The olfactory stimulation slows down the substance clearance in the extracellular space of the hippocampus in rat brain

https://doi.org/10.1016/j.bbrc.2019.05.159Get rights and content

Highlights

  • In this study, the effect of olfactory stimulation on substance clearance in the interstitial space of the hippocampus had been explored.

  • Eugenol can enhance the functions of neurons in the hippocampus, but it can slow down the ISF clearance.

  • Aqp4 is involved in the regulation of ISF drainage and influences the structure of ECS.

Abstract

Accelerating the clearance of toxin in the brain extracellular space (ECS) has grown a promising strategy for treating some central nervous system diseases. As oldest sensory system, we know little about the influence of olfaction on the brain, but preclinical studies such as treatment of neurological diseases through it are in the ascendant. This makes it important to clarify the effects of olfaction on brain ECS and interstitial fluid (ISF) drainage. In this study, the effect of olfactory stimulation (eugenol, EUG) on ISF flow in hippocampus and its association with aquaporin 4 (Aqp4) had been investigated. The results show that eugenol can significantly increase the activity of hippocampal neurons, but reduce the clearance and diffusion rates of Gd-DTPA and A-594 in hippocampus. Similarly, eugenol inhalation slows down the rate of Gd-DTPA in CSF entering the hippocampus and its clearance. And knockout of Aqp4 gene aggravated these processes. In vitro results showed that after Aqp4 gene silencing, astrocytes grew slowly, with significantly decreased cells number, less nuclei, atrophied bodies and shorter processes. These results concluded that olfactory stimulation can change the ECS structure of the hippocampus, slow down the ISF drainage, and improve the function of neurons, while Aqp4 plays important roles.

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).

References (30)

  • H. Mestre et al.

    Aquaporin-4-dependent glymphatic solute transport in the rodent brain

    eLife

    (2018)
  • L. Xie et al.

    Sleep drives metabolite clearance from the adult brain

    Science (New York, N.Y.)

    (2013)
  • M. Jucker et al.

    Neurodegeneration: amyloid-beta pathology induced in humans

    Nature

    (2015)
  • C. Shi et al.

    Transportation in the interstitial space of the brain can Be regulated by neuronal excitation

    Sci. Rep.

    (2015)
  • S. Fujisawa et al.

    Eugenol and its role in chronic diseases

    Adv. Exp. Med. Biol.

    (2016)
  • Cited by (4)

    • Preventive effects of a standardized flavonoid extract of safflower in rotenone-induced Parkinson's disease rat model

      2022, Neuropharmacology
      Citation Excerpt :

      ECS contains brain interstitial fluid (ISF) and occupies 15%–20% of the living brain by volume (Lei et al., 2017; Tønnesen et al., 2018). Substances in the brain ECS provide the living and working environments for neuronal activities (Colbourn et al., 2019; Pajevic, 2019; Patel et al., 2019), also control memory, sleep, cognition, and sensory formation (Li et al., 2020; Zhang et al., 2019; Zhao et al., 2020). DA-ergic neuronal loss, astrocyte activation, and protein deposition are the most common physiological processes of PD, thus, observing the changes in ECS diffusion parameters can provide important information for determining the pathogenesis and pathological changes of PD and further reveal the preventive measures of PD.

    View full text