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Title: Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Abstract

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman's space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)- dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman's space and all segments of themore » nephron within the imaging depth limit, about 100 µm below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman's space. We expect this technique to have wide applicability in renal physiologic investigation.« less

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Pacific Northwest National Lab. (PNNL), Richland, WA (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1501620
Report Number(s):
PNNL-SA-137928
Journal ID: ISSN 1940-087X; jove
DOE Contract Number:  
AC05-76RL01830
Resource Type:
Journal Article
Journal Name:
Journal of Visualized Experiments
Additional Journal Information:
Journal Volume: 140; Journal Issue: 140; Journal ID: ISSN 1940-087X
Publisher:
MyJoVE Corp.
Country of Publication:
United States
Language:
English

Citation Formats

Matsushita, Katsuyuki, Golgotiu, Kirsti, Orton, Daniel J., Smith, Richard D., Rodland, Karin D., Piehowski, Paul D., and Hutchens, Michael P. Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy. United States: N. p., 2018. Web. doi:10.3791/58206.
Matsushita, Katsuyuki, Golgotiu, Kirsti, Orton, Daniel J., Smith, Richard D., Rodland, Karin D., Piehowski, Paul D., & Hutchens, Michael P. Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy. United States. doi:10.3791/58206.
Matsushita, Katsuyuki, Golgotiu, Kirsti, Orton, Daniel J., Smith, Richard D., Rodland, Karin D., Piehowski, Paul D., and Hutchens, Michael P. Mon . "Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy". United States. doi:10.3791/58206.
@article{osti_1501620,
title = {Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy},
author = {Matsushita, Katsuyuki and Golgotiu, Kirsti and Orton, Daniel J. and Smith, Richard D. and Rodland, Karin D. and Piehowski, Paul D. and Hutchens, Michael P.},
abstractNote = {Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman's space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)- dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman's space and all segments of the nephron within the imaging depth limit, about 100 µm below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman's space. We expect this technique to have wide applicability in renal physiologic investigation.},
doi = {10.3791/58206},
journal = {Journal of Visualized Experiments},
issn = {1940-087X},
number = 140,
volume = 140,
place = {United States},
year = {2018},
month = {1}
}