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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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

Abstract

The lower urinary tract has two main functions, namely, periodic urine storage and micturition; these functions are mediated through central and peripheral neuroregulation. Although extensive research on the lower urinary tract nervous system has been conducted, most studies have focused on primary culture. This protocol introduces a method for the isolation and culture of bladder neurons and glia from Sprague–Dawley rats. In this method, the neurons and glia were incubated in a 37 °C, 5% CO2 incubator for 5–7 days. As a result, they grew into mature shapes suitable for related subsequent immunofluorescence experiments. Cells were morphologically observed using an optical microscope. Neurons, synaptic vesicles, and glia were identified by β-III-tubulin and MAP-2, Synapsin-1, and GFAP staining, respectively. Meanwhile, immunocytochemistry was performed on several neurotransmitter-related proteins, such as choline acetyltransferase, DYNLL2, and SLC17A9.

Introduction

The lower urinary tract has two main functions: periodic urine storage and micturition1. The lower urinary tract nervous system (LUTNS) controls these functions and is delicate and susceptible to many neuropathies, which can be innate (porphyria), acquired (Lyme disease), secondary to disease states (diabetic cystopathy), drug induced (hemorrhagic cystitis), surgery caused (abdominoperineal resection), or injury caused (traumatic spinal cord injury)2,3,4,5,6,

Protocol

All experimental protocols and animal procedures complied with the ethical principle guidelines of the National Research Council.

1. Preparation of materials

  1. Sterilize all instruments and ddH2O using an autoclave before performing the experiment. Instruments include but are not limited to surgical scissors, ophthalmic scissors, forceps, spoons nucleus divider, glass dishes (60–100 mm in diameter), and glass breakers.
  2. Prepare the Krebs solution as follows .......

Representative Results

In the process of primary cell culture, the cells acquired were round with bright and clear boundaries before the attached state. As the neurons grew, dendrites and axons started to be distinct. After 5–7 days of culture, the neurons reached a mature form with long projections, which were ideal for imaging or function studies. Although most of the impurities and cell debris could be removed due to changing media, certain residuals attached to poly-D-lysine and laminin coating were visible (Figu.......

Discussion

Plate Preparation
The use of glass coverslips in 6-, 12-, or 48-well culture plates for immunofluorescent or calcium imaging experiments is an economical and sample-sparing operation. Cells grow well in plates without coverslips during the preparation of primary cell cultures. Therefore, coverslips are dispensable in experiments, such as Western blot or polymerase chain reaction. Furthermore, coating is a necessary step before plating cells, with or without coverslips. Laminin and poly-D-lysine are.......

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Grant no. 81673676) and Dongguan Science and Technology Bureau (Grant no. 2019622101002). The authors thank Dr. Maryrose Sullivan (Assistant Professor in Surgery, Harvard Medical School) for technical consulting.

....

Materials

NameCompanyCatalog NumberComments
0.25% trypsinGibico15050065Enzyme digestion
48-well culture plateCorning3548Coating dish
antibiotic/antimycoticGibico15240062Culture media/Rinse media
Anti-Glial Fibrillary Acidic Protein AntibodySanta Cruzsc-33673ICC
B-27Gibico17504044Culture media
BSA Fraction VGibico332Enzyme digestion
Choline Acetyltransferase AntibodyAbcamab18736ICC
CO2 IncubatorHeraeusB16UUCells culture
Collagenase type IISigma2593923Enzyme digestion
DMEM/F-12Gibico11330032Rinse media
DYNLL2 AntibodySanta Cruzsc-13969ICC
Fetal Bovine SerumGibico10100147Culture media/Rinse media
ForcepsShanghai Jin Zhong Medical Devices138310 cm; Sterile operation
Glass breakersHuan Qiu Medical Devices110150 ml; Sterile operation
Glass coverslipsWHB ScientificWHB-48-CSCoating dish
Glass dishesHuan Qiu Medical Devices1177100 mm; Sterile operation
Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 488Southernbiotech3050-30ICC
Goat Anti-Rat IgG(H+L), Mouse ads-Alexa Fluor 555Southernbiotech3050-30ICC
Hoechst 33342BD561908ICC
Laminar flow benchSu Jie Medical DevicesCB 1400VSterile operation
LamininSigmaL2020Coating dish
L-glutamineGibico25030081Culture media
MAP-2 AntibodyAffinityAF5156ICC
Murine GDNFPeprotechAF45044Culture media
Neurobasal-A MediumGibico10888022Culture media
Ophthalmic scissorsShanghai Jin Zhong Medical DevicesJ2101012.5 cm; Sterile operation
PipettesEppendorf3120000240100-1000 ul; Reagent and sample pipetting
PipettesEppendorf312000026710-100 ul; Reagent and sample pipetting
Poly-D-lysineSigmaP7280Coating dish
Refrigerated centrifugePing Fan InstrumentTGL-16AEnzyme digestion
Shaking incubatorHaimen Kylin-Bell Lab InstrumentsT8-1Enzyme digestion
SLC17A9 AntibodyMBL InternationalBMP079ICC
Spoons nucleus dividerShanghai Jin Zhong Medical DevicesYZR03012 cm; Sterile operation
Substance P AntibodySanta Cruzsc-58591ICC
Surgical scissorsShanghai Jin Zhong Medical DevicesJ2113016 cm; Sterile operation
Surgical towelFu Kang Medical Devices500240 x 50 cm; Sterile operation
Synapsin-1 AntibodyCST5297TICC
Tubulin beta Antibody(β-III-tubulin)AffinityAF7011ICC

References

  1. Fowler, C. J., Griffiths, D., de Groat, W. C. The neural control of micturition. Nature Reviews Neuroscience. 9 (6), 453-466 (2008).
  2. Golbidi, S., Laher, I. Bladder dysfunction in diabetes mellitus. Frontiers in Pharmacology. 1, 136....

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