Inhalation of Atmospheric-Pressure Gas Plasma Attenuates Brain Infarction in Rats With Experimental Ischemic Stroke

Abstract

Previous studies suggest the potential efficacy of neuroprotective effects of gaseous atmospheric-pressure plasma (APP) treatment on neuronal cells. However, it remains unclear if the neuroprotective properties of the gas plasmas benefit the ischemic stroke treatment, and how to use the plasmas in the in vivo ischemic stroke models. Rats were subjected to 90 min middle cerebral artery occlusion (MCAO) to establish the ischemic stroke model and then intermittently inhaled the plasma for 2 min at 60 min MCAO. The regional cerebral blood flow (CBF) was monitored. Animal behavior scoring, magnetic resonance imaging (MRI), 2,3,5-triphenyltetrazolium chloride (TTC) staining, and hematoxylin and eosin (HE) staining were performed to evaluate the therapeutic efficacy of the gas plasma inhalation on MCAO rats. Intermittent gas plasma inhalation by rats with experimental ischemic stroke could improve neurological function, increase regional CBF, and decrease brain infarction. Further MRI tests showed that the gas plasma inhalation could limit the ischemic lesion progression, which was beneficial to improve the outcomes of the MCAO rats. Post-stroke treatment with intermittent gas plasma inhalation could reduce the ischemic lesion progression and decrease cerebral infarction volume, which might provide a new promising strategy for ischemic stroke treatment.

Keywords: atmospheric pressure plasma; ischemic stroke; magnetic resonance imaging; neuro-protection; plasma medicine.

FIGURE 1

APPJ device and treatment protocol. (A) Diagram of the experimental setup. (B) Experimental procedure. (C) Rats successfully received MCAO surgery were randomly divided into MCAO, MCAO + APPJ, and MCAO + He groups. Then rats in each group have to be further randomly divided into two subgroups for different experiments, rats in one subgroup sequentially received CBF monitor, and sacrificed for TTC staining. (D) Rats in another subgroup sequentially received the MRI test, the behavioral test, and sacrificed for HE staining at indicated time points. APPJ, atmospheric-pressure plasma jet; CBF, cerebral blood flow; HE, hematoxylin and eosin; MCAO, middle cerebral artery occlusion; TTC, 2,3,5-triphenyltetrazolium chloride.

FIGURE 2

Plasma process and characteristics ensure mild and effective treatment conditions in animal models. (A) Images of the plasma inhalation treatment of rats with experimental ischemic stroke. (B) OES of the plasma jet at the quartz tube nozzle when APPJ is ejected into the air (case 1) or into a rat’s nasal cavity (case 2). (C) Gas temperature estimates by fitting the experimental and the simulation spectra of OH [A²Σ⁺(υ′ = 0) → X²Π (υ″ = 0)] at case 2, T_gas ≈ T_rot = 310 K. APPJ, atmospheric-pressure plasma jet; OES, optical emission spectroscopy.

FIGURE 3

The therapeutic efficacy of the plasma inhalation on neurological severity scores, brain ischemic damage, and CBF after MCAO. (A). Effect of plasma inhalation on neurological severity scores in each group. (B). Representative TTC-stained sections from different groups, where the living tissues were stained red, whereas infarcted tissues were stained white. (C). Quantitative analysis of TTC-defined infarct size corrected for edema, represented by the lesion volume percentage. All points stand for raw data for respective rats, whereas horizontal lines are indicative of mean. ***p < 0.001. (D) The CBF in the MCA region was recorded from 30 min before MCAO surgery to 4 h after the MCAO surgery. Data were represented as % of baseline value and error bars stand for SEM. ***p < 0.001 vs. the MCAO + Helium group. CBF, cerebral blood flow; MCA, middle cerebral artery; MCAO, middle cerebral artery occlusion; TTC, 2,3,5-triphenyltetrazolium chloride.

FIGURE 4

Representative MRI images of different groups at different time points.

FIGURE 5

Quantitative analysis of the MRI results. (A) Temporal evolution of ADC-defined ischemic lesion volumes in each group. The difference of MCAO group was not significant compared with MCAO + Helium group at every time point. Values were the mean ± SEM. ***p < 0.001 compared with MCAO + APPJ group after APPJ inhalation. ^###p < 0.001 compared with MCAO + APPJ group at 24 h. (B–D) ADC-defined ischemic lesion volume changes at different time points in MCAO, MCAO + Helium, and MCAO + APPJ groups. The points stand for raw data for all respective rats, whereas the horizontal line indicates the mean value of each group. *p < 0.05, **p < 0.01, and ***p < 0.001. (E) Volume of T2WI lesion for all groups at 24-h post-MCAO. The points stand for raw data for all respective rats, whereas horizontal line is indicative of mean. ***p < 0.001. APPJ, atmospheric-pressure plasma jet; ADC, apparent diffusion coefficient; MCAO, middle cerebral artery occlusion.

FIGURE 6

Tissue damage in each group was examined using the HE staining at 24-h post-MCAO. The representative overview and × 200 HE staining images of each group were provided. The magnified view for the cerebral cortex (Cx) and basal ganglion (BG) regions was shown as indicated by the boxes and circles, respectively, in each group. Results showed that the gas plasma inhalation improved MCAO-induced pathological changes. Scale bars show 100 μm. HE, hematoxylin and eosin; MCAO, middle cerebral artery occlusion.