Ertugrul Kilic

Delayed post-ischaemic neuroprotection following systemic neural stem cell transplantation involves multiple mechanisms

Recent evidence suggests that neural stem/precursor cells (NPCs) promote recovery in animal models with delayed neuronal death via a number of indirect bystander effects. A comprehensive knowledge of how transplanted NPCs exert their therapeutic effects is still lacking. Here, we investigated the effects of a delayed transplantation of adult syngenic NPCs--injected intravenously 72 h after transient middle cerebral artery occlusion--on neurological recovery, histopathology and gene expression. NPC-transplanted mice showed a significantly improved recovery from 18 days post-transplantation (dpt) onwards, which persisted throughout the study. A small percentage of injected NPCs accumulated in the brain, integrating mainly in the infarct boundary zone, where most of the NPCs remained undifferentiated up to 30 dpt. Histopathological analysis revealed a hitherto unreported very delayed neuroprotective effect of NPCs, becoming evident at 10 and 30 dpt. Tissue survival was associated with downregulation of markers of inflammation, glial scar formation and neuronal apoptotic death at both mRNA and protein levels. Our data highlight the relevance of very delayed degenerative processes in the stroke brain that are intimately associated with inflammatory and glial responses. These processes may efficaciously be antagonized by (stem) cell-based strategies at time-points far beyond established therapeutic windows for pharmacological neuroprotection.

The phosphatidylinositol-3 kinase/Akt pathway mediates VEGF’s neuroprotective activity and induces blood brain barrier permeability after focal cerebral ischemia

Based on its trophic influence on neurons and vascular cells, vascular endothelial growth factor (VEGF) is a promising candidate for stroke treatment. VEGFs survival‐promoting effects are purchased at the expense of an increased blood brain barrier permeability, which potentially compromises tissue survival. The mechanisms via which VEGF protects the brain against ischemia remained unknown. We examined signaling pathways underlying VEGFs neuroprotective activity in our transgenic mouse line, which expresses human VEGF165 under a neuron‐specific enolase (NSE) promoter. We show that VEGF receptor‐2 (Flk‐1) is expressed on ischemic neurons and astrocytes and is activated by VEGF. Following 90‐min episodes of middle cerebral artery occlusion, VEGF increased phosphorylated (but not total) Akt and ERK‐1/‐2 and reduced phosphorylated mitogen activated protein kinase/p38 and c‐Jun NH2‐terminal kinase (JNK)‐1/‐2 levels, at the same time decreasing inducible NO synthase expression in ischemic neurons. Inhibition of Akt with Wortmannin reversed VEGFs neuroprotective properties, diminished brain swelling, and restored the vascular permeability induced by VEGF to below levels in WT animals. The aggravation of brain injury by Wortmannin was associated with the restitution of p38, but not of JNK‐1/‐2, ERK‐1/‐2, or inducible NOS (iNOS). Our data demonstrate that VEGF mediates both neuroprotection and blood brain barrier permeability via the phosphatidylinositol‐3 kinase (PI3K)/Akt pathway. Based on our observation that VEGF neuroprotection and vascular leakage depend on PI3K/Akt, which is putatively regulated by VEGF receptor‐2, we predict that it may not easily be possible to make use of VEGFs neuroprotective function without accepting its unfavorable consequence, the increased vascular permeability.—Kilic, E., Kilic, Ü., Wang, Y., Bassetti, C. L., Marti, H. H., Hermann, D. M. The phosphatidylinositol‐3 kinase/Akt pathway mediates VEGFs neuroprotective activity and induces blood brain barrier permeability after focal cerebral ischemia. FASEB J. 20, E307–E314 (2006)

Intravenous TAT-GDNF Is Protective After Focal Cerebral Ischemia in Mice

Background and Purpose— Delivery of therapeutic proteins into tissues and across the blood-brain barrier is severely limited by their size and biochemical properties. The 11-amino acid human immunodeficiency virus TAT protein transduction domain is able to cross cell membranes and the blood-brain barrier, even when coupled with larger peptides. The present studies were done to evaluate whether TAT–glial line-derived neurotrophic factor (GDNF) fusion protein is protective in focal cerebral ischemia. Methods— Anesthetized male C57BL/6j mice were submitted to intraluminal thread occlusion of the middle cerebral artery. Reperfusion was initiated 30 minutes later by thread retraction. Laser Doppler flow was monitored during the experiments. TAT-GDNF, TAT-GFP (0.6 nmol each), or vehicle was intravenously applied over 10 minutes immediately after reperfusion. After 3 days (30 minutes of ischemia), animals were reanesthetized and decapitated. Brain injury was evaluated by histochemical stainings. Results— Immunocytochemical experiments confirmed the presence of TAT-GDNF protein in the brains of fusion protein–treated nonischemic control animals 3 to 4 hours after TAT fusion protein delivery. TAT-GDNF significantly reduced the number of caspase-3–immunoreactive and DNA-fragmented cells and increased the number of viable neurons in the striatum, where disseminated tissue injury was observed, compared with TAT-GFP– or vehicle-treated animals. Conclusions— Our results demonstrate that TAT fusion proteins are powerful tools for the treatment of focal ischemia when delivered both before and after an ischemic insult. This approach may be of clinical interest because such fusion proteins can be intravenously applied and reach the ischemic brain regions. This approach may therefore offer new perspectives for future strategies in stroke therapy.

Intravenous TAT-Bcl-Xl is protective after middle cerebral artery occlusion in mice.

The delivery of proteins across the blood–brain barrier is severely limited by the proteins size and biochemical properties. Eleven–amino acid human immunodeficiency virus TAT protein is able to cross cell membranes even when coupled with larger peptides. We evaluated whether TAT–Bcl‐XL fusion protein is protective in focal ischemia. Mice underwent 30 or 90 minutes of intraluminal middle cerebral artery thread occlusion. TAT–Bcl‐XL, TAT–β‐galactosidase, or TAT‐GFP (0.6nmol each) were applied intravenously over 10 minutes either 1 hour before or immediately after ischemia. Additional animals received no TAT protein infusions. We show that the brain tissue is progressively transduced with TAT proteins within 3 to 4 hours after intravenous delivery. We provide evidence that TAT–Bcl‐XL treatment reduces infarct volume and neurological deficits after long ischemic insults lasting 90 minutes, when applied both before and after ischemia. After short insults, lasting only 30 minutes, TAT–Bcl‐XL further diminishes the number of caspase‐3–reactive and DNA fragmented cells and increases the number of viable neurons in the striatum. Our results indicate that TAT fusion proteins are elegant and powerful tools that might be of clinical interest for stroke treatment, because factors may be intravenously applied. Thus, fusion proteins may open fascinating perspectives for future research.

When Melatonin Gets on Your Nerves: Its Beneficial Actions in Experimental Models of Stroke:

This article summarizes the evidence that endogenously produced and exogenously administered melatonin reduces the degree of tissue damage and limits the biobehavioral deficits associated with experimental models of ischemia/reperfusion injury in the brain (i.e., stroke). Melatonins efficacy in curtailing neural damage under conditions of transitory interruption of the blood supply to the brain has been documented in models of both focal and global ischemia. In these studies many indices have been shown to be improved as a consequence of melatonin treatment. For example, when given at the time of ischemia or reperfusion onset, melatonin reduces neurophysio-logical deficits, infarct volume, the degree of neural edema, lipid peroxidation, protein carbonyls, DNA damage, neuron and glial loss, and death of the animals. Melatonins protective actions against these adverse changes are believed to stem from its direct free radical scavenging and indirect antioxidant activities, possibly from its ability to limit free radical generation at the mitochondrial level and because of yet-undefined functions. Considering its high efficacy in overcoming much of the damage associated with ischemia/reperfusion injury, not only in the brain but in other organs as well, its use in clinical trials for the purpose of improving stroke outcome should be seriously considered.

TLR-4 deficiency protects against focal cerebral ischemia and axotomy-induced neurodegeneration

The pattern recognition receptor toll-like receptor (TLR)-4 mediates innate danger signaling in the brain, being activated in response to lipopolysaccharide. Until now, its role in the degenerating brain remained unknown. We here examined effects of a loss-of-function mutation of TLR-4 in mice submitted to transient focal cerebral ischemia and retinal ganglion cell (RGC) axotomy, which are highly reproducible and clinically relevant in vivo models of acute and subacute neuronal degeneration. We show that TLR-4 deficiency protects mice against ischemia and axotomy-induced RGC degeneration. Decreased phosphorylation levels of the mitogen-activated kinases ERK-1/-2, JNK-1/-2 and p38 together with reduced inducible NO synthase levels in injured neurons of TLR-4 mutant mice suggests that TLR-4 deficiency downscales parenchymal stress responses, thereby enhancing neuronal survival. At the same time, densities of MPO+ neutrophils and Iba1+ microglial cells were increased in the brains of TLR-4 mutant animals, pointing towards a futile inflammatory response aiming to compensate lost functions. Our data indicate that innate immunity may represent an attractive target for neuroprotective treatments in stroke and neurodegeneration.

Aggravation of ischemic brain injury by prion protein deficiency : Role of ERK-1/-2 and STAT-1

The cellular isoform of prion protein, PrPc, may confer neuroprotection in the brain, according to recent studies. To elucidate the role of PrPc in stroke pathology, we subjected PrPc-knockout (Prnp(0/0)), wild-type and PrPc-transgenic (tga20) mice to 30 min of intraluminal middle cerebral artery occlusion, followed by 3, 24 or 72 h reperfusion, and examined how PrPc levels influence brain injury and cell signaling. In immunohistochemical experiments and Western blots, we show that PrPc expression is absent in the brains of Prnp(0/0) mice, detectable in wild-type controls and approximately 4.0-fold elevated in tga20 mice. We provide evidence that PrPc deficiency increases infarct size by approximately 200%, while transgenic PrPc restores tissue viability, albeit not above levels in wild-type animals. To elucidate the mechanisms underlying Prnp(0/0)-induced injury, we performed Western blots, which revealed increased activities of ERK-1/-2, STAT-1 and caspase-3 in ischemic brains of Prnp(0/0)mice. Our data suggest a role of cytosolic signaling pathways in Prnp(0/0)-induced cell death.

Signal transduction pathways involved in melatonin-induced neuroprotection after focal cerebral ischemia in mice

Abstract:u2002 Because of its favorable action profile in humans, melatonin is a particularly interesting candidate as a neuroprotectant in acute ischemic stroke. Until now, the signaling mechanisms mediating melatonins neuroprotective actions remained essentially uninvestigated. Herein, we examined the effects of melatonin, administered either orally for 9u2003wk as a stroke prophylactic (4u2003mg/kg/day) or intraperitoneally immediately after reperfusion onset (4u2003mg/kg), on the activation of signal transduction pathways in mice submitted to 90u2003min of intraluminal middle cerebral artery occlusion, followed by 24u2003hr of reperfusion. In these studies, melatonin significantly reduced ischemic infarct size by ∼30–35%, as compared with animals receiving diluent (sham) treatment, independent of whether the indole was administered prior to or after ischemia. Under both conditions, animals receiving melatonin exhibited elevated phosphorylated Akt levels in their brains, as determined by Western blots. Additionally, phosphorylation levels of mitogen‐activated protein kinase/extracellular‐regulated kinase (ERK)‐1/‐2 and Jun kinase (JNK)‐1/‐2 were increased following prophylactic, but not acute, melatonin treatment. Our data suggest a role of phosphatidyl inositol‐3 kinase/Akt signaling in acute melatonin‐induced neuroprotection, while ERK‐1/‐2 and/or JNK‐1/‐2 rather appear to be involved in melatonins long‐term effects.

Inhibition of multidrug resistance transporter-1 facilitates neuroprotective therapies after focal cerebral ischemia.

The blood-brain barrier possesses active transporters carrying brain-permeable xenobiotics back into the blood against concentration gradients. We demonstrate that multidrug resistance transporter (Mdr)-1 is upregulated on capillary endothelium after focal cerebral ischemia; moreover, Mdr-1 deactivation by pharmacological inhibition or genetic knockout preferably enhances the accumulation and efficacy of two neuroprotectants known as Mdr-1 substrates in the ischemic brain. We predict that Mdr-1 inhibition may greatly facilitate neuroprotective therapies.

Neural stem/precursor cells for the treatment of ischemic stroke

In ischemic stroke, the third most frequent cause of mortality in industrialized countries, therapeutic options have until now been limited to the first hours after disease onset. Cell transplantation has emerged in various neurological disorders, including experimental stroke, as a successful recovery-promoting approach also in the post-acute stroke phase. However, before envisaging any translation into humans of such promising cell-based approaches we still need to clarify: (i) the ideal cell source for transplantation, (ii) the most appropriate route of cell administration, and, last but not least, (iii) the best approach to achieve an appropriate and functional integration of transplanted cells into the host tissue. Here we discuss, with special emphasis on neural stem/precursor cells, potential mechanisms that may be involved in the action of cell-based therapies in stroke.