spatial-temporal-analysis-of-fieldwise-flow-in-microvasculature

Spatial Temporal Analysis of Fieldwise Flow in Microvasculature

Changes in blood flow velocity and distribution are vital in maintaining tissue and organ perfusion in response to varying cellular needs. Further, ...

Indiana University

Developing mammalian embryonic kidney becomes progressively more elaborate as the ureteric bud branches into undifferentiated mesenchyme. Morphological perturbations of nephrogenesis, such as those seen in inherited renal diseases or induced in transgenic animals, require careful and often tedious documentation by multiple methodologies. We have applied a relatively quick and simple approach combining two-photon microscopy and advanced three-dimensional (3-D) imaging techniques to visualize and evaluate these complex events. As compared with laser confocal microscopy, two-photon microscopy offers superior optical sectioning deep into biological tissues, permitting analysis of large, heterogeneous, 3-D structures such as developing mouse kidney. Embryonic and newborn mouse kidneys were fluorescently labeled with lectins, phalloidin, or antibody. Three-dimensional image volumes were then collected. The resulting volume data sets were processed using a novel 3-D visualization technique. Reconstructed image volumes demonstrate the dichotomous branching of ureteric bud as it progresses from a simple, symmetrical structure into an elaborate, asymmetrical collecting system of multiple branches. Detailed morphology of in situ cysts was elucidated in a transgene-induced mouse model of polycystic kidney disease. We expect this integration of two-photon microscopy with advanced 3-D image analysis will provide a powerful tool for illuminating a variety of complex developmental processes in multiple dimensions.

Three-dimensional imaging of embryonic mouse kidney by two-photon microscopy.

Cystic kidney disease has been linked to mutations in the Invs gene in mice with inversion of embryonic turning (inv/inv) and the INVS (NPHP2) gene in infants with nephronophthisis type 2 (NPHP2). The inv mouse model features multiorgan defects including renal cysts, altered left-right laterality, and hepatobiliary duct malformations transmitted in an autosomal recessive manner. Affected mice usually die of renal and liver failure by postnatal day 7. Although cardiopulmonary and liver anomalies have been carefully detailed, renal cysts have yet to be fully characterized in inv/inv. By use of three-dimensional visualization by two-photon microscopy, this study provides the first comprehensive analysis of in situ cyst formation and progression in inv/inv kidneys. At embryonic day 15, there is dilatation of Bowman's capsule followed temporally by corticomedullary cysts involving collecting ducts, proximal tubules, and thick ascending limbs. Collecting ducts of newborn inv/inv mice are uniformly and diffusely cystic from medulla to cortex, with normal diameters found only at their most proximal tips. Proximal tubules form fusiform cysts that alternate with segments of normal or narrowed caliber along torturous convolutions. Because defective cilia have been linked to situs inversus and cystogenesis, we examined inv/inv cilia by scanning and transmission electron microscopy. The former detected monocilia of expected length in cystic collecting ducts and proximal tubules; the latter demonstrated the usual 9 + 2 pattern in respiratory cilia. The inv mutant mouse has renal cysts resembling infantile NPHP2 and will provide broader insight into the role cilia play in renal cystogenesis.

Renal cysts of inv/inv mice resemble early infantile nephronophthisis.

Microvascular perfusion dynamics are vital to physiological function and are frequently dysregulated in injury and disease. Typically studies measure microvascular flow in a few selected vascular segments over limited time, failing to capture spatial and temporal variability. To quantify microvascular flow in a more complete and unbiased way we developed STAFF (Spatial Temporal Analysis of Fieldwise Flow), a macro for FIJI open-source image analysis software. Using high-speed microvascular flow movies, STAFF generates kymographs for every time interval for every vascular segment, calculates flow velocities from red blood cell shadow angles, and outputs the data as color-coded velocity map movies and spreadsheets. In untreated mice, analyses demonstrated profound variation even between adjacent sinusoids over seconds. In acetaminophen-treated mice we detected flow reduction localized to pericentral regions. STAFF is a powerful new tool capable of providing novel insights by enabling measurement of the complex spatiotemporal dynamics of microvascular flow.

Article	Year
Three-dimensional imaging of embryonic mouse kidney by two-photon microscopy. The American journal of pathology\| PubMed ID: 11141478	2001
Renal cysts of inv/inv mice resemble early infantile nephronophthisis. Journal of the American Society of Nephrology : JASN\| PubMed ID: 15213262	2004
A simple automated method for continuous fieldwise measurement of microvascular hemodynamics. Microvascular research\| PubMed ID: 30502365	2019

Adele Filson

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