The olfactory stimulation slows down the substance clearance in the extracellular space of the hippocampus in rat brain
Introduction
The glymphatic system is a pathway involved in the transport of metabolites and waste removal, consisting of extracellular space (ECS) in the brain. It is named glymphatic system because of its function similar to the lymphatic system in the body [1]. This pathway includes the para-artery pathway of cerebrospinal fluid (CSF) into the brain parenchyma, and the clearing pathway of interstitial fluid (ISF) from the brain and spinal cord. The clearance of soluble proteins, waste and excess extracellular fluid in the brain is achieved by flow of ISF, which is regulated by astrocyte-4 (Aquapin-4, Aqp4) [2]. In brain fluid, the extracellular fluid, ie interstitial fluid occupies 12%–20% of the total brain volume [3]. Normally, the ISF contains abundant nutrients, hormones, peptides, neurotransmitters and waste products. Therefore, ECS is pivotal for maintaining the homeostasis of brain. In some central nervous system (CNS) diseases, the poisonous substance released from various cells is accumulated in ECS, such as glutamate [4] and Aβ [5], which are harmful to the neural cells. How to eliminate these toxicants is a new hot spot for CNS disease treatment, consequently, the basic properties of ISF in brain have grown a prerequisite demand.
The ISF clearance can be modulated by the body's condition (e.g sleep/awake status) [3] and neuronal excitation (e.g painful stimulation) [6]. It is reported that the neuronal excitement produced by odorant molecules can be transmitted from olfactory fila to the hippocampus and facilitate the development of smell memory [7]. The pre-clinical studies of olfactory pathway used to treat neurological diseases have also been reported, such as Eugenol (EUG) has been proven effective and clinically useful in the treatment of stroke through the olfactory pathway [8]. However, the effects of this process on the ECS drainage are still to be cleared. Therefore, in this study, using Eugenol inhalation, we observed the effects of olfactory stimulation on the ISF clearance in hippocampus.
The ISF drainage is influenced by several factors, such as the tortuosity, volume fraction of ECS and extracellular matrix [9]. The boundary structure of ECS is made of the cell membrane and the wall of blood vessels [10]. Therefore, the volume of neural cells will definitely impact the ECS characters. Aqp4 is a kind of water channels, predominantly expressed on the astrocyte endfoot along the blood-brain barrier [11]. After neuroexcitation, K+ released from neurons is partly absorbed by astrocytes with aid of K+ transporters and Aqp4 [12], which accompany with osmotic water influx and astrocyte swelling. This process results in declined ECS volume and consequently accelerate the ISF clearance [13].
There are four methods to exploring the ECS in the living brain: ion-selective microelectrodes, microdialysis, integrative optical imaging and tracer-based MR imaging [14]. So far, MR imaging method, gadolinium-diethylene triamine pentaacetic acid (Gd-DTPA) as a tracer, is regard as the only method that can detect the dynamic ISF drainage in the whole brain. According to the classical diffusion equation, the diffusion coefficient, clearance coefficient and half-life of Gd-DTPA can be calculated from the concentration-time profile [15]. In this study, using MRI and integrative optical imaging methods, the effects of olfactory stimulation on the ISF clearance in hippocampus were observed, and its mechanism associated with Aqp4 was also explored.
Section snippets
Materials and methods
All the methods used in this study were assessed and approved by the Animal Care and Use Committee at Peking University Health Sciences Center, following the Guidelines for the Use of Animals in Neuroscience Research by the Society for Neuroscience (Beijing, Certificate No. SCXK 2002-0001).
Establishment of Aqp4 knockout rat model
The Aqp4−/− rat was established in this study. The genotype of the rat was determined using gene sequencing. The Aqp4−/− rats were lack of a 4-base pair segment (Fig. 1A–C). The immunohistochemistry staining showed that there was highly positive expression of Aqp4 in the endfeet of astrocyte surrounding the microvessel, which was absent in the Aqp4 knockout rats (Fig. 1D). The Western blot results indicated that the expression level of Aqp4 was significantly decreased in Aqp4 knockout rats (
Discussion
In this study, we found that the olfactory stimulation (EUG inhalation) slowed down the ISF clearance in hippocampus and consequently enhanced the neuron's functions, which might be partly modulated by Aqp4 protein.
In some CNS diseases, such as stroke, there are numerous toxicant released from the injured neurons and glial cells, such as glutamate [21] and inflammatory factors, which worsen the pathological process. In this study, we found that the Gd-DTPA in the hippocampus could be cleared
Conflict of interest
None.
Acknowledgments
This work had been supported by the National Natural Science Foundation of China (Grant No. 31471028) and the Seeding Grant for Medicine and Engineering Sciences of Peking University (Grant No.2014-ME-02).
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