Immunohistochemical expression of CD11b, RECA-1 and α7nAChR in the lung. A)

<p>Lungs harvested 6 hours after heat stress were stained for CD11b (green), RECA-1 (red), and α7nAChR (blue). The arrows indicate CD11b-positive cells expressing α7nAChR. Bars represent 20 µm, original magnification ×1200. <b>B)</b> Results of statistical analysis for the percentage of CD11b-positive cells in the lung. <b>C)</b> Results of statistical analysis for the percentage of CD11b-positive cells expressing α7nAChR in all CD11b-positive cells. *<i>p</i> <0.05, **<i>p</i> <0.01.</p

Serial changes in levels of serum TNF-α, IL-6, sTM, and sE-selectin after heat stress.

<p>Serum concentrations of TNF-α (<b>A</b>), IL-6 (<b>B</b>), sTM (<b>C</b>), and sE-selectin (<b>D</b>) in the sham group, HS-cont group (heatstroke rats without VNS treatment), and HS-VNS group (VNS-treated heatstroke rats) are shown. All data are expressed as mean ± standard error of the mean. n  =  5 at every time point in the sham group and n  =  10 at every time point in the HS-cont and HS-VNS groups. *<i>p</i> <0.05, **<i>p</i> <0.01.</p

Schema of experimental design and time schedule of heatstroke model.

<p>Schema of experimental design and time schedule of heatstroke model.</p

Survival rates during the first 7 days after heat stress.

<p>The dotted line represents the survival curve of the rats that underwent heat stress without VNS, and the solid line represents the VNS-treated heatstroke rats (each group, n  =  23). Treatment with VNS was associated with a significantly higher rate of survival against heat stress (<i>p</i>  =  0.016). Sham operation caused no mortality in the rats that did not undergo heat stress (n  =  8, data not shown).</p

Data_Sheet_1_Ultrasound stimulation of the vagal nerve improves acute septic encephalopathy in mice.docx

Septic encephalopathy (SE) is characterized by symptoms such as coma, delirium, and cognitive dysfunction, and effective therapeutic interventions for SE remain elusive. In this study, we aimed to investigate the potential alleviating effects of vagal nerve stimulation (VNS) on SE-associated signs. To evaluate our hypothesis, we utilized a mouse model of SE induced by intraperitoneal injection of lipopolysaccharide (0.3 mg per mouse) and administered noninvasive, high-frequency ultrasound VNS. To assess the efficacy of ultrasound VNS, we measured inflammation-related molecules, including the α7 nicotinic acetylcholine receptor (α7nAChR) expression in peritoneal macrophages and plasma interleukin 1β (IL-1β) levels. Consistent with our hypothesis, SE mice exhibited reduced α7nAChR expression in macrophages and elevated IL-1β levels in the blood. Remarkably, VNS in SE mice restored α7nAChR expression and IL-1β levels to those observed in control mice. Furthermore, we evaluated the effects of VNS on survival rate, body temperature, and locomotor activity. SE mice subjected to VNS demonstrated a modest, yet significant, improvement in survival rate, recovery from hypothermia, and increased locomotor activity. To investigate the impact on the brain, we examined the hippocampus of SE mice. In control mice, VNS increased the expression of c-fos, a marker of neuronal electrical excitability, in the hippocampus. In SE mice, VNS led to the restoration of aberrant firing patterns in hippocampal neurons. Additionally, proteomic analysis of hippocampal tissue in SE mice revealed abnormal increases in two proteins, tissue factor (TF) and acyl-CoA dehydrogenase family member 9 (ACAD9), which returned to control levels following VNS. Collectively, our findings support the value of exploring the beneficial effects of ultrasound VNS on SE.</p

Immunohistochemical expression of CD11b and α7nAChR in the spleen. A)

<p>Spleens harvested 6 hours after heat stress were stained for CD11b (green) and α7nAChR (blue). The open arrowheads indicate CD11b-positive cells expressing α7nAChR, and the closed arrowheads indicate CD11b-positive cells without expression of α7nAChR. Bars represent 100 µm, original magnification ×200. MZ, marginal zone; WP, white pulp; RP, red pulp. <b>B)</b> Results of statistical analysis for the percentage of CD11b-positive cells in the spleen. <b>C)</b> Results of statistical analysis for the percentage of CD11b-positive cells expressing α7nAChR in all CD11b-positive cells. **<i>p</i> <0.01.</p

Results of single logistic regression analysis.

<p>Logistic regression analysis was performed to identify the factors related to the presence of NET and Cit-H3 in the bloodstream. Only “the presence of bacteria in tracheal aspirate” (+) at the time of intubation was a significant factor associated with the presence of NET and Cit-H3 (<i>p</i> = .0112). NETs: neutrophil extracellular traps, Cit-H3: citrullinated histone H3, SIRS: systemic inflammatory response syndrome, cf-DNA: circulating free DNA, WBC: white blood cell, IL: interleukin, TNF: tumor necrosis factor, HMGB1: high mobility group box-1.</p><p>Results of single logistic regression analysis.</p

Diagnoses and the number of patients exhibiting neutrophil extracellular traps and citrullinated histone H3 in each diagnostic group.

<p>In the blood smears surveyed in this study, we identified NETs in 5 patients and Cit-H3 in 11 patients. Both NETs and Cit-H3 were identified concurrently in one patient with infection. We found no NETs or Cit-H3-positive cells in samples from patients with trauma (0/7) or heart disease (0/4). NETs: neutrophil extracellular trap, Cit-H3: citrullinated histone H3.</p><p>Diagnoses and the number of patients exhibiting neutrophil extracellular traps and citrullinated histone H3 in each diagnostic group.</p

Results of multiple logistic regression analysis of factors associated with the presence of neutrophil extracellular traps and/or citrullinated histone H3.

<p>Two methods of multiple regression analysis, backward and forward regression, yielded similar models. “The presence of bacteria in tracheal aspirate” was the only factor that was significantly related to the presence of neutrophil extracellular traps and/or citrullinated histone H3 in the bloodstream. The odds ratio for aspiration was 5.750. Coeff (β): coefficient; OR: odds ratio, Lower: lower level of 95% confidence interval, Upper: upper level of 95% confidence interval.</p><p>Results of multiple logistic regression analysis of factors associated with the presence of neutrophil extracellular traps and/or citrullinated histone H3.</p

Representative images of immunostaining using anti-CD66b antibody in the blood smear sample from a critically ill patient.

<p>Triple staining by DAPI, anti-CD66b antibody, and anti-citrullinated histone H3 was performed using the blood smear sample obtained from a critically ill patient. A. The CD66b-positive cells were subjected to citrullination of histone H3 in their nuclei. Citrullination of histone H3 was not detected in the CD66b-negative cell (arrow). B. Arrow indicates the occurrence of citrullination of histone H3 in a neutrophil that had immunoreactivity against CD66b. Arrowheads indicate NETs stained with CD66b, whose appearance was of a string-like structure extending from the cell body. Asterisk indicates a neutrophil that was beginning to release NETs from its ruptured cell body. Interestingly, freshly produced NETs (asterisk) held immunoreactivity against citrullination of histone H3. In contrast, elongated NETs (arrowheads) were not stained with anti- citrullinated histone H3 antibody. Blue, DAPI; Red, CD66b; Green, citrullinated histone H3. (Magnification ×400). Scale bar; 50 µm.</p