Real-Time Dynamic Collection of Hippocampal Extracellular Fluid from Conscious Rats Using a Microdialysis System

Summary

The protocol here provides a detailed real-time dynamic sampling of extracellular fluid from the hippocampus of awake rats using a microdialysis system.

Abstract

A variety of central nervous system (CNS) diseases are associated with changes in the composition of hippocampal extracellular fluid (HECF). However, difficulty in obtaining HECF in real time from conscious rats has long restricted the evaluation of CNS disease progression and the effectiveness of ethnomedicine therapy. Encouragingly, a brain microdialysis technique can be used for continuous sampling with the advantages of dynamic observation, quantitative analysis, and a small sampling size. This allows the monitoring of changes in the extracellular fluid content for compounds from traditional herbs and their metabolites in the brain of living animals. The aim of this study was thus to accurately implant a cerebrospinal fluid microdialysis probe into the hippocampal region of Sprague Dawley (SD) rats with a three-dimensional brain stereotaxic apparatus, cutting off molecular weights greater than 20 kDa. The high-quality HECF was then obtained from conscious rats using a microdialysis sampling control system with an adjustable sampling rate from 2.87 nL/min - 2.98 mL/min. In conclusion, our protocol provides an efficient, rapid, and dynamic method to obtain HECF in awake rats with the help of microdialysis technology, which provides us with unlimited possibilities to further explore the pathogenesis of CNS-related diseases and evaluate drug efficacy.

Introduction

Central nervous system (CNS) diseases with high morbidities, such as neurodegenerative disease, traumatic brain injury, high-altitude hypoxia-induced brain injury, and ischemic stroke, are crucial causes of the growing mortality worldwide^1,2,3. Real-time monitoring of cytokines and protein changes in specific brain regions contributes to the diagnostic accuracy of CNS diseases and brain pharmacokinetic studies after medication. Traditional scientific research uses brain tissue homogenate or a manual collection of animal interstitial brain fluid for the detection of specific s....

Protocol

The experimental protocol was conducted in accordance with the requirements of the Use of Laboratory Animals and Institutional Animal Care and Use Committee at Chengdu University of Traditional Chinese Medicine (Record number: 2021-11). Male Sprague Dawley (SD) rats (280 ± 20 g, 6-8 weeks old) were used for the present study.

1. Brain microdialysis probe implantation surgery

1. Use 3% and 1.5% isoflurane for the induction and maintenance of rat anesthesia, respectively, using an animal anesthesia system in an air-oxygen mixture at 0.6 L/min. Make sure that the rats are deeply anesthetized without both a pain r....

Results

Following the above experimental protocol and the sampling parameters set in Table 1, water-like, colorless, and transparent rat HECF was obtained at the set sampling rate (Figure 1K). The osmotic pressure of the obtained rat HECF was 290-310 mOsm/L, which can indirectly ensure the quality of samples^18,19.

Discussion

The pathogenesis of CNS diseases is still not fully understood, which hinders the development of new therapies and drugs. Studies have shown that most CNS diseases are closely related to hippocampal lesions^20,21,22. The proposed brain microdialysis technique can target specific regions of the brain, especially the hippocampus, which makes it stand out from the traditional approach of collecting HECF. Probes are placed in the CA1.......

Materials

Name	Company	Catalog Number	Comments
Air-drying oven	Suzhou Great Electronic Equipment Co., Ltd	GHG-9240A
Animal anesthesia system	Rayward Life Technology Co., Ltd	R500IE
Animal temperature maintainer	Rayward Life Technology Co., Ltd	69020
Artificial cerebrospinal fluid	Beijing leagene biotech. Co., Ltd	CZ0522
Brain microdialysis probe	CMA Microdialysis AB	T56518
Catheter	CMA Microdialysis AB	T56518
Covance infusion harness	Instech Laboratories, Inc.	CIH95
Denture base resins	Shanghai Eryi Zhang Jiang Biomaterials Co., Ltd	190732
Electric cranial drill	Rayward Life Technology Co., Ltd	78001
Electric shaver	Rayward Life Technology Co., Ltd	CP-5200
Free movement tank for animals	CMA Microdialysis AB	CMA120
Heparin sodium injection	Chengdu Haitong Pharmaceutical Co., Ltd	H51021208
Iodophor	Sichuan Lekang Pharmaceutical Accessories Co., Ltd	202201
Isofluran	Rayward Life Technology Co., Ltd	R510-22
Microdialysis catheter stylet	CMA Microdialysis AB	8011205
Microdialysis collection tube	CMA Microdialysis AB	7431100
Microdialysis collector	CMA Microdialysis AB	CMA4004
Microdialysis fep tubing	CMA Microdialysis AB	3409501
Microdialysis in vitro stand	CMA Microdialysis AB	CMA130
Microdialysis microinjection pump	CMA Microdialysis AB	788130
Microdialysis syringe (1.0 mL)	CMA Microdialysis AB	8309020
Microdialysis tubing adapter	CMA Microdialysis AB	3409500
Non-absorbable surgical sutures	Shanghai Tianqing Biological Materials Co., Ltd	S19004
Ophthalmic forceps	Rayward Life Technology Co., Ltd	F12016-15
Osmometer	Löser	OM 807
Sodium hyaluronate eye drops	URSAPHARM Arzneimittel GmbH	H20150150
Stereotaxie apparatus	Rayward Life Technology Co., Ltd	68025
Surgical scissors	Rayward Life Technology Co., Ltd	S14014-15
Surgical scissors	Shanghai Bingyu Fluid technology Co., Ltd	BY-103
Syringe needle	CMA Microdialysis AB	T56518
Trypsin solution	Boster Biological Technology, Ltd.	PYG0107
Ultrasonic cleaner	Guangdong Goote Ultrasonic Co., Ltd	KMH1-240W8101