title: Quantitative fluorescence microscopy provides high resolution imaging of passive diffusion and P-gp mediated efflux at the in vivo blood-brain barrier.
abstract: Quantitative fluorescent microscopy is an emerging technology that has provided significant insight into cellular dye accumulation, organelle function, and tissue physiology. However, historically dyes have only been used to qualitatively or semi-quantitatively (fold change) determine changes in blood-brain barrier (BBB) integrity. Herein, we present a novel method to calculate the blood to brain transfer rates of the dyes rhodamine 123 and Texas red across the in situ BBB. We observed that rhodamine 123 is subject to p-glycoprotein mediated efflux at the rat BBB and can be increased nearly 20-fold with p-glycoprotein inhibition. However, Texas Red appears to not be subject to MRP2 mediated efflux at the rat BBB, agreeing with literature reports suggesting MRP2 may lack functionality at the normal rat BBB. Lastly, we present data demonstrating that once dyes have crossed the BBB, diffusion of the dye molecule is not as instantaneous as has been previously suggested. We propose that future work can now be completed to (1) match BBB transfer coefficients to interstitial diffusion constants and (2) use dyes with specific affinities to cellular organelles or that have specific properties (e.g., subject to efflux transporters) to more fully understand BBB physiology.
authors.affiliation: Department of Pharmaceutical Sciences, School of Pharmacy, Texas Tech University HSC, Amarillo, TX 79106-1712, United States.
authors.forename: Rajendar K
authors.initials: RK
authors.lastname: Mittapalli
authors.forename: Vamshi K
authors.initials: VK
authors.lastname: Manda
authors.forename: Kaci A
authors.initials: KA
authors.lastname: Bohn
authors.forename: Chris E
authors.initials: CE
authors.lastname: Adkins
authors.forename: Paul R
authors.initials: PR
authors.lastname: Lockman
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000465:Algorithms; D000818:Animals; D001345:Autoradiography; D001812:Blood-Brain Barrier / Q000187:drug effects* / Q000502:physiology*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002478:Cells, Cultured; D003627:Data Interpretation, Statistical; D005456:Fluorescent Dyes; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D016014:Linear Models; D008297:Male; D008856:Microscopy, Fluorescence; D010477:Perfusion; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D051381:Rats; D011916:Rats, Inbred F344; D012015:Reference Standards; D020112:Rhodamine 123; D014700:Verapamil / Q000494:pharmacology; D014966:Xanthenes
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D002121:Calcium Channel Blockers; D005456:Fluorescent Dyes; D011422:Propionates; D011804:Quinolines; D014966:Xanthenes; D049452:Green Fluorescent Proteins; D020112:Rhodamine 123; C059141:verlukast; C034657:Texas red; D014700:Verapamil
keywords: Alzheimer's; Autoradiography; Blood–brain barrier; Drug delivery; Parkinson's; Quantitative fluorescent microscopy; Stroke; Transport
references.citation: J Cereb Blood Flow Metab. 1997 Nov;17(11):1239-53
references.pmid: 9390656
references.citation: J Cereb Blood Flow Metab. 2008 Jun;28(6):1222-34
references.pmid: 18349876
references.citation: J Cereb Blood Flow Metab. 1983 Mar;3(1):8-32
references.pmid: 6822623
references.citation: J Exp Biol. 2005 Dec;208(Pt 23):4363-76
references.pmid: 16339857
references.citation: Semin Cell Dev Biol. 2001 Jun;12(3):257-65
references.pmid: 11428918
references.citation: J Neurochem. 2001 Feb;76(4):1032-41
references.pmid: 11181822
references.citation: Pharmacol Rev. 2005 Jun;57(2):173-85
references.pmid: 15914466
references.citation: Biochem Biophys Res Commun. 1995 Jun 26;211(3):719-26
references.pmid: 7598699
references.citation: Neurosci Lett. 2010 Jan 22;469(2):224-8
references.pmid: 19963040
references.citation: J Pharmacol Exp Ther. 2008 Jan;324(1):95-102
references.pmid: 17947497
references.citation: Pharm Biotechnol. 1996;8:285-307
references.pmid: 8791815
references.citation: Int J Biol Macromol. 2006 Dec 15;40(1):1-8
references.pmid: 16769109
references.citation: J Neurosurg. 1965 Nov;23(5):484-7
references.pmid: 5858439
references.citation: J Neurochem. 2001 Nov;79(3):588-94
references.pmid: 11701762
references.citation: Brain Res. 2004 Jul 16;1014(1-2):221-7
references.pmid: 15213006
references.citation: J Pharmacol Exp Ther. 1995 Apr;273(1):298-303
references.pmid: 7714780
references.citation: Am J Physiol Cell Physiol. 2008 Aug;295(2):C440-50
references.pmid: 18524938
references.citation: Mol Pharmacol. 2000 Dec;58(6):1357-67
references.pmid: 11093774
references.citation: Eur Neurol. 1970;3(5):293-302
references.pmid: 4315475
references.citation: Pharmacol Rev. 2006 Jun;58(2):140-61
references.pmid: 16714484
references.citation: Acta Neuropathol. 1983;61(3-4):201-6
references.pmid: 6316707
references.citation: J Antimicrob Chemother. 2007 Nov;60(5):987-93
references.pmid: 17890284
references.citation: J Pharmacol Exp Ther. 2002 Aug;302(2):407-15
references.pmid: 12130697
references.citation: J Nucl Med. 2003 Nov;44(11):1845-54
references.pmid: 14602869
references.citation: Mol Pharmacol. 1990 Jun;37(6):801-9
references.pmid: 1972772
references.citation: J Neurosci Res. 2009 Mar;87(4):1023-36
references.pmid: 18855943
references.citation: J Clin Invest. 1998 Apr 1;101(7):1310-9
references.pmid: 9525973
references.citation: Int J Oncol. 2006 Dec;29(6):1413-9
references.pmid: 17088979
references.citation: Neuropharmacology. 2010 Jun;58(7):1019-32
references.pmid: 20080116
references.citation: Am J Physiol Regul Integr Comp Physiol. 2008 Oct;295(4):R1311-9
references.pmid: 18650317
references.citation: J Neurochem. 2003 Aug;86(3):627-34
references.pmid: 12859676
references.citation: Clin Cancer Res. 2010 Dec 1;16(23):5664-78
references.pmid: 20829328
references.citation: Arq Neuropsiquiatr. 1972 Mar;30(1):1-7
references.pmid: 5045075
references.citation: Brain Res. 2000 Sep 8;876(1-2):148-53
references.pmid: 10973603
references.citation: Environ Physiol Biochem. 1975;5(6):389-95
references.pmid: 1213027
references.citation: J Pharmacol Exp Ther. 2005 Aug;314(2):636-42
references.pmid: 15845856
references.citation: Am J Pathol. 1990 Jul;137(1):43-57
references.pmid: 2164776
references.citation: Acta Neuropathol. 1970;16(2):117-24
references.pmid: 4097399
references.citation: J Neurosurg. 1970 Jan;32(1):89-94
references.pmid: 5410802
references.citation: Ann N Y Acad Sci. 1986;481:186-201
references.pmid: 3468857
references.citation: Pharm Res. 2003 May;20(5):705-13
references.pmid: 12751624
references.citation: J Neurosci Methods. 2006 Mar 15;151(2):262-7
references.pmid: 16181683
references.citation: Nucl Med Biol. 2005 Oct;32(7):719-25
references.pmid: 16243647
references.citation: Brain Res. 1996 Nov 11;739(1-2):88-96
references.pmid: 8955928
references.citation: Arch Insect Biochem Physiol. 2006 Nov;63(3):123-34
references.pmid: 17048246
references.citation: Stroke. 1977 Mar-Apr;8(2):182-8
references.pmid: 847782
references.citation: J Neurochem. 2005 Jul;94(1):37-44
references.pmid: 15953347
references.citation: J Neuropathol Exp Neurol. 1971 Jan;30(1):139-40
references.pmid: 5542526
references.citation: J Neurol Sci. 1983 Jun;59(3):305-22
references.pmid: 6875604
references.citation: J Cell Biol. 1967 Jul;34(1):207-17
references.pmid: 6033532
references.citation: AIDS. 2005 Dec 2;19(18):2097-102
references.pmid: 16284458
references.citation: J Drug Target. 2004;12(9-10):635-41
references.pmid: 15621689
references.citation: Brain Res. 2007 Feb 23;1134(1):1-11
references.pmid: 17196184
references.citation: Nat Biotechnol. 1997 Apr;15(4):373-7
references.pmid: 9094141
references.citation: Am J Physiol. 1984 Sep;247(3 Pt 2):H484-93
references.pmid: 6476141
references.citation: Pharm Res. 2005 Jan;22(1):113-21
references.pmid: 15771237
references.citation: Epilepsy Res. 2006 Apr;69(1):1-14
references.pmid: 16504477
references.citation: Life Sci. 1996;59(18):1521-31
references.pmid: 8890933
references.citation: AMA Arch Neurol Psychiatry. 1956 Nov;76(5):457-67
references.pmid: 13371961
references.citation: Methods Cell Biol. 2008;85:497-538
references.pmid: 18155477
references.citation: Am J Physiol. 1985 Sep;249(3 Pt 2):H629-37
references.pmid: 3929626