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Article ; Online: Derivation of Sendai-Virus-Reprogrammed Human iPSCs-Neuronal Precursors:

Shigyo, Michiko / Kobayashi, Yoshiomi / Platoshyn, Oleksandr / Marsala, Silvia / Kato, Tomohisa / Takamura, Naoki / Yoshida, Kenji / Kishino, Akiyoshi / Bravo-Hernandez, Mariana / Juhas, Stefan / Juhasova, Jana / Studenovska, Hana / Proks, Vladimir / Ciacci, Joseph D / Marsala, Martin

Cell transplantation

2023 Volume 32, Page(s) 9636897231163232

Abstract: The critical requirements in developing clinical-grade human-induced pluripotent stem cells-derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, ... ...

Abstract	The critical requirements in developing clinical-grade human-induced pluripotent stem cells-derived neural precursors (hiPSCs-NPCs) are defined by expandability, genetic stability, predictable
MeSH term(s)	Humans ; Rats ; Animals ; Induced Pluripotent Stem Cells ; Neural Stem Cells ; Sendai virus/genetics ; Leukocytes, Mononuclear ; Neurons/metabolism ; Cell Differentiation
Language	English
Publishing date	2023-04-18
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1135816-6
ISSN	1555-3892 ; 0963-6897
ISSN (online)	1555-3892
ISSN	0963-6897
DOI	10.1177/09636897231163232
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Article ; Online: Expandable Sendai-Virus-Reprogrammed Human iPSC-Neuronal Precursors:

Kobayashi, Yoshiomi / Shigyo, Michiko / Platoshyn, Oleksandr / Marsala, Silvia / Kato, Tomohisa / Takamura, Naoki / Yoshida, Kenji / Kishino, Akiyoshi / Bravo-Hernandez, Mariana / Juhas, Stefan / Juhasova, Jana / Studenovska, Hana / Proks, Vladimir / Driscoll, Shawn P / Glenn, Thomas D / Pfaff, Samuel L / Ciacci, Joseph D / Marsala, Martin

Cell transplantation

2023 Volume 32, Page(s) 9636897221107009

Abstract: One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, ...

Abstract	One of the challenges in clinical translation of cell-replacement therapies is the definition of optimal cell generation and storage/recovery protocols which would permit a rapid preparation of cell-treatment products for patient administration. Besides, the availability of injection devices that are simple to use is critical for potential future dissemination of any spinally targeted cell-replacement therapy into general medical practice. Here, we compared the engraftment properties of established human-induced pluripotent stem cells (hiPSCs)-derived neural precursor cell (NPCs) line once cells were harvested fresh from the cell culture or previously frozen and then grafted into striata or spinal cord of the immunodeficient rat. A newly developed human spinal injection device equipped with a spinal cord pulsation-cancelation magnetic needle was also tested for its safety in an adult immunosuppressed pig. Previously frozen NPCs showed similar post-grafting survival and differentiation profile as was seen for freshly harvested cells. Testing of human injection device showed acceptable safety with no detectable surgical procedure or spinal NPCs injection-related side effects.
MeSH term(s)	Adult ; Animals ; Humans ; Rats ; Cell Differentiation/physiology ; Cellular Reprogramming/genetics ; Cellular Reprogramming/physiology ; Genetic Vectors/genetics ; Graft Survival/physiology ; Induced Pluripotent Stem Cells/physiology ; Induced Pluripotent Stem Cells/transplantation ; Injections, Spinal/adverse effects ; Injections, Spinal/instrumentation ; Injections, Spinal/methods ; Neural Stem Cells/physiology ; Neural Stem Cells/transplantation ; Sendai virus ; Specimen Handling/methods ; Stem Cell Transplantation/adverse effects ; Stem Cell Transplantation/instrumentation ; Stem Cell Transplantation/methods ; Swine ; Tissue and Organ Harvesting/methods ; Treatment Outcome ; Brain ; Spinal Cord
Language	English
Publishing date	2023-05-04
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	1135816-6
ISSN	1555-3892 ; 0963-6897
ISSN (online)	1555-3892
ISSN	0963-6897
DOI	10.1177/09636897221107009
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Article ; Online: Spinal parenchymal occupation by neural stem cells after subpial delivery in adult immunodeficient rats.

Marsala, Martin / Kamizato, Kota / Tadokoro, Takahiro / Navarro, Michael / Juhas, Stefan / Juhasova, Jana / Marsala, Silvia / Studenovska, Hana / Proks, Vladimir / Hazel, Tom / Johe, Karl / Kakinohana, Manabu / Driscoll, Shawn / Glenn, Thomas / Pfaff, Samuel / Ciacci, Joseph

Stem cells translational medicine

2019 Volume 9, Issue 2, Page(s) 177–188

Abstract: Neural precursor cells (NSCs) hold great potential to treat a variety of neurodegenerative diseases and injuries to the spinal cord. However, current delivery techniques require an invasive approach in which an injection needle is advanced into the ... ...

Abstract	Neural precursor cells (NSCs) hold great potential to treat a variety of neurodegenerative diseases and injuries to the spinal cord. However, current delivery techniques require an invasive approach in which an injection needle is advanced into the spinal parenchyma to deliver cells of interest. As such, this approach is associated with an inherent risk of spinal injury, as well as a limited delivery of cells into multiple spinal segments. Here, we characterize the use of a novel cell delivery technique that employs single bolus cell injections into the spinal subpial space. In immunodeficient rats, two subpial injections of human NSCs were performed in the cervical and lumbar spinal cord, respectively. The survival, distribution, and phenotype of transplanted cells were assessed 6-8 months after injection. Immunofluorescence staining and mRNA sequencing analysis demonstrated a near-complete occupation of the spinal cord by injected cells, in which transplanted human NSCs (hNSCs) preferentially acquired glial phenotypes, expressing oligodendrocyte (Olig2, APC) or astrocyte (GFAP) markers. In the outermost layer of the spinal cord, injected hNSCs differentiated into glia limitans-forming astrocytes and expressed human-specific superoxide dismutase and laminin. All animals showed normal neurological function for the duration of the analysis. These data show that the subpial cell delivery technique is highly effective in populating the entire spinal cord with injected NSCs, and has a potential for clinical use in cell replacement therapies for the treatment of ALS, multiple sclerosis, or spinal cord injury.
MeSH term(s)	Animals ; Neural Stem Cells/metabolism ; Parenchymal Tissue/cytology ; Parenchymal Tissue/metabolism ; Rats ; Rats, Sprague-Dawley
Language	English
Publishing date	2019-12-04
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2642270-0
ISSN	2157-6580 ; 2157-6564
ISSN (online)	2157-6580
ISSN	2157-6564
DOI	10.1002/sctm.19-0156
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Article ; Online: A First-in-Human, Phase I Study of Neural Stem Cell Transplantation for Chronic Spinal Cord Injury.

Curtis, Erik / Martin, Joel R / Gabel, Brandon / Sidhu, Nikki / Rzesiewicz, Teresa K / Mandeville, Ross / Van Gorp, Sebastiaan / Leerink, Marjolein / Tadokoro, Takahiro / Marsala, Silvia / Jamieson, Catriona / Marsala, Martin / Ciacci, Joseph D

Cell stem cell

2018 Volume 22, Issue 6, Page(s) 941–950.e6

Abstract: We tested the feasibility and safety of human-spinal-cord-derived neural stem cell (NSI-566) transplantation for the treatment of chronic spinal cord injury (SCI). In this clinical trial, four subjects with T2-T12 SCI received treatment consisting of ... ...

Abstract	We tested the feasibility and safety of human-spinal-cord-derived neural stem cell (NSI-566) transplantation for the treatment of chronic spinal cord injury (SCI). In this clinical trial, four subjects with T2-T12 SCI received treatment consisting of removal of spinal instrumentation, laminectomy, and durotomy, followed by six midline bilateral stereotactic injections of NSI-566 cells. All subjects tolerated the procedure well and there have been no serious adverse events to date (18-27 months post-grafting). In two subjects, one to two levels of neurological improvement were detected using ISNCSCI motor and sensory scores. Our results support the safety of NSI-566 transplantation into the SCI site and early signs of potential efficacy in three of the subjects warrant further exploration of NSI-566 cells in dose escalation studies. Despite these encouraging secondary data, we emphasize that this safety trial lacks statistical power or a control group needed to evaluate functional changes resulting from cell grafting.
MeSH term(s)	Adult ; Animals ; Cell Line ; Chronic Disease ; Female ; Humans ; Male ; Neural Stem Cells/cytology ; Neural Stem Cells/transplantation ; Rats ; Rats, Nude ; Spinal Cord Injuries/pathology ; Spinal Cord Injuries/surgery ; Spinal Cord Injuries/therapy ; Stem Cell Transplantation/adverse effects ; Young Adult
Language	English
Publishing date	2018-06-01
Publishing country	United States
Document type	Case Reports ; Clinical Trial, Phase I ; Journal Article
ZDB-ID	2375354-7
ISSN	1875-9777 ; 1934-5909
ISSN (online)	1875-9777
ISSN	1934-5909
DOI	10.1016/j.stem.2018.05.014
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Zs.A 6589: Show issues			Location: Je nach Verfügbarkeit (siehe Angabe bei Bestand) bis Jg. 2021: Bestellungen von Artikeln über das Online-Bestellformular ab Jg. 2022: Lesesaal (EG)
Zs.MG 75: Show issues

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Article ; Online: Time-dependent, bidirectional, anti- and pro-spinal hyper-reflexia and muscle spasticity effect after chronic spinal glycine transporter 2 (GlyT2) oligonucleotide-induced downregulation.

Kamizato, Kota / Marsala, Silvia / Navarro, Michael / Kakinohana, Manabu / Platoshyn, Oleksandr / Yoshizumi, Tetsuya / Lukacova, Nadezda / Wancewicz, Ed / Powers, Berit / Mazur, Curt / Marsala, Martin

Experimental neurology

2018 Volume 305, Page(s) 66–75

Abstract: The loss of local spinal glycine-ergic tone has been postulated as one of the mechanisms contributing to the development of spinal injury-induced spasticity. In our present study using a model of spinal transection-induced muscle spasticity, we ... ...

Abstract	The loss of local spinal glycine-ergic tone has been postulated as one of the mechanisms contributing to the development of spinal injury-induced spasticity. In our present study using a model of spinal transection-induced muscle spasticity, we characterize the effect of spinally-targeted GlyT2 downregulation once initiated at chronic stages after induction of spasticity in rats. In animals with identified hyper-reflexia, the anti-spasticity effect was studied after intrathecal treatment with: i) glycine, ii) GlyT2 inhibitor (ALX 1393), and iii) GlyT2 antisense oligonucleotide (GlyT2-ASO). Administration of glycine and GlyT2 inhibitor led to significant suppression of spasticity lasting for a minimum of 45-60 min. Treatment with GlyT2-ASO led to progressive suppression of muscle spasticity seen at 2-3 weeks after treatment. Over the subsequent 4-12 weeks, however, the gradual appearance of profound spinal hyper-reflexia was seen. This was presented as spontaneous or slight-tactile stimulus-evoked muscle oscillations in the hind limbs (but not in upper limbs) with individual hyper-reflexive episodes lasting between 3 and 5 min. Chronic hyper-reflexia induced by GlyT2-ASO treatment was effectively blocked by intrathecal glycine. Immunofluorescence staining and Q-PCR analysis of the lumbar spinal cord region showed a significant (>90%) decrease in GlyT2 mRNA and GlyT2 protein. These data demonstrate that spinal GlyT2 downregulation provides only a time-limited therapeutic benefit and that subsequent loss of glycine vesicular synthesis resulting from chronic GlyT2 downregulation near completely eliminates the tonic glycine-ergic activity and is functionally expressed as profound spinal hyper-reflexia. These characteristics also suggest that chronic spinal GlyT2 silencing may be associated with pro-nociceptive activity.
MeSH term(s)	Animals ; Down-Regulation/physiology ; Female ; Glycine Plasma Membrane Transport Proteins/metabolism ; Muscle Spasticity/metabolism ; Muscle Spasticity/physiopathology ; Rats ; Rats, Sprague-Dawley ; Reflex, Abnormal/physiology ; Spinal Cord/metabolism ; Spinal Cord/physiopathology ; Spinal Cord Injuries/metabolism ; Spinal Cord Injuries/physiopathology ; Thoracic Vertebrae ; Time Factors
Chemical Substances	Glycine Plasma Membrane Transport Proteins ; Slc6a5 protein, rat
Language	English
Publishing date	2018-03-30
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	207148-4
ISSN	1090-2430 ; 0014-4886
ISSN (online)	1090-2430
ISSN	0014-4886
DOI	10.1016/j.expneurol.2018.03.013
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Zs.A 184: Show issues

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Article ; Online: Precision spinal gene delivery-induced functional switch in nociceptive neurons reverses neuropathic pain.

Tadokoro, Takahiro / Bravo-Hernandez, Mariana / Agashkov, Kirill / Kobayashi, Yoshiomi / Platoshyn, Oleksandr / Navarro, Michael / Marsala, Silvia / Miyanohara, Atsushi / Yoshizumi, Tetsuya / Shigyo, Michiko / Krotov, Volodymyr / Juhas, Stefan / Juhasova, Jana / Nguyen, Duong / Kupcova Skalnikova, Helena / Motlik, Jan / Studenovska, Hana / Proks, Vladimir / Reddy, Rajiv /

Driscoll, Shawn P / Glenn, Thomas D / Kemthong, Taratorn / Malaivijitnond, Suchinda / Tomori, Zoltan / Vanicky, Ivo / Kakinohana, Manabu / Pfaff, Samuel L / Ciacci, Joseph / Belan, Pavel / Marsala, Martin

Molecular therapy : the journal of the American Society of Gene Therapy

2022 Volume 30, Issue 8, Page(s) 2722–2745

Abstract: Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory ... ...

Abstract	Second-order spinal cord excitatory neurons play a key role in spinal processing and transmission of pain signals to the brain. Exogenously induced change in developmentally imprinted excitatory neurotransmitter phenotypes of these neurons to inhibitory has not yet been achieved. Here, we use a subpial dorsal horn-targeted delivery of AAV (adeno-associated virus) vector(s) encoding GABA (gamma-aminobutyric acid) synthesizing-releasing inhibitory machinery in mice with neuropathic pain. Treated animals showed a progressive and complete reversal of neuropathic pain (tactile and brush-evoked pain behavior) that persisted for a minimum of 2.5 months post-treatment. The mechanism of this treatment effect results from the switch of excitatory to preferential inhibitory neurotransmitter phenotype in dorsal horn nociceptive neurons and a resulting increase in inhibitory activity in regional spinal circuitry after peripheral nociceptive stimulation. No detectable side effects (e.g., sedation, motor weakness, loss of normal sensation) were seen between 2 and 13 months post-treatment in naive adult mice, pigs, and non-human primates. The use of this treatment approach may represent a potent and safe treatment modality in patients suffering from spinal cord or peripheral nerve injury-induced neuropathic pain.
MeSH term(s)	Animals ; Gene Transfer Techniques ; Mice ; Neuralgia/etiology ; Neuralgia/therapy ; Nociceptors ; Posterior Horn Cells ; Spinal Cord ; Spinal Cord Dorsal Horn ; Swine
Language	English
Publishing date	2022-05-05
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Research Support, N.I.H., Extramural
ZDB-ID	2010592-7
ISSN	1525-0024 ; 1525-0016
ISSN (online)	1525-0024
ISSN	1525-0016
DOI	10.1016/j.ymthe.2022.04.023
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Zs.A 5640: Show issues

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Article ; Online: Surface N-glycoproteome patterns reveal key proteins of neuronal differentiation.

Tyleckova, Jirina / Valekova, Ivona / Zizkova, Martina / Rakocyova, Michaela / Marsala, Silvia / Marsala, Martin / Gadher, Suresh Jivan / Kovarova, Hana

Journal of proteomics

2015 Volume 132, Page(s) 13–20

Abstract: Pluripotent stem cell-derived committed neural precursors are an important source of cells to treat neurodegenerative diseases including spinal cord injury. There remains an urgency to identify markers for monitoring of neural progenitor specificity, ... ...

Abstract	Pluripotent stem cell-derived committed neural precursors are an important source of cells to treat neurodegenerative diseases including spinal cord injury. There remains an urgency to identify markers for monitoring of neural progenitor specificity, estimation of neural fate and follow-up correlation with therapeutic effect in preclinical studies using animal disease models. Cell surface capture technology was used to uncover the cell surface exposed N-glycoproteome of neural precursor cells upon neuronal differentiation as well as post-mitotic mature hNT neurons. The data presented depict an extensive study of surfaceome during neuronal differentiation, confirming glycosylation at a particular predicted site of many of the identified proteins. Quantitative changes detected in cell surface protein levels reveal a set of proteins that highlight the complexity of the neuronal differentiation process. Several of these proteins including the cell adhesion molecules ICAM1, CHL1, and astrotactin1 as well as LAMP1 were validated by SRM. Combination of immunofluorescence staining of ICAM1 and flow cytometry indicated a possible direction for future scrutiny of such proteins as targets for enrichment of the neuronal subpopulation from mixed cultures after differentiation of neural precursor cells. These surface proteins hold an important key for development of safe strategies in cell-replacement therapies of neuronal disorders. Biological significance: Neural stem and/or precursor cells have a great potential for cell-replacement therapies of neuronal diseases. Availability of well characterised and expandable neural cell lineage specific populations is critical for addressing such a challenge. In our study we identified and relatively quantified several hundred surface N-glycoproteins in the course of neuronal differentiation. We further confirmed the abundant changes for several cell adhesion proteins by SRM and outlined a strategy for utilisation of such N-glycoproteins in antibody based cell sorting. The comprehensive dataset presented here demonstrates the molecular background of neuronal differentiation highly useful for development of new plasma membrane markers to identify and select neuronal subpopulation from mixed neural cell cultures.
MeSH term(s)	Cell Differentiation/physiology ; Cell Line ; Cells, Cultured ; Humans ; Membrane Glycoproteins/metabolism ; Nerve Tissue Proteins/metabolism ; Neural Stem Cells/cytology ; Neural Stem Cells/metabolism
Chemical Substances	Membrane Glycoproteins ; Nerve Tissue Proteins
Language	English
Publishing date	2015-11-12
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2400835-7
ISSN	1876-7737 ; 1874-3919
ISSN (online)	1876-7737
ISSN	1874-3919
DOI	10.1016/j.jprot.2015.11.008
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Article ; Online: A scalable solution for isolating human multipotent clinical-grade neural stem cells from ES precursors.

Bohaciakova, Dasa / Hruska-Plochan, Marian / Tsunemoto, Rachel / Gifford, Wesley D / Driscoll, Shawn P / Glenn, Thomas D / Wu, Stephanie / Marsala, Silvia / Navarro, Michael / Tadokoro, Takahiro / Juhas, Stefan / Juhasova, Jana / Platoshyn, Oleksandr / Piper, David / Sheckler, Vickie / Ditsworth, Dara / Pfaff, Samuel L / Marsala, Martin

Stem cell research & therapy

2019 Volume 10, Issue 1, Page(s) 83

Abstract: Background: A well-characterized method has not yet been established to reproducibly, efficiently, and safely isolate large numbers of clinical-grade multipotent human neural stem cells (hNSCs) from embryonic stem cells (hESCs). Consequently, the ... ...

Abstract	Background: A well-characterized method has not yet been established to reproducibly, efficiently, and safely isolate large numbers of clinical-grade multipotent human neural stem cells (hNSCs) from embryonic stem cells (hESCs). Consequently, the transplantation of neurogenic/gliogenic precursors into the CNS for the purpose of cell replacement or neuroprotection in humans with injury or disease has not achieved widespread testing and implementation. Methods: Here, we establish an approach for the in vitro isolation of a highly expandable population of hNSCs using the manual selection of neural precursors based on their colony morphology (CoMo-NSC). The purity and NSC properties of established and extensively expanded CoMo-NSC were validated by expression of NSC markers (flow cytometry, mRNA sequencing), lack of pluripotent markers and by their tumorigenic/differentiation profile after in vivo spinal grafting in three different animal models, including (i) immunodeficient rats, (ii) immunosuppressed ALS rats (SOD1 Results: In vitro analysis of established CoMo-NSCs showed a consistent expression of NSC markers (Sox1, Sox2, Nestin, CD24) with lack of pluripotent markers (Nanog) and stable karyotype for more than 15 passages. Gene profiling and histology revealed that spinally grafted CoMo-NSCs differentiate into neurons, astrocytes, and oligodendrocytes over a 2-6-month period in vivo without forming neoplastic derivatives or abnormal structures. Moreover, transplanted CoMo-NSCs formed neurons with synaptic contacts and glia in a variety of host environments including immunodeficient rats, immunosuppressed ALS rats (SOD1G93A), or spinally injured minipigs, indicating these cells have favorable safety and differentiation characteristics. Conclusions: These data demonstrate that manually selected CoMo-NSCs represent a safe and expandable NSC population which can effectively be used in prospective human clinical cell replacement trials for the treatment of a variety of neurodegenerative disorders, including ALS, stroke, spinal traumatic, or spinal ischemic injury.
MeSH term(s)	Cell Line ; Flow Cytometry ; Humans ; Multipotent Stem Cells/cytology ; Neural Stem Cells/cytology
Language	English
Publishing date	2019-03-12
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, Non-U.S. Gov't
ZDB-ID	2548671-8
ISSN	1757-6512 ; 1757-6512
ISSN (online)	1757-6512
ISSN	1757-6512
DOI	10.1186/s13287-019-1163-7
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Article ; Online: Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice.

Tadokoro, Takahiro / Miyanohara, Atsushi / Navarro, Michael / Kamizato, Kota / Juhas, Stefan / Juhasova, Jana / Marsala, Silvia / Platoshyn, Oleksandr / Curtis, Erik / Gabel, Brandon / Ciacci, Joseph / Lukacova, Nada / Bimbova, Katarina / Marsala, Martin

Journal of visualized experiments : JoVE

2017 , Issue 125

Abstract: The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene ... ...

Abstract	The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 µL; 1.2 x 10
MeSH term(s)	Animals ; Brain/metabolism ; Dependovirus/genetics ; Female ; Genetic Vectors/genetics ; Genetic Vectors/metabolism ; Green Fluorescent Proteins/genetics ; Male ; Mice ; Mice, Inbred C57BL ; Microscopy, Fluorescence ; Spinal Cord/metabolism ; Video Recording
Chemical Substances	Green Fluorescent Proteins (147336-22-9)
Language	English
Publishing date	2017-07-13
Publishing country	United States
Document type	Journal Article ; Video-Audio Media
ISSN	1940-087X
ISSN (online)	1940-087X
DOI	10.3791/55770
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Article: Subpial adeno-associated virus 9 (aav9) vector delivery in adult mice

Journal of visualized experiments. 2017 July 13, , no. 125

2017

Abstract	The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 μL; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.
Keywords	Dependoparvovirus ; adults ; animal injuries ; animal models ; axons ; catheters ; gene expression ; genome ; green fluorescent protein ; mice ; motor cortex ; neurodegenerative diseases ; polyethylene ; rats ; spinal cord ; swine ; transgenic animals
Language	English
Dates of publication	2017-0713
Size	p. e55770.
Publishing place	Journal of Visualized Experiments
Document type	Article
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/55770
Database	NAL-Catalogue (AGRICOLA)

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