Differential caspase activation in a rodent model of resuscitated hemorrhagic shock
CCCF ePoster library. Gilbert K. Oct 26, 2015; 117315; P13
Dr. Kim Gilbert
Dr. Kim Gilbert
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Topic: Basic/Translational Science

Differential caspase activation in a rodent model of resuscitated hemorrhagic shock

Kim Gilbert, F. Baril, G. Rousseau, K. Gilbert, E. Charbonney

Medicine, Faculty of Medicine, University of Montreal, Montreal, Canada | Biomedical sciences, Faculty of Medicine, University of Montreal, Montreal, Canada | Pharmacology, Centre de recherche Hôpital du Sacré-Coeur, University of Montreal, Montreal, Canada | Facutly of Medicine, Centre de recherche Hôpital du Sacré-Cœur, University of Montreal, Montreal, Canada | Medicine, Centre de recherche Hôpital du Sacré-Coeur, University of Montreal, Montreal, Canada


After an ischemic event and reperfusion (I/R), tissue apoptosis is incriminated in the organ injury process, triggered by specific death-signaling activated pathways; the neutrophils, as well as soluble Fas ligand induced epithelial apoptosis, are incriminated in lung and kidney injury (1,2). However, the apoptosis itself is also responsible for the enhanced local inflammation in these organs (3,4). Classically, the initiator caspase-8 is linked to the pro-inflammatory pathways, when the downstream caspase-3, as final effector, is a stronger regulator of apoptosis. The long-term differential activation of theses two caspases is not explored yet.


The primary objective was to test and characterize the relative apoptotic signal (caspase 3 and 8) in the lung and kidney after ischemia-reperfusion, in a surviving animal model of I/R. The secondary objective was to characterize the inflammatory (Neutrophils myeloperoxidase) activity in tissues.


Ventilated Wistar rat, placed under general anesthesia and ventilated, were exposed to a controlled pressure-targeted hemorrhagic shock (MAP 25-30 mmHg) for 1 h, by blood withdrawal through inguinal catheters. They were then resuscitated by reinjection of the stored blood mixed with Ringer Lactate (1:1), in order to reach their initial MAP, then received fluid repeatedly in order to maintain MAP > 60mmHg. Shams were ventilated and the catheters were also inserted. The rats were euthanized at 2 hours, or wakened and euthanized at 24/72h. Four hours before their euthanasia at 24h, they were fed with marked (FITC) Dextran in order to measure their intestinal permeability, as a physiological marker of post-ischemic organ injury. In homogenized tissues extracts Caspase-3 and 8 activities were measured using spectrofluorometry, and myeloperoxidase (MPO) were measured using a colorimetric activity assay. Values were controlled for the total protein content.


The intestinal permeability, measured by the amount of circulating FITC-Dextran (mcg/mL), was elevated for the group with shock compared to Shams (4 fold at 24h and 3 fold at 72h). Caspase 3 (C3) and 8 (C8) increased significantly, even at the earlier time-point (2h), and remained elevated till 72h for C8, in both organs (Fig.1A and B). In the lung, C3 decreased significantly under the baseline activity, at 24h and 72h. In the kidney, the activity of both caspases remained higher than in the lung, despite a tendency for C3 to decrease, across time. C3 and C8 activities were highly correlated in the kidney at 2h (rho=0.95, p< 0.001; Spearman), but not in the lung (rho=0.45, p=0.13). The neutrophils infiltration at 24h, measured by MPO activity was higher in the Shock group, with an average magnitude of 28 fold in the lung and 11 fold in the kidney, compared to controls (Fig. 2A and B). MPO activity observed in the controls was higher in the lung than the kidney.


The caspases profile after I/R, across time, were different between the lung and kidney, particularly for the C3 activity drastic drop at 24h in the lung. The activities were more sustained in the kidney. The variation between organs might be explained by the difference in vascularisation, neutrophilic infiltration or other unknown factors.


1. Serrao, K L et al. AJP Lung Cell Mol Physiol. 2001; 280(2): 298-305

2. Nogae, S et al. JASN 1998; 9:620–631

3. Daemen, M.A. et al. JCI 1999; 104:541–549

4. Kitamura, Y et al. AJRCCM 2001; 163:762–769

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