Primer escenario de estudio

Although the ultimate therapeutic goal in ischemic stroke is addressed, as said, toward thrombolysis or mechanical recanalization, restoration of blood flow may at times collapse in the so-called “cerebral reperfusion injury”. Indeed, while reperfusion may reduce infarct size and improve clinical outcome in some subjects, a subset of patients paradoxically experience disastrous outcomes in the form of fatal edema or intracranial hemorrhage that extend beyond the sole failure of cellular energy supply to neurons (Chen and Nuñez, 2010; Eltzschig and Eckle, 2011) (Fig. 13).

Fig. 13

Reperfusion injury.

When a cerebral artery is occluded, brain tissue becomes ischemic (blue rectangle), and death spreads exponentially. If the vessel is not reperfused using appropriate treatment, a huge brain area will be injured (blue line). If reperfusion occurs in a reasonable time, the infarct size is reduced (pink line). However, if reperfusion occurs together with neuroprotection to reduce reperfusion injury (pink shading), the infarct size is further attenuated (red line).

(Adapted from Sanz-Rosa et al., 2012).

Reperfusion is characterized by vascular restoration and concomitant reoxygenation of ischemic tissues that may nevertheless undergo cellular changes including nuclear fragmentation, chromatin condensation and neuronal cell body shrinkage, often accompanied by phospholipidic membrane alteration (Balasubramanian and Schroit, 2003; Broughton et

28

al., 2009). Multiple pathological molecular mechanisms are believed to be implicated in this injurious process, involving both innate and adaptive immune responses as well as complement system, platelets and coagulation factors (Nour et al., 2013). As a consequence, cell death can occur either by necrosis or apoptosis (Hotchkiss et al., 2009), thereby further stimulating the inflammatory system and exacerbating the ongoing injury (Chen and Nuñez, 2010; Elliott et al., 2009).

In the frame of such impaired cellular milieu, activated leukocytes interact with endothelial cells and extravasate from capillaries, infiltrating brain tissue and releasing leukotrienes, prostaglandins and other proinflammatory cytokines.

Several studies also suggested a negative involvement of platelets after cerebral ischemia and reperfusion (Ishikawa et al., 2004; Pan et al., 2007). Beside the mechanical obstruction, platelets can cause vasospasm through serotonin, thromboxane A2, and free radicals release. In addition, activated platelets promote chemotaxis and migration of leukocytes, emphasizing the inflammatory cascade.

The plunge into the inflammatory cascade and the release of neutrophil-derived oxidants and proteolytic enzymes increase the permeability of the vascular bed and eventually disrupt BBB integrity, ultimately deteriorating also the salvageable ischemic penumbra.

Moreover, excitotoxicity-mediated inhibition of astrocytic repair function also disables the BBB, functionally correlating with the severity of patients outcome (Liu and Chopp, 2016; Schaller and Graf, 2004).

In this inward spiral, the depletion of cellular metabolic resources leads to anaerobic glycolysis and lactic acidosis, further release of glutamate, cytotoxic edema, and excessive generation of free radicals that overwhelm the system, collapsing in irretrievable cellular demise (Dirnagl et al., 1999; Lee et al., 2000).

Reactive radical oxide species play a major role in this hypoxic context (Olmez and Ozyurt, 2012). Inducible nitric oxide synthase (iNOS) is activated during cerebral ischemia, leading to nitric oxide and peroxynitrite production, DNA damage, fall of ATP production and, ultimately, either p53 upregulation or Bcl-2 downregulation in case of, respectively, necrotic or apoptotic cell death (Shen et al., 2003).

Besides the oxidant harm, ROS may directly alter BBB permeability, causing long-lasting edema and exacerbating the impedance to adequate tissue perfusion, possibly contributing to the hemorrhagic transformation of ischemic stroke (Bektas et al., 2010; Wang and Lo, 2003).

29

Although no study has so far finely quantified these events in stroke patients, animal stroke models have extensively recapitulated the concept of reperfusion injury at molecular and cellular level. Injury from reperfusion has been indeed observed to last beyond the period of ischemia and to extend wider in the penumbra, with evidence of sustained oxidative stress on pericytes in the microvasculature, and in spite of arterial recanalization in MCAo (Middle Cerebral Artery occlusion) murine models (Yemisci et al., 2009). Furthermore, neutrophil accumulation at the site of neuronal injury with consequent increase in infarct volume has been shown to occur earlier and to greater extent in reperfused areas than in tissue permanently deprived of blood supply, starting already 6 hours after restoration of cerebral circulation (Zhang et al., 1994).

As current revascularization approaches have become highly successful and continue to improve, reperfusion injury has obtained a prominent interest for patient treatment and supportive care. On purpose, a better understanding of the mechanisms undermining cerebral ischemia and reperfusion injury has provided several potential strategies to limit or rather prevent further brain damage during reperfusion. Clinical trials ranged from inhibition of apoptosis to promotion of angiogenesis, targeting T-cells, inhibiting reactive oxygen species production and modulating cellular metabolic apparatus (Eltzschig and Eckle, 2011).

Several interventions have targeted leukocyte infiltration by means of anti-neutrophil antiserum or anti-neutrophil monoclonal antibodies, showing improved recovery and reduced infarct size after cerebral reperfusion in both rats and rabbits (Pan et al., 2007). On the same

line, monoclonal antibodies anti-adhesion molecules like ICAM-1 (i.e. enlimomab®) reduced

neutrophil infiltration and lesion size after reperfusion in rats, although the translation of these promising agents into effective therapies in humans has been disappointing (Enlimomab Acute Stroke Trial Investigators, 2001).

Additionally, given the major role of free radicals in hypoxic reperfusion, several studies have tailored neuroprotective interventions to target these players. Among those, modulation of superoxide dismutase and NADPH oxidase proved to limit injury in animal models of ischemia-reperfusion (Kahles and Brandes, 2012; Radermacher et al., 2013).

Dextran sulfate, unfractionated heparin and fingolimod, a sphingosine-1 phosphate receptor agonist, also reduced infarct volume after reperfusion and provided improved functional neurological outcomes, paving a potential role for these agents alongside conventional therapeutic options (Wei et al., 2011).

30

Besides these drug therapies, management strategies including inhalation of hydrogen or nitric oxide gases, brain cooling and conditioned blood perfusion are under clinical evaluation due to the possibility to modulate several pathways involved in reperfusion injury (Stowe et al., 2011; Yenari and Hemmen, 2010).

In this context, neuroimaging is currently being effectively used to provide real-time, clear- cut, non-invasive, easily reproducible measures of potential success for therapeutic strategies implied in injury recovery (Nour et al., 2013; Pan et al., 2007).

However, despite preclinical success of several previously described agents, clinically convincing benefits are still far from attainment, therefore a deeper understanding of revascularization beyond the concept of recanalization alone is strongly required.