Ductos Biliares/patologia , Ductos Biliares/cirurgia , Quimioterapia Adjuvante , Terapia Combinada , Constrição Patológica , Desoxicitidina/administração & dosagem , Desoxicitidina/análogos & derivados , Feminino , Seguimentos , Humanos , Tumor de Klatskin/tratamento farmacológico , Laparotomia , Masculino , Pessoa de Meia-Idade , Intervalo Livre de Progressão , Estudos Retrospectivos , Resultado do Tratamento
10.
Sci Rep ; 10(1): 9149, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32499601

RESUMO

In gene electrotransfer and cardiac ablation with irreversible electroporation, treated muscle cells are typically of elongated shape and their orientation may vary. Orientation of cells in electric field has been reported to affect electroporation, and hence electrodes placement and pulse parameters choice in treatments for achieving homogeneous effect in tissue is important. We investigated how cell orientation influences electroporation with respect to different pulse durations (ns to ms range), both experimentally and numerically. Experimentally detected electroporation (evaluated separately for cells parallel and perpendicular to electric field) via Ca2+ uptake in H9c2 and AC16 cardiomyocytes was numerically modeled using the asymptotic pore equation. Results showed that cell orientation affects electroporation extent: using short, nanosecond pulses, cells perpendicular to electric field are significantly more electroporated than parallel (up to 100-times more pores formed), and with long, millisecond pulses, cells parallel to electric field are more electroporated than perpendicular (up to 1000-times more pores formed). In the range of a few microseconds, cells of both orientations were electroporated to the same extent. Using pulses of a few microseconds lends itself as a new possible strategy in achieving homogeneous electroporation in tissue with elongated cells of different orientation (e.g. electroporation-based cardiac ablation).


Assuntos
Forma Celular/fisiologia , Eletroporação/métodos , Animais , Linhagem Celular , Movimento Celular/fisiologia , Fura-2/química , Humanos , Microscopia de Fluorescência , Miócitos Cardíacos/citologia , Ratos
11.
Sci Rep ; 10(1): 10471, 2020 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-32591612

RESUMO

The permeabilization of the live cells membrane by the delivery of electric pulses has fundamental interest in medicine, in particular in tumors treatment by electrochemotherapy. Since underlying mechanisms are still not fully understood, we studied the impact of electric pulses on the biochemical composition of live cells thanks to label-free optical methods: confocal Raman microspectroscopy and terahertz microscopy. A dose effect was observed after cells exposure to different field intensities and a major impact on cell peptide/protein content was found. Raman measurements reveal that protein structure and/or environment are modified by the electric pulses while terahertz measurements suggest a leakage of proteins and other intracellular compounds. We show that Raman and terahertz modalities are a particularly attractive complement to fluorescence microscopy which is the reference optical technique in the case of electropermeabilization. Finally, we propose an analytical model for the influx and efflux of non-permeant molecules through transiently (electro)permeabilized cell membranes.


Assuntos
Membrana Celular/metabolismo , Eletroquimioterapia/psicologia , Eletroporação/métodos , Microscopia de Fluorescência/métodos , Animais , Linhagem Celular , Permeabilidade da Membrana Celular/fisiologia , Cães , Eletricidade , Eletroquimioterapia/métodos , Células Madin Darby de Rim Canino , Neoplasias/metabolismo , Proteínas/metabolismo
12.
J Vis Exp ; (160)2020 06 14.
Artigo em Inglês | MEDLINE | ID: mdl-32597854

RESUMO

In utero electroporation is an in vivo DNA transfer technique extensively used to study the molecular and cellular mechanisms underlying mammalian corticogenesis. This procedure takes advantage of the brain ventricles to allow the introduction of DNA of interest and uses a pair of electrodes to direct the entrance of the genetic material into the cells lining the ventricle, the neural stem cells. This method allows researchers to label the desired cells and/or manipulate the expression of genes of interest in those cells. It has multiple applications, including assays targeting neuronal migration, lineage tracing, and axonal pathfinding. An important feature of this method is its temporal and regional control, allowing circumvention of potential problems related with embryonic lethality or the lack of specific CRE driver mice. Another relevant aspect of this technique is that it helps to considerably reduce the economic and temporal limitations that involve the generation of new mouse lines, which become particularly important in the study of interactions between cell types that originate in distant areas of the brain at different developmental ages. Here we describe a double electroporation strategy that enables targeting of cell populations that are spatially and temporally separated. With this approach we can label different subtypes of cells in different locations with selected fluorescent proteins to visualize them, and/or we can manipulate genes of interest expressed by these different cells at the appropriate times. This strategy enhances the potential of in utero electroporation and provides a powerful tool to study the behavior of temporally and spatially separated cell populations that migrate to establish close contacts, as well as long-range interactions through axonal projections, reducing temporal and economic costs.


Assuntos
Encéfalo/metabolismo , DNA/administração & dosagem , Eletroporação/métodos , Embrião de Mamíferos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Neurais/metabolismo , Análise Espaço-Temporal , Animais , Encéfalo/citologia , DNA/genética , DNA/metabolismo , Embrião de Mamíferos/citologia , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/citologia , Neurogênese , Plasmídeos/administração & dosagem , Gravidez
13.
J Vis Exp ; (159)2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32510510

RESUMO

As genome-wide association studies shed light on the heterogeneous genetic underpinnings of many neurological diseases, the need to study the contribution of specific genes to brain development and function increases. Relying on mouse models to study the role of specific genetic manipulations is not always feasible since transgenic mouse lines are quite costly and many novel disease-associated genes do not yet have commercially available genetic lines. Additionally, it can take years of development and validation to create a mouse line. In utero electroporation offers a relatively quick and easy method to manipulate gene expression in a cell-type specific manner in vivo that only requires developing a DNA plasmid to achieve a particular genetic manipulation. Bilateral in utero electroporation can be used to target large populations of frontal cortex pyramidal neurons. Combining this gene transfer method with behavioral approaches allows one to study the effects of genetic manipulations on the function of prefrontal cortex networks and the social behavior of juvenile and adult mice.


Assuntos
Comportamento Animal , Eletroporação/métodos , Técnicas Genéticas , Animais , Estudos de Viabilidade , Camundongos , Camundongos Transgênicos , Plasmídeos/genética
14.
J Vis Exp ; (159)2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32510512

RESUMO

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.


Assuntos
Astrócitos/citologia , Córtex Cerebral/citologia , Eletroporação/métodos , Animais , Cor , Feminino , Camundongos , Neurogênese
15.
Anim Sci J ; 91(1): e13386, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32512638

RESUMO

This study was conducted to investigate the effect of seven concentrations of Cas9 protein (0, 25, 50, 100, 200, 500, and 1,000 ng/µl) on the development and gene editing of porcine embryos. This included the target editing and off-target effect of embryos developed from zygotes that were edited via electroporation of the Cas9 protein with guide RNA targeting Myostatin genes. We found that the development to blastocysts of electroporated zygotes was not affected by the concentration of Cas9 protein. Although the editing rate, which was defined as the ratio of edited blastocysts to total examined blastocysts, did not differ with Cas9 protein concentration, the editing efficiency, which was defined as the frequency of indel mutations in each edited blastocyst, was significantly decreased in the edited blastocysts from zygotes electroporated with 25 ng/µl of Cas9 protein compared with that of blastocysts from zygotes electroporated with higher Cas9 protein concentrations. Moreover the frequency of indel events at the two possible off-target sites was not significantly different with different concentrations of Cas9 protein. These results indicate that the concentration of Cas9 protein affects gene editing efficiency in embryos but not the embryonic development, gene editing rate, and non-specific cleavage of off-target sites.


Assuntos
Proteína 9 Associada à CRISPR , Eletroporação/métodos , Eletroporação/veterinária , Desenvolvimento Embrionário/genética , Edição de Genes , Marcação de Genes/veterinária , Miostatina/genética , RNA Guia , Suínos/embriologia , Suínos/genética , Zigoto , Animais , Blastocisto , Proteína 9 Associada à CRISPR/farmacologia , Relação Dose-Resposta a Droga
16.
J Cancer Res Ther ; 16(2): 280-285, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32474514

RESUMO

Context: The safety and efficacy of irreversible electroporation (IRE) for locally advanced pancreatic carcinoma (LAPC) are well established. However, whether adjuvant chemoradiotherapy after IRE increases, the survival rate remains unknown. Therefore, this study evaluated the effect of chemoradiotherapy combined with IRE in patients with LAPC. Subjects and Methods: We retrospectively analyzed 42 patients with LAPC between July 2015 and December 2016 at PLA General Hospital treated with IRE or IRE combined with radiation and/or chemotherapy. These patients were divided into the IRE group and the combined-therapy group. All patients underwent computed tomography (CT), magnetic resonance imaging, and positron-emission tomography-CT and no signs of metastases were found. The prognosis of these patients was observed. Results: The times after operation and after diagnosis in the combined-therapy group (304.20 ± 118.54) and (334.40 ± 114.07) days, respectively, were better those than in the IRE group (214.36 ± 95.68) and (244.68 ± 110.61) days, respectively. Moreover, patients in the combined-therapy group had a significantly better survival rate than the IRE group (80 vs. 45.45%, P < 0.05). Conclusions: IRE combined with radiotherapy or chemotherapy was superior to IRE alone for the treatment of LAPC, as it prolonged the survival time and improved the survival rate, making it worthy of wide dissemination and clinical application.


Assuntos
Quimiorradioterapia Adjuvante/mortalidade , Eletroporação/métodos , Neoplasias Pancreáticas/terapia , Quimiorradioterapia Adjuvante/métodos , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/patologia , Tomografia por Emissão de Pósitrons/métodos , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida , Tomografia Computadorizada por Raios X/métodos
17.
Biochem Biophys Res Commun ; 527(4): 1039-1042, 2020 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-32439162

RESUMO

Many genome-edited animals have been produced using the CRISPR/Cas system. Genome-edited strains were produced by introducing nucleases into pronuclear stage embryos. Recently, a new electroporation technique (TAKE: Technique for Animal Knockout system by Electroporation) was developed for the production of genome-edited animals by introducing nucleases into intact embryos using electroporation instead of the microinjection method. Furthermore, this method, which can introduce nucleases into intact embryos, enables genome editing of mouse embryos in the oviducts. However, the present protocol required improvements for low litter size and restriction of operation time. In this study, the influence on the development and genome editing of mouse embryos in the oviducts by electroporation and operation time was examined. The genome-editing rate was higher in the embryos electroporated at 16:00-17:00 (PM) (54%) on the following day of natural mating compared to that of embryos at 10:00-11:00 (AM) (27%). The embryos at AM formed a complex with cumulus cells, and cumulus cells were freed from embryos by treatment with hyalronidase before electroporation. The results showed that the genome-editing rate was significantly increased in the embryos treated with hyalronidase at AM, because the cumulus cells surrounding the embryos interfered with the introduction of nucleases into embryos. This study demonstrated that it was possible to adjust the operation time for the introduction of nucleases into embryos in the oviducts by treatment with hyalronidase before electroporation. However, litter size and development of embryos after electroporation was quite low in all experiments (5-7) compared with the control without operation (11).


Assuntos
Células do Cúmulo/citologia , Eletroporação/métodos , Edição de Genes/métodos , Oviductos/citologia , Animais , Sistemas CRISPR-Cas , Embrião de Mamíferos/metabolismo , Feminino , Masculino , Camundongos Endogâmicos ICR
18.
Updates Surg ; 72(4): 1089-1096, 2020 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32399592

RESUMO

BACKGROUND: Locally advanced pancreatic cancer (LAPC) is usually treated with chemoradiotherapy with poor results. The aim of the study was to assess whether intraoperative electrochemotherapy could be proposed as additional therapy in treating LAPC. METHODS: Observational study of patients affected by LAPC who underwent intraoperative electrochemotherapy (ECT) after chemoradiotherapy. Data at diagnosis, at restaging and short and long-term outcomes, including assessment of quality of life, were collected for each patient. RESULTS: Five patients underwent ECT: in four cases, the tumours were located in the head and, in one, in the body of the pancreas. Preoperative chemotherapy consisted mainly of six cycles of modified Folfirinox. At restaging, the serum value of carbohydrate antigen (Ca 19-9) and tumour size were reduced; however, the vascular involvement did not change. No downstaging was recorded. The ECT procedure was performed using at least four needles with a mean duration time of 27 min (range 15-40). No postoperative mortality or major complications were reported. The mean length of stay (LOS) was 8 days (range 5-14). Four patients were alive and well at the end of the study, while one patient died from disease progression. The mean follow-up was 20.8 months (range 9-34) from diagnosis and 9.4 months (range 2-19) from ECT. The quality of life was good and there was improvement in pain/discomfort. CONCLUSIONS: Electrochemotherapy could be proposed as a simple, feasible and safe palliative additional treatment in LAPC without progression after chemoradiotherapy. It seems to allow a good quality of life and pain improvement.


Assuntos
Antineoplásicos/administração & dosagem , Eletroquimioterapia/métodos , Eletroporação/métodos , Cuidados Intraoperatórios/métodos , Cuidados Paliativos/métodos , Pancreatectomia/métodos , Neoplasias Pancreáticas/terapia , Qualidade de Vida , Idoso , Bleomicina/administração & dosagem , Quimiorradioterapia , Terapia Combinada , Feminino , Humanos , Laparotomia , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/patologia , Cirurgia Assistida por Computador , Resultado do Tratamento
19.
Int J Hyperthermia ; 37(1): 486-505, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32423258

RESUMO

Introduction: Irreversible electroporation (IRE) is a relatively new ablation method for the treatment of unresectable cancers. Although the main mechanism of IRE is electric permeabilization of cell membranes, the question is to what extent thermal effects of IRE contribute to tissue ablation.Aim: This systematic review reviews the mathematical models used to numerically simulate the heat-generating effects of IRE, and uses the obtained data to assess the degree of mild-hyperthermic (temperatures between 40 °C and 50 °C) and thermally ablative (TA) effects (temperatures exceeding 50 °C) caused by IRE within the IRE-treated region (IRE-TR).Methods: A systematic search was performed in medical and technical databases for original studies reporting on numerical simulations of IRE. Data on used equations, study design, tissue models, maximum temperature increase, and surface areas of IRE-TR, mild-hyperthermic, and ablative temperatures were extracted.Results: Several identified models, including Laplace equation for calculation of electric field distribution, Pennes Bioheat Equation for heat transfer, and Arrhenius model for thermal damage, were applied on various electrode and tissue models. Median duration of combined mild-hyperthermic and TA effects is 20% of the treatment time. Based on the included studies, mild-hyperthermic temperatures occurred in 30% and temperatures ≥50 °C in 5% of the IRE-TR.Conclusions: Simulation results in this review show that significant mild-hyperthermic effects occur in a large part of the IRE-TR, and direct thermal ablation in comparatively small regions. Future studies should aim to optimize clinical IRE protocols, maintaining a maximum irreversible permeabilized region with minimal TA effects.


Assuntos
Eletroporação/métodos , Modelos Teóricos
20.
J Vis Exp ; (159)2020 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-32449731

RESUMO

Manipulation of gene expression in vivo during embryonic development is the method of choice when analyzing the role of individual genes during mammalian development. In utero electroporation is a key technique for the manipulation of gene expression in the embryonic mammalian brain in vivo. A protocol for in utero electroporation of the embryonic neocortex of ferrets, a small carnivore, is presented here. The ferret is increasingly being used as a model for neocortex development, because its neocortex exhibits a series of anatomical, histological, cellular, and molecular features that are also present in human and nonhuman primates, but absent in rodent models, such as mouse or rat. In utero electroporation was performed at embryonic day (E) 33, a midneurogenesis stage in ferret. In utero electroporation targets neural progenitor cells lining the lateral ventricles of the brain. During neurogenesis, these progenitor cells give rise to all other neural cell types. This work shows representative results and analyses at E37, postnatal day (P) 1, and P16, corresponding to 4, 9, and 24 days after in utero electroporation, respectively. At earlier stages, the progeny of targeted cells consists mainly of various neural progenitor subtypes, whereas at later stages most labeled cells are postmitotic neurons. Thus, in utero electroporation enables the study of the effect of genetic manipulation on the cellular and molecular features of various types of neural cells. Through its effect on various cell populations, in utero electroporation can also be used for the manipulation of histological and anatomical features of the ferret neocortex. Importantly, all these effects are acute and are performed with a spatiotemporal specificity determined by the user.


Assuntos
Eletroporação/métodos , Furões/metabolismo , Neocórtex/citologia , Células-Tronco Neurais/citologia , Animais , Feminino , Histerectomia , Neurônios/metabolismo , Gravidez
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