BACKGROUND: In this investigation, we explored the effects of pharmacological cholinergic stimulation on cardiac function and renal inflammation following acute myocardial infarction (AMI) in spontaneously hypertensive rats (SHRs).
METHODS: Adult male SHRs were randomized into three experimental groups: sham-operated; AMI + Veh (infarcted, treated with vehicle); and AMI + PY (infarcted, treated with the cholinesterase inhibitor, pyridostigmine bromide (PY)-40 mg/kg, once daily for seven days). Rats were euthanized 7 or 30 days post-surgery. The clinical parameters were assessed on the day before euthanasia. Subsequent to euthanasia, blood samples were collected and renal tissues were harvested for histological and gene expression analyses aimed to evaluate inflammation and injury.
RESULTS: Seven days post-surgery, the AMI + PY group demonstrated improvements in left ventricular diastolic function and autonomic regulation, and a reduction in renal macrophage infiltration compared to the AMI + Veh group. Furthermore, there was a notable downregulation in pro-inflammatory gene expression and an upregulation in anti-inflammatory gene expression. Analysis 30 days post-surgery showed that PY treatment had a sustained positive effect on renal gene expression, correlated with a decrease in biomarkers, indicative of subclinical kidney injury.
CONCLUSIONS: Short-term cholinergic stimulation with PY provides both cardiac and renal protection by mitigating the inflammatory response after AMI.

Acute respiratory distress syndrome (ARDS) is a critical illness complication that is associated with high mortality. ARDS is documented in severe cases of COVID-19. No effective pharmacological treatments for ARDS are currently available. Dysfunctional immune responses and pulmonary and systemic inflammation are characteristic features of ARDS pathogenesis. Recent advances in our understanding of the regulation of inflammation point to an important role of the vagus-nerve-mediated inflammatory reflex and neural cholinergic signaling. We examined whether pharmacological cholinergic activation using a clinically approved (for myasthenia gravis) cholinergic drug, the acetylcholinesterase inhibitor pyridostigmine alters pulmonary and systemic inflammation in mice with lipopolysaccharide (LPS)-induced ARDS. Male C57Bl/6 mice received one intratracheal instillation of LPS or were sham manipulated (control). Both groups were treated with either vehicle or pyridostigmine (1.5 mg/kg twice daily, 3 mg/day) administered by oral gavage starting at 1 h post-LPS and euthanized 24 h after LPS administration. Other groups were either sham manipulated or received LPS for 3 days and were treated with vehicle or pyridostigmine and euthanized at 72 h. Pyridostigmine treatment reduced the increased total number of cells and neutrophils in the bronchoalveolar lavage fluid (BALF) in mice with ARDS at 24 and 72 h. Pyridostigmine also reduced the number of macrophages and lymphocytes at 72 h. In addition, pyridostigmine suppressed the levels of TNF, IL-1β, IL-6, and IFN-γ in BALF and plasma at 24 and 72 h. However, this cholinergic agent did not significantly altered BALF and plasma levels of the anti-inflammatory cytokine IL-10. Neither LPS nor pyridostigmine affected BALF IFN-γ and IL-10 levels at 24 h post-LPS. In conclusion, treatments with the cholinergic agent pyridostigmine ameliorate pulmonary and systemic inflammatory responses in mice with endotoxin-induced ARDS. Considering that pyridostigmine is a clinically approved drug, these findings are of substantial interest for implementing pyridostigmine in therapeutic strategies for ARDS.

Systemic arterial hypertension and heart failure are cardiovascular diseases that affect millions of individuals worldwide. They are characterized by a change in the autonomic nervous system balance, highlighted by an increase in sympathetic activity associated with a decrease in parasympathetic activity. Most therapeutic approaches seek to treat these diseases by medications that attenuate sympathetic activity. However, there is a growing number of studies demonstrating that the improvement of parasympathetic function, by means of pharmacological or electrical stimulation, can be an effective tool for the treatment of these cardiovascular diseases. Therefore, this review aims to describe the advances reported by experimental and clinical studies that addressed the potential of cholinergic stimulation to prevent autonomic and cardiovascular imbalance in hypertension and heart failure. Overall, the published data reviewed demonstrate that the use of central or peripheral acetylcholinesterase inhibitors is efficient to improve the autonomic imbalance and hemodynamic changes observed in heart failure and hypertension. Of note, the baroreflex and the vagus nerve activation have been shown to be safe and effective approaches to be used as an alternative treatment for these cardiovascular diseases. In conclusion, pharmacological and electrical stimulation of the parasympathetic nervous system has the potential to be used as a therapeutic tool for the treatment of hypertension and heart failure, deserving to be more explored in the clinical setting.

Type I diabetes (T1D) is a T cell-driven autoimmune disease that results in the killing of pancreatic β-cells and, consequently, loss of insulin production. Using the multiple low-dose streptozotocin (MLD-STZ) model of experimental autoimmune diabetes, we previously reported that pretreatment with a specific acetylcholinesterase inhibitor (AChEI), paraoxon, prevented the development of hyperglycemia in C57BL/6 mice. This correlated with an inhibition of T cell infiltration into the pancreatic islets and a reduction in pro-inflammatory cytokines. The cholinergic anti-inflammatory pathway utilizes nicotinic and muscarinic acetylcholine receptors (nAChRs and mAChRs, respectively) expressed on a variety of cell types. In this study, we carried out a comparative analysis of the effect of specific antagonists of nAChRs or mAChRs on the development of autoimmune diabetes. Co-administration of mecamylamine, a non-selective antagonist of nAChRs maintained the protective effect of AChEI on the development of hyperglycemia. In contrast, co-administration of atropine, a non-selective antagonist of mAChRs, mitigated AChEI-mediated protection. Mice pretreated with mecamylamine had an improved response in glucose tolerance test (GTT) than mice pretreated with atropine. These differential effects of nAChR and mAChR antagonists correlated with the extent of islet cell infiltration and with the structure and functionality of the β-cells. Taken together, our data suggest that mAChRs are essential for the protective effect of cholinergic stimulation in autoimmune diabetes.

We tested the hypothesis that oscillatory airway smooth muscle (ASM) mechanics is governed by mechanosensitive energy loss and energy release elements that can be recruited by prestrain and cholinergic stimulation. We measured mechanical energy loss and mechanical energy release in unstimulated and carbachol-stimulated bovine ASM held at prestrains ranging from 0.3 to 1.0 Lo (reference length) and subjected to sinusoidal length oscillation at 1 hz with oscillatory strain amplitudes ranging from 0.1 to 1.5% Lo. We found that oscillatory ASM mechanics during sinusoidal length oscillation is governed predominantly by one class of nonlinear mechanosensitive energy loss element and one class of nonlinear mechanosensitive energy release element with differential mechanosensitivities to oscillatory strain amplitude. The greater mechanosensitivity of the energy loss element than energy release element may explain the bronchodilatory effect of deep inspiration. Prestrain, an important determinant of ASM responsiveness, differentially increased energy loss and energy release in unstimulated and carbachol-stimulated ASM. Cholinergic stimulation, an important cause of bronchoconstriction and airway inflammation, also differentially increased energy loss and energy release. When prestrain and cholinergic stimulation were combined, we found that prestrain and cholinergic stimulation synergistically increased energy loss and energy release by ASM. The relationship between recruitment of energy loss elements and recruitment of energy release elements was nonlinear, suggesting that energy loss and energy release elements are not coupled in ASM cells. These findings imply that large lung volume and cholinergic ASM activation would synergistically increase mechanical energy expenditure during inspiration and mechanical recoil of ASM during expiration. NEW & NOTEWORTHY We report for the first time that oscillatory airway smooth muscle mechanics is governed predominantly by one class of nonlinear mechanosensitive energy loss element and one class of nonlinear mechanosensitive energy release element with differential mechanosensitivities to oscillatory strain amplitude. Prestrain and cholinergic stimulation synergistically and differentially recruit energy loss and energy release elements. The greater mechanosensitivity of the energy loss element than the energy release element may explain the bronchodilatory effect of deep inspiration.

Tissue damage and pathogen invasion during surgical trauma have been identified as contributing factors leading to neuroinflammation in the hippocampus, which can be protected by stimulation of the cholinergic anti-inflammatory pathway using the acetylcholinesterase inhibitor physostigmine. Macroautophagy, an intracellular degradation pathway used to recycle and eliminate damaged proteins and organelles by lysosomal digestion, seems to be important for cell survival under stress conditions. This study aimed to examine the role of autophagy in physostigmine-mediated hippocampal cell protection in a rat model of surgery stress. In the presence or absence of physostigmine, adult Wistar rats underwent surgery in combination with lipopolysaccharide (LPS). Activated microglia, apoptosis-, autophagy-, and anti-inflammatory-related genes and -proteins in the hippocampus were determined by Real-Time PCR, Western blot and fluorescence microscopy after 1 h, 24 h and 3 d. Surgery combined with LPS-treatment led to microglia activation after 1 h and 24 h which was accompanied by apoptotic cell death after 24 h in the hippocampus. Furthermore, it led to a decreased expression of ATG-3 after 24 h and an increased expression of p62/ SQSTM1 after 1 h and 24 h. Administration of physostigmine significantly increased autophagy related markers and restored the autophagic flux after surgery stress, detected by increased degradation of p62/ SQSTM1 in the hippocampus after 1 h and 24 h. Furthermore, physostigmine reduced activated microglia and apoptosis relevant proteins and elevated the increased expression of TGF-beta1 and MFG-E8 after surgery stress. In conclusion, activation of autophagy may be essential in physostigmine-induced neuroprotection against surgery stress.

OBJECTIVE: This study used advanced optical mapping techniques to examine atrial fibrillation (AF) dynamics before and after 2 distinct electrogram-based ablation strategies: complex fractionated atrial electrograms (CFAEs) and DFmax/rotor ablation.
BACKGROUND: Among the electrogram analytical features proposed to unravel the atrial regions that perpetuate AF, CFAEs, highest dominant frequency sites (DFmax), and, more recently, phase analysis-enabled rotor mapping have received the largest attention. Still, the mechanisms by which these approaches modulate AF dynamics and lead to AF termination are unknown.
METHODS: In Langendorff-perfused sheep hearts, AF was maintained by the continuous perfusion of acetylcholine and high-resolution endocardial-epicardial optical videos were recorded from the left atrial free wall and the posterior left atrium. Then, DFmax/rotor regions (n = 7), or CFAE regions harboring the highest wavebreak density (HWD) (n = 5), were targeted with a 4F ablation catheter (5 to 15 W, 30 to 60 s/point). Thereafter, we examined the changes in AF dynamics and whether AF terminated.
RESULTS: DFmax/rotor point ablation resulted in a significant decrease in DFmax values. In 2 animals AF terminated, whereas in the remaining 5 animals the post-ablation DFmax domain remained in the vicinity of its pre-ablation location. However, after HWD/CFAEs density ablation, DFmax values did not change, AF did not terminate, and post-ablation DFmax domains relocated from the left atrial free wall to the pulmonary vein-posterior left atrium region. In another group of hearts (n = 12), we observed that upon a progressive increase in acetylcholine concentration-mimicking the acute electrophysiological changes occurring after ablation-3-dimensional rotors drifted from one atrial region to another along large gradients of myocardial thickness.
CONCLUSIONS: \"On-target\" DFmax/rotor ablation leads to the annihilation of the fibrillation-driving rotor. This translates into large decreases in AF frequency or AF termination. In contrast, \"nearly missed\" HWD/CFAEs ablation spares the fibrillation-driving rotor, and set the stage for rotor drift along large myocardial thickness gradients.

Pyridostigmine bromide (PB) is a reversible cholinesterase inhibitor. The aim of this study was to determine the effect of orally administration of single dose sustained-released tablet of pyridostigmine bromide (PBSR) on the frequency domain indices of heart rate variability (HRV). Thirty-two healthy young men were participated in this study. They were divided into 2 groups; the pyridostigmine group (n = 22) and the placebo group (n = 10). Electrocardiogram (ECG) was recorded at 10, 30, 60, 90, 120, 150, 180, 210, 240, 300 and 420 min after PBSR administration. At each time, simultaneously, a blood sample was prepared and PB plasma concentration was measured by high-performance liquid chromatography (HPLC) method. Statistical analysis showed that in different indices of HRV, there is a significant increase in low frequency (LF) band at 300 min, but no difference in high frequency band (HF). It also showed significant decreases in normalized high frequency band (Hfnu), normalized low frequency band (Lfnu) and LF/HF ratio at 120, 240 and 300 min after PBSR administration. Maximum plasma concentration of PB was 150 min after the administration. In conclusion, administration of a single dose PBSR can enhance the frequency domains indices of HRV and improvesympathovagal balance.