Disertacijos objektas yra koronarine širdies liga ir ryškiai sumažinta kairiojo skilvelio sistoline funkcija sergančių ligonių klinikinės būklės pokyčiai ir išgyvenimas vėlyvuoju pooperaciniu laikotarpiu po izoliuotų aorto-vainikinių arterijų apeinamųjų jungčių suformavimo operacijų (AVAJSO), bei kairio skilvelio tūrio ir formos atkūrimo operacijų (TFAO). Tai retrospektyvinis tyrimas. Analizuoti 216 ligonių sergančių koronarine širdies liga, kuriems atliktos AVAJSO ir 139 ligonių, kuriems kartu su revaskuliarizacija atliktos kairio skilvelio TFAO, duomenys. Nustatyta, kad ligonių, sergančių IŠL su KS sistoline disfunkcija, išgyvenimas vėlyvuoju – iki 7 metų laikotarpiu po AVAJSO bei 6 metų laikotarpiu po KS TFAO, yra geras. Nustatėme, kad operacijų, kurias nuspręsta atlikti ligoniams, turintiems krūtinės anginos simptomus, be miokardo gyvybingumo požymių įrodymo echokardiografiniu mažų dozių dobutamino krūvio mėginiu, pirmųjų 30 parų mirties rizika nebuvo didesnė. Rasta, kad ligonių, kurių funkcinė klasė vienerių metų laikotarpiu po AVAJSO buvo blogesnė, iki operacijos buvo EKG ilgesnė QRS komplekso trukmė, didesni KS galiniai diastoliniai dydžiai ir operacijos metu jiems buvo suformuota mažiau AVA jungčių. Išaiškinta, kad KS tūrio ir formos atkūrimo operacijos ligoniams su toli pažengusia KS remodeliacija atliekamos saugiai, su ne didesne pirmųjų 30 parų mirties rizika nei atliekant vien AVAJSO. Išaiškinta, kad tų ligonių, kurių funkcinė būklė nepagerėjo vienerių metų... [toliau žr. visą tekstą] / ANNOTATION OF DISERTATION Subject of disertation: Surgical treatment of patients suffering from ischemic heart disease with significantly decreased left ventricle systolic function: clinical evaluation. Objective - evaluation of clinical status and long-term postoperative survival of patients suffering from ischemic heart disease and decreased left ventricle systolic function (LV EF ≤ 35%) after isolated coronary by-pass grafting and left ventricle volume and shape surgical restoration operations. Retrospective study. The study group included 216 patients for whom coronary artery bypass grafting (CABG) and 139 patients who underwent surgical ventricular restoration (SVR). It was postulated that the long-term survival (up to 7 years) in patients after CABG and long-term survival (6 years) in patients after SVR is good. It was found out that the risk of fatal outcome during the first 30 days after CABG in patients 0suffering from ischemic LV dysfunction and symptoms of angina pectoris with viable myocardium, statistically reliably did not differ from patients with nonviable myocardium. It was stated that patients with greater functional class during one year after CABG, preoperatively had longer duration of ECG QRS complex, higher end-diastolic findings and had lower number of by-passes during the operation. We also demonstrated that LV shape and volume restoration operations were being performed for patients with highly advanced LV remodeling safely and postoperative risk... [to full text]

<p>Otkrivanje embolusa u cerebralnoj cirkulaciji na egzaktan način moguće je samo upotrebom transkranijalnog doplera. Istraživanje je obuhvatilo 150 ispitanika, obolelih od akutnog ishemijskog cerebrovaskularnog događaja (ishemijskog moždanog udara i tranzitornog ishemijskog ataka) u zoni vaskularizacije a. cerebri medie (ACM), a lečenih na Klinici za neurologiju, Kliničkog centra Vojvodine. Ciljevi istraživanja su obuhvatili određivanje prevalence i frekvence MES kod bolesnika sa akutnim ishemijskim cerebrovaskularnim događajem (TIA, IMU) tokom serijskog monitoringa, utvrđivanje povezanost pojave MES u odnosu na etiologiju ishemijske epizode, procenjivanje efekata terapije (antiagregacione i antikoagulantne) na pojavu MES tokom serijskog monitoringa, i utvrđivanje prediktivnog značaja MES na dalji tok bolesti tj, rani povratni embolizam unutra tri meseca. Utvrdili smo da se mikroembolusi kao markeri aktivne embolizacije mogu registrovati primenom transkranijalnog doplera u akutnoj fazi moždanog udara u određenoj meri. U ispitivanom uzorku metodom transkranijalne detekcije kod 52 (34,7%) bolesnika je registrovana pojava cerebralnih mikroembolusa. Ovi ispitanici su činili MES (+) grupu pacijenata. Kod 98 (65,3%) bolesnika nisu registrovani ES, oni su činili MES (-) grupu pacijenata. Detekcija je vr&scaron;ena u prvih 72h od vremena nastanka IMU ili TIA. Zaključili smo da se serijskim monitoringom registruje smanjenje prevalence i frekvence embolijskih signala. Utvrdili smo da su starija životna dob, hipertenzija i dijabetes statistički značajno povezani sa pojavom mikroembolusnih signala. Najveća zastupljenost mikroembolusa registrovana je u aterotrombotičnom podtipu ishemijskog moždanog udara. Utvrđen je prediktivni značaj aterosklerotske bolesti velikih krvnih sudova na pojavu MES. Registrovana je statistički značajno če&scaron;ća pojava MES kod simptomatske karotidne stenoze, visokog stepena (70-90%), neravne i ulcerisane povr&scaron;ine plaka. Nije utvrđena statistički značajna povezanost pojave MES, kliničkih manifestacija bolesti i neuroradiolo&scaron;kog nalaza. Nije registrovan uticaj antitrombotičke terapije na pojavu mikroembolusnih signala. Zabeležena je veća stopa recidiva IMU i TIA kod bolesnika sa registrovanim cerebralnim mikroembolusima. Utvrđen je prediktivni značaj MES na pojavu recidiva IMU ali ne i prediktivni značaj na pojavu letalnog ishoda.</p> / <p>Detection of emboli in the cerebral circulation to the exact way it is possible only by using transcranial doppler. The study included 150 patients of acute ischemic cerebrovascular events (ischemic stroke and TIA) in a zone of vascularization a. cerebri media (ACM), and treated at the Clinic of Neurology, Clinical Center of Vojvodina Research objectives included the determination of the prevalence and frequency of MES in patients with acute ischemic cerebrovascular accident (TIA, IMU) during serial monitoring, establishing the link between the appearance MES in relation to the etiology of ischemic episodes, assessing the effects of therapy<br />(antiplatelet and anticoagulant) on the occurrence of MES during serial monitoring and determine the predictive value MES in the further course of the disease, ie. return early embolism within three months. We have found that microemboli as markers of active embolization can register by using transcranial Doppler in the acute phase of stroke in certain extent. In the examined sample using transcranial detection with 52 (34.7%) patients the occurrence of cerebral microemboli is registered. These respondents are accounted for MES (+) group of patients. With 98 patients (65.3%) is not registered EC, they account for MES (-) group of patients. Detection was performed during 72 hours from the time of occurrence of ischemic stroke or TIA. We concluded that serial monitoring registers decrease in prevalence and frequency of embolic signals. We found that older age, hypertension, and diabetes are significantly associated with the appearance of microembolic signals. The highest incidence of microemboli was registered in atherothrombotic ischemic stroke subtype. It is determined the predictive significance of atherosclerotic disease of large blood vessels on the occurrence of MES. More common MES is significantly registered with symptomatic carotid stenosis, greater degree (70-90%), uneven surfaces and ulcerated plaque. There was no statistically significant correlation between the occurrence of MES, clinical manifestations and neuroradiological findings. It is not registered impact of antithrombotic therapy on the incidence of microembolic signals. We are noticed thet the higher rate of recurrence of ischemic stroke and TIA patients with cerebral microemboli is registered. The predictive significance of MES in recurrence of ischemic stroke is determined, but not predictive significance of the occurrence of a lethal outcome.</p>

The major physiological function of hemoglobin (Hb) is to bind, transport and deliver oxygen to tissues; made efficient by endogenous effectors, such as protons and 2,3-diphosphoglycerate. Synthetic allosteric effectors of Hb (AEHs) are also known to modulate Hb oxygen affinity, showing potential for the treatment of sickle cell disease (SCD) and ischemic-related diseases. In this project, AEHs which increase Hb affinity for oxygen, including derivatives of the anti-sickling compounds, 5HMF and benzaldehydes, as well as an AEH that decreases Hb affinity for oxygen, RSR-13, were synthesized for their effects on Hb oxygen binding property and their capability to release NO from substituted nitrate ester moieties. Compounds that were found to increase Hb affinity for oxygen were further tested for their anti-sickling activities. Structural studies were carried out to gain insight into the compound’s mode of action. Development of these agents could be a therapeutic strategy for SCD or ischemic-related diseases.

Ce travail de thèse a permis, dans un premier temps, de mettre en évidence de nouveaux grandsréarrangements dans trois pathologies étudiées au laboratoire : la mucoviscidose, la pancréatitechronique et l’hémochromatose. En particulier, ces travaux ont permis de trouver de nouveaux CNVs(Copy Number Variations) pathologiques dans le gène CFTR (Cystic Fibrosis Transmembraneconductance Regulator), de mieux comprendre les mécanismes d’un remaniement complexe dansPRSS1 (Protease Serine 1) et d’aider à caractériser finement un réarrangement dans HFE(Hemochromatosis). Ces études ont donc servi de preuve de concept pour l’utilisation de puces à ADN àl’échelle d’un gène et dans des zones difficiles car riches en séquences répétées.Dans un second temps, la recherche de facteurs de susceptibilité génétiques aux infarctus cérébraux(AICs) du sujet jeune a été réalisée chez 168 cas et 200 témoins âgés de moins de 40 ans. Dans notrepopulation, l’hypertension, les migraines, le tabac, et la prise de stupéfiants sont des facteurs de risqueimportants, multipliant respectivement par 35, 3,8, 4 et 2,8 le risque d’AIC. Notre étude pangénomiquepar CGH-array (Comparative Genomic Hybridization-array) a mis en évidence 98 régionspolymorphiques dans le génome humain. Parmi elles, la délétion d’une partie du gène NOTCH2, pourraitjouer un rôle protecteur dans la survenue des AICs (OR=0,11 [0,01-0,87] ; p=0,013) mais qui ne dépassepas le seuil fixé par la correction de Bonferroni). Ce travail a également mis en évidence environ 400CNVs rares, dont deux récurrents chez les cas, l'un portant les gènes CRELD2 (cysteine-rich with EGFlikedomains 2) et AGL12 (asparagine-linked glycosylation 12, alpha-1, 6-mannosyltransferase) (p=0,02)et le deuxième situé en 5’ du gène VBP1 (von Hippel-Lindau binding protein 1) (p=0,04). Enfin, uneapproche gènes candidats a été effectuée sur les gènes NOTCH2 et ALOX5AP (5-lipoxygenaseactivating protein) sans donner de résultats significatifs. Ceci a également été réalisé sur les mutationsprincipales de trois gènes de la coagulation (Facteur II, Facteur V Leiden et MTHFR). Une associationsignificative a été mise en évidence entre la C677T du gène MTHFR (5,10-methyltetrahydrofolate) et lesinfarctus cérébraux du sujet jeune (OR=2,39, p=0,02 pour le génotype TT). Ce travail de thèse a permisde confirmer l’existence de facteurs de risque environnementaux et génétiques déjà connus mais surtoutd’émettre de nouvelles hypothèses génétiques dans la survenue des AICs du sujet jeune. / The use of locus-specific array-CGH (Comparative Genomic Hybridization) has allowed us to detect largerearrangements in three pathologies of our laboratory: cystic fibrosis, chronic pancreatitis andhemochromatosis. We successfully observed new pathological CNV (Copy Number Variations) in theCFTR (Cystic Fibrosis Transmembrane conductance Regulator) gene and characterized complex eventsin PRSS1 (Protease Serine 1) and HFE (Hemochromatosis) genes, showing that the use of thistechnique is possible even in regions with high sequence homologies.We also confirmed that hypertension, migraine, tobacco and drugs are high significant risk factors forischemic strokes (IS) in young population (under 40 years) (OR=35, 3.8, 4 and 2.8, respectively). Then,we tried to identify new genetic susceptibility loci using a pangenomic approach. Among the 98 copynumber polymorphisms (CNP) observed, an interstitial NOTCH2 deletion is candidate for a protective rolein IS (OR=0.11 [0.01-0.87] ; p=0.013 before Bonferonni correction). We also observed approximately 400uncommon CNV, two of them being particularly reccurent in patients: a 22q13.31 duplication containingCRELD2 (cysteine-rich with EGF-like domains 2) and AGL12 (asparagine-linked glycosylation 12, alpha-1, 6-mannosyltransferase) genes (p=0.02) and a Xq28 deletion localised in the 5’ region of the VBP1 (vonHippel-Lindau binding protein 1) gene (p=0.04). We also applied a candidate-gene approach onNOTCH2, ALOX5AP (5-lipoxygenase activating protein) and coagulation genes (Factor II, Factor VLeiden and MTHFR). A significant association was found for the C677T in the MTHFR gene (5,10-methyltetrahydrofolate) and young ischemic strokes (OR=2.39, p=0.02 for TT genotype). In conclusion,this study confirmed the implication of environmental and genetic factors in ischemic strokes before 40years and suggests new genetic risk factors for IS.

Stroke is the 5th leading cause of death in the U.S. with 130,000 deaths and around 800,000 affected annually. Currently, there is a significant disconnect between basic stroke research and clinical stroke therapeutic needs. Few animal models of stroke target the large vessels that produce cortical deficits seen in the clinical setting. Also, current routes of drug administration, intraperitoneal and intravenous, do not mimic the clinical route of intra-arterial drug administration. To bridge this divide, we have retro-engineered a mouse model of stroke from the current standard of care for emergent large vessel occlusion (ELVO) stroke, endovascular thrombectomy, to include selective intra-arterial pharmacotherapy administration. Using the tandem transient common carotid and middle cerebral artery occlusion (MCAo) model to induce stroke, we threaded micro-angio tubing into the external carotid artery (ECA) towards the bifurcation of the common carotid and internal carotid arteries (CCA/ICA) allowing for the delivery of agents to the site of acute ischemia. Our model was optimized through a flow rate and injection volume study using carbon black ink injected through the intra-arterial model at different flow rates and injection volumes. The purpose of this study was to demonstrate that our injections were arriving at the site of ischemia and to improve injection volumes for future dosing while mitigating systemic side effects by preventing or minimizing systemic distribution. We determined that a flow rate of 2.5 µl/minute and injection volume of 10 µl was optimal. Next, we tested potential neuroprotective compounds nitroglycerin, verapamil, and a combination of verapamil and lubeluzole. Compounds were chosen for drug synergy and to target specific pathways in either an acute or delayed manner. Acute treatments included nitroglycerin and/or verapamil while delayed treatment included lubeluzole. The known mechanism of action for FDA approved nitroglycerin is through vessel dilation that results in increased blood flow to the treated region. A secondary mechanism of nitroglycerin is the production of nitric oxide, which has demonstrated antioxidant and anti-apoptotic effects when processed and released from cells surrounding the blood vessels. Verapamil, a calcium channel blocker, also FDA-approved for cerebral artery vasospasm: is thought to act by blocking the L-type calcium channels on the cell membrane from opening following membrane depolarization after insult. Finally, lubeluzole, also FDA-approved, is proposed to work as an NMDA modulator inhibiting the release of glutamate and nitric oxide synthase and blocking sodium and calcium channels. Through our stroke model we were able to demonstrate that each drug(s) showed a significant decrease in infarct volume and improved functional recovery while simultaneously minimizing potential systemic side effects suggesting that our stroke model may improve the preclinical validation of potential stroke therapies and help bridge the bench to bedside divide in developing new stroke therapies.

No description available.

Title: Functional consequences of perinatal hypoxia-ischemia in rat Objectives: The aim of this diploma thesis is to design a set of behavioral tests which provide an effective assessment of motor and cognitive-behavioural deficits in adults rats after experimental hypoxic-ischemic insult during the perinatal period (P7). Supposed benefit is to establish a model of motor and cognitive-behavioural abilities of individuals after this procedure. Methods: The present thesis has a theoretical-empirical character. The practical part describes how the experiment was performed. 32 long Evans Rats were randomly devided into two groups: experimental group (HIE) and control group (Ctrl). The method to produce hypoxic-ischemic brain damage in the 7 day-old rats consisted of right common carotid ligation followed by systemic hypoxia by the inhalation of 8% oxygen and 92% nitrogen. The adult animals (55-75 days old) were tested by the following list of behavioral tests: Bar holding test, Rotarod test, Ladder rung walking test, Reaching test, Open field test and Morris water maze test. Sigma Plot and Microsoft Excel 2010 were the programs used for statistical analysis. Results: Results of Open field test, Ladder rung walking test and Morris water maze test confirmed that hypoxic-ischemic insult affects the...

<p>Intricate neural circuits underlie all brain functions. However, these neural circuits are highly dynamic. The ability to change, or the plasticity, of the brain has long been demonstrated at the level of isolated single synapses under artificial conditions. Circuit organization and brain function has been extensively studied by correlating neuronal activity with information input. The primary visual cortex has become an important model brain region for the study of sensory processing, in large part due to the ease of manipulating visual stimuli. Much has been learned from studies of visual cortex focused on understanding the signal-processing of visual inputs within neural circuits. Many of these findings are generalizable to other sensory systems and other regions of cortex. However, few studies have directly demonstrated the orchestrated neural-circuit plasticity occurring during behavioral experience. </p> <p>It is vital to measure the precise circuit connectivity and to quantitatively characterize experience-dependent circuit plasticity to understand the processes of learning and memory formation. Moreover, it is important to study how circuit connectivity and plasticity in neurological and psychiatric disease states deviates from that in healthy brains. By understanding the impact of disease on circuit plasticity, it may be possible to develop therapeutic interventions to alleviate significant neurological and psychiatric morbidity. In the case of neural trauma or ischemic injury, where neurons and their connections are lost, functional recovery relies on neural-circuit repair. Evaluating whether neurons are reconnected into the local circuitry to re-establish the lost connectivity is crucial for guiding therapeutic development.</p> <p>There are several major technical hurdles for studies aiming to quantify circuit connectivity. First, the lack of high-specificity circuit stimulation methods and second, the low throughput of the gold-standard patch-clamp technique for measuring synaptic events have limited progress in this area. To address these problems, we first engineered the patch-clamp experimental system to automate the patching process, increasing the throughput and consistency of patch-clamp electrophysiology while retaining compatibility of the system for experiments in <i>ex vivo </i>brain slices. We also took advantage of optogenetics, the technology that enables control of neural activity with light through ectopic expression of genetically encoded photo-sensitive channels in targeted neuronal populations. Combining optogenetic stimulation of pre-synaptic axonal terminals and whole-cell patch-clamp recording of post-synaptic currents, we mapped the distribution and strength of synaptic connections from a specific group of neurons onto a single cell. With the improved patch-clamp efficiency using our automated system, we efficiently mapped a significant number of neurons in different experimental conditions/treatments. This approach yielded large datasets, with sufficient power to make meaningful comparisons between groups.</p> <p>Using this method, we first studied visual experience-dependent circuit plasticity in the primary visual cortex. We measured the connectivity of local feedback and recurrent neural projections in a Fragile X syndrome mouse model and their healthy counterparts, with or without a specific visual experience. We found that repeated visual experience led to increased excitatory drive onto inhibitory interneurons and intrinsically bursting neurons in healthy animals. Potentiation at these synapses was absent or abnormal in Fragile X animals. Furthermore, recurrent excitatory input onto regular spiking neurons within the same layer remained stable in healthy animals but was depressed in Fragile X animals following repeated visual experience. These results support the hypothesis that visual experience leads to selective circuit plasticity which may underlie the mechanism of visual learning. This circuit plasticity process is impaired in a mouse model of Fragile X syndrome. </p> <p>In a separate study, in collaboration with the laboratory of Dr. Gong Chen, we applied the circuit-mapping method to measure the effect of a novel brain-repair therapy on functional circuit recovery following ischemic injury, which locally kills neurons and creates a glial scar. By directly reprogramming astrocytes into neurons within the region of the glial scar, this gene-therapy technology aims to restore the local circuit and thereby dramatically improve behavioral function after devastating neurological injury. We found that direct reprogramming converted astrocytes into neurons, and importantly, we found that these newly reprogrammed neurons integrated appropriately into the local circuit. The reprogramming also improved connections between surviving endogenous neurons at the injury site toward normal healthy levels of connectivity. Connections formed onto the newly reprogrammed neurons spontaneously remodeled, the process of which resembled neural development. By directly demonstrating functional connectivity of newly reprogrammed neurons, our results suggest that this direct reprogramming gene-therapy technology holds significant promise for future clinical application to restore circuit connectivity and neurological function following brain injury.</p>

O fator derivado do epitélio pigmentado (PEDF) é um fator neurotrófico que possui um grande potencial trófico nos neurônios motores da medula espinal, bem como é capaz de modular o microambiente da lesão. Desta forma, analisamos a capacidade do tratamento com PEDF em promover a neuroplasticidade da medula espinal após lesão isquêmica. Ratos Wistar adultos foram submetidos à lesão medular isquêmica do tipo fototrombótica, segundo o método de Rose Bengal, na altura do 11° segmento torácico e foram imediatamente tratados com inoculação local de PEDF (grupo PEDF) ou solvente (grupo Salina). Ratos submetidos à cirurgia simulada (grupo Sham) receberam a injeção do solvente. Ao término do procedimento cirúrgico, os ratos foram submetidos a testes neurofuncionais durante 6 semanas. Após esse período, os animais sofreram eutanásia e o tecido medular foi dividido entre as técnicas de imunoistoquímica, western blot e PCR em tempo real. Foi analisada na região lombar anterior da medula espinal a modulação das CSPGs, a expressão dos fatores neurotróficos NT-3, GDNF, BDNF e FGF-2, bem como os níveis das moléculas associadas à angiogênese e apoptose (laminina e Bcl-2), das proteínas relacionadas à neuroplasticidade (MAP-2, GAP-43 e sinaptofisina) e do sistema Eph/efrina e a RhoA, que são capazes de modular o crescimento de fibras. Os resultados mostraram uma recuperação parcial e espontânea do comportamento sensório-motor dos animais que foram submetidos à lesão fototrombótica, onde o tratamento com PEDF foi capaz de potencializar alguns desses parâmetros. A análise da região lombar anterior da medula espinal, caudal à lesão, mostrou uma diminuição das CSPGs nos dois grupos lesados, o que pode ter favorecido os eventos de neuroplasticidade. O tratamento com PEDF foi capaz de promover a regulação dos fatores neurotróficos NT-3 e GDNF, diminuir a angiogênese local (diminuição da laminina) e potencializar o processo de neuroplasticidade (aumento da MAP-2) nessa região. A lesão medular isquêmica foi capaz de modular a expressão do receptor EphA4 e da efrina-B1 e o tratamento com PEDF possivelmente regulou o estado de ativação da efrina-A2 e da efrina-B3 e certamente modulou a ativação da efrina-B2. Ainda, os receptores Eph e as efrinas foram observados diferentemente nos neurônios e astrócitos. Nossos resultados confirmam a capacidade plástica da medula espinal após lesão e mostra que o tratamento com PEDF foi capaz de potencializar esse processo / The pigment epithelium derived factor (PEDF) is a neurotrophic factor that has a great trophic potential in the motor neurons of the spinal cord, and is able to modulate the lesion microenvironment. We analyzed the capacity of the treatment with PEDF to promote the neuroplasticity after ischemic spinal cord injury. Adult male Wistar rats were underwent to photothrombotic ischemic spinal cord injury, according to the Rose Bengal method, at the level of 11° thoracic segment, and were immediately treated with local injection of PEDF (PEDF group) or solvent (Saline group). Rats underwent to a sham surgery (Sham group) received solvent injection. At the end of surgery, the rats were submitted to neurofunctional tests during 6 weeks. After this period, the animals were euthanized, and the anterior lumbar region of the spinal cord tissue was submitted to immunohistochemistry, western blot and real-time PCR analyses. The inhibitory response of CSPGs, the expression of neurotrophic factors (NT-3, GDNF, BDNF and FGF-2), the molecules associated with angiogenisis and apoptosis (laminin and Bcl-2), the proteins related to neuroplasticity (MAP-2, GAP-43 and synaptophysin), as well the Eph/ephrin system and the RhoA, which is able to modulate the fibers growth, were evaluated. The results showed a spontaneous, and parcial, recovery of the sensory motor behavior of the animals that were underwent to a photothrombotic injury, and the treatment with PEDF was able to potentiate some of these parameters. The analysis of the anterior lumbar region of the spinal cord, caudally to the lesion, showed a decrease of CSPGs, which may have favored the neuroplasticity events. The treatment with PEDF was able to promote the regulation of NT-3 and GDNF, as well the reduction of laminin and the increase of MAP-2 in that region. In relation to the Eph/ephrin system, the ischemic spinal cord injury was able to modulate the EphA4 receptor and ephrin-B1 expression, and the treatment with PEDF possibly regulated the activation state of ephrin-A2 and ephrin-B3 and certainly modulated the ephrin-B2 activation. Eph receptors and ephrins have been found specifically in neurons and astrocytes. Our results confirmed the plastic capacity of the spinal cord after injury and showed that the treatment with PEDF was able to enhance this process

Introdução: O trauma é um fenômeno que cursa com lesão tecidual, sendo que o trauma cirúrgico (TC) apresenta a referida lesão como consequência de um ato cirúrgico. A isquemia seguida de reperfusão (IR) é um evento comum em várias condições patológicas, bem como em diversos procedimentos cirúrgicos, principalmente transplantes. É frequente o desenvolvimento de lesões teciduais locais e remotas após trauma e após a I/R, parte de um fenômeno conhecido como síndrome da resposta inflamatória sistêmica (SRIS), frequentemente seguida pela falência de múltiplos órgãos (FMO). Estudos provaram o envolvimento do neutrófilo em tais síndromes como resultado da ação de enzimas proteolíticas secretadas a partir de grânulos citoplasmáticos, radicais livres produzidos por explosão respiratória e citocinas liberadas após a infiltração nos tecidos. Nesse contexto, foi provado que o pré-condicionamento isquêmico (PCI), definido como curtos episódios de isquemia precedendo a IR, protege contra essas lesões, com menor ativação de neutrófilos. No entanto, o conhecimento a respeito dos mecanismos operantes nos neutrófilos após o trauma cirúrgico, a isquemia seguida de reperfusão ou o pré-condicionamento isquêmico, ainda são preliminares. Objetivo: Analisar com maior profundidade o impacto dessas condições (TC, IR e PCI) no proteoma e fosfoproteoma do neutrófilo. Métodos: Foi realizada a análise de parâmetros hematológicos juntamente com a análise proteômica e fosfoproteômica de neutrófilos de ratos submetidos a TC, IR e PCI, comparados ao grupo controle. A análise proteômica foi realizada em sistema de nLC-MS/MS orbitrap de alto desempenho, usando marcação com iTRAQ, enriquecimento de fosfopeptídios e pré-fracionamento por HILIC. A análise estatística baseada em clusters utilizando scripts em R mostrou proteínas com abundância relativa diferencial em todas as condições. Resultados: A avaliação dos parâmetros hematológicos antes e depois de TC, IR e IPC demonstrou alterações no número, forma e tamanho de linfócitos, hemácias, plaquetas e, principalmente, neutrófilos (granulócitos). Observou-se um claro aumento na contagem de neutrófilos após TC e IR, sendo que tal aumento foi prevenido pelo PCI. Um total de 393 proteínas foram determinadas como reguladas para abundância relativa entre o grupo controle e o grupo TC. A maioria das proteínas encontradas como reguladas em comum nos grupos TC e IR estão relacionadas à apoptose (caspase-3), motilidade celular (PAK2), transdução de sinal (IL-5, IL-6 e TNF) e degradação pelo sistema proteassoma no neutrófilo. Maior produção de espécies reativas de oxigênio e disfunção da migração direcional de neutrófilos (PKC-delta) com aumento do tempo de vida dos neutrófilos são eventos iniciais importantes que podem resultar em mais dano tecidual e em infecção. A análise proteômica de neutrófilos de ratos após PCI levou à identificação de 2437 grupos de proteínas atribuídos a 5 clusters diferentes, contendo proteínas de abundância relativa significativamente aumentada ou diminuída em IR e PCI. O estudo de vias desses clusters baseado no KEGG revelou aumento nas vias de fagocitose mediada por Fc-gama R, sinalização por quimiocinas, adesão focal e migração transendotelial, citoesqueleto de actina, metabolismo e diminuição nas vias ribossomais, de transporte de RNA, de processamento de proteínas. A regulação da fosforilação de proteínas após IR e PCI mostrou algumas vias como quimiocinas, Fc-gama, GPCR, migração celular e vias pró e antiapoptóticas, sendo que a via de splicing alternativo foi a que apresentou regulação mais evidente (p < 0.0001). A regulação da abundância, bem como da fosforilação, presença de motivos e de domínios levou à identificação de fosfatases, como Fgr, GRK2, PKC delta, ptpn6 e ptprc reguladas por IR, bem como stk38, pkn1, syk e inpp5d reguladas por PCI. A interação mais marcante entre proteínas foi demonstrada como sendo entre os receptores de Fgr e Ptp. Conclusão: Concluímos que as alterações causadas por TC, IR e PCI levaram a intenss alterações na abundância de algumas proteínas e em eventos de fosforilação em neutrófilos, levando ao efeito destrutivo observado após a IR e ao efeito protetor consequente ao PCI / Introduction: Trauma is a phenomenon that involves tissue injury, whereas the surgical trauma (ST) has such injury as a consequence of a surgery. Ischemia reperfusion is common event in many surgical procedures, especially in transplants, as well as in many pathological conditions. Local and remote tissue injuries usually develop after trauma and ischemic reperfusion, part of a phenomenon known as systemic inflammatory response syndrome, frequently followed by multiple organ failure (MOF). Studies have proven the involvement of the neutrophil in all these injuries as a result of proteolytic enzymes secreted from cytoplasmic granules, free radicals produced by respiratory burst, cytokines released after tissue infiltration. In that context, ischemic preconditioning (IPC), that are short episodes of ischemia before ischemia reperfusion, was proved to be protective against these injuries with less activation of neutrophils. However the knowledge about the underlying mechanism operating in the neutrophil after surgical trauma, ischemia reperfusion and preconditioning is preliminary. Objective: To deeply analyze the impact of these conditions (ST, IR and IPC) on the neutrophil proteome and phosphoproteome. Methodology: We did hematological analysis along proteomics and phospho proteomics through high throughput nLC-MS/MS analysis by orbitrap using iTRAQ labeling, phospho peptide enrichments, and HILIC pre-fractionation. Neutrophils from control, ST, IR and IPC conditions after extraction were processed for proteomic analysis. Statistical package using R based on cluster analysis led to the detection of differentially regulated proteins in all conditions. Results: The evaluation of the hematological parameters before and after ST, IR or IPC on blood cells stated alteration in size, number and shape of lymphocytes, RBCs, platelets and specially neutrophils (granulocytes). In the analysis, a clear increase in neutrophil count after ST and IR with such increase prevented by IPC. A total of 393 proteins were found differentially regulated between control and trauma groups. Most of the common proteins found regulated in trauma and IR seem to be related to apoptosis (caspase-3), cell motility (PAK2) and signal transduction in IL5, IL6 and TNF and proteasomal degradation in neutrophil. Higher oxygen species production and dysfunction of directional neutrophil migration (PKC delta) with increase in the life span of neutrophils are early important events that can finally result into more tissue damage and infection. The total proteomic analysis of rat neutrophils after IPC led to the identification of 2437 protein groups assigned to five different clusters with significantly up and downregulated proteins in IR and IPC. Cluster based KEGG pathways analysis revealed up-regulation of chemokine signaling, focal adhesion, leukocyte transendothelial migration, actin cytoskeleton, metabolism and Fc gamma R mediated phagocytosis, whereas downregulation in ribosome, spliceosome, RNA transport, protein processing in endoplasmic reticulum and proteasome, after intestinal ischemic preconditioning. The phosphoregulated proteins containing domains and motifs in the regulated peptides after IR and IPC led to the identification of some of important players such as chemokine, Fc gamma, GPCR, migration and pro/anti-apoptotic pathways. The phosphoproteins from alternative splicing was the pathway presenting the most remarkable regulation with a p-value of 0.0001. The regulation in expression as well as in phosphorylation, the presence of motifs and domains led to the identification of kinases and phosphatases including Fgr, GRK2, PKC delta, ptpn6 and ptprc in neutrophils after IR whereas stk38, pkn1, syk, and inpp5d in neutrophil due to IPC. The highest protein-protein interaction was shown by Fgr and Ptp receptors. Conclusion: We concluded that the changed stimulus produced after ST, IR and IPC led to the huge alteration in proteins expression and phosphorylation events in the neutrophil proteome as mentioned in our work, that leads to final destructive and protective phenotype of neutrophils respectively