Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Pazyra-Murphy, Maria F.; Segal, Rosalind A.

doi:10.3791/951

Cited by 18 publications

(14 citation statements)

References 6 publications

(5 reference statements)

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“…16,[24][25][26] On day 1, trigeminal neuronal cells (1 · 10 3 ) were plated in the central compartment. The side compartments were filled with a mixture that was identical except that it also contained cytosine arabinoside (AraC, 0.3 lM; Sigma).…”

Section: In Vitro Experimentsmentioning

confidence: 99%

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Corneal Neurotoxicity Due to Topical Benzalkonium Chloride

Sarkar¹,

Chaudhary²,

Namavari³

et al. 2012

Invest. Ophthalmol. Vis. Sci.

116

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PURPOSE. The aim of this study was to determine and characterize the effect of topical application of benzalkonium chloride (BAK) on corneal nerves in vivo and in vitro.METHODS. Thy1-YFP+ neurofluorescent mouse eyes were treated topically with vehicle or BAK (0.01% or 0.1%). Widefield stereofluorescence microscopy was performed to sequentially image the treated corneas in vivo every week for 4 weeks, and changes in stromal nerve fiber density (NFD) and aqueous tear production were determined. Whole-mount immunofluorescence staining of corneas was performed with antibodies to axonopathy marker SMI-32. Western immunoblot analyses were performed on trigeminal ganglion and corneal lysates to determine abundance of proteins associated with neurotoxicity and regeneration. Compartmental culture of trigeminal ganglion neurons was performed in Campenot devices to determine whether BAK affects neurite outgrowth.RESULTS. BAK-treated corneas exhibited significantly reduced NFD and aqueous tear production, and increased inflammatory cell infiltration and fluorescein staining at 1 week (P < 0.05). These changes were most significant after 0.1% BAK treatment. The extent of inflammatory cell infiltration in the cornea showed a significant negative correlation with NFD. Sequential in vivo imaging of corneas showed two forms of BAK-induced neurotoxicity: reversible neurotoxicity characterized by axonopathy and recovery, and irreversible neurotoxicity characterized by nerve degeneration and regeneration. Increased abundance of beta III tubulin in corneal lysates confirmed regeneration. A dose-related significant reduction in neurites occurred after BAK addition to compartmental cultures of dissociated trigeminal ganglion cells. Although both BAK doses (0.0001% and 0.001%) reduced nerve fiber length, the reduction was significantly more with the higher dose (P < 0.001).CONCLUSION. Topical application of BAK to the eye causes corneal neurotoxicity, inflammation, and reduced aqueous tear production. (Invest Ophthalmol Vis Sci.

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Section: In Vitro Experimentsmentioning

confidence: 99%

“…The side compartments were filled with a mixture that was identical except that it also contained cytosine arabinoside (AraC, 0.3 lM; Sigma). 26 …”

Section: In Vitro Experimentsmentioning

confidence: 99%

Corneal Neurotoxicity Due to Topical Benzalkonium Chloride

Sarkar¹,

Chaudhary²,

Namavari³

et al. 2012

Invest. Ophthalmol. Vis. Sci.

116

View full text Add to dashboard Cite

show abstract

“…Cultures were set up following an established protocol (Pazyra-Murphy and Segal, 2008), modified to increase growth into the axonal compartment, and using permanox chamber slides (Nalge Nunc International). Further to the reported method, 10 g/ml laminin was added to the methyl cellulose bridge.…”

Section: Antibody Uptake Using Compartmented Drg Culturesmentioning

confidence: 99%

Rab11 and Its Effector Rab Coupling Protein Contribute to the Trafficking of β1 Integrins during Axon Growth in Adult Dorsal Root Ganglion Neurons and PC12 Cells

Eva¹,

Dassie²,

Caswell³

et al. 2010

J. Neurosci.

118

144

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Integrins play an important part in axon growth, but integrin traffic in neurons is poorly understood. Expression of the tenascin-Cbinding integrin ␣9 promotes axon regeneration. We have therefore studied the mechanism by which ␣9 integrin and its partner ␤1 are trafficked along axons and at the growth cone using adult DRG neurons and PC12 cells. We have focused on the small GTPase Rab11 and its effector Rab coupling protein (RCP), as they are involved in the long-range trafficking of ␤1 integrins in other cells. Rab11 colocalizes with ␣9 and other ␣ integrins and with ␤1 integrin in growth cones and axons, and immunopurified Rab11 vesicles contain ␣9 and ␤1. Endocytosed ␤1 integrins traffic via Rab11. However, Rab11 vesicles in axons are generally static, and ␣9 integrins undergo bouts of movement during which they leave the Rab11 compartment. In growth cones, ␣9 and ␤1 overlap with RCP, particularly at the growth cone periphery. We show that ␤1 integrin trafficking during neurite outgrowth involves Rab11 and RCP, and that manipulation of these molecules alters surface integrin levels and axon growth, and can be used to enhance ␣9 integrin-dependent neurite outgrowth. Our data suggest that manipulation of trafficking via Rab11 and RCP could be a useful strategy for promoting integrin-dependent axonal regeneration.

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“…Compartmentalized cultures, such as Campenot chambers [2, 9] and microfluidic devices ([10], and described herein), separate neuronal cell bodies and proximal neurites from distal projections. This provides an in vitro model of in vivo neuronal architecture, and facilitates specific treatment of the distal projections.…”

Section: Methodsmentioning

confidence: 99%

Isolation, Purification, and Culture of Primary Murine Sensory Neurons

Katzenell¹,

Cabrera²,

North³

et al. 2017

Innate Antiviral Immunity

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Cultured primary neurons have been of extraordinary value for the study of neuronal anatomy, cell biology, and physiology. While use of neuronal cell lines has ease and utility, there are often caveats that arise due to their mitotic nature. This methods article presents detailed methodology for the preparation, purification, and culture of adult murine sensory neurons for the study of herpes simplex virus lytic and latent infections. While virology is the application for our laboratory, these cultures also have broad utility for neurobiologists and cell biologists. While these primary cultures have been highly informative, the methodology is challenging to many investigators. Through publication of this highly detailed protocol, it is our hope that the use of this culture system can spread in the field to allow more rapid progress in furthering our understanding of neurotropic virus infection.

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Preparation and Maintenance of Dorsal Root Ganglia Neurons in Compartmented Cultures

Cited by 18 publications

References 6 publications

Corneal Neurotoxicity Due to Topical Benzalkonium Chloride

Corneal Neurotoxicity Due to Topical Benzalkonium Chloride

Rab11 and Its Effector Rab Coupling Protein Contribute to the Trafficking of β1 Integrins during Axon Growth in Adult Dorsal Root Ganglion Neurons and PC12 Cells

Isolation, Purification, and Culture of Primary Murine Sensory Neurons

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