Novel pharmacological strategies for neuroprotection in stroke: from bench to bedside

Trabalho Final de Mestrado Integrado, Ciências Farmacêuticas, 2021, Universidade de Lisboa, Faculdade de Farmácia.O acidente vascular cerebral (AVC) é uma das principais causas de mortalidade e morbilidade a longo prazo no mundo. Esta doença tem uma forte repercussão não apenas nos doentes, mas também nos seus cuidadores, visto que os sobreviventes se tornam frequentemente incapazes de realizar tarefas diárias básicas de forma independente. O AVC pode ser dividido em duas grandes categorias: isquémico ou hemorrágico, sendo que o AVC isquémico é responsável por cerca de 87% da totalidade de casos. O grande impacto negativo provocado por esta doença justifica a necessidade de desenvolver novas e melhores estratégias que permitam melhorar a qualidade de vida dos sobreviventes. Atualmente o “gold standard” para tratamento do AVC é a trombólise através da administração intravenosa de trombolíticos (tPA e derivados recombinantes). Quando administrado nas primeiras horas após o AVC, estes fármacos permitem aumentar a probabilidade de recuperação dos doentes. Infelizmente, muitos doentes não cumprem os critérios necessários para realizar este tratamento, o que justifica a necessidade de desenvolver novas estratégias. Nas últimas décadas, o conceito de neuroprotecção tem recebido muita atenção por parte dos investigadores, e inúmeros agentes têm sido testados em contexto pré-clínico, e alguns em ensaios clínicos. A melhor compreensão da cascata isquémica que leva ao dano neuronal permitiu a identificação de novos alvos para estratégias de neuroprotecção. Muitos dos ensaios pré-clínicos com estes compostos têm tido resultados encorajadores, mas que falham em demonstrar benefício na translação para contexto clínico. Alguns autores têm identificado razões para esta falha de translação. Apesar dos desafios que têm surgido neste campo, muitos compostos foram testados em ensaios clínicos nos últimos 5 anos, alguns dos quais ainda estão a decorrer. Independentemente das falhas que ocorreram no passado, a neuroprotecção tem um futuro promissor, e novas terapêuticas têm emergido como a adropina, a tirosina fosfatase STEP, o verapamilo e os microRNAs. A terapêutica combinada tem também um grande potencial, visto que poderia aumentar o número de doentes elegíveis para tratamento com trombolíticos. É muito provável que nos próximos anos surjam descobertas no tratamento do AVC, e os investigadores devem manter em mente os erros que foram cometidos no passado de forma a prevenir que se voltem a repetir no futuro.Stroke is among the main causes of death worldwide and it is a serious cause of long-term disability. This disease has an important impact not only in patients, but also in care providers, since often stroke survivors become unable to perform basic daily tasks on their own. Stroke can be divided into two major categories: ischemic and hemorrhagic. Ischemic stroke is the most common, being responsible for around 87% of all stroke cases. The huge burden associated with this disease, justifies the need to develop new and better therapeutic strategies that can improve survivor’s quality of life. Currently, the gold standard for treatment of stroke is recombinant tissue plasminogen activator (rtPA) administered intravenously. Given within the first hours of symptoms onset, rtPA can considerably increase the chances of stroke recovery. Unfortunately, a lot of patients do not meet the criteria to be eligible for this treatment, thus the need to develop new therapeutic strategies. In the last decades, a lot of attention has been drawn to the concept of neuroprotection and numerous agents were tested in preclinical studies and some of them in clinical trials. A better understanding of the ischemic cascade that leads to neuronal damage has enabled the development of novel therapeutic targets for neuroprotective strategies. Numerous compounds have undergone preclinical trials with exciting results but have failed to translate into clinical benefit. Some reasons behind the gap between bench and bedside have been identified by authors. Despite the challenges of translation, multiple compounds have been tested in clinical trials in the last 5 years, some of which are still ongoing. Regardless of the failures that occurred in the past, neuroprotection has a promising future with new emergent treatments such as adropin, tyrosine phosphatase STEP, verapamil, and microRNAs. Combination therapy holds great potential, since it could amplify the number of patients eligible for treatment with rtPA. In the next years, is likely that new discoveries arise in stroke research, and investigators must keep in mind the failures that happened in the past, to prevent them from happening in the future

The role of the autophagy in the differential vulnerability and the integrated stress response to the cerebral ischemia = El papel de la autofagia en la vulnerabilidad diferencial y en la respuesta integrada al estrés en la isquemia cerebral

142 p.El coste social y económico del accidente cerebrovascular (CVA) está continuamente en aumento y hasta la fecha hay pocas posibilidades de reducir la mortalidad o la discapacidad derivadas de esta patología. Por ello la búsqueda de nuevas dianas terapéuticas para su aplicación en clínica constituye una necesidad prioritaria. En el presente estudio se estudió la respuesta autofágica a la isquemia cerebral y su relación con el estrés del retículo. El objetivo era profundizar en el conocimiento de estos mecanismos, que constituyen potenciales dianas terapéuticas contra el CVA. En el primer capítulo, se realizó un estudio comparativo de la respuesta autofágica entre dos estructuras cerebrales (corteza cerebral e hipocampo) que presentan una vulnerabilidad diferencial a la isquemia. Para ello, se utilizó un modelo ex vivo de isquemia basado en el uso de secciones cerebrales. Este modelo permite la comparación de respuestas de diferentes estructuras en idénticas condiciones experimentales (en particular, se utilizaron 30 minutos de privación de oxígeno y glucosa [OGD] seguido de 3 horas en condiciones normóxicas [RL, reperfusion like]. Para caracterizar la actividad autofágica se midieron los niveles tanto de marcadores de inducción de autofagia como de sustratos específicos de autofagia (estimando así el flujo autofágico). Los resultados obtenidos permitieron concluir que la OGD induce una respuesta autofágica rápida en la corteza cerebral que no se observó en el hipocampo. El papel neuroprotector de la autofagia en la corteza cerebral parece implicar el control de los niveles de poliubiquitina y de la liberación de glutamato. El segundo capítulo compara la respuesta autofágica entre estructuras utilizando un modelo de isquemia cerebral global. Este modelo permitió extender los resultados descritos en el capítulo anterior a un modelo in vivo. Se analizó tanto la respuesta autofágica a la isquemia como su relación con la respuesta a las proteínas mal plegadas (UPR) inducida por el estrés de retículo endoplásmico. Tanto en la corteza cerebral como en la estructura hipocampal CA1 se observó actividad autofágica que no pudo ser detectada en la región hipocampal CA3. La respuesta autofágica en CA1 no parece suficiente para combatir el estrés de retículo, lo que explica la presencia de secuestosomas (agregados de proteínas mal plegadas) observados en las neuronas de esta estructura. La activación farmacológica de la UPR incrementó la actividad autofágica, eliminando los secuestosomas y reduciendo el daño isquémico. El tercer capítulo analiza la relación entre autofagia y la vía PERK de la UPR en un modelo de cultivos organotípicos de hipocampo sometidos a OGD y RL. Este modelo permite controlar la concentración de fármaco que llega a las neuronas y obtener muestras de una forma rápida, lo que permite estudiar diferencias en las respuestas entre tiempos muy cortos. El estudio mostró que la fosforilación de eIF2α (marcador de la activación de la vía PERK) presentó una respuesta rápida y de corta duración. Cuando está fosforilación se mantuvo a lo largo de la RL, se produjo un efecto neuroprotector mediado por ATF4 (activating transcription factor 4). Se ha descrito que ATF4 es capaz de producir una respuesta citoprotectora conocida como respuesta integrada al estrés (ISR, integrated stress response). La ISR incluye la activación de la autofagia y el aumento de la actividad antioxidante. Al contrario de lo observado en otros modelos de hipoxia, en el modelo aquí utilizado el efecto neuroprotector de ATF4 se debe a una respuesta antioxidante más que a una activación de la autofagia. Esto permitió concluir que el peso específico de los distintos componentes de la ISR depende del contexto celular y del modelo experimental

The Role of Wnt3a in Ischemic Stroke

After ischemic stroke, apoptosis of neurons is a primary factor in determining outcome. Wnt3a is a naturally occurring protein that has been shown to have protective effects in the brain for traumatic brain injury. Although wnt3a has been investigated in the phenomena of neurogenesis, anti-apoptosis, and anti-inflammation, it has never been investigated as a therapy for stroke. We hypothesized that the potential neuroprotective agent wnt3a would reduce infarction and improve behavior following ischemic stroke by attenuating neuronal apoptosis and promoting cell survival through the Frizzled-1/PIWI1a/FOXM1 pathway in MCAO rats. 229 Sprague-Dawley rats were assigned to male, female, and aged 9-month male MCAO or sham groups followed by reperfusion 2 hours after MCAO. Animals assigned to MCAO were either given wnt3a or its control. To explore the downstream signaling of wnt3a, the following interventions were given: Frizzled-1 siRNA, PIWI1a siRNA, and PIWI1a-CRISPR, along with the appropriate controls. Post-MCAO assessments included neurobehavioral tests, infarct volume, Western blot, and immunohistochemistry. Endogenous levels of wnt3a, Frizzled-1/PIWI1a/FOXM1, were lowered after MCAO. The administration of intranasal wnt3a, 1 h post MCAO, increased PIWIL1a and FOXM1 expression through Frizzled-1, reducing brain infarction and neurological deficits at 24 and 72 hours. Frizzled-1 and PIWI1a siRNAs reversed the protective effects of wnt3a post MCAO. Restoration of PIWIL1a after knockdown of Frizzled-1 increased FOXM1 survival protein and reduced Cleaved Caspase-3 levels. In summary, wnt3a decreases neuronal apoptosis and improves neurological deficits through Frizzled-1/PIWI1a/FOXM1 pathway after MCAO in rats. Therefore, wnt3a is a novel intranasal approach to decrease apoptosis after stroke

Brain Injury

The present two volume book "Brain Injury" is distinctive in its presentation and includes a wealth of updated information on many aspects in the field of brain injury. The Book is devoted to the pathogenesis of brain injury, concepts in cerebral blood flow and metabolism, investigative approaches and monitoring of brain injured, different protective mechanisms and recovery and management approach to these individuals, functional and endocrine aspects of brain injuries, approaches to rehabilitation of brain injured and preventive aspects of traumatic brain injuries. The collective contribution from experts in brain injury research area would be successfully conveyed to the readers and readers will find this book to be a valuable guide to further develop their understanding about brain injury

Studies on Vascular Changes and Immune Cell Role in the Ischemic Brain By <i>In Vivo</i> Two-Photon Microscopy

Mechanisms contributing to the inflammatory cascade, including vascular modifications and immune cell recruitment/activation, act with high dynamism within a three-dimensional space, thus being ideally investigated by in vivo two-photon microscopy (2-PM). In this thesis I applied in vivo 2-PM and quantitative bright field and confocal microscopy to explore cerebrovascular remodelling, immune cell dynamism and phenotype in two models of ischemia in mice achieved by transient or permanent middle cerebral artery occlusion. To specifically visualize microglia and T-cells, I used cx3crl eGFP and hCD2_eGFP mice, respectively. I imaged animals before and at different time points after ischemia, and quantified blood flow velocity and extravasation in individual vessels. After reperfusion, blood flow exhibited >80% drop in most vessels and extravasation established as early as 20min after ischemia onset. In this ischemic territory, I analyzed motility and morphology of GFP+ microglia. Microglia were stationary and became ameboid at 24h after injury. The absence of fractalkine receptor (CX3CR1) prevented the ameboid switch and favoured a protective M2 microglia polarization, characterized by decreased CD68 and iNOS expression and increased Yml expression. To assess the temporal evolution of microglia/macrophage polarization in the ischemic brain, I investigated the expression and coexpression pattern of CD11b, CD45, CD68, Yml, CD206 and iNOS after ischemia by conventional immunohistochemistry. Microglia/macrophages showed multiple polarization states, with a specific pattern of distribution and association with globular or ramified CD11b morphology. M2 microglia/macrophage peaked at 24h after injury, whereas Ml cells peaked at 48h. The phagocytic activity (CD68), mainly confined at the lesion borders at 6h-48h, dramatically increased and occupied all the ischemic territory at 7d. I finally investigated T-cells dynamism within the ischemic territory by in vivo 2-PM. I described two populations characterized by different track velocities and found that motile cells preferentially moved along the perivascular space, where they contacted astrocytes and perivascular macrophages. These data provide novel information on the inflammatory response after stroke and pave the way for developing strategies resulting in promotion of a protective inflammatory phenotype

Gene Expression Profiling in Human Acute Ischemic Stroke

Background: Gene expression profiling of human acute ischemic stroke (AIS) has the potential to identify a diagnostic panel for differential diagnosis of AIS early in the treatment phase. Purpose: The objective of this dissertation was to identify peripheral blood biomarkers that could be further explored for use in differential diagnosis of AIS and the design of stroke therapeutics.Methods: A prospective gene expression profiling study of 39 patients and 25 healthy controls was conducted. Peripheral blood samples were collected in Paxgene Blood RNA tubes from patients who were ≥18 years of age with MRI diagnosed AIS after differential diagnosis and controls who were Non-stroke neurologically healthy. In stroke patients, blood was redrawn 24 hours following onset of symptoms to determine changes in gene expression profiles over time. RNA was hybridized to Illumina humanRef-8v2 bead chips. Validation was performed using Taqman Gene expression polymerase chain reaction on significant targets. Results: A nine gene profile has been identified for AIS. Five of these nine genes were identified in the previously published whole blood gene expression profiling study of stroke and therefore play a likely role in the response to AIS in humans. One of these nine genes (s100A12) was significantly associated with increasing age and therefore may be non-specific for stroke. Three genes were identified as the whole blood expression profile change over time (LY96, IL8, and SDPR). Pathway analysis revealed a robust innate immune response, with toll like receptor (TLR) signaling as a highly significant pathway present in the peripheral whole blood of AIS patients.Conclusion: The findings of this study support the claim that gene expression profiling of peripheral whole blood can be used to identify diagnostic markers of AIS. A plausible case for innate immunity through the activation of TLR4 as a mediator of response to AIS has been made from the results of this study. This study and those conducted by Moore et al (2005) and Tang et al (2006) provide the foundation of data that support the use of peripheral whole blood for future blood profiling studies of neurological disease; which significantly opens the door of opportunity

Therapeutic modulation of liver ischaemia reperfusion injury

Liver Ischaemia Reperfusion Injury (IRI) leads to production of reactive oxygen species and cytokines, which affects hepatocellular function following liver resection and transplantation. This thesis examines 2 hypotheses: 1) The role of intravenous glycine in amelioration of liver IRI in a in vivo animal model of partial lobar liver IRI. 2) Does prophylactically administered N-acetylcysteine prevent liver IRI in patients undergoing elective liver resection. Materials and Methods 1) A rabbit model of hepatic lobar IRI was used to evaluate glycine. 3 groups (n=6) Sham group (laparotomy alone), ischaemia reperfusion (I/R) group (1 hour ischaemia and 6 hours of reperfusion), and glycine I/R group (IV glycine 5 mg/kg prior to the I/R protocol) were used. Portal blood flow, bile flow and bile was analysed by H1NMR spectroscopy. Hepatic microcirculation, intracellular tissue oxygenation, serum TNFα, IL-8, ALT, AST were measured at 1, 2, 4 and 6 hours following reperfusion. 2) A randomised double blind clinical trial was conducted to assess the effect of NAC on liver IRI following liver resections. The main outcomes were: morbidity and mortality, ICAM-1 expression in liver tissue, liver function tests. Patients were randomised to receive NAC as IV infusion (NACG) or a placebo group (PG) which received 5% dextrose only. Immunohistochemistry for ICAM-1 was carried out on perioperative liver biopsies. Results 1) Glycine normalised the bile flow, reduced phosphatidylcholine shedding, lactate surge, and stimulated bile acid, pyruvate, glucose and acetoacetate release. Glycine improved portal blood flow, hepatic microcirculation by the 2nd hour, and hepatic intracellular tissue oxygenation by the 4th hour of reperfusion. Glycine ameliorated serum TNFα at 1, 2 and 4 hours and serum Il- 8, AST and ALT up to 6 hours post reperfusion as compared to the I/R alone group. 2) Of the 43 patients, 15 received NAC, 16 were randomised to the PG, 12 were excluded due to inoperable tumours. Serum ALT was reduced in NACG (p=0.001), while serum ALP was higher in the NACG (p=0.003). ICAM-1 expression was up-regulated in 6/16 patients in the PG and in 3/15 patients in NACG. ICAM-1 was down-regulated in 1/15 patients in the NACG and none in the PG, the difference was not significant. Conclusions 1) Glycine ameliorated liver IRI, improved bile flow and composition. 2) NAC ameliorated parenchymal liver injury and enhanced liver regeneration in patients undergoing elective liver resection

Determination of Lipid Peroxidation Associated with a Focal Seizure Model through In Vivo Microdialysis Sampling

Two methods were developed to evaluate the extent of lipid peroxidation in an animal model of focal seizures. Microdialysis was used to sample from the extracellular fluid in specific regions of the hippocampus. The first method was a CE-fluorescence method for the thiobarbituric acid derivatized malondialdehyde (MDA) fluorophore. The second was an HPLC-MS method for seven eicosanoids; five products of the cyclooxygenase (COX) pathway (6-ketoprostaglandin F1α, thromboxane B2, prostaglandin F2, prostaglandin E2 and prostaglandin D2), one product of the lipooxygenase pathway (leukotriene B4), and one free radical byproduct of prostaglandin formation (8-isoprostaglandin F2). In a focal seizure model, a microdialysis probe was implanted in either the CA1 or CA3 region of the hippocampus. The chemical convulsant 3-mercaptopropionic acid (3-MPA) was dosed directly into the hippocampus by perfusion through the microdialysis probe for 50 minutes to produce a focal seizure. There were significant increases (p 0.001 compared to control) in MDA from 20 minutes after the start through 30 minutes after the end of dosing. It is hypothesized that these increases in MDA were related to the COX pathway. Therefore, an LC-MS method was developed and applied to the same animal model. Interestingly, there were decreases in the detectable prostaglandins (E2, D2, F2, and TXB2) during the 3-MPA dosing followed by immediate increases after dosing. It is thought that these prostaglandins are rapidly depleted during dosing from excessive oxidative damage

Elucidation of the Molecular Mechanisms of Electrically-Induced Cardioprotection

Cardiovascular disease is the leading cause of death worldwide. A myocardial infarction (MI), commonly known as a heart attack, is a major event in cardiovascular disease characterized by reduced blood flow to the heart. The ischemia and reperfusion (I/R) injury associated with an MI results in a region of dead tissue in the heart called an infarct, the size of which influences patient prognosis. In the 1980s, it was discovered that short, non-lethal episodes of I/R, termed ischemic preconditioning (IPC), can protect the heart against a subsequent MI. Ischemic preconditioning demonstrated the phenomenon of endogenous cardioprotection. Cardioprotection has great potential to reduce myocardial cell death and improve patient outcomes after MI, and yet most cardioprotective strategies have had limited success in clinical scenarios. Remote nociceptor-induced cardioprotection (NIC) elicits the most powerful reduction of cell death ever reported. Electrical stimulation (ES) administered via cutaneous patches offers a clinically feasible way to induce cardioprotection via NIC. Our previous work demonstrates that the transcription factor nuclear factor k-light- chain-enhancer of activated B cells (NF-kB) regulates many cardioprotective genes in the heart following IPC, including the heat shock proteins (HSPs), which act in concert with each other and their cofactors to assist in protein folding, repair, and degradation following myocardial injury. Little is known about the molecular mechanisms of electrically-induced cardioprotection, which is a barrier to the therapy\u27s optimization and successful translation to the clinic. Based on our work in IPC, we hypothesized that electrical stimulation of the skin is cardioprotective and requires the synthesis of NF-kB-dependent distal mediators of cardioprotection. In the studies herein, a cutaneous, 5-volt electrical stimulus applied to the abdomen reduces infarct size in a mouse surgical model of MI. Genetic blockade of NF-kB activation demonstrates the requirement of NF-kB in electrically-induced cardioprotection, yet RT-qPCR revealed small, nonsignificant changes in HSP mRNA and protein. Next-generation sequencing on mRNA and microRNA identified a unique transcriptome associated with electrically-induced cardioprotection that includes both recognized mediators and novel transcripts. Confirmatory studies on select molecular candidates were performed by RT-qPCR and Western blotting, and the functional role of the NF-kB-dependent gene nitric oxide synthase 2 (NOS2) was demonstrated in vivo. Results support that an electrical stimulus is cardioprotective in multiple paradigms of cardioprotection. Cardioprotection occurs without a concurrent increase in HSPs, but NF-kB and the NF-kB-dependent gene nitric oxide synthase 2 (NOS2) are required for ES- induced cardioprotection. Electrical stimulation also reduced cardiac levels of miR-10b, a circulating microRNA with no previously known role in cardioprotection. These changes were validated by RT-qPCR. In conclusion, electrically-induced cardioprotection is a novel and translational strategy to reduce cell death following MI. The molecular mechanisms of ES are cardioprotective via unique transcriptomic changes involving NF-kB and the NF-kB-dependent gene NOS2. The effect might be regulated epigenetically by microRNA

Developing novel fluorescent probe for peroxynitrite: implication for understanding the roles of peroxynitrite and drug discovery in cerebral ischemia reperfusion injury

Session 7 - Oral PresentationsSTUDY GOAL: Peroxynitrite (ONOO‐) is a cytotoxic factor. As its short lifetime, ONOO‐ is hard to be detected in biological systems. This study aims to develop novel probe for detecting ONOO‐ and understand the roles of ONOO‐ in ischemic brains and drug discovery ABSTRACT: MitoPN‐1 was found to be a ONOO‐ specific probe with no toxicity. With MitoPN‐1, we studied the roles of ONOO‐ in hypoxic neuronal cells in vitro and MCAO …postprin