To investigate the efficacy of varying doses of methylprednisolone (MP) on mice with acute exacerbations of chronic obstructive pulmonary disease (AECOPD) induced with influenza A virus (IAV).

Mouse model of COPD was established using LPS combined with smoking for 12 weeks, and then these COPD mice were treated with administration of 40 μL IAV via nasal drip to establish a AECOPD model. A total of 15 AECOPD mice were randomly divided into low-, medium- and high-dose MP groups, oseltamivir group and blank group. The body weight and survival time were monitored within 10 d after IAV infection. On days 1, 3, and 5 post-treatment, lung function was assessed using whole-body plethysmography (WBP), inflammatory factors in bronchoalveolar lavage fluid (BALF) were quantified with ELISA, viral titers in BALF were determined using plaque assays, and colony-forming units were evaluated with blood agar plates. Immunofluorescence analysis: ① Pulmonary immunofluorescence assay: Mice were randomly categorized into (n=4): LPS 1-day group, LPS 3-day group, and LPS+MP treatment group. All groups received an initial dose of LPS via atomization; subsequently, the LPS+MP treatment group received a single gavage dose of MP. Lung tissues were harvested from the 1-day LPS group on 1 d post-treatment, and from the 3-day LPS and LPS+MP groups on 3 d for immunofluorescence staining. ② Cellular immunofluorescence assay: Mouse bone marrow neutrophils were classified into blank control (no intervention), LPS stimulation (LPS group), MP intervention with LPS stimulation (LPS+MP group), and MP intervention alone (MP group). The above cells were collected in 4 h after corresponding interventions for subsequent cellular immunofluorescence analysis.

① The medium-dose MP group demonstrated the most significant improvement in survival rate, weight recovery, and lung function when compared to other groups (P< 0.05). ② Treatment of medium-dose MP obviously reduced the levels of IL-6 and neutrophil extracellular traps (NETs) (P< 0.05), while, elevated inflammatory factors and NETs were observed in the high-dose MP group on day 5 post-treatment. ③ Notable decline in the lung injury score was found in the medium-dose MP group than the other groups (P< 0.05). ④ The high-dose MP group exhibited substantial bacterial proliferation and delayed viral clearance since day 5 after treatment.

Medium-dose MP shows best efficacy in treatment of IAV-induced AECOPD, and the dose neither delays viral clearance nor increases the risk of bacterial infection following viral infection.

To probe the spatial distribution of respiratory-related nuclei throughout the whole brain, and to investigate the circuit and projection links between the paraventricular hypothalamic nucleus (PVN) and the preBötzinger complex (preBötC) in mice.

Using a retrograde tracing strategy from peripheral to central, pseudorabies viruses (PRV) were microinjected into the diaphragm of mice to retrogradely label the upstream inputs brain regions from spinal respiratory phrenic motor neurons. The anterograde non-transsynaptic tracing viruses (AAV2/9-EGFP) were microinjected into the PVN to label the downstream brain region of the PVN; And using the Cre/loxP gene expression strategy, Dio-EGFP and Retro-Cre viruses were injected in the PVN and preBötC, respectively, to validate the PVN-preBötC circuit and the projection relationship between the two.

The results of PRV-mediated retrograde tracing showed that PRV-EGFP-positive neurons were distributed in several brain regions such as PVN, preBötC, nucleus tractus solitarii (NTS), locus coeruleus (LC) and periaqueductal grey (PAG). Except for the medulla oblongata region, the PVN brain region had the highest percentage of PRV-EGFP-positive neurons (P<0.0001). After AAV2/9-EGFP viruses were injected in the PVN, green neurofibrils were observed in the preBötC brain region after viral infection; Dio-EGFP and Retro-Cre viruses were injected in the PVN and preBötC, respectively, and green neuronal cells were seen in the brain region of the PVN after viral infection.

PVN is an important upstream input nucleus of spinal respiratory phrenic motor neurons, and has the potential to regulate respiration through direct projections from PVN-preBötC circuit.