eoe sub articles

EoE Sub Articles

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparations
NOTE: The solutions and tools described in this section can be made well ahead of time and used on the day of dissection and recording.
<ol>
	<li>Prepare Liebovitz media supplemented with 10 mM HEPES (L-15 solution). The following steps describe making 1 L of L-15 solution.
	<ol>
		<li>Pour 990 mL o...

isolating vestibular ganglion neurons from a mouse inner ear

Source: Iyer, M. R. et al., <a href="https://app.jove.com/v/64908">Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings.</a>&#160;J. Vis. Exp. (2023).
This video showcases a method for isolating vestibular neurons from a mouse pup&#39;s inner ear. It meticulously outlines the steps involved in dissecting the inner ear, harvesting the vestibular ganglion, and subsequently dissociating the ganglion into single cells. These cells are then cultured on glass-bottom dishes to establish a vestibular neuron culture.

Isolating Vestibular Ganglion Neurons From a Mouse Inner Ear

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
1. Preparations
NOTE: The solutions ...

Take one-half of a mouse pup&#39;s head and remove the brain.
Cut the top of the skull and excise the surrounding tissue to visualize the otic capsule, a bony outer layer protecting the inner ear structures.
Transfer the tissue to a culture plate containing a buffered medium.
Separate the superior and inferior vestibular ganglia, which contain the vestibular nerve cell bodies.
Shave off the bony ridge and harvest the underlying superior ganglion.
Transfer the superior ganglion to a fresh plate containing the buffered medium.
Remove adherent tissues from the surface to clean the isolated ganglion.
Place the ganglion in a solution containing proteolytic enzymes.
The enzymes degrade the tissue&#39;s extracellular matrix, loosening the cells.
Transfer the ganglion to a polymer-coated glass-bottom dish containing culture medium and mechanically dissociate the tissue to release the cells.
Incubate, allowing the cells to adhere and establish a vestibular ganglion neuron culture.

isolating-vestibular-ganglion-neurons-from-a-mouse-inner-ear

Research

JoVE Encyclopedia of Experiments

Neuroscience

Neuronal Culture Techniques

Primary Neuronal Culture

A Protocol for Decellularizing Mouse Cochleae for Inner Ear Tissue Engineering

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current regenerative medicine strategies have focused on transplanting stem cells or genetic manipulation of surrounding support cells in the inner ear to encourage replacement of damaged stem cells to correct hearing loss. Yet, the extracellular matrix (ECM) may play a vital role in inducing and maintaining function of hair cells, and has not been well investigated. Using the cochlear ECM as a scaffold to grow adult stem cells may provide unique insights into how the composition and architecture of the extracellular environment aids cells in sustaining hearing function. Here we present a method for isolating and decellularizing cochleae from mice to use as scaffolds accepting perfused adult stem cells. In the current protocol, cochleae are isolated from euthanized mice, decellularized, and decalcified. Afterward, human Wharton&#39;s jelly cells (hWJCs) that were isolated from the umbilical cord were carefully perfused into each cochlea. The cochleae were used as bioreactors, and cells were cultured for 30 days before undergoing processing for analysis. Decellularized cochleae retained identifiable extracellular structures, but did not reveal the presence of cells or noticeable fragments of DNA. Cells perfused into the cochlea invaded most of the interior and exterior of the cochlea and grew without incident over a duration of 30 days. Thus, the current method can be used to study how cochlear ECM affects cell development and behavior.

The goal of this protocol is to demonstrate an effective method to decellularize and decalcify mouse cochleae for utilization as scaffolds for tissue engineering applications.

In mammals, mechanosensory hair cells that facilitate hearing lack the ability to regenerate, which has limited treatments for hearing loss. Current ...

JoVE Journal

Bioengineering

Isolation and Culture of Chick Ciliary Ganglion Neurons

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the eye. This ganglion is constituted by a homogenous population of ciliary and choroidal neurons that innervate striated and smooth muscle fibers, respectively. Each of these neuronal types regulate specific eye structures and functions. Over the years, neuronal cultures of the chick ciliary ganglia were shown to be effective cell models in the study of muscle-nervous system interactions, which communicate through cholinergic synapses. Ciliary ganglion neurons are, in its majority, cholinergic. This cell model has been shown to be useful comparatively to previously used heterogeneous cell models that comprise several neuronal types, besides cholinergic. Anatomically, the ciliary ganglion is localized between the optic nerve (ON) and the choroid fissure (CF). Here, we describe a detailed procedure for the dissection, dissociation and in vitro culture of ciliary ganglia neurons from chick embryos. We provide a step-by-step protocol in order to obtain highly pure and stable cellular cultures of CG neurons, highlighting key steps of the process. These cultures can be maintained in vitro for 15 days and, hereby, we show the normal development of CG cultures. The results also show that these neurons can interact with muscle fibers through neuro-muscular cholinergic synapses.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system. Neuronal cultures of chick CG neurons were shown to be effective cell models in the study of nerve muscle interactions. We describe a detailed protocol for the dissection, dissociation and in vitro culture of CG neurons from chick embryos.

Chick ciliary ganglia (CG) are part of the parasympathetic nervous system and are responsible for the innervation of the muscle tissues present in the ...

Coculture of Axotomized Rat Retinal Ganglion Neurons with Olfactory Ensheathing Glia, as an In Vitro Model of Adult Axonal Regeneration

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory bulb. Throughout adult life, they are key for axonal growing of newly generated olfactory neurons, from the lamina propria to the ONL. Due to their pro-regenerative properties, these cells have been used to foster axonal regeneration in spinal cord or optic nerve injury models.
We present an in vitro model to assay and measure OEG neuroregenerative capacity after neural injury. In this model, reversibly immortalized human OEG (ihOEG) is cultured as a monolayer, retinas are extracted from adult rats and retinal ganglion neurons (RGN) are cocultured onto the OEG monolayer. After 96 h, axonal and somatodendritic markers in RGNs are analyzed by immunofluorescence and the number of RGNs with axon and the mean axonal length/neuron are quantified.
This protocol has the advantage over other in vitro assays that rely on embryonic or postnatal neurons, that it evaluates OEG neuroregenerative properties in adult tissue. Also, it is not only useful for assessing the neuroregenerative potential of ihOEG but can be extended to different sources of OEG or other glial cells.

We present an in vitro model to assess olfactory ensheathing glia (OEG) neuroregenerative capacity, after neural injury. It is based on a coculture of axotomized adult retinal ganglion neurons (RGN) on OEG monolayers and subsequent study of axonal regeneration, by analyzing RGN axonal and somatodendritic markers.

Olfactory ensheathing glia (OEG) cells are localized all the way from the olfactory mucosa to and into the olfactory nerve layer (ONL) of the olfactory ...

Isolating Vestibular Ganglion Neurons From a Mouse Inner Ear

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