AAST 2014 PLENARY PAPER
A pharmacologic approach to vagal nerve stimulation prevents mesenteric lymph toxicity after hemorrhagic shock Koji Morishita, MD, Todd W. Costantini, MD, Akinori Ueno, PhD, Vishal Bansal, MD, Brian Eliceiri, PhD, and Raul Coimbra, MD, PhD, San Diego, California
BACKGROUND: Electrical stimulation of the vagus nerve (VN) prevents gut and lung inflammation and mesenteric lymph (ML) toxicity in animal models of injury. We have previously shown that treatment with CPSI-121, a guanylhydrazone-derived compound, prevents gut barrier failure after burn injury. While the structure of CPSI-121 predicts that it will activate parasympathetic signaling, its ability to stimulate the VN is unknown. The aims of this study were to (1) measure the ability of CPSI-121 to induce VN activity, (2) determine whether CPSI-121 causes significant hemodynamic effects, and (3) further define the potential for CPSI-121 to limit the systemic inflammatory response to injury. METHODS: Male Sprague-Dawley rats were given 1-mg/kg CPSI-121 intravenously while blood pressure, heart rate, and efferent VN electrical activity were recorded. Rats were also assigned to sham or trauma/hemorrhagic shock (T/HS). T/HS was induced by laparotomy and 60 minutes of HS (mean arterial pressure, 35 mm Hg) followed by fluid resuscitation. A separate cohort of animals received CPSI-121 after the HS phase. Gut and lung tissues were harvested for histologic analysis. Lung wet-dry ratios were also evaluated. The ability of ML to prime neutrophils was assessed by measuring in vitro oxidative burst using flow cytometry. RESULTS: Blood pressure was not altered after treatment with CPSI-121, while heart rate decreased only slightly. Recording of efferent VN electrical activity revealed an increase in discharge rate after administration of CPSI-121. T/HS caused gut and lung injury, which were prevented in animals treated with CPSI-121 (p G 0.05). Treatment with CPSI-121 following T/HS attenuated neutrophil priming after exposure to ML (p G 0.05). CONCLUSION: CPSI-121 causes efferent VN output and limits shock-induced gut and lung injury as well as ML toxicity. CPSI-121 is a candidate pharmacologic approach to VN stimulation aimed at limiting the inflammatory response in patients following T/HS. (J Trauma Acute Care Surg. 2015;78: 52Y59. Copyright * 2015 Wolters Kluwer Health, Inc. All rights reserved.) KEY WORDS: Hemorrhagic shock; mesenteric lymph; CPSI-121; vagal nerve stimulation; rats.
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rauma/hemorrhagic shock (T/HS) leads to gut barrier failure that initiates a systemic inflammatory response resulting in acute lung injury (ALI) and multiple-organ dysfunction syndrome.1 After T/HS, the injured gut releases proinflammatory mediators into the mesenteric lymph (ML) that activate inflammatory and endothelial cells.2 Thus, ML is considered to be central in the pathogenesis of ALI/multiple-organ dysfunction syndrome following T/HS. We have previously shown that electrical vagal nerve stimulation (VNS) prevents intestinal barrier failure and alters inflammatory cell trafficking in the ML.3Y5 Previous studies in our laboratory and others have also demonstrated that VNS attenuates injury-induced ALI through its ability to limit gut inflammation, rather than via direct effects on the lung.6Y9 Although evidence supporting the use of electrical VNS in burn, sepsis, and T/HS is compelling in preclinical animal
Submitted: August 1, 2014, Revised: September 8, 2014, Accepted: October 2, 2014. From the Division of Trauma, Surgical Critical Care, Burns, and Acute Care Surgery, Department of Surgery, University of California San Diego Health Sciences, San Diego, California. This study was presented at the 73rd annual meeting of the American Association for the Surgery of Trauma, September, 10Y13, 2014, in Philadelphia, Pennsylvania. Address for reprints: Todd W. Costantini, MD, Division of Trauma, Surgical Critical Care, Burns, and Acute Care Surgery, Department of Surgery, University of California San Diego Health Sciences, 200 W Arbor Dr, #8896, San Diego, CA 92103Y8896; email: [email protected]. DOI: 10.1097/TA.0000000000000489
models, direct electrical stimulation of the vagus nerve in acutely injured patients currently is impractical. A pharmacologic approach to VNS would be a more clinically feasible and rapidly deployable strategy in the clinical setting. We have previously demonstrated that treatment with CPSI-121, a divalent guanylhydrazone, prevents gut barrier failure after burn injury with antiinflammatory effects that are comparable with direct, electrical VNS.10 While the structure of CPSI-121 predicts that it will activate parasympathetic signaling, its ability to stimulate the vagus nerve is unknown. In this series of experiments, we administered CPSI-121 following T/HS to determine its ability to prevent gut and lung injury. We hypothesized that CPSI-121 would induce vagus nerve electrical activity, decrease the toxicity of ML, and limit distant organ injury, demonstrating the capacity for CPSI-121 to limit the systemic inflammatory response to injury.
MATERIALS AND METHODS T/HS Model All animal experiments were approved by the University of California San Diego Institutional Animal Care and Use Committee. Male Sprague-Dawley rats weighing 280 g to 300 g were obtained from Harlan Laboratories (Placentia, CA). Animals were anesthetized with ketamine (75 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (10 mg/kg; Sigma J Trauma Acute Care Surg Volume 78, Number 1
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Chemical, St. Louis, MO), and the left femoral artery and vein were cannulated with a polyethylene tube (PE-50). The mean arterial pressure (MAP) was continuously monitored using the femoral arterial catheter (Philips V24/26, Andover, MA). Heart rate was recorded using the Biopac MP36 Acquisition System (Santa Barbara, CA). Each animal’s body temperature was maintained at 37-C. A right medial visceral rotation was performed through a midline laparotomy (trauma), and then, the abdomen was closed. HS was induced via withdrawal of blood from the femoral vein catheter until the MAP was reduced to 35 mm Hg and maintained for 60 minutes. At the end of HS, animals in the T/HS group were resuscitated with shed blood + two times shed blood volume in normal saline (NS) (Baxter, Deerfield, IL). Sham animals underwent the identical anesthesia and cannulation of the femoral vessels without trauma and hemorrhage. A separate cohort of animals was treated with CPSI-121 (Ferring, San Diego, CA) diluted in sterile water immediately after injury. CPSI-121 was administered at 1 mg/kg intravenously based on our previous experiments demonstrating gut barrier protection after injury using this dose.10
ML was collected on ice during the pre-HS phase (30 minutes), HS phase (60 minutes), and post-HS phase (120 minutes, Fig. 1C). The ML was centrifuged at 2,000 rpm for 5 minutes at 4-C, and the supernatant was stored at j80-C until further analysis.
Collection of ML
Histologic Evaluation
As previously described,11 the ML duct was exposed, and the efferent ML duct was cannulated using PE50 tubing.
At 24 hours after injury, segments of distal ileum and NS-perfused lungs were removed and fixed in 10% buffered
Respiratory Burst The biologic activity of ML was measured via its ability to activate PMNs in vitro.12 Whole blood was obtained via a femoral artery catheter from naive male rats then treated with RBC lysis buffer (BD Pharmingen, San Diego, CA). Freshly thawed lymph samples (n = 3Y5 animals per group) were added to the remaining leukocyte pellet (5% vol./vol.) and then placed into an incubator for 5 minutes. Dihydrorhodamine 123 (15 ng/mL) (Molecular Probes, Inc., Grand Island, NY) was added to the tubes for 5 minutes before stimulation with Phorbol 12-myristate 13-acetate (PMA, Sigma). Samples were incubated for 15 minutes at 37-C. The PMN respiratory burst was measured by flow cytometry using a BD Accuri C6 (BD Bioscience, San Jose, CA). The forward and side scatter detectors were used to identify the PMN population, and the fluorescence gain was adjusted using unstimulated and stimulated control samples.
Figure 1. Hemodynamic effect of CPSI-121. To determine whether CPSI-121 causes significant hemodynamic effect, we continuously measured MAP and heart rate after CPSI-121administration (1 mg/kg). Heart rate (A) and MAP (B) were measured continuously throughout the experiment. C, A model of T/HS was induced by laparotomy and 60 minutes of HS (MAP, 35 mm Hg), followed by fluid resuscitation. A separate cohort of animals received CPSI-121 (1 mg/kg) after the HS phase (D). * 2015 Wolters Kluwer Health, Inc. All rights reserved.
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formalin, embedded in paraffin, and sectioned (n = 4Y5 per group). Hematoxylin and eosin staining was performed by the University of California San Diego Histology Core Services. An investigator blinded to the experimental groups analyzed multiple fields from each section of tissue with a light microscope. The ileum and lung sections were classified according to the degree of tissue injury according to histologic grading scores as previously described.13,14
Wet-to-Dry Lung Weight Ratios The wet-to-dry ratio of the lung was determined as a measure of lung edema.15 Lungs were excised after injury (n = 3Y5 animals per group) and weighed to obtain the wet weight. Lungs were then dried in an oven at 60-C for 7 days to obtain the dry weight and calculate the wet-to-dry ratio ([wet weight j dry weight] / dry weight).
Recording of Vagus Nerve Activity Animals were anesthetized with isoflurane, the vagus and femoral nerves were exposed, both positive and negative electrodes were applied to the target nerve with a certain distance, and a ground electrode was inserted under the skin. Signals from the electrodes were passed through a band-pass filter (100Y2,000 Hz), and filtered nerve activity was recorded using a Biopac MP36 Acquisition System. Efferent VN activity was recorded for 20 minutes following systemic administration of either saline or CPSI-121.
Statistical Analysis Data are expressed as the mean T SEM. Analysis of variance with Student-Newman-Keuls post hoc analysis or Student’s t test was performed where appropriate. Statistical significance was determined based on p G 0.05.
RESULTS Hemodynamic Effect of CPSI-121 To determine whether CPSI-121 causes significant hemodynamic effects, we continuously measured MAP and heart rate after CPSI-121 administration. MAP was not altered after treatment with CPSI-121 (Fig. 1B and D), while heart rate decreased only slightly from 300 beats per minute to 260 beats per minute (Fig. 1A).
CPSI-121 Induces Vagus Nerve Electrical Activity Nerve activity was recorded for 20 minutes after administration of NS or CPSI-121. Injection of NS did not increase electrical activity from the cervical vagus nerve (Fig. 2A). Administration of CPSI-121 increased electrical activity in the vagus nerve at both the cervical (Fig. 2B) and abdominal (Fig. 2C) locations. Nerve activity increased within minutes of CPSI-121 treatment and persisted for up to 30 minutes. There was no significant electrical activity observed in the femoral nerve after CPSI-121 treatment, suggesting some specificity for the vagus nerve (Fig. 2D). These results demonstrate that CPSI-121 stimulates vagus nerve electrical activity.
CPSI-121 Prevents T/HS-Induced Gut Injury and Attenuates the Biologic Activity of the ML T/HS caused histologic gut injury characterized by mucosal injury and shortening of the villi at 24 hours after injury (Fig. 3B). CPSI-121 attenuated T/HS-induced histologic gut injury (Fig. 3C), with histologic injury scores from CPSI-121Ytreated animals similar to those of sham (Fig. 3D). The ability of the injured gut to generate biologically active ML was assessed by its ability to prime PMNs. The ML collected from animals following T/HS caused a significant increase in PMN oxidative burst (3.8 T 0.5 105) when
Figure 2. CPSI-121 induces vagus nerve electrical activity. Rats were given CPSI-121 (1 mg/kg) or NS intravenously, while vagus nerve and femoral nerve electrical activities were recorded using a Biopac MP36 Acquisition System. A, Representative nerve output tracings from the cervical vagus nerve after NS administration. Treatment with CPSI-121 increases electrical activity from both the cervical vagus nerve (B) and the abdominal vagus nerve (C). CPSI-121 does not increase electrical activity in the femoral nerve, suggesting specificity for the vagus nerve (D). 54
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Figure 3. CPSI-121 prevents T/HS-induced gut injury. Sections of distal ileum were harvested 24 hours after T/HS, fixed, and stained with hematoxylin and eosin. Representative sections from animals following (A) sham, (B) T/HS, and (C) T/HS + CPSI-121 administration. D, Histologic gut injury was scored according to a histology grading scale. Data are expressed as mean T SEM (n = 4Y5, *p G 0.05 compared with T/HS).
compared with pre-HS (0.8 T 0.3 105, p G 0.05). Treatment with CPSI-121 after injury attenuates T/HS-induced PMN oxidative burst (1.7 T 0.8 105, p G 0.05) (Fig. 4A and B).
CPSI-121 Prevents T/HS-Induced ALI At 24 hours after injury, the histologic appearance of lung sections harvested after T/HS demonstrated interstitial edema and inflammatory cell infiltrates when compared with
the lung sections from the sham animals (Fig. 5A). Treatment with CPSI-121 abolished T/HS-induced histologic lung injury and significantly decreased the lung histology grading score compared with that of the T/HS group (p G 0.05) (Fig. 5B). To further evaluate the effect of CPSI-121 on pulmonary microvascular permeability, we measured wet-dry ratios as an indicator of pulmonary edema. Wet-dry ratios were increased after T/HS (3.9 T 0.1) compared with the sham (3.6 T 0.1, p G0.05).
Figure 4. ML from CPSI-121Ytreated T/HS rats has reduced PMNs oxidative burst. The ability of ML to prime PMNs was assessed by measuring in vitro oxidative burst. Lymph samples (5% vol./vol.) from each experimental group were added to PMNs isolated from the blood of naive rats. PMN respiratory burst was measured by flow cytometry after incubation with PMA. A, Representative histogram demonstrating that CPSI-121 attenuated T/HS-induced PMN oxidative burst. B, Graph demonstrating mean fluorescence intensity (MFI) as a measure of PMN respiratory burst. Data are expressed as mean T SEM (n = 3Y5, *p G 0.05 compared with T/HS). * 2015 Wolters Kluwer Health, Inc. All rights reserved.
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Figure 5. CPSI-121 prevents T/HS-induced ALI. Lung specimens were collected 24 hours after injury and stained with hematoxylin and eosin. A, Representative section from sham, T/HS, and T/HS + CPSI-121. B, Lung injury was scored according to a histology grading scale. Data are expressed as mean T SEM (n = 3Y5, *p G 0.05 compared with T/HS). C, Lung wet-dry ratios were measured to determine lung edema. Data are expressed as mean T SEM (n = 3Y5, *p G 0.05 compared with T/HS).
Animals treated with CPSI-121 following T/HS had decreased lung edema compared with T/HS alone (3.5 T 0.1, p G0.05), with wet-dry ratios similar to those of the sham (Fig. 5C).
DISCUSSION Major trauma is known to disrupt the gut barrier, leading to an intestinal inflammatory response at early time points following injury.16 Biologically active mediators leave the gut, traffic through the ML, and can lead to a robust systemic inflammatory response and distant organ injury. A common site of organ dysfunction after injury is the lung, where the innate immune response promotes inflammatory cell recruitment and proinflammatory cytokine release leading to microvascular permeability, disruption of the alveolar-capillary interface, and lung edema.17 The development of ALI after major trauma is a source of significant morbidity and mortality during the subacute recovery from injury. Although a number of therapeutic strategies aimed at limiting the systemic inflammatory response to injury have been investigated in preclinical models, their success has been limited in the clinical setting. The vagus nerve plays an important role in mediating the immune response through its ability to both sense the peripheral inflammatory state of the host through its afferent limb, while signaling through its efferent arm to attenuate inflammation, thus limiting damage to host tissues and promoting a return to homeostasis.18 Stimulating the vagus nerve to cause efferent antiinflammatory signaling has been proposed as a strategy to limit the immune response to infection and injury. VNS has been shown to limit both the circulating immune response and through direct effects on tissues such as the gut.3,19Y21 Work in our 56
laboratory and others have demonstrated the ability of direct, electrical VNS to prevent gut barrier failure, intestinal inflammation, the production of biologically active ML, and ALI in animal models of trauma and burn injury.4,6,7,21,22 While direct, electrical stimulation of the vagus nerve is safe and is an approved treatment for patients with drug-resistant epilepsy,23 its potential for deployment in the treatment of severely injured patients is currently limited. The major limitation to the clinical use of VNS is in the invasiveness of stimulator placement, especially within the first 1 hour to 2 hours after injury where a therapeutic window exists.24 While future advances in bioengineering and nanotechnology may allow for a rapid, minimally invasive approach to direct stimulation of the vagus nerve, we considered a pharmacologic approach to VNS that could be portable and immediately be administered as an adjunct to standard resuscitation strategies. CPSI-121 is a guanylhydrazone-derived compound that was developed as a pharmacologic vagal agonist. CPSI-121 is structurally related to CNI-1493 (Semapinod), a tetravalent guanylhydrazone-derived compound, which is known to induce an increase in efferent vagus nerve activity and inhibits the synthesis of proinflammatory mediators.25 While CPSI-121 is a parasympathetic agonist that is predicted to cause vagal anti-inflammatory signaling based on its structure, its ability to cause vagal electrical activity was previously unknown. Therefore, we tested whether CPSI-121 would induce vagal electrical activity and limit the inflammatory response to T/HS. In the present study, we found that CPSI-121 induced an increase in the electrical discharge rate in the cervical and abdominal vagus nerve without harmful hemodynamic effects. This study is the first to confirm the ability of CPSI-121 to * 2015 Wolters Kluwer Health, Inc. All rights reserved.
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induce vagal electrical activity. This vagal activity seen after treatment with CPSI-121 was associated with gut protection as demonstrated by a decrease in T/HS-induced histologic gut injury. This is in agreement with our previously published study showing the gut-protective effects of CPSI-121 after burn injury.11 We found that CPSI-121, similar to electrical VNS, decreased the toxicity of the ML as measured by its ability to induce neutrophil oxidative burst. Generation of biologically active ML is a key mediator in igniting the systemic inflammatory response to injury.26,27 Therefore, the ability of CPSI-121 to limit the toxicity of the ML, as seen in this model, suggests its potential to limit distant organ injury. The decreased biologic activity of the ML in animals treated with CPSI-121 was associated with decreased histologic lung injury and less lung edema compared with animals given standard resuscitation, further supporting the ability of this pharmacologic vagal agonist to limit distant organ injury after T/HS. Although the exact mechanism of CPSI-121 is less well understood in acute models of injury, guanylhydrazone-derived compounds are known to have a potent anti-inflammatory properties. CNI-1493 is a powerful inhibitor of tumor necrosis factor and interleukin 1 synthesis28 and acts to inhibit cytokine synthesis via inhibition of p38-MAP kinase phosphorylation in lipopolysaccharide-stimulated macrophages.29,30 Websky et al.31 reported that CPSI-2364, another guanylhydrazonederived compound, ameliorates ischemia-reperfusion injury after experimental small bowel transplantation by inhibition of proinflammatory cytokine synthesis and suppression of nitric oxide production in macrophages. We have previously shown that the protective effects of CPSI-121 on gut injury are lost when the vagus nerve connection to the gut is disrupted by surgical abdominal vagotomy.10 This suggests that the gut protection provided by treatment with CPSI-121 are due to its ability to cause efferent vagus nerve signaling rather than through direct, anti-inflammatory effects on gut tissue. Further mechanistic studies are planned to determine the signaling pathways through which CPSI-121 attenuates the inflammatory response in this model. In summary, CPSI-121 is a pharmacologic approach to VNS that prevents gut barrier failure, limits the toxicity of the ML, and prevents ALI in a preclinical model of major trauma. A pharmacologic approach to VNS may be ideal because it is a noninvasive, easily administered therapy that could be used in the clinical setting and administered in the early period after injury. The portability of a pharmacologic vagal agonist would allow for treatment during transport, upon arrival to the resuscitation room, or in the military setting. Treatment with CPSI-121 to augment efferent VN signaling following injury may represent a strategy to alter the immune response to T/HS and potentially improve outcomes in severe trauma patients. AUTHORSHIP K.M. performed the animal model and sample collection. K.M. and A.U. recorded the VN activity. K.M. and B.E. performed the data collection and analysis of flow cytometry. K.M., T.W.C., B.E., V.B., and R.C. conceived the study and participated in its design and coordination. K.M., A.U., T.W.C., B.E., and R.C. drafted the manuscript. All authors read and approved the final manuscript.
DISCLOSURE The authors declare no conflicts of interest.
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acetylcholine receptor in the enteric nervous system: a cholinergic agonist prevents gut barrier failure after severe burn injury. Am J Pathol. 2012; 181:478Y486. Reys LG, Ortiz-Pomales YT, Lopez N, Cheadle G, de Oliveira PG, Eliceiri B, Bansal V, Costantini TW, Coimbra R. Uncovering the neuroentericpulmonary axis: vagal nerve stimulation prevents acute lung injury following hemorrhagic shock. Life Sci. 2013;92:783Y792. Al-Otaibi FA, Hamani C, Lozano AM. Neuromodulation in epilepsy. Neurosurgery. 2011;69:957Y979; discussion 79. Krzyzaniak M, Peterson C, Loomis W, Hageny AM, Wolf P, Reys L, Putnam J, Eliceiri B, Baird A, Bansal V, et al. Postinjury vagal nerve stimulation protects against intestinal epithelial barrier breakdown. J Trauma. 2011;70:1168Y1175; discussion 1175Y1176. Borovikova LV, Ivanova S, Nardi D, Zhang M, Yang H, Ombrellino M, Tracey KJ. Role of vagus nerve signaling in CNI-1493-mediated suppression of acute inflammation. Auton Neurosci. 2000;85:141Y147. Deitch EA, Xu D, Kaise VL. Role of the gut in the development of injuryand shock induced SIRS and MODS: the gut-lymph hypothesis, a review. Front Biosci. 2006;11:520Y528. Magnotti LJ, Upperman JS, Xu DZ, Lu Q, Deitch EA. Gut-derived mesenteric lymph but not portal blood increases endothelial cell permeability and promotes lung injury after hemorrhagic shock. Ann Surg. 1998;228:518Y527. D’Souza MJ, Oettinger CW, Milton GV, Tracey KJ. Prevention of lethality and suppression of proinflammatory cytokines in experimental septic shock by microencapsulated CNI-1493. J Interferon Cytokine Res. 1999; 19:1125Y1133. Cohen PS, Nakshatri H, Dennis J, Caragine T, Bianchi M, Cerami A, Tracey KJ. CNI-1493 inhibits monocyte/macrophage tumor necrosis factor by suppression of translation efficiency. Proc Natl Acad Sci U S A. 1996; 93:3967Y3971. Cohen PS, Schmidtmayerova H, Dennis J, Dubrovsky L, Sherry B, Wang H, Bukrinsky M, Tracey KJ. The critical role of p38 MAP kinase in T cell HIV-1 replication. Mol Med. 1997;3:339Y346. Websky M, Fujishiro J, Ohsawa I, Praktiknjo M, Wehner S, Abu-Elmagd K, Kitamura K, Kalff JC, Schaefer N, Pech T. The novel guanylhydrazone CPSI-2364 ameliorates ischemia reperfusion injury after experimental small bowel transplantation. Transplantation. 2013;95:1315Y1323.
DISCUSSION Dr. William G. Cioffi (Providence, Rhode Island): I’d like to congratulate Dr. Coimbra and his lab for a continuation of their studies on the effect of vagal nerve stimulation on postinjury organ dysfunction. This study is important because they now use a pharmacologic agent to attempt vagal nerve stimulation. Importantly, they have studied both trauma hemorrhage models, as in this paper, and a burn injury models which they showed in their preliminary data. Intravenous CPSI appears to have the same effect as direct vagal nerve stimulation. In addition, they note increased vagal nerve activity, suggesting this as the mechanism. The results they have shown us are straightforward and valid. However, before we suggest, potentially, a human trial, a few more questions need to be answered. First, in both of your models of hemorrhage and burn injury there is a significant amount of gut dysfunction. Is this an amplification of the human condition or are rats just different in the degree of gut dysfunction they show after these relatively simple injuries? Your model is carefully controlled, as it should be, for basic science work. But what happens when other physiologic variables are deranged, which often happens in our patients, such as temperature, oxygenation and the like? Do 58
you get the same result as you show in the careful models that you have done? Third is dosing. This dose was figured out by your burn injury models but is this enough or is it too much? What are the long-term consequences of this drug? Do you have any data suggesting effects of continuous administration of the drug on other organ function? You gave the drug right after hemorrhage but before resuscitation. It would be a difficult thing to potentially do in our patients, to get it in that quick. What happens with later administration of the drug? I think potentially the most important question to answer, though, is what is the mechanism? Although you clearly show increased vagal nerve activity, CPSI is also known to have a direct anti-inflammatory effect. So how do you truly know the mechanism of the drug and is it via vagal nerve stimulation, as you suggested? Despite these questions, this is a nice piece of work and important as it moves us closer to a potential clinical treatment. And, again, I’d like to congratulate the lab on continuing the work with vagal nerve stimulation. Dr. Tom Granchi (Iowa City, Iowa): This is a very elegant study, good physiology. Does this just mimic the effects of enteral feeding? We know there is a benefit in trauma and burn patients. And have you run the studies with an arm for enteral and no-enteral feedings? Dr. Koji Morishita (San Diego, California): Thank you very much for your questions, Dr. Cioffi, and your discussion of our paper. I will attempt to address all of these wonderful questions. You asked about the differences in the gut response to injury in rats versus human. As you know, the gut is believed to be the source of the systemic inflammatory response to injury in humans. Therefore, our goal is to limit gut barrier failure at early time points after injury to limit mesenteric lymph toxicity. While the kinetics of gut injury and repair are likely different between rats and humans, this rat model allows us to collect specimens such as mesenteric lymph that are not readily accessible from severely injured patients. You highlight the importance of developing animal models that recapitulate the human injury response as closely as possible, this is an active area of investigation in our lab. You asked a question about whether we see similar protective effects when using CPSI in the face of other physiologic derangements such as hypoxia or hypothermia. As you mention, our injury model controlled for these variables in this initial series of experiments. It will be important to add additional variables to our model to better simulate clinical scenarios as we attempt to advance the use of this drug to the treatment of injured patients. You asked about the mechanism of action of CPSI 121 and how it functions in relation to vagus nerve stimulation versus direct anti-inflammatory effects. Guanylhydrazone compounds were originally developed to inhibit inflammatory mediators such as TNF and NO. Prior experiments with the older generation compound have shown that it inhibits production of pro-inflammatory cytokines in macrophages by inhibiting the phosphorylation of P38 MAPK. * 2015 Wolters Kluwer Health, Inc. All rights reserved.
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In this study, we found that CPSI 121 caused electrical vagus nerve output and prevented gut inflammation and mesenteric lymph toxicity. We previously administered CPSI 121 after abdominal vagotomy to define whether the protective effects of CPSI were mediated by vagus nerve activity or through direct effects of CPSI on the cells of the gut wall. We found that the protective effects of CPSI 121 were lost after vagotomy in our burn model, therefore it seems that the anti-inflammatory effects on the gut require an intact vagus nerve. You asked about the dosing of CPSI 121. Based on our previous experiments, CPSI 121 was administrated using a single 1 mg/kg dose that is given post-injury. We believe this is a clinically relevant strategy simulating administration of the drug upon arrival to the trauma center. We have not yet tested other dosing strategies but it will be interesting to study the
anti-inflammatory effects of repeated administration of the drug as we consider treatment strategies that can be translated to the clinical setting. Dr. Granchi, you asked a question if our results mimic the effects of enteric feeding. While the beneficial immune effects of enteral nutrition are clear, we believe that CPSI 121 can be delivered more rapidly after injury and is likely to exert greater anti-inflammatory effects. Our prior studies suggest that vagal agonists need to be delivered within the first 90 minutes of injury in our animal models. It would be unlikely to see similar effects from enteral feeding alone during that therapeutic window. Unfortunately, we do not have data regarding the effects of CPSI 121 in the presence of enteral feeding post-injury, but this will be interesting to consider in future studies. Thank you very much.
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A pharmacologic approach to vagal nerve stimulation prevents mesenteric lymph toxicity after hemorrhagic shock Koji Morishita, MD, Todd W. Costantini, MD, Akinori Ueno, PhD, Vishal Bansal, MD, Brian Eliceiri, PhD, and Raul Coimbra, MD, PhD, San Diego, California
BACKGROUND: Electrical stimulation of the vagus nerve (VN) prevents gut and lung inflammation and mesenteric lymph (ML) toxicity in animal models of injury. We have previously shown that treatment with CPSI-121, a guanylhydrazone-derived compound, prevents gut barrier failure after burn injury. While the structure of CPSI-121 predicts that it will activate parasympathetic signaling, its ability to stimulate the VN is unknown. The aims of this study were to (1) measure the ability of CPSI-121 to induce VN activity, (2) determine whether CPSI-121 causes significant hemodynamic effects, and (3) further define the potential for CPSI-121 to limit the systemic inflammatory response to injury. METHODS: Male Sprague-Dawley rats were given 1-mg/kg CPSI-121 intravenously while blood pressure, heart rate, and efferent VN electrical activity were recorded. Rats were also assigned to sham or trauma/hemorrhagic shock (T/HS). T/HS was induced by laparotomy and 60 minutes of HS (mean arterial pressure, 35 mm Hg) followed by fluid resuscitation. A separate cohort of animals received CPSI-121 after the HS phase. Gut and lung tissues were harvested for histologic analysis. Lung wet-dry ratios were also evaluated. The ability of ML to prime neutrophils was assessed by measuring in vitro oxidative burst using flow cytometry. RESULTS: Blood pressure was not altered after treatment with CPSI-121, while heart rate decreased only slightly. Recording of efferent VN electrical activity revealed an increase in discharge rate after administration of CPSI-121. T/HS caused gut and lung injury, which were prevented in animals treated with CPSI-121 (p G 0.05). Treatment with CPSI-121 following T/HS attenuated neutrophil priming after exposure to ML (p G 0.05). CONCLUSION: CPSI-121 causes efferent VN output and limits shock-induced gut and lung injury as well as ML toxicity. CPSI-121 is a candidate pharmacologic approach to VN stimulation aimed at limiting the inflammatory response in patients following T/HS. (J Trauma Acute Care Surg. 2015;78: 52Y59. Copyright * 2015 Wolters Kluwer Health, Inc. All rights reserved.) KEY WORDS: Hemorrhagic shock; mesenteric lymph; CPSI-121; vagal nerve stimulation; rats.
T
rauma/hemorrhagic shock (T/HS) leads to gut barrier failure that initiates a systemic inflammatory response resulting in acute lung injury (ALI) and multiple-organ dysfunction syndrome.1 After T/HS, the injured gut releases proinflammatory mediators into the mesenteric lymph (ML) that activate inflammatory and endothelial cells.2 Thus, ML is considered to be central in the pathogenesis of ALI/multiple-organ dysfunction syndrome following T/HS. We have previously shown that electrical vagal nerve stimulation (VNS) prevents intestinal barrier failure and alters inflammatory cell trafficking in the ML.3Y5 Previous studies in our laboratory and others have also demonstrated that VNS attenuates injury-induced ALI through its ability to limit gut inflammation, rather than via direct effects on the lung.6Y9 Although evidence supporting the use of electrical VNS in burn, sepsis, and T/HS is compelling in preclinical animal
Submitted: August 1, 2014, Revised: September 8, 2014, Accepted: October 2, 2014. From the Division of Trauma, Surgical Critical Care, Burns, and Acute Care Surgery, Department of Surgery, University of California San Diego Health Sciences, San Diego, California. This study was presented at the 73rd annual meeting of the American Association for the Surgery of Trauma, September, 10Y13, 2014, in Philadelphia, Pennsylvania. Address for reprints: Todd W. Costantini, MD, Division of Trauma, Surgical Critical Care, Burns, and Acute Care Surgery, Department of Surgery, University of California San Diego Health Sciences, 200 W Arbor Dr, #8896, San Diego, CA 92103Y8896; email: [email protected]. DOI: 10.1097/TA.0000000000000489
models, direct electrical stimulation of the vagus nerve in acutely injured patients currently is impractical. A pharmacologic approach to VNS would be a more clinically feasible and rapidly deployable strategy in the clinical setting. We have previously demonstrated that treatment with CPSI-121, a divalent guanylhydrazone, prevents gut barrier failure after burn injury with antiinflammatory effects that are comparable with direct, electrical VNS.10 While the structure of CPSI-121 predicts that it will activate parasympathetic signaling, its ability to stimulate the vagus nerve is unknown. In this series of experiments, we administered CPSI-121 following T/HS to determine its ability to prevent gut and lung injury. We hypothesized that CPSI-121 would induce vagus nerve electrical activity, decrease the toxicity of ML, and limit distant organ injury, demonstrating the capacity for CPSI-121 to limit the systemic inflammatory response to injury.
MATERIALS AND METHODS T/HS Model All animal experiments were approved by the University of California San Diego Institutional Animal Care and Use Committee. Male Sprague-Dawley rats weighing 280 g to 300 g were obtained from Harlan Laboratories (Placentia, CA). Animals were anesthetized with ketamine (75 mg/kg; Fort Dodge Animal Health, Fort Dodge, IA) and xylazine (10 mg/kg; Sigma J Trauma Acute Care Surg Volume 78, Number 1
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Chemical, St. Louis, MO), and the left femoral artery and vein were cannulated with a polyethylene tube (PE-50). The mean arterial pressure (MAP) was continuously monitored using the femoral arterial catheter (Philips V24/26, Andover, MA). Heart rate was recorded using the Biopac MP36 Acquisition System (Santa Barbara, CA). Each animal’s body temperature was maintained at 37-C. A right medial visceral rotation was performed through a midline laparotomy (trauma), and then, the abdomen was closed. HS was induced via withdrawal of blood from the femoral vein catheter until the MAP was reduced to 35 mm Hg and maintained for 60 minutes. At the end of HS, animals in the T/HS group were resuscitated with shed blood + two times shed blood volume in normal saline (NS) (Baxter, Deerfield, IL). Sham animals underwent the identical anesthesia and cannulation of the femoral vessels without trauma and hemorrhage. A separate cohort of animals was treated with CPSI-121 (Ferring, San Diego, CA) diluted in sterile water immediately after injury. CPSI-121 was administered at 1 mg/kg intravenously based on our previous experiments demonstrating gut barrier protection after injury using this dose.10
ML was collected on ice during the pre-HS phase (30 minutes), HS phase (60 minutes), and post-HS phase (120 minutes, Fig. 1C). The ML was centrifuged at 2,000 rpm for 5 minutes at 4-C, and the supernatant was stored at j80-C until further analysis.
Collection of ML
Histologic Evaluation
As previously described,11 the ML duct was exposed, and the efferent ML duct was cannulated using PE50 tubing.
At 24 hours after injury, segments of distal ileum and NS-perfused lungs were removed and fixed in 10% buffered
Respiratory Burst The biologic activity of ML was measured via its ability to activate PMNs in vitro.12 Whole blood was obtained via a femoral artery catheter from naive male rats then treated with RBC lysis buffer (BD Pharmingen, San Diego, CA). Freshly thawed lymph samples (n = 3Y5 animals per group) were added to the remaining leukocyte pellet (5% vol./vol.) and then placed into an incubator for 5 minutes. Dihydrorhodamine 123 (15 ng/mL) (Molecular Probes, Inc., Grand Island, NY) was added to the tubes for 5 minutes before stimulation with Phorbol 12-myristate 13-acetate (PMA, Sigma). Samples were incubated for 15 minutes at 37-C. The PMN respiratory burst was measured by flow cytometry using a BD Accuri C6 (BD Bioscience, San Jose, CA). The forward and side scatter detectors were used to identify the PMN population, and the fluorescence gain was adjusted using unstimulated and stimulated control samples.
Figure 1. Hemodynamic effect of CPSI-121. To determine whether CPSI-121 causes significant hemodynamic effect, we continuously measured MAP and heart rate after CPSI-121administration (1 mg/kg). Heart rate (A) and MAP (B) were measured continuously throughout the experiment. C, A model of T/HS was induced by laparotomy and 60 minutes of HS (MAP, 35 mm Hg), followed by fluid resuscitation. A separate cohort of animals received CPSI-121 (1 mg/kg) after the HS phase (D). * 2015 Wolters Kluwer Health, Inc. All rights reserved.
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formalin, embedded in paraffin, and sectioned (n = 4Y5 per group). Hematoxylin and eosin staining was performed by the University of California San Diego Histology Core Services. An investigator blinded to the experimental groups analyzed multiple fields from each section of tissue with a light microscope. The ileum and lung sections were classified according to the degree of tissue injury according to histologic grading scores as previously described.13,14
Wet-to-Dry Lung Weight Ratios The wet-to-dry ratio of the lung was determined as a measure of lung edema.15 Lungs were excised after injury (n = 3Y5 animals per group) and weighed to obtain the wet weight. Lungs were then dried in an oven at 60-C for 7 days to obtain the dry weight and calculate the wet-to-dry ratio ([wet weight j dry weight] / dry weight).
Recording of Vagus Nerve Activity Animals were anesthetized with isoflurane, the vagus and femoral nerves were exposed, both positive and negative electrodes were applied to the target nerve with a certain distance, and a ground electrode was inserted under the skin. Signals from the electrodes were passed through a band-pass filter (100Y2,000 Hz), and filtered nerve activity was recorded using a Biopac MP36 Acquisition System. Efferent VN activity was recorded for 20 minutes following systemic administration of either saline or CPSI-121.
Statistical Analysis Data are expressed as the mean T SEM. Analysis of variance with Student-Newman-Keuls post hoc analysis or Student’s t test was performed where appropriate. Statistical significance was determined based on p G 0.05.
RESULTS Hemodynamic Effect of CPSI-121 To determine whether CPSI-121 causes significant hemodynamic effects, we continuously measured MAP and heart rate after CPSI-121 administration. MAP was not altered after treatment with CPSI-121 (Fig. 1B and D), while heart rate decreased only slightly from 300 beats per minute to 260 beats per minute (Fig. 1A).
CPSI-121 Induces Vagus Nerve Electrical Activity Nerve activity was recorded for 20 minutes after administration of NS or CPSI-121. Injection of NS did not increase electrical activity from the cervical vagus nerve (Fig. 2A). Administration of CPSI-121 increased electrical activity in the vagus nerve at both the cervical (Fig. 2B) and abdominal (Fig. 2C) locations. Nerve activity increased within minutes of CPSI-121 treatment and persisted for up to 30 minutes. There was no significant electrical activity observed in the femoral nerve after CPSI-121 treatment, suggesting some specificity for the vagus nerve (Fig. 2D). These results demonstrate that CPSI-121 stimulates vagus nerve electrical activity.
CPSI-121 Prevents T/HS-Induced Gut Injury and Attenuates the Biologic Activity of the ML T/HS caused histologic gut injury characterized by mucosal injury and shortening of the villi at 24 hours after injury (Fig. 3B). CPSI-121 attenuated T/HS-induced histologic gut injury (Fig. 3C), with histologic injury scores from CPSI-121Ytreated animals similar to those of sham (Fig. 3D). The ability of the injured gut to generate biologically active ML was assessed by its ability to prime PMNs. The ML collected from animals following T/HS caused a significant increase in PMN oxidative burst (3.8 T 0.5 105) when
Figure 2. CPSI-121 induces vagus nerve electrical activity. Rats were given CPSI-121 (1 mg/kg) or NS intravenously, while vagus nerve and femoral nerve electrical activities were recorded using a Biopac MP36 Acquisition System. A, Representative nerve output tracings from the cervical vagus nerve after NS administration. Treatment with CPSI-121 increases electrical activity from both the cervical vagus nerve (B) and the abdominal vagus nerve (C). CPSI-121 does not increase electrical activity in the femoral nerve, suggesting specificity for the vagus nerve (D). 54
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Figure 3. CPSI-121 prevents T/HS-induced gut injury. Sections of distal ileum were harvested 24 hours after T/HS, fixed, and stained with hematoxylin and eosin. Representative sections from animals following (A) sham, (B) T/HS, and (C) T/HS + CPSI-121 administration. D, Histologic gut injury was scored according to a histology grading scale. Data are expressed as mean T SEM (n = 4Y5, *p G 0.05 compared with T/HS).
compared with pre-HS (0.8 T 0.3 105, p G 0.05). Treatment with CPSI-121 after injury attenuates T/HS-induced PMN oxidative burst (1.7 T 0.8 105, p G 0.05) (Fig. 4A and B).
CPSI-121 Prevents T/HS-Induced ALI At 24 hours after injury, the histologic appearance of lung sections harvested after T/HS demonstrated interstitial edema and inflammatory cell infiltrates when compared with
the lung sections from the sham animals (Fig. 5A). Treatment with CPSI-121 abolished T/HS-induced histologic lung injury and significantly decreased the lung histology grading score compared with that of the T/HS group (p G 0.05) (Fig. 5B). To further evaluate the effect of CPSI-121 on pulmonary microvascular permeability, we measured wet-dry ratios as an indicator of pulmonary edema. Wet-dry ratios were increased after T/HS (3.9 T 0.1) compared with the sham (3.6 T 0.1, p G0.05).
Figure 4. ML from CPSI-121Ytreated T/HS rats has reduced PMNs oxidative burst. The ability of ML to prime PMNs was assessed by measuring in vitro oxidative burst. Lymph samples (5% vol./vol.) from each experimental group were added to PMNs isolated from the blood of naive rats. PMN respiratory burst was measured by flow cytometry after incubation with PMA. A, Representative histogram demonstrating that CPSI-121 attenuated T/HS-induced PMN oxidative burst. B, Graph demonstrating mean fluorescence intensity (MFI) as a measure of PMN respiratory burst. Data are expressed as mean T SEM (n = 3Y5, *p G 0.05 compared with T/HS). * 2015 Wolters Kluwer Health, Inc. All rights reserved.
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Figure 5. CPSI-121 prevents T/HS-induced ALI. Lung specimens were collected 24 hours after injury and stained with hematoxylin and eosin. A, Representative section from sham, T/HS, and T/HS + CPSI-121. B, Lung injury was scored according to a histology grading scale. Data are expressed as mean T SEM (n = 3Y5, *p G 0.05 compared with T/HS). C, Lung wet-dry ratios were measured to determine lung edema. Data are expressed as mean T SEM (n = 3Y5, *p G 0.05 compared with T/HS).
Animals treated with CPSI-121 following T/HS had decreased lung edema compared with T/HS alone (3.5 T 0.1, p G0.05), with wet-dry ratios similar to those of the sham (Fig. 5C).
DISCUSSION Major trauma is known to disrupt the gut barrier, leading to an intestinal inflammatory response at early time points following injury.16 Biologically active mediators leave the gut, traffic through the ML, and can lead to a robust systemic inflammatory response and distant organ injury. A common site of organ dysfunction after injury is the lung, where the innate immune response promotes inflammatory cell recruitment and proinflammatory cytokine release leading to microvascular permeability, disruption of the alveolar-capillary interface, and lung edema.17 The development of ALI after major trauma is a source of significant morbidity and mortality during the subacute recovery from injury. Although a number of therapeutic strategies aimed at limiting the systemic inflammatory response to injury have been investigated in preclinical models, their success has been limited in the clinical setting. The vagus nerve plays an important role in mediating the immune response through its ability to both sense the peripheral inflammatory state of the host through its afferent limb, while signaling through its efferent arm to attenuate inflammation, thus limiting damage to host tissues and promoting a return to homeostasis.18 Stimulating the vagus nerve to cause efferent antiinflammatory signaling has been proposed as a strategy to limit the immune response to infection and injury. VNS has been shown to limit both the circulating immune response and through direct effects on tissues such as the gut.3,19Y21 Work in our 56
laboratory and others have demonstrated the ability of direct, electrical VNS to prevent gut barrier failure, intestinal inflammation, the production of biologically active ML, and ALI in animal models of trauma and burn injury.4,6,7,21,22 While direct, electrical stimulation of the vagus nerve is safe and is an approved treatment for patients with drug-resistant epilepsy,23 its potential for deployment in the treatment of severely injured patients is currently limited. The major limitation to the clinical use of VNS is in the invasiveness of stimulator placement, especially within the first 1 hour to 2 hours after injury where a therapeutic window exists.24 While future advances in bioengineering and nanotechnology may allow for a rapid, minimally invasive approach to direct stimulation of the vagus nerve, we considered a pharmacologic approach to VNS that could be portable and immediately be administered as an adjunct to standard resuscitation strategies. CPSI-121 is a guanylhydrazone-derived compound that was developed as a pharmacologic vagal agonist. CPSI-121 is structurally related to CNI-1493 (Semapinod), a tetravalent guanylhydrazone-derived compound, which is known to induce an increase in efferent vagus nerve activity and inhibits the synthesis of proinflammatory mediators.25 While CPSI-121 is a parasympathetic agonist that is predicted to cause vagal anti-inflammatory signaling based on its structure, its ability to cause vagal electrical activity was previously unknown. Therefore, we tested whether CPSI-121 would induce vagal electrical activity and limit the inflammatory response to T/HS. In the present study, we found that CPSI-121 induced an increase in the electrical discharge rate in the cervical and abdominal vagus nerve without harmful hemodynamic effects. This study is the first to confirm the ability of CPSI-121 to * 2015 Wolters Kluwer Health, Inc. All rights reserved.
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induce vagal electrical activity. This vagal activity seen after treatment with CPSI-121 was associated with gut protection as demonstrated by a decrease in T/HS-induced histologic gut injury. This is in agreement with our previously published study showing the gut-protective effects of CPSI-121 after burn injury.11 We found that CPSI-121, similar to electrical VNS, decreased the toxicity of the ML as measured by its ability to induce neutrophil oxidative burst. Generation of biologically active ML is a key mediator in igniting the systemic inflammatory response to injury.26,27 Therefore, the ability of CPSI-121 to limit the toxicity of the ML, as seen in this model, suggests its potential to limit distant organ injury. The decreased biologic activity of the ML in animals treated with CPSI-121 was associated with decreased histologic lung injury and less lung edema compared with animals given standard resuscitation, further supporting the ability of this pharmacologic vagal agonist to limit distant organ injury after T/HS. Although the exact mechanism of CPSI-121 is less well understood in acute models of injury, guanylhydrazone-derived compounds are known to have a potent anti-inflammatory properties. CNI-1493 is a powerful inhibitor of tumor necrosis factor and interleukin 1 synthesis28 and acts to inhibit cytokine synthesis via inhibition of p38-MAP kinase phosphorylation in lipopolysaccharide-stimulated macrophages.29,30 Websky et al.31 reported that CPSI-2364, another guanylhydrazonederived compound, ameliorates ischemia-reperfusion injury after experimental small bowel transplantation by inhibition of proinflammatory cytokine synthesis and suppression of nitric oxide production in macrophages. We have previously shown that the protective effects of CPSI-121 on gut injury are lost when the vagus nerve connection to the gut is disrupted by surgical abdominal vagotomy.10 This suggests that the gut protection provided by treatment with CPSI-121 are due to its ability to cause efferent vagus nerve signaling rather than through direct, anti-inflammatory effects on gut tissue. Further mechanistic studies are planned to determine the signaling pathways through which CPSI-121 attenuates the inflammatory response in this model. In summary, CPSI-121 is a pharmacologic approach to VNS that prevents gut barrier failure, limits the toxicity of the ML, and prevents ALI in a preclinical model of major trauma. A pharmacologic approach to VNS may be ideal because it is a noninvasive, easily administered therapy that could be used in the clinical setting and administered in the early period after injury. The portability of a pharmacologic vagal agonist would allow for treatment during transport, upon arrival to the resuscitation room, or in the military setting. Treatment with CPSI-121 to augment efferent VN signaling following injury may represent a strategy to alter the immune response to T/HS and potentially improve outcomes in severe trauma patients. AUTHORSHIP K.M. performed the animal model and sample collection. K.M. and A.U. recorded the VN activity. K.M. and B.E. performed the data collection and analysis of flow cytometry. K.M., T.W.C., B.E., V.B., and R.C. conceived the study and participated in its design and coordination. K.M., A.U., T.W.C., B.E., and R.C. drafted the manuscript. All authors read and approved the final manuscript.
DISCLOSURE The authors declare no conflicts of interest.
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DISCUSSION Dr. William G. Cioffi (Providence, Rhode Island): I’d like to congratulate Dr. Coimbra and his lab for a continuation of their studies on the effect of vagal nerve stimulation on postinjury organ dysfunction. This study is important because they now use a pharmacologic agent to attempt vagal nerve stimulation. Importantly, they have studied both trauma hemorrhage models, as in this paper, and a burn injury models which they showed in their preliminary data. Intravenous CPSI appears to have the same effect as direct vagal nerve stimulation. In addition, they note increased vagal nerve activity, suggesting this as the mechanism. The results they have shown us are straightforward and valid. However, before we suggest, potentially, a human trial, a few more questions need to be answered. First, in both of your models of hemorrhage and burn injury there is a significant amount of gut dysfunction. Is this an amplification of the human condition or are rats just different in the degree of gut dysfunction they show after these relatively simple injuries? Your model is carefully controlled, as it should be, for basic science work. But what happens when other physiologic variables are deranged, which often happens in our patients, such as temperature, oxygenation and the like? Do 58
you get the same result as you show in the careful models that you have done? Third is dosing. This dose was figured out by your burn injury models but is this enough or is it too much? What are the long-term consequences of this drug? Do you have any data suggesting effects of continuous administration of the drug on other organ function? You gave the drug right after hemorrhage but before resuscitation. It would be a difficult thing to potentially do in our patients, to get it in that quick. What happens with later administration of the drug? I think potentially the most important question to answer, though, is what is the mechanism? Although you clearly show increased vagal nerve activity, CPSI is also known to have a direct anti-inflammatory effect. So how do you truly know the mechanism of the drug and is it via vagal nerve stimulation, as you suggested? Despite these questions, this is a nice piece of work and important as it moves us closer to a potential clinical treatment. And, again, I’d like to congratulate the lab on continuing the work with vagal nerve stimulation. Dr. Tom Granchi (Iowa City, Iowa): This is a very elegant study, good physiology. Does this just mimic the effects of enteral feeding? We know there is a benefit in trauma and burn patients. And have you run the studies with an arm for enteral and no-enteral feedings? Dr. Koji Morishita (San Diego, California): Thank you very much for your questions, Dr. Cioffi, and your discussion of our paper. I will attempt to address all of these wonderful questions. You asked about the differences in the gut response to injury in rats versus human. As you know, the gut is believed to be the source of the systemic inflammatory response to injury in humans. Therefore, our goal is to limit gut barrier failure at early time points after injury to limit mesenteric lymph toxicity. While the kinetics of gut injury and repair are likely different between rats and humans, this rat model allows us to collect specimens such as mesenteric lymph that are not readily accessible from severely injured patients. You highlight the importance of developing animal models that recapitulate the human injury response as closely as possible, this is an active area of investigation in our lab. You asked a question about whether we see similar protective effects when using CPSI in the face of other physiologic derangements such as hypoxia or hypothermia. As you mention, our injury model controlled for these variables in this initial series of experiments. It will be important to add additional variables to our model to better simulate clinical scenarios as we attempt to advance the use of this drug to the treatment of injured patients. You asked about the mechanism of action of CPSI 121 and how it functions in relation to vagus nerve stimulation versus direct anti-inflammatory effects. Guanylhydrazone compounds were originally developed to inhibit inflammatory mediators such as TNF and NO. Prior experiments with the older generation compound have shown that it inhibits production of pro-inflammatory cytokines in macrophages by inhibiting the phosphorylation of P38 MAPK. * 2015 Wolters Kluwer Health, Inc. All rights reserved.
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In this study, we found that CPSI 121 caused electrical vagus nerve output and prevented gut inflammation and mesenteric lymph toxicity. We previously administered CPSI 121 after abdominal vagotomy to define whether the protective effects of CPSI were mediated by vagus nerve activity or through direct effects of CPSI on the cells of the gut wall. We found that the protective effects of CPSI 121 were lost after vagotomy in our burn model, therefore it seems that the anti-inflammatory effects on the gut require an intact vagus nerve. You asked about the dosing of CPSI 121. Based on our previous experiments, CPSI 121 was administrated using a single 1 mg/kg dose that is given post-injury. We believe this is a clinically relevant strategy simulating administration of the drug upon arrival to the trauma center. We have not yet tested other dosing strategies but it will be interesting to study the
anti-inflammatory effects of repeated administration of the drug as we consider treatment strategies that can be translated to the clinical setting. Dr. Granchi, you asked a question if our results mimic the effects of enteric feeding. While the beneficial immune effects of enteral nutrition are clear, we believe that CPSI 121 can be delivered more rapidly after injury and is likely to exert greater anti-inflammatory effects. Our prior studies suggest that vagal agonists need to be delivered within the first 90 minutes of injury in our animal models. It would be unlikely to see similar effects from enteral feeding alone during that therapeutic window. Unfortunately, we do not have data regarding the effects of CPSI 121 in the presence of enteral feeding post-injury, but this will be interesting to consider in future studies. Thank you very much.
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