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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., (Copyright © 2013. Published by Elsevier B.V.)

The aims of the present study were as follows: (i) to perform the first (87)Rb MRI in live rats with focal ischemic stroke; and (ii) to test the hypothesis that K(+) egress from the brain in this model is quantifiable in individual animals by high-field (7-T) K/Rb substitution MRI. Rats preloaded with dietary Rb(+) (resulting in Rb/(K + Rb) replacement ratios of 0.1-0.2 in the brain) were subjected to permanent occlusion of the middle cerebral artery, and (87)Rb MRI was implemented with 13-min temporal resolution using a dedicated RF coil and a spiral ultrashort-TE sequence (TR/TE = 3/0.07 ms). The ischemic core was localized by apparent diffusion coefficient mapping, by microtubule-associated protein-2 immunohistochemistry and by changes in surface reflectivity. [K], [Na] and [Rb] were determined independently in the micropunched samples by post-mortem flame photometry. Both techniques were generally in agreement in the nonischemic cortex; however, the MRI-assessed [K(+) + Rb(+)] drop in ischemic brain was less pronounced (average efflux rate of 4.8 ± 0.2 nEq/mm(3) /h versus 10 ± 1 nEq/mm(3)/h by flame photometry; p < 0.0001). The use of higher field gradients for better spatial resolution, and hence more accurate quantification, is suggested., (Copyright © 2011 John Wiley & Sons, Ltd.)

The delineation of early infarction in large gyrencephalic brain cannot be accomplished with triphenyl-tetrazolium chloride (TTC) due to its limitations in the early phase, nor can it be identified with microtubule-associated protein 2 (MAP2) immunohistochemistry, due to the fragility of large thin sections. We hypothesize that MAP2 immunostaining of thick brain sections can accurately identify early ischemia in the entire monkey brain. Using ischemic brains of one rat and three monkeys, a thick-section MAP2 immunostaining protocol was developed to outline the infarct region over the entire non-human primate brain. Comparison of adjacent thick and thin sections in a rat brain indicated complete correspondence between ischemic regions (100.4mm(3)+/-1.2%, n=7, p=0.44). Thick sections in monkey brain possessed the increased structural stability necessary for the extensive MAP2 immunostaining procedure permitting quantification of the ischemic region as a percent of total monkey brain, giving infarct volumes of 11.4, 16.3, and 19.0% of total brain. Stacked 2D images of the intact thick brain tissue sections provided a 3D representation for comparison to MRI images. The infarct volume of 16.1cm(3) from the MAP2 sections registered with MRI images agreed well with the volume calculated directly from the stained sections of 16.6 cm(3). Thick brain tissue section MAP2 immunostaining provides a new method for determining infarct volume over the entire brain at early time points in a non-human primate model of ischemic stroke.

Purpose: To test the hypotheses that (i) the regional heterogeneity of brain sodium concentration ([Na(+)](br)) provides a parameter for ischemic progression not available from apparent diffusion coefficient (ADC) data, and (ii) [Na(+)](br) increases more in ischemic cortex than in the caudate putamen (CP) with its lesser collateral circulation after middle cerebral artery occlusion in the rat., Materials and Methods: (23)Na twisted projection MRI was performed at 3 Tesla. [Na(+)](br) was independently determined by flame photometry. The ischemic core was localized by ADC, by microtubule-associated protein-2 immunohistochemistry, and by changes in surface reflectivity., Results: Within the ischemic core, the ADC ratio relative to the contralateral tissue was homogeneous (0.63 +/- 0.07), whereas the rate of [Na(+)](br) increase (slope) was heterogeneous (P < 0.005): 22 +/- 4%/h in the sites of maximum slope versus 14 +/- 1%/h elsewhere (here 100% is [Na(+)](br) in the contralateral brain). Maximum slopes in the cortex were higher than in CP (P < 0.05). In the ischemic regions, there was no slope/ADC correlation between animals and within the same brain (P > 0.1). Maximum slope was located at the periphery of ischemic core in 8/10 animals., Conclusion: Unlike ADC, (23)Na MRI detected within-core ischemic lesion heterogeneity., ((c) 2009 Wiley-Liss, Inc.)

Purpose: To validate (23)Na twisted projection magnetic resonance imaging (MRI) as a quantitative technique to assess local brain sodium concentration ([Na(+)](br)) during rat focal ischemia every 5.3 minutes., Materials and Methods: The MRI protocol included an ultrashort echo-time (0.4 msec), a correction of radiofrequency (RF) inhomogeneities by B(1) mapping, and the use of 0-154 mM NaCl calibration standards. To compare MRI [Na(+)](br) values with those obtained by emission flame photometry in precision-punched brain samples of about 0.5 mm(3) size, MR images were aligned with a histological three-dimensional reconstruction of the punched brain and regions of interest (ROIs) were placed precisely over the punch voids., Results: The Bland-Altman analysis of [Na(+)](br) in normal and ischemic cortex and caudate putamen of seven rats quantitated by (23)Na MRI and flame photometry yielded a mean bias and limits of agreement (at +/-1.96 SD) of 2% and 43% of average, respectively. A linear increase in [Na(+)](br) was observed between 1 and 6 hours after middle cerebral artery occlusion., Conclusion: (23)Na MRI provides accurate and reliable results within the whole range of [Na(+)](br) in ischemia with a temporal resolution of 5.3 minutes and precisely targeted submicroliter ROIs in selected brain structures.

An animal model was developed to monitor [K(+)] in the brain using partial K(+) replacement with Rb(+) and (87)Rb MRI. Fifty-one rats were given 0-80 mM of RbCl in the drinking water for up to 90 days. Focal cerebral ischemia was produced in 15 of the animals. Na, K, and Rb content in precision-guided submilligram samples of cortical brain were determined by emission flame photometry. Multinuclear (87)Rb/(23)Na/(1)H MRI was performed on phantoms and rats at 3T using a twisted projection imaging (TPI) scheme for (87)Rb/(23)Na, and custom-built surface or parallel cosine transmit/receive coils. Brain [Rb(+)] was safely brought up to 17-25 mEq/kg within 2-3 weeks of feeding. The characteristic patterns of [K(+)] decrease (with a sharp drop at 3-4 hr of ischemia) and [Na(+)] increase (at a rate of 31%/hr) observed previously in animals without Rb/K substitution were reproduced in ischemic cortex. The Rb/(Rb+K) ratio increased over time in ischemic areas (R = 0.91, P < 0.001), suggesting an additional index of ischemia progression. Preliminary (87)Rb MRI gave an estimate of 20-25 mEq Rb/kg brain weight (N = 2). In conclusion, brain Rb(+) is detectable by (87)Rb MRI and does not significantly interfere with ion dynamics in ischemic brain, which enables (87)Rb MRI studies of K(+) in ischemia.

Background and Purpose: Thrombolytic therapy with intravenous tPA must be administered within 3 hours after stroke onset. However, stroke onset time cannot be established in 20% to 45% of potential patients. We propose that the rate of increase of the brain concentration of sodium ([Na+]br) after stroke, monitored using sodium MRI in a rat model of cortical ischemia, is linear in each individual animal, can locate the ischemic region, and can be used to estimate onset time., Methods: After induction of focal cortical ischemia in rats under isoflurane anesthesia, [Na+]br time course maps were acquired continuously on a 3 T whole body scanner from 2 to 7 hours after occlusion followed by T2-weighted proton images. Microtubule-associated protein-2 immunostained brain sections were used to verify the location of the infarct., Results: The ischemic region identified with microtubule-associated protein-2 corresponded to the region of maximum [Na+]br increase (P<0.001; n=5), and all of the animals demonstrated high linearity. [Na+]br increased at a mean rate of 25+/-4.7%/h in ischemic tissue (P=0.013) but not in normal cortex (1.0+/-1.1%/h; P=0.42). The mean onset time error was 1+/-4 minutes (n=4)., Conclusions: These results of sodium MRI show that the region of maximum [Na+]br increase corresponds to the ischemic region. Although [Na+]br increases at a different rate in each animal, the increase is linear, and, therefore, onset time can be estimated. These findings suggest that this method can be used as a ticking clock to estimate time elapsed after vascular occlusion.

Extracellular adenosine 5'-triphosphate (ATP) provides excitatory transmission in the central nervous system. Stimulation by ATP of ionotropic ligand-gated ion channel purinoceptors (P(2X)) leads to increased intracellular calcium levels, and activation of P(2X) receptors may be involved in the process of excitotoxic neuronal injury caused by stroke. Suramin, as an agent that is known to block P(2X) receptors at a specific concentration, was assessed for its neuroprotective potential in a model of experimental stroke in the rat. We propose that the effectiveness of suramin is limited to those concentrations where it is an effective P(2X) receptor antagonist. Focal brain ischemia was produced by unilateral occlusion and transection of the middle cerebral artery (MCAT) and bilateral occlusion of the common carotid arteries (CCA). Thirty-four male Sprague-Dawley rats were randomly separated into five groups. Changes in regional cerebral blood flow in the ischemic region were verified by laser Doppler flowmetry. All rats received, over a period of 30 min before MCAT, a dose of suramin at 0 (saline), 25, 50, 100, or 250 mg/kg intravenously in a volume of 1 ml using an infusion pump. Six hours after ischemia onset, evaluation of the neurologic status and cerebral blood flow was followed by morphometric analysis of infarct volume. During the surgical procedure mean arterial pressure, blood gases (PaCO(2), PaO(2)), and pH were monitored. A dose-dependent decrease in infarct volume (slope -0.049, SE 0.009, P<0.001) was observed in groups treated over the dose range of 0 to 100 mg/kg (r(2)=0.55). Suramin at a dose of 100 mg/kg significantly decreased infarct volume (n=9, P<0.001) and edema volume (P=0.003). The neuroprotective effect of suramin at a dose of 100 mg/kg was supported by an improved neurologic score in this group (median 0) compared with a median of 3 in control animals (P=0.02). These findings indicate that suramin at 100 mg/kg is an effective pre-treatment neuroprotective agent. As the estimated brain concentration of 10 microM (from McNally et al., 2000, Life Sci 67:1847-1857) is the IC(50) for suramin-mediated P(2X) antagonism, these results suggest that interference with the ATP excitatory system could provide neuroprotection from brain ischemia.