STUDIO ANATOMICO E FUNZIONALE DELLA REGIONE DI PENOMBRA IN UN MODELLO DI ISCHEMIA IN VITRO

ANATOMICAL AND FUNCTIONAL STUDY OF THE PENUMBRA REGION IN AN IN VITRO MODEL OF FOCAL CEREBRAL ISCHEMIA Experimental data have shown that the ischemic brain region is characterized by a highly damaged core surrounded by a rim of reversibly altered tissue, commonly referred to as the ischemic penumbra. The core region is characterized by a severely compromised CBF, whereas in the penumbra CBF is reduced but cellular metabolism is still preserved (Hossmann, 2008). One possible approach to identify core and penumbra is based on the evaluation of both metabolism and perfusion of the cerebral tissue. In principle, MRI could be utilized to solve this issue. Still, the significance of the MRI changes observed in the early phase of focal cerebral ischemia is a matter of discussion (Neumann-Hafelin et al., 2000; Kidwell et al., 2003; Rivers et al., 2006). Understanding the functional and structural correlates of MRI findings will help to characterize the pathological status of brain tissue that continuously change during the early few hours that follow an ischemic stroke. The model of the isolated brain facilitates these objectives because permits to induce a highly reproducible infarction by the simple ligation of the MCA and allows to perform multiple fine electrophysiological recordings before, during and after occlusion. In the guinea pig the medial cerebral artery (MCA) supplies the entire piriform cortex (PC), the Olfactory Tubercle (OT) is perfused, also, by collaterals of the anterior cerebral artery, especially in its medial part. This vascular condition puts the lateral Olfactory Tubercle (l-OT) as an area of watershed between the ischemic core and the normally perfused tissue. We analyzed and correlated neurophysiological, morphological and MRI changes induced by transient (30-60 minutes) and permanent occlusion of the medial cerebral artery (MCAo) in the isolated guinea pig brain. Multi-site extracellular recordings demonstrated prolonged ischemic depolarizations (ID) and the abolition of evoked responses in the piriform cortex served by MCA, but not in the olfactory tubercle, supplied by the anterior cerebral artery. Here evoked responses were transiently reduced and brief peri-infarctual depolarizations (PID) could be observed during the transient MCA occlusion. Diffusion weighted images performed on brains fixed 4 hours after transient ischemia and immunostaining for a microtubule-associated protein, MAP-2, showed overlapping changes due to acute brain injury, restricted to the piriform cortex. Our compared analysis of neurophysiological, MR and anatomical data demonstrate that DW-MRI sequences underestimate the extension of brain tissue damage induced by focal ischemia and do not include the area of PIDs generation, namely the penumbra region Hence the penumbra is a region that maintains the capability to be excited. Hypothetically the ionic imbalance successive to the ischemic condition may prelude to a seizure that is frequent consequence of a stroke. In a consecutive study we verified whether anoxic ischemia per se, without intracranial hemorrhagic complication and in the absence of blood-borne elements responsible for brain infarction, is able to induce early changes in excitability that may prelude to the generation of seizures, and, ultimately could prime epileptogenesis. We used the multimodal approach herein described to identify the penumbra region in the very acute post-ischemic phase. To evaluate the effect of ischemia on cortical excitability we focused on ventral cortical structures (PC and OT) that have been extensively analyzed in this preparation (Biella & de Curtis, 1995; Carriero et al., 2009). The major input to these olfactory-limbic regions is carried by the lateral olfactory tract (LOT) that originates from neurons in the olfactory bulb (Haberly & Price, 1978). LOT is supplied by the medial cerebral artery (MCA). Since we aimed at evaluating synaptic excitability changes that occur acutely after ischemia, we needed to preserve the LOT as source of stimulation to evoke field responses in the olfactory cortex. Therefore, we chose to perform permanent bilateral occlusion of the anterior cerebral arteries (ACAo). We recorded, in olfactory cortices, slow potentials and evoked responses using specific patterns of stimulation in order to evaluate excitability changes in the acute phase after ischemia. The ACAo induces a core area located in the shell of nucleus accumbens and a region of penumbra in the underlying olfactory cortices, where characteristic slow potential shifts (i.e PIDs), but no reduction of diffusion tensor MR signal and MAP-2 immunostaining (typical of ischemic core) were observed. Recording of responses evoked by low- and high-frequency stimulations of the lateral olfactory tract showed no excitability changes in the early hours that follow ischemia in the olfactory cortical areas supplied by ACA. The absence of early hyperexcitability changes in an isolated whole brain model of ischemia, strongly suggests that brain anoxia per se does not contribute to the generation of early seizures. These findings support the view that blood-borne events (such as hemorrhage and inflammation) may play a major role in early post-ischemic seizures.