Moderate Hypothermia Provides Better Protection of the Intestinal Barrier than Deep Hypothermia during Circulatory Arrest in a Piglet Model: A Microdialysis Study

Abstract

Introduction: This study aimed to assess the effects of different temperature settings of hypothermic circulatory arrest (HCA) on intestinal barrier function in a piglet model.

Methods: Twenty Wuzhishan piglets were randomly assigned to 40 min of HCA at 18°C (DHCA group, n = 5), 40 min of HCA at 24°C (MHCA group, n = 5), normothermic cardiopulmonary bypass (CPB group, n = 5) or sham operation (SO group, n = 5). Serum D-lactate (SDL) and lipopolysaccharide (LPS) levels were determined. Microdialysis parameters (glucose, lactate, pyruvate and glycerol) in the intestinal dialysate were measured. After 180 min of reperfusion, intestinal samples were harvested for real-time polymerase chain reaction and western blotting measurements for E-cadherin and Claudin-1.

Results: Higher levels of SDL and LPS were detected in the DHCA group than in the MHCA group (P < 0.001). Both MHCA and DHCA groups exhibited lower glucose levels, higher lactate and glycerol levels and a higher lactate to pyruvate (L/P) ratio compared with the CPB group (p<0.05); the DHCA group had higher lactate and glycerol levels and a higher L/P ratio (p<0.05) but similar glucose levels compared to the MHCA group. No significant differences in E-cadherin mRNA or protein levels were noted. Upregulation of claudin-1 mRNA levels was detected in both the DHCA and MHCA animals' intestines (P < 0.01), but only the DHCA group exhibited a decrease in claudin-1 protein expression (P < 0.01).

Conclusion: HCA altered the energy metabolism and expression of epithelial junctions in the intestine. Moderate hypothermia (24°C) was less detrimental to the markers of normal functioning of the intestinal barrier than deep hypothermia (18°C).

Fig 1. Experimental protocol.

CA40, circulatory arrest for 40 min; RP30, reperfusion for 30 min; RP60, reperfusion for 60 min; RP 90, reperfusion for 90 min; RP 120, reperfusion for 120 min; RP 150, reperfusion for 150 min; and RP 180, reperfusion for 180 min.

Fig 2. Hematoxylin-eosin staining of the intestinal tissue.

The mucosal villi and glands were normal in the intestinal epithelia of the SO and CPB groups. Marked edema of the mucosal villi, increased gaps between the epithelial cells and inflammatory cell infiltration were observed in the MHCA and DHCA groups. Magnification: 100 folds; the scale bars indicate 100 μm.

Fig 3. Serum D-lactate levels at 180 min of reperfusion.

SO, sham operation group; CPB, cardiopulmonary bypass group; DHCA, deep hypothermic circulatory arrest group; MHCA, moderate hypothermic circulatory arrest group. Mean±SD. n = 5. *P < 0.001 DHCA vs. CPB (t = 7.330 at 60 min of reperfusion and t = 15.32 at 180 min of reperfusion); # P < 0.001 MHCA vs. CPB (t = 6.898 at 60 min of reperfusion and t = 9.415 at 180 min of reperfusion); and ^ P < 0.05 DHCA vs. MHCA (t = 5.515 at 180 min of reperfusion).

Fig 4. Serum lipopolysaccharide (LPS) levels at 180 min of reperfusion.

SO, sham operation group; CPB, cardiopulmonary bypass group; DHCA, deep hypothermic circulatory arrest group; MHCA, moderate hypothermic circulatory arrest group. Mean±SD; n = 5. *P < 0.001 DHCA vs. CPB (t = 8.414 at 60 min of reperfusion and t = 18.710 at 180 min of reperfusion); # P < 0.001 MHCA vs. CPB (t = 6.363 at 60 min of reperfusion and t = 13.601 at 180 min of reperfusion); and ^ P < 0.05 DHCA vs. MHCA (t = 5.105 at 180 min of reperfusion).

Fig 5

a-e. Microdialysis parameters in interstitial fluid of small intestine. Fig 5a, Glucose; Fig 5b, Glycerol; Fig 5c, Lactate; Fig 5d, Pyruvate; Fig 6e, Lactate/ Pyruvate ratio. CPB, cardiopulmonary bypass group; DHCA, deep hypothermic circulatory arrest group; MHCA, moderate hypothermic circulatory arrest group. ^ P<0.05 MHCA vs CPB; * P<0.05 DHCA vs CPB; # P<0.05 DHCA vs MHCA. ECA, end of cardiac arrest; RP60, reperfusion for 60min; RP120, reperfusion for 120min; RP180, reperfusion for 180min.

Fig 6

a-b. e-cadherin and claudin-1 mRNA levels determined by real-time PCR. (a) e-cadherin mRNA levels at 180 min of reperfusion. The relative fold changes of e-cadherin in the SO, CPB, MHCA and DHCA groups were 1.001 ± 0.057, 0.925 ± 0.207, 1.607 ± 0.775 and 1.837 ± 1.002, respectively. (b) claudin-1 mRNA levels at 180 min of reperfusion. The relative fold changes of claudin-1 in the SO, CPB, MHCA and DHCA groups were 1.092 ± 0.555, 1.291 ± 0.535, 10.664 ± 1.903 and 9.106 ± 0.623, respectively. Mean±SD; n = 5. ^P < 0.05 DHCA vs. CPB; * P < 0.05 MHCA vs. CPB.

Fig 7. E-cadherin protein expression levels determined by western blot.

The relative fold changes of E-cadherin in the SO, CPB, MHCA and DHCA groups were 1.000 ± 0.090, 0.998 ± 0.103, 0.971 ± 0.121 and 0.886 ± 0.071, respectively. Mean ± SD; n = 5.

Fig 8. Claudin-1 protein expression levels determined by western blot.

The relative folds changes of Claudin-1 in the SO, CPB, MHCA and DHCA groups were 1.000 ± 0.130, 0.992 ± 0.069, 0.956 ± 0.062 and 0.784 ± 0.054, respectively. Mean ± SD; n = 5. **P < 0.05 DHCA vs. CPB; ^ P < 0.05 DHCA vs. MHCA.