Background Corticotropin-releasing factor (CRF) mediates our body’s overall responses to stress. The role of central CRF in stress-stimulated colonic motility is well characterized. We hypothesized that transient perturbation in expression of enteric CRF is sufficient to change stress-induced colonic motor and secretory responses. Methods Sprague-Dawley rats (adult, male) were subjected to 1-h partial restraint stress (PRS) and euthanized at 0, 4, 8, and 24 h. CRF mRNA and peptide levels in the colon were quantified by real-time RT-PCR, enzyme immunoassay, and immunohistochemistry. Double-stranded RNA (dsRNA) designed to target CRF (dsCRF) was injected into the colonic wall to attain RNA interference (RNAi)-mediated inhibition of CRF mRNA expression. DsRNA for β-globin was used as a control (dsControl). Four days after dsRNA injection, rats were subjected to 1-h PRS. Fecal output was measured. Ussing chamber techniques were used to assess colonic mucosal ion secretion and transepithelial tissue conductance. Key Results Exposure to PRS elevated CRF expression and increased CRF release in the rat colon. Injection of dsCRF inhibited basal CRF expression and prevented the PRS-induced increase in CRF expression, whereas CRF expression in dsControl-injected colons remained high after PRS. In rats treated with dsControl, PRS caused a significant increase in fecal pellet output, colonic baseline ion secretion, and transepithelial tissue conductance. Inhibition of CRF expression in the colon prevented PRS-induced increase in fecal output, baseline ion secretion, and transepithelial tissue conductance. Conclusions & Inferences These results provide direct evidence that transient perturbation in peripherally expressed CRF prevents colonic responses to stress.
Thirteen-lined ground squirrels (Ictidomys tridecemlineatus) enter hibernation as a survival strategy during extreme environmental conditions. Typical ground squirrel hibernation is characterized by prolonged periods of torpor with significantly reduced heart rate, blood pressure, and blood flow, interrupted every few weeks by brief interbout arousals (IBA) during which blood flow fluctuates dramatically. These physiological conditions should increase the risk of stasisinduced blood clots and myocardial ischemia. However, ground squirrels have adapted to survive repeated bouts of torpor and IBA without forming lethal blood clots or sustaining lethal ischemic myocardial damage. The purpose of this study was to determine if ground squirrels are resistant to thrombosis and myocardial ischemia during hibernation. Blood markers of coagulation, fibrinolysis, thrombosis, and ischemia, as well as histological markers of myocardial ischemia were measured throughout the annual hibernation cycle. Hibernating ground squirrels were also treated with isoprenaline to induce myocardial ischemia. Thrombin-antithrombin complex levels were significantly reduced (p < 0.05) during hibernation, while D-dimer level remained unchanged throughout the annual cycle both consistent with an antithrombotic state. During torpor the ground squirrels were in a hyperfibrinolytic state with an elevated ratio of tissue plasminogen activator complexed with plasminogen activator inhibitor to total plasminogen activator inhibitor (p < 0.05). Histological markers of myocardial ischemia were reversibly elevated during hibernation with no increase in markers of myocardial cell death in the blood. These data suggest that ground squirrels do not form major blood clots during hibernation through suppression of coagulation and a hyperfibrinolytic state. These animals also demonstrate myocardial resistance to ischemia.
During hibernation, a ground squirrel (Spermophilus tridecemlineatus) has decreased blood flow, which could lead to the formation of stasis blood clots. Ground squirrels have been shown to suppress blood clotting in order to survive hibernation. However, the stability of blood clots and the degradation process through fibrinolysis have not been determined. Preliminary results indicate no significant difference in plasminogen levels during hibernation. To see if fibrinolytic pathways were being activated during hibernation, plasmin activity was measured, and found to be 4‐fold higher in pooled hibernating versus pooled non‐hibernating plasma. These results suggest that fibrinolysis may be activated during hibernation, suggesting that some clots are still forming in spite of the suppression of hemostasis by the ground squirrels. A clot retraction assay was used to compare the stability of clots, and human platelet clots retracted in 45 minutes on average, compared with 25 minutes for non‐hibernating ground squirrels. The stability of blood clots in hibernating and non‐hibernating squirrels will be compared with the clot retraction assay when hibernating ground squirrel blood becomes available in the winter. A decreased stability of platelet clots may also protect ground squirrels from the formation of clots during hibernation. This research was funded by an NIH R15 grant.
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