Traumatic brain injury (TBI) results in over 50,000 deaths and 80,000 disabilities each year. Current treatment strategies aim to alleviate acute disturbances, but are not able to address the chronic disorders associated with TBI. Neural transplantation is one potential treatment that will provide multifaceted sustained therapy to degenerating injured tissue. Transplantation of multipotent neural stem cells (NSCs) has been shown to enhance functional recovery in TBI models; however, poor cell survival and integration with host tissue potentially restrict the efficacy of such transplants. This limitation may be due to the absence of inherent NSC pro-survival cues (e.g., cell-ECM interactions). Furthermore, the neural injury environment presents cell death factors to transplanted NSCs. It is hypothesized that a 3-D scaffold presenting specific CNS adhesive moieties will enhance donor cell survival and promote differentiation and migration. This project encompassed material development and in vitro characterization. Results highlighted the importance of ligand tethering chemistry and density and also the mechanical integrity of cell scaffold systems. Furthermore, the developed scaffold provides a controlled microenvironment to assess the influence of LN on NSC survival, migration, and differentiation. Lastly, co-delivering NSC with the MC-LN tissue engineered scaffold into a mechanically injured neural co-culture test-bed or in vivo TBI model confirmed the importance of ECM cues for NSC survival and migration, respectively.

Although spinal cord injury (SCI) is a debilitating condition that presents a large socioeconomic problem in the United States, there is currently no treatment that reliably reduces morbidity and mortality. Current research is aimed at identifying mechanisms involved in the pathophysiology of SCI and using this knowledge to develop rational treatments. We have observed plasma membrane compromise in the acute (within 10 minutes), sub-acute (3 days), and chronic phases (5 weeks) in a rat model of contusion SCI and postulate that it negatively affects neurological outcome. Holes/tears in the plasma membrane were assessed with a dye exclusion assay, in which a fluorescent cell-impermeant dye was injected into the cerebrospinal fluid prior to sacrifice; therefore, cellular uptake of the dye is indicative of plasma membrane compromise. As early as 10 minutes after SCI, widespread uptake of permeability markers was evident in neuronal cell bodies as well as axonal projections. The number of permeable cells and the size of the membrane breaches (measured by using permeability markers of various sizes) varied with distance from the injury site, with larger disruptions located closer to the epicenter. Greater cellular uptake was observed when the impact force was increased (200 > 150 > 100 kdyn > sham). At longer time points (3 days and 5 weeks), substantial permeability marker uptake was observed in axons but not in cell bodies. Cells with increased permeability displayed a variety of pathomorphological alterations, including swelling, blebbing, retraction bulb formation, neurofilament loss, and fragmentation, suggesting that increased plasma membrane permeability is detrimental to cell survival and function. We therefore investigated a clinically-relevant treatment strategy designed to restore plasma membrane integrity. Animals were treated with citicoline, a molecule utilized in the endogenous synthesis of phosphatidylcholine (the major membrane component in mammalian cells). Citicoline has been shown to be beneficial in numerous studies of neurological disease, improving overall outcome by increasing phospholipid synthesis and attenuating phospholipid destruction (by reducing phospholipase A2 activity). However, these mechanisms have not been explored in a model of SCI. When compared to injured animals receiving vehicle (saline) injections, citicoline treatment after SCI did not have a statistically significant effect on cytoplasmic PLA2 activity (at 24h post-injury), the density of permeable axons (at 3 days post-injury), or the lesion volume (at 3 days post-injury). Since citicoline may improve neurological outcome after SCI through mechanisms we did not directly assess, we then conducted a longer-term study to evaluate the overall efficacy of citicoline treatment in terms of longer-term functional and histological consequences. Citicoline did not have a biologically significant effect on behavioral recovery (evaluated during open field locomotion, grid walk and hyperalgesia testing weekly for up to 5 weeks post-injury) or lesion volume (at 5 weeks post-injury). The lack of citicoline-mediated effect may be attributed to experimental parameters (e.g., dosing or sensitivity of outcome measures) or biological inefficacy. Although we were not able to demonstrate that citicoline improves outcome after SCI, the finding that plasma membrane damage occurs in a persistent fashion and is associated with pathophysiological cellular alterations may provide fundamental knowledge necessary for developing treatments targeted at membrane repair. Future work examining the complex mechanisms causing prolonged membrane damage after SCI and evaluating strategies for manipulating these pathways (potentially using citicoline in combination with other pharmacological agents) may lead to a clinically effective therapy.

Sialylation is an important carbohydrate modification of glycoconjugates, which introduces sialic acids (SA). The relatively large nine-carbon, negatively charged sugars are typically located at the termini of carbohydrate chains. SA's are often required for functionally important molecular and cellular interactions including virus-host interactions, tumor progression and malignancy, immune system development and function, and nervous system development and function. However, the study of sialylation in vertebrates, including man, encounters serious obstacles associated with the complexity of vertebrates' biology and limitations of available experimental approaches. Drosophila is a useful model system with many advantages including quick generation time, a large number of progeny, simplified glycosylation and neurophysiology, and ease of genetic manipulations. The primary focus of this thesis is on the functions of Drosophila melanogaster CMP sialic acid synthetase (DmCSAS) and sialyltransferase (DSiaT) in the central nervous system (CNS). A combination of genetic, immunostaining, and neurobiology approaches were used to characterize the functions of DmCSAS and DSiaT in Drosophila. This investigation revealed the expression of DmCSAS and suggested that it plays an important role in a specialized and developmentally regulated process in the nervous system of Drosophila. Further experiments examined sub-cellular localization of DmCSAS revealing that this protein has a complex mostly Golgi-associated distribution within the cell in vivo. I discovered a novel link between Drosophila sialylation and circadian rhythm regulation. I also characterized the electrophysiological phenotypes of DmCSAS mutants and compared them to the corresponding defects associated with DSiaT mutations. My experiments also revealed that the relationship between DmCSAS and DSiaT are more complex than originally thought; these genes may have independent functions while also participating in the same pathway. Taken together, these results elucidate the sialylation pathway in Drosophila and shed more light on the role of sialylation in the nervous system. My experiments provide a unique evolutionary perspective on the sialylation pathway in animals and suggest that the neural function of SA in Drosophila can be conserved in vertebrates, including humans.

The abuse of anabolic androgenic steroids (AAS) is becoming increasingly common and may result in a range of physiological as well as psychological effects such as altered behavior in terms of increased aggression, cognitive dysfunction and addictive behavior. AAS comprise testosterone and its derivatives, of which nandrolone is one of the more common. Previous studies have shown nandrolone-induced effects in male rats on peptide levels within the Substance P (SP) system and the dynorphinergic system; these effects may be linked to some of the reported behavior alterations. The studies presented in this thesis aimed to investigate the mechanisms underlying these peptide alterations and also to further investigate neuropeptidergic effects attributed to nandrolone administration. The results display significant effects on the enzymatic conversion of SP and Dynorphin A into their bioactive metabolites SP(1-7) and Leu-enkephalin-Arg6, respectively, as a result of nandrolone treatment. More profound investigations on the dynorphinergic system displayed effects on the kappa opioid receptor density in various brain regions. There was also a significant increase in the expression of the gene transcript of prodynorphin in the hippocampus, a brain region associated with cognitive processes. In addition, impaired spatial learning and memory in the Morris water maze task following nandrolone administration was encountered. The results provide further understanding regarding neuropeptidergic mechanisms underlying AAS-induced behavioral effects.

Proteins offer a prominent group of compounds which may be ubiquitously affected in disease and used as biomarkers for early diagnosis, assessing treatment or drug development. Clinical proteomics aim to screen for protein biomarkers by a comprehensive analysis of all proteins expressed in a biological matrix during a certain pathology. Characterization of thousands of proteins in a complex biological matrix is from an analytical point of view a challenging task. Hence, sophisticated methods that are sensitive, specific and robust in a high-throughput manner are required. Mass spectrometry (MS) is able to perform this to a wide extent is. A prominent source for finding protein biomarkers related to neurological diseases is the central nervous system (CNS) due to close proximity of the pathogenesis. Neuroproteomic analysis of CNS tissue samples is thus likely to reveal novel biomarkers. Cerebrospinal fluid (CSF) bathes the entire CNS and offers a good balance between clinical implementation and usefulness. Both matrices put further requirements on the methodology due to a high dynamic range, low protein concentration and limited sample amount. The central objective of this thesis was to develop, assess and utilize analytical methods to be used in combination with MS to enable protein biomarker discovery in the CNS. The use of hexapeptide ligand libraries was exemplified on CSF from patients with traumatic brain injury and demonstrated the ability to compress the dynamic range to enable protein profiling in the order of mg/mL to pg/mL. Further, a method based on cloud-point extraction was developed for simultaneous enrichment and fractionation of hydrophobic/hydrophilic proteins in brain tissue. Comparison between label and label-free MS based strategies were carried out, mimicking the true conditions with a few differentially expressed proteins and a bulk of proteins occurring in unchanged ratio. Finally, a clinical application was carried out to explore the molecular mechanism underlying the analgesic effect of spinal cord stimulation (SCS) in patients with neuropathic pain. The CSF concentration of Lynx1 was found to increase upon SCS. Lynx1, acting as a specific modulator of the cholinergic system in the CNS, may act as a potential important molecular explanation of SCS-induced analgesia.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Diss. (sammanfattning) Umeå : Univ., 2008. / Härtill 3 uppsatser.

Made available in DSpace on 2014-06-11T19:32:56Z (GMT). No. of bitstreams: 0 Previous issue date: 2010-08-28Bitstream added on 2014-06-13T19:43:50Z : No. of bitstreams: 1 miguel_tt_dr_arafo.pdf: 1534577 bytes, checksum: a92938fef6b90d6df76ee4d3b3896a21 (MD5) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Os confrontos dos animais com situações que induzem medo e ansiedade resultam em uma série de respostas comportamentais defensivas (ex. luta, fuga, imobilidade, vocalização, etc.), ativação neurovegetativa (ex. taquicardia, hipertensão, defecação, etc.), antinocicepção, além de influenciar o comportamento agressivo e aumentar a vulnerabilidade à dependência e recaída ao uso de drogas. Com base no potencial efeito ansiogênico dos neurotransmissores glutamato (via ativação do complexo receptor NMDA-óxido nítrico) e fator liberador de corticotrofina (via receptores CRF1 e CRF2), este estudo investigou o papel desses mediadores, através de injeções sistêmicas, na matéria cinzenta periaquedutal (MCP) ou no núcleo dorsal da rafe (NDR), nas respostas apontadas acima. Os seguintes modelos foram utilizados: labirinto em cruz elevado (LCE, ansiedade), injeção de formalina a 2,5% (nocicepção), conflito intruso-residente (agressão) e estresse de derrota social (dependência à cocaína). Os resultados indicaram: a) o efeito ansiogênico do agonista de receptores NMDA (N-metil-D-aspartato; NMDA) na MCP foi antagonizado pela inibição da enzima de síntese de NO, b) os efeitos ansiogênico e antinociceptivo do CRF na MCP foram via ativação de receptores CRF1 (mas não CRF2) e independentes de NO, c) os efeitos aversivo e antinociceptivo do NO (via administração de um doador de NO) na MCP mostraram-se sensíveis ao bloqueio de receptores CRF1, d) a ativação de receptores CRF2 intra-NDR reduziu o comportamento agressivo induzido pelo conflito intruso-residente, e) o tratamento sistêmico com antagonista CRF1 bloqueou a sensibilização comportamental à cocaína e atenuou o aumento do consumo da mesma induzidos pelo estresse da derrota social / Animal confrontation against fear/anxiety-induced situations results in a repertory of behavioral defensive responses (e.g., fight, flight, immobility, vocalization), neurovegetative activation (e.g., tachycardia, hypertension, defecation), antinociception, as well as affects aggressive behavior and increases animals vulnerability to addiction and relapse to drug take. Based on the potential anxiogenic effect elicited by glutamate (via activation of NMDA-nitric oxide receptor complex) and corticotropin releasing factor (via CRF1 and CRF2 receptors), this study investigated the effect of systemic, intra-periaqueductal gray (PAG) or intradorsal raphe nucleus (DRN) injections of these mediators on the above described responses. The following animal models were used: elevated plus maze (EPM, anxiety test), formalin 2.5% injection (nociceptive test), resident-intruder conflict (aggression test) and social defeat stress (to induce cocaine addiction). Results indicated that: a) the anxiogenic effect elicited by intra-PAG injection of glutamate NMDA (N-methyl-D-aspartate; NMDA) receptor agonist was antagonized by prior local infusion an NO synthase inhibitor, b) the anxiogenic and antinociceptive effects elicited by intra-PAG CRF were mediated by CRF1 (but not CRF2) receptor activation and did not depend on NO synthesis, c) the aversive and antinociceptive effects of NO production (induced by intra-PAG injection of a NO donor) were sensitive to CRF1 blockade, d) activation of the CRF2 receptor within the DRN attenuated aggressive behavior elicited by resident-intruder conflict, e) systemic treatment with CRF1 receptor antagonist inhibited cocaine behavioral sensitization and social-defeat stress-induced cocaine consumption

Le virus de la rougeole (MV) provoque une maladie sévère chez les enfants qui induit une forte immunosuppression et peut dans certains cas infecter le système nerveux central (SNC). La protéine CD150, principal récepteur cellulaire du virus, permet l’entrée du MV en se liant à l’hémagglutinine (MV-H). L’altération fonctionnelle des cellules dendritiques (DC) étant considérée comme essentielle dans l’immunopathogénèse du MV, nous avons analysé les conséquences de l’interaction de MV-H avec les DC. Nous avons développé un modèle expérimental qui nous permet d’étudier l’interaction directe entre CD150 et MV-H hors contexte infectieux. Nos résultats montrent que cette interaction provoque une diminution de l’expression des molécules de surface CD80, CD83, CD86, et HLA-DR, de la production d’IL-12 par les DC, et de la capacité des DC à stimuler la prolifération des lymphocytes T. L’interaction CD150-MV-H a inhibé la phosphorylation des protéines kinases JNK1/2 dans les DC et les lymphocytes B (LB) induite par la stimulation de CD150 par un anticorps spécifique, mais pas celle des kinases ERK1/2. Par ailleurs MV-H seule induit la phosphorylation d’Akt via CD150 dans les DC et les LB. La liaison de CD150 par MV-H a réduit la réponse inflammatoire chez les souris transgéniques exprimant CD150 humain, ce qui confirme l’effet de l’interaction de CD150 et MV-H in vivo. Nos études ont révélé l’expression de CD150 dans les tumeurs du SNC et l’existence d’une nouvelle isoforme de CD150. Cette isoforme contient un exon supplémentaire de 83 pb et est exprimée dans les cellules lymphoïdes et les DC en plus des tumeurs du SNC. Bien que l’expression de CD150 soit uniquement intracellulaire dans les cellules tumorales, elle peut représenter un nouveau marqueur pour les tumeurs cérébrales humaines. Cette étude apporte un éclairage nouveau sur le rôle immunorégulateur de CD150 et sur la diversité de ses isoformes, et ouvre ainsi de nouvelles perspectives pour leurs applications thérapeutiques. / Measles virus (MV) causes an acute childhood disease, associated in certain cases with the infection of the central nervous system (CNS). MV induces a profound immunosuppression, resulting in high infant mortality. The major cellular receptor for MV is CD150, which binds MV hemagglutinin (MV-H). As dendritic cell (DC) dysfunction is considered to be essential for the MV immunopathogenesis, we analyzed consequences of MV-H interaction with DCs. We developed an experimental model allowing us to analyze the direct CD150-MV-H interaction in the absence of infectious context. This interaction caused the downregulation of surface expression of CD80, CD83, CD86 and HLA-DR molecules and inhibition of IL-12 production in DCs. DCs also failed to activate T cell proliferation. The CD150-MV-H interaction in DCs and B cells decreased the phosphorylation of JNK1/2, but not ERK1/2 kinases, after subsequent CD150 ligation with anti-CD150 antibodies. Moreover, MV-H by itself induced Akt phosphorylation via CD150 in DCs and B cells. Engagement of CD150 by MV-H in mice transgenic for human CD150 decreased the inflammatory reaction, contact hypersensitivity response, confirming the immunosuppressive effect of CD150-MV-H interaction in vivo. Furthermore, our studies revealed the CD150 expression in CNS tumors and identified the novel CD150 isoform, containing an additional 83bp exon expressed in lymphoid cells, DCs and CNS tumors. Although its isoforms remain intracellular in tumor cells, CD150 may represent a new marker for human brain tumors. Novel mechanism of CD150-induced immunosuppression and new CD150 isoform identified in these studies shed new light on its immunoregulatory role and CD150 isoform diversity and open perspectives for their clinical applications.

Abstract The repair of thoracoabdominal aortic aneurysms carries a substantial risk of ischaemic perioperative spinal cord injury. Although several protective methods have been developed, the risk of paraplegia has not been eliminated. Moreover, aortic aneurysms, including arch aneurysms, are complex clinical challenges requiring cerebral protection with hypothermic circulatory arrest (HCA). Hypothermia lowers the rate of cerebral metabolism and allows a temporary halt of the systemic circulation. However, there is still a risk for cerebral damage and a need for additional neuroprotective methods. During the last 15 years, our research group has used a porcine model to investigate a variety of neuroprotective tools. In this thesis, an animal model was utilized to study the efficacy of remote ischaemic preconditioning (RIPC) to ameliorate ischaemic damage to the central nervous system, and to shed light on the potential mechanism. Moreover, diazoxide, the pharmacological mimetic of RIPC, was tested in the HCA animal model. In the first Study (I), RIPC showed beneficial effect on the spinal cord against ischaemic insult as recorded with motor-evoked potentials. Strikingly, the beneficial effect of RIPC was observed even before the ischaemia. In the second Study (II), some beneficial effect of RIPC was seen in the immunohistochemical analysis of the spinal cord ischemia but the result remains inconclusive. Similarly, the diazoxide-treated animals had better hemodynamic status postoperatively and mildy better antioxidant activity of the brain in the third Study (III). The fourth study (IV) was a review of the current knowledge of RIPC from the cardiovascular point of view. Our studies indicate that RIPC might be a potential adjunct for preventing neuronal ischaemic injury in the setting of thoracoabdominal aortic surgery. Our result indicates that further preclinical studies with diazoxide are required before studies can be conducted in humans. / Tiivistelmä Torakoabdominaalisen aortan aneurysman kirurginen korjaaminen sisältää riskin iskeemiselle selkäytimen vauriolle. Vaikka useita suojaavia tekniikoita on kehitetty, paraplegian riskiä ei ole saatu poistettua kokonaan. Kirurgisen korjaamisen haasteellisuus moninkertaistuu, jos aneurysma on laajentunut myös aortan kaareen. Tällöin vaaditaan hypotermista verenkierron pysäytystä (HCA). Hypotermia alentaa aivojen metabolista aktiivisuutta merkittävästi ja siten verenkierron väliaikainen pysäytys on mahdollista. Tästä huolimatta hypotermiseen verenkierron pysäytykseen liittyy riski aivokudoksen vauriolle. Meidän tutkimusryhmämme on tutkinut useita keskushermostoa suojaavia tekniikoita ja lääkeaineita viimeisen 15 vuoden aikana. Käytämme sikaa koe-eläin mallina, jota on tämänkin väitöskirjan osajulkaisuissa käytetty. Tämän väitöskirjatyön tarkoituksena on ollut tutkia sekä esialtistavan raajaiskemian (RIPC) että farmakologisen mimeetin, diazoxiden, keskushermostoa suojaavia vaikutuksia sekä niiden mahdollista vaikutusmekanismia. Ensimmäisessä osajulkaisussa esialtistava raajaiskemia paransi selkäytimen iskemian sietokykyä, mikä näkyi alaraajojen motorisissa herätepotentiaaleissa. Merkittävintä tutkimuksessa oli, että esialtistavan raajaiskemian edulliset vaikutuksen selkäytimeen oli nähtävissä jo ennen iskemiaa. Toisessa osajulkaisussa esialtistava raajaiskemialla oli nähtävissä edullisia, mutta ei varauksettomia, vaikutuksia selkäytimen iskemian immunohistokemiallisessa analyysissä. Kolmannessa osatyössä diazoxidin vaikutukset iskemiaa vastaan nähtiin parempana hemodynaamisena tilana ja antioksidatiivisen aktiivisuuden lisääntymisenä aivoissa, mutta tulos on myös tulkinnanvarainen. Neljäs osajulkaisu kokosi tämän hetken tietämyksen esialtistavasta raajaiskemiasta. Tutkimuksissamme osoitimme, että esialtistava raajaiskemiassa on potentiaalia tulla yhdeksi välineeksi keskushermoston iskemiaa vastaan torakoabdominaalisen aortan kirurgiassa. Lisäksi diazoxidin mahdolliset neuroprotektiiviset vaikutukset vaativat lisää koe-eläintutkimuksia ennen ihmiskokeisiin siirtymistä.