CECUM LIGATION vs. INTRAPERITONEAL METHODS: A Comparative Study for Surgical Techniques

Introduction

Sepsis is a significant condition marked by a strong inflammatory response due to infection. It often leads to cardiac issues, especially atrial fibrillation (AF), which is common among septic patients. AF that develops during sepsis is known as new-onset AF. Patients with new-onset AF during sepsis have a higher risk of hospitalization for heart failure within five years compared to those without AF. Moreover, these patients face greater risks of mortality, heart failure, ischemic stroke, and recurring AF after hospital discharge. The exact causes for this new-onset AF during sepsis are unclear and may differ from AF in other conditions. Creating animal models to study sepsis-induced AF has proven difficult. Current methods either involve complex genetic modifications or electrical stimulation in large animals, making research costly and complicated. The two primary sepsis models used are cecum ligation and puncture (CLP) and lipopolysaccharide (LPS) injection. These models show that sepsis can lead to arrhythmias, including AF. However, determining the best model for studying new-onset AF in sepsis remains a challenge.

Materials and Methods

Animal Preparation and Study Protocol

Forty-seven Sprague-Dawley rats were used for the study. All experiments were approved and performed according to ethical guidelines. Electrocardiograms were recorded before the modeling. Rats were divided into four groups: control (CT), sham operation, CLP, and LPS treatment. The CLP group underwent fasting before cecum ligation and fecal extrusion to induce infection. The LPS group received injections of LPS at varying doses. On the fourth day after modeling, under anesthesia, atrial electrophysiology studies were conducted on all rats to evaluate heart function.

Measurement of Effective Atrial Refractory Period

The effective atrial refractory period (AERP) was measured through electrical stimulation in both atria. A specific stimulation protocol was used, and AERP was defined as the longest interval without a response. Each rat underwent multiple trials, and average results were recorded.

Measurement of AF Induction Rate

To assess AF induction, burst stimulation was applied. The presence of AF was recorded post-stimulation. AF lasting over 1000 ms was documented, and the AF induction rate was calculated as a percentage of successful inductions.

Measurement of Left Atrial Electrical Conduction Velocity and Heterogeneity

Microelectrode array recordings were employed to assess conduction velocity in the left atrium. A median sternotomy was performed to access the heart, and a flexible microelectrode array was placed on the atrium’s surface. Data were analyzed to produce conduction maps, with calculations of mean conduction velocities.

Echocardiography

Echocardiography was conducted to visualize heart structure and function.

Histological Analysis

Left atrial tissues were stained for analysis. Inflammation and fibrosis were evaluated through microscopy, and image analysis quantified these parameters.

ELISA Assay

Plasma samples were analyzed for IL-18 concentration using an ELISA kit. The process included standard dilutions and optical density measurements.

Western Blotting

Protein extraction from left atrial tissues was performed for Western blot analysis. Proteins were separated and transferred to membranes for antibody incubation and detection.

Statistical Analysis

Data were analyzed using statistical software. Differences between groups were determined using appropriate tests, with significance set at p

Results

The Most Stable Model of New-Onset AF in Sepsis Induced by Intraperitoneal Injection of 10 mg/kg LPS

Compared to the CT group, rats in the CLP and LPS groups displayed clear signs of illness and higher inflammatory markers. New-onset AF occurred in both CLP and LPS groups. The incidence did not differ significantly among CLP subgroups but was distinct in the LPS groups at a dose of 10 mg/kg.

Echocardiography Findings of AF in the LPS Group

Echocardiograms showed significant left atrial enlargement in CLP and LPS groups, unlike the CT and sham groups. CLP and LPS groups showed incoordination in ventricular motion, and key echocardiographic measures indicated impaired heart function.

Reduced Effective Atrial Refractory Period and Enhanced AF Susceptibility in the CLP and LPS Groups

ERPs in the CT and sham groups ranged from 70-80 ms, while the CLP and LPS groups had significantly lower ERPs. Rates of AF induction were significantly higher in the CLP and LPS groups compared to controls.

Impaired Electrical Impulse Conduction in the LPS and CLP Groups

Electrical impulse conduction was disordered in CLP and LPS groups, indicated by irregular conduction patterns. Conduction velocity was notably lower in both the LPS and CLP groups.

Enhanced Inflammatory Response and Atrial Fibrosis in the LPS Group Relative to the CLP Group

Significantly greater inflammatory cell infiltration and fibrosis were observed in the LPS group compared to the CLP group, pointing to a more pronounced inflammatory response.

Elevated NLRP3 Inflammasome and S1P/S1P2 Signaling in the LPS Group Relative to the CLP Group

Western blot analyses showed increased levels of NLRP3 inflammasomes and S1P signaling components in both the CLP and LPS groups, with the LPS group having higher expression levels.

Discussions

The CLP model is effective for simulating sepsis but is technically demanding. The LPS model offers advantages, including ease of use and reproducibility. Electrocardiography is vital for diagnosing AF and assessing related cardiac changes. Structural changes, such as left atrial enlargement, promote AF by extending conduction pathways. A shorter ERP leads to increased excitability and risk for AF due to reentrant circuits. Inflammation from sepsis releases cytokines that affect ion channel function, leading to increased AF susceptibility. NLRP3 inflammasome activation releases inflammatory cytokines, further increasing AF risk. Treatments targeting inflammation may help reduce AF occurrences in sepsis.

Limitations

The mechanisms behind new-onset AF in sepsis require further exploration. Future studies should investigate the role of NLRP3 inflammasomes and signaling pathways while examining inflammatory cell types involved in AF development.

Conclusion

Injecting 10 mg/kg LPS effectively induced new-onset AF in septic conditions, demonstrating a high incidence of AF with low mortality. This model shows relevant electrical and structural heart changes typical of AF, along with increased NLRP3 and S1P signaling in the LPS group.