Activation of Cholinergic Pathway via CCK-8 or Direct Vagal Stimulation Protects Hepatic Ischemia-Reperfusion Injury

Research Article

J Cardiovasc Disord. 2016; 3(2): 1027.

Activation of Cholinergic Pathway via CCK-8 or Direct Vagal Stimulation Protects Hepatic Ischemia-Reperfusion Injury

Elahe T Crockett¹*, Shadi Dowlatshahi² and Kolammal Nageswari³

¹Division of Cardiovascular, Department of Medicine, Michigan State University, USA

²Department of Internal Medicine, University of Southern California, USA

³IIT Bombay, Indian Institute of Technology Bombay, India

*Corresponding author: Elahe T Crockett, Department of Medicine, Michigan State University, East Lansing, MI 48824, USA

This work was supported by National Institutes of Health Grant R21HL75475 to E.T. Crockett, USA

Received: December 30, 2015; Accepted:March 28, 2016; Published: March 30, 2016


Background: Cholecystokinin-8 (CCK-8) at physiological levels stimulates the release of acetylcholine (ACh) via cholinergic pathway. We previously reported ACh receptor agonists protected the liver from Ischemia-Reperfusion (IR) injury, suggesting a potential role of the cholinergic anti-inflammatory pathway. This study examined whether administration of CCK-8 or direct vagal stimulation would have a protective effect on hepatic-IR injury.

Methods: Adult male mice underwent 90min of partial liver ischemia followed by reperfusion. CCK-8 was administered i.p. prior to ischemia, followed by a second dose at reperfusion time. A second group of mice were subjected to bilateral vagotomy prior to CCK-8, while a third group were subjected to electrical vagal stimulation for 20min, followed by hepatic IR. Plasma alanine aminotransferase (ALT) levels and liver histopathology were assessed for liver injury. Plasma cytokine Tumor Necrosis Factor (TNF)-α, Macrophage Inflammatory Protein-2 (MIP-2), Monocyte Chemoattractant Protein-1 (MCP-1) and Interleukin-6 (IL-6) were measured.

Results: CCK-8 pretreated mice had significantly lower ALT levels (87%) and hepatic injury, as compared to saline- treated mice. This protective effect was absent in the vagotomized mice, indicating a role for an intact cholinergic pathway. Vagal stimulation confirmed this observation as ALT levels and liver injury were significantly decreased in these mice. The injury was associated with marked elevation of plasma cytokines in vagotomized mice and saline treated mice, which were significantly decreased in CCK-8/electrically stimulated groups.

Conclusion: Activation of the cholinergic pathway through humoral and electromechanical stimuli provides a protective effect on hepatic IR injury, which could present therapeutic means for the treatment of inflammatory diseases.

Keywords: Cholinergic pathway; Anti-Inflammatory; Tissue injury; Vagotomy


ACh: Acetylcholine; AChR: Acetylcholine Receptor; CCK-8: Cholecystokinin-8; ELISA: Enzyme Linked Immunosorbent Assay; IR: Ischemia/Reperfusion; MIP-2: Macrophage Inflammatory Protein-2; MCP-1: Monocyte Chemoattractant Protein-1; TNF-α: Tumor Necrosis Factor-α


The phenomenon, hepatic Ischemic-Reperfusion (IR) injury, is a feature of many clinically important events, including hepatic surgery, transplantation, trauma, and hemorrhagic shock [1]. The injury occurs in two different phases; the acute injury phase (early phase), is associated with kupffer cell activation, release of proinflammatory cytokines, and generation of reactive oxygen species, which is followed by a second phase (late phase) characterized by massive neutrophil infiltration and further production of the inflammatory mediators [2]. The Central Nervous System (CNS) regulates systemic inflammatory responses to various stimuli through humoral mechanisms. For example, pro- inflammatory mediators (e.g., TNF, IL-1) activate afferent pathways in the vagus nerve, which stimulates the release of the pituitary adrenocorticotropic hormone; the resultant increase in plasma corticosteroids suppresses cytokine release to prevent excessive inflammation [3]. In addition to this sensory function of the vagus nerve in systemic inflammation, there exists an efferent or motor vagus neural mechanism by which Acetylcholine (ACh) inhibits cytokine release from resident tissue macrophages [4]. Studies have shown that both pharmacological and electrical stimulation of the efferent vagus nerve significantly inhibited the development of inflammatory response to a wide range of stimuli including IR [5-7]. The inhibitory effect was associated with decreased levels of the pro-inflammatory cytokine, Tumor Necrosis Factor (TNF)-α. It appears that CNS-derived motor output through the cholinergic anti-inflammatory pathway regulates cytokine release to prevent potentially damaging inflammation and maintain homeostasis -- this neural reflex is referred to as the “inflammatory reflex” [8].

Cholecystokinin, a neuropeptide hormone that is released from the small intestinal endocrine cells upon dietary fat intake, can stimulate vagal sensory fibers and neural ACh release [9]. Cholecystokinin, and, in particular, the cleaved octapeptide Cholecystokinin-8s (CCK- 8s) induces vagal-mediated gastric relaxation, increases pancreatic exocrine secretion, and induces short-term satiety [9-11]. Apart from its role in digestion and satiety, CCK-8 mediates the communication between nervous and immune systems, and is expressed in neurons, monocytes/macrophages and lymphocytes, where it regulates neurological and immune activities [12-15]. The anti-inflammatory activity of CCK-8 has been demonstrated in various disease models, including sepsis, endotoxic shock, hemorrhagic shock, and traumatic shock [16-20], suggesting that CCK-8 is a promising therapeutic drug candidate for inflammatory diseases. Studies have shown that the vagal-mediated effects of CCK-8 are almost exclusively due to CCK- 8 paracrine action on peripheral capsaicin- sensitive vagal afferent fibers [21-23].

Borovikova et al. have reported that direct electrical stimulation of the peripheral vagus nerve in vivo during lethal endotoxemia in rats inhibited TNF synthesis in liver, attenuated peak serum TNF levels, and prevented the development of shock [4]. Furthermore, vagal activation inhibited cytokine release and improved the disease endpoints in experimental sepsis, ileus, hemorrhagic shock, pancreatitis, and a number of other inflammatory models [reviewed in 7]. The cholinergic anti-inflammatory pathway can be activated chemically or electrically, which effectively suppresses systemic TNF release and attenuates organ damage. In our previous study, we showed protection of early phase hepatic IR injury by pharmacological modulation of the cholinergic pathway [6]. The aim of our current study was to evaluate the protective effect of the endogenous cholinergic pathway stimulation on hepatic IR. Two different approaches were applied; 1) direct electrical stimulation of the vagus nerve and 2) CCK-8 induced vagal stimulation. To accomplish this, an in-vivo model of vagal stimulation and vagotomy in the mouse was developed. The inflammatory response and liver injury were determined by measurement of the plasma cytokines and ALT levels, and histopathology examination.

Materials and Methods

All chemicals were purchased from the Sigma Chemical Co. (St. Louis, MO), unless otherwise noted. All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85-23, revised 1985). Experimental protocols were reviewed and approved by the Michigan State University Animal Use and Care Committee. Adult (8-10wk.) male C57BL/6 mice (Charles River Laboratories, Portage, MI) weighing between 24-29g were fed a standard diet and acclimated in the animal housing area for 1wk before experimentation. Three (3) sets of experiments were carried out. In the first set, mice were pretreated with vehicle (i.e. normal saline), or CCK-8 (i.p., 200μl 10min prior to surgery and 100μl at start of reperfusion). The test group underwent 90min hepatic ischemia and 3h reperfusion, while the sham group underwent the same surgical protocol but without vascular occlusion (see below for IR protocol). In the second set of experiment, the mice were subjected to bilateral vagotomy 24h prior to hepatic IR. In the third set of experiment, mice were subjected to vagal stimulation for 20min (5mV, 2ms, 5Hz) followed by 90min of ischemia and 3h of reperfusion.

Experimental animal model of hepatic ischemia and reperfusion

The partial hepatic IR model used in this study induces severe ischemic insult to the liver without inducing hypertension and subsequent bacterial translocation into the portal venous blood [24]. The mice were anesthetized by an i.p. injection of 35mg/kg sodium pentobarbital (Abbott Laboratories, North Chicago, IL). A midline laparotomy was performed, the portal circulation to the median and left lateral lobes of the liver was carefully dissected, and an a traumatic vascular clip (Accurate Surgical and Scientific Instruments Corp. Westbury, NY) was placed on the vessels, interrupting the portal venous, and hepatic arterial blood supply to these lobes. Five drops of sterile saline were applied over the abdominal viscera to keep the organs moist and to compensate for the fluid loss. The abdomen was temporarily closed with sterile staple sutures to prevent dehydration and possible contamination. The animal was kept in the recovery room under close supervision. After 90min of partial hepatic ischemia, the clamp was removed and reperfusion was resumed. The abdomen was closed in a double layer using 5-O nylon, and 0.8ml sterile lactated Ringer’s Solution (Abbott Laboratories, North Chicago, IL) was administered subcutaneously to compensate for operative fluid loss. During the reperfusion, the mice were kept in clean cages with controlled warm temperature (i.e., 23-25°C), with no further administration of anesthesia or analgesics. After reperfusion, mice were euthanized, and blood and tissue samples were collected as described below.

All the surgical procedures were performed under aseptic conditions. In this model, the caudate and right lateral lobes, as well as the papillary and quadrate processes retained an intact portal and arterial inflow and venous outflow to prevent intestinal venous congestion. This resulted in the induction of ischemia to approximately 70% of the liver. There was no to minimal bleeding during the surgical operation (bleeding <1+ of scale of 0-5+ considered minimal). If a small (2+) to moderate (3+) amount of bleeding occurred due to blood vessel puncture, the animal was excluded from the group and euthanized. The bleeding scale (0 – 5+) was arbitrarily based on saturation of each cotton swab that held about 150-200μl of blood. This was considered as 1+ bleeding. The liver IR model with 90min of ischemia induces a severe, isolated, and reproducible liver injury with very minimal animal mortality within one week (i.e. 0-5%).

Electric stimulation of the vagus nerve

Following anesthesia, a small midline incision was made on the ventral surface of the mouse neck, the vagus nerve (right) was dissected, isolated, and placed across a bipolar platinum stimulator electrodes connected to a stimulator module (Model SI USA, Grass S88 Stimulator). An optimal stimulatory condition was determined, and a constant voltage stimulus (5v, 2ms, 5Hz) was applied to the nerve for 20min to provide an isolated voltage stimulus to the nerve, followed by hepatic IR (90min of ischemia and 3h of reperfusion). Sham-operated animals underwent right vagus nerve dissection, isolation and exposure without stimulation.

Bilateral vagotomy

A ventral cervical incision was made and both vagus trunks were exposed. The vagus nerve was ligated at both cephalic and caudal ends using 4-0silk-suture and cut in half using a surgical scissors. In the sham-operated animals, both vagal trunks were dissected and exposed, but not ligated, and were left intact.

Drug administration

Animals received an i.p. injection of the CCK-8 (50μg/Kg b.w.); 200μl of CCK-8 at10min before the induction of ischemia and 100μl at start of reperfusion. Saline-treated mice received an equal volume of vehicle (saline).

Peripheral blood and tissue procurement

Following anesthesia, blood samples were obtained from the right ventricle via a left anterior thoracotomy at the conclusion of reperfusion. The blood was collected in a sterile syringe containing 50μl of heparin (100 USP Units/ml), and centrifuged to separate the plasma. The plasma samples were stored at -700C until use for cytokine and ALT assays. A portion of the ischemic and non-ischemic liver lobes were fixed in buffered 10% formalin, embedded in paraffin, and used for Hematoxylin and Eosin (H&E) staining.

Measurement of plasma alanine aminotransferase levels

The plasma ALT levels were determined spectrophotometrically, as previously described [6, 24]. The ALT values are expressed in International Units per Liter (IU/L).


H&E staining was performed on tissue sections prepared at 5-μm intervals from paraffin-embedded liver tissue. A pathologist, blinded to the experimental conditions, examined the liver tissue sections.

Plasma cytokine concentrations

Plasma TNF-α, IL-6, MCP-1 and MIP-2 were determined in a 96-well Nunc-Immuno microplate (VWR Scientific, Chicago, IL), using a sandwich Enzyme-Linked Immunosorbent Assay (ELISA) technique, as previously described [6]. The capture antibody was a polyclonal anti-mouse TNF-α, IL-6, MCP-1 or MIP-2 specific goat IgG (R&D Systems, Minneapolis, MN) and the detection antibody was a biotinylated polyclonal anti-mouse TNF-α, IL-6, MCP-1 or MIP-2 specific goat IgG, (R&D Systems). All plasma samples were tested in duplicate. The minimal detectable protein concentration was 20 pg/ml.

Statistical analysis

The data is expressed as means ± standard error of the mean (Mean ± SEM). Comparisons between two groups were performed using an unpaired t-test. Comparisons between multiple groups and various time points were analyzed using ANOVA followed by a Fisher’s PLSD post-hoc test. P ≤ 0.05 was considered statistically significant. All data were analyzed using the StatView version 5.0.1 software for Windows.


Effects of CCK-8 induced vagal stimulation on hepatocellular injury

Our previous published work has shown that 90min of hepatic ischemia followed by reperfusion resulted in hepatocellular injury in a time-dependent fashion, as demonstrated by plasma ALT level [6,24]. After 90min of ischemia followed by 3h of reperfusion, there was moderate to severe liver injury histologically that increased by 6h of reperfusion, and this injury was associated with significant plasma ALT elevation. The current study was focused on the effect of CCK-8 on the early phase of liver injury, i.e. 90min ischemia and 3h of reperfusion. The optimal dose that was determined for the study (50ug/Kg b.w.) did not produce any clinical signs (i.e., altered breathing, heart rate). Figure 1 depicts the changes in the plasma ALT level in the different animal groups studied. The results showed that CCK-8 pretreatment (10min before the induction of ischemia and at the start of reperfusion) significantly reduced plasma ALT levels (i.e., Mean ± SEM, Saline IR= 4422 ± 544 vs. CCK-8 IR= 456 ± 130 IU/L, p < 0.0001, ANOVA). The protective effect of CCK-8 on hepatic injury via the cholinergic pathway was confirmed by another group of mice, which underwent bilateral vagotomy 24h before subjected to CCK-8 pretreatment and hepatic IR (i.e., ALT levels, Mean + SEM, Saline IR= 4422 ± 544 and in vagotomized CCK-8 IR= 4846 ± 395 IU/L). The results showed that vagotomy completely inhibited the protective effect of CCK-8 pretreatment. Further, the sham group demonstrated that either the injection of CCK-8 alone or vagotomy did not cause any hepatic injury as judged by the ALT release (Figure 1).