Rozprawy doktorskie na temat „Cerebral ischemia – Treatment”

Ischemic stroke is a devastating cerebrovascular disease resulting in high mortality rate and distressing sequelae such as hemiplegia, ataxia and even visual impairment. Retinal ischemia refers to a common pathological feature shared by many blinding diseases including retinal vascular occlusions, diabetic retinopathy, glaucoma, and retinopathy of prematurity. Ischemia/reperfusion injury is implicated in both of these pathological conditions, which greatly impact on one’s daily life. The eventual consequence of the insult is irreversible neuronal cell death and functional deterioration. Apart from current symptomatic treatment for these diseases, researchers and clinicians are dedicated to look for ideal neuroprotectant to meet the clinical needs. Traditional Chinese medicine has been received accumulating attention in recent years, and Lycium barbarum is one of them. The polysaccharides (LBP) utilized in the present study are the rich extracts of the fruit of Lycium barbarum that has been shown to exert many biological effects. This study aims to evaluate its protective effects in cerebral and retinal ischemia, which has not yet been fully investigated. A well-established rodent model, middle cerebral artery occlusion, was utilized in the present study to mimic cerebral and retinal ischemia/reperfusion injury. In the study of cerebral ischemia, both pre-treatment and post-treatment of LBP were explored. Seven-day LBP pre-treatment revealed significant protection against neurological deficits and cerebral infarction. Besides, it attenuated cerebral edema and glial activation, as well as preserved blood-brain barrier integrity. Further study showed that these beneficial effects of LBP pre-treatment might act via anti-apoptosis, antioxidation and anti-inflammation. However, similar findings were not noted in LBP post-treatment experiments, possibly due to the timing of intervention. In the investigation of retinal ischemia, the observation time was prolonged to 7 days after the insult. Electroretinogram was used to evaluate the functional alternation of retinal neurons. Sustained retinal dysfunction was induced by two-hour ischemia. LBP pre-treatment with continuous daily supplementation effectively alleviated visual dysfunction and protected the retina from morphological impairment including neuronal death, glial activation and blood-retinal barrier disruption. Similarly, these protective effects might be associated with the involvement of attenuation of apoptosis and oxidative stress. In conclusion, LBP pre-treatment with continuous daily supplementation protected the brain and retina, both functionally and morphologically, from ischemia/reperfusion injury. This dosing regimen hold great promise in serving as a prophylactic neuroprotectant in patients at high risk for ischemic stroke, as well as preserving normal visual function and reducing irreversible neuronal death in ischemic retinopathies. Further studies on the active ingredients and underlying mechanisms would be informative for better application of LBP in clinical situation.
published_or_final_version
Ophthalmology
Doctoral
Doctor of Philosophy

 Ischemic stroke is the leading cause of death and disability in human diseases all around the world. As effective treatment for ischemic stroke is still absent, seeking for new therapy is of great interest. Currently, several key pathological cascades following cerebral ischemia have been explored to develop further therapies. Among them, reactive nitrogen species (RNS) has been indicated to play a critical role in cerebral ischemia reperfusion injury. As one of the RNS, peroxynitrite contributes to the neural cell death and subsequent brain dysfunction in the process. Thus, development of antioxidants targeting on peroxynitrite could be an important strategy for the treatment of cerebral ischemia-reperfusion injury. Baicalin is a polyphenolic compound isolated from roots of Scutellaria baicalensis. Baicalin exerted protective effects against cerebral ischemia-reperfusion injury but the mechanisms are not clear yet. In this study, we investigated the free radical scavenging ability and neuroprotective effects of baicalin. According to our results, baicalin neutralized DPPH radicals effectively. By using electron paramagnetic resonance (EPR) spin trapping technology and fluorescent probe DAF-2DA, we found that baicalin dose-dependently scavenged superoxide, but had very low effect on elimination of nitric oxide. The immunofluoresent results revealed that baicalin at the concentration of 50 M completely suppressed the nitrotyrosine formation induced by 3-morpholinylsydnoneimine chloride (SIN-1, a peroxynitrite donor) in neuroblastoma SH-SY5Y cells. Mass spetrum provided direct evidence of the peroxynitrite scavenging ability of baicalin. Using MTT assays, we found that baicalin totally reversed peroxynitrite-induced cytotoxicity in SH-SY5Y cells and protected SH-SY5Y cells in oxygen glucose deprivation (OGD) and following reoxygenation injury. Furthermore, in vivo experiments revealed that intravenous injection of baicalin exerted better neuroprotective effect than intraperitoneal administration in rats underwent middle cerebral artery occlusion (MCAO). After cerebral ischemia reperfusion, rats treated with 3 mg/kg of peroxynitrite decomposition catalyst (FeTMPyP) or 25 mg/kg of baicalin revealed a smaller size of infarction volume, suppressed neural cell death and reduced nitrotyrosine formation than MCAO rats. However, baicalin did not alter the expression of tight junction proteins, claudin-5 and ZO-1, in brain endothelial bEnd3 cell line treated with OGD following reoxygenation. In cerebral ischemia reperfusion rats, administration of FeTMPyP at the dosage of 3 mg/kg diminished the Evans blue leakage caused by blood brain barrier disruption, whereas treatment of baicalin did not show significant effect. In conclusion, this study suggests that baicalin can scavenge peroxynitrite and protect neural cells from peroxynitrite-induced injury. Furthermore, baicalin could prevent brains from cerebral ischemia-reperfusion injury and the neuroprotective mechanisms are associated with the scavenging effects on peroxynitrite. These findings provide new insights into the antioxidant and neuroprotective properties of baicalin and indicate the potential application of baicalin for the treatment of ischemic stroke.
published_or_final_version
Chinese Medicine
Master
Master of Philosophy

Stroke is a devastating neurological disease with limited treatment opportunities. Recent advances in understanding the underlying pathogenesis of cerebral ischemia support the involvement of multiple biochemical pathways in the development of the ischemic injury. The work reported in this thesis was undertaken to investigate the hypothesis that fenamate NSAIDs have neuroprotective properties against ischemic stroke and to explore the underlying mechanisms for any efficacy. Fenamates are non-selective inhibitors of cyclooxygenases. In addition, fenamates are antagonists of non-selective cation channels, subtype-selective modutators of GABAA receptors, weak inhibitors of glutaniate receptors and activators of some potassium channels, all potentially important in the pathogenesis of ischemic stroke, Mefenamic acid, a prototype fenamate, administered by intracerebroventricular (ICV) infusion, reduced the ischemic brain damage and edema volume in the middle cerebral artery occlusion model in male rats. Consistent with these results; systemic administration of mefenamic acid, by multiple intravenous injections, also reduced the ischemic damage and edema volume measured by morphometric analysis and as a function of brain water content. These are the first set of experiments to demonstrate a significant neuroprotective effect of a fenamate against an in vivo model of ischemic stroke. In vitro, mefenamic acid was also shown to reduce glutamate-evoked cell death (excitotoxicity) in a concentration-dependent manner in cultured embryonic rat hippocampal neurons. Similarly, selected other fenamates also reduced excitotoxicity in the rank order (from highest): mefenamic acid > flufenamic acid ≥ meclofenamic acid > niflumic acid supporting the idea that this is a drug class action. Three pharmacological properties of fenamates, cyclooxygenase inhibition, GABAA receptor modulation and potassium channel activation were investigated as the potential mechanism(s} for the neuroprotective effects of mefenamic acid against excitotoxicity. The experimental results suggest that these are not the primary mechanisms for neuroprotective effects of mefenamic acid against glutamate-evoked cell death. Collectively, these data support the hypothesis that fenamate NSAIDs are neuroprotective against experimental models of cerebral ischemia and suggest they should be further investigated as potential pharmacological treatments for stroke.

[Truncated abstract] Clinical therapeutic agents to directly inhibit ischaemic neuronal death are presently unavailable. One approach to developing therapeutics is based upon the identification of proteins up-regulated by 'preconditioning', a natural adaptive response utilised by the neural cells to counter damaging insults, such as ischaemia. Thus, my project aimed to firstly identify proteins differentially expressed following erythropoietin (EPO) mediated neuronal preconditioning and secondly to assess whether any of these proteins possessed neuroprotective activity using in vitro ischaemia like models. To achieve the first aim, it was shown that in vitro neuronal EPO preconditioning could: (i) induce cell signal changes in neuronal cultures, (ii) protect neurons against in vitro ischaemia and (iii) induce differential protein expression. Overall, 40 differentially expressed proteins were identified in cortical neuronal cultures following EPO preconditioning. In order to investigate the neuroprotective or neurodamaging activity of proteins induced by EPO preconditioning I developed an adenoviral expression system for use in neuronal cultures. To this end, I assessed the suitability of four promoters (cytomegalovirus [CMV], rous sarcoma virus [RSV], human synapsin 1 [hSYN1], rat synapsin 1 [rSYN1]) previously used to express proteins in neuronal cultures and demonstrated the superiority of the RSV promoter for this purpose. ... Finally, in order to validate this adenoviral expression system, I over-expressed the anti-apoptotic protein Bcl-XL in neuronal cultures and subsequently confirmed its neuroprotective activity in the in vitro ischaemia and oxidative stress models used in my project. Using this adenoviral vector system and the in vitro oxidative stress model I assessed a number of proteins up-regulated by EPO preconditioning. The results of this preliminary study indicated that cyclophilin A (CyPA), peroxiredoxin 2 (PRDX2) and superoxide dismutase 1 (SOD1) over-expression were neuroprotective. It was subsequently verified that adenoviral mediated over-expression of CyPA and PRDX2, v but not SOD1 in cortical neuronal cultures could protect neurons from in vitro ischaemia. I also confirmed that CyPA mRNA increased in the rat hippocampus in response to 3 minutes of global cerebral ischaemia. Interestingly, an increase in CyPA, PRDX2 or SOD1 protein was not observed in the same experimental paradigm. To investigate CyPA's mode of action I confirmed that cultured neurons, but not astrocytes, express the CyPA receptor, CD147. It was also demonstrated that administration of exogenous CyPA protein to neuronal cultures could protect neurons against oxidative and ischaemic injury. I further demonstrated that exogenous administration of CyPA induces a rapid and transient activation of the extracellular signal-regulated kinase (ERK) 1/2 pathway in neuronal cultures. From this observation, I have proposed that the extracellular mediated neuroprotective activity of CyPA occurs via CD147 receptor signalling and activation of ERK1/2 pro-survival pathways. Based on the findings reported in this thesis, the neuroprotective activities of PRDX2 and CyPA warrant further investigation as targets for the development of new therapies to treat cerebral ischaemia.

Stroke is the 3rd leading cause of death and the leading cause of major disability worldwide. Currently, there are no neuroprotective drugs approved for the acute treatment of ischaemic stroke. The vast majority of stroke therapeutics failed in clinical trials due to toxic side effects and/or a clinically irrelevant therapeutic window. This thesis is focused on exploiting the delayed neurodegeneration that occurs in the compromised penumbra, as these cells may be capable of being saved by therapeutic intervention in a clinically obtainable window. In order to investigate the ischaemic cascade and be able to draw conclusions that are applicable to humans, the international gold standard animal model for cerebral ischaemia, the filament insertion middle cerebral artery occlusion (MCAO) model, was established at the University of Otago. This model was validated under new laboratory conditions and employed adult male Sprague Dawley rats. After testing multiple occlusion lengths, it was concluded that a 2hr ischaemic period was sufficient to produce a consistent infarct of optimal size. It has been well documented that neuroinflammation contributes to much of the delayed progression of neural injury post-stroke. Therefore, the catechin (-)-epigallocatechin gallate (EGCG), which is an anti-inflammatory, anti-oxidant and free-radical scavenging agent was investigated in the MCAO model of stroke. 50mg/kg i.p. of EGCG or saline was administered immediately post-MCAO and animals were sacrificed at 72hr post-filament insertion. The results confirmed that treatment with EGCG was neuroprotective and non-toxic. However, EGCG also induced an over 50% increase in the risk of haemorrhagic conversions. The anti-platelet effects of EGCG and lack of toxicity suggests that the catechin may prove to be an efficacious prophylactic for stroke. The contrary findings for EGCG treatment led to the re-evaluation of the neuroinflammatory pathway for alternate mechanisms to target therapeutic interventions. The temporal profile of the primary inducible enzymes nitric oxide synthase (NOS), cyclooxygenase (COX) and arginase (and their isoforms) were quantified 0, 3 and 7 days post-stroke. In both hemispheres, total NOS activity exhibited a significant and sustained up-regulation to 7 days post-occlusion. In the ipsilateral hemisphere at least half of the total increase was accounted for by inducible NOS (iNOS) expression. Arginase, which competes with NOS for L-arginine, demonstrated a delayed but significant increase in activity by day 7 in the infarcted hemisphere, thereby correlating well with the downward slope of NOS activity (illustrating the switch in the conversion pathway). COX activity was observably increased in the ipsilateral hemisphere, but the up-regulation did not reach significance by day 7. Alternately, the contralateral hemisphere displayed a significant decrease in activity by day 3. These results give conclusive evidence that the contralateral hemisphere is NOT an appropriate internal control and imply that NOS and COX inhibitors may prove to be efficacious for a much longer therapeutic window than current treatments. However, the delayed induction of COX activity may also indicate that this enzyme has a finite therapeutic window, as it may also stimulate remodelling of surviving neural networks. The prolonged up-regulation of inflammatory mediators implies that there may be an induction of an autoimmune component to the response. Therefore, the thymus (T) lymphocyte activation was quantified up to 14 days post-stroke. Cluster of differentiation (CD) 3⁺ T lymphocytes (equally contributed to by CD4⁺ and CD8⁺ T cells) exhibited a significant and sustained up-regulation in the infarcted region from day 3 up to at least day 14 post-ischaemia. Quantitative analysis of all cells present post-stroke determined that immune cells make up an average of 73% of all cells present in the 'peak' ischaemic areas. The CD4⁺ T helper cell response was delineated by double immunohistochemical labelling. Interferon-γ positively labelled with CD4⁺ T cells at days 3, 7 and 14 post-insult detailing a Th1-driven pro-inflammatory response. This evidence indicates that the autoimmune response is critical post-ischaemia and that it may be highly susceptible to modification by anti-inflammatory therapeutic intervention. The primary downstream effect of the pro-inflammatory/immune cascade is apoptosis. The main organelle responsible for the 'go, no go' response to apoptotic factors is the mitochondria. In order to distinguish whether mitochondrial dysfunction was initiated shortly after ischaemia induction or if it was delayed, like the inflammatory/immune response, to a clinically relevant window, the temporal profile of mitochondrial complex inactivation was studied. It was found that mitochondrial membrane viability was impaired by day 3, followed by a significant decrease in respiratory complex activation and an increase in tissue injury by oxidative stress by 7 days post-ischaemia. These results indicate that targeting the early decrease in membrane viability or mitochondrial permeability transition pore opening combined with anti-apoptotic therapeutics, may attenuate the proceeding mitochondrial impairment in oxidative phosphorylation, reactive oxygen species generation and subsequent cell death cascades. The current investigations into the temporal profile and quantitative contributions of the inflammatory, immune and apoptotic mechanisms post-stroke highlight potential strategies for modulation by acute stroke therapeutics. Furthermore, the general knowledge amassed from these studies dictates the necessity of a new approach to therapeutic intervention. The acknowledgement of so many contributing systems suggests that in addition to a thrombolytic, a combination therapy involving multiple neuroprotectants should be employed to account for the multifaceted nature of the sequelae of ischaemic stroke.

Les défauts de translation des études précliniques vers les essais cliniques dans le domaine des AVC ischémiques et l'échec des développements thérapeutiques pourraient être expliqués par trois aspects : (1) le manque de compréhension des mécanismes des deux formes de rtPA, le traitement pharmacologique de l'AVC; (2) le manque d'outils adaptés d'imagerie de perfusion chez le petit animal et (3) l'influence de l'anesthésie sur l’effet des traitements testés dans les modèles animaux.Le tPA utilisé en clinique (Actilyse®) est un mélange de deux formes de tPA: une forme chaîne simple (sc-rtPA) et une double chaîne (tc-rtPA). Malgré des activités fibrinolytiques similaires, ces deux formes de tPA exercent des fonctions cérébrales distinctes influençant différemment la récupération des patients. Ainsi, nous avons décidé d'étudier en détail dans un modèle murin d'AVC thromboembolique les mécanismes pouvant expliquer ces effets divergents. Nous avons confirmé ces observations à savoir que sc-rtPA est bénéfique lorsqu'il est perfusé tôt après le début de l'AVC, alors que le tc-rtPA est délétère en raison d'une altération de la barrière hémato-encéphalique.L'imagerie en temps réel de la perfusion de l'ensemble du cerveau est un atout pour les études cliniques et précliniques. L'émergence des ultrasons ultra-rapides a conduit au développement du Doppler ultra-rapide et de la Microscopie de Localisation à Ultrasons (ULM), deux méthodes avec différents profils de résolutions spatio-temporelles et une excellente sensibilité aux petits flux sanguins. Nous avons combiné ces deux méthodes pour fournir un suivi longitudinal en 3D de la perfusion cérébrale dans un modèle murin d’AVC thromboembolique avec fibrinolyse par rtPA. Nos données montrent que le FUS et l’ULM présentent un intérêt majeur pour le pronostic précoce de l'AVC ischémique et de la réponse au traitement, avec une corrélation étroite entre la reperfusion précoce à 2h et la récupération tissulaire à 24h.Enfin, l’anesthésie utilisée en laboratoire interfère sur la lésion ischémique et les effets des molécules thérapeutiques testées. Nous nous sommes affranchis de ces effets en développant un nouveau modèle d’AVC ischémique chez des souris totalement éveillées. Le débit sanguin cérébral régional a été suivi par laser Doppler avant, pendant et 45min après le début de l’AVC. Le traitement par rtPA (à 20 min) est bénéfique dans les modèles d’AVC vigile et anesthésié, mais l'anesthésie est associée à un manque de corrélation entre la recanalisation et les volumes de lésion post-ischémie. Nous testons actuellement une molécule neuroprotectrice, qui était prometteuse avant d’échouer lors des essais cliniques (NXY-059), afin d’évaluer la pertinence de ce modèle novateur d’AVC pour les futures études pharmacologiques. Dans l’ensemble, ce travail fournit un panel de données précliniques innovantes pour améliorer nos chances de translation en clinique, incluant un modèle pertinent d'AVC thromboembolique chez des animaux vigiles et une méthode d'imagerie du pronostic précoce de réponse aux traitements vasculaires
The lack of translation between preclinical studies and clinical trials in the field of ischemic stroke and the failure of therapeutic developments could be explained by three aspects: (1) the lack of understanding the mechanism of the two forms of tPA, the pharmacological treatment in stroke; (2) the lack of optimized perfusion imaging tools for small animal and (3) the influence of anesthesia on treatment tested in animal models.tPA used in the clinical setting (Actilyse®) is a mix of two forms of tPA: single chain form (sc-rtPA) and two chains form (tc-rtPA). Despite similar fibrinolytic activities, these two forms exert distinct brain functions therefore influencing differentially the outcome patients. We then decided to further investigate in a relevant model of thromboembolic stroke in rodents, the mechanisms that can explain these differential effects. Here, we have confirmed differential outcomes of the two forms: whereas sc-rtPA is clearly beneficial when infused shortly after stroke onset, tc-rtPA is deleterious due to an increased alteration of the blood brain barrier integrity.Live imaging of cerebral perfusion of the whole brain is an asset for both clinical and preclinical studies. The emergence of ultrafast ultrasound led to the development of ultrafast Doppler (fUS) and Ultrasound Localization Microscopy (ULM), two methods with different sets of spatio-temporal resolutions and excellent sensitivity to small blood flows. We combined these two methods to provide a longitudinal monitoring of whole brain perfusion using the thromboembolic stroke model in mice with rtPA-induced reperfusion. Our data show that fUS and ULM are of major interest for early prognosis of ischemic stroke and response to treatment, with a tight correlation between early reperfusion at 2h and tissue recovery at 24h. Finally, we develop a relevant awake ischemic stroke model to test new therapies, avoiding interferences due to anesthesia commonly used during in vivo studies mice. The patern of the MCA was followed using Laser Doppler monitoring before, during and 45 min after the stroke onset. Although rtPA treatment is beneficial in both awake and anesthetized stroke models, anesthesia is associated with a lack of correlation between recanalization and stroke outcome. We are now testing a neuroprotective molecule, which was promising before failing in clinical trials (NXY-059), to assess the relevance of this innovative stroke model for future pharmacological studies. Altogether, we provide here a set of innovative pre-clinical data to improve our chance of translation to clinic, including a relevant model of thromboembolic stroke in awake animals and an early prognosis imaging method of response to vascular treatments

碩士
國立成功大學
醫學工程研究所碩博士班
94
The neuroprotective properties of cinnamophilin, a strong natural antioxidant and free radical scavenger which may enhance cerebral hemodynamics, decrease platelet aggregation and lipid peroxidation, and prevent energy depletion during ischemia, will be evaluated in Sprague-Dawley rats subjected to 1-hour transient middle cerebral artery (MCA) occlusion. A series of experiments with delayed treatment of CINN was employed to examine whether CINN is neuroprotective following a short period of recovery using a model of reversible MCA occlusion which includes ischemic brain damage not only caused by grey matter but also by white matter damages. Postmortem infarct volumes will be determined by quantitative image analysis of Nissl-stained brain sections and by immunohistochemical methods. In addition, postischemic electrophysiological recovery will be evaluated. The possible mechanisms of action relevant to short-term neuroprotective efficacy by which CINN may exhibit the neuroprotective effects was evaluated in the model. The data not only extend our original findings to the ischemic-reperfusion injury but also look at prolonged outcomes, thus, may provide a potential outlook to treat patients against ischemic stroke. Delayed intravenous administration with CINN at 120 minutes after ischemia both enhances electrophysiological and neurobehavioral outcomes and reduces cortical and striatal infarction after cerebral ischemia-reperfusion. Since CINN is noted to be lack of toxicity and has persistent neuroprotection, it is worthwhile further investigating for potential benefits for those patients who will undergo planned cerebrovascular occlusion during neurosurgical surgery and for possible applications for ischemic stroke patients.

碩士
國立成功大學
醫學工程研究所
93
In the current study, magnesium sulfate (750μmol/kg) was given via intracarotid infusion at the beginning of reperfusion in a rat model of transient focal cerebral ischemia. Much evidence implicated neuroprotective mechanisms of Mg2+ to include inhibition of excitatory neurotransmitter release, blockade of voltage-dependent NMDA channels, and blockade of L- and N-type voltage-operated calcium channels. In addition to reduced brain infarction, this study emphasized the importance of electrophysiological and neurobehavioral outcomes. Improvement of functional outcome is more important than reduced brain damage with regard to stroke patients through treatment. The results showed that the P1-N1 amplitude of somatosensory evoked potentials (SSEP) recorded from ischemic fore- and hindpaw cortical fields decreased to 18% and 26% of baselines in vehicle-injected rats at 72 hr after the onset of reperfusion. Respectively, the P1-N1 amplitude of SSEP recorded from cerebral ischemia rats treated with MgSO4 improved to 24% and 40% of baselines. Additionally, MgSO4 also improved the neurobehavioral outcomes.

碩士
國立成功大學
醫學工程研究所
91
In general, cerebral ischemia elicits calcium accumulation and production of free radicals leading to irreversible cell damage and neurobehavioral dysfunction. Thus an effective neuroprotective agent is needed to protect ischemic brain after thrombolytic therapies. Melatonin is well known a direct free radical scavenger and an indirect antioxidant, and was reported to reduce infarct volume recently. In the current study, we investigate weather delayed treatment with melatonin (5mg/kg iv) could improve electrophysiology and neurobehavioral function in the transient cerebral ischemia model. Because for stroke patients through treatment, the improvement of functional outcome is more important than reduced brain damage In controls, the amplitude of somatosensory evoked potentials (SSEPs) recorded from fore- and hindpaw cortical fields decreased to 39% and 36% of baselines. Respectively, the amplitude of SSEPs recorded from cerebral ischemia rats treat with melatonin improved to 65% and 74% of baselines. Additionally, melatonin also reduced cerebral infarction volume. In conclusion, delayed treatment with melatonin could improve the electrophysiology and neurobehavioral outcomes and reduce brain damage induced by brain ischemia and reperfusion.

Early deterioration and long-term recurrence were common after stroke or transient ischemic attach (TIA), however, it is unclear whether they were correlated with active embolization and the consequent new cerebral infarct in acute phase. By employing TCD and diffusion weighted imaging (DWI), we studied the significance of the progression of MES and infarcts during acute phase on the clinical outcomes. We found that the disappearance of MES was correlated with better improvement on day 7 of recruitment; for the long-term outcome, occurrence of exacerbating infarct tended to predict recurrent stroke. Treatment aiming to reduce MES and prevent infarct exacerbation in acute phase may improve the prognosis after stroke.
Finally, one study was performed to assess the changes of hemodynamic parameters after stenting of severe stenosis in the MCA. We aimed to investigate whether TCD can reflect the lumen changes after revascularization and detect hyperperfusion. The findings showed that the velocity of stented MCA in most patients normalized within 24 hours after procedure, but the role of TCD in detecting restenosis in long run needed to be verified; no one suffered from hyperperfusion during the period of our study. The long-term outcomes of patients with normalized velocity versus those with persistently high velocity needed to be further studied. Apart from the velocity changes, changes of the collateral flow after intervention may also be an important part of hemodynamic changes. (Abstract shortened by UMI.)
It was suggested that anti-platelet therapy can reduce the MES, but little was known about the efficacy of low molecular weight heparin (LMWH) although in theory LMWH can reduce the red fibrin-dependent thromboemboli. As a sub-analysis of Fraxiparine in Ischemic Stroke (FISS)-tris study, our study did not show advantages of LMWH in eliminating MES compared with aspirin.
Previous studies showed the accuracy of TCD in diagnosis of middle cerebral artery (MCA) stenosis was variable and the positive predictive value (PPV) was less than 50% in a recent report. One of the important reasons was that most criteria were based on the velocity-only method, ignoring other non-velocity information. Thus, we tried to establish new diagnostic criteria by means of designing an assessment form which integrated more characteristics apart from the velocity acceleration. A composite score for each MCA was calculated according to following parameters in the form: Velocity Scale (score 0-6 for peak systolic velocities<140 to ≥300cm/s), Hemodynamic Scale (score 0-5 for focal or diffuse velocity increase; score 0-6 for differences between bilateral MCA; score 17 for damping velocity), Spectrum Scale (score 0-2 for normal spectrum, turbulence and musical murmurs). Our results showed that compared with the previously reported criteria, the score calculated from the assessment form yielded much more balanced accuracy against magnetic resonance angiography (MRA) and digital subtraction angiography (DSA). However, the composition of the assessment form was only based on personal experience and need to be further modified. Multicenter studies with large sample size are also needed to confirm the advantages of this new method.
Second, we performed three studies to investigate the relationship between the progression of MES and the short or long-term outcome and the relationship between MES and different treatments.
Hao, Qing.
Adviser: Ka Sing Wong.
Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3419.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2008.
Includes bibliographical references (leaves 155-181).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

碩士
中臺科技大學
醫學影像暨放射科學系暨研究所
100
This study investigated the influence of a large-area irradiation of low-level laser on the proliferation and differentiation of inductions of adipose-derived stem cells (i-ADSCs) into nerve cells. We then applied this approach to treat ischemic stroke in rats in the evaluation of various treatment mechanisms. DAPI staining showed that the number of cells did not increase as a result of irradiation; MTT assays indicated no significant difference between the number of laser-treated and non-laser-treated cells. Our results demonstrate that wide-area irradiation using a low power laser did not have a significant impact on the differentiation of i-ADSCs. Immunofluorescent staining indicated a significant increase in the neural stem cell marker Nestin following exposure to low power laser irradiation. No significant difference was observed between the glial cell marker GFAP and neuronal precursor cell protein DCX because the differentiation agent used in this study induced differentiation into nerve cells, rather than glial cells. Moreover, DCX can only be detected after neural stem cells form neurons in later stages; therefore, we conclude that the irradiation had no influence on the expression of this protein. Western blotting also showed that following irradiation, the expression of Nestin was significantly increased. The experiment results indicate that a large-area irradiation of low-level laser had no particular influence on the proliferation of i-ADSCs but still facilitated the differentiation of i-ADSCs considerably. This study employed stem cell implantation to treat rats following stroke. In the recovery of motor functions, the rats implanted with i-ADSCs (LS+) began regaining running and gripping functions on the 14th day following treatment. In comparison, stroke rats implanted with i-ADSCs (LS-) presented symptoms of dysfunction. At 28 days post treatment, the motor functions of the rats treated with i-ADSCs (LS+) did not differ greatly from those in the sham group, while the motor functions of the rats treated with i-ADSCs (LS-) remained somewhat dysfunctional. H&E-stained brain tissue samples from the rats treated with i-ADSCs (LS+) exhibited near-complete recovery with almost no brain tissue damage, whereas tissue from the rats treated with i-ADSCs (LS-) displayed necrotic scarring and voids. Western blotting revealed a significant expression of Oligo-2, a protein produced by the myelin-forming oligodendrocytes of the central nervous system, in the rats treated with i-ADSCs (LS+) as well as the sham group. From this, we can determine that treatment involving the implantation of i-ADSCs (LS+) is capable of repairing brain tissue damaged by ischemia. The results of this experiment also demonstrate that a large-area irradiation of low-level laser has a positive influence on the differentiation of i-ADSCs and can be employed to treat rats suffering from ischemic stroke to regain motor functions.

Han, Jinghao.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2007.
Includes bibliographical references (p. 182-204).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.

More than 15 million people worldwide will suffer a stroke each year two thirds will die or be left permanently disabled. Accordingly, stroke represents an enormous financial burden on the community, due to the cost of hospitalisation, treatment and rehabilitation of stroke patients. Despite the significance of this public health problem, a safe and widely applicable stroke therapeutic remains elusive. Cerebral oedema is widely recognised as a common and often fatal complication of stroke that is associated with worsened outcome. However, the exact mechanisms of oedema formation remain unclear, with current therapies largely ineffective in addressing the mechanisms of cerebral swelling, and also being associated with their own negative side-effect profile. This thesis characterises the role of neurogenic inflammation and the neuropeptide, substance P (SP), in mediating the development of blood brain barrier breakdown, cerebral oedema and resultant functional deficits following stroke, using a rodent model of reversible cerebral ischaemia. The findings of this thesis demonstrate that increased SP immunoreactivity, particularly of the penumbral tissue vasculature, is a feature of tissue perfusion following stroke, but not in non-reperfused infarcts. The central role for SP in the breakdown of the BBB following stroke and the associated deleterious effects of such breakdown was confirmed by studies using an NK₁ receptor antagonist. These antagonists conferred a profound attenuation of BBB breakdown, cerebral oedema formation, neuronal death and injury, and the associated development of functional deficits following reversible stroke. Similarly, depletion of all neuropeptides by capsaicin pre-treatment also reduced both histological abnormalities and functional deficits following stroke, confirming the central role of neuropeptides in the secondary injury process after stroke. The NK₁ receptor antagonist was able to be safely combined with the currently approved treatment for stroke, tPA, producing a synergistic effect of greater protection from the ischaemic insult. In particular, histological and functional outcome were markedly improved, as well as a reduction in the risk of intracerebral haemorrhage and death. Furthermore, the NK₁ receptor antagonist was effective even when administered up to 8 h following the onset of ischaemia, and in a variety of stroke severities. We conclude that SP plays a central role in the secondary injury that occurs following stroke, in particular, the genesis of BBB breakdown and cerebral oedema. Accordingly, combination therapy of tPA and an NK₁ receptor antagonist may offer a novel therapeutic strategy for the clinical management of ischaemic stroke of varying severity.
http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1298280
Thesis (Ph.D.) -- The University of Adelaide, School of Medical Sciences, 2007

More than 15 million people worldwide will suffer a stroke each year two thirds will die or be left permanently disabled. Accordingly, stroke represents an enormous financial burden on the community, due to the cost of hospitalisation, treatment and rehabilitation of stroke patients. Despite the significance of this public health problem, a safe and widely applicable stroke therapeutic remains elusive. Cerebral oedema is widely recognised as a common and often fatal complication of stroke that is associated with worsened outcome. However, the exact mechanisms of oedema formation remain unclear, with current therapies largely ineffective in addressing the mechanisms of cerebral swelling, and also being associated with their own negative side-effect profile. This thesis characterises the role of neurogenic inflammation and the neuropeptide, substance P (SP), in mediating the development of blood brain barrier breakdown, cerebral oedema and resultant functional deficits following stroke, using a rodent model of reversible cerebral ischaemia. The findings of this thesis demonstrate that increased SP immunoreactivity, particularly of the penumbral tissue vasculature, is a feature of tissue perfusion following stroke, but not in non-reperfused infarcts. The central role for SP in the breakdown of the BBB following stroke and the associated deleterious effects of such breakdown was confirmed by studies using an NK₁ receptor antagonist. These antagonists conferred a profound attenuation of BBB breakdown, cerebral oedema formation, neuronal death and injury, and the associated development of functional deficits following reversible stroke. Similarly, depletion of all neuropeptides by capsaicin pre-treatment also reduced both histological abnormalities and functional deficits following stroke, confirming the central role of neuropeptides in the secondary injury process after stroke. The NK₁ receptor antagonist was able to be safely combined with the currently approved treatment for stroke, tPA, producing a synergistic effect of greater protection from the ischaemic insult. In particular, histological and functional outcome were markedly improved, as well as a reduction in the risk of intracerebral haemorrhage and death. Furthermore, the NK₁ receptor antagonist was effective even when administered up to 8 h following the onset of ischaemia, and in a variety of stroke severities. We conclude that SP plays a central role in the secondary injury that occurs following stroke, in particular, the genesis of BBB breakdown and cerebral oedema. Accordingly, combination therapy of tPA and an NK₁ receptor antagonist may offer a novel therapeutic strategy for the clinical management of ischaemic stroke of varying severity.
Thesis (Ph.D.) -- The University of Adelaide, School of Medical Sciences, 2007

Lin, Wenhua.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2011.
Includes bibliographical references (leaves 162-190).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

博士
國立中興大學
食品暨應用生物科技學系所
105
The impact of ascorbate (vitamin C, ascorbic acid) on oxidative stress-related diseases is moderate because of its limited oral bioavailability and rapid clearance. However, parenteral administration can increase the benefit of ascorbate supplementation as is evident in critically ill patients. Heatstroke is defined as a form of excessive hyperthermia associated with a systemic inflammatory response that results in multiple organ dysfunctions in which central nervous system disorders such as delirium, convulsions, and coma are predominant. The thermoregulatory, immune, coagulation and tissue injury responses of heatstroke closely resemble those observed during sepsis and are likely mediated by similar cellular mechanisms. This study was performed by using the characteristic high lethality rate and sepsis-mimic systemic inflammatory response of a murine model of heat stroke to test our hypothesis that supra-physiological doses of ascorbate may have therapeutic use in critical care. We demonstrated that parenteral administration of ascorbate abrogated the lethality and thermoregulatory dysfunction in murine model of heat stroke by attenuating heat stroke-induced accelerated systemic inflammatory, coagulation responses and the resultant multiple organ injury, especially in hypothalamus. Stroke is a major public health problem and ranks third most common cause of death in adults worldwide. Focal ischemic stroke can cause permanent disability and lead to severe neurological sequelae. In this study, we hypothesized that high- dose ascorbate may exert its therapeutic effects through attenuating oxidative stress driven cerebral ischemia-reperfusion injury in a rat model of transient focal cerebral ischemia. Indeed, we did demonstrate that administration of supra-physiological doses of ascorbate significantly improved neurological deficits, the sequelae of brain infarction and edema, in a rat model of transient focal cerebral ischemia by attenuating cerebral neuronal apoptosis and blood brain barrier disruption. Overall, our findings support the hypothesis and notion that supra-physiological doses of ascorbate may have therapeutic use in critical care. Parenteral administration of high-dose ascorbate provides an inexpensive, strong and multifaceted antioxidant therapy, especially robust for resuscitation of the circulation. In critically ill patients, future research should focus on the use of short-term high-dose intravenous ascorbate as a resuscitation drug, or combined with other antioxidants to intervene as early as possible in the oxidant cascade in order to optimize macrocirculation and microcirculation and to limit cellular injury.

Hung, Sze Man.
Thesis (M.Phil.)--Chinese University of Hong Kong, 2013.
Includes bibliographical references (leaves 186-192).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts also in Chinese.

Serotonin (5-hydroxytryptamine, 5-HT) is a neurotransmitter involved in the embryonic neural development and adult neurogenesis. But the effects of 5-HT on stem cells are not fully known. In this study, the effects and underlying signal pathways of 5- HT on proliferation and neural differentiation of mouse embryonic stem (ES) cells, neural progenitor (NP) cell line C 17.2 and embryonic neural stem (NS) cells were explored. Molecular analysis, immunostaining and western blotting revealed that NP/NB cells expressed the rate-limiting enzyme tryptophan hydroxylase (TPH) and produced endogenous 5-HT. While mouse ES cells showed no expression of TPH. Quantitative PCR demonstrated that ES cells and NPINS cells expressed majority of 5-HT receptor sUbtypes. In serum free propagation culture, WST1, BrdU incorporation and neural colony forming cell assay demonstrated that 5-HT enhanced proliferation of ES cells and NPINS cells in a dose-dependent manner. Tryptophan hydroxylase (TPH) inhibitor para-chlorophenylalanine (PCPA) which can inhibit biosynthesis of endogenous 5-HT decreased viability of mouse NP/NS cells. Mouse ES cells derived embryoid bodies (EB) and NS/NP cells were subjected to neural induction in serum-free medium with and without 5-HT or PCPA. On day 8 of EB cultures, immunofluorescence staining displayed a less percentage of SSEA-1+ cells derived from cultures supplemented with 5-HT. Nestin positivity are comparable. Quantitative PCR analysis suggested that supplement of 5-HT in EB culture inhibit neural differentiation of ES cells and induce mesodermal commitment. On day 21 of ES cells neural induction, compared to cultures without 5-HT treatment, a significantly less number of ß-tubulin III+ neurons, GEAP+ astrocytes and GaIC+ oligodendrocytes were noted in 5-HT -supplemented cultures. For NS/NP cells, the inhibitory effects of 5-HT on neuronal and oligodendrocytic commitment were also observed. And the application of PCPA exerted a promoting effect on neural differentiation of NS cells. Manipulating 5-HT level can affect the expression level of key genes which involved in 5-HT metabolism. ES and NS/NP cells treated with 5-HT showed decreased production of endogenous reactive oxygen species (ROS). 5-HT demonstrated a significant anti-apoptotic effect on NP cells and this antiapoptotic effect may be mediated by up-regulated expression of anti-apoptotic gene Bel- 2. Whole genome cDNA microarray analysis and quantitative RT-PCR revealed that notch signal pathway was involved in mediating the biological effects of 5-HT. Western blotting further confirmed that 5-HT treatment up-regulated the protein level of NICD and notch downstream effectors Hes-l and Hes-5. Finally, the therapeutic effects of ES cell-derived neural cells were testified in a mouse model of global ischemia. Two weeks post-transplantation, BrdU labeled ES cell-derived neural cells survived and migrated throughout brain parenchyma. A majority of transplanted cells remained nestin positive. The cognitive functions of cell transplanted groups showed significant recovery compared with untransplanted arms, but no significant difference was observed between transplanted groups treated with and without 5-HT. Taken together, data of this study indicated 5-HT play an important role in neural development and ES cell-derived neural cells might be applicable in the treatment of stroke.
Li, Jin.
"November 2011."
Thesis (Ph.D.)--Chinese University of Hong Kong, 2012.
Includes bibliographical references (leaves 195-241).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.
Abstracts in English.
ACKNOWLEDGEMENTS --- p.i
LIST OF PUBLICATIONS --- p.ii
ABSTRACT --- p.iii
ABSTRACT [in Chinese] --- p.v
TABLE OF CONTENT --- p.vi
LISTS OF FLOWCHARTS --- p.xii
LISTS OF FIGURES --- p.xiii
LIST OF TABLES --- p.xvi
LIST OF EQUIPMENTS --- p.xvii
LIST OF ABBREVATIONS --- p.xvii
Chapter Chapter1 --- Introduction --- p.1
Chapter 1.1 --- Central nervous system disorder --- p.1
Chapter 1.1.1 --- Stroke --- p.1
Chapter 1.1.2 --- Spinal cord injuries --- p.4
Chapter 1.1.3 --- Parkinson's disease --- p.6
Chapter 1.1.4 --- Amyotrophic Lateral Sclerosis --- p.8
Chapter 1.2 --- Stem cell therapy --- p.10
Chapter 1.2.1 --- General considerations in stem cell therapy --- p.11
Chapter 1.2.2 --- Stem cell therapy for stroke --- p.11
Chapter 1.2.3 --- Stem cell therapy for spinal cord injury --- p.15
Chapter 1.2.4 --- Stem cell therapy for Parkinson's disease --- p.16
Chapter 1.2.5 --- Stem cell therapy for ALS --- p.18
Chapter 1.3 --- Stem cells --- p.20
Chapter 1.3.1 --- Embryonic stem cells --- p.21
Chapter 1.3.1.1 --- Derivation and characterization --- p.21
Chapter 1.3.1.2 --- Biology of ES cells --- p.21
Chapter 1.3.1.2.1 --- Pluripotency of ES cells --- p.21
Chapter 1.3.1.2.2 --- Differentiation of ES cells to multiple lineages --- p.24
Chapter 1.3.1.2.2.1 --- Ectodermal differentiation --- p.25
Chapter 1.3.1.2.2.2 --- Mesodermal differentiation --- p.27
Chapter 1.3.1.2.2.3 --- Endodermal differentiation --- p.28
Chapter 1.3.2 --- Neural stem cells --- p.30
Chapter 1.3.2.1 --- Derivation and characterization --- p.30
Chapter 1.3.2.2 --- Biology of NS cells --- p.32
Chapter 1.3.3 --- Induced pluripotent stem cells --- p.34
Chapter 1.3.4 --- Mesenchymal stem cells --- p.35
Chapter 1.4 --- Serotonin (5-HT) --- p.36
Chapter 1.4.1 --- Distribution --- p.37
Chapter 1.4.2 --- Metabolism --- p.37
Chapter 1.4.3 --- Biological effects of 5-HT --- p.38
Chapter 1.4.4 --- Serotonin receptor subtypes and receptor signal transduction pathways --- p.40
Chapter Chapter2 --- Aim --- p.43
Chapter 2.1 --- Hypothesis and study objectives --- p.43
Chapter Chapter3 --- Materials and Methods --- p.49
Chapter 3.1 --- Chemicals and Reagents --- p.49
Chapter 3.1.1 --- Cell culture --- p.49
Chapter 3.1.2 --- Serotonin, serotonin receptor subtypes specific agonists/antagonists and drugs that regulate serotonin metabolism --- p.51
Chapter 3.1.3 --- Cell proliferation assay --- p.52
Chapter 3.1.4 --- Cell apoptosis assay --- p.52
Chapter 3.1.5 --- Immunohistochemistry and staining --- p.52
Chapter 3.1.6 --- Western blotting --- p.55
Chapter 3.1.7 --- Molecular biology --- p.56
Chapter 3.1.8 --- Whole genome cDNA micro array --- p.58
Chapter 3.1.9 --- MAO activity assay --- p.58
Chapter 3.1.10 --- Endogenous ROS production assay --- p.58
Chapter 3.2 --- Consumable --- p.58
Chapter 3.3 --- Cells --- p.60
Chapter 3.3.1 --- Feeder cell --- p.60
Chapter 3.3.1.1 --- Mouse embryonic fibroblasts --- p.60
Chapter 3.3.2 --- ES cells --- p.61
Chapter 3.3.2.1 --- ES cell D3 --- p.61
Chapter 3.3.2.2 --- ES cell-E14TG2a --- p.61
Chapter 3.3.3 --- NS cells --- p.61
Chapter 3.3.3.1 --- Neural progenitor cells line C172 --- p.61
Chapter 3.3.3.2 --- Mouse embryonic neural stem cells --- p.61
Chapter 3.4 --- In-house prepared solutions --- p.62
Chapter 3.4.1 --- Stock solution ofInsulin, Transferrin, Selentine (ITS) Supplement --- p.63
Chapter 3.4.2 --- Gelatin solution 01% --- p.62
Chapter 3.4.3 --- Paraformaldehyde solution 4% (PFA) --- p.62
Chapter 3.4.4 --- Tritox X-lOO solution 03% --- p.63
Chapter 3.4.5 --- Popidium iodide solution 1 ug/ml (PI) --- p.63
Chapter 3.4.6 --- Poly-L-ornithine solution --- p.63
Chapter 3.4.7 --- Laminin solution --- p.64
Chapter 3.4.7 --- MEF Maintenance medium --- p.64
Chapter 3.4.9 --- Cryopreservation Media for MEF and C172 (2X) --- p.64
Chapter 3.4.10 --- Cryopreservation Media for mouse ES cell (2X) --- p.65
Chapter 3.4.11 --- Cryopreservation Media for mouse NS cell (2X) --- p.65
Chapter 3.4.12 --- Serum based maintenance medium for C172 --- p.65
Chapter 3.4.13 --- Serum free maintenance medium for C172 --- p.66
Chapter 3.4.14 --- Serum-based propagation medium for ES cells --- p.66
Chapter 3.4.15 --- Serum-free propagation medium forES cells --- p.67
Chapter 3.4.16 --- Serum-free induction medium for ES cells --- p.67
Chapter 3.4.16.1 --- Serum-free induction medium I --- p.67
Chapter 3.4.16.2 --- Serum-free induction medium II --- p.68
Chapter 3.4.16.3 --- Serum-free induction medium III --- p.68
Chapter 3.4.17 --- Tris-HCl (1 M), pH 74 --- p.68
Chapter 3.4.18 --- Tris-HCl (1 M), pH 87 --- p.69
Chapter 3.4.19 --- Tris-HCI (1 M), pH 69 --- p.69
Chapter 3.4.20 --- APS 10% (wt/vol) --- p.69
Chapter 3.4.21 --- Protease inhibitor (10X) --- p.70
Chapter 3.4.22 --- RIPA --- p.70
Chapter 3.4.23 --- Resolving buffer (8X) --- p.70
Chapter 3.4.24 --- Stacking buffer (4X) --- p.71
Chapter 3.4.25 --- Protein running buffer (lOX) --- p.71
Chapter 3.4.26 --- Transfer buffer (10X) --- p.72
Chapter 3.4.27 --- Transfer buffer (IX) --- p.72
Chapter 3.4.28 --- Blocking buffer (lOX) --- p.72
Chapter 3.4.29 --- TBS (10X) --- p.73
Chapter 3.4.30 --- TBS-T (IX) --- p.73
Chapter 3.4.31 --- Stacking gel --- p.73
Chapter 3.4.32 --- Resolving gel --- p.74
Chapter 3.5 --- Methods --- p.75
Chapter 3.5.1 --- Cell culture --- p.75
Chapter 3.5.1.1 --- Preparation of acid washed cover slips --- p.75
Chapter 3.5.1.2 --- Preparation of gelatinized culture wares --- p.75
Chapter 3.5.1.3 --- Poly-L-omithine and laminin coating --- p.76
Chapter 3.5.1.4 --- Thawing cryopreserved cells --- p.76
Chapter 3.5.1.5 --- Passage of culture --- p.77
Chapter 3.5.1.5 --- 6 Cell count --- p.78
Chapter 3.5.1.7 --- Cytospin --- p.78
Chapter 3.5.1.8 --- Trypan blue dye exclusion test --- p.78
Chapter 3.5.1.9 --- Cryopreservation --- p.79
Chapter 3.5.1.10 --- Derivation and culture of mouse embryonic fibroblasts (MEF) --- p.79
Chapter 3.5.1.11 --- Propagation of ES cells in serum-based/free medium --- p.81
Chapter 3.5.1.12 --- Neural differentiation ofES cells --- p.83
Chapter 3.5.1.13 --- Propagation ofNP cell C172 in serum-based or serum-free medium --- p.84
Chapter 3.5.1.14 --- Neural differentiation ofC172 --- p.85
Chapter 3.5.1.15 --- Derivation and propagation of embryonic NS cells --- p.85
Chapter 3.5.1.13 --- Neural differentiation of embryonic NS cells --- p.86
Chapter 3.5.1.17 --- BrdU labeling of the ES cells derived products --- p.87
Chapter 3.5.2 --- Cell proliferation assay --- p.87
Chapter 3.5.2.1 --- Cell morphology --- p.87
Chapter 3.5.2.2 --- WST-1 assay --- p.88
Chapter 3.5.2.3 --- BrdU incorporation assay --- p.88
Chapter 3.5.2.4 --- NCFC assay --- p.89
Chapter 3.5.3 --- Conventional and quantitative RT-PCR --- p.89
Chapter 3.5.3.1 --- RNA extraction --- p.89
Chapter 3.5.3.2 --- RNA quantitation --- p.90
Chapter 3.5.3.3 --- Reverse Transcription ofthe First Strand complementary DNA --- p.90
Chapter 3.5.3.4 --- Polymerase chain reaction --- p.91
Chapter 3.5.3.5 --- RNA Integrity Check --- p.91
Chapter 3.5.3.6 --- Electrophoresis and visualization of gene products --- p.91
Chapter 3.5.3.7 --- Real-time quantitative PCR --- p.92
Chapter 3.5.4 --- Microarray --- p.94
Chapter 3.5.5 --- Immunofluoresent staining --- p.94
Chapter 3.5.6 --- Western blot --- p.95
Chapter 3.5.6.1 --- Harvesting samples --- p.95
Chapter 3.5.6.2 --- Protein extraction --- p.96
Chapter 3.5.6.3 --- Protein quantification --- p.96
Chapter 3.5.6.4 --- SDS-PAGE --- p.97
Chapter 3.5.6.5 --- Wet transfer of protein to PVDF membrane --- p.97
Chapter 3.5.6.6 --- Blocking the membrane --- p.97
Chapter 3.5.6.7 --- Immunoblotting --- p.97
Chapter 3.5.6.8 --- Signal detection --- p.98
Chapter 3.5.7 --- Cell apoptosis assay --- p.98
Chapter 3.5.7.1 --- ANNEXINV-FITC apoptosis detection --- p.98
Chapter 3.5.7.2 --- TUNEL --- p.99
Chapter 3.5.8 --- Endogenous ROS assay --- p.100
Chapter 3.5.9 --- In vivo studies --- p.101
Chapter 3.5.9.1 --- Induction of cerebral ischemia in mice --- p.101
Chapter 3.5.9.2 --- Transplantation --- p.101
Chapter 3.5.9.3 --- Assessment of learning ability and memory --- p.102
Chapter 3.5.10 --- Histological analysis --- p.103
Chapter 3.5.10.1 --- Animal sacrifice for brain harvest --- p.103
Chapter 3.5.10.2 --- Cryosectioning --- p.103
Chapter 3.5.10.3 --- Haematoxylin and eosin staining --- p.104
Chapter 3.6 --- Data analysis --- p.104
Chapter Chapter4 --- Results --- p.113
Chapter 4.1 --- Expression profile of 5-HT receptors and metablism of endogenous 5-HT --- p.113
Chapter 4.1.1 --- Expression profiles of 5-HT receptors in stem cells --- p.113
Chapter 4.1.2 --- Biosynthesis of endogenous 5-HT --- p.115
Chapter 4.2 --- Effects of 5-HT on proliferation of mouse ES cells and NS cells --- p.115
Chapter 4.2.1 --- Effects of 5-HT on proliferation ofES cells --- p.115
Chapter 4.2.2 --- Effects of 5-HT on proliferation ofNP and NS cells --- p.117
Chapter 4.3 --- Effects of 5-HT on differentiation of mouse ES cells and NS cells --- p.119
Chapter 4.3.1 --- Neural differentiation ofES cells --- p.119
Chapter 4.3.2 --- Effects of 5-HT on differentiation ofES cells --- p.119
Chapter 4.3.3 --- Neural differentiation ofNP and NS cells --- p.120
Chapter 4.3.4 --- Effects of 5-HT on differentiation ofNP and NS cells --- p.121
Chapter 4.4 --- 5-HT metabolism in mouse ES cells and NS cells --- p.122
Chapter 4.4.1 --- Expression of key 5-HT metablic genes in stem cells --- p.122
Chapter 4.4.2 --- Detection ofROS generation in mouse NS cells --- p.123
Chapter 4.4.3 --- Effects of 5-HT on expression level of MAO-A, MAO-B and SERT --- p.123
Chapter 4.5 --- Anti-apoptotic effect of 5-HT on NP and NS cells in neural induction --- p.127
Chapter 4.6 --- Potential signaling pathways mediated by 5-HT --- p.130
Chapter 4.7 --- Therapeutic effects of 5-HT treated mouse ES cell-derived cells in a stoke model --- p.130
Chapter 4.7.1 --- Induction of global ischemia by transient BCCAO --- p.130
Chapter 4.7.1.1 --- HE staining of post ischemic brain --- p.131
Chapter 4.7.1.2 --- TUNEL analysis of cell apoptosis at post ischemia day 3 --- p.132
Chapter 4.7.2 --- Cell labelling --- p.132
Chapter 4.7.3 --- Cognition monitoring post transplantation --- p.133
Chapter 4.7.4 --- Survival, migration and differentiation of transplanted neural cells --- p.135
Chapter Chapter5 --- Discussion --- p.180
Chapter Chapter6 --- Conclusions --- p.192
References --- p.195

Baicalin, which is a flavonoid, was previously shown to exert neuroprotective effects against ischemic injury and oxidative insults. In this study, baicalin was found to induce neuronal differentiation on both C17.2 NSC and primary mouse NSC originated from hippocampuses of E14.5 mouse embryos. The baicalin-mediated differentiation of C17.2 NSC was noted in dose- and time-dependent manners. Baicalin-treated NSC displayed long processes of neurites. The gene expression of neuronal markers, NF-L, TUBB3 and MAP2 was also significantly increased after treated with 20 to 50 muM baicalin on C17.2 NSC. Treating C17.2 NSC with baicalin significantly increased the number of TUBB3 positive cells by 300%. A significant increase in the gene expression of TUBB3 was also observed on primary NSC upon baicalin treatment at 5 to 10 muM. The number of TUBB3 positive cells was increased by 100% after treating with 10 muM baicalin. C17.2 NSC treated with baicalin also increased the gene expression of GABAergic and serotonergic neuronal subtype specific enzymes GAD1 and TPH1.
Nature provides a vast pool of natural compounds with neuroprotection and neurotrophism. A few of these compounds can induce the differentiation of neural stem cells (NSC). There are ample opportunities to discover more natural compounds with differentiation inducing effect on NSC. One of the objectives of this project is to look for novel natural compounds showing neurogenic effect on NSC. This project has established a platform for screening medicinal materials and natural compounds with neural differentiation promoting effect on C17.2 mouse neural stem cell line. Screening results identified total Sanqi saponins, total Renshen saponins, Huangqin extracts and baicalin as potent candidates for inducing this differentiation of NSC.
This project also aims at characterizing the mechanisms involved in the neuronal differentiation effect of baicalin on NSC. Annotation from microarray analysis indicated that baicalin treatment on C17.2 NSC is related to development of tissue and nervous system. qPCR study attested the increased gene expression of nerve growth factor-beta, neurotrophin-3, pro-neural transcriptional factors Ngn1, Ngn2 and NeuroD2. Western blotting showed that baicalin activated ERK1/2 MAP kinase but not JNK and p38 MAP kinases.
This project demonstrated the neurogenic potential of natural resources on NSC. A novel neuronal induction effect of baicalin on NSC was also demonstrated with its mechanisms characterized. This project also revealed that baicalin can be used for promoting functional recovery of post-ischemia animals.
This study showed for the first time that baicalin exerts neuronal differentiation inducing effect on NSC. Another objective of this project is to study whether baicalin can promote functional recovery of animals with ischemia brain injury. Mice having undergone transient occlusion of the bilateral common carotid arteries with blood-reperfusion to induce global cerebral ischemia were treated with baicalin and/or EGFP-NSC. Ischemia animals received implantation of EGFP-NSC into the caudate putamen and/or intravenous injection of baicalin on alternate days for two-week on day seven post-ischemia displayed significant improvement of the cognitive function in terms of the incident of error and escape time in the water T-maze task compared to the control arm of ischemia mice. Data of the study suggested that the therapeutic effect of baicalin would be comparable to that of neural stem cell transplant in improving the cognitive function in a mouse ischemic stroke model.
Li, Ming.
Adviser: P. C. Shaw.
Source: Dissertation Abstracts International, Volume: 73-01, Section: B, page: .
Thesis (Ph.D.)--Chinese University of Hong Kong, 2009.
Includes bibliographical references (leaves 199-232).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstract also in Chinese.

Neonatal hypoxic-ischemic encephalopathy (HIE) is a common clinical problem. Tanshinone IIA is a compound purified from the Chinese herb Danshen ( Radix Salviae Miltiorrhiza Bge). Thrombopoietin (TPO) and Erythropoietin (Epo) are hematopoietic growth factors. The effects of tanshinone IIA, EPO and TPO on hypoxia-ischemia brain injury were investigated in this study, using in vitro model of neural cell culture and an in vivo model of hypoxic-ischemic brain damage.
Our observation provided the first evidence showing the expression of functional TPO receptor c-mpl in central nervous system. It revealed that novel agents TPO, EPO and tanshinone IIA have neuroprotection effects against brain injury induced by hypoxia-ischemia in neonatal rats, and these agents could be developed for clinical applications.
To investigate the effect of TPO, EPO and tanshinone IIA on in-vivo neural protection, a neonatal rat model of hypoxic-ischemic brain damage was established. Our results demonstrated significant and sustained brain injury in the hypoxic-ischemic and vehicle-treated group, measured by the reduction in relative weights of the ipsilateral (right) to the contralateral (left) brain at 1 and 3 weeks post-surgery, compared with those of sham-operated animals. At 3 weeks post-surgery, the hypoxic-ischemic animals had decreased cortical neuron density quantified by neuron-specific enolase (NSE) staining, and compromised sensorimotor functions in response to the postural reflex test. Treatment with TPO, EPO and tanshinone IIA significantly reduced the severity of brain injury, as indicated by the significantly increased ipsilateral brain weight and neuron density. Recoveries of sensorimotor functions (p < 0.05) and histopathology were also observed in animals that received TPO, EPO and tanshinone IIA. The plasma of tanshinone IIA-treated animals exhibited higher antioxidant activities (oxygen radical absorbance capacity assay) than those from vehicle-treated rats.
TPO and TPO receptor (c-mpl) mRNA was identified in human cerebral hemispheres, cerebellum, mouse neural progenitor cell line C17.2 and four neuroblastoma cell lines (SK-N-MC, MHH-NB-11, SK-N-AS and SH-SY-5Y) using RT-PCR methods. TPO proteins were detected in human cerebrospinal fluid (CSF) and plasma by ELISA. Furthermore, TPO receptor c-mpl was confirmed in human cerebral hemispheres, hippocampus, cerebellum, brainstem and spinal cord using immunohistostaining. TPO had a stimulating effect on the growth of neural progenitor cell C17.2 in culture via the phosphoinositide 3-kinase (PI3K)/Akt signaling pathway as demonstrated by Western blot. The anti-apoptotic effects of TPO, EPO on C17.2 cells were demonstrated by staining with Annexin-V and PI. EPO exerted a protective effect against SHSY-5Y cell damage induced by NMDA (N-methyl-d-aspartate), as demonstrated by the MTT and LDH assay. The anti-oxidative property of tanshinone IIA was studied in the C17.2 cell line. Tanshinone IIA increased the viability of these cells subjected to 2,2'-azobis (2-amidino propane hydrochloride) (AAPH)-induced oxidative stress.
by Xia Wen-Jie.
"May 2005."
Advisers: Kwok-Pui Fung, Tai-Fai Fok.
Source: Dissertation Abstracts International, Volume: 67-01, Section: B, page: 0126.
Thesis (Ph.D.)--Chinese University of Hong Kong, 2005.
Includes bibliographical references (p. 126-146).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Abstracts in English and Chinese.
School code: 1307.

碩士
國立陽明大學
物理治療學系暨研究所
95
Background and Purpose: Brain following transient focal cerebral ischemia develops a complex series of pathophysiological events. As a consequence of ischemia and subsequent reperfusion, the reactive free radicals react with cellular components (carbohydrates, amino acids, DNA, and phospholipids) and damage them. Hyperbaric oxygenation (HBO) has a characteristic in dramatically increasing the oxygen content of blood by physically dissolving oxygen and theoretically penetrates to deeper and poorly perfused ischemic areas. Thus, HBO appears to be a neuroprotective procedure for treating the cerebral ischemia. However, the neurophysiological mechanisms underlying such neuroprotective effects are still uncertain. It is suggested that some of the effects of HBO on neuronal survival are mediated indirectly by astrocyte. Moreover, astrocytes are believed to play a crucial part in the antioxidant defense of the brain, due to containing the high concentrations of antioxidants as well as providing the neurons with substrates for important antioxidants such as glutathione (GSH). The purpose of this study was to investigate whether HBO can promote the neuronal antioxidant defenses by activation the astrocytes in transient focal cerebral ischemic rats. Methods: 48 male Spraque-Dawley rats were subjected to 60 minutes middle cerebral artery occlusion (MCAO). Animals were randomly assigned to the HBO group or the control group (n=24 in each group). HBO was administered at a pressure of 3 atm absolute (3 ATA) for 1 hour with 100% oxygen, starting at 3 hours after MCAO in the HBO group. For the control group, animals were placed in the chamber and exposed to normobaric room air. All rats were sacrificed 24 hours after the MCAO procedure. The infarct volume, activation of astrocyte, glutathione, and the degree of lipid peroxidation were assessed for both groups. Comparisons of infarct volume, level of activated astrocyte, glutathione and lipid peroxidation between groups were carried out by independent t-test. Statistical comparisons of the glutathione and lipid peroxidation among the four cerebral areas were analyzed by one-way analysis of variance (ANOVA) and the post hoc Bonferroni test for pairwise comparisons if a significant difference was found. A probability value <0.05 was considered significant. Results: Total infarct volume was reduced by 35% (control group: 21.69±4.27% versus HBO group: 13.96±2.33%; P<0.01). Furthermore, HBO treatment enhanced the activation of astrocytes in the affected brain area compared with that of the control group, especially in the cerebral cortex. The GSH levels of right cortex (mean±SD, 2.5061±0.6470 nmol/mg tissue) and right striatum (mean±SD, 1.7419±0.5526 nmol/mg tissue) were increased after HBO treatment, especially in right cortex (P<0.05) compared with that of control group. Additionally, oxidative injuries due to cerebral ischemia were also significantly attenuated in the HBO-treated rats compared to those of the control rats. Conclusions: In the present investigation, we demonstrated a single HBO treatment after a transient ischemic insult in rats resulted in a significant reduction of the infarct volume. Our study also revealed astrocyte activation and decrease in oxidative injury in HBO rats. According to our findings, we suggest that HBO can promote the antioxidant defenses by activation the astrocytes and results in decreasing infarct volume in transient focal cerebral ischemic rats. Clinical Relevance: The present results provide evidence for possible neuroprotective mechanisms of the hyperbaric oxygen treatment, which is a clinical treatment for ischemic stroke patients.