Jens Weise

Deletion of Cellular Prion Protein Results in Reduced Akt Activation, Enhanced Postischemic Caspase-3 Activation, and Exacerbation of Ischemic Brain Injury

Background and Purpose— The physiological function of cellular prion protein (PrPc) is not yet understood. Recent findings suggest that PrPc may have neuroprotective properties, and its absence increases susceptibility to neuronal injury. The purpose of this study was to elucidate the role of PrPc in ischemic brain injury in vivo. Methods— PrP knockout (Prnp0/0) and Prnp+/+ wild-type (WT) mice were subjected to 60-minute transient or permanent focal cerebral ischemia followed by infarct volume analysis 24 hours after lesion. To identify effects of PrPc deletion on mechanisms regulating ischemic cell death, expression analysis of several proapoptotic and antiapoptotic proteins was performed at 6 and 24 hours after transient ischemia and in nonischemic controls using Western blot or immunohistochemistry. Results— Prnp0/0 mice displayed significantly increased infarct volumes after both transient or permanent ischemia when compared with WT animals (70.2±23 versus 13.3±4 mm3; 119.8±24 versus 86.4±25 mm3). Expression of phospho-Akt (Ser473) was significantly reduced in Prnp0/0 compared with WT animals both early after ischemia and in sham controls. Furthermore, postischemic caspase-3 activation was significantly enhanced in the basal ganglia and the parietal cortex of Prnp0/0 mice. In contrast, expression of total Akt, Bax, and Bcl-2 did not differ between both groups. Conclusions— These results demonstrate that PrPc deletion impairs the antiapoptotic phosphatidylinositol 3-kinase/Akt pathway by resulting in reduced postischemic phospho-Akt expression, followed by enhanced postischemic caspase-3 activation, and aggravated neuronal injury after transient and permanent cerebral ischemia.

Hematopoietic Stem Cells Reduce Postischemic Inflammation and Ameliorate Ischemic Brain Injury

Background and Purpose— Systemic injection of hematopoietic stem cells after ischemic cardiac or neural lesions is one approach to promote tissue repair. However, mechanisms of possible protective or reparative effects are poorly understood. In this study we analyzed the effect of lineage-negative bone marrow-derived hematopoietic stem and precursor cells (Lin−-HSCs) on ischemic brain injury in mice. Methods— Lin−-HSCs were injected intravenously at 24 hours after onset of a 45-minute transient cerebral ischemia. Effects of Lin−-HSCs injection on infarct size, apoptotic cell death, postischemic inflammation and cytokine gene transcription were analyzed. Results— Green fluorescent protein (GFP)-marked Lin−-HSCs were detected at 24 hours after injection in the spleen and later in ischemic brain parenchyma, expressing microglial but no neural marker proteins. Tissue injury assessment showed significantly smaller infarct volumes and less apoptotic neuronal cell death in peri-infarct areas of Lin−-HSC–treated animals. Analysis of immune cell infiltration in ischemic hemispheres revealed a reduction of invading T cells and macrophages in treated mice. Moreover, Lin−-HSC therapy counter-regulated proinflammatory cytokine and chemokine receptor gene transcription within the spleen. Conclusions— Our data demonstrate that systemically applied Lin−-HSCs reduce cerebral postischemic inflammation, attenuate peripheral immune activation and mediate neuroprotection after ischemic stroke.

Upregulation of cellular prion protein (PrPc) after focal cerebral ischemia and influence of lesion severity

The pathological isoform of the prion protein (PrP(Sc)) has been identified to mediate transmissible spongiform encephalopathies like Creutzfeldt-Jakob disease (CJD). In contrast, the physiological function of the normal cellular prion protein (PrP(c)) is not yet understood. Recent findings suggest that PrP(c) may have neuroprotective properties and that its absence increases susceptibility to oxidative stress and neuronal injury. To determine whether PrP(c) is part of the cellular response to neuronal injury in vivo, we investigated PrP(c) regulation after severe and mild focal ischemic brain injury in mice using the thread occlusion stroke model. Western Blot and ELISA analysis showed a significant upregulation of PrP(c) in the ischemic hemisphere at 4 and 8h after onset of permanent focal ischemia, which was no longer detectable at 24h after lesion induction when compared to control animals. In contrast, transient focal ischemia (60 min) did only lead to slightly but not significantly elevated PrP(c) levels in the ischemic hemisphere when compared to controls. These results demonstrate that cerebral PrP(c) is upregulated early in response to focal cerebral ischemia. The extent of upregulation, however, seems to depend on the severity of ischemia and may therefore reflect the extent of ischemia induced neuronal damage. Given the known neuroprotective effects of PrP(c) in vitro, ischemia-induced upregulation of cerebral PrP(c) supports the hypothesis that, as part of an early adaptive cellular response to ischemic brain injury, PrP(c) may be involved in the regulation of ischemia-induced neuronal cell death in vivo.

MRI characteristics of acute and subacute brainstem and thalamic infarctions: value of T2- and diffusion-weighted sequences.

Abstract MRI including diffusion-weighted sequences (DW-MRI) has demonstrated its high sensitivity for acute supratentorial ischemic lesions. In this study we examined the sensitivity of different MRI sequences for the detection of acute brainstem and isolated thalamic infarctions. Diffusion- and T2-weighted MRI of 45 consecutive patients with signs and symptoms of infratentorial and thalamic infarction between 6/1997 and 1/2000 were analysed. The time between the onset of symptoms and the first MRI varied between 2 hours to 7 days with a median of 2 days. MRI repeats were performed in 4 patients in whom the clinical brainstem infarction had not been detected initially. Lesion detectability and size were evaluated for different brainstem and thalamic localizations. An acute brainstem or thalamic infarction as defined by the clinical condition could be identified in all patients by comparison of DW-MRI and T2-weighted images. Pons infarctions were the largest, followed by midbrain and thalamic lesions. Medulla oblongata infarctions were small in comparison. Pons, midbrain and thalamic infarctions were reliably identified beginning 12 hours after the onset of symptoms. In contrast, detectability of medulla oblongata infarctions varied within the first 24 hours and their overall visibility was worse than that of other brainstem infarctions corresponding to their small size. However, regardless of location, none of the 3 infarctions examined within the first 5 hours after the onset of symptoms could be identified. These lesions were demonstrated in follow-up examinations. In conclusion, pontine, midbrain and thalamic infarctions can reliably be visualized by a combination of DW-MRI and T2-weighted images beginning 12 hours after the ischemic attack. However, sensitivity seems to be lower earlier than 12 hours after ischemia and for medulla oblongata lesions.

TAT-Hsp70-mediated neuroprotection and increased survival of neuronal precursor cells after focal cerebral ischemia in mice

Cerebral ischemia stimulates endogenous neurogenesis within the subventricular zone and the hippocampal dentate gyrus of the adult rodent brain. However, such newly generated cells soon die after cerebral ischemia. To enhance postischemic survival of neural precursor cells (NPC) and long-lasting neural regeneration, we applied the antiapoptotic chaperone heat shock protein 70 (Hsp70) fused to a cell-penetrating peptide derived from the HIV TAT to ensure delivery across the blood-brain barrier and the cell membrane. After transient focal cerebral ischemia in mice, TAT-Hsp70 was intravenously injected concomitant with reperfusion and additionally on day 14 after stroke. TAT-Hsp70 treatment resulted in smaller infarct size (27.1 ± 9.0 versus 109.0 ± 14.0 and 88.5 ± 26.0 mm3 in controls) and in functional improvement as assessed by the rota rod, tight rope, and water maze tests when compared with saline- and TAT-hemagglutinin-treated controls. In addition, postischemic survival of endogenous doublecortin (Dcx)-positive NPC was improved within the lesioned striatum of TAT-Hsp70-treated animals for up to 4 weeks after stroke without changing overall cell proliferation of BrdU+ cells. Thus, TAT-Hsp70 treatment after stroke may be a promising tool to act neuroprotective and improve postischemic functional outcome, and also to increase survival of endogenous NPC after stroke.

Transduction of Neural Precursor Cells with TAT‐Heat Shock Protein 70 Chaperone: Therapeutic Potential Against Ischemic Stroke after Intrastriatal and Systemic Transplantation

Novel therapeutic concepts against cerebral ischemia focus on cell‐based therapies in order to overcome some of the side effects of thrombolytic therapy. However, cell‐based therapies are hampered because of restricted understanding regarding optimal cell transplantation routes and due to low survival rates of grafted cells. We therefore transplanted adult green fluorescence protein positive neural precursor cells (NPCs) either intravenously (systemic) or intrastriatally (intracerebrally) 6 hours after stroke in mice. To enhance survival of NPCs, cells were in vitro protein‐transduced with TAT‐heat shock protein 70 (Hsp70) before transplantation followed by a systematic analysis of brain injury and underlying mechanisms depending on cell delivery routes. Transduction of NPCs with TAT‐Hsp70 resulted in increased intracerebral numbers of grafted NPCs after intracerebral but not after systemic transplantation. Whereas systemic delivery of either native or transduced NPCs yielded sustained neuroprotection and induced neurological recovery, only TAT‐Hsp70‐transduced NPCs prevented secondary neuronal degeneration after intracerebral delivery that was associated with enhanced functional outcome. Furthermore, intracerebral transplantation of TAT‐Hsp70‐transduced NPCs enhanced postischemic neurogenesis and induced sustained high levels of brain‐derived neurotrophic factor, glial cell line‐derived neurotrophic factor, and vascular endothelial growth factor in vivo. Neuroprotection after intracerebral cell delivery correlated with the amount of surviving NPCs. On the contrary, systemic delivery of NPCs mediated acute neuroprotection via stabilization of the blood‐brain‐barrier, concomitant with reduced activation of matrix metalloprotease 9 and decreased formation of reactive oxygen species. Our findings imply two different mechanisms of action of intracerebrally and systemically transplanted NPCs, indicating that systemic NPC delivery might be more feasible for translational stroke concepts, lacking a need of in vitro manipulation of NPCs to induce long‐term neuroprotection. STEM CELLS2012;30:1297–1310

TAT-Bcl-xL improves survival of neuronal precursor cells in the lesioned striatum after focal cerebral ischemia

Cerebral ischemia activates endogenous neurogenesis in the subventricular zone (SVZ) and the dentate gyrus. Consecutively, SVZ-derived neural precursors migrate towards ischemic lesions. However, functional relevance of activated neurogenesis is limited by poor survival of new-born precursors. We therefore employed the HI-virus-derived fusion protein TAT-Bcl-x(L) to study the effects of acute anti-apoptotic treatment on endogenous neurogenesis and functional outcome after transient cerebral ischemia in mice. TAT-Bcl-x(L) treatment led to significantly reduced acute ischemic cell death (128+/-23 vs. 305+/-65 TUNEL+ cells/mm(2) in controls) and infarct volumes resulting in less motor deficits and improved spatial learning. It significantly increased survival of doublecortin (Dcx)-positive neuronal precursors (389+/-96 vs. 213+/-97 Dcx+ cells in controls) but did not enhance overall post-ischemic cell proliferation or lesion-specific neuronal differentiation 28 days after ischemia. Our data demonstrate that post-stroke TAT-Bcl-x(L)-treatment results in acute neuroprotection, improved functional outcome, and enhanced survival of lesion-specific neuronal precursor cells after cerebral ischemia in mice.

Transplantation of TAT-Bcl-xL-transduced neural precursor cells: long-term neuroprotection after stroke.

Neural precursor cells (NPC) are an interesting tool in experimental stroke research, but their therapeutic potential is limited due to poor long-term survival. We therefore in vitro transduced subventricular zone-(SVZ)-derived NPC with the anti-apoptotic fusion protein TAT-Bcl-x(L) and analyzed NPC survival, differentiation, and post-stroke functional deficits after experimental ischemia in mice. Survival of TAT-Bcl-x(L)-transduced NPC, which were injected at day 7 post-stroke into the ischemic striatum, was significantly increased at 4 weeks after stroke. Increased survival of NPC was associated with reduced infarct injury and decreased post-stroke functional deficits. Animals grafted with TAT-Bcl-x(L)-transduced NPC showed an increased number of immature cells expressing the neuronal marker doublecortin. Since mature neuronal differentiation of NPC was not observed, reduced post-stroke injury cannot be attributed to enhanced neuronal regeneration, but rather to indirect by-stander effects of grafted NPC. In line with this, NPC-mediated neuroprotection of cortical neurons in vitro was associated with increased secretion of growth factors. Thus, in vitro transduction of cultivated NPC with TAT-Bcl-x(L) results in enhanced resistance of transplanted NPC followed by long-term neuroprotection and ameliorated functional deficits after transient focal cerebral ischemia in mice.

Cellular prion protein promotes post-ischemic neuronal survival, angioneurogenesis and enhances neural progenitor cell homing via proteasome inhibition

Although cellular prion protein (PrPc) has been suggested to have physiological roles in neurogenesis and angiogenesis, the pathophysiological relevance of both processes remain unknown. To elucidate the role of PrPc in post-ischemic brain remodeling, we herein exposed PrPc wild type (WT), PrPc knockout (PrP−/−) and PrPc overexpressing (PrP+/+) mice to focal cerebral ischemia followed by up to 28 days reperfusion. Improved neurological recovery and sustained neuroprotection lasting over the observation period of 4 weeks were observed in ischemic PrP+/+ mice compared with WT mice. This observation was associated with increased neurogenesis and angiogenesis, whereas increased neurological deficits and brain injury were noted in ischemic PrP−/− mice. Proteasome activity and oxidative stress were increased in ischemic brain tissue of PrP−/− mice. Pharmacological proteasome inhibition reversed the exacerbation of brain injury induced by PrP−/−, indicating that proteasome inhibition mediates the neuroprotective effects of PrPc. Notably, reduced proteasome activity and oxidative stress in ischemic brain tissue of PrP+/+ mice were associated with an increased abundance of hypoxia-inducible factor 1α and PACAP-38, which are known stimulants of neural progenitor cell (NPC) migration and trafficking. To elucidate effects of PrPc on intracerebral NPC homing, we intravenously infused GFP+ NPCs in ischemic WT, PrP−/− and PrP+/+ mice, showing that brain accumulation of GFP+ NPCs was greatly reduced in PrP−/− mice, but increased in PrP+/+ animals. Our results suggest that PrPc induces post-ischemic long-term neuroprotection, neurogenesis and angiogenesis in the ischemic brain by inhibiting proteasome activity.

Early diffusion-weighted MRI predicts regional neuronal damage in generalized status epilepticus in rats treated with diazepam

We applied diffusion-weighted MRI (DWI) in the pilocarpine-induced status epilepticus (SE) model to investigate the evolution of acute phase changes in brain diffusion with and without early anticonvulsive therapy correlated to long-term SE-induced neuronal cell loss. Hereby, DWI was performed before (baseline) and serially between 3 and 120 min after onset of SE in untreated and treated animals (n=15 in each group). Anticonvulsive-treated animals received 20 mg/kg diazepam at 15 min after onset of SE. Apparent diffusion coefficients (ADC) were calculated for the parietal, temporal and piriform cortex, thalamus, hippocampus and amygdala and compared to baseline. Neuronal cell loss was quantified at 2 weeks after onset of SE utilizing cresyle-violet-staining. The results of ADC-mapping demonstrated a significant transient increase in ADC (to 116+/-4% of baseline) in the very acute phase starting 3 min after SE onset, lasting for 10 min in both groups. In untreated animals, there was a significant gradual decline in ADC to 75+/-12% of baseline while this decline in diazepam-treated animals was significantly less pronounced (P<0.05) and ADC recovered to 93+/-6% of baseline. There was good correlation between neuronal cell loss in specific brain regions at 2 weeks after SE and maximal decrease in ADC (r>0.79). In conclusion, serial DWI is a sensitive noninvasive technique for early detection, monitoring and prediction of SE-induced neuronal alterations. Using ADC-mapping, verification of early anticonvulsive therapy in SE seems to be possible as there is good correlation between the maximal decrease in ACD in the acute phase of SE and late neuronal cell loss.