Resistance to systemic inflammation and multi organ damage after global ischemia/reperfusion in the arctic ground squirrel

Abstract

Introduction: Cardiac arrest (CA) and hemorrhagic shock (HS) are two clinically relevant situations where the body undergoes global ischemia as blood pressure drops below the threshold necessary for adequate organ perfusion. Resistance to ischemia/reperfusion (I/R) injury is a characteristic of hibernating mammals. The present study sought to determine if arctic ground squirrels (AGS) are protected from systemic inflammation and multi organ damage after CA- or HS-induced global I/R and if, for HS, this protection is dependent upon their hibernation season.

Methods: For CA, rats and summer euthermic AGS (AGS-EU) were asphyxiated for 8 min, inducing CA. For HS, rats, AGS-EU, and winter interbout arousal AGS (AGS-IBA) were subject to HS by withdrawing blood to a mean arterial pressure of 35 mmHg and maintaining that pressure for 20 min before reperfusion with Ringers. For both I/R models, body temperature (Tb) was kept at 36.5-37.5°C. After reperfusion, animals were monitored for seven days (CA) or 3 hrs (HS) then tissues and blood were collected for histopathology, clinical chemistries, and cytokine level analysis (HS only). For the HS studies, additional groups of rats and AGS were monitored for three days after HS to access survival and physiological impairment.

Results: Rats had increased serum markers of liver damage one hour after CA while AGS did not. For HS, AGS survived 72 hours after I/R whereas rats did not survive overnight. Additionally, only rats displayed an inflammatory response after HS. AGS maintained a positive base excess, whereas the base excess in rats was negative during and after hemorrhage.

Conclusions: Regardless of season, AGS are resistant to organ damage, systemic inflammation, and multi organ damage after systemic I/R and this resistance is not dependent on their ability to become decrease Tb during insult but may stem from an altered acid/base and metabolic response during I/R.

Figure 1. Experimental protocols for ischemia/reperfusion.

CA, cardiac arrest; CPR, cardiopulmonary resuscitation; ROSC, return of spontaneous circulation; HS, hemorrhagic shock. Core body temperature and head temperature were maintained between 36.5 and 37.5°C with a warm water blanket under the animal and heat lamps above the animalfrom the start of surgical preparation until the start of post ROSC or post HS monitoring. Arrows indicate blood sampling timepoints.

Figure 2. The mean arterial blood pressure and average heart rate decrease during cardiac arrest.

Both mean arterial pressure (MAP; top) and heart rate (HR; bottom) decrease similarly for both AGS and rat and return to near normal after CA. Darkened area represents period of asphyxia. MAP and HR were recorded immediately before asphyxiation (Before CA) and one hour after CA (After CA). Data shown as mean ± SEM, * Tukey, p<0.05 between rat and AGS. For both groups n = 5.

Figure 3. Blood serum markers for liver damage after cardiac arrest.

AGS did not show an increase in blood serum markers for liver damage one hour after CA. Rats had an increase in both ALT (top, * Tukey, p<0.05) and AST (bottom, * Tukey, p<0.05) one hour after CA compared to all other groups. Dark bars indicate baseline values. Light bars are values one hour after CA. Raw data is shown as mean ± SEM. Statistical analysis was done on the difference between baseline and one hour after CA. For all groups n = 5–6.

Figure 4. Percent survival after hemorrhagic shock.

Extended survival assessed up to 72(MAP ∼35 mmHg for 20 min) in euthermic AGS, interbout arousal AGS, and rats (control; n = 4 for all groups; A). Survival up to three hours after 55–60% blood loss in euthermic- (n = 4) and interbout arousal (n = 4) AGS (B).

Figure 5. Mean arterial pressure during hemorrhagic shock.

Mean arterial pressure decreases to the same mmHg for all groups during HS and is restored to varying degrees with Ringers reperfusion. Shaded region indicates period of hemorrhage. * Tukey, p<0.05 between rats and both EU- and IBA-AGS, ** Tukey, p<0.05 between all groups. Data shown as mean±SEM, n = 6–7 for all groups.

Figure 6. Rats have a higher average heart rate before, during and after hemorrhagic shock than AGS.

Shaded region indicates period of hemorrhage. Rats differed from both AGS groups (a, Tukey, p<0.05), just the AGS-IBA (b, Tukey, p<0.05), or AGS-EU (c, Tukey p<0.05). AGS-EU and –IBA did not differ from each other at any timepoint. Data shown as mean±SEM, n = 6–7 for all treatment groups.

Figure 7. Serum markers of organ damage after hemorrhagic shock.

Serum markers indicate that the AGS do not sustain kidney damage as indicated by increased serumcreatinine. Dark areas indicate baseline levels obtained at the start of hemorrhage. Light areas are the levels three hours after end of resuscitation. Raw data is shown and expressed as mean ± SEM Statistical analysis with an ANOVA was performed on the difference between three hours after resuscitation and baseline. *Tukey, p<0.05 between SHS and HS; n = 4–8.

Figure 8. Fold changes in circulating cytokine levels after hemorrhagic shock.

AGS do not have a significant fold change in plasma cytokine levels immediately prior to hemorrhage and three hours after resuscitation. Cytokines levels assessed for: IL-1 alpha (A), IL-1 beta (B), IL-6 (C), IL-10 (D), TNF-alpha (E), and INF-gamma (F); *p<0.05, Tukey versus corresponding sham; n = 6–8 for each group.

Figure 9. AGS maintain lower blood lactate∶glucose ratio throughout hemorrhagic shock and reperfusion.

Blood glucose, lactate, and lactate∶glucose ratio, before, during and after HS Solid lines are SHS, dashed lines are HS. * Tukey, p<0.05 between SHS and HS. Naive values for glucose were 162.29±10.94, 153.88±10.69, 205.67±7.78 mg/dL; lactate 1.66±0.29, 1.48±0.25, 1.92±0.20 mmol/L; for AGS-EU, AGS, IBA, and rats, respectively.

Figure 10. AGS maintain positive whole blood base excess values before, during, and after hemorrhagic shock.

All animals initially had positive BE values with the AGS (both seasons) having a starting BE higher than the rats. Data presented as mean±SEM; * Tukey, p<0.05 between AGS-EU Sham HS (SHS) and HS, ** Tukey, p<0.05 between rat SHS and HS.