This study reveals three major findings: (i) mild intestinal barrier damage could be detected within 24 h in a rat model of 6 min CA. (ii) only mild local intestinal inflammation could be shown within 24 h after CA. (iii) a systemic inflammation and thus a potential contribution of the small intestine to systemic inflammation could not be simulated after 6 min of CA in rats.
Intestinal barrier damage
Although 6 min of transient global ischaemia and subsequent reperfusion led to mucosal damage and decreased thickness of the muscularis in the small intestine, jejunal tissue concentrations of IL-1α, IL-1β, IL-10 and TNF-α were only slightly affected. This result may be due to several reasons.
First, the duration of 6 min CA in the present study that is sufficient to show cerebral damage [
21] is inadequate to initiate a systemic inflammatory response, which is a clear limitation of this study. As shown by Qian, exceeding the duration of CA of 6 min may extend intestinal damage and influence serum cytokine concentrations. In detail, intestinal microcirculatory blood flow was significantly decreased accompanied by mild elevated serum concentrations of TNF-α and IL-6 during 8 min of CA in pigs [
22]. In contrast, significant inflammatory response following local ischaemia and reperfusion of the small intestine was shown to be initiated after a duration of at least 15–30 min [
23]. However, models of focal ischaemia and reperfusion are not capable of being translated to conditions of systemic ischaemia and reperfusion. In fact, CA leads to a complex systemic ischaemia-reperfusion-injury with contribution of multiple independent tissues, which are integrated into a complicated cascade of cell-death and systemic inflammation [
5,
6].
Secondly, intestinal repair mechanisms seem to be commenced immediately after CA. A normalization of leukocyte-endothelial interaction as well as the wall shear rate was reported to be initiated within 120 min after CA, which is a period not mirrored in our study [
24]. However, reports show that cytokines such as IL-1ra, IL-6, IL-8, IL-10 and TNF-α are expressed within 3 h [
5,
6,
25] to 6 h [
24,
26] and peak within the first 2 days after CA [
5,
6]. This is in line with studies reporting a massive up-regulation of cytokines after ischaemic brain injury [
2,
27]. Due to oxidative stress, NF-КB is up-regulated and orchestrates the release of a number of cytokines such as IL-1ß, IL-2, IL-4, IL-5, IL-6 and IL-10, IL-12, IL-13 and TNF-α [
28,
29]. Accordingly, we collected blood (sera) and tissues at early stages including 6 h and 24 h post-ROSC and chose a similar cytokine profile to be investigated. However, in clinical studies, it is often proposed that the PCAS may result from a systemic inflammatory activation persisting for days [
5,
30]. Therefore, cytokine concentrations in jejunum and sera were also evaluated within the first 7 days after CA in the present study.
Third, as repeatedly shown, simulation of a PCAS is difficult in rats and accompanied by a high failure rate of more than 50% [
24,
31]. Thus, on the basis of previous scientific findings, we conclude, that severely injured rats that would develop a PCAS initially died during resuscitation procedures. With a survival rate of only 29.5% in the present study, we conclude that this model can be utilized to a limited extent to reproduce a PCAS or peripheral tissue damage, respectively. Nevertheless, ROSC rates are comparable to previous experiments performed in our group, which are able to adequately determine cerebral damage [
16,
18,
21,
24,
32‐
34]. In this pilot study, our main objective was to evaluate peripheral tissue potentially injured due to CA. Notably, we did not lose any animals after ROSC, which is advantageous since post-ROSC mortality is known to be 38% within an observation period > 48 h [
35]. Interestingly, Vognsen et al. recently showed that only 12% of animal studies sufficiently report outcome parameters according to the Utstein Guidelines, which was a strong criterion to increase the validity of this study [
35].
Given the clinical phenomenon of bacteremia after CA [
7,
8], intestinal damage seems to be conclusive even after short periods of ischaemia. Congruently, our results point towards a mild local intestinal damage, which is in line with Pan et al. who reported similar Chiu scorings 24 h after CA of 6 min duration [
36]. Likewise, Teschendorf et al. showed a 3–4-fold stronger plasma extravasation from post-capillary mesenteric venules at 120 min after CA, which is a characteristic sign of endotoxaemia [
24]. Another factor taken into consideration is the short initial tissue hyperperfusion followed by a sustained hypoperfusion of intestinal tissue after CA [
37,
38]. This inevitably leads to a prolonged period of relative ischaemia where the intestine receives only 5% of cardiac output [
39] and fosters further tissue damage. Interestingly, we found a significant correlation between the duration of CA and the Chiu-grade in the 7 d group. However, these results should be considered with caution because significant mucosal damage was also shown with a shorter duration of CA in the 6 h group (Additional file
1).
It must be noted that mucosal damage and cytokines were not analyzed in animals that could not be resuscitated. However, results were compared to a control group serving as reference. Since achievement of ROSC is mainly dependent on heart function, significant intestinal damage detectable immediately after ceasing CPR was not assumed. As reported, mucosal damage develops within the first 6 to 24 h after cardiac arrest [
36] and may contribute to the development of the PCAS, which was the main objective in this investigation.
Cytokine expression in tissue and serum
A systemic ischaemia-reperfusion-injury causes a so-called sterile inflammation, which is associated with influx of neutrophils and macrophages, leading to the production of inflammatory cytokines [
40]. IL-1α, present in gastrointestinal epithelial and endothelial cells [
41], acts via initiation of the inflammatory cascade and is thus a valuable parameter for the determination of inflammatory processes. In the present study, IL-1α showed a significant increase in the jejunum, resulting in the highest concentrations at 24 h post-ROSC. At the same timepoint, IL-1α concentrations in serum were close to detection limits. It has been reported that in vitro circulating IL-1α was released from endothelial cells [
41] but was barely detectable in patients suffering from severe inflammation [
42]. This suggests that jejunal and serum IL-1α concentrations do not necessarily match, as observed in the present study.
The IL-10 family acts protectively during intestinal inflammation by induction of anti-inflammatory effects and inhibition of pro-inflammatory cytokines such as IL-1β, IL-6 and TNF-α [
43]. In our study, in jejunal tissues, IL-1α, IL-1β and TNF-α increased significantly at 24 h post-ROSC indicating that increasing IL-10 concentrations at the same time might counteract the increase of pro-inflammatory cytokines such as IL-1α, IL-1β and TNF-α.
Overall, serum cytokine concentrations were not significantly altered except for a significant increase in IL-1β after 24 h post-ROSC. Additionally, IL-1α, IL-1β, IL-10 and TNF-α concentrations were lower in serum than in the jejunum. This may indicate that the observed intestinal inflammation may not be associated with systemic inflammation.
Notably, intestinal cytokine increments were in accordance with morphological changes of the intestinal mucosa. Intestinal tissue damage peaked at 6 h post-ROSC and decreased subsequently at 24 h and 72 h. Tissue repair mechanisms accompanied by increases in intestinal inflammatory cytokine release seem to have been initiated. Consequently, desquamation of villus tips, development of a Gruenhagen’s space, hydropic generation of epithelial cells and changes in muscular layer thickness were observed.
The mechanism, which induces a further increase in intestinal tissue damage 7 d post-ROSC, accompanied by rising cytokine concentrations of IL-1β and IL-10 at the same timepoint, requires further research. As the duration between 72 h and 7 d post-ROSC is comparably long, it might be possible that further changes in cytokine concentrations remained undetected.