Thèses sur le sujet « Blood Brain Barriers »

The passage of peptides across the blood-brain or blood-cerebrospinal fluid barrier is extremely limited. Peptides can be hindered from entering the central nervous system due to the hydrophilic nature of peptides and their susceptibility to enzymatic degradation by various peptidases. This limitation can be overcome through chemical modifications of opioid peptides with the goal of increasing biological stability and blood-central nervous system permeation. In the present studies, an in vitro bovine brain microvessel endothelial cell model of the blood-brain barrier was characterized both functionally and enzymatically. This primary culture model was found to be reflective of the in vivo blood-brain barrier in reference to predicting a peptides relative lipophilicity. Bovine brain microvessel endothelial cells were also found to be quite active enzymatically as far as the peptidases known to be involved in the degradation of methionine enkephalin. The conformationally stable analog of methionine enkephalin, DPDPE, was also characterized for its ability to enter the CNS using the in situ brain perfusion technique. DPDPE was found to enter the brain by both saturable and non-saturable uptake mechanisms. Chlorohalogenation was also found to significantly improve the central nervous system entry as well as biological stability of a potent opioid agonist, biphalin. In addition, the mu-opioid receptor selective antagonist, CTAP, was also evaluated for its ability to enter the CNS. The amount of CTAP that crossed both the blood-brain and blood-cerebrospinal fluid barrier was quantitatively comparable to the mu-selective agonist, morphine. Biphalin was found to enter both spinal and supra-spinal sites that have been shown previously to express mu and delta opioid receptors. In situ brain perfusion experiments identified a saturable component that contributes to the brain entry of [¹²⁵125I-Tyr¹]biphalin. Further experiments revealed that [¹²⁵125I-Tyr¹]biphalin was entering the CNS by the large neutral amino acid transporter and not by the leucine enkephalin uptake system or DPDPE transport system. This research has provided important preliminary work for the characterization of peptide transport into the brain. The importance of using neuropharmaceutical drug delivery vectors in modern medicine needs attention for the evolution of successful drug design targeted for CNS entry.

Thesis advisor: Cyril P. Opeil
The physiology of the vasculature in the central nervous system (CNS) which includes the blood-brain-barrier (BBB) and other factors, prevents the transport of most anticancer agents to the brain and restricts delivery to infiltrating brain tumors. The heterogeneous vascular permeability in tumor vessels (blood-tumor barrier; BTB), along with several other factors, creates additional hurdles for drug treatment of brain tumors. Different methods have been used to bypass the BBB/BTB, but they have their own limitations such as being invasive, non-targeted or requiring the formulation of new drugs. Magnetic Resonance Imaging guided Focused Ultrasound (MRIgFUS), when combined with circulating microbubbles, is an emerging noninvasive method to temporarily permeabilize the BBB and BTB. The purpose of this thesis was to use this alternative approach to deliver chemotherapeutic agents through the BBB/BTB for brain tumor treatment in a rodent model to overcome the hinderances encountered in prior approaches tested for drug delivery in the CNS. The results presented in thesis demonstrate that MRIgFUS can be used to achieve consistent and reproducible BBB/BTB disruption in rats. It enabled us to achieve clinically-relevant concentrations of doxorubicin (~ 4.8±0.5 µg/g) delivered to the brain with the sonication parameters (0.69 MHz; 0.55 MPa; 10 ms bursts; 1 Hz PRF; 60 s duration), microbubble concentration (Definity, 10 µl/kg), and liposomoal doxorubicin (Lipo-DOX) dose (5.67 mg/kg) used. The resulting doxorubicin concentration was reduced by 32% when the agent was injected 10 minute after the last sonication. Three weekly sessions of FUS and Lipo-DOX appeared to be safe in the rat brain, despite some minor tissue damage. Importantly, the severe neurotoxicity seen in earlier works using other approaches does not appear to occur with delivery via FUS-BBB disruption. The resuls from three weekly treatments of FUS and Lipo-DOX in a rat glioma model are highly promising since they demonstrated that the method significantly inhibits tumor growth and improves survival. Animals that received three weekly sessions of FUS + Lipo-DOX (N = 8) had a median survival time that was increased significantly (P<0.001) compared to animals who received Lipo-DOX only (N = 6), FUS only (N = 8), or no treatment (N = 7). Median survival for animals that received FUS + Lipo-DOX was increased by 100% relative to untreated controls, whereas animals who received Lipo-DOX alone had only a 16% improvement. Animals who received only FUS showed no improvement. No tumor cells were found in histology in 4/8 animals in the FUS + Lipo-DOX group, and only a few tumor cells were detected in two animals. Tumor doxorubicin concentrations increased monotonically (823±600, 1817±732 and 2432±448 ng/g) in the control tumors at 9, 14 and 17 days respectively after administration of Lipo-DOX. With FUS-induced BTB disruption, the doxorubicin concentrations were enhanced significantly (P<0.05, P<0.01, and P<0.0001 at days 9, 14, and 17, respectively) and were greater than the control tumors by a factor of two or more (2222±784, 3687±796 and 5658±821 ng/g) regardless of the stage of tumor growth. The transfer coefficient Ktrans was significantly (p<0.05) enhanced compared to control tumors only at day 9 but not at day 14 or 17. These results suggest that FUS-induced enhancements in tumor drug delivery for Lipo-DOX are relatively consistent over time, at least in this tumor model. These results are encouraging for the use of large drug carriers, as they suggest that even large/late-stage tumors can benefit from FUS-induced drug enhancement. Corresponding enhancements in Ktrans were found variable in large/late-stage tumors and not significantly different than controls, perhaps reflecting the size mismatch between the liposomal drug (~100 nm) and Gd-DTPA (molecular weight: 938 Da). Overall, this thesis research provides pre-clinical data toward the development of MRIgFUS as a noninvasive method for the delivery of agents such as Lipo-DOX across the BBB/BTB to treat patients with diseases of the central nervous system
Thesis (PhD) — Boston College, 2014
Submitted to: Boston College. Graduate School of Arts and Sciences
Discipline: Physics

2006/2007
INTRODUZIONE A basse concentrazioni la bilirubina non coniugata (unconjugated bilirubin, UCB) prodotta dalla degradazione dell’emoglobina, sembra essere un potente anti-ossidante, mentre è estremamente dannosa ad alte concentrazioni, causando encefalopatia nei neonati con severo ittero. Il 70% dei bambini che presentano kernittero muoiono entro sette giorni di vita, mentre il 30% dei sopravvissuti manifesta irreversibili conseguenze come sordità, ritardo mentale e danni cerebrali permanenti. L’encefalopatia dovuta ad alti livelli di bilirubina rappresenta oggi la maggior causa di riammissione ospedaliera nei neonati entro il primo mese di vita. Storicamente gli studi riguardanti le modalità di ingresso della bilirubina nel sistema nervoso centrale si sono concentrati sulla barriera emato-encefalica (blood brain barrier, BBB), costituita dai microvasi e dai capillari del cervello (micro vessels, MV). Tali studi hanno dimostrato come solamente la bilirubina non coniugata e non legata all’albumina del sangue, definita “bilirubina libera” (free bilirubin, Bf) sia capace di attraversare le membrane cellulari e diffondere nel tessuto. Tuttavia i microvasi non sono l’unica interfaccia sangue-tessuto presente nel cervello. Una seconda barriera è costituita dai plessi coroidei (CP). Questi, collocati nei ventricoli del cervello, mediano il passaggio delle molecole dal sangue al liquido cefalorachidiano e viceversa, posseggono una ampia superficie di scambio, il più alto flusso sanguigno del sistema nervoso centrale ed un fenotipo barriera meno restrittivo rispetto ai microvasi del parenchima. L’ingresso della bilirubina nel cervello sembra essere attivamente controllato da due trasportatori appartenenti alla famiglia delle “ATP dependent transporters”, Mrp1 e Pgp. Tali trasportatori potrebbero mantenere bassa la concentrazione della bilirubina limitandone l’ ingresso a livello di barriere o agendo direttamente a livello delle cellule del parenchima. Nonostante l’ impatto di questi trasportatori sulla disponibilità nel sistema nervoso centrale non solo della bilirubina ma egualmente di atre molecole potenzialmente tossiche, così come dei principi attivi, la loro espressione e localizzazione nelle interfacce sangue-cervello non sono del tutto chiare. Per tali motivi il lavoro di questi tre anni di tersi è stato incentrato a: Ia) chiarire il livello di espressione proteica relativa di Mrp1 e Pgp nelle due principali barriere cerebrali, la BBB (blood brain barrier, barriera emano encefalica) e la BCSFB (blood CerebroSpinal Fluid barrier, barriera emato liquorale); Ib) Definire l’andamento della loro espressione nel corso dello sviluppo post-natale in situazione fisiologica. II) Valutare l’effetto di elevati livelli serici di bilirubina sull’espressione di Mrp1 e Pgp nelle barriere emato encefaliche, come prima linea di difesa verso la bilirubina nel kernittero. Per raggiungere questo secondo obiettivo abbiamo utilizzato il ratto Gunn, considerato il modello in vivo per la sindrome di Crigler-Najjar e il kernittero. I ratti Gunn presentano elevati livelli di bilirubina serica ed un quadro clinico simile a quanto si riscontra nell’uomo. L’iperbilirubinemia, nel ratto, è dovuta ad una mutazione nell’enzima responsabile della coniugazione del pigmento, passaggio fondamentale per la sua successiva eliminazione. Nell’omozigote (jj) la bilirubina totale nel sangue (TBS) è molte volte più alta che nell’eterozigote (Jj) in cui l’allele non mutato codifica per l’enzima nella sua forma attiva, sufficiente a mantenere livelli di bilirubina normali. RISULTATI Ia) Attraverso una quantificazione relativa dell’ espressione proteica, ottenuta tramite Western blot, abbiamo dimostrato una espressione speculare dei due trasportatori nelle interacce sangue cervello. Mentre i microvasi sono caratterizzati dalla forte espressione di Pgp, ed i livelli di Mrp1 sono 15-20 volte inferiori rispetto ai plessi, questi ultimi presentano una elevata espressione di Mrp1 ed una quasi completa assenza di Pgp. Per quanto riguarda l’espressione di Mrp1 nei plessi coroidei (CP), abbiamo potuto evidenziare una differenza, con la massima espressione nel plesso del 4° ventricolo rispetto ai ventricoli laterali. Tramite immunofluorescenza abbiamo poi evidenziato per entrambe i trasportatori una localizzazione lato sangue, con Pgp luminale nei vasi e Mrp1 baso-laterale nel plessi coroidei. Ib) Anche l’andamento dell’espressione durane lo sviluppo post-natale differisce. Mentre Mrp1 è sin dalla nascita (2 giorni di vita) altamente espresso in entrambe le barriere, Pgp è inizialmente espresso a livelli più bassi (4,6 volte meno) rispetto all’ adulto (60 giorni). Contemporaneamente anche la densità dei vasi nel parenchima aumenta. II) Nel modello iperbilirubinemico rappresentato dal ratto Gunn, la TBS (jj) e molte volte più alta che nell’ eterozigote (Jj) e tale differenza permane per tutto l’arco di tempo esaminato (0-60 giorni dalla nascita). Al contrario la bilirubina libera (calcolata) è elevata solo nelle prime due settimane di vita, quando il rapporto bilirubina-albumina nel sangue è superiore all’unità. Poi, il rapido aumento della concentrazione ematica di albumina determina un significativo calo della Bf. Mentre l’analisi degli effetti (macroscopici) dell’iperbilirubinemia sullo sviluppo degli emisferi cerebrali non evidenzia differenze tra Jj e jj; in questi ultimi la crescita del cervelletto è severamente inibita. Già a 17 giorni di vita l’ipoplasia del cervelletto si manifesta con una differenza nel peso del 50% nei jj rispetto agli animali normo bilirubinemici di pari età. Durante tale periodo anche l’espressione dei due trasportatori nelle barriere è modificata. L’espressione proteica di Pgp nella BBB degli animali iperbilirubinemici è aumentata ad ogni età presa in esame. Tuttavia tale incremento non modifica in maniera importante la quantità del trasportatore nei MV durante lo sviluppo post natale, rimanendo quindi poco espresso (5 volte meno rispetto all’adulto) almeno fino ai 17 giorni di vita. Contemporaneamente la presenza Mrp1 nella BCSFB è inibita. Già a 9 giorni nel plesso del 4° ventricolo Mrp1 è il 50% rispetto al controllo (pari età, Jj). Anche se nei plessi dei ventricoli laterali l’inibizione dell’espressione è inferiore, nell’insieme la quantità di Mrp1 è fortemente ridotta negli animali iperbilirubinemici. Contrariamente, nei ratti Jj, Mrp1 ha un andamento simile a quello descritto nella sezione (Ib). CONCLUSIONI I risultati da noi ottenuti sottolineano importanti differenze tra le due barriere. La barriera emato-encefalica si sviluppa durante il primo periodo post-natale, in un ambiente caratterizzato dalla forte presenza di membrane cellulari. Similarmente l’espressione di Pgp è inizialmente bassa ed incrementa molto durante lo sviluppo post-natale. Al contrario I plessi coroidei appaiono precocemente in età embrionale, contribuiscono allo sviluppo del cervello e posseggono il più alta espressione di enzimi di fase II, coinvolti nel metabolismo di potenziali sostanze tossiche, del cervello. Un alto livello di Mrp1 sin dalla nascita suggerisce un suo coinvolgimento nel trasporto di qualche sostanza importante nello sviluppo del cervello o in un suo precoce coinvolgimento nel mantenimento dello stato ossido riduttivo, o nell’eliminazione di metabolici dal sistema nervoso centrale. Elevati livelli di bilirubina, come nel modello Gunn, modulano sia l’espressione di Pgp nella BBB, che di Mrp1 nella BCSFB. Tuttavia l’incremento nell’espressione di Pgp nei microvasi non sembra essere sufficiente a contrastare efficacemente l’ingresso della bilirubina libera, molto elevata fino al 17 giorno di vita. La simultanea riduzione di Mrp1 nei plessi coroidei, può facilitare l’ingresso o ridurre l’efflusso della bilirubina nel liquido cefalo rachidiano, consentendo l’accumulo e conseguente danno dei tessuti esposti.
................................................................ ....................... .. .BACKGROUND The unconjugated bilirubin (UCB), a heme degradation product, has been suggested to be a potent antioxidant at low concentration while it seems to be extremely dangerous at higher concentrations, causing encephalopathy in severely jaundiced neonates. Around 70% of children with kernicterus die within seven days, while the 30% survivors usually suffer irreversible sequels, including hearing loss, paralysis of upward gaze, mental retardation, and cerebral palsy with athetosis. Bilirubin encephalopathy is actually the leading cause of hospital readmission of newborns within the first month after birth. Historically the studies concerning the bilirubin entry the central nervous system have focused on the blood brain barrier (BBB), located at the level of the endothelial cells forming the brain micro vessels (MV), leading to the “free bilirubin theory”. It consists in the idea that only the free unconjugated bilirubin, the part of bilirubin exceeding the binding ability of the serum albumin, is able to cross the cell membranes and diffuse in tissue. In brain a second blood brain barrier is present. It is located at the level of the epithelial cells forming the choroids plexuses, between the blood and the cerebrospinal fluid (blood-cerebrospinal fluid barrier, BCSFB). Despite the largest surface area available for the exchanges, the high blood flux, the strategically position between two circulating fluids and the more leaky phenotype, limited studies have been made concerning its role in limiting the bilirubin entry the brain. Two ATP dependent transporters, the Multidrug Resistance-associated Protein 1 (Mrp1) and the MultiDrug resistance Protein (Pgpor MDR1), appear to be actively involved in UCB trafficking. The transporters play an important role in keeping extra cellular bilirubin concentration, such as potentially toxic compounds, below toxic levels by limiting the entry of UCB from blood to brain, or else in controlling intracellular bilirubin levels in parenchyma cells. Despite the importance of Mrp1 and Pgp on BBI their pattern of expression and cellular localization remains still unsettled. Based on these considerations - The first aim of the thesis was clarify the relative protein expression of these transporters at the two major BBI protecting the brain from toxic insults (Ia), and to identify their post-natal developmental profile of expression and cellular localisation (Ib). Similarly, no data about the Mrp1 and Pgp expression on BBI during the bilirubin encephalopathy are available. - The second aim of the thesis was investigate a relation between the high level of blood bilirubin and Mrp1 and Pgp expression in brain barriers in vivo using the Gunn rat (II), in witch the symptoms closely correlate to the human kernicterus and Crigler-Najjar syndrome type I. In this animal model, a mutation in the enzyme responsible for the conjugation and subsequent elimination of bilirubin, leads to the total absence of the enzymatic activity in the homozygous animals (jj), causing a severe life long hyperbilirubinemia. In the heterozygous Gunn rats (Jj), the enzymatic activity, until if reduced, is present and result in normal serum bilirubin levels. RESULTS Ia) By quantitative Western blot, we have demonstrated a mirroring expression of the two transporters at the blood brain interfaces in the adult rat. On the BBB the Pgp is strongly expressed and the Mrp1 amount is 15-20 times lower than in CPs. At the contrary, the CPs are characterized by the high expression of Mrp1, with a difference between the lateral ventricle (LV) and the 4th ventricle (4thV) CP, the former being a lower Mrp1 expression than in the last. In both LV and 4thV CPs, Pgp is virtually absent. By immunofluorescence we revealed that both ABC transporters are located at the blood side, the Pgp luminal on MV, and Mrp1 basal on CPs. Ib) With respect to the post-natal development, the Mrp1 expression is high since the early post-natal age and do not change significantly from birth to adult life in both barriers. By contrast, Pgp expression is weak a P9 and increase 4.6 fold with maturation on MV. Synchronously the density of Pgp stained MV in parenchyma seems increasing. II) In the homozygous Gunn rat (jj) the total bilirubin in serum is several time higher than in the heterozygous (Jj) animals all life long. By contrast the (calculated) free bilirubin is extremely elevated until the first week of life, when the bilirubin-albumin ratio exceed the unit, then drop due to the developmental increasing albumin concentration in blood. While no differences in Cx weight have been found between Jj and jj rat at every postnatal age, the cerebellum development is strongly impaired by the bilirubin toxic effect, displaying a Summarymarked hypoplasia, with about the 50% of weight loss respect the Jj control at 17 days after the birth. Concerning the ABC transporters, the differential pattern of expression between blood brain interfaces is maintained. But, in jj Gunn rats, the Pgp expression at the BBB is up-regulated at every post natal age analysed, also if this increase do not seems to be sufficient to confer protection at list until P17, when the amount of the transporter in the MV is about 5 times lower than in adult (P60). At the same time the Mrp1 expression on the BCSFB is down regulated. Since P9 the amount of Mrp1 in the 4thV CP of jj rats drops around to the 50% respect the amount in the littermates. In the LV CP the decrease is less marked, but in any case the Mrp1expression in both CPs is strongly impaired. This down regulation seems to be post-transcriptional. In the Jj animals, the Mrp1 relative expression is already high in both plexuses at early postnatal stages. A significant difference was noted only between LV CP (76%) and 4thV CP at P60 (100%). CONCLUSIONS All together these results indicate that the two barriers differ: The BBB develops after the birth and is surrounded by the lipid rich parenchyma environment, in agreement with the transporter preference for the lipid compounds and the strong post-natal developmental increase of the Pgp amount on MV. The CPs develops early in the foetal life, are involved in the guidance of the brain development and posses the highest phase II metabolising enzymes in the brain. The Mrp1 amount in CPs is similar to the adult level since the birth and may be involved in the transport of some compounds important in the brain development, in the detoxification or in the maintenance of the redox state (GS- sulfo- conjugates, LC4, etc.). In Gunn rats, as model for Kernicterus and Crigler-Najjar syndrome type I, the Pgp offered protection is not sufficiently modulate until P17, when the amount of the free bilirubin is elevated and could cross the brain barriers. The simultaneously down regulation of Mrp1 at the BCSFB may facilitate the entry of the bilirubin or strongly impair their clearance in the central nervous system, leading to the accumulation in brain and subsequent damage of tissue.
1974

Bibliography: leaves 318-367. Examines the blood-brain barrier in normal and pathological conditions induced by intravascular and extravascular insults. Intravascular insults were induced by administration of Clostridium perfringens prototoxin; extravascular insults were induced by an impact acceleration model for closed head injury to induce traumatic brain injury. Also examines the integrity of the blood-brain barrier ultrastructurally and by its ability to exclude endogenous and exogenous tracers. Also studies the expression of 2 blood-brain barrier specific proteins, endothelial barrier antigen (EBA) and glucose transporter 1 (GLUT1)

During development, netrin guidance cues control cell motility and cell adhesion. Cell-adhesion between endothelial cells at the blood-brain barrier makes the endothelium impermeable to blood-derivatives and immune cells. To establish and maintain this barrier during development, and adulthood, and as well as during disease, brain endothelial cells must develop and sustain these strong adhesive contacts, through expression of tight junction molecules. However, we do not know whether netrins support inter-endothelial cell adhesion at the blood-brain barrier. Given this, we hypothesize that netrin tightens the blood-brain barrier during development, adulthood, and protects it during disease. Methods: To test this, we used both human adult primary brain-derived endothelial cells and newborn netrin-1 knockout mice and evaluated netrin's effect on inter-endothelial cell adhesion and barrier permeability. We also assessed netrins' therapeutic potential to maintain the barrier and limit immune cell infiltration into the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE). Results: Our results demonstrate that brain endothelial cells express netrins where they function in three ways. They help to form a tighter blood-brain barrier during development. They also maintain and protect the adult barrier by increasing the expression of endothelial junction molecules, thus promoting inter-endothelial adhesion and reducing protein leakage across the barrier. Netrins also reduce blood-brain barrier breakdown and diminish initial myeloid cell infiltration into the brain and spinal cord during EAE, which delays disease onset and ameliorates disease severity. However, during the chronic phase of EAE, netrin-1 treated mice have higher numbers and more activated T cells in their CNS and exhibit an ataxic gait and limb spasticity. Discussion: We conclude that netrins enhance BBB stability, but have dual functions on immune responses during neuroinflammatory disease. These findings favour the hypothesis that if netrin function was to be manipulated as a therapeutic, early short-term approaches would likely be the most effective.
Au cours du développement, les molécules de la famille des nétrines contribuent à la morphologénèse des organes en contrôlant la motilité et l'adhérence cellulaire. L'adhérence cellulaire entre les cellules endothéliales est une caractéristique importante de la barrière hémato-encéphalique (BHE), ce qui rend l'endothélium imperméable aux molécules sanguines et aux cellules immunitaires. Pour établir et maintenir cette barrière au cours du développement, à l'âge adulte et au cours de la maladie, les cellules endothéliales du cerveau doivent développer et maintenir ces contacts adhésifs en exprimant des molécules de jonction serrées. Cependant, nous ne savons pas si les molécules de la famille des nétrines influencent l'adhérence cellulaire inter-endothéliale de la BHE. Nous avons donc émis l'hypothèse que les nétrines resserrent la BHE au cours du développement, à l'âge adulte, et la protège au cours de la maladie.Méthodes: Pour valider notre hypothèse, nous avons utilisé des cellules endothéliales primaires dérivées des cerveaux humains adultes ou des cerveaux de souris nouveau-nés déficientes en nétrine-1 et évalué l'effet de la nétrine sur l'adhésion cellulaire endothéliale et inter-perméabilité de la barrière. Nous avons également évalué le potentiel thérapeutique des nétrines a restaurer la barrière et l'infiltration de cellules immunitaires limite dans le système nerveux central (SNC) pendant encéphalomyélite allergique expérimentale, un modèle animal de sclérose en plaques. Résultats: Nos résultats démontrent que les nétrines sont exprimées par les cellules endothéliales du cerveau, exprimes nétrines. Au cours du développement les nétrines aident à assurer l'étanchéité de la BHE. Chez les adultes, ils maintiennent et protègent la barrière adulte en augmentant l'expression des molécules de jonctions serrées, favorisant ainsi l'adhérence inter-endothéliale et diminuant les fuites de protéines à travers la BHE. Dans la pathologie de l'EAE, le rôle des nétrins diffère en fonction de la phase de la maladie. Au cours de la phase aigue, les nétrines atténuent la perte de l'intégrité de la BHE et diminuent l'infiltration des cellules myéloïdes dans le SNC. Ceci retarde l'apparition de la maladie et réduit sa sévérité. Au cours de la phase chronique de l'EAE, les souris traitées avec netrin-1 ont un plus grand nombre des cellules T activées dans leurs SNC et présentent une démarche ataxique ainsi qu'une spasticité des membres. Discussion: Nous concluons que les nétrins améliorent la stabilité de la BHE. Ces résultats suggèrent que les nétrines peuvent être envisagée comme agent thérapeutique dans les maladies neuroinflammatoire. Dans ce cas une approche précoce et à court terme serait probablement plus efficace.

Thesis (S.M.)--Massachusetts Institute of Technology, Dept. of Nuclear Science and Engineering, 2007.
Includes bibliographical references (p. 53-56).
Selective vascular irradiation enables the critical examination of the vasculature and its role in the onset of late radiation effects. It is a novel approach to expose the endothelial cells to much higher levels of ionizing radiation relative to normal cells by utilizing the boron neutron capture reaction. When boron-containing compounds are restricted to the lumen of the blood vessel, the resulting high-LET alpha and lithium particles cannot deposit their energy in the normal cells beyond the vasculature after the target is exposed to thermal neutrons. This allows for a 2- to 3-fold increase in the calculated dose to the endothelial cells. However, this technique has been criticized because there is no direct evidence that the endothelial cells receive an absorbed dose from the selective vascular irradiation. The objective of this work is to provide corroborating experimental evidence that selective vascular irradiation physically damages the endothelial cells. An established assay utilizing blood-brain barrier disruption was adopted to quantify the radiation damage to the endothelial cells in female BALB/C mice, 8-12 weeks of age. A dye that attaches to the plasma proteins in the blood and that is ordinarily kept out of the brain by the blood-brain barrier is injected into the blood supply before the irradiation, and following irradiation, damage to the vasculature will result in disruption of the blood-brain barrier that allows blood stained with the dye to enter the brain. After sacrificing, the blood in the vessel lumen is cleared by performing a trans-cardiac perfusion, and the brain is homogenized and prepared for analysis. The absorbance of the resulting supernatant of each brain sample is measured with a spectrophotometer at the optimal wavelength of the dye.
(cont.) The absorbance is related to the quantity of blood that leaked through the blood-brain barrier, which is also related to the damage caused to the vasculature from exposure to ionizing radiation. Increased leakage through the blood-brain barrier was observed for those mice exposed to selective vascular irradiation, indicating a direct relationship between the leakage through the blood-brain barrier and the 10B concentration in the blood. The most significant increase in the leakage through the blood-brain barrier (p<0.002) was observed at the highest lOB concentration in the blood (161 ppm). The compound biological effectiveness (CBE) for sulfhydryl borane (BSH) was calculated to be 0.28, which is consistent with the published value of the CBE for BSH in the rat spinal cord. This suggests that the assumptions used for calculating the absorbed doses for selective vascular irradiation are reasonable and approximate to what the endothelial cells receive.
by Rebecca L. Raabe.
S.M.

The blood-brain barrier (BBB) is located at the level of the cerebral microvasculature and is critical to maintain central nervous system (CNS) homeostasis. The tight junction (TJ) protein complexes between endothelial cells at the BBB are primarily responsible for limiting paracellular diffusion of substances from the blood to the CNS. The BBB’s functional integrity is compromised in a number of disease states which affect the CNS, suggesting BBB dysfunction causes or contributes to many diseases of the CNS. A common component of most of these diseases is oxidative stres. Oxidative stress is associated with hypoxia-reoxygenation (HR) and peripheral inflammatory pain (PIP). Both HR and PIP have been shown to compromise BBB functional integrity. Using in vivo rat models of HR and PIP, we examined the role of ROS on BBB permeability as well as the TJ protein occludin using the free radical scavenger tempol. First, we subjected rats to HR with or without pre-treatment with tempol (200 mg/kg). We showed that tempol prevents up-regulation of the cellular stress marker heat shock protein 70 at the BBB during HR. Next we showed tempol reverses HR-mediated BBB permeability increase to ¹⁴C-sucrose, a marker of BBB paracellular permeability. Tempol also attenuated changes in the structure and localization of occludin, suggesting ROS produced during HR alter occludin and lead to disruption of BBB. We then investigated whether ROS production have similar effects on occludin and BBB permeability during PIP by administering 3% λ-carrageenan into the hind paw of rats. We found tempol attenuated carrageenan-induced increase in paw edema and thermal hyperalgesia. Tempol also attenuated up-regulation of the cellular stress marker NF-κB in cerebral microvessels. Tempol significantly decreased BBB permeability to ¹⁴C sucrose during PIP. We found PIP reduces disulfide bonds in occludin oligomeric assemblies thought to be important in maintaining the structural integrity of the BBB. Tempol significantly inhibited disulfide bond reduction, suggesting ROS mediate BBB disruption during inflammatory pain by reducing occludin disulfide bonding. Taken together, these findings show the involvement of ROS during HR and PIP contributes to BBB dysfunction by altering the structure of high molecular weight occludin oligomeric assemblies.

Dans les systèmes biologiques, les barrières tissulaires permettent le transport sélectif de molécules du sang au tissu approprié. Un exemple de barrière tissulaire est la barrière hémato-encéphalique (BHE). La BHE protège le cerveau du sang et maintient l'homéostasie du microenvironnement du cerveau, ce qui est essentiel à l'activité et à la fonction neuronale. La caractérisation de cette BHE est importante, car un dysfonctionnement de cette barrière est souvent révélateur de toxicité ou de maladie. Bien que le nombre d'articles publiés dans le domaine du développement et de la caractérisation de la BHE ait été multiplié ces dernières années, la validité des modèles utilisés est encore un sujet de débat. L'avènement de l'électronique organique a créé une occasion unique pour coupler les mondes de l'électronique et de la biologie, à l'aide de dispositifs tels que le transistor électrochimique organique (OECT). OECT constitue un outil très sensible et économique pour diagnostiquer l’intégrité d’une barrière tissulaire. Dans cette étude, nous avons tout d’abord développé trois différents modèles de BHE. Nous avons optimisé l’adhésion des cellules endothéliales cérébrales sur la matière active du transistor. Nous avons ainsi pu établir l'intégration des OECTs avec des cellules immortalisées humaines micro vasculaires cérébrales endothéliales (h CMEC/D3) en tant que modèle in vitro de BHE. Nous avons démontré que la fonction de tissu de la BHE peut être détectée en utilisant cette nouvelle technique. En outre, par cette technique, une perturbation de la barrière (par exemple, provoquée par un composé toxique) pourra être détectée électriquement au moyen d'une mesure de courant
In biological systems many tissue types have evolved a barrier function to selectively allow the transport of matter from the lumen to the tissue beneath; one example is the Blood Brain Barrier (BBB). The BBB protects the brain from the blood and maintains homeostasis of the brain microenvironment, which is crucial for neuronal activity and function. Characterization of the BBB is very important as its disruption or malfunction is often indicative of toxicity/disease. Though the number of published papers in the field of in vitro BBB has multiplied in recent years, the validity of the models used is still a subject of debate.The advent of organic electronics has created a unique opportunity to interface the worlds of electronics and biology, using devices such as the Organic ElectroChemical Transistor (OECT), which provide a very sensitive way to detect minute ionic currents in an electrolyte as the transistor amplifies the gate current.In this study, we test three different type of BBB in order to develop a stable BBB model. We optimize the adhesion of brain endothelial cell on OECT conducting polymer. We show the integration of OECTs with immortalized human cerebral microvascular endothelial cells as a model of human blood brain barrier, and demonstrate that the barrier tissue function can be detected. Moreover, by this technique, a disruption in the barrier (e.g. caused by a toxic compound) is assessed electrically through a measurement of the drain current. Results show the successful development and validation of an in vitro BBB model. Dynamic monitoring of the barrier properties of the BBB barrier tissue was possible using the OECT

Compte tenu de l'absence de traitement pharmacologique efficace contre la maladie d'Alzheimer (MA), le développement d'approches thérapeutiques alternatives telles que le régime cétogène (« ketogenic diet » : KD) pourrait être envisagé. Le KD est un régime riche en graisses, basé sur la production de corps cétoniques (« ketone nodies » : KB) dans le sang. En raison des effets bénéfiques du KD sur le système nerveux central et de l'absence de données publiées sur la barrière hémato-encéphalique (BHE), nous avons utilisé une approche in vivo / in vitro pour étudier l'effet du KD et des KB sur la BHE. Pour l'étude in vivo, le sang de souris 129Sv a été récolté afin d’effectuer le dosage du beta-hydroxybutyrate et du glucose. Les capillaires cérébraux ont été isolés de cortex des souris, et des RT-qPCR ont été effectuées pour évaluer l'expression de l'ARNm des transporteurs / récepteurs impliqués dans la synthèse et le transport de KB, de glucose et du peptide bêta amyloïde. Les analyses transcriptionnelles ont été réalisées également dans un modèle in vitro de BHE, composé de cellules endothéliales dérivées de cellules souches hématopoïétiques (BLECs) en état de cétose. Après confirmation de l'intégrité des jonctions cellulaires des BLECs, Enfin, des expériences de transport de peptides beta amyloïde fluorescents après traitement avec les KBs ont été réalisées in vitro. Nos résultats montrent que les KBs modulent la physiologie de la BBB et l'expression de certains transporteurs et récepteurs du peptide bêta amyloïde, renforcent ainsi notre motivation à décrypter les mécanismes moléculaires et cellulaires au niveau vasculaire et plus précisément au niveau de la BHE
Given the current absence of an effective pharmacologic treatment for Alzheimer’s disease (AD), the development of alternative therapeutic approaches (such as the ketogenic diet, KD) might be considered. The KD is a low-carbohydrate, high-fat diet based on the production of ketone bodies (KBs) in the blood. In view of the KD’s beneficial effects on the central nervous system and the lack of published data on the blood brain barrier (BBB), we used an in vivo/in vitro approach to investigate the effect of the KD and KBs on the BBB. For the in vivo study, blood from 129Sv mice was assayed for beta-hydroxybutyrate and glucose dosage. Brain capillaries were isolated from mouse cortices, and RT-qPCR assays were used to evaluate the mRNA expression of transporters/receptors involved in the synthesis and transport of KBs, glucose and beta-amyloid peptide. The mRNA assays were also performed in an in vitro BBB model, based on brain-like endothelial cells (BLECs). After a ketotic state had been established and the BLECs’ integrity had been confirmed, we evaluated the mRNA expression of KB-, glucose- and amyloid-beta-related genes. Lastly, the transport of fluorescently labelled beta-amyloid peptide across the BBB was studied after treatment with KBs. Our results showed that KBs modulate the physiology of the BBB by regulating the expression of certain beta-amyloid peptide transporters/receptors and amyloid peptide-synthesizing enzymes. These data suggest that it is possible to modulate key molecular players in beta-amyloid peptide transport and synthesis at the BBB, and thus open up new perspectives for studying KB-related therapeutic approaches

The work of this thesis is based on research on peptides able to cross the blood-brain barrier and their use as tools to enable the delivery of drugs into the brain. The blood-brain barrier (BBB) is a permeable but selective barrier that tightly regulates the transport into the central nervous system (CNS). In this regard, therapeutic treatments at the CNS are hampered by the presence of this barrier (BBB). Thus, diverse strategies have been developed to overcome it. Blood-brain barrier shuttles are peptides able to cross this barrier and deliver drugs into the brain. Peptides are privileged structures from the therapeutic point of view, they share properties from small organic molecules and large biologics: the synthesis through solid-phase peptide synthesis (SPPS) enables a straightforward method to obtain them with high purity and at the same time they can be purified and characterized like small organic compound. In addition, their structure is present in nature and thus the risk of toxicity is lower or more predictable compared with organic compounds, and their larger structure enables to obtain more selective and stronger interactions with targets. In addition, peptides have been shown to cross the BBB by diverse transport mechanisms and thus enabling to select the best one for each therapy and drug. In this thesis a family of BBB shuttles crossing by passive diffusion (based on phenylproline) have been improved from a parent peptide shuttle (based on N-methyl-phenylalanine). The solubility was three orders of magnitude superior and the transport capacity was maintained upon cargo attachment. In addition, the role of stereochemistry in passive diffusion in biological membranes was demonstrated. A method which combined the use of MALDI-TOF MS and in vitro cell-based models of the BBB enabled the increase in sensitivity for transport quantification of three orders of magnitude compared to RP-HPLC-PDA. Additionally, a BBB shuttle library was evaluated and quantified by this novel methodology. Two new analogs showed better performance when evaluated in these in vitro cell-based models. Immunogenicity of BBB shuttle peptides made by L- or D-amino acids was evaluated and compared. Both peptide shuttles showed low immunogenic response in mice, however, the response to those made with D- amino acids was lower. Finally, the applicability of these peptide shuttles for a therapeutic use was considered for Friedreich’s Ataxia, a monogenic recessive disease. Both a protein replacement therapy and a gene therapy for the central nervous system were attempted by coupling covalently BBB shuttles to the affected protein or viruses, respectively. The protein replacement therapy was impeded by the high rate of proteolysis of the protein used. On the other hand, novel methods of conjugation of BBB shuttles into enveloped viruses (Herpes simplex Virus type 1; HSV- 1) were developed. These modified viral particles were subsequently characterized through a range of methods comprising molecular biology tools (SDS-PAGE, western blots), proteomics (mass spectrometry) and biophysical tools (dynamic light scattering and z-potential).
La barrera hematoencefàlica (BHE) actua com a protecció del sistema nerviós central (SNC) regulant el transport de molècules d’una manera selectiva. Això dificulta el tractament de malalties que afecten al SNC, ja que la BHE també evita que fàrmacs que serien efectius no siguin transportats al cervell. Per això, s’estan desenvolupant mètodes que permetin enviar selectivament fàrmacs a través de la BHE. És el cas dels pèptids llançadora. Aquests es poden dissenyar per creuar per algun dels mecanismes de transport existents en la BHE. En aquesta tesi es desenvolupen uns pèptids que creuen per difusió passiva (basats en fenilprolines), que respecte al disseny anterior (basats en N‐ metilfenilalanines) milloren la solubilitat en aigua en tres ordres de magnitud i al transport un cop s’hi enganxa el fàrmac. Per una altra banda, es desenvolupa una metodologia per a la quantificació del transport basada en la combinació d’espectrometria de masses MALDI‐TOF amb models de BHE in vitro (cel∙lulars), millorant la sensibilitat respecte a la detecció per RP‐HPLC‐PDA en tres ordres de magnitud. L’avaluació d’una peptidoteca derivada d’un pèptid llançadora mitjançant aquesta metodologia permet el descobriment de dos anàlegs del pèptid original que milloren el transport. Addicionalment, s’estudia la immunogenicitat de pèptids llançadora formats per aminoàcids L o D. S’observa que encara que ambdós mostren una baixa immunogenicitat, la resposta dels pèptids amb aminoàcids D és encara menor. Finalment, s’estudia de forma preliminar la possibilitat de desenvolupar una teràpia de reemplaçament proteic i una teràpia gènica per atàxia de Friedreich al SNC mitjançant l’ús de pèptids llançadora.

Cerebral small vessel pathology is now known to be associated with the development of cognitive impairment and mild motor impairments such as gait disturbance in a variety of neurodegenerative diseases. This dissertation explores the hypothesis that blood brain barrier dysfunction is an early event in cerebral ischemia and contributes to the development of cerebral small vessel disease (CSVD). A common rodent model of CSVD is permanent bilateral common carotid artery occlusion in the rat. This model was used to study several aspects of the progression of CSVD including the timecourse of blood brain barrier permeability changes following the onset of ischemia, gait disturbance, the expression of tight junction proteins and cytokine expression. It was determined that BBB permeability was elevated for 2 weeks following BCCAO and ischemic rats displayed lower gait velocity. There was no change in expression of TJ proteins. However, ischemic rats had higher levels of some proinflammatory cytokines and chemokines in brain tissue with no obvious changes in plasma levels. The mechanisms underlying the increase in BBB permeability were studied in vitro using artificial barriers made of confluent rat brain microvascular endothelial cells. Cerebral ischemia has been reported to cause an increase in plasma toxicity, likely by elevating the numbers of circulating microparticles (MPs). MPs isolated from the plasma of ischemic rats were applied to artificial barriers where it was found that they act mainly as vectors of TNF-α signaling. MPs induce activation of caspase-3 and the Rho/Rho kinase pathways. It is concluded that most of the increase in barrier permeability is due to apoptosis and disassembly of actin cytoskeleton and disruption of adherens junctions IV and not an increase in transcellular transport. The effects of treatment with the type III phosphodiesterase inhibitor cilostazol on dye extravasation in the brain, glial activation, white matter damage and motor performance were evaluated. It was determined that cilostazol could improve the increased BBB permeability and gait disturbance and microglial activation in optic tract following BCCAO. Also, the effects of treatment with cilostazol on plasma toxicity in vivo (24h and 14d following BCCAO) and artificial barriers (in vitro) were assessed. It was found that cilostazol could reduce plasma toxicity at 24h and improve increased endothelial barrier permeability that is induced by MP treatment respectively. In summary BBB dysfunction occurs in the rat model of chronic cerebral hypoperfusion with no differences in expression of TJ proteins. There is a mild motor disturbance in the form of lower gait velocity following BCCAO. Cytokines released in brain tissue may be associated with pathological consequences following BCCAO while there is no significant difference in plasma levels and circulating MPs may play a role in BBB dysfunction.

A major product of HIV-infected and cytokine-stimulated monocytic-lineage cells is quinolinic acid (QUIN), a neurotoxic metabolite of the kynurenine pathway (KP) of L-tryptophan (L-Trp) metabolism. Despite the large number of neurotoxins found in HIV patients with AIDS Dementia Complex (ADC), only QUIN correlates with both the presence and severity of ADC. With treatment, cerebrospinal fluid (CSF) QUIN concentrations decrease proportionate to the degree of clinical and neuropsychological improvement. As endothelial cells (EC) of the blood-brain barrier (BBB) are the first brain-associated cell that a bloodborne pathogen would encounter, this project examined the BBB response to KP metabolites, as these are implicated in damage of the CNS associated with ADC. Using RT-PCR and HPLC/gas chromatographymass spectrometry (GC-MS), I found that cultured primary human BBB EC and pericytes constitutively expressed the KP. EC synthesised kynurenic acid (KA) constitutively, and after immune activation, kynurenine (KYN). Pericytes produced small amounts of picolinic acid and after immune activation, KYN. An SV40-transformed BBB EC showed no KP expression. By contrast, human umbilical vein EC only expressed low levels of KA after immune activation, however human dermal microvascular EC showed a similar constitutive and inducible KP to that in BBB EC. As T cells are central to primary HIV infection, I also examined KP expression in two CD4+ and one CD4- cell lines, but none showed either constitutive or inducible KP expression. I next examined how QUIN might interact with BBB EC. There was no binding of 3H-QUIN to cultured primary human BBB EC, however a biologically relevant concentration of QUIN induced changes in gene expression which adversely affected EC function, possibly mediated by lipid peroxidation and oxidative stress. The upregulated genes were of the heat shock protein family, and the downregulated genes were associated with regulation of cell adhesion, tight junction and cytoskeletal stability, metalloproteinase (MMP) regulation, apoptosis and G protein signaling. Immunofluorescence showed that QUIN induced morphological changes in BBB EC consistent with the changes in gene expression. Gelatin zymography showed that this was not mediated by MMPs, as constitutive MMP expression was unchanged. These data provide strong evidence for QUIN directly damaging the BBB in the context of HIV infection.

Coenzyme Q10 (CoQ10) deficiencies are unique among mitochondrial respiratory chain (MRC) disorders in that they are potentially treatable. While there is clear evidence, both clinically and biochemically, for the improvement of peripheral abnormalities associated with CoQ10 deficiency following CoQ10 supplementation, neurological symptoms are only partially ameliorated. The reasons for the refractory nature of the neurological sequelae associated with a CoQ10 deficiency are as yet unknown and may be a consequence of irreversible damage prior to CoQ10 supplementation, the retention of CoQ10 in the blood-brain barrier (BBB) itself, or simply reflect poor transport of CoQ10 across the BBB. This thesis presents the first isolated investigations into the mechanisms that govern bi-directional BBB transport of CoQ10 and its synthetic analogue, idebenone, using normal and pathophysiological cell models relevant to disorders of CoQ10 biosynthesis. The mouse BBB endothelial cell line bEnd.3 and porcine primary brain endothelial cells (PBECs) co-cultured with primary astrocytes were used to assess transcytosis from 'blood-to-brain' or 'brain-to-blood', revealing that although CoQ10 can traverse the BBB, CoQ10 is being effluxed back to the blood, which could explain the refractory nature of CoQ10 therapy, whereas, idebenone appeared to cross the BBB passively. Using inhibitors of known transport systems for lipoproteins, the circulatory bio-carriers of CoQ10 in vivo, three systems mediating the BBB transport of lipoprotein-bound CoQ10 were identified. Inhibitors of the scavenger receptor class B type 1 (SR-B1), BLT-1, and the receptor for advanced glycation end products (RAGE), FPS-ZM1, reduced uptake of lipoprotein-bound CoQ10 towards the brain, implicating RAGE and SR-B1 as modes for CoQ10 brain uptake. In the reverse direction, the low-density lipoprotein receptor-related protein-1 (LRP-1) inhibitor, RAP, reduced efflux of lipoprotein-bound CoQ10 towards the blood, implicating LRP-1 as a major impediment to brain entry of CoQ10. This study is the first to generate a BBB endothelial cell model of CoQ10 deficiency, using para-aminobenzoic acid (pABA) to pharmacologically induce a depletion of cellular CoQ10 status, resulting in a global reduction of MRC enzyme activities. The CoQ10 deficient BBB models were leakier to large permeability markers, with poor BBB tight-junction formation, and altered CoQ10 transport dynamics in favour of an increased net efflux towards the blood, suggesting BBB pathophysiology is key to the neurological presentation and refractory nature of CoQ10 supplementation in symptomatic patients. In addition, the effects of vitamin E, a common clinical co-therapy in the 'mito-cocktail', and simvastatin were assessed. Interestingly, vitamin E co-administration reduced net efflux of CoQ10 from the brain. It is unknown why this occurs, but oxidative effects on the BBB transporters and/or carrier-lipoproteins may be factors to consider. In-line with its deleterious effect on CoQ10 biosynthesis, simvastatin therapy appeared to disrupt BBB integrity, increasing the paracellular leak of the BBB. This would be detrimental to normal brain homeostasis, particularly given the BBBs major role in limiting brain entry of the small molecule plasma excitotoxins, calcium, and glutamate. Throughout this study CoQ10 was quantified using a novel and rapid mass spectrometric method (ESI+ LC-MS/MS), which could potentially enable detection of CoQ10 in the CSF of patients presenting with neurological symptoms, perhaps providing a new analytical tool for the diagnosis of CoQ10 deficiencies in clinical laboratories. In conclusion, this thesis has demonstrated for the first time the pathophysiological consequences of a CoQ10 deficiency on the BBB. It has highlighted the impact of a deficit in CoQ10 status on CoQ10 delivery to the brain parenchyma and has elucidated some of the mechanisms by which CoQ10 is transported across the BBB, which are ultimately dictated by lipoprotein interactions. Additionally, this thesis outlines the potential dangers of statin therapy in patients with an underlying or established MRC dysfunction. Overall, this thesis provides insights into the limitations of CoQ10 supplementation as a therapy for neurological disorders associated with MRC dysfunction and indicates that further work will be required to improve the delivery of exogenous CoQ10 across the BBB, alongside a need for further investigations into the composition of the widely administered 'mito-cocktail'.

Increased cerebrovascular permeability is an important factor responsible for the development of ischemic brain injury and edema formation associated with stroke pathophysiology. Extensive studies of stroke research have centered primarily on the response of neurons and astrocytes to hypoxic or ischemic insult. The response of cerebral capillary endothelial cells to hypoxia is not well understood. Damage to the blood-brain barrier (BBB) induced by hypoxia/ aglycemia may influence BBB permeability and transport mechanisms, thereby contributing to the development and severity of stroke. The development of a low flow in situ brain perfusion model was used in this study to illustrate the effect of ischemia/hypoperfusion coupled with hypoxia and aglycemia on BBB transport mechanisms. Three transport markers were used in various combinations of low flow, hypoxia, and aglycemia to characterize BBB transport mechanisms. The results of this study suggest BBB basal permeability is not com promised during low flow perfusion, however in the presence of hypoxia/ aglycemia, a significant change in BBB permeability is observed among the three transport markers. Thus, the effects of ischemia as produced by low flow, hypoxia, and aglycemia alter BBB permeability due to the probable impaired action of many transport systems under these adverse conditions.

[ES] El tratamiento de enfermedades neurológicas está muy limitado por la ineficiente penetración de los fármacos en el tejido cerebral dañado debido a la barrera hematoencefálica (BHE), lo que imposibilita mejorar la salud del paciente. La BHE es un mecanismo de protección natural para evitar la difusión de agentes potencialmente peligrosas para el sistema nervioso central. No obstante, la BHE se puede inhibir mediante ultrasonidos focalizados e inyección de microburbujas de forma segura, localizada y transitoria, una tecnología empleada mundialmente. La principal ventaja es su carácter no invasivo, siendo así muy atractiva y cómoda para el paciente. Normalmente, la zona cerebral enferma se trata en su parte central empleando un único foco. Sin embargo, enfermedades como el Alzheimer o el Parkinson requieren un tratamiento sobre estructuras de geometría compleja y tamaño elevado, situadas en ambos hemisferios cerebrales. Por tanto, la tecnología actual está muy limitada al no cumplir dichos requisitos. Esta tesis doctoral tiene como objetivo el desarrollo de una técnica novedosa, basada en hologramas acústicos, para resolver las limitaciones presentes en los tratamientos neurológicos empleando ultrasonidos. Se estudian las lentes acústicas holográficas impresas en 3D, que acopladas a un transductor mono-elemento, permiten el control preciso del frente de onda ultrasónico tanto para (1) compensar las distorsiones que sufre el haz hasta alcanzar el cerebro, como (2) focalizarlo simultáneamente en regiones múltiples y de geometría compleja o formando de vórtices acústicos, proporcionando así efectividad en tiempo y coste. Por ello, la investigación desarrollada en esta tesis abre un camino prometedor en el campo de la biomedicina que permitirá mejorar los tratamientos neurológicos, además de aplicaciones en neuroestimulación o ablación térmica del tejido.
[CA] El tractament de malalties neurològiques està molt limitat per la ineficient penetració del fàrmac en el teixit cerebral danyat a causa de la barrera hematoencefàlica (BHE), i així no és possible una millora de salut del pacient. La BHE és un mecanisme de protecció natural per a evitar la difusió d'agents potencialment perillosos per al Sistema Nervios Central. No obstant això, aquesta barrera es pot inhibir mitjancant una tecnologia emprada mundialment basada en ultrasons focalitzats i injeccio de microbombolles. El principal avantatge és el seu caràcter no invasiu, sent així molt atractiva i còmoda per al pacient, i permet obrir la BHE de manera segura, localitzada i transitòria. Normalment, la zona cerebral malalta es tracta en la seua part central, emprant un unic focus. No obstant això, malalties com l'Alzheimer o el Parkinson requereixen un tractament al llarg d'estructures de geometria complexa i grandària elevada, situades en tots dos hemisferis cerebrals. Per tant, la tecnologia actual està fortament limitada al no complir amb aquests requeriments. Aquesta tesi doctoral està enfocada a investigar i desenvolupar una tècnica nova, basada en hologrames acústics, per a solucionar les limitacions presents en els tractaments neurològics. Una lent acústica holograca de baix cost impresa en 3D acoblada a un transductor d'element simple permet el control precs del front d'ona ultrasònic punt per a (1) compensar les distorsions que pateix el feix en el seu camí cap al cervell, i (2) focalització simultània del feix en regions multiples i de geometria complexa, proporcionant aix un tractament efectiu en temps i cost. Per això, la investigació desenvolupada en aquesta tesi demostra la possibilitat de realitzar qualsevol tractament neurològic, a més d'aplicacions en la neuroestimulació o l'ablació tèrmica dins del camp biomèdic.
[EN] Treatments for neurological diseases are strongly limited by the inefficient penetration of therapeutic drugs into the diseased brain due to the blood-brain barrier (BBB), and therefore no health improvement can be achieved. In fact, the BBB is a protection mechanism of the human body to avoid the diffusion of potentially dangerous agents into the central nervous system. Nevertheless, this barrier can be successfully inhibited by using a worldwide spread technology based on microbubble-enhanced focused ultrasound. Its main advantage is its non-invasive nature, thus defining a patient-friendly clinical procedure that allows to disrupt the BBB in a safe, local and transient manner. Conventionally, the diseased brain structure has been targeted in its center, with a single focus. However, Alzheimer's or Parkinson's Diseases do require that ultrasound is delivered to entire, complex-geometry and large-volume structures located at both hemispheres of the brain. Therefore, current technology presents several limitations as it does not fulfill these requirements. This doctoral thesis aims to develop a novel technique based on using focused ultrasound acoustic holograms to solve the existing limitations to treat neurological diseases. In this dissertation, we study 3D-printed holographic acoustic lenses coupled to a single-element transducer that allow to accurately control the acoustic wavefront to both (1) compensate distortions suffered by the beam in its path to the brain, and (2) simultaneous focusing in multiple and complex-geometry structures or acoustic vortex generation, providing a time- and cost- efficient procedure. Therefore, the research carried out throughout this thesis opens a promising path in the biomedical field to improve the treatment for neurological diseases, neurostimulation or tissue ablation applications.
Acknowledgments to the Spanish institution Generalitat Valenciana, which funding grant allowed me to develop this doctoral thesis, and as well funded my research stay at Columbia University. The development of the entire thesis was supported through grant Nª. ACIF/2017/045. Particularly, the research carried out in Chapter 3 and Chapter 4 was possible thanks to and supported through grant BEFPI/2019/075. Action co-financied by the Agència Valenciana de la Innovació through grant INNVAL10/19/016 and by the European Union through the Programa Operativo del Fondo Europeo de Desarrollo Regional (FEDER) of the Comunitat Valenciana 2014-2020 (IDIFEDER/2018/022).
Jiménez Gambín, S. (2021). Transcranial Ultrasound Holograms for the Blood-Brain Barrier Opening [Tesis doctoral]. Universitat Politècnica de València. https://doi.org/10.4995/Thesis/10251/171373
TESIS

Thesis (Ph.D.)--George Mason University, 2009.
Vita: p. 102. Thesis director: Serguei G. Popov. Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biosciences. Title from PDF t.p. (viewed June 10, 2009). Includes bibliographical references (p. 84-101). Also issued in print.

The aims of this research project were to manufacture and characterise PDPA-based pH-sensitive functionalised polymersomes using a medium-high content screening method, suitable for CNS drug delivery. Angiopep-2 functionalised polymersome formulations have been found that are able to penetrate the blood-brain barrier (BBB) effectively in both in vitro models and in vivo. Using Transmission Electron Microscopy, Dynamic Light Scattering, FACS analysis, and 2D in vitro screening gave information on the physical and biological features of polymersomes based on their different chemistries, including size distribution, architecture, topology, cellular localisation, cellular uptake kinetic and immune response. The studies showed the possibility of controlling cellular internalization and cargo destinations by manipulating polymersome surface chemistry and specific ligand(s). The subsequent in vitro and in vivo studies built on these screening results. Using extensive Confocal Laser Microscopy and image analysis, Ang-POEGMA-PDPA polymersomes showed effective receptor-mediated transcytosis (RMT) in the 3D in vitro BBB model established, while the RVG- functionalised formulation did not. Further in vivo studies showed that the Ang-functionalised polymersomes were able to penetrate the mouse BBB via effective RMT. Moreover, primary cargo delivery studies showed successful IgG transport into brain by Angiopep-2-functionalised POEGMA-PDPA polymersomes. The results in this thesis can provide a useful platform for further examination of CNS delivery of polymersomes and their cargos.

The blood-brain barrier (BBB) is located at the level of the cerebral microcapillaries, and functions to maintain environmental homeostasis by allowing the neurons access to the required nutrients and enabling the exchange of metabolic waste. BBB dysfunction has been observed in a number of pathophysiologic statres including peripheral inflammatory pain (Huber et al., 2001b). Using the lamda-carrageenan inflammatory pain (CIP) model, we observed alterations in the tight junction (TJ) proteins paralleled by an increase in BBB permeability to [14C] sucrose. The mechanisms by which these perturbations occurred remain to be elucidated. In the current study, we investigate the central mechanism for the BBB perturbations under CIP. It is our hypothesis that the modulations of the BBB under CIP, are mediated via a central signaling pathway. First, to investigate the involvement of neuronal input from pain activity on alterations in BBB, we developed a method for inhibiting the nociceptive input from the paw. Using a perineural injection of 0.75% bupivacaine into the right hind leg prior to CIP, we were able inhibit development thermal hyperalgesia induced by CIP, as tested by infrared heat stimulus, without effecting edema formation 1 h post CIP. Upon inhibition of nociception under CIP, there was an attenuation of both the changes in permeability and the changes in tight junction protein expression, with both returning to control levels. Next, we investigated intercellular adhesion molecule-1 (ICAM-1), a key signaling protein at the BBB, which in the presence of proinflammatory mediators, increases in expression leading to the activation of signaling pathways as well as morphological changes. We found a region specific increase in ICAM-1 mRNA and protein expression following CIP which directly correlated with increased expression of activated microglia. Finally, we investigated the influence activated microglia had on BBB permeability. Using an 0.150 mg intrathecal bolus injection of minocycline, a potent inhibitor of microglia activation (Klein and Cunha, 1995), we were able to inhibit the increased expression of activated microglia, and saw an attenuation of permeability to control levels. These findings suggest CIP induced BBB disruption is localized and has a central-mediated component independent of peripheral influence.

Hypertension is involved in the exacerbation of stroke. Increased blood-brain barrier (BBB) permeability and cerebral edema formation are potentially lethal complications of cerebral infarction. It is unclear how BBB tight junction (TJ), ion transporter, and protein kinase C (PKC) signaling pathway proteins critical for maintaining brain homeostasis contribute to cerebral infarction during hypertension development. The hypothesis of this study is that hypertension leads to molecular changes in the BBB which predispose the brain to increased cerebral infarct damage following ischemic stroke. Studies were undertaken to investigate the effect of hypertension development on (1) physiological parameters of the spontaneously hypertensive rat (SHR) and on the expression levels of BBB TJ, ion transporter, and PKC proteins potentially involved in ischemia-induced infarct damage; (2) ischemia-induced infarct volume following permanent middle cerebral artery occlusion (MCAO); and (3) the effect of inhibition of Na+/H+ exchanger isoform 1 (NHE-1) on ischemia-induced infarct volume following permanent MCAO in hypertensive SHR (15 weeks). Early hypertension development was determined in SHR and compared to normotensive, age-matched Wistar-Kyoto (WKY) rats at 5 (pre-hypertension), 10 (early stage hypertension), and 15 (later stage hypertension) weeks of age. Characterization of BBB TJ and ion transporter proteins known to contribute to edema and fluid volume changes in the brain show differential protein expression patterns during hypertension development. Western blot analysis of TJ zonula occludens-2 (ZO-2) showed decreased expression while ion transporter, NHE-1 was markedly increased in hypertensive SHR (15 weeks) compared to age-matched controls. Hypertensive SHR (10 and 15 weeks) showed greatly increased necrotic volume with impaired neurological deficits and edema formation. Increased NHE-1 expression in hypertensive SHR (15 week) suggests a potential role for this ion transporter in the promotion of ischemic brain injury. Selective inhibition of NHE-1 using 5-(N,N-Dimethyl)amiloride (DMA) showed significant attenuation in ischemia-induced infarct volume in hypertensive SHR following MCAO. These data suggest a novel role for NHE-1 at the BBB/neurovascular unit in the regulation of ischemia-induced infarct volume in hypertensive SHR suggesting that modulation of NHE-1 may be a factor important in the potentiation of MCAO infarct size and a novel therapeutic target in the prevention of ischemic stroke.

Thesis (Ph. D.)--School of Pharmacy. University of Missouri--Kansas City, 2006.
"A dissertation in pharmaceutical sciences and pharmacology." Advisor: Srikumaran Melethil. Typescript. Vita. Title from "catalog record" of the print edition Description based on contents viewed Dec. 20, 2007. Includes bibliographical references (leaves 159-192). Online version of the print edition.

Drug delivery to the central nervous system (CNS) is significantly hindered by thepresence of the blood brain barrier (BBB) and blood cerebrospinal fluid barrier(BCSFB) and associated drug efflux transporter proteins. The aim of this work was to modulate the expression of breast cancer resistance protein (BCRP) at each barrier site using phytochemical modulators. In-vitro cellular models of both the BBB (PBMEC/C1-2) and BCSFB (Z310) were utilised and 18 phytochemical modulators screened for their cellular toxicity with IC50 values for the majority of phytochemicals being in excess of 100 μM. Intracellular accumulation of H33342 was assessed in each barrier cell line to determine short-term modulation of BCRP efflux or long-term modulation of protein expression. Incubations with modulators demonstrated significant inhibition of BCRP efflux activity for a range of modulators in both cell lines with TMF (1-100 μM) demonstrating a > 6 fold increase in intracellular accumulation. Similarly, many modulators demonstrated proposed protein-level modulation of BCRP resulting in increases or decreases in H33342 accumulation following a 24 hour exposure. Western blotting subsequently confirmed that quercetin and naringin for PBMEC/C1-2 and baiclain and flavone for Z310 induced BCRP expression (to 2-3 fold of control) whereas curcumin and 17-β-estradiol for PBMEC/C1-2 and silymarin, quercetin and 17-β-estradiol for Z310 down-regulated BCRP expression (to 0.24-0.4 fold of control). This was further confirmed in substrate transport studies using permeable insert models which demonstrated functional changes in the permeabilityof BCRP substrates across both barrier models. Subsequently the regulation of BCRP by AhR was confirmed through siRNAknockdown of AhR, which resulted in a significant decrease in BCRP geneexpression in both cell lines. Furthermore the induction/down-regulation effects on BCRP were, in general, diminished following AhR knockdown, suggesting AhR plays an important role in mediating the genomic/proteomic alterations in BCRP expression when exposed to phytochemicals.

This study employed a new model of mild-to-moderate head trauma to specifically identify the role of dural mast cell (MC) histamine in trauma-induced increased permeability in the blood-brain barrier (BBB). A single line was scored partially through the left dorsal parietal skull. Immediately following the trauma, degranulation was seen in 39% of the MCs on the left and in 2% on the right. After a 20 min survival period, left duras showed 55% with MC degranulation (fewer with complete degranulation) compared to 34% on the right. In the other experiments two parallel lines were scored following the injection of Evan's blue. Histamine assay showed histamine increased in the left cortex to 154% at 5 min, 174% at 10 min, and 151% at 20 min. Fluorescent quantitation of extravasated Evan's blue at 20 min following the trauma gave an increase of 1385% over the value measured for the right cortex. Zolantidine, a selective histamine H2 receptor antagonist, administered at 10- and 20- mg/kg 30 min before the trauma blocked 65% of the Evan's blue extravasation compared with the control and 2.5 mg group.

Introduction: The survival rate of neurological diseases such as brain cancer has remained poor at 1% over the last 30 years despite improvements in technology and novel medicines entering the market. The key obstacle in the treatment of any neurological disease is the blood brain barrier (BBB), a restrictive barrier which ensures the homeostasis of the central nervous system. Developments in lipid-based nanoparticles have presented the opportunity to deliver medicine across the BBB due to their size and ability to tune the ideal properties required to cross. By using human physio-mimicking models for the BBB, better effect and safety of the nanoparticles can be evaluated compared to traditional in vitro testing. Methods: Solid lipid nanoparticles (SLN) were fabricated and purified. The characteristics of size, charge, morphology and structure were determined using techniques of Dynamic Light Scattering (DLS); Nano Flow Cytometry (NanoFCM); Atomic Force Microscopy (AFM); and Fourier-transform Infrared Spectroscopy (FTIR). Blood-brain barrier models were developed using traditional 2D in vitro studies as well as two human physio-mimicking 3D models to determine the impact of appropriate models for clinical translation. Impact of nanoparticles on 2D models were done using assays for cell viability (CCK-8), cell cytotoxicity (LDH), cell migration (cell tracking analysis) and cell morphology changes (Nanolive). Two types of 3D human physio-mimicking models were fabricated and optimised. Non-invasive real-time testing was done using Trans-endothelial resistance (TEER) output, FTIC-dextran permeability and cell viability (CCK-8) assays and end-point immunofluorescence images were taken to validate the establishment of the 3D BBB models. Finally, SLN were applied to the developed models and above biological assays determined the effect. Results and discussion: SLN were successfully fabricated, purified and characterised for properties of size, charge, shape and physiochemical composition. It was also found that the SLN were sensitive to ultrasound, introducing the possibility of targeted drug delivery using SLN propelled by ultrasound to be investigated in the future. The SLN were applied to human brain endothelial cells and their impact was assessed. It was concluded using cell viability, cytotoxicity, migration tracking and changes in morphology that the SLN impacted the cells with increasing concentration. Two 3D in vitro human physio-mimicking models of the BBB were developed: BBB-PhysioMimix-OOC and PDMS BBB-on-chip and formation of a barrier was optimised using barrier integrity testing. When SLN were applied to BBB-PhysioMimix-OOC the preliminary 2D in vitro trend was mirrored in the results for barrier distruption, thus validated this model. Validation of the PDMS BBB-on-chip experienced limitations. The two physio-mimicking BBB models were compared for their qualities of low-throughput and high-throughput. Conclusion: Solid lipid nanoparticles can be used as a strategy to pass the blood-brain barrier; and drug testing on human physio-mimicking in vitro models of the blood-brain barrier must be done to ensure better clinical translation of therapeutics for neurological diseases.

Upon an inflammation the immune system signals the brain by secreted cytokines to elicit central nervous responses such as fever, loss of appetite and secretion of stress hormones. Since the blood brain barrier, (BBB) protects the brain from unwanted material, molecules like cytokines are not allowed to cross the barrier and enter the brain. However, it is clear that they in some way can signal the brain upon an inflammation. Many suggestions concerning this signaling has been made, one being that cytokines bind to receptors on the endothelial cells of the blood vessels of the brain and trigger the production of prostaglandins that can cross the BBB. This conversion is catalyzed by the enzyme cyclooxygenase-2, (COX-2), which is induced by transcription factors like NF-κB in response to cytokines. One of the central nervous responses to inflammatory stimuli is activation of the HPA-axis whose main purpose is glucocorticoid production. Glucocorticoids inhibit the inflammatory response by suppressing gene transcription of pro-inflammatory genes including those producing prostaglandins through direct interference with transcription factors such as NF-κB or initiation of transcription of anti-inflammatory genes like IκB or IL-10. It has however not been clear if glucocorticoids can target the endothelial cells of the brain in order to provide negative feed-back on the immune-to-brain signaling, and in that way inhibit central nervous inflammatory symptoms. An anatomical prerequisite for such a mechanism would be that the induced prostaglandin production occurs in cells expressing GR. This has however never been demonstrated. Here I show that a majority of the brain endothelial cells expressing the prostaglandin synthesizing enzyme COX-2 in response to immune challenge also express the glucocorticoid receptor, (GR). This indicates that immune-to-brain signaling is a target for negative regulation of inflammatory signaling executed by glucocorticoids and identifies brain endothelial GR as a possible future drug target for treatment of central nervous responses to inflammation such as fever and pain.

The developmental aspect of drug transport across the blood-brain barrier (BBB) was investigated. Microdialysis was used to study unbound morphine BBB transport at different ages in sheep. An in vitro study was performed to find differentially expressed genes in brain capillary-rich fractions of the brain in rats of different ages. Microdialysis and brain-to-plasma ratios were used to study the contribution of breast cancer resistance protein (Bcrp) to the transport of nitrofurantoin (NTF) across the BBB of rats during development as well as in adult rats and mice. A method of analysing morphine and its metabolites in plasma and microdialysis samples was developed and validated. The in vivo recovery of deuterated morphine, used as a calibrator in microdialysis experiments, was not affected by the presence of morphine in the tissue. A net influx of morphine was observed in premature lambs and adult sheep, in contrast to the efflux seen in other species. This influx decreased with age, indicating that the morphine transport across the BBB changes with age. In contrast, the transport of the morphine metabolite morphine-3-glucuronide (M3G) did not change with age. Microarray data indicated that several active transporters are differentially expressed with age. Moreover, the mRNA expression levels of Abcg2 (Bcrp) and Slc22a8 (organic anion transporter 3) changed with age when quantified using real-time polymerase chain reaction. In contrast, the expression of Abcb1 (P-glycoprotein) and occludin (a tight junction protein) did not change with age. In rats, the brain distribution of NTF decreased with age due to increased protein binding in plasma. The concentration ratio of unbound NTF across the BBB was low in the adult rat, due to intra-brain metabolism and/or efflux by other transporters. Bcrp did not appear to have a significant contribution in the developing rat or in knock-out mice compared to wild-type controls with regard to NTF BBB transport. In conclusion, in vitro studies showed that the expression levels of some genes changed with age, presumably affecting subsequent drug distribution to the brain. Further, in vivo studies showed that distribution across the BBB changed with age for morphine but not for M3G or NTF.

T cells recognizing myelin auto-antigens penetrate into the CNS to induce inflammatory autoimmune disease following complex sequential interactions with individual components of the vascular blood-brain barrier (BBB), particularly endothelial cells, and perivascular phagocytes. To determine the functional consequences of these processes, two-photon intravital imaging was performed to compare the behavior of three myelin-specific GFP-expressing T cell lines with different potentials for transferring Experimental Autoimmune Encephalomyelitis. Imaging documented that, irrespective of their pathogenic potential, all T cell lines reached the CNS and interacted with vascular endothelial cells indistinguishably, crawling on the luminal surface, preferably against blood flow, before crossing the vessel wall. In striking contrast, after extravasation the T cell motility and their interactions with perivascular antigen presenting cells (APCs) varied dramatically. While highly encephalitogenic T cells showed a low motility, made stable contacts with local APCs and became activated, the corresponding contacts of weakly encephalitogenic T cells remained short, their motility high and their activation marginal. Supplying auto-antigen, via either local injection or by transfer of antigen-pulsed meningeal APCs, lowered their motility and prolonged the contact duration of weakly encephalitogenic T cells to values characteristic for highly pathogenic ones. Only after exogenous antigen supply, the weakly encephalitogenic T cells became activated, infiltrated the CNS parenchyma, and triggered clinical EAE, suggesting that the strength of the antigen-dependent signals received by immigrating effector T cells from leptomeningeal APCs is crucial for their pathogenic effect within the target tissue. To directly correlate the activation of encephalitogenic T cells with their dynamic behavior in the CNS, a truncated fluorescent derivative of nuclear factor of activated T cells (NFAT) was introduced as a real-time activation indicator. Two-photon imaging documented the activation of the auto-reactive T cells extravasated into the perivascular space, but not within the vascular lumen. Activation correlated with reduced T cell motility, and it was related to contacts with the local APCs. However, it did not necessarily lead to a long-lasting arrest, as individual, activated T cells SUMMARY 2 were able to sequentially contact other APCs. A spontaneous cytosol-nuclear translocation of the marker was noted only in T cells with a high pathogenic potential. The translocation implied the presentation of an auto-antigen, as the weakly pathogenic T cells, which remained silent in the untreated hosts, were activated upon the instillation of exogenous auto-antigen. It is proposed here that the presentation of local auto-antigen by BBB-associated APCs provides stimuli that guide autoimmune T cells to the CNS destination and enable them to attack the target tissue. In addition, a theoretical, physicist approach was used for modeling T cell activation in the leptomeningeal space. Assuming that T cells have evolved to gain their activation signal in a way that is energetically optimal for them, two possible scenarios for T cell activation were compared. The first one assumes that, after finding an APC presenting the epitope of interest, the T cell will stop and interact with the APC until it becomes fully activated. The second model considers the possibility that a T cell can accumulate activation signals from different APCs while scanning them without stopping, until a certain threshold is exceeded and the T cell becomes activated. Using this approach, it is proposed that the T cells in EAE are more likely to become activated following the first scenario. However, in a more natural environment such as a lymph node, the second scenario could give them some advantages

Dynamic contrast enhanced (DCE-) MRI data can be analyzed using a two-compartment pharmacokinetic model to measure biophysical parameters such as blood-brain barrier (BBB) permeability. This work focuses on developing a DCE-MRI based technique to accurately estimate BBB permeability in multiple sclerosis (MS) lesions. The impact of the temporal resolution of the DCE-MRI acquisition on the accuracy and precision of permeability measurements is evaluated experimentally and through simulations, and a new protocol based on a two-phase acquisition is shown to reduce the errors in permeability estimates. BBB permeability maps in MS patients are also examined for correlations with differences in regions of enhancement observed following two non-dynamic protocols: a standard and an optimized one. Results indicate that voxels only enhancing with the optimized protocol are associated with lower BBB permeability values than voxels enhancing with both protocols, which supports the hypothesis that each non-dynamic enhancement protocol has its own sensitivity threshold. This indicates that using a quantitative DCE-MRI technique, such as the one presented here, provides a more complete view of MS lesion pathology by enabling quantitative measurement of BBB permeability rather than simple binary classification as enhancing or non-enhancing.
Les données d'IRM dynamique avec agent de contraste (DCE-MRI) peuvent être analysées à l'aide d'un modèle pharmaco-cinétique à deux compartiments, afin de mesurer des paramètres biophysiques tels que la perméabilité de la barrière hémato-encéphalique (BHE). Notre étude vise à développer une technique de DCE-MRI qui permette de mesurer avec exactitude la perméabilité de la BHE dans les lésions de sclérose en plaques (SEP). L'impact de la résolution temporelle de l'acquisition sur l'exactitude et la précision des mesures de perméabilité est évalué au moyen d'expériences et de simulations, et un nouveau protocole en deux temps permettant de réduire l'erreur dans les mesures est proposé. Sont analysées ensuite les corrélations entre les cartes de perméabilité et les différences de réhaussement entre deux protocoles non dynamiques: l'un standard et l'autre optimisé. Les résultats préliminaires montrent que les valeurs de perméabilité sont plus basses dans les voxels réhaussés uniquement par le protocole optimisé que dans ceux réhaussés par les deux. Ces résultats soutiennent l'hypothèse d'un seuil de sensibilité intrinsèque à chaque protocole non-dynamique avec contraste. L'utilisation d'une technique quantitative de DCE-MRI, telle que celle présentée ici, apporterait une vision plus complète de la pathologie des lésions de SEP par la mesure de la perméabilité de la BHE, comparée à la classification binaire en régions réhaussant ou non.

The blood-brain barrier (BBB) is a dynamic interface, mainly consisting of highly specialized brain microvascular endothelial cells (BMECs) that segregate the central nervous system (CNS) from the peripheral circulation. Impairment of the BBB, due to disruption of tight junction (TJ) proteins and inflammatory responses, may initiate and/or contribute to the progress of CNS disorders, including stroke. Stroke is the second leading cause of death worldwide. It has been shown that aging and environmental pollutants can induce brain endothelium dysfunction, and are considered as risk factors for stroke. Deficiency of telomerase is highly linked with aging-associated vascular diseases. Evidence indicates that patients with shorter telomere length are at higher risk of heart disease or stroke. Results in this dissertation address the influence of telomerase reverse transcriptase (TERT), a key component of telomerase, on the BBB integrity in the context of ischemic stroke induced brain injury. Our results indicate that aging-related BBB alterations aggregate the stroke outcomes by inducing oxidative stress and stimulating proinflammatory responses on the brain microvessels. The ability of the BBB to protect the brain from harmful compounds indicates that the BBB may be targeted by chemical toxicants in the peripheral circulation. Polychlorinated biphenyls (PCBs) are persistent organic pollutants that frequently bind to nanoparticles (NPs) in the environment. Our results demonstrate that binding PCB153, one of the most abundant PCB congeners in the environment, to silica nanoparticles (PCB153-NPs) potentiates cerebrovascular toxicity and stroke outcomes via stimulation of inflammatory responses and disruption of BBB integrity. These events are mediated by activation of toll-like receptor 4 (TLR4), which subsequently recruits tumor necrosis factor-associated factor 6 (TRAF6) and initiates the production of multiple inflammatory mediators. Research presented in this dissertation demonstrates that aging and environmental pollutants play crucial roles in modifying the function of the BBB through alterations of inflammatory responses and TJ protein expression, which further contribute to the progression of stroke-induced cerebral ischemic injury.

Aim: The general aim of this research is development and evaluation of novel methods for predicting active transport over the human blood-brain-barrier (BBB), while this project specifically aims to i) review the literature and select suitable methods and substrates, ii) develop models for determining in vitro kinetic properties of selected compounds, analyze the in vitro data using the developed models and to use Maximum Transport Activity (MTA) approach (Karlgren et al., 2012), iii) perform Physiology Based Pharmacokinetic (PBPK) modelling and compare to in vivo literature data. Background: Drug permeation to the brain through blood circulation is primarily limited by blood-brain barrier (BBB), due to existence of tight junctions in endothelial cells of blood vessels as well as active efflux and influx transporters in the barrier. Toxicity and CNS related side effects can be caused by peripheral targeted drugs crossing BBB. Hence, prediction of BBB permeability and estimation of drug concentration in the brain tissue are challenging in drug discovery. To resolve this, estimating the human BBB permeability using improved in vitro and in silico predictive models can be a facilitator. Methods: In vitro data provided by the Drug Delivery research group was used to develop in vitro predictive models for BBB penetration of Verapamil, Risperidone, and Prazosin using R-studio 1.2.5. The MTA approach was adjusted for extrapolation of BBB in vitro transporter activity to in vivo condition. For PBPK modelling, we took advantage of PK-Sim® to simulate drug disposition and time profile of Risperidone in human and animal species. Results: It was shown that MDR1 is the major transporter for efflux transport of Prazosin and Risperidone in brain while both BCRP and MDR1 have similar impact on transport of Verapamil. Furthermore, it was presented in PBPK models that the predicted brain concentration of Risperidone increases in rat and nonhuman primate (NHP) when MDR1 And BCRP are knocked out while the brain concentration of Risperidone in dog is not affected by expression level of the efflux transporters. Conclusion: Both MDR1 and BCRP are contributing in efflux transport of Verapamil, Risperidone, and Prazosin across the BBB. Additionally, expression of the efflux transporters shown to have an impact on brain exposure of Risperidone in animal PBPK models.

La résistance aux médicaments antiépileptiques (MAEs) est un des problèmes majeurs des épilepsies infantiles, comme par exemple le syndrome de Dravet. La pharmacoresistance de l’épilepsie pourrait s’expliquer par une diminution du passage des MAEs dans le cerveau, à travers la Barrière Hémato-Encéphalique (BHE). La BHE comporte des transporteurs des familles « ATP-binding cassette » (ABC) et « SoLute Carrier » (SLC) localisés au niveau de la membrane des cellules endothéliales qui contrôlent leur passage entre le sang et le cerveau. La pharmacoresistance des épilepsies a été associée à ces transporteurs car des MAEs ont été identifiés comme substrats de transporteurs comme la glycoprotéine-P (P-gP) et la « Breast Cancer Resistance Protein » (BCRP). L’hypothèse de cette relation est confortée par l’observation de l’augmentation de l’expression de ces transporteurs d’efflux dans le foyer épileptogène et par l’identification des polymorphismes dans les gènes des transporteurs chez des patients pharmacorésistants. L’interaction au cours du développement cérébral entre les cellules endothéliales et les neurones et astrocytes pourrait modifier le profil des transporteurs de la BHE. Les MAEs sont aussi connus pour être soit des inducteurs, soit des inhibiteurs des enzymes du métabolisme des médicaments et des transporteurs membranaires. Ces données nous permettent de faire les hypothèses suivantes: 1) La BHE en développement présente un profil de transporteurs différent de la BHE mature qui pourrait modifier le passage des MAEs vers le cerveau ; et 2) le traitement chronique administré au cours du syndrome de Dravet pourrait changer le phénotype des transporteurs de la BHE en développement. Nous résultats ont montré que la P-gP et la BCRP augment leur expression au cours du développement. La maturation de la BHE a aussi un impact sur le passage des MAEs étudiés. Nous avons constaté une augmentation de l’expression des différents transporteurs ABC et SLC étudiés pendant le développement de la BHE, suite au traitement chronique avec la thérapie du Syndrome de Dravet. L’acide valproïque, un des MAEs utilisé dans ce traitement, diminue l’activité d’efflux de la P-gP chez les rats en développement et adultes, ce qui a été confirmé dans un modèle in-vitro de BHE immature. Ces résultats mettent en évidence l’interaction entre la BHE en développement et le traitement chronique par les MAEs peut modifier leur distribution au niveau du cerveau et la réponse aux MAEs
Resistance to Antiepileptic Drugs (AEDs) has been a major concern in infantile epilepsies such as for example the Dravet Syndrome. One hypothesis concerning the pharmacoresistance in epilepsy is that a decreased delivery of these drugs to the brain may occur in relation to changes in the Blood-Brain Barrier (BBB) function. BBB exhibits ATP-binding cassette (ABC) and SoLute Carrier (SLC) transporters at the surface of endothelial cells that control the blood-brain transport. Pharmacoresistance in epilepsy may be linked to changes in the functions of these transporters since some AEDs are substrates of the P-glycoprotein (P-gP) and Breast Cancer Resistance Protein (BCRP) transporters. The increased expression of efflux transporters in epileptogenic tissue and the identification of polymorphisms in the efflux transporters genes of resistant patients further support this potential relationship. The interaction of endothelial cells with astrocytes and neurons during brain development could change the pattern of transporters in the BBB. AEDs are also known as either inducers or inhibitors of drug metabolic enzymes and membrane transporters. Taken together, these facts led us to test the following hypothesis: 1) the developing BBB in immature animals presents a different pattern of transporters that could change AEDs disposition in the brain of immature subjects; and 2) the chronic pharmacotherapy used in infantile epilepsies such as the Dravet Syndrome may change the transporters phenotype of the BBB. Our work showed that the expression of P-gP and BCRP increases during development as a function of age. We also showed the maturation of the BBB has an impact on brain disposition of the studied AEDs. We finally observed an increase in the expression of various ABC and SLC transporters induced by the pharmacotherapy of the Dravet Syndrome in immature animals. One of the drugs used, valproic acid, appeared nonetheless to reduce the efflux activity of P-gP in developing and adult animals, which was confirmed in an in-vitro model of the immature BBB. Taken together, these results demonstrated that the interaction between the developing BBB and the AEDs chronic treatment may lead to differences in brain disposition of the AEDs that may impact on the response to AEDs

The immune-to-brain signaling is a critical survival factor when the body is confronted by pathogens, and in particular by microorganisms. During infections, the ability of the immune system to engage the central nervous system (CNS) in the management of the inflammatory response is just as important as its ability to mount a specific immune response against the pathogen, since the CNS can provide a systemic negative feed-back to the immune activation by release of stress hormones and also can prioritize the usage of the energy resources by the vital organs. Prostaglandin E2 (PGE2) and proinflammatory cytokines were among the first mediators to be identified to participate in the immuneto-brain signaling, a process that is clinically recognized by the development of manifestations of common illness such as fever, anorexia, decreased social interactions, lethargy, sleepiness, and hyperalgesia. In this thesis the contribution of PGE2 to the immune-to-brain signaling was further characterized at the blood-brain-barrier (BBB) and in the anterior preoptic area (POA) of the hypothalamus (i.e. the thermoregulatory region or, in sickness, the fever generating region). BBB is the major interface region between peripheral circulating cytokines and the neuronal parenchyma and a critical source of PGE2. Using chimeric mice lacking the inducible enzyme for PGE2 synthesis, microsomal PGE synthase-1 (mPGES-1), in either hematopoietic or non-hematopoietic cells, we demonstrate in paper I that brain endothelial cells are the critical source of PGE2 in BBB during peripheral inflammation. For the demonstration of the mPGES-1 expression in the BBB cells we developed in paper I a method for enzymatic dissociation of these cells, followed by fluorescence activated cell sorting (FACS). Using the same method, we show in paper II that the BBB response to immune stimuli is towards an increased production of PGE2 in endothelial cells and an increased sensitivity of these cells for pro-inflammatory cytokines. These changes are supported by decreased PGE2 degradation and decreased synthesis of other prostanoids in perivascular macrophages, which hence respond in concordance with the endothelial cells in enhancing PGE2 signaling. Once released in the neuronal tissue, PGE2 has been shown to be critical for the fever response by acting on the type 3 PGE2 receptors (EP3) within POA. By laser capture microdissection (LCM) we extracted the EP3 receptor expressing region in POA, defined by in situ hybridization histochemistry, from mouse brain sections. We demonstrate in paper III that the predominant subtypes of the EP3 receptor in POA are EP3α and EP3γ. In paper IV we further analyze the effect of PGE2 on the LCM dissected EP-rich POA using gene expression microarrays. We demonstrate that PGE2 has a limited effect on the gene expression changes within POA, suggesting that the neuronal activity is modulated by PGE2 in a transcription-independent manner and that the profound gene expression changes that are seen in the CNS during inflammation are accordingly PGE2-independent.

L'efficacité thérapeutique de la majorité des molécules développées pour traiter les pathologies du système nerveux central (SNC) n’est pas optimale en raison des caractéristiques anatomiques et physiologiques uniques de la barrière hémato-encéphalique (BHE) qui joue un rôle majeur dans la protection du parenchyme nerveux. Une stratégie permettant de délivrer des agents pharmacologiques dans le cerveau est de détourner un système physiologique : la transcytose régulée par des récepteurs (RMT). Cette stratégie implique la conjugaison d’agents pharmacologiques à des vecteurs développés pour cibler spécifiquement des récepteurs présents au niveau de la BHE et impliqués dans des processus de RMT. Nous avons eu pour objectif de développer de nouveaux vecteurs spécifiques de récepteurs exprimés au niveau de la BHE. Nous avons tout d’abord construit une banque de phages présentant les vecteurs afin de réaliser différentes stratégies de sélection par phage display. A l’issue d’étapes de criblage, les vecteurs sélectionnés ont été produits et caractérisés (affinités pour les récepteurs, compétitions avec les ligands naturels…). Nous avons ensuite évalué le potentiel des vecteurs les plus prometteurs à transporter une biomolécule. Pour ce faire, deux vecteurs par récepteur ont été fusionnés à la région Fc d’une IgG1 humaine et la capacité des conjugués à traverser un modèle de BHE in vitro a été évaluée. Enfin, nous avons évalué la distribution cérébrale des vecteurs, chez la souris in vivo. L’ensemble de nos résultats démontre l'intérêt du ciblage des récepteurs choisis et le potentiel des de nos vecteurs pour des applications de délivrance cérébrale
The vast majority of the molecules developed to treat neurological diseases do not reach their targets efficiently enough because of the unique anatomical and physiological features of the blood-brain barrier (BBB) which plays a major protective role. Hijacking a physiological system named receptor-mediated transcytosis (RMT) is considered a promising route for brain delivery of pharmacological. This strategy involves the conjugation of pharmacological agents to vector molecules developed to specifically target receptors present at the BBB and involved in RMT processes. We aimed to develop new vectors specific for receptors expressed at the BBB. First, we constructed a phage library presenting vectors in order to perform various phage display selection strategies. Following screening steps, the selected vectors were produced and characterized (affinities for the receptors, competitions with natural ligands…). Then, we assessed the potential of the most promising vectors to carry a biomolecule. Two vectors per receptor were fused to a human IgG1 Fc region and the fusion proteins were tested for their ability to cross an in vitro model of the BBB. Finally, we evaluated the brain distribution of the vectors, in mice in vivo. Our results demonstrate the potential of our receptor-targeting vectors for brain drug delivery

SPARC (secreted protein acidic and rich in cysteine) is a cell-matrix modulating protein involved in angiogenesis and endothelial barrier function, yet a potential role in cerebrovascular repair and inflammatory responses in the central nervous system (CNS) has not previously been characterized. The inflammatory demyelinating disease, multiple sclerosis (MS) is characterized pathologically by inflammatory infiltrates, demyelination and axonal damage/loss and aberrant alterations in blood-brain barrier (BBB) integrity. We hypothesize that SPARC expression may be influenced by inflammatory or repair processes during MS, and that SPARC itself may influence BBB integrity. This study examined SPARC expression in cultured human cerebral microvascular endothelial cell (hCMEC/D3), an in vitro model of the BBB, under steady state conditions or those modeling an inflammatory milieu by immunoblotting and immunocytochemistry. hCMEC/D3s constitutively express SPARC during proliferative growth and downregulate SPARC as cells establish a BBB phenotype. SPARC expression in cerebral endothelia directly correlated with the cell proliferation marker Ki-67, consistent with a role for SPARC in CNS angiogenesis. Proinflammatory cytokines associated with inflammation and immune activation differentially regulate SPARC expression in cerebral endothelia. Tumor necrosis factor alpha (TNF-α) cytokine or lipopolysaccharide (LPS) endotoxin treatment significantly increased SPARC protein levels. TNF-α and interferon gamma (IFN-γ) cotreatment abrogated SPARC induction compared to TNF-α alone, suggesting divergent roles for each cytokine in regulating SPARC expression in cerebral endothelia. Compared to cultures replenished with media lacking exogenously supplied SPARC, addition of a physiological SPARC concentration observed in healthy individuals (0.1μg/ml) increased tight junction protein expression of zonula occludens 1 (ZO-1) and occludin by approximately thirty percent, suggesting a role in BBB maintenance. Paradoxically, functional assays show recombinant human SPARC applied exogenously increased the transendothelial permeability of hCMEC/D3 monolayers. In agreement, barrier hCMEC/D3s exposed to increased SPARC concentrations (1-10 μg/ml) associated with pathological conditions in vivo, reduced ZO-1 and occludin by one-third. Together, these data support a role for SPARC in BBB maintenance under normal physiological conditions and BBB alterations during inflammatory conditions. In this regard, SPARC levels may play a key role in regulating BBB integrity and serve to alter processes of CNS inflammation and repair.

Towards the development of nanoparticulate carriers that cross the blood-brain barrier, a series of alkylglyceryl-modified chitosans with systematically varied degrees of grafting were prepared through synthetic steps that involved the protection of amino moieties via the formation of phthaloyl chitosan. The modified chitosans were formulated into nanoparticle using an ionic gelation technique employing sodium tripolyphosphate. Polymers were characterised by FTER, ¹H- and ¹³C-NMR, and by viscometry and GPC techniques. The size distribution profiles of nanoparticles were determined by dynamic light scattering.