Relevance of Porcine Stroke Models to Bridge the Gap from Pre-Clinical Findings to Clinical Implementation.

In the search of animal stroke models providing translational advantages for biomedical research, pigs are large mammals with interesting brain characteristics and wide social acceptance. Compared to rodents, pigs have human-like highly gyrencephalic brains. In addition, increasingly through phylogeny, animals have more sophisticated white matter connectivity; thus, ratios of white-to-gray matter in humans and pigs are higher than in rodents. Swine models provide the opportunity to study the effect of stroke with emphasis on white matter damage and neuroanatomical changes in connectivity, and their pathophysiological correlate. In addition, the subarachnoid space surrounding the swine brain resembles that of humans. This allows the accumulation of blood and clots in subarachnoid hemorrhage models mimicking the clinical condition. The clot accumulation has been reported to mediate pathological mechanisms known to contribute to infarct progression and final damage in stroke patients. Importantly, swine allows trustworthy tracking of brain damage evolution using the same non-invasive multimodal imaging sequences used in the clinical practice. Moreover, several models of comorbidities and pathologies usually found in stroke patients have recently been established in swine. We review here ischemic and hemorrhagic stroke models reported so far in pigs. The advantages and limitations of each model are also discussed.

Targeting Pro-Oxidant Iron with Exogenously Administered Apotransferrin Provides Benefits Associated with Changes in Crucial Cellular Iron Gate Protein TfR in a Model of Intracerebral Hemorrhagic Stroke in Mice.

We have previously demonstrated that the post-stroke administration of iron-free transferrin (apotransferrin, ATf) is beneficial in different models of ischemic stroke (IS) through the inhibition of the neuronal uptake of pro-oxidant iron. In the present study, we asked whether ATf is safe and also beneficial when given after the induction of intracerebral hemorrhage (ICH) in mice, and investigated the underlying mechanisms. We first compared the main iron actors in the brain of IS- or collagenase-induced ICH mice and then obtained insight into these iron-related proteins in ICH 72 h after the administration of ATf. The infarct size of the IS mice was double that of hemorrhage in ICH mice, but both groups showed similar body weight loss, edema, and increased ferritin and transferrin levels in the ipsilateral brain hemisphere. Although the administration of human ATf (hATf) to ICH mice did not alter the hemorrhage volume or levels of the classical ferroptosis GPX4/system xc- pathways, hATf induced better neurobehavioral performance, decreased 4-hydroxynonenal levels and those of the second-generation ferroptosis marker transferrin receptor (TfR), and restored the mRNA levels of the recently recognized cytosolic iron chaperone poly(RC) binding protein 2. In addition, hATf treatment lowered the ICH-induced increase in both endogenous mouse transferrin mRNA levels and the activation of caspase-2. In conclusion, hATf treatment provides neurobehavioral benefits post-ICH associated with the modulation of iron/oxidative players.

Establishment of a reproducible and minimally invasive ischemic stroke model in swine.

The need for advances in the management/treatment options for ischemic stroke patients requires that upcoming preclinical research uses animals with more human-like brain characteristics. The porcine brain is considered appropriate, although the presence of the rete mirabile (RM) prevents direct catheterization of the intracranial arteries to produce focal cerebral ischemia. To develop a reproducible minimally invasive porcine stroke model, a guide catheter and guide wire were introduced through the femoral artery until reaching the left RM. Using the pressure cooker technique, Squid-12 embolization material was deposited to fill, overflow, and occlude the left RM, the left internal carotid artery, and left circle of Willis wing up to the origins of the middle cerebral arteries (MCAs), mimicking the occlusion produced in the filament model in rodents. Longitudinal multimodal cerebral MRI was conducted to assess the brain damage and cerebral blood supply. The technique we describe here occluded up to the origins of the MCAs in 7 of 8 swine, inducing early damage 90 minutes after occlusion that later evolved to a large cerebral infarction and producing no mortality during the intervention. This minimally invasive ischemic stroke model in swine produced reproducible infarcts and shows translational features common to human stroke.

Comparative Proteomics Unveils LRRFIP1 as a New Player in the DAPK1 Interactome of Neurons Exposed to Oxygen and Glucose Deprivation.

Death-associated protein kinase 1 (DAPK1) is a pleiotropic hub of a number of networked distributed intracellular processes. Among them, DAPK1 is known to interact with the excitotoxicity driver NMDA receptor (NMDAR), and in sudden pathophysiological conditions of the brain, e.g., stroke, several lines of evidence link DAPK1 with the transduction of glutamate-induced events that determine neuronal fate. In turn, DAPK1 expression and activity are known to be affected by the redox status of the cell. To delineate specific and differential neuronal DAPK1 interactors in stroke-like conditions in vitro, we exposed primary cultures of rat cortical neurons to oxygen/glucose deprivation (OGD), a condition that increases reactive oxygen species (ROS) and lipid peroxides. OGD or control samples were co-immunoprecipitated separately, trypsin-digested, and proteins in the interactome identified by high-resolution LC-MS/MS. Data were processed and curated using bioinformatics tools. OGD increased total DAPK1 protein levels, cleavage into shorter isoforms, and dephosphorylation to render the active DAPK1 form. The DAPK1 interactome comprises some 600 proteins, mostly involving binding, catalytic and structural molecular functions. OGD up-regulated 190 and down-regulated 192 candidate DAPK1-interacting proteins. Some differentially up-regulated interactors related to NMDAR were validated by WB. In addition, a novel differential DAPK1 partner, LRRFIP1, was further confirmed by reverse Co-IP. Furthermore, LRRFIP1 levels were increased by pro-oxidant conditions such as ODG or the ferroptosis inducer erastin. The present study identifies novel partners of DAPK1, such as LRRFIP1, which are suitable as targets for neuroprotection.