Elsevier

Neuroscience

Volume 128, Issue 1, 2004, Pages 209-216
Neuroscience

Capillary level imaging of local cerebral blood flow in bicuculline-induced epileptic foci

https://doi.org/10.1016/j.neuroscience.2004.07.002Get rights and content

Abstract

Local hemodynamics of the cerebral cortex is the basis of modern functional imaging techniques, such as fMRIand PET. Despite the importance of local regulation of the blood flow, capillary level quantification of cerebral blood flow has been limited by the spatial resolution of functional imaging techniques and the depth penetration of conventional optical microscopy. Two-photon laser scanning microscopic imaging technique has the necessary spatial resolution and can image capillaries in the depth of the cortex. We have loaded the serum with fluorescein isothiocyanate dextran and quantified the flow of red blood cells (RBCs) in capillaries in layers 2/3 of the mouse somatosensory cortex in vivo. Basal capillary flux was quantified as approximately 28.9±13.6 RBCs/s (n=50, mean±S.D.) under ketamine–xylazine anesthesia and 26.7±16.0 RBCs/s (n=31) under urethane anesthesia. Focal interictal (epileptiform) activity was induced by local infusion of bicuculline methochloride in the cortex. We have observed that capillary blood flow increased as the cortical local field events developed into epileptiform in the vicinity of GABA receptor blockade (<300 μm from the administration site). Local blood flow in the interictal focus increased significantly (42.5±18.5RBCs/s, n=52) relative to the control conditions or to blood flow measured in capillaries at distant (>1 mm from the focus) sites from the epileptic focus (27.8±12.9 RBCs/s, n=30). These results show that hyper-synchronized neural activity is associated with increased capillary perfusion in a localized cortical area. This volume is significantly smaller than the currently available resolution of the fMRI signal.

Section snippets

Subjects and surgery

Male and female C57BL6 mice (20 g–30 g) were used. Animals were deeply anesthetized with either urethane (1.7 g/kg) or ketamine–xylazine cocktail (25 mg ketamine, 6.3 mg xylazine, 0.25 mg acepromazine per 1.0 ml solution, cocktail dosage=3 ml/kg). Two small stainless micro-screws were driven into the bone above the cerebellum. These screws serve as the ground and reference electrodes for the differential recording of local field potential. An insulated metallic head frame (approximately 1 cm×4 cm,

Visualization and measurement of capillary level hemodynamics

Blood flow was imaged and quantified in a total of 163 capillaries from 26 animals. This included eight animals under urethane anesthesia and 18 animals under ketamine–xylazine anesthesia. Among the ketamine–xylazine-anesthetized population, IISs were induced in 12 animals. Microvessels that had the inner diameter (i.e. FITC filled portion) of less than or equal to 6 μm are considered to be capillaries.

Capillary blood flow can be quantified in terms of the flux (number of traveling RBCs per unit

Discussion

Two-photon imaging of capillaries filled with fluorescently labeled serum yields the highest time and spatial resolution of blood flow among currently available techniques for blood flow measurements. We have utilized 2-PLSM to investigate capillary level blood flow in layer II/III of the somatosensory cortex in both basal conditions and bicuculline-induced epileptic conditions. As a result, we found a reliable relationship between activity changes of neurons and intracortical blood

Acknowledgments

We thank A. G. Hudetz, D. L. Buhl, and A. Sirota for their critical comments on an earlier version of this manuscript. This work was supported by National Institutes of Health grant NS043157 (G.B.) and by the Epilepsy Foundation (H.H.).

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