Automated and quantitative image analysis of ischemic dendritic blebbing using in vivo 2-photon microscopy data

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Abstract

Ischemia induces a ‘blebbing’ of dendrites, a structural alteration where dendrites take on a ‘beads on a string’ appearance. We developed a toolkit program, BlebQuant, for quantitative automated bleb analysis to chart the morphology of dendrites labeled with GFP/YFP under normal conditions and after ischemia-induced damage. In vivo 2-photon data from mouse layer 5 neurons with apical dendritic tufts extending to the cortical surface were examined before, during, and after global ischemia. To quantify changes in dendritic structure, we used morphometric tools that exploit characteristic features of blebbing, distinguished as localized regions of spherical or ellipsoid swellings. By comparing acquired images during ischemia and reperfusion to a pre-ischemia reference image, our automated approach detected blebs based on defined eccentricity and area thresholds and quantified the percentage of blebbed dendrites based on a block-selection method. Our results indicate that the automated morphometric indices we employ yield results that correlate with manual assessment. The automated approach permits rapid and effective analysis of dendritic structure and may facilitate the study of ischemic dendritic damage.

Research highlights

BlebQuant was developed for automated detection and quantification of blebs. ▶ Results from the automated approach correlate with manual work. ▶ Automation will facilitate future analysis of dendrite structure.

Introduction

Transgenic mice expressing fluorescent proteins have recently become a tool to examine real time changes in synapses and dendrites in models of stroke and neurodegenerative disease (Davalos et al., 2005, Feng et al., 2000, Spires et al., 2005). In particular, studies have investigated alterations of dendritic spines in stroke (Brown et al., 2007, Brown et al., 2008, Brown and Murphy, 2008, Mostany et al., 2010, Risher et al., 2010). In addition to changes to spines, under certain conditions, the dendritic branch can also be structurally dynamic. During ischemia, dendrites take on a distinct pattern of structural damage termed ‘blebbing’. Blebbing is characterized as localized regions of spherical or ellipsoid (‘varicose’) swellings, resembling a ‘beads on a string’ appearance (Hasbani et al., 2001, Li and Murphy, 2008, Murphy et al., 2008, Obeidat et al., 2000, Park et al., 1996, Zhang et al., 2005, Zhang and Murphy, 2007).

We have previously reported the rapid induction of blebbing within minutes of both focal and global models of stroke in apical dendrites of layer 5 neurons using in vivo 2-photon (2P) imaging (Li and Murphy, 2008, Murphy et al., 2008, Zhang and Murphy, 2007). Interestingly, this structural damage may not always be permanent as dendrites show a certain degree of recovery with reperfusion (Murphy et al., 2008), and those within a penumbra are able to recover between periods of spontaneous peri-infarct spreading depolarization (Risher et al., 2010).

While there has been much work describing dendritic blebbing, to this date, procedures for the identification and quantification of blebs have relied on a manual approach which is labor intensive and can be subjective. In this paper, we describe our approaches in developing BlebQuant, a toolkit program which allows automated examination of changes in dendritic structure induced by ischemia in mouse somatosensory cortex in vivo (Li and Murphy, 2008, Murphy et al., 2008, Zhang et al., 2005, Zhang and Murphy, 2007). Our approach relies on exploiting morphological features of blebs such as eccentricity and changes in area of blebbed regions. Fig. 1 summarizes our MATLAB-based approach (MATLAB R2008a, the MathWorks, Natick, MA) for the detection and quantification of blebs. We report that this approach provides a quantitative unbiased appraisal of ischemic damage that is comparable with blinded manual analysis.

Section snippets

Image acquisition

Male C57BL6 (2–5 months of age) yellow fluorescent protein (YFP) and green fluorescent protein (GFP) expressing transgenic mice (H and M lines) (Feng et al., 2000) were used in this study. Transgenic mice were bred at the University of British Columbia animal facilities. All the animal preparations (anesthesia, surgery and physiological maintenance) were similar to previous reports (Murphy et al., 2008). Briefly, anesthesia was induced with intraperitoneal injection of urethane (0.12% w/w) and

Morphological features of ischemia

The experimental results presented here are from a model of global ischemia (Murphy et al., 2008). In both manual and automated approaches, we identified dendrites as blebbed if they exhibited regularly spaced and rounded herniations that were punctuated by inter-bleb regions with lower, or in some cases nearly undetectable fluorescence (Murphy et al., 2008). If bleb identification relied solely on meeting the coarse selection size threshold of 6 < ST < 625, many detected objects have small areas

Discussion

We have provided an automated approach to perform analysis of dendritic blebbing as a result of ischemia. Previously, reports have described various algorithms for identification and quantification of spines (Cheng et al., 2007, Koh et al., 2002, Yuan et al., 2009, Zhang et al., 2007, Zhang et al., 2010). Here, we add to the study of dendrite structure by describing an approach for evaluating ischemia-induced dendritic blebbing.

Under fluorescence microscopy, blebbed dendrites are distinguished

Information sharing statement

The BlebQuant toolkit package and some of the image stacks for validation are available online from http://www.neuroscience.ubc.ca/faculty/murphy_software.

Acknowledgments

The authors would like to thank Pumin Wang and Cindy Jiang for assistance with surgical preparations and Alexander Goroshkov and Jamie Boyd for technical assistance. This work was supported by a Heart and Stroke Foundation of BC and Yukon grant in aid to THM, in part by a CIHR operating grant (MOP49586) to THM, and an NSERC studentship to ST.

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