Endothelin-1-Mediated Brainstem Glial Activation Produces Asthmatic Airway Vagal Hypertonia via Enhanced ATP-P2X4 Receptor Signaling

Neurology Medicine Respiratory System Basic Medical Sciences Life Sciences Immunology
Endothelin-1 Brainstem glial activation Airway vagal hypertonia ATP-P2X4 receptor Asthma Sprague–Dawley rats

Endothelin-1 Mediated Brainstem Glial Activation Produces Asthmatic Airway Vagal Hypertonia via Enhanced ATP-P2X4 Receptor Signaling

Academic Background

Asthma is one of the major non-communicable diseases globally, causing a heavy social and economic burden. Its main clinical symptoms include airflow obstruction, chronic airway inflammation, increased sensitivity, and hyperresponsiveness. The occurrence of these symptoms is closely related to airway vagal hypertonia and enhanced airway vagal reflexes. In recent years, research has found that the activity of bronchial branches is a key factor in the enhancement of airway vagal reflexes in asthma patients. However, the central nervous mechanism remains unclear.

In animal models, increased excitability of central neurons in the airway vagal reflex arc not only amplifies reflex output but may also lead to “asthmatic airway vagal hypertonia,” causing asthma symptoms even in the absence of airway stimulation. Previous studies have found that in allergic asthma rat models, brainstem extracellular ATP (adenosine triphosphate) concentrations increase, mainly related to decreased expression and activity of ecto-5’-nucleotidase (CD73), an enzyme associated with extracellular ATP degradation. Extracellular ATP may enhance the excitability of the airway vagal reflex arc in the central nervous system of asthma patients by activating purinergic P2X receptors (P2X4R) to increase the excitability of airway vagal preganglionic neurons.

Paper Source

This paper, titled “Endothelin-1 mediated brainstem glial activation produces asthmatic airway vagal hypertonia via enhanced ATP-P2X4 receptor signaling in Sprague-Dawley rats,” was published in the Journal of Neuroimmune Pharmacology (2024) 19:13. The authors include Yun Lin, Tian Liu, Hong Chen, et al., all from the Department of Basic Medical Sciences at Fudan University. The paper was received on November 29, 2022, and accepted on April 1, 2024.

Research Details

The experimental design of this study includes using ovalbumin (OVA) to prepare an asthmatic rat model, evaluating airway vagal tone, and observing brainstem changes. The experiment is divided into several steps, as follows:

Research Process

  1. Experimental Animals: Seven-week-old male Sprague-Dawley rats (180±10 g) were purchased from the Shanghai Experimental Animal Center of the Chinese Academy of Sciences.

  2. Asthmatic Rat Model Preparation: Rats were divided into control and OVA sensitization groups. Sensitized rats received intraperitoneal injections of OVA (10 mg) and Al(OH)3 (2 mg) on days 0 and 7, followed by daily 5% OVA aerosol challenge from days 14 to 28. The control group received an equal volume of saline.

  3. Experimental Procedures: In some experiments, some rats received injections of the bromide fungicide bq788 or adeno-associated virus (AAV) carrying shRNA, aimed at inhibiting P2X4 receptors.

  4. Detection of Airway Vagal Tone: The intensity of recurrent laryngeal nerve discharge (RLD) was evaluated by electrophysiological recording methods. A catheter was placed for immediate cerebrospinal fluid (CSF) sample collection and ATP concentration determination.

Experimental Results

  1. Enhanced ET-1/EDNRB and ATP/P2X4R Signaling: ELISA experiments showed significantly increased ET-1 content in the brainstem of OVA-sensitized rats. RT-qPCR and Western blot results showed significantly increased expression of EDNRB and P2X4R receptors.

  2. Glial Cell and Neuron Activation: Immunofluorescence staining showed that EDNRB is mainly expressed in astrocytes, while P2X4R is expressed in neurons and significantly increased in both control and OVA groups. OVA-sensitized rats showed significant glial cell activation, with enhanced signals for astrocytes (GFAP) and microglia (IBA1).

  3. Airway Vagal Hypertonia: Electrophysiological recordings showed significantly increased RLD intensity during the inspiratory phase in OVA-sensitized rats, which could be significantly inhibited by P2X4R antagonist (5-BDBD) and brainstem P2X4R gene knockdown therapy.

  4. Significant Effects of Bq788 Treatment: Chronic intraperitoneal injection of Bq788 significantly alleviated allergically enhanced RLD intensity and lung function decline, while also reversing changes in P2X4R characterization, CD73 activity, and ATP concentration in CSF.

Conclusions and Significance

This is the first direct electrophysiological recording of recurrent laryngeal nerve discharge to detect airway vagal hypertonia in an asthma model. The results indicate that EDNRB plays a key role through astrocyte activation, and enhanced ATP/P2X4R signaling in central neurons leads to enhanced airway vagal reflexes. P2X4R antagonists may have potential in alleviating asthma symptoms in the future.

Research Highlights

  1. Clear EDNRB and P2X4R Signaling: EDNRB and P2X4R signaling were significantly enhanced in OVA-sensitized rats, clarifying their roles in asthmatic airway vagal reflexes.

  2. Effective Drug Intervention: Chronic Bq788 treatment effectively reversed allergically activated glial cells and reduced CD73 activity, showing great potential for future asthma treatment.

  3. Methodological Innovation: The method of electrophysiologically recording recurrent laryngeal nerve discharge provides a new sensitive technique for detecting experimental airway vagal hypertonia, which is highly innovative.

This study not only provides new clues for understanding the central mechanism of asthma but also offers new ideas for future asthma treatment.