228. Autologous bone marrow cell administration 24 h following MCAO reduces behavioral deficits and lesion size in a novel large animal model
J. Boltze1,2,3, A. Förschler4,5, H. Barthel6, B. Nitzsche1, A. Dreyer1, V. Zeisig1, T. Von Geymüller1, C. Boltze1, O. Sabri6, D. Lobsien5, A. Hoffmann7, A. Reischauer8, F. Emmrich2,3,9 and U. Gille10
1Neurorepair Research Group, Fraunhofer Institute for Cell Therapy and Immunology; 2Institute for Clinical Immunology and Transfusion Medicine; 3Translational Centre for Regnerative Medicine, Leipzig; 4Department of Neuroradiology, University Hospital rechts der Isar, Technical University of Munich, Munich; 5Centre for Diagnostic Radiology; 6University Clinic of Nuclear Medicine, University of Leipzig; 7Department of Anatomy, Histology and Embryology, Faculty of Veterinary Medicine, University of Leipzig; 8Institute for Veterinary Pathology, University of Leipzig; 9Fraunhofer Institute for Cell Therapy and Immunology; 10VITA34 Inc., Leipzig, Germany
Objectives and background: The purpose of our study was to evaluate the therapeutic efficacy of autologous bone marrow cell (BMC) application in the sub-acute stage of stroke in large animals by long term behavioral phenotyping and multimodal brain imaging. The model itself was designed on close approximation to the situation of human stroke patients, potentially providing enhanced translational power for experimental protocols including cell therapies.
The promising therapeutic potential of BMC therapies for stroke has recently been demonstrated in numerous rodent trials. As many experimental protocols, showing promising results in small animal studies, failed in clinical trials, transfer to clinical application requires close-to-practice large animal models of stroke. We evaluated benefit of autologous BMC transplantation 24 h upon stroke onset in a novel sheep model of focal cerebral ischemia which also allows for control of lesion size and subsequent functional deficits.
Methods: 30 adult rams weighting 51 to 104 kg were subjected to permanent middle cerebral artery occlusion (MCAO) for stroke induction. 100 mL of bone marrow were harvested from the iliac crest and BMCs were subsequently obtained by density centrifugation. A minimum of 4.0 × 10E6 cells gained per kilogram bodyweight (kgBW) was defined as inclusion criterion for cell treated subjects. Following baseline behavioral phenotyping, magnetic resonance imaging (MRI) and positron emission tomography (PET), 15 animals were randomly subjected to intravenous autologous nuclear BMC treatment 24 h after MCAO. 15 sheep served as controls. Functional outcome was continuously observed by behavioral phenotyping. Lesion size development and brain atrophy were monitored by MRI as well as 15O-water- and 18F-Deoxyglucose PET performed at days 14 and 42 before brains were removed for further histological investigation (which is actually still ongoing).
Results: In 4 animals, less than 2.0 × 10E6 cells per kgBW were obtained. Those subjects were excluded from the treatment group, but also monitored by MRI for 42 days. In BMC treated animals (n = 11), an enhanced functional improvement was observed as compared to control animals (P<0.01). Despite a spontaneous tendency of motorfunction improvement, non-treated animals suffered from moderate to severe motor and sensory dysfunctions like ataxia, absent startle reflexes and spatial hemineglect for the entire observation period. MRI investigations showed similar lesion size in both groups at day 1 (P = 0.59) and reduction of lesion size/hemispherical atrophy in cell treated rams 42 days upon MCAO (P<0.01), but not at day 14. These findings could be confirmed by 15O-water- and 18F-Desoxyglucose PET (P<0.05) and macroscopic pathological lesion volumetry (P<0.05). Interestingly, transplantation of less than 4.0x10E6 BMCs failed to induce lesion size reduction. No tumor formation was observed upon BMC administration.
Conclusions: Autologous BMC administration 24 h following stroke is safe and effective in sheep and might therefore be evaluated as a novel treatment option for stroke in upcoming clinical trials. The study revealed first indications that the therapeutic effect is related to a cell-dose-dependent neuroprotection. However, further examinations are required to elucidate the role of neural replacement and/or activation of the endogenous restorative potential of the brain in this approach.
543. Transplantation of human neural progenitor cells induces axonal plasticity in the host brain after stroke
R.H. Andres1, N. Horie1, G. Sun1, H. Shichinohe1, E. Mcmillan2, C.N. Svendsen1,2, B.T. Schaar1, T.M. Bliss1 and G.K. Steinberg1
1Department of Neurosurgery and Stanford Stroke Center, Stanford University, Stanford, California, USA; 2The Waisman Center, University of Wisconsin—Madison, Madison, Wisconsin, USA
Objectives: Cell transplantation therapy using human neural progenitor cells (hNPCs) has emerged as a promising new experimental treatment approach for stroke. However, many questions regarding the mode of action of the transplanted cells remain unanswered. In addition to directly replacing lost cells, transplanted hNPCs might enhance endogenous repair mechanisms that occur after cerebral ischemia. In the present study, we addressed the hypothesis that grafted hNPCs enhance axonal plasticity in the host brain after stroke and tried to elucidate the underlying mechanisms by functionally testing whether hNPCs secrete factors that enhance axonal plasticity in vitro.
Methods: hNPCs derived from the fetal cortex were grown as neurospheres. hNPCs or buffer were transplanted into the ischemic cortex of NIH Nude rats 7 days after distal middle cerebral artery occlusion (dMCAO). Five weeks post-transplantation, animals were injected with the anterograde axonal tracer biotinylated dextran (BDA) into the contralesional cortex and sacrificed 2 weeks thereafter. The extent of axonal sprouting towards the lesioned cortex from the contralateral side was quantified in different brain regions by confocal image analysis.
To investigate the underlying mechanisms of axonal plasticity in vitro, cortical and striatal progenitor cells were isolated from E14 rat embryos and grown as dissociated cultures. Indirect co-culture with hNPCs was performed during days 3 to 7 in vitro, and immunodepletion studies were carried out with either neutralizing antibodies against vascular endothelial growth factor (VEGF) and thrombospondin (TSP)-1/2, or Slit-neutralizing soluble ROBO-Fc chimeras. Species-matched antibody isotypes were used as controls. Axons were visualized by staining for neurofilament and axonal outgrowth was quantified using automated high-throughput image analysis software.
Results: hNPC-grafted rats showed significantly greater extension of BDA-labeled host axons from the contralateral hemisphere towards the damaged cortex after dMCAO than controls (P<0.05). This finding correlated with neurological recovery in the vibrissae-elicited forelimb placing test (P<0.05).
Indirect co-culture of cortical and striatal progenitor cells with hNPCs resulted in enhanced axonal outgrowth after 5 days in culture (P<0.01). This effect was partially abolished when neutralizing antibodies against VEGF or TSP-1/2 were present (P<0.01). Neutralization of Slit by exposure of the cultures to ROBO-Fc resulted in similar effects (P<0.01).
Conclusions: In sum, our findings suggest that transplanted hNPCs significantly enhance axonal rewiring from the contralesional side after stroke. VEGF, TSP-1/2, and Slit were identified as at least partially responsible mediators of these effects in vitro. Understanding how transplanted hNPCs augment host brain plasticity might help us to improve cell transplantation approaches for stroke.
522. Distinct advantages of recombinant human versus plasma derived C1-inhibitor in brain ischemic injury
E.R. Zanier1, R. Gesuete1, F. Orsini1, C. Storini1, A. Fantin1, M. Stravalaci1, H. Vietsch2, B. Ziere2, M.L.M. Mannesse2, M. Gobbi1 and M.G. De Simoni1
1Mario Negri Institute, Milano, Italy; 2Pharming Technologies B.V, Leiden, The Netherlands
Background and aims: C1-inhibitor (C1-INH) is an endogenous multifaced anti-inflammatory molecule, known to act as inhibitor of complement and kinin systems. We have previously shown that C1-INH has potent neuroprotective properties in mouse models of cerebral ischemia and traumatic brain injury, where plasma-derived (pd) C1-INH attenuated acute neurobehavioral deficits, anatomical damage, and neurodegeneration. However pdC1-INH had a narrow therapeutic window, since no protective effects were observed when it was administered 1 h after the onset of acute brain injury.1–5
Methods: We evaluated the effects of the recently available recombinant human C1-INH (rhC1-INH), administered intravenously to C57Bl/6 mice undergoing both transient and permanent ischemia, on infarct volume and neurodegeneration. We then compared the localization of rhC1-INH and pdC1-INH in the ischemic brain tissue by immunohistochemistry and confocal analysis and evaluated their binding profiles using Surface Plasmon Resonance (SPR). Having found a selective binding of rhC1-INH to MBL we explored the susceptibility of mannose binding lectin (MBL) deficient mice to transient ischemia.
Puropose: To explore the efficacy and the therapeutic window of rhC1-INH and to investigate its possible mechanism of action in comparison with that of pdC1-INH.
Results: rhC1-INH markedly reduced cerebral damage when administered up to 18 h from the beginning of transient ischemia and up to 6 h after permanent ischemia, thus showing a surprisingly wide therapeutic window. We found that rhC1-INH was confined to cerebral vessels while pdC1-INH diffused into the brain parenchyma. Furthermore rhC1-INH, which has a different glycosylation pattern, showed a different binding profile. In particular, rhC1-INH, but not pdC1-INH, binds MBL with high affinity thus revealing a novel target of C1-INH action. Finally a significantly lower susceptibility to ischemic damage was observed in MBL−/− compared to WT mice.
Conclusions: rhC1-INH, which possesses a distinct glycosylation pattern and binding profile, is superior to the corresponding plasmatic protein in the protection against cerebral injury. This may possibly be due to a direct binding to MBL deposited on endothelial cells early after ischemia thus preventing lectin-complement pathway activation and providing a long lasting protection towards ischemic damage. The surprisingly wide time-window of efficacy of rhC1-INH represents a crucial aspect for the possible therapeutic use of this drug in the treatment of stroke. Noteworthy, C1-INH is already used in humans for the therapy of C1-INH deficiency.
1072. Perlecan domain V improves stroke outcome
D. Clarke, B. Lee and G.J. Bix
Texas A&M College of Medicine, College Station, Texas, USA
Stroke is a significant world-wide cause of death and serious long-term disability. However, while advances have been made in acute stroke treatment, our understanding of the mechanisms underlying brain self-repair after stroke remains poor. Therefore, the problem of brain repair and stroke rehabilitation is an emerging research priority, with the underlying goal of identifying and improving brain reparative processes. Brain repair occurs in a close temporal-spatial neurovascular niche of revascularization (angiogenesis) and neuronal repopulation (neurogenesis), processes that both involve extracellular matrix (ECM) remodeling and subsequent generation of ECM fragments.
Objectives: We sought to demonstrate whether one of these ECM fragments, perlecan domain V (DV), could improve functional stroke outcome by stimulating angiogenesis, neurogenesis and neurovascular niche formation. Our objectives were based on previous observations that stroke rapidly generates bioactive fragments of perlecan,1 and perlecan is required for both angiogenesis and neurogenesis.2
Methods: Rats underwent left middle cerebral artery occlusive stroke, or sham surgery control, via stereotactic injections of endothelin-1 (or PBS vehicle control)3 followed by intraperitoneal injections of human recombinant DV (0.5 mg/kg) or PBS vehicle control on post-stroke days 1, 3, 5 and 7 (n = 5 for each group, experiment repeated 3 times). Functional use of the affected contralateral limb was assessed via the cylinder test4 and groups were statistically analyzed via ANOVA. Additionally, rat brain tissue was immunohistochemically processed to detect administered DV, as well as to measure post-stroke angiogenesis and neurogenesis. In parallel experiments, the effects of DV on angiogenesis and neurogenesis were studied in vitro with isolated rat brain microvascular endothelial cells, cortical neurons and neurospheres, respectively.
Results: From post-stroke day 3 onward, stroked rats treated with a total of 2 doses or more of DV were indistinguishable (P = 0.3) from sham surgery controls in the spontaneous use of the contralaterally affected limb, while untreated stroked rats remained significantly impaired throughout testing (P<0.005). Brain immunohistochemistry demonstrated that administered DV specifically homed to the ischemic core and peri-infarct cortex, depositing in a perivascular distribution, and increased both peri-infarct angiogenesis and neurogenesis. Additionally, a significant majority of these newborn neurons (P<0.001) were closely associated with new blood vessels where administered DV deposited. In vitro studies further demonstrated that DV enhanced several aspects of angiogenesis including endothelial cell proliferation, migration and tube formation. Likewise, DV enhanced both neuronal migration and neurosphere formation.
Conclusions: In a stroke animal model, perlecan DV homes to stroked brain tissue and improves functional stroke outcome by enhancing post-stroke angiogenesis and neurogenesis. Our results suggest that DV could be a promising new stroke therapy.
689. Impact of genetic variant of BDNF (Val66Met) polymorphism on stroke-induced neovascularization
L. Qin1, R. Ratan1,2, E. Kim1, F. Lee2 and S. Cho1,2
1Burke Medical Research Institute, White Plains; 2Weill Cornell Medical College, New York, New York, USA
Objective: The extent of angiogenesis affects stroke outcome and recovery. Brain derived neurotrophic factor (BDNF), a widely expressed neurotrophin in the mammalian nervous system, plays a key role in angiogenesis. BDNF is shown to promote endothelial cell survival and revascularization in ischemic limbs and newly described as a mediator of angiogenesis in ischemic tissue.1 Recently, a common single nucleotide polymorphism (SNP) in the pro-domain of the bdnf, that leads to a methionine (Met) substitution for valine (Val) at codon 66 (Val66Met) has been identified in human. The mutation is associated with altered brain anatomy and memory and deficits in regulated BDNF secretion.2,3 By using mice with genetic knock-in of the humanized bdnf variant in both allele (BDNFMet/Met) and wild type (BDNFVal/Val), this study investigates the impact of genetic variant of BDNF (Val66Met) polymorphism on stroke outcome and stroke-induced neovascularization.
Methods: Male BDNFMet/Met and BDNFVal/Val (10 to 12 week old) mice were subjected to 30 mins middle cerebral artery occlusion (MCAO) using an intraluminal thread method.4 Mice were killed 3/7 days after stroke. Brain sections were used for quantification of infarct volume. Enzyme-linked immunoadsorbant assay (ELISA) was used to evaluate the protein level of BDNF. Double fluorescence immunohistochemistry of CD31 (endothelial cell marker) and Ki-67 (cell proliferation marker) was performed to visualize proliferating endothelial cell (neovascularization).
Results: Infarct volume (IV) and hemispheric swelling assessed 3 days after ischemia revealed no statistical difference between the strains (BDNFVal/Val versus BDNFMet/Met, IV: 36.9±4.2, 27.7±5.4 mm3; Swelling: 11.5±2.7, 7.9±3.5%, n = 9 to 15). However, stroke-induced BDNF secretion was significantly reduced in BDNFMet/Met brain at both 3 and 7 days post-ischemia (BDNFVal/Val versus BDNFMet/Met: 3d, 79.8±19.3, 42.8±14.6 pg/mg. 7d, 52.5±19.3, 30.9±8.8 pg/mg. n = 5 to 10). The decreased BDNF secretion in BDNFMet/Met mice was associated with less neovascularization at 3 days post-ischemia (See figure, BDNFVal/Val versus BDNFMet/Met, 13.6±2.2, 8.2±1.7 cell/mm2).
Conclusion: The data demonstrate that stroke-induced BDNF secretion is not involved in the acute infarct development and BDNF secretion deficits in BDNFMet/Met mice is associated with reduced stroke-induced neovascularization. The study implicate translational approach that aim at reversing BDNF secretion deficits to promote stroke-induced angiogenesis. (Supported by NIH HL82511 and Burke Foundation).
450. Influence of chronic arterial hypertension on the evolution of perfusion-diffusion mismatch
A. Letourneur, S. Roussel, J. Toutain, M. Bernaudin and O. Touzani
UMR CI-NAPS 6232, CERVOxy Team ‘Hypoxia and Cerebrovascular Pathophysiology’, CNRS, CEA, Université de Caen Basse-Normandie and Université Paris Descartes, Caen, France
Objectives: Chronic arterial hypertension (CAH) increases the risk of stroke as well as the severity of the resultant lesion. In spite of this fact, arterial hypertension is rarely taken into consideration during preclinical investigations. To better define the therapeutic window in hypertensive subjects, the aim of our study was to analyse the impact of CAH on the spatio-temporal evolution of the ischemic lesion in the acute phase. Special attention was paid to the ischemic penumbra defined by the mismatch between perfusion and diffusion as visualised by magnetic resonance imaging (MRI).
Methods: Six sequential MRI examinations (7 Teslas, Bruker) were undertaken from 30 mins up to 4 h after intraluminal permanent Middle Cerebral Artery Occlusion (MCAO) in isoflurane-anaesthetised spontaneously hypertensive rats (SHR, n = 11) and their normotensive control rats (WKY, n = 12). Mean arterial pressure (MAP), heart rate, and blood gases were monitored throughout the experiment.
In each MRI session, Apparent Diffusion Coefficient (ADC) maps were generated based on echo planar images (Diffusion Weighted Imaging (DWI), repetition time (TR) = 3500 ms, echo time (TE) = 40.53 ms, matrix = 128 × 128). Perfusion Weighted Imaging (PWI) examinations have been realized using the first pass of a gadolinium chelate (Gradient Echo EPI; TR = 500 ms, TE = 10.32 ms, 120 repetitions, matrix: 256 × 256). The integrity of the blood brain barrier (BBB), the occurrence of haemorrhage and the status of major cerebral arteries were also imaged sequentially with specific sequences (respectively T1, T2*, Angiography). Animals were euthanized 24 h later and infarct volume was measured.
Results: MAP, as measured in awake rats, was higher in SHR than in WKY (170±11 mm Hg; 130±18 mm Hg; P<0.001). This difference in blood pressure was also seen in the anesthetised rats during MRI examination.
MRI studies showed that the ischemic lesion, defined by a significantly decreased ADC relative to the mean value of the contralateral hemisphere, was significantly larger in hypertensive rats than in normotensive ones at all the time points analysed (ANOVA followed by Fischer PLSD (P<0.005 at each time)). Analysis of the mismatch between perfusion and diffusion interestingly showed that WKY displayed a significant penumbra up to 240 mins post occlusion, whereas in SHR, the penumbra disappeared as soon as 60 mins following the occlusion (Figure). MRI angiography confirmed MCAO in all animals. Neither major BBB disruption nor haemorrhage was observed irrespective of the strain studied.
Conclusion: Hypertensive rats present larger lesions and a rapid disappearance of the penumbra compared to normotensive ones. This suggests that even if a treatment is precociously administered, the ischemic damage will remain greater in hypertensive subjects. This supports the integration of CAH in pre-clinical studies relative to the treatment of stroke.
Representation of the diffusion-perfusion mismatch in a representative animal of each strain at 30 mins post MCAO.
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