Increasing evidence suggests that the intestinal microbiota composition could play a role in specific pathologies such as hypertension, obesity and diabetes. This study aims to demonstrate that the intestinal microbiota modulated by a diet creating dysbiosis increased the size of the myocardial infarction and that probiotics could attenuate this effect. To do this, microbiota transplants from rats fed a dysbiotic or non-dysbiotic diet in the presence or absence of probiotics were performed for 10 days on rats whose microbiota had been previously suppressed by antibiotic therapy. Then, the anterior coronary artery of the transplanted rats was occluded for 30 min. Infarct size was measured after 24 h of reperfusion, while signaling pathways were evaluated after 15 min of reperfusion. Intestinal resistance, plasma concentration of LPS (lipopolysaccharides), activation of NF-κB and Akt and composition of the microbiota were also measured. Our results demonstrate a larger infarct size in animals transplanted with the dysbiotic microbiota without probiotics compared to the other groups, including those that received the dysbiotic microbiota with probiotics. This increase in infarct size correlates with a higher firmicutes/bacteroidetes ratio, NF-kB phosphorylation and plasma LPS concentration, and a decrease in intestinal barrier resistance and Akt. These results indicate that dysbiotic microbiota promotes an increase in infarct size, an effect that probiotics can attenuate.

Secondary brain injury following hemorrhagic shock (HS) is a frequent complication in patients, even in the absence of direct brain trauma, leading to behavioral changes and more specifically anxiety and depression. Despite preclinical studies showing inflammation and apoptosis in the brain after HS, none have addressed the impact of circulating mediators. Our group demonstrated an increased uric acid (UA) circulation in rats following HS. Since UA is implicated in endothelial dysfunction and inflammatory response, we hypothesized UA could alter the blood-brain barrier (BBB) and impact the brain. Male Wistar rats were randomly assigned to: SHAM, HS (hemorrhagic shock) and HS + U (hemorrhagic shock + 1.5 mg/kg of uricase). The uricase intervention, specifically targeting UA, was administered during fluid resuscitation. It prevented BBB dysfunction (fluorescein sodium salt permeability and expression of intercellular adhesion molecule-1) following HS. As for neuroinflammation, all of the results obtained (MPO activity; Iba1 and GFAP expression) showed a significant increase after HS, also prevented by the uricase. The same pattern was observed after quantification of apoptosis (caspase-3 activity and TUNEL) and neurodegeneration (Fluoro-Jade). Finally, the forced swim, elevated plus maze, and social interaction tests detected anxiety-like behavior after HS, which was blunted in rats treated with the uricase. In conclusion, we have identified UA as a new circulatory inflammatory mediator, responsible for brain alterations and anxious behavior after HS in a murine model. The ability to target UA holds the potential of an adjunctive therapeutic solution to reduce brain dysfunction related to hemorrhagic shock in human.

BACKGROUND Multiorgan failure is a consequence of severe ischemia-reperfusion injury after traumatic hemorrhagic shock (HS), a major cause of mortality in trauma patients. Circulating uric acid (UA), released from cell lysis, is known to activate proinflammatory and proapoptotic pathways and has been associated with poor clinical outcomes among critically ill patients. Our group has recently shown a mediator role for UA in kidney and lung injury, but its role in liver and enteric damage after HS remains undefined. Therefore, the objective of this study was to evaluate the role of UA on liver and enteric injury after resuscitated HS. METHODS A murine model of resuscitated HS was treated during resuscitation with a recombinant uricase, a urate oxidase enzyme (rasburicase; Sanofi-Aventis, Canada Inc, Laval, Canada), to metabolize and reduce circulating UA. Biochemical analyses (liver enzymes, liver apoptotic, and inflammatory markers) were performed at 24 hours and 72 hours after HS. Physiological testing for enteric permeability and gut bacterial product translocation measurement (plasma endotoxin) were performed 72 hours after HS. In vitro, HT-29 cells were exposed to UA, and the expression of intercellular adhesion proteins (ZO-1, E-cadherin) was measured to evaluate the influence of UA on enteric permeability. RESULTS The addition of uricase to resuscitation significantly reduced circulating and liver UA levels after HS. It also prevented HS-induced hepatolysis and liver apoptotic/inflammatory mediators at 24 hours and 72 hours. Hemorrhagic shock-induced enteric hyperpermeability and endotoxemia were prevented with uricase. CONCLUSIONS After resuscitated HS, UA is an important mediator in liver and enteric injury. Uric acid represents a therapeutic target to minimize organ damage in polytrauma patients sustaining HS.

BACKGROUND Multiple organ failure can develop after hemorrhagic shock (HS). Uric acid (UA) is released from dying cells and can be proinflammatory. We hypothesized that UA could be an alternative mediator of organ apoptosis and inflammation after HS. METHODS Ventilated male Wistar rats were used for the HS model. Two durations of shock (5 minutes vs. 60 minutes) were compared, and shams were instrumented only; animals were resuscitated and observed for 24 hours/72 hours. Caspases-(8/3), myeloperoxidase (MPO), TNF-α were measured in lungs and kidneys. Plasma UA and cytokine (IL-1β, IL-18, TNF-α) were measured. A second set of animals were randomized to vehicle versus Rasburicase intraperitoneal intervention (to degrade UA) during resuscitation. Another group received exogenous UA intraperitoneally without HS. Measures mentioned above, in addition to organs UA, were performed at 24 hours. In vitro, caspases-(8/3) activity was tested in epithelial cells exposed to UA. RESULTS Hemorrhagic shock increased organ (kidney and lung) TNF-α, MPO, and caspases activity in various patterns while caspase-8 remained elevated over time. Hemorrhagic shock led to increased plasma UA at 2 hours, which remained high until 72 hours; TNF-α and IL-18 were elevated at 24 hours. The exogenous UA administration in sham animals reproduced the activation of caspase-8 and MPO in organs, and TNF-α in the lung. The increased plasma and organ UA levels, plasma and lung TNF-α, as well as organ caspase-(8/3) and MPO, observed at 24 hours after HS, were prevented by the administration of Rasburicase during resuscitation. In vitro, soluble UA induced caspases-(3/8) activity in epithelial cells. CONCLUSION Uric acid is persistently high after HS and leads to the activation of caspases-8 and organ inflammation; these can be prevented by an intervention to degrade UA. Therefore, UA is an important biomarker and mediator that could be considered a therapeutic target during HS resuscitation in human.

Caspase-3 activation in the limbic system and depressive-like symptoms are observed after an acute myocardial infarction (MI) and studies suggest that inflammation may play a significant role. Combined treatment with the probiotic strains Bifidobacterium longum and Lactobacillus helveticus in rats has been shown to attenuate caspase-3 activation and depressive-like behaviour together with a reduction in pro-inflammatory cytokines. The present study was designed to determine the respective contribution of these two strains on caspase-3 activity in the limbic system and on depressive-like behaviour. Sprague-Dawley rats were assigned to one of four groups: Vehicle, L. helveticus R0052, B. longum R0175 and L. salivarius HA-118, administered orally for 14 days (109CFU daily) before inducing MI by occlusion of the left anterior descending artery for 40 min followed by 14 days of reperfusion. Animals were then tested for socialisation, passive avoidance and forced swim test to assess depressive-like behaviour. At day 18 the animals were sacrificed; infarct size was estimated, plasma C-reactive protein concentration and brain caspase-3 activity were measured. Results indicated that infarct size did not vary across the different treatments. Rats treated with B. longum spent more time socializing, learned more rapidly the passive avoidance test and spent less time immobile in the forced swim test compared to the vehicle groups. Caspase-3 activity and plasma C-reactive protein concentrations were reduced in the lateral and medial amygdala as well as in the dentate gyrus of B. longum-supplemented animals. The only significant effect in the two groups receiving Lactobacilli compared to vehicle was that rats receiving L. salivarius learned more rapidly in the step-down passive avoidance test. In conclusion, most of the beneficial effects that we previously reported with the combination of two probiotic strains in our experimentation regarding post-myocardial infarction depression are related to Bifidobacterium longum.

Myocardial infarction (MI) in animal models induces cognitive deficits as well as the activation of caspase in the limbic system; both can be blocked by 2 weeks of treatment following MI using tricyclic antidepressants or selective serotonin uptake blockers. Here we used three different treatment schedules to test the short- and long-term effects of the combined serotonin-norepinephrine reuptake inhibitor desvenlafaxine on post-MI-associated cognitive deficits and caspase activation. MI was induced in 39 young adult rats, and 39 rats served as sham-operated controls. Desvenlafaxine (3 mg/kg/day, i.p.) or saline was administered according to one of three schedules: (1) for 2 weeks, starting right after surgery; (2) for 16 weeks, starting 2 weeks after surgery; (3) for 16 weeks, starting right after surgery. Behavior was tested 2 weeks (social interaction, passive avoidance) and 16 weeks (forced swimming, Morris water maze) after surgery. Caspase-3 and caspase-6 activities were measured 16 weeks after surgery. At 2 and 16 weeks post-surgery, saline-treated MI rats displayed performance deficits compared to desvenlafaxine-treated rats, regardless of the treatment schedule. Caspase-3 activity was higher in the amygdala (medial and lateral) and hippocampal CA3 region in untreated MI rats, whereas caspase-6 activity was higher in the CA1 region. Caspase-6 activity correlated positively with deficits in the Morris water maze. These results indicate that, independently of treatment schedules, various treatment schedules with desvenlafaxine can prevent MI-associated cognitive deficits and decrease caspase activities in the limbic system.

The present study was designed to ascertain the effects of 3 diets with different omega-3/6 fatty acid ratios on infarct size and the modifications that these diets induce in the lipid composition of cardiac tissue. Sprague-Dawley rats were fed omega-3/6 fatty acid diets with 1:1, 1:5, or 1:20 ratios for at least 10 days, followed by occlusion of the left anterior descending artery for 40 min and 24 h of reperfusion. Infarct size was significantly smaller in the 1:1 group than in the other groups. Significantly higher concentrations of the omega-3 fatty acids eicosapentaenoic acid, docosapentaenoic acid, and docosahexaenoic acid were found in the 1:1 group than in the other groups. Omega-6 polyunsaturated fatty acid levels were similar between groups, although they were higher in the 1:5 and 1:20 groups than in the 1:1 group. Margaric acid concentrations were higher in the 1:1 group than in the other groups. Docosahexaenoic acid levels in cardiac tissue and infarct size were significantly correlated with no other significant links being apparent. The present study indicated that a 1:1 omega-3/6 fatty acid ratio protected against ischemia and was associated with increased omega-3 fatty acid composition of cardiac tissue.

The high-fat diet of North Americans has a major impact on cardiovascular disease occurrence. Notably, fatty acids have been identified as important factors that could modulate such diseases, especially myocardial infarction (MI). Experimentally, omega-3 polyunsaturated fatty acids (PUFA) have demonstrated positive effects on cardiovascular disorders and have also shown cardioprotection by decreasing MI size. Although many animal experiments have clearly established the benefits of omega-3 PUFA, clinical studies have not reached similar conclusions. In fact, the findings of recent clinical investigations indicate that omega-3 PUFA play only a minor role in cardiovascular health. This dichotomy between experimental and clinical studies may be due to different parameters that are not taken into account in animal experiments. We have recently observed that the high consumption of omega-6 PUFA results in significant attenuation of the beneficial effect of omega-3 PUFA on MI. We believe that part of the dichotomy between experimental and clinical research may be related to the quantity of omega-6 PUFA ingested. This review of the data indicates the importance of considering omega-6 PUFA consumption in omega-3 PUFA studies.

Background: Haemostasis and correction of hypovolemia are the pillars of early haemorrhage shock (HS) management. Vasopressors, which are not recommended as first-line therapy, are an alternative to aggressive fluid resuscitation, but data informing the risks and benefits of vasopressor therapy as fluid-sparing strategy is lacking. We aimed to study its impact on end organs, in the setting of a haemodynamic response to the initial volume resuscitation. Methods: Following controlled HS (60 min) induced by blood withdrawal, under anaesthesia and ventilation, male Wistar rats (N = 10 per group) were randomly assigned to (1) sham, (2) HS with fluid resuscitation only [FR] and (3) HS with fluid resuscitation to restore haemodynamic (MAP: mean arterial pressure) then norepinephrine [FR+NE]. After a reperfusion time (60 min) during which MAP was maintained with fluid or norepinephrine, equipment was removed and animals were observed for 24 h (N = 5) or 72 h (N = 5) before euthanasia. Besides haemodynamic parameters, physiological markers (creatinine, lactate, pH, PaO2 ) and one potential contributor to vasoplegia (xanthine oxidase activity) were measured. Apoptosis induction (caspase 3), tissue neutrophil infiltration (MPO: myeloperoxidase) and illustrative protein markers were measured in the lung (Claudin-4), kidney (KIM-1) and brain amygdala (Iba1). Results: No difference was present in MAP levels during HS or reperfusion between the two resuscitation strategies. FR required significantly more fluid than FR+NE (183% vs 106% of bleed-out volume; p = 0.003), when plasma lactate increased similarly. Xanthine oxidase was equally activated in both HS groups. After FR+NE, creatinine peaked higher but was similar in all groups at later time points. FR+NE enhanced MPO in the lung, when Claudin-4 increased significantly after FR. In the brain amygdala, FR provoked more caspase 3 activity, MPO and microglial activation (Iba1 expression). Conclusion: Organ resuscitation after controlled HS can be assured with lesser fluid administration followed by vasopressors administration, without signs of dysoxia or worse evolution. Limiting fluid administration could benefit the brain and seems not to have a negative impact on the lung or kidney.

Myocardial infarction (MI) has dramatic mid- and long-term consequences at the physiological and behavioral levels, but the mechanisms involved are still unclear. Our laboratory has developed a rat model of post-MI syndrome that displays impaired cardiac functions, neuronal loss in the limbic system, cognitive deficits and behavioral signs of depression. At the neuronal level, caspase-3 activation mediates post-MI apoptosis in different limbic regions, such as the amygdala – peaking at 3 days post-MI. Cognitive and behavioral impairments appear 2-3 weeks post-MI and these correlate statistically with measures of caspase-3 activity. The protocol described here is used to induce MI, collect amygdala tissue and measure caspase-3 activity using spectrofluorometry. To induce MI, the descending coronary artery is occluded for 40 min. The protocol for evaluation of caspase-3 activation starts 3 days after MI: the rats are sacrificed and the amygdala isolated rapidly from the brain. Samples are quickly frozen in liquid nitrogen and kept at -80 °C until actual analysis. The technique performed to assess caspase-3 activation is based on cleavage of a substrate (DEVD-AMC) by caspase-3, which releases a fluorogenic compound that can be measured by spectrofluorometry. The methodology is quantitative and reproducible but the equipment required is expensive and the procedure for quantifying the samples is time-consuming. This technique can be applied to other tissues, such as the heart and kidneys. DEVD-AMC can be replaced by other substrates to measure the activity of other caspases.

We previously observed that resolvin D1 (RvD1), a metabolite of the omega-3 polyunsaturated fatty acid (PUFA) docosahexaenoic acid, reduces infarct size by a mechanism involving the PI3-K/Akt pathway. In parallel, the beneficial effect of a high omega-3 PUFA diet on infarct size can be attenuated by increased omega-6 PUFA consumption. The present study was designed to determine if augmented linoleic acid (LA), an omega-6 PUFA administered at the same time, attenuates the cardioprotective action of RvD1. Male Sprague-Dawley rats received 0.1μg RvD1 alone or with one of three LA doses (1, 5 or 10μg) directly into the left ventricle chamber 5min before ischemia. The animals underwent 40min of ischemia by occlusion of the left descending coronary artery followed by 30min or 24h of reperfusion. Infarct size and neutrophil accumulation were evaluated after 24h of reperfusion, while caspase-3, -8 and -9 and Akt activities were assessed at 30min of reperfusion. LA attenuated cardioprotection afforded by RvD1, resulting in significantly increased infarct size. Neutrophil accumulation and Akt activity were similar between groups. Caspase activities, especially caspase-9, which could be activated by ischemia, were stimulated in the presence of LA, suggesting that this omega-6 PUFA accentuates ischemia intensity. The present results indicate that LA significantly attenuates the beneficial effect of RvD1 on infarct size. Therefore, reduction of omega-6 intake should be considered to maintain the protection afforded by RvD1.

Abstract Intestinal stem cells (ISCs) are a long-lived, dynamic cell population at the base of intestinal crypts that adjust their molecular phenotypes as necessary to meet the maintenance and regenerative needs of the epithelium. The long-life, plasticity and proliferative potential of ISCs underlie the necessity for tight regulation, as these cells have been implicated as initiators of colorectal tumorigenesis. In that context, guanylyl cyclase C (GCC) and its effector, cGMP, regulate intestinal epithelial proliferation, differentiation, and susceptibility to intestinal insult and tumor formation. However, the specific role of this signaling axis in ISCs remains undefined. Here, utilizing transgenic mouse models and immunodetection techniques, we reveal that GCC mediates ligand-dependent cGMP signal transduction in ISCs. Further, the absence of GCC alters expression of key ISC markers, decreasing the active ISC marker Lgr5 and increasing the reserve ISC marker Bmi1. Endoplasmic reticulum (ER) stress, which is typically absent from ISCs and has been associated with inflammation and tumorigenesis, is elevated throughout the crypt base in the absence of GCC. Conversely, administration of cGMP or the chemical chaperone taurourdeoxycholic acid eliminates ER stress and restores the normal crypt ISC phenotype. Finally, ISCs exhibit an impaired regeneration response following irradiation in the absence of GCC, highlighting the importance of this signaling pathway in ISC survival and epithelial repair. Together, these observations suggest that GCC signaling modulates ER stress as part of the essential machinery defending the integrity of the ISC niche. Citation Format: Crystal Kraft, Jieru Lin, Adam Snook, Gilbert Kim, Scott Waldman. Intestinal stem cell integrity is preserved through modulation of endoplasmic reticulum stress by guanylyl cyclase C. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1712.

The aim of this project was to investigate the impact of two dietary omega-3 polyunsaturated fatty acids (PUFAs), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), alone or in combination, on infarct size.

Despite a well‐characterized epidemiological association, mechanisms linking colorectal cancer with obesity remain incompletely defined. An emerging paradigm suggests that colorectal cancer arises by silencing the GUCY2C tumor suppressor through loss of the paracrine hormone guanylin, a gene product universally eliminated in intestinal transformation. The impact of diet‐induced obesity on ER stress and the unfolded protein response (UPR) in extra‐intestinal tissues is well‐characterized. Here, we demonstrate that obesogenic diets induce intestinal ER stress, suppressing guanylin expression and silencing GUCY2C. In that context, both pharmacologic and genetic induction of ER stress silenced guanylin expression in intestinal cells in vitro and in vivo. Diet‐induced disruption of the GUCY2C‐guanylin axis increased epithelial dysfunction, amplifying colorectal tumorigenesis. Conversely, pharmacologic inhibition of ER stress using taurodeoxycholic acid (TUDCA) relieved the UPR and restored guanylin expression. Inhibition of a specific ER stress pathway through pharmacologic and genetic inhibition of PERK restored guanylin expression suppressed by tunicamycin. Importantly, conditional intestine‐specific expression of guanylin eliminated epithelial dysfunction and colorectal tumorigenesis in obese transgenic mice. Together, these observations suggest that the guanylin‐GUCY2C axis is a principle mechanistic link between obesity and colorectal cancer, and highlight a unique therapeutic opportunity for cancer prevention in obese patients by oral replacement of GUCY2C ligands.

Although controversial, some data suggest that omega-3 polyunsaturated fatty acids (PUFA) are beneficial to cardiovascular diseases, and could reduce infarct size. In parallel, we have reported that the administration of Resolvin D1 (RvD1), a metabolite of docosahexaenoic acid, an omega-3 PUFA, can reduce infarct size. The present study was designed to determine if the inhibition of two important enzymes involved in the formation of RvD1 from omega-3 PUFA could reduce the cardioprotective effect of omega-3 PUFA. Sprague-Dawley rats were fed with a diet rich in omega-3 PUFA during 10 days before myocardial infarction (MI). Two days before MI, rats received a daily dose of Meloxicam, an inhibitor of cyclooxygenase-2, PD146176, an inhibitor of 15-lipoxygenase, both inhibitors or vehicle. MI was induced by the occlusion of the left coronary artery for 40min followed by reperfusion. Infarct size and neutrophil accumulation were evaluated after 24h of reperfusion while caspase-3, -8 and Akt activities were assessed at 30min of reperfusion. Rats receiving inhibitors, alone or in combination, showed a larger infarct size than those receiving omega-3 PUFA alone. Caspase-3 and -8 activities are higher in ischemic areas with inhibitors while Akt activity is diminished in groups treated with inhibitors. Moreover, the study showed that RvD1 restores cardioprotection when added to the inhibitors. Results from this study indicate that the inhibition of the metabolism of Omega-3 PUFA attenuate their cardioprotective properties. Then, resolvins seem to be an important mediator in the cardioprotection conferred by omega-3 PUFA in our experimental model of MI.

Myocardial infarction (MI) induces an inflammatory process that is associated with increased apoptosis in the limbic system of rats and the development of post-MI depressive symptoms. Resolvin D1 (RvD1), an omega-3 fatty acid metabolite, is known for its pro-resolution properties; it reduces infarct size and attenuates post-MI depression-like symptoms. The present study was designed to determine if a single RvD1 dose could abate caspase-3 activation, a marker of apoptosis, in the limbic system. Male Sprague-Dawley rats underwent 40min of myocardial ischemia, followed by 24-h reperfusion. Five min before MI, the animals received a single intra-cardiac RvD1 injection (0.01, 0.1 or 0.3μg) or vehicle (saline). Infarct size was assessed and caspase-3 activity measured in the amygdala and hippocampus. Rats receiving 0.1 or 0.3μg RvD1 showed significantly decreased infarct size. Caspase-3 activity was significantly attenuated in the lateral amygdala and dentate gyrus with 0.1μg RvD1 and in the CA1 region of the hippocampus and medial amygdala with 0.3μg RvD1. In conclusion, RvD1 could reduce infarct size and caspase-3 activity in the amygdala and hippocampus.

This study was designed to determine if Resolvin D1 (RvD1), a pro-resolution metabolite of the omega-3 polyunsaturated fatty acid docosahexaenoic acid, could decrease myocardial infarct size with delivered at the onset of ischemia. Male Sprague Dawley rats underwent 40 minutes of myocardial ischemia followed by reperfusion. These animals received 1 intraventricular injection of RvD1 (0.01, 0.1, or 0.3 μg RvD1) or vehicle (saline) before coronary occlusion. Infarct size and neutrophil accumulation were evaluated 24 hours after the onset of reperfusion. Caspase-3, caspase-8, protein kinase B (Akt) activities were evaluated 30 minutes after the reperfusion. Rats receiving 0.1 or 0.3 μg RvD1 showed a significant decrease of infarct size and caspase-3 and caspase-8 activities compared with the vehicle controls. Neutrophil accumulations were reduced in rats administered RvD1 compared with vehicle, independently of dose level. Akt activation was increased only in animals receiving 0.1 or 0.3 μg, whereas no change was observed in the 0.01 μg group. When they were treated with LY-294002, a phosphoinositide 3-kinase (PI3K)/Akt inhibitor, cardioprotection by RvD1 was abrogated. RvD1 treatment at the onset of ischemia decreases infarct size by a mechanism involving the PI3K/Akt pathway.