Current Neurovascular Research (v.12, #1)

Platelets in the Alzheimerµs Disease Brain: do they Play a Role in Cerebral Amyloid Angiopathy? by Kathrin M. Kniewallner, Daniela Ehrlich, Andreas Kiefer, Josef Marksteiner, Christian Humpel (4-14).
Alzheimer's disease (AD) is characterized by extracellular beta-amyloid plaques and intracellular tau tangles. AD-related pathology is often accompanied by vascular changes. The predominant vascular lesions in AD are cerebral amyloid angiopathy (CAA) and arteriosclerosis. Platelets circulate along the vessel wall responding immediately to vascular injury. The aim of the present study was to explore the presence and migration of platelets (thrombocytes) to sites of small vascular bleedings and/or to beta-amyloid plaques in the brain. We infused fluorescently labeled red PKH26 mouse platelets into transgenic Alzheimer mice overexpressing APP with Swedish/Dutch/Iowa mutations (APP_SDI) and explored if platelets migrate into the brain. Further we studied whether platelets accumulate in the vicinity of β-amyloid plaques. Our animal data shows that infused platelets are found in the liver and partly in the lung, while in the brain platelets were visible to a minor degree. In mice, we did not observe a significant association of platelets with beta-amyloid plaques or vessels. In the brain of Alzheimer postmortem patients platelets could be detected by immunohistochemistry for CD41 and CD62P, but the majority was found in vessels with or without beta-amyloid load, and only a few single platelets migrated deeper into the brain. Our findings suggest that platelets do not migrate into the brains of Alzheimer disease but are concentrated in brain vessels.

Thromboxane A2 Receptor Polymorphism in Association with Cerebral Infarction and its Regulation on Platelet Function by Jiaxiang Shao, Yi Fu, Wanlin Yang, Jing Yan, Jing Zhao, Shengdi Chen, Weiliang Xia (15-24).
Binding of thromboxane A2 (TXA2) to its receptor TXA2R modulates thrombosis/hemostasis and plays a vital role in the pathogenesis of cerebral infarction (CI). In this study, we investigated the relationship between TXA2R polymorphisms and CI in Chinese Han population and the effect on platelet function by these polymorphisms. Polymerase chain reaction and ligase detection reaction (PCR-LDR) was performed in 230 CI patients and 143 healthy volunteers to examine four single nucleotide polymorphisms (SNPs) in human TXA2R gene (C795T, T924C and G1686A in the exon region, and rs768963 in the promoter region). We found that rs768963 polymorphism was significantly more frequent in the CI group than in the non-CI group and the T-T-G-T haplotype of C795T-T924C-G1686A-rs768963 was significantly less frequent in the CI subjects (0.238 versus 0.339; OR 0.617 [95%CI 0.444-0.856]). In the meantime, we constructed wild-type and mutant (C795T, G910A and T924C) eukaryotic expression plasmids, and transfected these plasmids into human embryonic kidney (HEK) 293T cells or Chinese hamster ovary (CHO) cells stably expressing human TXA2R (GP b/a- CHO). C795T and T924C variants of TXA2R led to increased ligand binding-induced intracellular calcium influx and fibrinogen-integrin conjugation, while dominant negative mutant G910A abolished the signal enhancement. Together these data show that TXA2R polymorphisms may affect platelet function and the risk of developing cerebral ischemia.

Identification of Target Antigens of Antiendothelial Cell Antibodies Against Human Brain Microvascular Endothelial Cells in Healthy Subjects by Akio Kimura, Takeo Sakurai, Nobuaki Yoshikura, Akihiro Koumura, Yuichi Hayashi, Hirofumi Ohtaki, Mitsuhiro Chousa, Mitsuru Seishima, Takashi Inuzuka (25-30).
Antiendothelial cell antibodies (AECAs) have been detected in patients who have autoimmune and inflammatory diseases. Previous studies showed that AECAs against human umbilical vein endothelial cells were detected in healthy subjects. In the present study, we evaluated AECAs against human brain microvascular endothelial cells (HBMEC) in serum. We detected 250 antigen spots that reacted with AECAs in serum samples from 30 healthy subjects by 2-dimensional immunoblot using primary cultured HBMEC as the antigen source. There were 10 spots that corresponded to common target antigen spots and reacted with AECAs in serum samples from > 25% of the 30 healthy subjects. We identified 7 proteins that corresponded to 8 of the 10 spots by mass spectrometry: 78-kDa glucose-regulated protein, dihydropyrimidinase-related protein 2, heterogeneous nuclear ribonucleoprotein L, vimentin, perilipin 3, alpha-enolase, and annexin A2. The results suggest that these 7 HBMEC proteins are major target antigens of natural AECAs.

Carbon Monoxide and Iron Modulate Plasmatic Coagulation in Alzheimerµs disease by Vance G. Nielsen, Etheresia Pretorius, Janette Bester, Wayne K. Jacobsen, Patrick K. Boyle, Joao P. Reinhard (31-39).
Alzheimer's disease (AD) is a significant source of morbidity and mortality for millions of people worldwide, and multiple potential etiologies have been postulated to contribute to AD. Among these, spontaneous cerebral emboli and increased cerebral and circulating heme oxygenase (Hmox) activity in AD patients are of particular interest, as two of the products of Hmox activity, carbon monoxide (CO) and iron enhance plasmatic coagulation and modify the ultrastructure of thrombi. We hypothesized that patients afflicted with AD would have coagulation kinetics modulated by CO and iron. Using viscoelastic assessments of coagulation, it was determined with a small cohort (n=11) of AD patients that all had enhancement of coagulation by CO, iron, or both. In a complementary fashion, it was determined that a separate cohort (n=12) of AD patients had thrombi with ultrastructural features consistent with iron and CO exposure as assessed with scanning electron microscopy. Further, when stratified by normal or abnormally increased serum ferritin concentrations (which can be increased by Hmox), the AD patients with abnormal ferritin concentrations had significantly thinner fibrin fiber diameters, not unlike that noted when normal plasma is mixed with iron or CO. In sum, AD patients were noted to have plasmatic coagulation kinetic and thrombus ultrastructural changes consistent with exposure to CO and iron. Future investigation of CO and iron in the pathogenesis of Alzheimer's disease is warranted.

This study investigated the correlation of serum cystatin C levels with cerebral infarction (CI) and different subtypes of CI, the severity of CI and the risk of recurrent CI. Totally 1017 patients with CI and 534 healthy controls were included, with clinical and biochemical characteristics documented and analyzed. The results showed serum cystatin C were 0.93 ± 0.21 mg/l and 1.19 ± 0.5 mg/l in control and CI, respectively (p < 0.001). The significant risk factors for CI were age (p < 0.001, OR = 1.048), presence of diabetes (p = 0.007, OR = 2.397), cystatin C (p = 0.001, OR = 1.021), uric acid (p = 0.017, OR = 4.226), and systolic blood pressure (p = 0.021, OR = 1.003). The risk of CI increased with cystatin C levels. Also, cystatin C levels were associated with different subtypes of CI, with large-artery atherosclerosis (LAA) subtype having the highest level of cystatin C. Moreover, cystatin C levels were significantly and positively correlated with Essen Stroke Risk Score (ESRS). The higher level of cystatin C the higher risk of recurrent stroke. In contrast, correlation between cystatin C levels and National Institute of Health stroke scale (NIHSS) score was not significant. In conclusion, cystatin C was an independent risk factor for CI. LAA subtype of CI was more closely related to increased cystatin C level. Furthermore, cystatin C levels predict risk of recurrent CI but not the severity of CI.

Low Baseline Urine Creatinine Excretion Rate Predicts Poor Outcomes among Critically Ill Acute Stroke Patients by Chia-Yu Hsu, Yi-Ling Wu, Chun-Yu Cheng, Jiann-Der Lee, Ying-Chih Huang, Ming-Hsueh Lee, Chih-Ying Wu, Huan-Lin Hsu, Ya-Hui Lin, Yen-Chu Huang, Hsin-Ta Yang, Jen-Tsung Yang, Meng Lee, Bruce Ovbiagele (47-52).
Urinary creatinine excretion rate (CER) is an established marker of muscle mass. Low CER has been linked to poor coronary artery disease outcomes, but a link between CER and acute stroke prognosis has not been previously explored. We prospectively collected data from patients with acute stroke (ischemic or hemorrhagic) within 24 hours from symptom onset in a Neurological and Neurosurgery Intensive Care Unit in Taiwan. Baseline CER (mg/d) was calculated by urine creatinine concentration in morning spot urine multiplies 24-hour urine volume on the second day of admission. Patients were divided into 3 tertiles with highest, middle, and lowest CER. Primary endpoint was poor outcome defined as modified Rankin Scale 3-6 at 6 months. Among 156 critically ill acute stroke patients meeting study entry criteria, average age was 67.9 years, and 83 (53.2%) patients had ischemic stroke. Patients with lowest CER (vs. highest CER) had a high risk of poor outcome at 6-month after adjustment (odds ratio 4.96, 95% confidence interval 1.22 to 20.15, p value = 0.025). In conclusion, low baseline CER, a marker of muscle mass, was independently associated with poor 6-month outcome among critically ill acute stroke patients. We speculate that preservation of muscle mass through exercise or protein-energy supplement might be helpful for improving prognosis in severe stroke patients.

Melatonin Leads to Axonal Regeneration, Reduction in Oxidative Stress, and Improved Functional Recovery Following Sciatic Nerve Injury by Yasemin Kaya, Kamil Savas, Levent Sarikcioglu, Nazmi Yaras, Doychin N. Angelov (53-62).
Despite recent advances in microsurgical techniques and equipments, recovery of function following repair of transected nerves often remains suboptimal. Contrary to traumatic injuries vascular damage that causes peripheral nerve injury has not been well-documented in the literature. In the present study a total of 40 female rats were randomly divided into four groups: Group 1: intact controls (n: 10), Group 2: sham-operated (n: 10), Group 3: vehicle-treated (n: 10), Group 4: melatonin-treated (n: 10). Sciatic nerve damage was created by stripping of the epineurial vessels around the nerve. 50 mg/kg Melatonin was injected intraperitoneally immediately after epineurial stripping in Group 4 in the course of 4 postoperative weeks. We found that melatonin administration after stripping of the epineurial vessels exerted a beneficial effect on axonal regeneration and functional recovery was confirmed by functional (sensory-motor, biochemical, and electrophysiological analyses) and morphological (light microscopic and ultrastructural analyses) data. In the light of these results we concluded that melatonin in a model of sciatic nerve injury leads to axonal regeneration, reducing in oxidative stress, and improved functional recovery.

HspB8 is Neuroprotective during Oxygen Glucose Deprivation and Reperfusion by Binbin Yang, He Zhang, Xiaoye Mo, Han Xiao, Zhiping Hu (63-72).
Heat shock protein B8 (HspB8) is a chaperone protein that is highly and constitutively expressed in the brain, cardiac tissue and many other organs. Recently, it has been shown that HspB8 can enhance cardiac function and render cardioprotection. However, the potential benefits of HspB8 action on ischemic stroke and the underlying mechanism(s) are largely unknown. To investigate whether HspB8 exerts protective effects on in vitro ischemia/ reperfusion (I/R) injury, mouse neuroblastoma cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R). At first, we investigated and described the HspB8 expression and distribution in N2A cells exposed to OGD/R. Expressions of HspB8 were upregulated in mouse N2A cells after OGD/R, both at mRNA and protein levels. The level of HspB8 began increased after the OGD/R and peaked at 12 hour (12h) after the reperfusion, then declined at 24-hour time points, however, the level of HspB8 was still significantly increased compared to controls. Immunofluorescence analysis revealed that the expressed HspB8 was constitutively localized in the cytoplasm and at the periphery of the nucleus. Nuclear HspB8 levels increased after OGD/R compared with levels in the control, beginning as early as 4h reperfusion, the most conspicuous nuclear HspB8 staining was observed after 24h of reperfusion. Furthermore, overexpression of HspB8 reduced OGD/R -induced apoptosis by reducing the release of cytochrome c from mitochondria to cytosol. In conclusion, our data demonstrated that increased HspB8 expression and its nuclear shift in mouse N2A neuroblastoma cells protected against I/R injury, resulting in reduced apoptosis with the decrease of the release of cytochrome c from mitochondria to cytosol. Therefore, HspB8 might play a fundamental role in opposing and alleviating I/R injury in primary myocardial cells, and it may constitute a new therapeutic target for cerebral ischemic diseases.

Ketamine Treatment Partly Reverses Alterations in Brain Derived- Neurotrophic Factor, Oxidative Stress and Energy Metabolism Parameters Induced by an Animal Model of Depression by Gislaine Z. Réus, Marcos Paulo Nacif, Helena M. Abelaira, Débora B. Tomaz, Maria Augusta B. dos Santos, Anelise S. Carlessi, Beatriz I. Matias, Jaine R. da Luz, Amanda V. Steckert, Gabriela C. Jeremias, Giselli Scaini, Meline O.S. Morais, Emilio L. Streck, João Quevedo (73-84).
Studies have suggested that ketamine, a nonselective NMDA receptor antagonist, could be a new drug in the treatment of major depression, but the way ketamine presents such effects remains to be elucidated. Therefore, the objective of this paper was to evaluate the effects of ketamine treatment on parameters related to depression in the brain of adult rats subjected to an animal model of depression. The animals were divided into: non-deprived + saline; non-deprived + ketamine; deprived + saline; deprived + ketamine. Treatments involving ketamine (15 mg/kg) were administered once a day during 14 days in the animal's adult phase. After treatment, the brain derived-neurotrophic factor (BDNF) levels, oxidative stress and energy metabolism activity were evaluated in brain structures of rats involved in the circuit of depression. In the amygdala, hippocampus and nucleus accumbens (NAc), a reduction in BDNF levels was observed in deprived rats, but the animals treated with ketamine reversed the effects of this animal model only in the amygdala and NAc. In addition to this, the complex I activity, in deprived rats, was diminished in the prefrontal cortex (PFC) and amygdala; in the PFC and hippocampus, the complex II-III was diminished in deprived rats; still the administration of ketamine increased the complex IV activity in the PFC and amygdala of rats submitted to the maternal deprivation. In deprived rats, the creatine kinase activity was reduced in the PFC and amygdala, however the administration of ketamine reversed this decrease in the amygdala. The malondialdehyde (MDA) equivalents were increased in non-deprived rats treated with ketamine in the PFC and NAc. Carbonyl levels in the PFC were diminished in control rats that received saline. Though ketamine treatment reversed this effect in deprived rats in the PFC and hippocampus. Still, in NAc, the carbonyl levels were diminished in deprived rats. The superoxide dismutase (SOD) activity was increased in control rats that received ketamine in the PFC and NAc, and were diminished in deprived rats that received saline or ketamine in the PFC and hippocampus. These findings may help to explain that dysfunctions involving BDNF, oxidative stress and energy metabolism within specific brain areas, may be linked with the pathophysiology of depression, and antidepressant effects of ketamine can be positive, at least partially due to the control of these pathways.

Factor VIII Levels are Associated with Ischemic Stroke, Stroke Subtypes and Neurological Worsening by Chih-Yu Kuo, Chun-Hsien Lin, Ya-Wen Kuo, Yen-Chu Huang, Huan-Lin Hsu, Ya-Hui Lin, Chih-Ying Wu, Ying-Chih Huang, Meng Lee, Hsin-Ta Yang, Chia-Yu Hsu, Yi-Ting Pan, Jiann-Der Lee (85-90).
The role played by hemostasis in the pathogenesis of ischemic stroke is still controversial. In the present study, we looked for a possible association of ischemic stroke with the high clotting activity of factor VIII (FVIII). The study population consisted of 116 patients with acute ischemic stroke who had been admitted between September 2013 and September 2014 to the Department of Neurology, Chiayi Chang Gung Memorial Hospital, and 76 control subjects with no history of stroke. FVIII levels were higher in stroke patients as compared to controls (127.5 ± 52.5 vs. 108.4 ± 49.0 IU/dL; P = 0.012). In a univariate risk analysis, FVIII at levels above 150 IU/dL was associated with an odds ratio (OR) for ischemic stroke of up to 2.55 (95% CI, 1.20-5.42, P = 0.013). In a multivariate analysis using a logistic regression model including age, hypertension, low density lipoprotein cholesterol level, estimated glomerular filtration rate, and high FVIII (< 150 IU/dL), high FVIII was still found to significantly add to the risk of ischemic stroke (OR = 3.26 with 95% CI, 1.38-7.68, P = 0.007). As for the stroke subtypes, mean FVIII level was significantly higher in patients with cardioembolic stroke than patients with noncardioembolic stroke (156.0 ± 51.5 IU/dL vs. 124.3 ± 51.9 IU/dL). High levels of FVIII were also associated significantly with neurological worsening (OR = 3.66 with 95% CI, 1.24-10.82, P = 0.019). A high plasma level of FVIII is a prevalent and independent risk factor for ischemic stroke and neurological worsening after acute stroke.

Telmisartan Promotes Potential Glucose Homeostasis in Stroke-Resistant Spontaneously Hypertensive Rats via Peroxisome Proliferator-Activated Receptor γ Activation by Yoshio Omote, Kentaro Deguchi, Tomoko Kurata, Toru Yamashita, Kota Sato, Nozomi Hishikawa, Koji Abe (91-97).
An angiotensin 2 type 1 receptor blocker (ARB) telmisartan possesses not only an antihypertensive effect but also an anti-metabolic syndrome effect due to peroxisome proliferator-activated receptor γ (PPAR-γ) activation. In the present study, we examined the effects of telmisartan on the angiotensin 2 type 1 receptor (AT1R), PPAR-γ, and insulin receptor (IR) in stroke-resistant spontaneously hypertensive rats (SHR-SR), comparing them with Wistar rats. Three-months-old SHR-SR rats were divided into three treatment groups, i.e., vehicle (SHR/Ve), low-dose telmisartan (0.3 mg/kg/day, SHR/Low), and high-dose telmisartan (3 mg/kg/day, SHR/High). Compared with Wistar rats, SHR/Ve increased the staining of AT1R, PPAR-γ and IR in the cerebral cortical neurons. On the other hand, telmisartan dose-dependently suppressed the excessive expression of AT1R and IR, but enhanced PPAR-γ activation. Low-dose telmisartan showed these effects even without lowering blood pressure (BP), while high-dose telmisartan lowered BP and showed further effects. The present study suggests that even a low dose of telmisartan decreased AT1R and IR, and increased PPAR-γ in the cerebral cortex of SHR-SR without lowering BP, probably by improving glucose homeostasis. The high dose of telmisartan showed further decreases in AT1R and IR, and further PPAR-γ activation while lowering BP, suggesting an additive benefit to lowering BP, namely the improvement of glucose homeostasis.

The Cellular Protective Effects of Rosmarinic Acid: From Bench to Bedside by Seyed Fazel Nabavi, Gian Carlo Tenore, Maria Daglia, Rosa Tundis, Monica Rosa Loizzo, Seyed Mohammad Nabavi (98-105).
Rosemary (Rosmarinus officinalis L.) is one of the most common household herbs, used as spices in a variety of foods, and employed in traditional medicine for its healing properties. Rosemary is a rich source of active antioxidant constituents such as phenolic diterpenes, flavonoids and phenolic acids. Caffeic acid and rosmarinic acid are the most important bioactive constituents. Rosmarinic acid is the ester of caffeic acid and 3,4-dihydroxyphenyllactic acid and is widely identified in different plant species. Chemical structure of rosmarinic acid contains two phenolic rings: one of them is derived from phenylalanine via caffeic acid and the other from tyrosine via dihydroxyphenyl-lactic acid. Its large-scale production is obtained from plant cell cultures of Coleus blumei Benth. It is easily absorbed through gastrointestinal tract as well as the skin. Rosmarinic acid is one of the most important and well known natural antioxidant compounds, which possesses neuroprotective effects in different models of neuroinflammation, neurodegeneration, as well as chemicalinduced neurotoxicity and oxidative stress. Therefore, in present review, we aim to discuss about chemistry, sources, biotechnological production and neuroprotective actions of rosmarinic acid with emphasis on its possible molecular mechanisms of neuroprotection.