Current Neurovascular Research (v.10, #2)

The pigmented ciliary epithelium (PCE) of mammalian eye harbors resident population of stem cells that lie inapposition with endothelial cells which release vascular endothelial growth factor (VEGF) that may influence the fate andfunction of these stem cells in ways that remain unclear. We examined the role of VEGF in proliferation of PCE stemcells and expression of Notch, Jagged, N-Cadherin and ?-Catenin which are known to maintain proliferation state ofneural stem cells. We cultured human PCE cells obtained from 12-20 weeks old fetal eyes. The neurospheres wereanalyzed for the proliferation capacity of PCE stem cells in presence of VEGF on 3,6 and 9 day. Real time PCR was usedto quantitate the mRNA expression of above mentioned genes on PCE derived neurospheres on 3,6 and 9 day. We foundincreased number of neurospheres when PCE stem cells were stimulated with VEGF alongwith epidermal growth factor(EGF) and basic fibroblast growth factor (bFGF) than EGF and bFGF. BrdU immunostaining was done to analyze theproliferation of CE cells and presence of neural and retinal progenitor markers such as Nestin and Pax6 were alsoinvestigated. An increased Notch and Jagged mRNA was observed on 6<sup>th</sup> day in VEGF, EGF and bFGF treated PCE cellsas compared to 0,3 and 9 day. A similar pattern was noticed with N-cadherin and ?-catenin mRNA levels. These findingsmay clarify the role of VEGF on PCE stem cell proliferation with possible involvement of Notch, Jagged, N-cadherin and?-Catenin. The data may suggest importance of harvesting 6<sup>th</sup> day neurospheres for transplantation purposes in preclinicalinvestigations pertaining to retinal degenerative diseases, however, additional studies are needed to substantiate thefindings.

Human Mesenchymal Stem Cells Increases Expression of α-Tubulin and Angiopoietin 1 and 2 in Focal Cerebral Ischemia and Reperfusion by Xue-ling Ma, Kang-ding Liu, Fu-chun Li, Xin-mei Jiang, Lai Jiang, Hu-lun Li (103-111).
Angiogenesis is associated with improved neurologic recovery after cerebral ischemia. Human bone marrowmesenchymal stem cells (hMSCs) have been successfully used to treat ischemic stroke and were shown to induce theexpression of a number of neurotrophic factors including VEGF, epidermal growth factor (EGF) and basic fibroblastgrowth factor (bFGF) in a rat middle cerebral artery occlusion (MCAO) ischemia model. In this study, we aimed tounderstand the mechanism underlying the improvement of neurological function following hMSCs transplantation intoMCAO rats. We established a rat MCAO model and used immunofluorescence to evaluate &#945;-tubulin expression in thehippocampus. We used RT-PCR to determine the expression of Ang-1 and Ang-2 mRNAs after transplantation of hMSCsinto MCAO rats. We showed a significant decrease in &#945;-tubulin expression in rats with cerebral ischemia, suggesting that&#945;-tubulin is a protective protein in cerebral ischemia Transplantation of hMSCs significantly upregulated &#945;-tubulin levelsin the hippocampus. Transplantation of hMSCs also resulted in a significant upregulation of Ang-1 and Ang-2 mRNAs inMCAO rats. Ang-2 expression was upregulated earlier than Ang-1, suggesting that 1) transplantation of hMSCs promotesangiogenesis and that 2) Ang-2 may be an initiator of angiogenesis. Our results provide a theoretical basis for thetherapeutic use of hMSCs in cerebral ischemia.

Increased Neuronal Injury in Clock Gene Per-1 Deficient-Mice after Cerebral Ischemia by Nina Wiebking, Erika Maronde, Abdelhaq Rami (112-125).
Transient, severe global ischemia that arises in humans as a consequence of cardiac arrest or cardiac surgery orthat is induced experimentally in animals, leads to selective and delayed neuronal death, particularly in the hippocampus.Especially, in this brain structure, clock genes are rhythmically expressed, for instance the inducible and archetypicalclock gene is Period1 (Per1). An eventual involvement of its trans-activating protein products in the daytime-dependentseverity of ischemia-induced cell damage is not excluded. Probably, neurons may exhibit endogenously a daytimedependentvariation in the expression of predictive cell death proteins. We therefore compared the cell death machinery inthe hippocampus between Per1<sup>-/-</sup>- and wildtype (WT) mice upon cerebral ischemia. Neuronal death in the hippocampalCA1-subfield, was observed in both types of mice, but the density of damaged cells in Per1<sup>-/-</sup>-mice was increased bymore than 23% as compared to wildtype mice. To explore the mechanisms underlying the excessive vulnerability of thehippocampus in Per1<sup>-/-</sup>-mice and to address if hippocampal susceptibility inherits a daytime component, the expression ofboth, apoptotic and autophagic predictors of cell death was monitored. In Per1<sup>-/-</sup>-mice, the expression ofapoptotic/autophagic markers are altered and higher levels of the proapoptotic factors such as cytochrome c and Apaf-1were observed as compared to WT mice. Moreover, the autophagy marker LC3B was dramatically reduced in Per1<sup>-/-</sup>-mice. Our data suggests that basal activities of apoptosis and autophagy seem to be modulated by PER1, and that theautophagic machinery is probably slowed down when this clock gene is absent. These alterations may be causal for theobserved innate vulnerability of Per1<sup>-/-</sup>-mice to cerebral ischemia.

Functional Effects of Levosimendan in Rat Basilar Arteries In Vitro by Juergen Konczalla, Jan Mrosek, Stefan Wanderer, Patrick Schuss, Erdem Guresir, Volker Seifert, Hartmut Vatter, Johannes Platz (126-133).
Levosimendan is a novel calcium sensitizer that is an established treatment for congestive heart failure. Incoronary vessels, levosimendan has a vasorelaxant, endothelium-independent effect and an antagonistic effect onendothelin-1 (ET-1). There is also some data for a neuroprotective effect in a traumatic brain injury model, andlevosimendan can prevent the reduction of the luminal area of the basilar artery. We considered that patients who sufferheart attack after subarachnoid hemorrhage (SAH) might respond well to levosimendan, which might also be useful toinduce hypertension in patients with cerebral vasospasm.

However, the functional effects of levosimendan in the cerebrovasculature are unknown. Here, we investigated thefunctional role of levosimendan on rat basilar artery by assessing vasocontractile reactivity in response to ET-1,sarafotoxin S6c, acetylcholine, sodium nitroprusside, cGMP, and prostaglandin F2? (PGF).

Contrary to observations in coronary vessels, levosimendan did not affect the ET-1 system in cerebral arteries; neitherET(A)-receptor-induced contraction nor ET(B)-receptor-dependent relaxation were changed. For the nitric oxide (NO)pathway, only a slight increase was detected. Rather, levosimendan caused significant and dose-dependent relaxation afterPGF precontraction.

To our knowledge, this is the first report that describes levosimendan-induced functional changes of cerebrovascularcontractility and relaxation. Under physiological conditions, levosimendan did not influence ET(A)/ET(B)-receptorsignaling or the NO pathway. Interestingly, levosimendan seemed to affect the prostaglandin system and dosedependentlyreversed PGF- induced contraction. We did not detect a vasospastic potential for levosimedan in cerebralarteries, suggesting that it would be safe for use in SAH patients.


The Neurovascular Protection Afforded by Delayed Local Hypothermia after Transient Middle Cerebral Artery Occlusion by Jong-Heon Kim, Minchul Seo, Hyung Soo Han, Jaechan Park, Kyoungho Suk (134-143).
Therapeutic hypothermia is a robust therapeutic tool in experimental stroke models but its clinical applicationsare limited. Furthermore, optimal conditions for therapeutic hypothermia, such as, temperature and the initiation andduration of cooling must be individualized. Here, we evaluated the therapeutic effects of delayed local hypothermia,administered for 44 hr after 4 hr of reperfusion in a rat model of transient middle cerebral artery occlusion (tMCAo), usinga cooling device that allowed controlled local hypothermia (31

Soy Isoflavone Alleviates A?1-42-Induced Impairment of Learning and Memory Ability Through the Regulation of RAGE/LRP-1 in Neuronal and Vascular Tissue by Yuan-Di Xi, Xiao-Ying Li, Juan Ding, Huan-Ling Yu, Wei-Wei Ma, Lin-Hong Yuan, Jian Wu, Rong Xiao (144-156).
The neuroprotective properties of soy isoflavone (SIF) have been demonstrated by our previous studies andothers, but its potential mechanism is not clear. Because of the key role of neurovascular dysfunction in the pathogenesisof Alzheimer&#039;s disease (AD), we hypothesized neurovascular tissue might be one neuroprotective target of SIF. In thepresent study, learning and memory ability, ?-amyloid (A?) expressions both in neurovascular tissue and plasma, thereceptor for advanced glycation end products (RAGE), low-density lipoprotein receptor-related protein (LRP)-1, nuclearfactor-?B p65 (NF-?B p65), tumor necrosis factor-? (TNF-?) and interleukin-1? (IL-1?) expressions in neurovasculartissue were measured in Wistar rats following lateral cerebral ventricle administration of A?1-42 by miniosmotic pumpwith or without intragastric administration of SIF from 14 days before surgery to the end of experiment. The resultsshowed that SIF could improve the impairment of learning and memory of rats induced by A?1-42, maintain A?homeostasis in brain, regulate the disordered expressions of RAGE/LRP-1 and restrain RAGE related NF-?B andinflammatory cytokines activation in neurovascular structure. These results suggested that SIF could protect A?-impairedlearning and memory in rats, and its mechanism might be associated with the regulation of vascular A? transportation andvascular inflammatory reaction.

Cervicocranial Arterial Dissection: An Analysis of the Clinical Features, Prognosis, and Treatment Efficacy by Jiali Jin, Jingjing Guan, Lai Qian, Mei-Juan Zhang, Yujie Huang, Yongbo Yang, Dening Guan, Hui Zhao, Yongjuan Lin, Zhibin Chen, Weiyun Zhang, Jingwei Li, Yun Xu (157-163).
Clinical features and therapeutic strategies of cervicocranial arterial dissection (CCAD) are still unclear. Aretrospective review was conducted on 71 CCAD patients. Diagnosed by DSA and outcome evaluation was through mRSscores follow-up 12 months. All patients were allocated into three groups according to clinical situation: 1) subarachnoidhemorrhage (SAH), 2) ischemic symptoms and 3) mass effect. CCAD with anterior circulation arterial dissection (ACAD)had higher ischemia than that with posterior circulation arterial dissection (PCAD) (p=0.023). The non-aneurysmaldissection (NAD) patients were susceptible to ischemia (p=0.00) and patients with aneurismal dissection (AD) were moresusceptible to SAH (p=0.001); The outcome of patients with SAH was significantly worse than patients with othermanifestations (p=0.012). Following up one year, the outcome of CCAD involving posterior inferior cerebellar artery(PICA) was significantly worse than the other area (p=0.035). There was no statistically significant difference in mRSscores between endovascular treatment and conservative treatment (p=0.052) at one year follow-up. Patients sufferingfrom SAH that received endovascular treatment experienced improved outcomes than patients with conservative treatment(p=0.033). The patients in the ACAD, NAD and extracranial CCAD groups were more likely to suffer from ischemia,while patients in the AD group were susceptible to SAH. CCAD with SAH or involving PICA had poor prognoses. Thetherapeutic efficacy of conservative treatment is nearly equal to endovascular treatment in CCAD patients follow up 12months; however, endovascular treatment may decrease the risk of mortality for the patient with SAH.

Bi-3-Azaoxoisoaporphine Derivatives have Antidepressive Properties in a Murine Model of Post Stroke-Depressive Like Behavior by Seyed Fazel Nabavi, Eduardo Sobarzo-Sanchez, Seyed Mohammad Nabavi, Antoni Sureda, Akbar Hajizadeh Moghaddam (164-171).
In the present study, three bi-3-azaoxoisoaporphine derivatives were synthesized and intracerebroventricularlyadministrated to BALB/c mice. The antidepressant actions in stroke-induced depressive like behavior in mice wereexamined using despair swimming test and tail suspension test. The results reported that bilateral common carotid arteriesocclusion caused a significant abnormality of the normal behaviors. Behavioral models demonstrated that synthesizedcompounds showed antidepressant action. The most antidepressant active compound was DIME2 (4,4&#039;-dimethyl-7H,7&#039;H-[6,6&#039;-bibenzo[e]perimidine]-7,7&#039;-dione), which decreased the immobility time and increased the swimming and climbingtimes in despair swimming model. DIME2 also showed similar results in decreasing the immobility time in the tailsuspension model. In open field tests, DIME2 at 0.1&mu;g/&mu;l showed a significant activity in the modification of the distancemovement and the number and duration of rearing versus bilateral common carotid arteries occlusion (P<0.001).Furthermore, bilateral common carotid arteries occlusion caused a significant increase in the water consumption andsignificant decreasing in the sucrose consumption which are indicated as a state of anhedonia, a well known commonsymptom of transient ischemic stroke-induced depressive like behavior, versus normal group (P<0.001). In conclusion, bi-3-azaoxoisoaporphine derivatives can be considered as antidepressant agents for post stroke-induced depressive likebehavior therapy.

Therapeutic Effects of Renal Denervation on Renal Failure by Yutang Wang, Sai-Wang Seto, Jonathan Golledge (172-184).
Sympathetic nerve activity (SNA) is increased in both patients and experimental animals with renal failure. Thekidney is a richly innervated organ and has both efferent and afferent nerves. Renal denervation shows protective effectsagainst renal failure in both animals and humans. The underlying mechanisms include a decrease in blood pressure, adecrease in renal efferent SNA, a decrease in central SNA and sympathetic outflow, and downregulation of the reninangiotensinsystem. It has been demonstrated that re-innervation occurs within weeks after renal denervation in animalsbut that no functional re-innervation occurs in humans for over two years after denervation. Renal denervation might notbe renal protective in some situations including bile duct ligation-induced renal failure and ischemia/reperfusion-inducedacute kidney injury. Catheter-based renal denervation has been applied to patients with both early and end stage renalfailure and the published results so far suggest that this procedure is safe and effective at decreasing blood pressure. Theeffectiveness of renal denervation in improving renal function in patients with renal failure needs to be furtherinvestigated.

Computational Insights into the Role of Glutathione in Oxidative Stress by Caitlin E. Presnell, Gaurav Bhatti, Lidya S. Numan, Mitchell Lerche, Salem K. Alkhateeb, Muhannad Ghalib, Mohammed Shammaa, Mahendra Kavdia (185-194).
Excess reactive oxygen species (ROS) generation and oxidative stress in vascular tissue is associated withmany diseases. Glutathione (GSH), one of the most abundant low molecular weight non-protein thiols, modulatesphysiological levels of ROS and is involved in the cell