Elsevier

NeuroImage

Volume 56, Issue 3, 1 June 2011, Pages 1001-1010
NeuroImage

Oscillating neuro-capillary coupling during cortical spreading depression as observed by tracking of FITC-labeled RBCs in single capillaries

https://doi.org/10.1016/j.neuroimage.2011.02.078Get rights and content

Abstract

Coupling between capillary red blood cell (RBC) movements and neuronal dysfunction during cortical spreading depression (CSD) was examined in rats by employing a high-speed camera laser-scanning confocal fluorescence microscope system in conjunction with our Matlab domain software (KEIO-IS2). Following microinjection of K+ onto the surface of the brain, changes in electroencephalogram (EEG), DC potential and tissue optical density were all compatible with the occurrence of a transient spreading neuronal depression. RBC flow in single capillaries was not stationary. Unpredictable redistribution of RBCs at branches of capillaries was commonly observed, even though no change in diameter was apparent at the reported site of the capillary sphincter and no change of arteriolar-venule pressure difference was detected. There appeared to be a slow morphological change of astroglial endfeet. When local neurons were stunned transiently by K+ injection, the velocity and oscillation frequency of RBCs flowing in nearby capillaries started to decrease. The flow in such capillaries was rectified, losing oscillatory components. Sluggish floating movements of RBCs in pertinent capillaries were visualized, with occasional full stops. When CSD subsided, RBC movements recovered to the original state. We postulate that neuronal depolarization blocks oscillatory signaling to local capillaries via low-shear plasma viscosity increases in the capillary channels, and a complex interaction between the RBC surface and the buffy coat on the capillary wall surface increases the capillary flow resistance. Then, when CSD subsides and oscillatory neuronal function is recovered, the normal physiological conditions are restored.

Graphical abstract

Research highlights

► RBC flow in single capillaries was not stationary. ► RBC redistribution at branches of capillaries without change in diameter. ► RBC flow in capillaries was decreased, occasionally stopped, by K+-induced CSD. ► Sluggish floating movements of RBCs in pertinent capillaries were visualized. ► Slow morphological change of astroglial endfeet was observed after K+ administration.

Introduction

The various flow control systems of cerebral vessels serve mainly to supply blood to the capillaries at levels sufficient to meet local neuronal requirements. RBC capillary flow behavior is especially important, since RBCs are the predominant oxygen carrier from the lung to the brain tissue. However, RBCs in blood do not behave like solutes, since their dimensions are of the same order as capillaries, so that their passage is constrained by local changes in viscosity, morphological transformation and so on. To visualize capillary flow in brain tissue in vivo at a depth of a few hundred micrometers below the pia mater, highly spatially-resolved two-photon microscopy (Chaigneau et al., 2003, Kleinfeld et al., 1998, Kleinfeld and Denk, 2000, Takano et al., 2007a, Takano et al., 2007b) has been employed, but rapid RBC movements in response to neuronal activation are difficult to examine because of the limited time resolution of this method. By employing a traditional intravital microscopic technique combined with novel use of fluorescein isothiocyanate (FITC)-labeled RBCs in the circulating blood, Biswal et al. observed low-frequency spontaneous oscillations in RBC capillary flow. After nitric oxide synthase inhibition, they found that the mean RBC velocity in the capillaries decreased to about half, but the 4–8 cpm (cycles per minute) frequency oscillations in velocity increased by about two-fold. They claimed that this, for the first time, provided direct evidence for low-frequency synchronous oscillations of RBC flow velocity in the cerebral capillary network (Biswal and Hudetz, 1996). However, the concept of capillary vasomotion was not new, since August Krogh had drawn attention to the influence of neuronal regulation on nearby capillary blood flow much earlier (Krogh, 1919). Furthermore, we had observed 4–6 cpm fluctuations in optical recordings of local cerebral blood volume (including arterioles, capillaries and venules) (Tomita et al., 1981) in cats. Introducing a confocal laser-scanning technique for intravital microscopy improved the spatial resolution (Seylaz et al., 1999; Villringer et al., 1994) and the velocity changes and even changes in the direction of flow of RBCs in capillaries could be traced.

Recently, the concept of the neurovascular unit, in which the activity of neurons modulates regional and local cerebral blood flow (CBF), has been proposed (del Zoppo, 2010). It is suggested that neurons and microvessels bi-directionally communicate with each other, with the participation of the intervening astrocytes. However, it has been difficult to directly observe microvascular flow, astrocyte function and neuronal activity in vivo because of methodological limitations.

To examine microcirculation at the level of capillaries, we improved the time resolution of our method by developing a high-speed camera laser-scanning confocal fluorescence microscopy system (Tomita et al., 2008a, Unekawa et al., 2008). One of our findings was that the RBC velocity was often independent of upstream arteriolar blood flow or tissue perfusion in the surrounding microvasculature as measured with, for example, the tissue hemodilution technique (Schiszler et al., 2000). When local blood flow was dramatically increased by topical application of nitric oxide (NO) on the brain surface, the capillary RBC flow was unexpectedly rather decreased (Tomita et al., 2009a). Application of exogenous NO causes excessive oxygenation in the tissue (Yanamoto et al., 2003), which would be toxic to neurons (Tomita et al., 1994), and this apparently impeded capillary flow via unknown mechanisms. Severe hypocapnia is known to enhance the heterogeneity of brain capillary perfusion, indicating a potential disturbance of cerebral microcirculation (Vogel et al., 1996). Thus, capillary RBC flow seemed to be locally and actively controlled by allowing the entry of only a limited portion of the blood volume in the arteriole (which may serve as a reservoir) into the capillaries in response to neuronal requirements. Furthermore, we found that under physiological and pathological conditions, the left–right distribution of RBCs at a branching site of capillaries was complex and unpredictable, as if RBCs were sucked up by a discontinuous force from the capillary, even though it has no muscle cells and showed no appreciable change in diameter. Nakai et al. also observed left–right RBC distribution in a closed capillary loop and attributed it to a precapillary sphincter (Nakai et al., 1981), but we could not confirm the presence of such a sphincter. These observations are in conflict with the historical arteriolar stopcock theory (Mchedlishvili, 1986) of parallel passive capillary flow distribution. It is therefore of great interest to know whether or not the local capillary flow is regulated by neurons adjacent to the capillaries.

The aim of this study was to examine comprehensively the hypothesis that direct neuro-capillary coupling occurs, by using a high-speed camera laser-scanning confocal fluorescence microscope which can detect variations of RBC flow in single capillaries within a certain region of interest (ROI). First, control RBC movements in single capillaries of the cortical tissue were measured, and then changes in RBC movements during cortical spreading depression (CSD) were evaluated, to obtain insight into potential negative effects of neurons on RBC flow in adjacent capillaries.

Section snippets

General procedures

Animals were used with the approval of the Animal Ethics Committee of Keio University (Tokyo, Japan), and all experimental procedures were in accordance with the university's guidelines for the care and use of laboratory animals. Male Wistar rats of 10–15 weeks old (mean body weight 326 ± 31 g) were anesthetized with urethane (1 g/kg ip and added as necessary). The methods of opening a skull window for the continuous recording of FITC-labeled RBCs in the cortical microvasculature and the techniques

General observations

The object of this study was to explore local RBC movements occurring in a limited area of the hemispheric cerebral cortex during the transition from physiological status to pathological K+-induced CSD. Changes in physiological parameters observed here during CSD were broadly compatible with those previously reported by us (Osada et al., 2006, Tomita et al., 2005a, Tomita et al., 2008b, Tomita et al., 2002, Unekawa et al., 2009).

Fig. 2 illustrates the physiological parameter changes over

Discussion

The findings of this study with our high-speed camera system were that RBC flows in single capillaries under physiological conditions were as high as 2 mm/s (Unekawa et al., 2008), and that flow was not stationary (Mchedlishvili, 1998), but rather high-frequency oscillatory in nature (capillary vasomotion). Employing two-photon microscopy, Kleinheld found that RBC flow in individual capillaries within the specific area sensitive to vibrissae was quite variable with a speed of 0.8 ± 0.5 mm/s and an

Conclusions

The observed RBC flow behavior changes in intraparenchymal capillaries during CSD suggest the presence of a regulatory mechanism of cerebral capillary flow, presumably via hemorheological factors, that may involve a direct or indirect coupling between neurons and nearby capillaries via astroglial elements.

The first author, Minoru Tomita, passed away on 17th January 2010. This work was completed by his co-authors.

The following are the supplementary materials related to this article.

Acknowledgments

This work was supported by JSPS Grants-in-Aid # 17390255 (Suzuki, N) and # 19591008 (Tomita, Y). The authors also thank Otsuka Pharmaceutical Co., Ltd. for financial support.

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