Our knowledge of the fine structure of the Human Spiral Ganglion (HSG) is still inadequate and new treatment techniques for deafness using electric stimulation, call for further information and studies on the neuronal elements of the human cochlea. This thesis presents results of analyses of human cochlear tissue and specimens obtained during neurosurgical transpetrosal removal of life-threatening meningeomas. The use of surgical biopsies produced a well-preserved material suitable for ultrastructural and immunohistochemical studies on the human inner ear. The SG was studied with respect to fine structure, using TEM technique and the immunostaining pattern of synaptophysin, which is an integral membrane protein of neuronal synaptic vesicles. The immunostaining patterns of the tight junctional protein ZO-1 and the gap junctional proteins Cx26 and Cx43 in the human cochlea were also studied. The ultrastructural morphology revealed an absence of myelination pattern in the HSG, thus differing from that in other species. Furthermore, formation of structural units as well as signs of neural interaction between the type 1 neurons could be observed. Type 1 cells were tightly packed in clusters, sharing the ensheathment of Schwann cells. The cells frequently made direct physical contact in Schwann cell gaps in which membrane specializations appeared. These specializations consisted of symmetrically or asymmetrically distributed filamentous densities along the apposed cell membranes. The same structures were also present between individual unmyelinated nerve fibres and the type 1 cells. Synapses were observed on the type 2 neurons, with nerve fibres originating from the intraganglionic spiral bundle. Such synapses, though rare, were also observed on the type 1 cells. The ultrastructural findings were confirmed by the synaptophysin study. A 3-D model of a Schwann cell gap between two type 1 cells was constructed, describing the distribution pattern of membrane specializations. In the immunohistochemical studies on the human cochlea, ZO-1 was expressed in tissues lining scala media, thus contributing to the formation of a closed compartment system, important for the maintenance of the specific ionic composition of the endolymph. Protein Cx26 could be identified in non-sensory epithelial cells of the organ of Corti, in connective tissue cells of the spiral ligament and spiral limbus, as well as in the basal and intermediate cell layers of stria vascularis. Cx26 in this region may be involved in the recycling of potassium. Protein Cx43 stained weakly in the spiral ligament, but intense staining in the SG may indicate that gap junctions exist between these neurons. A different functional role for the HSG can be assumed from the morphological characteristics and the signs of a neural interaction between the SG cells. This might be relevant for neural processing mechanisms in speech coding and could have implications for cochlear implant techniques.

Colorectal carcinoma is the second most common type of cancer in both men and women in Sweden. Cancer of the colon and rectum are often considered together and their ten year survival rate is approximately 50 – 60 % depending on sex and location. Different histopathological characteristics of such cancers, including the complexity of growth, are of importance for prognosis. This thesis has compared different morphometric methods in order to achieve a quantitative and objective measurement of the invasive front of colon carcinoma. Since the growth pattern is dependent on the cell adhesiveness of different proteins we studied the distribution and localization of E-cadherin, Beta-catenin, Claudin 1,2,7 and Occludin as well as screened the genes for mutations. We found a perturbed protein expression of E-cadherin, Beta-catenin, Claudin 1,2,7 and Occludin in tumor sections compared to normal mucosa, but no relation to tumor volume or growth pattern could be seen. The tumor volume was found to be correlated to the growth pattern but not responsible to the perturbed protein expression. In the mutation screening we found a SNP in exon 13 the E-cadherin gene in the tumor, as well as in exon 2 of Claudin 1 and exon 4 of Claudin 7 in both tumor and normal mucosa. No correlation between mutations and growth pattern or tumor volume was found. In conclusion, this thesis shows that the computer image analysis with estimation of fractal dimension and number of free tumor cell clusters is superior to the semi quantitative visual grading of tumor invasive complexity. The aberrant expression of cell adhesion proteins in the tumor compared to normal mucosa as well as polymorphisms in the cell adhesion genes CLDN1 and CLDN7 in both tumor and normal mucosa can suggest that these aberrations are important in the tumorigenesis of colon carcinoma.

TRAF4 est un gène fréquemment surexprimé dans les carcinomes suggérant qu’il y joue un rôle. Tandis que la protéine TRAF4 est majoritairement localisée dans les jonctions serrées (JS) des cellules épithéliales mammaires (CEM) normales, elle s’accumule dans le cytoplasme des CEM malignes. Dans cette étude, nous montrons que TRAF4 possède un nouveau domaine liant les phosphoinositides (PIP) et que ce dernier est requis pour son recrutement aux JS. Des analyses moléculaires et structurales ont montré que le domaine TRAF de TRAF4 forme un trimère pouvant lier jusqu’à trois molécules de lipides grâce à des résidus basiques présents à la surface. Des études cellulaires indiquent que TRAF4 régule négativement les JS et augmente la migration cellulaire. Ces deux fonctions sont dépendantes de sa capacité à lier les PIPs. Notre travail suggère que la surexpression de TRAF4 pourrait contribuer à la progression des cancers du sein en déstabilisant les JS et en favorisant la migration cellulaire. / TRAF4 (tumor necrosis factor (TNF) receptor-associated factor 4) is frequently overexpressed in carcinomas suggesting a specific role in cancer. While TRAF4 protein is predominantly found at tight junctions (TJ) in normal mammary epithelial cells (MEC), it accumulates in the cytoplasm of malignant MEC. How TRAF4 is recruited and functions at TJ is unclear. Here we show that TRAF4 possesses a novel phosphoinositide (PIP)- binding domain crucial for its recruitment to TJ. Molecular and structural analyses revealed that the TRAF domain of TRAF4 exists as a trimer which binds up to 3 lipids using basic residues exposed at its surface. Cellular studies indicated that TRAF4 acts a negative regulator of TJ and increases cell migration. These functions are dependent from its ability to interact with PIPs. Our results suggest that TRAF4 overexpression might contribute to breast cancer progression by destabilizing TJ and favoring cell migration.

Abstract It is a generally accepted dogma that sepsis disturbs skin function and wound healing, but surprisingly there is only remote pathophysiological evidence available behind that presumption. As the skin is the largest defensive barrier, the skin dysfunction in severe sepsis deserves more attention. In this study, the suction blister model was used to create experimental wounds. The study population included 44 patients with severe sepsis and 15 controls. The blister fluid was collected to analyse cytokine profile of the skin. The transepidermal water loss and blood flow from the wound were measured. A 4mm biopsy was taken under local anaesthesia on the first and the eighth day of the study from the healthy looking skin. Then 15 healing suction blisters were excised. Serum samples were also collected on the first day of the study. The barrier restoration was diminished, and the inflammation in the wound was more intense in severe sepsis than in the controls. The expression of the basement membrane components Laminin-332 and type IV collagen decreased during the septic disease, but increased over the next 3 months without achieving the level oft he controls. The expression of tight junction proteins remained nearly intact in the healing wound in severe sepsis compared to the controls. The expression of occludin on the leading edge of the migrating keratinocytes was more restricted and late in severe sepsis compared to the controls. The levels of the tumour necrosis factor (TNF), interleukin-10 (IL-10) and IL-6 in skin blister fluid were higher in the sepsis compared to controls. The blister fluid and serum cytokine response in the sepsis differed since the levels of epidermal growth factor, vascular endothelial growth factor, TNF and basic fibroblastic growth factor (bFGF) in the blister fluid did not correlate with the levels of serum. The septic patients with multiple organ failure had higher levels of several cytokines than patients without organ failure. Survivors had lower levels of IL-10 and bFGF in blister fluid than the non-survivors. This study offers novel findings for skin and wound healing in sepsis. Together, all the findings suggest that skin dysfunction in severe sepsis exists even when the most profound structures remain intact. Understanding these mechanisms of impaired wound healing can improve future treatments, such as the timing of surgery. / Tiivistelmä Sepsiksen ajatellaan heikentävän haavanparanemista, mutta tieteellistä näyttöä on niukasti. Iholla on keskeinen osa elimistön puolustuksessa ja tasapainon ylläpidossa, joten sen toiminnan häiriintyminen systeemisessä tulehduksessa ansaitsee suuremman huomion. Imurakkulahaavat tehtiin 44 septiselle potilaalle ja 15 kontrollille. Haavoista mitattiin veden haihtumista ja veren virtausta sekä otettiin imurakkulaneste näytteeksi sytokiinimäärityksiä varten. Tutkimuksen ensimmäisenä ja kahdeksantena päivänä otettiin 4mm biopsiat terveeltä iholta ja 15 potilaalta poistettiin näytteeksi paraneva imurakkulahaava. Seeruminäytteet otettiin tutkimuksen ensimmäisenä päivänä. Veden haihtuminen haavalta oli voimakkaampaa eli ihon barrierin palautuminen oli hidastunut septisillä potilailla verrattuna kontrolleihin. Haavassa havaittu tulehdus oli sepsiksessä voimakkaampi. Tyvikalvon komponenttien Laminiini-332:n ja tyypin IV kollageenin ilmentyminen oli vähäisempää sepsiksen aikana ja lisääntyi 3kk kohdalla, mutta ei kuitenkaan saavuttanut kontrollien tasoa. Tiivisliitosproteiinien ilmentyminen oli lähes muuttumatonta sepsiksessä kontrolleihin verrattuna. Okludiinin ilmentyminen sen sijaan paranevassa haavassa vaeltavien keratinosyyttien etureunassa oli rajoittuneempaa ja myöhäisempää sepsiksessä kuin kontrolleilla. Sytokiineistä tuumorinekroositekijä (TNF), interleukiini-10 (IL-10) ja IL-6 olivat koholla imurakkulanesteessä verrattuna kontrolleihin. Epidermaalinen kasvutekijä, verisuonten endoteelikasvutekijä, TNF ja perusfibroplastinen kasvutekijä (bFGF) pitoisuudet rakkulanesteessä erosivat seerumin pitoisuuksista eli ihon sytokiiniprofiili erosi systeemisestä sytokiiniprofiilista. Potilailla, joilla oli monielinvaurio, todettiin korkeampia sytokiinipitoisuuksia. Potilailla, jotka menehtyivät 30 vrk kuluessa, oli korkeammat pitoisuudet IL-10 ja bFGF rakkulanesteessä. Tämä tutkimus tarjoaa uutta tietoa ihosta ja haavanparanemisesta sepiksessä. Tulosten perusteella voidaan todeta, että ihon toimintahäiriö on sepsiksessä todellinen, vaikka kaikkein perustavimmat rakenteet säilyvät muuttumattomina. Toimintahäiriön mekanismien ymmärtäminen voisi auttaa septisen potilaan hoidossa, kuten kirurgisten toimenpiteiden ajoittamisessa paranemisen kannalta mahdollisimman otolliseen aikaan.

Abstract Lung cancers and chronic obstructive pulmonary disease (COPD) are the most common smoking-related lung diseases and both have high mortality rate. Tight junctions (TJ) are apical junctions between epithelial cells that regulate the permeability of epithelium and form the tight junction along with occludin. Dysfunction of the TJ and dysregulation of TJ proteins leads to a loss of cell-cell adhesion and a loss of cohesion as well as epitheliomesenchymal transition. These increase invasion of lung carcinomas and possibly predispose to the exacerbations in COPD. Therefore, the aim of this thesis was to study expression and regulation of claudins in different lung carcinomas and COPD. Carcinomas expressed claudins 1, 2, 3, 4, 5 and 7 in different variations. Claudin 5 expression was weak in all carcinoma types. Strong claudin 1, 4 and 7 expression was associated with better survival in squamous cell carcinoma and adenocarcinoma. Claudin 3 expression was associated with COPD in large airways. Claudins 3 and 4 was found to be stronger in small airways of smokers and COPD patients than in non-smokers. Transcription factor snail had prognostic value in lung carcinomas. Negative snail expression was associated with longer life expectancy in lung carcinoma patients. Negative snail expression was associated with up-regulated claudin 5 and 7 expression, while strong expression was associated with low claudin 1 and 3 expression. Transcription factors slug and twist were inversely associated with claudins 3 and 4 in small and large airways. Slug expression was higher in non-smokers than in COPD patients and smokers. Transcription factor knockdown increased claudin expression in normal bronchial cell line. Except for claudin 2 and 7, which were decreased. Adenocarcinoma-like cell line was not affected by snail knockdown and in squamous cell carcinoma-like cell line claudin 3, 4 and 7 expression was increased. Transcription factor snail knockdown inhibited invasion of cell lines. Twist knockdown increased transepithelial resistance in normal bronchial cell line indicating higher barrier function in cell layer. / Tiivistelmä Keuhkosyöpä ja keuhkoahtaumatauti ovat yleisiä tupakoinnin aiheuttamia keuhkosairauksia, joissa on korkea kuolleisuus. Tupakointi aiheuttaa muutoksia keuhkojen epiteelisoluissa ja solujen välisissä liitoksissa. Tiivisliitokset solujen välillä säätelevät epiteelin rakennetta ja läpäisevyyttä. Klaudiinit ovat proteiineja, jotka muodostavat tiivisliitoksen yhdessä okkludiinin kanssa. Tiivisliitos proteiinien toimintahäiriöt voivat johtaa solujen välisten liitosten katoamiseen ja epiteelin hajoamiseen sekä epiteelisolujen muuntumiseen mesenkymaalisten solujan kaltaisiksi. Nämä seikat lisäävät invaasiota keuhkosyövissä ja saattavat altistaa pahenemisvaiheisiin keuhkoahtaumataudissa. Väitöskirjassa tutkittiin klaudiinien ilmentymistä ja säätelyä keuhkosyövässä ja keuhkoahtaumataudissa. Klaudiinien1, 2, 3, 4, 5 ja 7 esiintyminen keuhkosyövän histologisissa alatyypeissä vaihteli. Klaudiinien 1, 4 ja 7 voimakas ilmentyminen voitiin yhdistää pidempään elinikään potilailla, joilla oli levyepiteeli- tai adenokarsinooma. Klaudiini 3:n ilmentyminen liittyi keuhkoahtaumatautiin suurissa hengitysteissä. Klaudiinien 3 ja 4 voimakas ilmeneminen pienissä ilmateissä oli yleisempää keuhkoahtaumatautipotilailla ja tupakoitsijoilla kuin tupakoimattomilla henkilöillä. Transkriptiotekijä snailin puuttuminen keuhkosyövässä liittyi potilaiden pidempään elinaikaan. Klaudiinien 5 ja 7 ilmeneminen oli voimakkaampaa, kun snailin määrä oli vähäinen. Klaudiinien 1 ja 3 ilmeneminen väheni snail:in ollessa voimakas keuhkosyövässä. Traskriptiotekijöiden (slug ja twist) ilmeneminen liittyi käänteisesti klaudiinien ilmentymiseen pienissä ja suurissa ilmateissä. Slugin ilmeneminen oli voimakkaampaa tupakoimattomilla henkilöillä kuin tupakoivilla tai keuhkoahtaumatautia sairastavilla. Transkriptiotekijöiden snail, slug ja twist toiminnan estäminen lisäsi klaudiinien määrää normaaleissa keuhkon epiteelisoluissa. Poikkeuksen muodostivat klaudiinit 2 ja 7, joiden määrä väheni kun snail:in toiminta oli estetty. Adenokarsinooma-soluissa snailin estolla ei ollut vaikutusta, ja levyepiteelisyövän soluissa klaudiinien 3, 4 ja 7 määrä kasvoi. Snail myös vähensi solujen invaasiota. Transkriptiotekijä twistin toiminnan esto normaaleissa keuhkoepiteelisoluissa nosti solumaton läpi kulkevan sähkön resistenssiä, mikä on osoitus tiiviistä solujen välisistä liitoksista.

La barrière épidermique du stratum corneum (SC) est doublée par une barrière secondaire des jonctions serrées (JS) qui influent sur la formation de barrière principale. Dans mes travaux, je me suis concentré sur l'étude de la présence et l'évolution des éléments jonctionnelles composants ces deux barrières ; les cornéodesmosomes au niveau du SC et les JSs au niveau de la granuleuse. En plus, je me suis intéressé à l'implication des glycanes dans la fonction barrière épidermique. Ces travaux ont été réalisés soit dans un contexte physiologique soit par la modulation de la barrière épidermique par des facteurs intrinsèques et extrinsèques. Nos résultats confirment que les JSs jouent un rôle subalterne par rapport à la barrière du SC et montrent que les glycanes persistent à la surface des cornéocytes humains. La composition et la répartition utlrastructurale des glycanes évoluent à travers les assises du SC jusqu'à la desquamation d'une manière concordante avec la répartition des cornéodesmosomes. Certaines modifications intrinsèques naturelles lors du vieillissement ou pathologiques notamment l'état pelliculaire et la dermatite atopique, ont permis d'appréhender le rôle de ces composants dans la cohésion du SC et la prestance d'une barrière fonctionnelle. Les modifications extrinsèques de la barrière par l'application de solvants, d'excipients ou de formulations perméabilisantes montrent l'importance de l'organisation utlrastructurale des composants jonctionnelles et non jonctionnelles du SC dans le maintien d'une barrière efficace / The stratum corneum (SC) barrier is doubled by the secondary barrier of tight junctions which influences the formation of the main barrier. In my work, I focused on the study of the junctional elements composing those two barriers; corneodesmosomes in the SC and the tight junction at the granular layer level. In addition, I got interested in the involvement of glycans in the epidermal barrier function. This work was carried out either in skin physiological conditions or by the modulation of the epidermal barrier by intrinsic or extrinsic factors. Our results confirm that tight junctions play a subordinate role compared to the SC barrier and that glycans remain present at the surface of human corneocytes. The composition and the ultrastructure distribution of glycans evolve from the SC compactum to the SC disjunctum, towards desquamation in a comparable manner to the repartition of corneodesmosomes. Natural intrinsic changes during aging and pathological changes, including dandruff and atopic dermatitis, helped us to understand the role of those components in the cohesion of the SC and the conservation of functional barrier. Extrinsic modulation of the barrier by the application of solvents, excipients or topical formulations shows the importance of the ultrastructural organization of junctional and non-junctional SC components in maintaining an effective barrier

Inflammatory bowel disease (IBD), which includes the subtypes Crohn's disease (CD) and ulcerative colitis (UC), is a common disease particularly in the Western world. IBD is characterised by inflammation of the small intestine and/or colon. The two subtypes affect different gut locations but both show an increased intestinal permeability or the 'leaky gut syndrome'. This led to the hypothesis that tight junction (TJ) proteins expressed in the epithelium may affect the intestinal permeability as a cause or effect of IBD.Initially, variants in the CARD15, IL23R and ATG16L1 genes, previously associated with an increased risk of IBD, were genotyped in a cohort of 500 IBD (295 CD and 205 UC) patients and 877 matched controls. These variants were significantly associated in our cohort. A random effects meta-analysis was undertaken on all previously reported CD associations with the variant rs2241880 from ATG16L1 (n=25, p=0.0017, OR: 1.36 95% CI 1.12-1.66) and with rs11209026 from IL23R (n=26, p=0.0006, OR: 0.37 95% CI 0.21-0.67), showing pooled odds ratios consistent with those reported in our cohort. Individuals carrying >1 CARD15 mutant variant were found to have a 2.5 fold increased risk of CD (p=0.0001). Candidate TJ proteins were chosen on the basis of previous reported associations and through the investigation of the claudin proteins which are abundant at TJs. Twenty one candidate genes were selected and 79 variants successfully genotyped in up to 1063 IBD (502 CD and 478 UC) and 870 control patients. Significant associations were detected with variants in the CLDN1, CLDN5 and CDH1 genes with CD; CLDN5, CLDN8 and CDH1 variants were associated to IBD; and the rs7791132 variant (between CLDN4 and ELN) and a CDH1 variant were associated to UC. The CLDN1 rs6809685 variant trended towards association in a Toronto ascertained IBD replication cohort (genotypic p=0.04, allelic p=0.06) suggesting this may be a novel IBD susceptibility variant. Small intestinal biopsies from CD patients with known rs6809685 genotypes showed a dose dependent reduced immunohistochemical staining of claudin 1 with carriage of the mutant G allele. Claudin 1 helps seal TJs and reduced levels may increase risk of CD.Peroxisome proliferator activator receptors (PPARs) can directly affect TJ proteins and could therefore affect intestinal permeability. Twelve PPARγ variants were genotyped in up to 1050 IBD (502 CD and 467 UC) and 725 control patients. Significant genotypic associations were found with the rs2067819 variant in CD (p=0.05) and IBD (p=0.02), and also the rs13099634 variant in UC (P=0.02). There was a strong gender difference particularly for rs2067819 and rs4135247, where allelic associations were highly significant and increased risk of IBD in men (p=0.01 and p=0.007 respectively). However no significant associations were found in the female cohort. Troglitazone a PPARα agonist increased Caco2 cell transepithelial electrical resistance (TEER), a marker of TJ integrity, and increased expression of claudins -3 and -4. In contrast, the PPARα antagonist GW6471 reduced the TEER without causing cell death and PPARγ ligands did not affect TEER measurements. In summary, using a robust cohort of cases and controls the data indicates that variants in genes encoding TJ proteins may affect susceptibility to IBD and that PPARs can regulate these proteins altering intestinal permeability.

No description available.

Les bénéfices attribués aux probiotiques sont nombreux : effets bénéfiques sur le microbiote intestinal, confort digestif, modulation du système immunitaire et prévention des infections intestinales. Ces effets divers et variés sont souches-spécifiques. Actuellement, de nombreuses études visent à mieux caractériser ces effets probiotiques.Ces travaux avaient pour objectif de déterminer et d’analyser les effets probiotiques de trois souches de la collection Danone : la souche de Lactobacillus paracasei CNCM I-3689, la souche de L. rhamnosus CNCM I-3690 ainsi que la souche de Bifidobacterium animalis subps. lactis CNCM I-2494. Dans un premier temps, leurs effets ont été caractérisés dans des modèles in vitro pour des propriétés anti-pathogènes, des propriétés d’immuno-modulation ainsi que pour des propriétés de protection de la barrière épithéliale intestinale. Dans un second temps, leurs effets de la protection de la barrière ont été confirmés dans un modèle murin de faible inflammation.Une analyse des mécanismes sous-jacents à ces effets à la fois chez la souche probiotique et chez l’hôte a été initiée par la construction et l’analyse fonctionnelle de banques génomiques bactériennes ainsi que par l’étude de la modulation des gènes impliqués dans le maintien de la barrière intestinale chez l’hôte. / Health benefits of probiotic bacteria are numerous: beneficial effects on the intestinal microbiota, digestive comfort, modulation of the immune system and prevention of winter infection. These diverse and various effects are strain-specific. Nowadays, numerous studies aim at better characterizing those probiotics effects.This project aimed at identifying and analyzing the probiotic effects of three strains from Danone collection: Lactobacillus paracasei CNCM I-3689, L. rhamnosus CNCM I-3690 and Bifidobacterium animalis subsp. lactis CNCM I-2494. First, their effects were assessed in in vitro models for immunomodulation properties, antipathogens activity and intestinal barrier protection. Secondly, their beneficial effects were confirmed in low-grade inflammation murine model.The analysis of the underlying mechanisms has been initiated both in the bacterial strains by the construction and the functional analysis of genomic libraries and in the host by measuring the modulation of the genes involved in the intestinal barrier maintain.

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.