Correlated sodium and potassium imbalances within the ischemic core in experimental stroke: a 23Na MRI and histochemical imaging study

Abstract

This study addresses the spatial relation between local Na(+) and K(+) imbalances in the ischemic core in a rat model of focal ischemic stroke. Quantitative [Na(+)] and [K(+)] brain maps were obtained by (23)Na MRI and histochemical K(+) staining, respectively, and calibrated by emission flame photometry of the micropunch brain samples. Stroke location was verified by diffusion MRI, by changes in tissue surface reflectivity and by immunohistochemistry with microtubule-associated protein 2 antibody. Na(+) and K(+) distribution within the ischemic core was inhomogeneous, with the maximum [Na(+)] increase and [K(+)] decrease typically observed in peripheral regions of the ischemic core. The pattern of the [K(+)] decrease matched the maximum rate of [Na(+)] increase ('slope'). Some residual mismatch between the sites of maximum Na(+) and K(+) imbalances was attributed to the different channels and pathways involved in transport of the two ions. A linear regression of the [Na(+)]br vs. [K(+)]br in the samples of ischemic brain indicates that for each K(+) equivalent leaving ischemic tissue, 0.8±0.1 Eq, on average, of Na(+) enter the tissue. Better understanding of the mechanistic link between the Na(+) influx and K(+) egress would validate the (23)Na MRI slope as a candidate biomarker and a complementary tool for assessing ischemic damage and treatment planning.

Keywords: (23)Na MRI; 3D; ADC; BBB; Focal ischemia; MAP2; MCA, MCAO, MCAT; Permanent MCAO; RF; ROI; Rat brain; Tissue potassium; Tissue sodium; [Na(+)](br) and [K(+)](br); apparent diffusion coefficient; blood–brain barrier; brain tissue sodium and potassium concentration, respectively; microtubule-associated protein 2; middle cerebral artery, MCA occlusion, and MCA transection with bilateral common carotid artery occlusion, respectively; radiofrequency; region of interest; three-dimensional.

Figure 1

Region-of-interest (ROI) analysis of Na⁺ accumulation and K⁺ drop in a rat brain after MCAO. Coronal images of the brain (at approximately bregma −0.4 mm) of the rat #5 are shown. (a) ADC map where ADC < 500 µm²/s in the ischemic area (left-hand side of the image). (b) Pseudocolor-coded parametric image of the rate of ²³Na signal increase (‘slope’) superimposed over the grayscale ¹H spin-echo MR image as an anatomic reference. Reference tubes with NaCl solutions were external to the rat head in the magnet and are not shown in MR images. (c) Cut-face photograph of the brain in the cryostat after sampling showing punch holes. A millimeter scale is shown at the top. Punch samples were analyzed using emission flame photometry. (d) Cross-section of the 3D reconstruction of the brain from the 35-µm-thick slices cut at 4.4 h after MCAO. The change in surface reflectivity of ischemic tissue shows the infarct location (outlined by a red dotted line). Cylindrical ROIs (yellow circles) were placed over the punch holes and are shown in images (a, b and e) as white or colored circles. (e) K⁺-stained slice taken at the sampling plane and used to calibrate optical density against [K⁺]. (f) The absence of staining in the MAP2-stained slice (separated by 0.67 mm from the sampling plane) indicates the ischemic lesion.

Figure 2

Spatial match of Na⁺ accumulation and K⁺ drop in a rat brain after MCAO. (a–d) Coronal images of the brain of rats #2–4 and 6, respectively. Top row: pseudocolor-coded parametric image of the rate of ²³Na signal increase (‘slope’) superimposed over the grayscale 3D brain reconstructions from histological 35-µm-thick sections. Middle row: the same planes of 3D brain reconstructions from the K⁺-stained sections. Lack of staining (white areas) indicate low [K⁺]_br in the ischemic lesion. Bottom row: ²³Na slope (black) and [K⁺]_br (red) profiles along the ribbons in the ischemic hemisphere (white or color arcs in the images) in the dorsal-to-ventral direction. Black dots in the images show holes in the brain after taking punch samples for calibration of Na⁺ and K⁺ images.

Figure 3

Na⁺ and K⁺ mapping of the ischemic brain (rat #5). (a,b) Pseudocolor images of the ²³Na slope superimposed over the grayscale 3D reconstruction of the brain from histological 35-µm-thick sections. (c,d) K⁺-stained slices cut at 4.4 h post MCAO. (e,f) ²³Na slope (black) and [K⁺]_br (red) profiles along the ribbons in the ischemic hemisphere (white or color arcs in (a)–(d)) in the dorsal-to-ventral direction. The coronal sections were taken at the rostral (bregma −0.4 mm, a,c,e) and caudal (bregma −3 mm, b,d,f) levels. (1) Dorsal ischemic edge; (2) central ischemic core; (3) ventral ischemic edge. Estimated [Na⁺] and [K⁺] in ipsilateral and homotopic contralateral ROIs (white or color squares in (a)–(d)) in regions 1–3 are given in Table 3. (g) 3D reconstruction of the brain from 21 co-registered K⁺-stained coronal slices (0.7-mm separation) as viewed from the front-right perspective. Regions with [K⁺]_br below 48 ± 4 mEq/kg (based on Gaussian-filtered images, to improve low-K⁺ ROI contiguity) are shown in yellow to highlight the peripheral ischemic core surrounding the central ischemic core. ROI of maximum ²³Na slope corresponding to the slope range of 11.4 to 15.6%/h and overlapping the low-K⁺ ROI is shown in red.

Figure 4

Linear fit of [Na⁺]_br vs. [K⁺]_br in ischemic tissue. [Na⁺]_br and [K⁺]_br were determined by emission flame photometry in the micropunch samples obtained from all 8 animals between 2.5 and 6 hours after MCAO. Insets: coding of rat numbers by different symbols (top), and fit parameters (bottom). Dashed lines show 95% confidence limits.

Figure 5

Calibration of K⁺ staining intensity by emission flame photometry. Horizontal axis: [K⁺]_br determined in punched samples by flame photometry. Vertical axis: digitized image intensity (GV, 8-bit gray value) averaged over ROIs precisely corresponding to the punch locations in immediately adjacent stained brain sections. A representative calibration curve for the rat #5 is shown. Calibration parameters for all rats are given in Table 2.