Disassembly and Mislocalization of AQP4 in Incipient Scar Formation After Experimental Stroke

There is an urgent need to better understand the mechanisms involved in scar formation in brain. It is well known that astrocytes are critically engaged in this process. Here we analyze in-cipient scar formation one week after a discrete ischemic insult to the cerebral cortex. We show that the infarct border zone is characterized by pronounced changes in the organization and subcellular localization of the major astrocytic protein AQP4. Specifically there is a loss of AQP4 from astrocytic endfoot membranes that anchor astrocytes to pericapillary basal laminae and a disassembly of the supramolecular AQP4 complexes that normally abound in these membranes. This disassembly may be mechanistically coupled to a downregulation of the newly discovered AQP4 isoform AQP4ex. AQP4 has adhesive properties and is assumed to facilitate astrocyte mo-bility by permitting rapid volume changes at the leading edges of migrating astrocytes. Thus, the present findings provide new insight in the molecular basis of incipient scar formation.

Mitofusin 2 Integrates Mitochondrial Network Remodelling, Mitophagy and the Renewal of Respiratory Chain Proteins in Neurons After Oxygen and Glucose Deprivation.

In the efforts to develop effective therapeutic strategies limiting post-ischemic injury, mitochondria emerge as key element in determining the fate of the neurons. Mitochondrial damage can be alleviated by various mechanisms including mitochondrial network remodelling, mitochondrial elimination and mitochondrial protein biogenesis. However, the mechanisms regulating the relationship between these phenomena are poorly understood. Here we hypothesize that mitofusin 2 (Mfn2), a mitochondrial GTPase, involved in mitochondrial fusion, mitochondria trafficking and mitochondria and endoplasmic reticulum (ER) tethering, may act as a linking and regulatory factor in neurons following ischemic insult. To verify this assumption, we performed a temporal oxygen and glucose deprivation (OGD) on rat cortical primary culture to determine whether Mfn2 protein reduction may affect the onset of mitophagy, subsequent mitochondrial biogenesis and thus neuronal survival. In our study we found that Mfn2 knock-down increased the susceptibility of the neurons to the OGD. Mfn2 protein reduction prevented mitochondrial network remodelling and resulted in the prolonged mitophagosomes formation in response to the insult. Further on, Mfn2 protein reduction was accompanied by a reduced level of Parkin protein and an increased Parkin accumulation with mitochondria. As for Mfn2-expressing neurons, the OGD insult was followed by an elevated mtDNA content and an increase in the respiratory chain proteins. Neither of this phenomena were observed for Mfn2-reduced neurons. Collectively, our findings show that Mfn2 in neurons is involved in their response to mild and transient OGD stress, balancing the extent of elimination of defective mitochondria and positively influencing mitochondrial respiratory proteins levels. Our study confirms that Mfn2 is an essential element of the neuronal response to ischemic insult, necessary for the neuronal survival.

Tissue-type plasminogen activator induces TNF-α-mediated preconditioning of the blood-brain barrier

Ischemic tolerance is a phenomenon whereby transient exposure to a non-injurious preconditioning stimulus triggers resistance to a subsequent lethal ischemic insult. Despite the fact that not only neurons but also astrocytes and endothelial cells have a unique response to preconditioning stimuli, current research has been focused mostly on the effect of preconditioning on neuronal death. Thus, it is unclear if the blood-brain barrier (BBB) can be preconditioned independently of an effect on neuronal survival. The release of tissue-type plasminogen activator (tPA) from perivascular astrocytes in response to an ischemic insult increases the permeability of the BBB. In line with these observations, treatment with recombinant tPA increases the permeability of the BBB and genetic deficiency of tPA attenuates the development of post-ischemic edema. Here we show that tPA induces ischemic tolerance in the BBB independently of an effect on neuronal survival. We found that tPA renders the BBB resistant to an ischemic injury by inducing TNF-α-mediated astrocytic activation and increasing the abundance of aquaporin-4-immunoreactive astrocytic end-feet processes in the neurovascular unit. This is a new role for tPA, that does not require plasmin generation, and with potential therapeutic implications for patients with cerebrovascular disease.

Hemostasis system in elderly patients with ischemic insult in the system of carotid arteries

There was performed investigation of rhelogic properties, of coagulation and anticoagulation blood systems in 88 patients of senile age with ischemic insult in the carotid system. It was determined that for elderly patients highly expressed tendencies for hyperaggregation and suppression of their own fibrinolytic blood activity are characteristic features. Initially increased level of aggregation and hypercoagulation, combined with presence in blood fibrin-monomer complexes were accompanied by progressing DIC syndrome, and as a rule, were associated with steady neurology deficiency and high incidence of mortality. Possible correction ways were considered for hemostasis system disorders in elderly patients.

HDAC1 deregulation promotes neuronal loss and deficit of motor function in stroke pathogenesis

Stroke is a common cause of death worldwide and leads to disability and cognitive dysfunction. Ischemic stroke and hemorrhagic stroke are major categories of stroke, accounting for 68% and 32% of strokes, respectively. Each year, 15 million people experience stroke worldwide, and the stroke incidence is rising. Epigenetic modifications regulate gene transcription and play a major role in stroke. Accordingly, histone deacetylase 1 (HDAC1) participates in DNA damage repair and cell survival. However, the mechanisms underlying the role of HDAC1 in stroke pathogenesis are still controversial. Therefore, we investigated the role of HDAC1 in stroke by using a rat model of endothelin-1-induced brain ischemia. Our results revealed that HDAC1 was deregulated following stroke, and its expressional level and enzymatic activity were decreased. We also used MS-275 to inhibit HDAC1 function in rats exposed to ischemic insult. We found that HDAC1 inhibition promoted the infarct volume, neuronal loss, DNA damage, neuronal apoptosis after stroke, and levels of reactive oxygen species and inflammation cytokines. Additionally, HDAC1 inhibition deteriorated the behavioral outcomes of rats with ischemic insult. Overall, our findings demonstrate that HDAC1 participates in ischemic pathogenesis in the brain and possesses potential for use as a therapeutic target.

Heparanase inhibition preserves the endothelial glycocalyx in lung grafts and improves lung preservation and transplant outcomes

The endothelial glycocalyx (eGC) is considered a key regulator of several mechanisms that prevent vascular injury and disease. Degradation of this macromolecular layer may be associated with post-transplant graft dysfunction. In this study, we aimed to demonstrate the benefits of eGC protection via heparanase inhibition on graft quality. We established rat models of lung grafts with damaged or preserved eGC using ischemic insult and transplanted the grafts into recipients. Lung grafts were also subjected to normothermic ex vivo lung perfusion for detailed assessment under isolated conditions. Physiologic parameters and eGC-associated cellular events were assessed in grafts before and after reperfusion. Structurally degraded eGC and highly activated heparanase were confirmed in lungs with ischemic insult. After transplant, lungs with damaged eGC exhibited impaired graft function, inflammation, edema, and inflammatory cell migration. Increased eGC shedding was evident in the lungs after reperfusion both in vivo and ex vivo. These reperfusion-related deficiencies were significantly attenuated in lungs with preserved eGC following heparanase inhibition. Our studies demonstrated that eGC plays a key role in maintaining lung graft quality and function. Heparanase inhibition may serve as a potential therapeutic to preserve eGC integrity, leading to improved post-transplant outcomes.

MO1016MULTIPLE-ORGAN DAMAGE FOLLOWING PERINATAL ASPHYXIA IN RAT MODEL

Background and Aims Perinatal asphyxia (PA) is associated with more than half a million mature newborn deaths yearly. It may lead to severe complications including hypoxic encephalopathy, renal- hepatic- and cardiovascular injury, as well as respiratory distress, Basic research and clinical trials mainly focus on mitigating central nervous system damage by selective head or whole body cooling, which is currently the only routinely used treatment in clinical practice. However, the extent of PA-associated multi-organ damage is not clarified yet and effective therapies are lacking. Our aim was to investigate the acute renal, hepatic and cardiac impairment following PA and to identify pathways involved in the pathomechanism. In addition, we aimed to explore long-term effects of PA on permanent organ damage and susceptibility to ischemia/-reperfusion injury in adulthood. Method Postnatal 7 day-old male Wistar rat pups (n=5-10/group) were randomly grouped as follows: (i) Baseline; (ii) Control; (iii) PA. The PA group was separated from the dam and received asphyxic gas mixture (4% O2; 20% CO2 in N2) for 15 minutes, while Control animals received normal air following separation. Serum and tissue samples were collected after 4 (T4) or 24 (T24) hours. In a second experiment 35 min bilateral renal ischemic insult was performed on control and PA rats aged 6 months (n=6-7/group). Serum and tissue samples were collected 24 (T24 IR) hours after reperfusion (Figure 1). Serum levels of electrolytes, kidney and liver functional parameters, and myocardial ischemic protein Troponin I were determined. Highly selective and sensitive tubular injury markers (Kim1, Ngal) were measured. Expressions of hypoxic (Hif1a, Hif2a) inflammatory (Il1α, Il1β, Il6, Tnfα, Mcp1, Tlr2), apoptotic (Bax, Bcl-2) and angiogenic genes (Vegf, Epo) and heat shock proteins (Hsp27, Hsp72) were investigated. Periodic-Acid Schiff stained kidney sections and Hematoxylin & Eosin stained liver sections were evaluated for structural injury. Results Blood urea nitrogen (BUN) and serum GPT were elevated at T4 following PA. Kim1, Ngal and heat shock protein expressions were increased, inflammatory and angiogenic pathways were activated in the kidney after PA. In the liver hypoxic and apoptotic pathways were activated at T24 in controls and after asphyxia, but not in the Baseline group. Vacuolisation, cytoplasmic degradation, and the onset of necrosis were observed in the liver following PA. Serum Troponin I was elevated indicating myocardial damage, moreover inflammatory cytokines and heat shock proteins increased in the heart. In adult PA rats BUN levels were elevated, suggesting a long-term detrimental effect of PA on renal function. In addition, adult PA rats were more susceptible to renal ischemic insult, confirmed by higher serum creatinine and GPT levels, as well as increased expression of tubular injury, hypoxic and inflammatory markers compared to Control rats subjected to ischemia. Conclusion Acute renal, hepatic and myocardial impairment was observed after PA. These results may justify the need for clinical follow-up and novel treatment strategies for possible multi-organ damage. The molecular pathways described here are potential targets for therapeutic intervention. In addition, birth asphyxia may increase sensitivity to renal injury in adulthood, which may be worth considering in clinical situations with potential renal impairment.

LDH and PDH Activities in the Ischemic Brain and the Effect of Reperfusion—An Ex Vivo MR Study in Rat Brain Slices Using Hyperpolarized [1-13C]Pyruvate

Ischemic stroke is a leading cause for neurologic disability worldwide, for which reperfusion is the only available treatment. Neuroimaging in stroke guides treatment, and therefore determines the clinical outcome. However, there are currently no imaging biomarkers for the status of the ischemic brain tissue. Such biomarkers could potentially be useful for guiding treatment in patients presenting with ischemic stroke. Hyperpolarized 13C MR of [1-13C]pyruvate is a clinically translatable method used to characterize tissue metabolism non-invasively in a relevant timescale. The aim of this study was to utilize hyperpolarized [1-13C]pyruvate to investigate the metabolic consequences of an ischemic insult immediately during reperfusion and upon recovery of the brain tissue. The rates of lactate dehydrogenase (LDH) and pyruvate dehydrogenase (PDH) were quantified by monitoring the rates of [1-13C]lactate and [13C]bicarbonate production from hyperpolarized [1-13C]pyruvate. 31P NMR of the perfused brain slices showed that this system is suitable for studying ischemia and recovery following reperfusion. This was indicated by the levels of the high-energy phosphates (tissue viability) and the chemical shift of the inorganic phosphate signal (tissue pH). Acidification, which was observed during the ischemic insult, has returned to baseline level following reperfusion. The LDH/PDH activity ratio increased following ischemia, from 47.0 ± 12.7 in the control group (n = 6) to 217.4 ± 121.3 in the ischemia-reperfusion group (n = 6). Following the recovery period (ca. 1.5 h), this value had returned to its pre-ischemia (baseline) level, suggesting the LDH/PDH enzyme activity ratio may be used as a potential indicator for the status of the ischemic and recovering brain.