Brain Poster Session: Angiogenesis

52. Brain probes for MRI of de novo neural progenitor cells in live brains after global cerebral ischemia

P.K. Liu¹, Z. You² and C.H. Liu¹

¹Massachusetts General Hospital/Harvard Medical School; ²Pediatrics, MGH/HMS, Charlestown, Massachusetts, USA

Cardiac arrest induces global cerebral ischemia (GCI) and hypothermia is the only treatment to reproducibly reduce neurological deficits after cardiac arrest. Gliogenesis, revascularization (angiogenesis), and neurogenesis are three major events thought to contribute to brain repair. The interaction of these three processes after GCI in the brain is not totally understood. Gene expression at the transcription level during brain repair that follows GCI may be related to metabolic response and plasticity, and all involve the presence of neural progenitor cells (NPC); therefore, understanding the changes that occur throughout the repair process will aid translation of gene targeting for therapies that will benefit patients. However, detection of de novo NPC in the brain is not routinely performed clinically because the techniques used to track de novo NPC rely on the use of biopsy or autopsy samples. The biopsy procedure to obtain brain tissue severely limits the utility of these methods because they remove the same cells that we wish to save in vivo, and often clearly precludes longitudinal therapeutic evaluation. To overcome these problems, we developed an alternative method that uses brain probes for magnetic resonance imaging (MRI); this novel method provides a powerful and less invasive means of in vivo detection of gene action in brain cells. Superparamagnetic iron oxide nanoparticles (SPION, a T₂ susceptibility MR contrast agent) are linked to phosphorothioate-modified antisense oligodeoxynucleotides (sODN) to target endogenous gene transcripts of astroglia in live C57black6 mouse brains. Because sODN are charged molecules, they are taken up by the brain cells along with SPION; sODN retention is determined by sequence homology with cellular mRNA targets. By virtue of its coupling with the complementary sODN, SPION retention in living brains can thus imaged by MRI. We induced GCI by transient bilateral carotid occlusion for 60 min. We detected abnormal water diffusion by way of hyperintense diffusion-weighted imaging and calculated significant reduction using the apparent diffusion coefficient (rADC) at one day of reperfusion. BBB leakage was detected in the region of rADC at four weeks after GCI. We detected angiogenesis using MR probe targeting cells with high actin gene transcript in the 8th week, and gliosis using MR probe targeting astroglia in the 9th week post GCI. We found gliosis and angiogenesis in close and adjacent regions, but not in the same cells. The MRI data were confirmed by immunohistology in the 10th week. Nestin antigen was found in microvasculature that expressed Actin, but not in GFAP-expressing astroglia. This finding is consistent with the presence of pericytes during angiogenesis in the brain. This method has applications for cell typing based on specific mRNA and exclusion of unbound probe by living cells in vivo, and has potential for translation from bench to bedside application. These brain probes can detect various cell types before enough proteins are translated to enable detection using antibodies. Supported by NINDS (R01NS45845; R21NS057556), NIA (R21DA 024235), NCRR (P41RR14075).

338. Distribution of transfused bone marrow cells following endothelial damage of a cortical artery

S. Yamada, H. Toriumi, H. Kimura, Y. Tomita, Y. Itoh, H. Hoshino and N. Suzuki

Department of Neurology, Keio University School of Medicine, Tokyo, Japan

Objective: Bone marrow cells (BMCs) are reported to supply ‘endothelial progenitor cells’ as well as possible microglia and may be beneficial in treatment of cerebral infarction. To investigate possible repair function of BMCs transfused after endothelial damage in a cerebral artery, endothelial cells in a cortical artery were selectively injured and recruitment of BMC at the injured site was continually observed through cranial window in mice.

Methods: Endothelial injury was inflicted in a cortical branch of middle cerebral artery via photochemical reaction between systemically injected rose bengal and green laser light (wavelength: 540 nm, diameter: 150 micro m, power: 150 micro W) transilluminated through a cranial window for 150 sec. BMCs were prepared from a C57BL/6 CAG-EGFP transgenic mouse, which express green fluorescent protein (GFP) under the control of a chicken beta-actin promoter and cytomegalovirus enhancer in all of the tissues except for erythrocytes and hair. The femurs and tibias were flushed with Hanks' buffered salt solution and BMCs were isolated with a separation medium (Lympholyte M). BMCs were transfused through tail vein 3 h after endothelial damage. Distribution of BMCs was periodically observed through a cranial window with a confocal microscopy. Binding of fluorescein isothiocyanate (FITC)-labeled Ulex europaeus agglutinin 1 (UEA-1) lectin was used to identify endothelial cells.

Results: Vasospasm as well as thrombus formation was observed at the injured artery following rose bengal infusion/laser illumination, suggesting endothelial damage. Hemostasis was observed in cortical vessels peripheral to the injured artery. Major thrombus was degraded within several hours, starting recirculation in most cortical vessels. Immediately after transfusion, BMCs circulating through the cortical brain vessels was observed via cranial window. Some BMCs attached on the cortical vein and migrated to the parenchyma as well as to the subarachnoid space (Figure). The upper cell migrated through a vessel wall, whereas the lower cell stayed on the luminal surface. (Movie will be presented.) Most of the BMCs in the parenchyma were later identified as GFP/Iba-1 double-positive microglia by immunohistochemistry. Some cells stayed on the luminal surface of the cortical vein and elongated toward the direction of blood flow. In contrast, only a few GFP-positive cells adhered to the injured cortical artery. Circulating BMCs were observed as late as 7 days after infusion.

Conclusion: These results suggest that BMC transfused after endothelial damage does not contribute to endothelial repair, at least within 7 days. In contrast, most BMCs trapped in the brain vessels may migrate into the parenchyma and function as microglia.

364. The insight of revascularization mechanism based on angiogenesis and arteriogenesis from the experimental and clinical works in moyamoya disease

H. Imai¹, M. Nakamura¹, C. Kubota¹, S. Puentes¹, A. Faried¹, Y. Yoshimoto¹ and N. Saito²

¹Department of Neurosurgery, Gunma University, Maebashi; ²Department of Neurosurgery, Tokyo University, Tokyo, Japan

Objectives: We focused on unveiling the mechanism of revascularization after indirect bypass surgery, encephalo-myo-synangiosis (EMS) in moyamoya disease, which is progressive occlusive cerebrovascular disease of the internal carotid arteries (ICAs). This treatment is believed to form vascular anastomoses between the extracranial tissue and the brain to maintain blood flow to the ischemic tissue. However, the underlying mechanism is not completely understood. Research has concentrated on both experimental and clinical works for revascularization mechanism in terms of two types of vessel growth, angiogenesis and arteriogeneis.

Methods: Experiments: Fourteen miniature pigs underwent transcranial surgery for EMS after ICA occlusion. Animals were allowed to recover at 1 week (n = 4) or 4 weeks (n = 7) after EMS. Control group animals were treated in the same way without occlusion (n = 3). MR imaging, angiography, and histopathology were performed. Patients: Total 29 hemispheres of 23 patients with moyamoya disease who were performed with angiography after direct or indirect bypass surgery were enrolled for the investigation. The eventual patterns of revascularization were categorized into 4 classes based on the dominancy of donor arteries.

Results: Experiments. One week after EMS, histopathology of both ICA occlusion and control groups showed the transplanted temporal muscle had adhered to the arachnoid via the fibrous coat where a number of newly formed small vessels (angiogenesis) in connective tissue were found. Four weeks after EMS, angiography and histopathology of ICA occlusion group showed the vascular anastomoses grew larger and more robust to construct patent anastomoses (arterogenesis) between the external carotid artery and the cortical arteries. In contrast, histopathology of the control group found cicatrized tissue of the fibrous coat. Patients: In cases of direct anasomosis, there are 4 hemispheres with dominant superficial temporal artery (STA) compared with EMS (STA>EMS), 7 hemispheres with dominant EMS compared with STA (EMS>STA). 5 hemispheres with nearly equal dominancy of both STA and EMS (STA = EMS), and 1 hemisphere without any anastomoses. In cases of indirect anastomosis, There are no hemisphere with (STA>EMS), 5 hemispheres with (EMS>STA), and 7 hemispheres with (STA = EMS).

Conclusions: The experiments disclosed functional revascularization for EMS required 2 distinct processes, angiogenesis and arteriogenesis. ¹ The angiogenesis is the initial step to generate the new vessels in the newly formed connective tissues, which resembles the process of wound healing associated with repair processes. However, the next step, arteriogenesis required specific and critical condition such as hypoperfusion of the cortex which boosts net forward flow from the donor artery to recipient pial artery due to the pressure gradient between the interconnecting arterial networks. Also clinical results showed EMS provide compensative blood supply via revascularization even in case of direct bypass as well as in indirect bypass surgery. EMS is reasonable surgical treatment for moyamoya disease because flexible revascularization developed adequately according with the requirement of blood supply in the ischemic lesion.

751. Role of soluble epoxide hydrolase in post-ischemic angiogenesis

W. Zhang, M. Grafe, J. Palmateer, P. Herson and N. Alkayed

Oregon Health & Sciences University, Portland, Oregon, USA

Objectives: Ischemia induces cerebral angiogenesis, which is part of a vascular remodeling response that necessary for regeneration and functional recovery. The mechanisms of post-ischemic angiogenesis remain unknown. Epoxyeicosatrienoic acids (EETs), P450 eicosanoids produced in brain by astrocytes, play an important role in blood flow regulation and protection after cerebral ischemia, and have been shown to exhibit angiogenic properties in multiple in vitro models of angiogenesis. EETs' actions are terminated by soluble epoxide hydrolase (sEH). We here tested the hypothesis that sEH gene deletion promotes post-ischemic angiogenesis and functional recovery after focal cerebral ischemia in mice.

Methods: Mice with targeted deletion of sEH (sEH knockout, sEHKO) and their wild type (WT) littermates were subjected to 45 min of middle cerebral artery occlusion (MCAO), followed by 8 days of recovery. A battery of somatosensory and cognitive neurobehavioral tests were performed during recovery. At the end of the experiment on day 8, mice were perfused, and brains were fixed, sections and stained for CD34, which identifies endothelial cells, as well as endothelial progenitor cells. Unbiased, stereology-based estimates of vascular density were obtained from the density of CD34-positive vascular profiles in these brain sections using computer-assisted optical dissector probe.

Results: Microvascular density was numerically higher in sEHKO compared to WT mice both within infarct zone (272±50 versus 239±31 profiles/mm², mean±sem), and contralateral cerebral cortex (262±40 versus 181±26, n = 4 per group).

Conclusion: The preliminary results are consistent with higher post-ischemic angiogenic capacity in sEHKO versus WT mice, consistent with higher EETs-mediated angiogenesis. Ongoing studies will determine if increased vascular density is associated with improved functional recovery.

946. Sympathetic denervation differentially modulates direct trophic effects of VEGF on contractile differentiation in ovine fetal middle cerebral arteries

S. Butler, J. Abrassart, J. Williams and W. Pearce

Center for Perinatal Biology, Loma Linda University School of Medicine, Loma Linda, California, USA

Objectives: Perivascular nerves dramatically influence the growth and functional maturation of cerebral arteries, ¹ but the mechanisms involved remain uncertain. Norepinephrine (NE) released from perivascular nerves may directly stimulate the gene transcription and protein synthesis governing vascular maturation. ³ Alternatively, patterns of vascular activation may predominantly influence cerebrovascular development, as predicted by the ‘excitation-transcription’ hypothesis; ² NE release could indirectly ‘prime’ arterial reactivity to growth factors, such as VEGF, which in turn could regulate transcription. The present study tested this hypothesis.

Methods: In fetal lambs at 124d gestation (term≈143d) the left superior cervical ganglion was preganglionically denervated. After denervation the fetuses were returned to the womb and maintained for 14 days, at which time their middle cerebral arteries were harvested.

Contractile characteristics of intact and denervated fetal middle cerebral arteries were determined immediately after sacrifice, and after 48 h of organ culture. In organ culture, arteries were serum starved in DMEM with: 30 ng/ml VEGF; 10 μmol/L NE; 30 ng/ml VEGF plus 10 μmol/L NE; or no additions (control). The contractility protocol measured myogenic stresses (dynes/mm²), active stresses (120 mmol/L potassium), and passive stresses (5 mmol/L EGTA) at graded stretch. Young's Modulus was calculated to quantitate artery stiffness. After contractility measurements, segments were sectioned and imaged following fluorescent immunohistochemical staining for smooth muscle α-actin and Myosin Light Chain Kinase to identify smooth muscle phenotypic changes.

Results: Sympathectomy generally had opposite effects on VEGF- and NE-induced changes in culture. It enhanced VEGF-induced loss of stiffness, but enhanced NE-induced increases in stiffness. Sympathectomy enhanced loss of myogenic tone during serum starvation, eliminated VEGF-induced increases in myogenic tone but enhanced NE-induced increases. Sympathectomy enhanced active tone after serum starvation, enhanced VEGF-induced depression of active tone, and attenuated NE-induced increases in active tone. The effects of VEGF and NE on stiffness, myogenic tone, and active tone were additive in control arteries, but competitive following denervation. Imaging revealed that serum starvation enhanced smooth muscle proliferation and contractile dedifferentiation, and that these effects were amplified by VEGF but attenuated by NE in control arteries. Sympathectomy further enhanced these competitive effects of VEGF and NE on smooth muscle phenotype.

Conclusion: These results demonstrate that perivascular NE release stimulates contractile maturation and increased stiffness in cerebral arteries though both direct effects on contractile protein expression, and through indirect inhibition of VEGF-induced proliferative influences. Because NE-mediated influences were interrupted by preganglionic denervation, physiological patterns of perivascular NE release appear critical for normal vessel development; this helps explain why attempts to restore trophic effects of NE by readdition have been unsuccessful. These results also suggest that both the direct effect ³ and ‘excitation-transcription’ ² hypotheses apply to cerebrovascular maturation. These findings have important consequences for neonates with cerebrovascular injury sufficient to compromise the perivascular adrenergic innervation, and thus the normal development of cerebral artery structure and function.

1066. F-18 FDG PET in Parkinson's syndrome patients with urinary dysfunction

K.H. Hwang, W. Choe and M.K. Lee

Nucl Med, Gachon Univ. Gil Hosp, Incheon, South Korea

Introduction: Recently, it was reported that a relationship between dopaminergic degeneration and symptoms of urinary dysfunction. We investigated the difference in cerebral metabolism between Parkinson's syndrome (PS) patients with and without urinary dysfunction.

Methods: This study included 7 PS male patients without urinary dysfunction (mean age: 71 yr) and 3 PS patients with urinary dysfunction (mean age: 75 yr) who performed F-18 FDG Brain PET. Using SPM2 (Statistical parametric Mapping 2, Wellcome Department of Cognitive Neurology, London, UK) software, all the images were spatially normalized into the standard template, smoothed with 10 mm FWHM isotropic Gaussian kernel. The count of each voxel was normalized versus the total count for the brain (proportional scaling in SPM) to remove global cerebral metabolic differences between the individuals. Images of PS patients with urinary disturbance were compared with those of PS patients without in a voxel wise manner using SPM2 in group-to-group analysis (uncorrected P<0.01).

Results: Decreased regional cerebral metabolism was demonstrated in bilateral superior cerebellar cortices and Rt parietal cortex in Parkinson's disease patients with urinary dysfunction, compared to those without. These findings are consistent with some study reports,^1,2 suggestive of connection of those areas to pontine micturition center.

Conclusions: Although the number of patients studied is small, these results suggest that urinary dysfunction in patients with Parkinson's disease may be associated with bilateral Cerebellum and Rt Parietal cortex.