In the present study, we observed that in neonates undergoing cardiac surgery, cerebral ischemia during deep hypothermia elicited an immediate inflammatory response in the cerebral circulation. Not only were there significant changes in the soluble milieu; neutrophils and monocytes acquired an activated phenotype and were even increased in number upon leaving the cerebral circulation. This was not the case in the situation where the brain was continuously perfused. This phenomenon has not been reported on previously and may hold important clues for further research on the inflammatory response to stroke.
Previous studies involving cerebral ischemia and inflammation, both in neonates and adults, have found changes in peripheral immune cells within hours after the ischemic insult
[1, 3, 8, 10–14, 16]. In the same time frame, leukocytes are reported to start their migration into the ischemic lesion
[2–4]. However, to our knowledge, the immediate effect of ischemia on the cerebral circulation has not been assessed previously. There have been reports of other models of ischemia in other organ systems, for example in limb ischemia and reperfusion during orthopedic surgery. These have found neutrophils and monocytes to acquire a more activated phenotype after approximately 30 minutes of ischemia
[25, 26]. In contrast, during coronary bypass operations in adults, less activated cells were observed distal to the ischemic coronary region than proximally
. Here, we report the opposite (more activated cells and increased cell numbers leaving the ischemic brain), which may suggest that this is an organ-specific effect.
The most striking finding in this study is the abundance of ‘non-classical’ (CD16intermediate and CD16+) monocytes in venous blood directly after cerebral ischemia. The concentration is much higher than the concentration of cells in the arterial sample, which was drawn simultaneously. The question arises as to where these cells have come from. To systematically address this question, a number of options are possible: 1) these cells have undergone proliferation, 2) the venous sample has a higher viscosity, which increases the concentration of immune cells, and/or 3) cells have migrated from the parenchyma into the intravascular compartment. The first option (proliferation) seems highly unlikely in the short period of half an hour that the increase was observed. The second option, of a temporarily more viscous venous sample, could be an option for example by leakage of plasma into the cerebral parenchyma, or an uptake of fluid by other cells. However, both hematocrit and thrombocyte numbers show stable values and thus argue against this. Therefore, the most valid explanation would be that the cells have migrated into the intravascular compartment, from elsewhere. The cells may have come from the ‘perivascular space’, which is located outside of the endothelial cell layer and inside the parenchymal basal membrane. Histopathologic studies of the brain have revealed that the perivascular space contains macrophages, neutrophils and possibly also lymphocytes
[6, 28, 29]. We hypothesize that the neutrophils and monocytes migrate from their perivascular location, into the vascular space and thereby are increased in the venous sample. The increased permeability of the blood–brain barrier due to the cessation of blood flow may facilitate the migration of cells into the circulation
[30–32]. A similar phenomenon has been observed in models of exercise, where within 30 minutes after infusion of adrenaline, amongst others, the ‘nonclassical’ monocytes (CD16intermediate and CD16+) are dramatically increased in the systemic circulation
. In this case, the release of these monocytes from the perivascular space is thought to be a direct effect of adrenaline, whereas in the current study, it seems plausible that the ischemia has initiated this process. The phenomenon has also been described in transendothelial models of monocyte trafficking, where the specifically the nonclassical monocyte populations ‘reverse-migrate’ from the tissue into the vascular lumen
The decreased concentrations of neutrophils and monocytes during deep hypothermic CPB may play a role in the above described perivascular phenomenon. As is depicted in Figure
2, neutrophil and monocyte concentrations were much lower than may be expected due to the expansion of the circulating volume by connection to the CPB circuit. Hence, cells may be migrating out of the circulation in response to another, as yet unknown, trigger. At this point, these cells may occupy the perivascular space, only to be released again promptly after cerebral ischemia has occurred.
Regarding the soluble effects of cerebral ischemia, although subtle, significant differences were found after DHCA in IL-6 and sVCAM-1. The higher IL-6 concentration in venous blood after DHCA is presumably a direct effect of ischemia on the endothelium and the intra- and perivascular leukocytes. In line with the activated phenotypes of neutrophils and monocytes, these may rapidly release vast amounts of IL-6. The lower sVCAM-1 concentration may represent the uptake of sVCAM by these cells, as their activated subsets are known to highly express the receptor CD49d
We cannot predict if the observed changes in inflammation are of any clinical relevance. Generally, a more proinflammatory milieu in the first hours of reperfusion predisposes to a more detrimental cerebral outcome
[5, 35]. However, the difference in inflammation between DHCA and ACP was limited to the time point immediately after the insult, suggesting that the effect of the ischemia was temporary. Of note, this may only be the case in this specific context, as the effect of other inflammatory triggers during surgery (such as the ongoing surgical damage, the use of CPB and cyanosis) are likely overwhelming
We acknowledge that this work has important limitations. First, this is a model of cerebral ischemia during deep hypothermia, a temperature not directly applicable to the clinical situation in which adults or children have a stroke. Similarly, although still a matter of debate, neonates may have a different permeability of the blood–brain barrier than adults
[38, 39]. Second, as dexamethasone was administered to all patients before the start of CPB, we cannot exclude that this may have mitigated the inflammatory response to the ischemia
. Finally, due to the many triggers for systemic inflammation that accompany the surgery of these patients, later effects of the ischemia are impossible to tease out. In an animal model, a more ‘clean’ model of cerebral ischemia can be performed, and the evolution of the inflammation can be studied at all desired time points. However, the translation to the human situation is fraught with difficulties, making studies like the current study essential to gain insight into these complex mechanisms.