This work was supported by the National Natural Science Foundation of China (82104533), the Science &#38; Technology Department of Sichuan Province (2021YJ0175), and the China Postdoctoral Science Foundation (2020M683273). The authors would like to thank Mr. Yuncheng Hong, a senior equipment engineer at Tri-Angels D&#38;H Trading Pte. Ltd. (Singapore) for providing technical services for the microdialysis technique.

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 &#177; 20 g, 6-8 weeks old) were used for the present study.
1. Brain microdialysis probe implantation surgery
<ol>
	<li>Use 3% and 1.5% isoflurane for the induction and maintenance of rat anesthesia, respec...

<ol>
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The authors have nothing to disclose.

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 lesions20,21,22. 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...

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 worldwide1,2,3. 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 substances and for pharmacokinetic studies. However, this has some shortcomings, such as a limited sample size, the inability to dynamically observe the changes of indicators, and uneven sampling quality4,5,6. Cerebrospinal fluid, an interstitial fluid, protects the brain and spinal cord from mechanical damage. Its composition is different from that of the serum due to the existence of the blood-brain barrier (BBB)7. Direct analysis of cerebrospinal fluid samples is more conducive to disclosing the mechanism of CNS lesions and drug discovery. Inevitably, the cerebrospinal fluid samples, manually obtained directly from the cisterna magna and cerebral ventricles through a syringe, have disadvantages of blood contamination, a random chance of sample collection, uncertainty of quantity, and almost no multiple retrieval possibility8,9. More notably, conventional interstitial brain fluid sampling methods cannot obtain samples from damaged brain regions, which hinders the exploration of the pathogenesis of CNS diseases in specific brain regions and the efficacy evaluation of targeted ethnomedicine therapies9,10.
Brain microdialysis is a technique for sampling interstitial brain fluid in awake animals11. The microdialysis system imitates vascular permeability with the help of a probe implanted in the brain. The microdialysis probe is armed with a semi-permeable membrane and is implanted in specific brain regions. After perfusion with isotonic artificial cerebrospinal fluid (ACSF), the dialyzed interstitial brain fluid can be favorably collected with the benefits of small sample sizes, continuous sampling, and dynamic observation12,13. In terms of location, brain microdialysis probes can be selectively implanted into brain structures or cranial cisterns of interest14. An observation of abnormal levels of an endogenous substance in the hippocampal extracellular fluid (HECF) suggests the occurrences of CNS diseases or the pathogenesis of disease. Several studies have shown that the biomarkers for CNS diseases, such as D-amino acids in schizophrenia, &#946;-amyloid and tau proteins in Alzheimer&#39;s disease, neurofilament light chains in traumatic brain injury, and ubiquitin carboxy-terminal hydrolase L1s in hypoxic ischemia encephalopathy, can be analyzed in cerebrospinal fluid15,16,17. A chemical analysis method based on the brain microdialysis sampling technique can be used to monitor dynamic changes of exogenous compounds such as active ingredients of ethnomedicine, which diffuse and distribute in specific brain regions14.
This article presents the specific process of dynamic HECF acquisition in awake rats and measures its osmotic pressure to ensure sample quality.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
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			<td>8011205</td>
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			<td>Microdialysis collection tube</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>7431100</td>
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			<td>Microdialysis collector</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>CMA4004</td>
			<td></td>
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			<td>Microdialysis fep tubing</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>3409501</td>
			<td></td>
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			<td>&#160;CMA Microdialysis AB</td>
			<td>CMA130</td>
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			<td>Microdialysis microinjection pump</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>788130</td>
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			<td>Microdialysis syringe (1.0 mL)</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>8309020</td>
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			<td>Microdialysis tubing adapter</td>
			<td>&#160;CMA Microdialysis AB</td>
			<td>3409500</td>
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			<td>Shanghai Tianqing Biological Materials Co., Ltd</td>
			<td>S19004</td>
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			<td>F12016-15</td>
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			<td>L&#246;ser</td>
			<td>OM 807</td>
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			<td>URSAPHARM Arzneimittel GmbH</td>
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			Biological Technology, Ltd.</td>
			<td>PYG0107</td>
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real-time dynamic collection of hippocampal extracellular fluid from conscious rats using a microdialysis system

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.

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 samples18,19.
<img alt="Figure 1" class="xfigimg" src="...

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

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

A variety of central nervous system (CNS) diseases are associated with changes in the composition of hippocampal extracellular fluid (HECF). However, ...

Hippocampal extracellular fluid and crowd by brain microdialysis technique contributes to monitor the disease specific micro of central nervous diseases. Brain microdialysis technical achieves dynamic and the continuous inquisition of interstitial brain fluid in specific brain regions of non-anesthetized rats. Begin by fixing the anesthetized rat on a stereotaxic brain locator and wipe the shaved surgical area with a povidone iodine and ethanol swab three times before the operation.Then make a 1.5 centimeter cranial facial incision down the middle with surgical scissors and remove the periosteum using surgical scissors and ophthalmic forceps. Considering the bregma as the basal position, using a cranial drill pierce the endocranium to drill a two millimeter aperture at the hippocampal CA1 region. Now fix the catheter stilette on the gripper of a stereotaxic brain locator and adjust the position of the microdialysis casing at the anteroposterior, mediolateral, and dorsal ventral positions.Adjust the dorsoventral valve of the brain stereo locator and implant the microdialysis casing into the CA1 region at a depth of 3.5 millimeters. Drill three more apertures with a two millimeter diameter such that the three apertures form a triangle in which the probe aperture is centrally-located. Implant screws into the apertures at a depth of one millimeter and fix the probe catheter with dental cement.Then use a 4-O surgical suture to close the skin. Connect the microdialysis pump microsyringe with ACSF awake activity device and cryogenic sample collector according to the manufacturer's instructions. Next, to discharge the air in the pipeline, set the microdialysis pump at a rate of one microliter per minute.Then connect the pipeline and brain microdialysis probe and operate the microdialysis pump at a rate of one microliter per minute to inject ACSF into the probe until the surface of the probe is slightly moist. Immerse the probe in a heparin sodium injection solution for subsequent use. To collect the HECF from the awake rat, insert the brain microdialysis probe into the probe catheter and place the rat in a chamber with padding to ensure the rats are free to move around.Turn on the multi-channel swivel controller to avoid intertwining the microdialysis pipelines during the free movement of the rat. Then turn on the microsyringe pump and pump the ACSF at a rate of one microliter per minute. After a 60 minute equilibration of the microdialysis HECF collection system, collect HECF periodically.After sampling termination, take the brain microdialysis probe out from the probe catheter. Immerse the probe in deionized water and lavage with deionized water for 12 hours to remove stranded salt depositions from the pipeline and the probe. Remove the probe and place it into a 0.05%tripsin solution at four degrees Celsius.Hippocampal extracellular fluid acquired by the brain microdialysis technique is water-like, colorless, and transparent at the set sampling rate. The most important procedures are implanting the microdialysis carefully into the CA1 region and inserting the probe into the pop catheter. The microdialysis simply technique makes it possible to assist the dynamical change of disease markers in specific brain regions, the substance of drug's efficacy, and the tissue catabolism of drug components.

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Research

JoVE Journal

Neuroscience

2.2K vistas.  Chengdu University of Traditional Chinese Medicine. El líquido extracelular del hipocampo y la multitud mediante la técnica de microdiálisis cerebral contribuyen a monitorear la enfermedad micro específica de las enfermedades nerviosas centrales. La técnica de microdiálisis cerebral logra la inquisición dinámica y continua de líquido cerebral intersticial en regiones cerebrales específicas de ratas no anestesiadas. Comience fijando la rata anestesiada en un localizador cerebral estereotáxico y limpie el área quirúrgica afeitada co...