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Differences in gene expression in the developing rat brain using cDNA microarray and real-time PCR
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy. (Margareta Hammarlund-Udenaes)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Division of Pharmacokinetics and Drug Therapy. (Margareta Hammarlund-Udenaes)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Toxicology. (Lennart Dencker)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences, Toxicology. (Lennart Dencker)
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

The blood-brain barrier (BBB) is formed by endothelial cells, connected by tight junction proteins restricting paracellular transport. Transport of substances into the brain may also be limited due to active efflux transporters. Not much is known about the development of tight junction proteins and active transporters in the BBB, and how this affects drug distribution to the brain at different ages. The aim of this study was to analyze possible differences in expression of tight junction proteins and active transporters in the BBB at different postnatal ages in the rat. Brain capillary-rich fractions (BCRF) were collected and gene expression levels were compared at three postnatal ages using microarray analysis. BCRF was also analyzed for the mRNA expression levels of P-gp Abcb1 (P-gp), Abcg2 (Bcrp), Slc22a8 (Oat3) and occludin using real-time PCR at 12 different postnatal ages between postnatal Day 1 and Adult. In the array analysis, 28 genes of interest were found to be differentially expressed. The mRNA levels of Abcg2 decreased to one seventh from postnatal Day 1 to Day 15. The levels of Slc22a8 increased from birth and reached a plateau at Day 3 - 12 followed by a decrease to Day 15. These findings show that active transporters are differentially expressed in the developing BBB, possibly affecting drug distribution to the brain. No change in the mRNA levels for Abcb1 or occludin was observed.

Keyword [en]
Blood-brain barrier, development, active transport, tight junctions, microarray, real-time PCR
National Category
Pharmaceutical Sciences
Research subject
Pharmacokinetics and Drug Therapy
Identifiers
URN: urn:nbn:se:uu:diva-109367OAI: oai:DiVA.org:uu-109367DiVA: diva2:272142
Available from: 2009-10-14 Created: 2009-10-14 Last updated: 2010-01-14
In thesis
1. Developmental Aspects of Drug Transport Across the Blood-Brain Barrier
Open this publication in new window or tab >>Developmental Aspects of Drug Transport Across the Blood-Brain Barrier
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 60 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 110
Keyword
Blood-brain barrier, development, active transport, tight junction proteins, microdialysis, recovery, morphine, nitrofurantoin, Bcrp, microarray, real-time PCR, in vitro, in vivo, LC-MS/MS
National Category
Pharmaceutical Sciences
Research subject
Pharmacokinetics and Drug Therapy
Identifiers
urn:nbn:se:uu:diva-108374 (URN)978-91-554-7627-4 (ISBN)
Public defence
2009-11-26, B42, Biomedicinskt Centrum, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-11-04 Created: 2009-09-17 Last updated: 2011-05-11Bibliographically approved

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