Mouse hippocampal explant culture system to study isolated axons

Pathak, Gunja K.; Aranda‐Espinoza, Helim; Shah, Sameer B.

doi:10.1016/j.jneumeth.2014.05.018

Cited by 3 publications

(8 citation statements)

References 34 publications

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“…To examine the axonal expression and regulation of transcripts involved in CNS RIS, we developed a mouse hippocampal explant system, which enables injury and analysis of isolated axons [20]. For this study, we cultured P1 explants for seven days and cut the axons on the seventh day to study axonal response to injury at time points up to 24 hours (Figures 1(a) and 1(b)).…”

Section: Resultsmentioning

confidence: 99%

“…Immunofluorescence evaluation of an axonal marker, phosphorylated neurofilament (SMI-31), and a dendritic marker, microtubule-associated protein 2 (MAP-2A), confirmed the axonal nature of long projections from the explant (Figure 1(c); cf. [20]). The exclusion of nuclear or cell body markers was assessed in the axonal lysate of each sample, and contaminated samples were excluded from analysis.…”

Section: Resultsmentioning

confidence: 99%

“…Details and validation of the explant culture system, including assessment of axonal purity, have been published [20]. Briefly, C57/Black 6 mouse neonates (P1) were euthanized.…”

Section: Methodsmentioning

confidence: 99%

“…For severed axons, tissue was collected both proximal and distal to the injury site, to enable comparison with corresponding control axons. We performed additional analysis on each sample to assess the exclusive axonal nature of the preparation, as described previously [20]. For RT-PCR assays, we confirmed the absence of γ -actin mRNA by PCR, which resides only in the soma but is restricted from axons [21–23].…”

Section: Methodsmentioning

confidence: 99%

“…A key unknown is whether and by what mechanisms levels of importin β 1 and other RIS-associated transcripts are altered locally in the axon in response to injury. The goal of this study was to perform an initial examination of importin β 1-dependent RIS mechanisms in the CNS, through the use of a new hippocampal explant system [20], which enables examination of axonal mRNA and protein expression independent of neuronal cell bodies. Our results suggest a biphasic axonal response, in which levels of several axonal transcripts, including those associated with RIS, increase rapidly in axons after injury.…”

Section: Introductionmentioning

confidence: 99%

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Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury

et al. 2016

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Axons in the peripheral nervous system respond to injury by activating retrograde injury signaling (RIS) pathways, which promote local axonal protein synthesis (LPS) and neuronal regeneration. RIS is also initiated following injury of neurons in the central nervous system (CNS). However, regulation of the localization of axonal mRNA required for LPS is not well understood. We used a hippocampal explant system to probe the regulation of axonal levels of RIS-associated transcripts following axonal injury. Axonal levels of importin β1 and RanBP1 were elevated biphasically at 1 and 24 hrs after axotomy. Transcript levels for β-actin, a prototypic axonally synthesized protein, were similarly elevated. Our data suggest differential regulation of axonal transcripts. At 1 hr after injury, deployment of actinomycin revealed that RanBP1, but not importin β1, requires de novo mRNA synthesis. At 24 hrs after injury, use of importazole revealed that the second wave of increased axonal mRNA levels required importin β-mediated nuclear import. We also observed increased importin β1 axonal protein levels at 1 and 6 hrs after injury. RanBP1 levels and vimentin levels fluctuated but were unchanged at 3 and 6 hrs after injury. This study revealed temporally complex regulation of axonal transcript levels, and it has implications for understanding neuronal response to injury in the CNS.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

“…Details and validation of the explant culture system, including assessment of axonal purity, have been published [20]. Briefly, C57/Black 6 mouse neonates (P1) were euthanized.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury

et al. 2016

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In Vitro Systems in Neurotoxicological Studies

Harry¹

2018

Comprehensive Toxicology

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Advances in ex vivo models and lab-on-a-chip devices for neural tissue engineering

et al. 2019

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The technologies related to ex vivo models and lab-on-a-chip devices for studying the regeneration of brain, spinal cord, and peripheral nerve tissues are essential tools for neural tissue engineering and regenerative medicine research. The need for ex vivo systems, lab-on-a-chip technologies and disease models for neural tissue engineering applications are emerging to overcome the shortages and drawbacks of traditional in vitro systems and animal models. Ex vivo models have evolved from traditional 2D cell culture models to 3D tissue-engineered scaffold systems, bioreactors, and recently organoid test beds. In addition to ex vivo model systems, we discuss lab-on-a-chip devices and technologies specifically for neural tissue engineering applications. Finally, we review current commercial products that mimic diseased and normal neural tissues, and discuss the future directions in this field.

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Mouse hippocampal explant culture system to study isolated axons

Cited by 3 publications

References 34 publications

Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury

Increases in Retrograde Injury Signaling Complex-Related Transcripts in Central Axons following Injury

In Vitro Systems in Neurotoxicological Studies

Advances in ex vivo models and lab-on-a-chip devices for neural tissue engineering

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