Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

Rui Wang; Zi-tong Huang; Wen-kang Ren; Jiao Zhang; Yao Zhang; Bo Tan; Ping Huang; Hong-ying Cao

doi:10.3791/61177

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

JoVE Journal
Neuroscience

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JoVE Journal Neuroscience

Isolation and Culture of Primary Neurons and Glia from Adult Rat Urinary Bladder

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06:18 min

May 23, 2020

DOI: 10.3791/61177-v

Rui Wang¹, Zi-tong Huang³, Wen-kang Ren¹, Jiao Zhang¹, Yao Zhang¹, Bo Tan³, Ping Huang^1,2, Hong-ying Cao^1,2

¹School of Pharmaceutical Sciences,Guangzhou University of Chinese Medicine, ²Dongguan & Guangzhou University of Chinese Medicine Cooperative Academy of Mathematical Engineering for Chinese Medicine,Guangzhou University of Chinese Medicine, ³School of Basic Medical Sciences,Guangzhou University of Chinese Medicine

Overview

This protocol provides a detailed method for isolating primary neurons and glial cells from rat bladder, serving as a foundation for further cellular experiments. The study aims to understand bladder neuron dysfunctions by utilizing a co-culture approach, enhancing cellular survival and efficiency.

Key Study Components

Area of Science

Cellular biology
Neuroscience
Physiological dysfunction studies

Background

Investigating neuron-glia interactions is crucial for understanding bladder physiology.
Existing protocols lack efficiency and reliability in cellular isolation.
There is a need for in vitro models to study bladder neuron dysfunction.
Research in this area may reveal insights into various disease mechanisms.

Purpose of Study

To develop a repeatable and efficient protocol for isolating bladder neurons and glia.
To facilitate further study of bladder neuron dysfunctions.
To provide a reliable in vitro model for cellular physiology experiments.

Methods Used

Isolation of primary neurons and glia from rat bladder tissues.
A detailed co-culture approach was implemented using surgical techniques for tissue extraction.
The protocol emphasizes rapid cellular digestion and efficient media handling.
Key timelines include a six-hour total isolation time and specific incubation periods for digestion.
Immunostaining techniques were employed to identify neuronal and glial cell types post-isolation.

Main Results

Successful isolation and culture of mature neurons exhibiting classical growth characteristics.
Distinct identification of different neuronal subtypes through immunohistochemistry.
Significant structural and functional maturation of neurons observed over five to seven days.
Validation of protocol efficacy demonstrated by synaptic spine development observed via immunostaining.

Conclusions

This study establishes a reliable protocol for isolating bladder neurons and glia.
The method enables exploration of physiological dysfunctions in bladder neurons.
Findings have implications for understanding neuronal mechanisms involved in bladder disorders.

Frequently Asked Questions

What are the advantages of this isolation protocol?

This protocol allows for high-efficiency isolation of bladder neurons and glia, completing the entire process in six hours.

How is the tissue extracted from the rat?

Rats are surgically prepared, and the bladder is carefully isolated and placed in a cold solution to enhance cell survival.

What types of cells can be isolated using this method?

The protocol allows for the isolation of primary neurons and glial cells from rat bladder tissue, facilitating further studies on their interactions.

How can the isolated cells be used in future studies?

These cells can be cultured and analyzed to explore physiological dysfunctions, neuronal mechanisms, and potential therapeutic interventions.

What important steps are involved in the digestion process?

The digestion involves careful segmentation and incubation protocols to ensure optimal recovery of viable cells for culture.

What techniques are used to identify the cells post-isolation?

Immunostaining methods allow for the identification of different neuronal subtypes and glia using specific antibodies.

Are there any limitations to this protocol?

Considerations such as the need for meticulous surgical techniques and proper timing in the steps are crucial for successful outcomes.

This protocol attempts to establish a repeatable protocol for primary neurons and glia isolation from rat bladder for further cellular experiments.

This co-culture method may lead towards cellular understanding of physiological dysfunction in phenotypes, such as bladder neuron dysfunctions. The main advantage of this protocol is high efficiency. The entire isolation can be completed in six hours.

Demonstrating the procedure will be Jiao Zhang, a postgraduate student from my laboratory. Begin by placing rats on a sterilized surgical towel and exposing the abdomen. Use scissors and forceps to open the abdominal cavity and reveal the bladder.

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