Anthony W. Majors and Stephen C. Jones Yang Wang ...

Brain Tissue Sodium Is a Ticking Clock Telling Time After Arterial Occlusion in Rat Focal Cerebral Ischemia Yang Wang, Weixing Hu, Alejandro D. Perez-Trepichio, Thian C. Ng, Anthony J. Furlan, Anthony W. Majors and Stephen C. Jones Stroke. 2000;31:1386-1392 doi: 10.1161/01.STR.31.6.1386 Stroke is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231 Copyright © 2000 American Heart Association, Inc. All rights reserved. Print ISSN: 0039-2499. Online ISSN: 1524-4628

The online version of this article, along with updated information and services, is located on the World Wide Web at: http://stroke.ahajournals.org/content/31/6/1386

Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Stroke can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: http://www.lww.com/reprints Subscriptions: Information about subscribing to Stroke is online at: http://stroke.ahajournals.org//subscriptions/

Downloaded from http://stroke.ahajournals.org/ by guest on June 6, 2013

Brain Tissue Sodium Is a Ticking Clock Telling Time After Arterial Occlusion in Rat Focal Cerebral Ischemia Yang Wang, MS; Weixing Hu, MD; Alejandro D. Perez-Trepichio, MD; Thian C. Ng, PhD; Anthony J. Furlan, MD; Anthony W. Majors, PhD; Stephen C. Jones, PhD Background and Purpose—Many patients with acute stroke are excluded from receiving thrombolysis agents within the necessary time limit (3 or 6 hours from stroke onset) because they or their family members are unable provide the time of stroke onset. Brain tissue sodium concentration ([Na⫹]) increases gradually and incessantly during the initial hours of experimental focal cerebral ischemia but only in severely damaged brain regions. We propose that this steady increase in [Na⫹] can be used to estimate the time after arterial occlusion in the rat middle cerebral artery occlusion model of ischemic stroke. Methods—Sixteen anesthetized Sprague-Dawley rats underwent permanent middle cerebral artery occlusion combined with bilateral common artery occlusion. After 100 to 450 minutes, diffusion-weighted MRI was used to generate apparent diffusion coefficient (ADC) maps, cerebral blood flow (CBF) was determined with 14C-iodoantipyrine (in a subset of 7 animals), and the brain was frozen. Autoradiographic CBF sections and punch samples for Na⫹ analysis were obtained from the brain at the same level of the MR image. Severely at risk regions were identified with an ADC of ⬍520 ␮m2/s and, in the subset, with both ADC of ⬍520 ␮m2/s and CBF of ⬍40 mL 䡠 100 g⫺1 䡠 min⫺1. Results—Both CBF and the ADC dropped quickly and remained stable in the initial hours after ischemic onset. Linear regression revealed strong linearity between [Na⫹] and time after onset, with a slope of 0.95 or 1.00 (mEq/kg DW)/min, with both ADC and ADC-plus-CBF criteria, respectively. The 95% CIs at 180 and 360 minutes were between 41 and 52 minutes. Conclusions—The time after ischemic onset can be estimated with this 2-step process. First, ADC and CBF are used to identify severely endangered regions. Second, the [Na⫹] in these regions is used to estimate time after onset. The favorable 95% CIs at the time limits for thrombolytic therapy and the availability of measurements of ADC, CBF, and [Na⫹] in humans through the use of MRI suggest that this time-estimation scheme could be used to assess the appropriateness of thrombolysis for patients who do not know when the stroke occurred. (Stroke. 2000;31:1386-1392.) Key Words: cerebral blood flow 䡲 cerebral ischemia 䡲 magnetic resonance imaging, diffusion-weighted 䡲 middle cerebral artery occlusion 䡲 sodium 䡲 stroke 䡲 rats

S

everal thrombolytic treatments for acute ischemic stroke have been shown to be efficacious.1–3 These treatments require administration during the hyperacute period within 6 hours of stroke onset. For example, if intravenous tissue plasminogen activator (tPA) administration is delayed past 3 hours, the risk of cerebral hemorrhage outweighs the benefit of thrombolysis.4 However, one fifth to one half of potential subjects cannot be treated with these agents because they or a family member were not aware or were asleep when the stroke occurred and the time of stroke onset could not be determined accurately or at all.5–7 Brain tissue sodium concentration ([Na⫹]), including intracellular, extracellular, and interstitial concentrations, increases gradually and incessantly during the initial hours of experimental focal cerebral ischemia.8 MRI Na⫹ measurements have been

See Editorial Comment, page 1392 advocated as possessing a threshold that, if exceeded, indicates irreversible tissue damage.9 In contrast to the persistent increase in [Na⫹], cerebral blood flow (CBF) and the apparent diffusion coefficient (ADC) of water drop very quickly and remain relatively constant during this same several-hour period.10 We propose that by measuring several physiological and biochemical parameters, an objective estimate of the time after onset can be determined. By using the initial drop in ADC or in ADC and CBF to select endangered brain tissue, we hypothesize that [Na⫹] in these selected regions can be used to estimate the time after arterial occlusion. Although we use an experimental model of ischemic stroke in the rat and brain tissue sampling, all of the crucial measurements can be

Received December 9, 1999; final revision received February 29, 1999; accepted February 29, 2000. From the Department of Anesthesiology (S.C.J.), Allegheny General Hospital, Pittsburgh, Pa; and the Cleveland Clinic Foundation (Y.W., W.H., A.D.P.-T., T.C.N., A.J.F., A.W.M.), Cleveland, Ohio. Correspondence to Stephen C. Jones, PhD, Department of Anesthesiology, Allegheny General Hospital, 320 East North Avenue, Pittsburgh, PA 15212-4772. E-mail: [email protected] © 2000 American Heart Association, Inc. Stroke is available at http://www.strokeaha.org

1386 Downloaded from http://stroke.ahajournals.org/ by guest on June 6, 2013

Wang et al

Time After Ischemic Stroke and Brain Tissue Sodium

made in humans with MRI,9 which suggests that this scheme for estimation of time after onset could be used to make an inclusion decision for thrombolytic therapy.

Materials and Methods Sixteen Sprague-Dawley rats weighing 307⫾7 g (mean⫾SEM) were prepared with anesthesia of 1% to 2% isoflurane and 70% N2O (balance O2) administered via an endotracheal tube and artificial respiration (model 681; Harvard Apparatus). Rectal temperature was maintained at 37°C with a servo-controlled heat lamp (YSI 74; Yellow Springs). Femoral arterial and venous catheters were inserted bilaterally. Gallamine triethiodide (Davis-Geck) in normal saline was infused intravenously at 10 mg 䡠 kg⫺1 䡠 h⫺1. The head of the animal was then placed in a frame adapted for temporal exposure and underwent permanent direct occlusion of the right middle cerebral artery, with bipolar electrocautery followed by transection, and both common carotid arteries, with previously placed ligatures.11 The time between the occlusion of the middle cerebral artery and that of the common carotid arteries was 10⫾1.3 minutes. The time at which the common carotid arteries were ligated was used as time zero to calculate the time from onset to decapitation (Ta). We used the time of occlusion of both common carotid arteries as time zero because at this time, the flow is reduced to ischemic levels; the subsequent persistence of ischemia and consequent infarction then follows.11 Isoflurane was reduced to a maintenance level of ⬇0.8% through monitoring of the blood pressure response to tail pinch. These procedures were in accordance with the institutional guidelines for studies with animals. Arterial blood gases (PaCO2, PaO2, and pH) and hemoglobin concentration [Hb] were determined with a blood gas analyzer (model ABL3; Radiometer America). Arterial blood pressure was continuously monitored from a femoral artery with a strain gauge transducer (model DT-XX; Viggo-Spectramed). Mean arterial blood pressure (MBP) and end-tidal CO2 were recorded on a polygraph (Gould, Inc). MBP and blood gases were recorded just after the arterial occlusion, at the end of the MRI, and just before CBF determination or death.

MRI Studies MRI experiments were performed with a 4.7-T General Electric CSI-II 40-cm horizontal-bore imaging spectrometer that was equipped with GE Acustar self-shielded gradient coils (maximum 200 mT/m). A revised saddle coil with a diameter of 35 mm and a window length of 40 mm was used for both transmittal and receipt. The B1 homogeneity of the revised saddle coil is similar to that of a birdcage coil. The coil was mounted on a plastic cradle, and the anesthetized rat was placed in the supine position with the head centered in the coil. The proper body temperature was maintained throughout the MRI scanning period with circulating temperaturecontrolled water under the rats. A sagittal gradient recalled echo image through the midline of the brain was acquired to position the coronal images for the study. Two slices, each 2 mm thick and 1 approximately at the level of bregma and the other 3 mm posterior, were selected for acquisition of coronal diffusion-weighted images. A spin echo sequence with interleaved b values12 was used. The field of view was 32.67 mm, the matrix size was 128⫻64 (zero filled to 128⫻128 before Fourier transformation), and voxel sizes were 0.255⫻0.255⫻2 mm. ADC maps were obtained from the diffusion-weighted MR images (spin warp; TR⫽1500 ms/TE⫽35 ms; b⫽0, 261, 586, and 1042 s/mm2, 12.5 ms diffusion time, ⌬⫽15 ms, ␦⫽7.5 ms) with a total acquisition time of 20 minutes. The time between the start of the ADC determination and decapitation was 52⫾3.7 minutes.

CBF Determination In 7 of the 16 animals, after 100 to 450 minutes from occlusion, quantitative CBF was determined with 14C-iodoantipyrine autoradiography.13,14 A background arterial blood sample was taken, and 14 C-iodoantipyrine (100 ␮Ci/kg) was infused into the femoral vein continuously for 45 seconds. Multiple arterial blood samples were

1387

collected and analyzed for 14C with liquid scintillation counting. After 45 seconds, the anesthetized animal was decapitated. To preserve the spatial characteristics of the brain so the MR and CBF images could be digitally superimposed and aligned, the head was frozen immediately in dry ice and stored in a freezer (⫺80°C). In this subset of 7 animals, the time between the start of the ADC determination and decapitation was 54⫾5.9 minutes.

Brain Processing The brain was chipped out of the skull in a ⫺20°C cold box and aligned in the cryostat (⫺20°C) to the same coordinates as for MRI. A 20-␮m CBF autoradiographic section was taken at the rostral face of the 2-mm ADC image, and 2-mm-deep samples were punched between the rostral and caudal ADC image boundaries. Punch sampling was directed in relation to low ADC10 from an ADC guide image. After cutting to a smooth surface, a second CBF autoradiographic section was taken. The brain sections were dried on a 60°C hot plate and exposed for 4 days to x-ray film (Kodak SB5) together with 8 precalibrated 14C-methyl-methacrylate standards.

Flame Photometry The samples were placed in predried and preweighed vials. Wet tissue samples were weighed on a microbalance (ATI Cahn model C-44; Analytical Technology, Inc) to obtain the wet weight with 0.001-mg precision. The average sample weight was 1.935⫾0.331 mg. Tissue samples were dried in an oven at 90°C for 3 days and then placed in a desiccating chamber for 1 hour before measurement of the dry weight. Samples were turned into ash by heating at 400°C for 24 hours, and [Na⫹], expressed as milliequivalents per kilogram of dry weight (mEq/kg DW), were determined with flame photometry15 (IL943 Automatic Flame Photometer; Instrumentation Laboratory).

Data Processing and Analysis CBF autoradiograms were digitized with a quantitative image analysis system (MCID model M1; Imaging Research). CBF images were produced by first converting optical density to 14C concentration with use of the 14C-methyl-methacrylate standards and then to CBF with use of the 14C concentration-versus-time arterial curve.13,14 The MR data were postprocessed with a SUN SPARC I workstation (SUN Microsystems). The ADC map was obtained by fitting the 4 diffusion-weighted images, pixel by pixel, with a linear leastsquares calculation, as the slope of the fits. ADC images were further converted into the digital format of the MCID image analysis system.

Image Alignment CBF and ADC images and the digitized photographic slides of the brain in the cryostat were aligned so that CBF and ADC values were from the same regions that were punch-sampled for Na⫹, as shown in Figure 1. The physiological image (CBF) and the position of the punches for the biochemical parameter [Na⫹] were coregistered to the MR image (ADC). Photographs of the brain surface both before and after the punch sampling were used to locate the punches in the CBF and ADC images. Because the punching process distorted the dorsal surface of the brain, the ventral surface of the brain from the prepunch image (Figure 1A) was used as an intermediary to align the positions of the punch samples in the postpunch image (Figure 1B) with the CBF (Figure 1C) and ADC (Figure 1D) images. The CBF image was aligned with the ADC image by positioning the autoradiographic film to coincide with the ADC image during digitization. CBF values were averaged from the rostral and caudal autoradiographic sections from both faces of the 2-mm-thick ADC image, and [Na⫹] in the punch samples was determined with flame photometry.15 These alignment procedures ensured that the values of CBF, ADC, and [Na⫹] were directly comparable.

Region Selection Threshold CBF and ADC values were used to select the regions that fulfilled the characteristics of ischemic core. Two strategies were used: the first used only an ADC threshold in all 16 animals, and the


1388

Stroke

June 2000

Figure 1. Prepunch (A), postpunch (B), CBF (C), and ADC (D) images at the same coronal level. The prepunch image (A) shows the cut face of the brain in the cryostat. The prepunch (A) and postpunch (B) images were photographed in the cryostat with a millimeter scale placed along the dorsal surface. The prepunch image (A) was aligned with the ADC image (D), and its ventral surface was aligned with the postpunch image (B). The CBF image (C) was aligned to the ADC image (D) by positioning the autoradiographic film before digitization. The locations of the punches in the postpunch image (B) were used to locate the regions (white circles) selected in the CBF (C) and ADC (D) images.

second used both ADC and CBF thresholds in the subset of 7 animals in which CBF was determined. The values of [Na⫹] from these selected regions of interest were averaged per animal and, as independent variables, were correlated to the time after occlusion, Ta. The 95% CIs of Ta at 180 and 360 minutes were used as a gauge of effectiveness, because these are the critical times for tPA1 and prourokinase (pro-UK) administration.2

Statistical Analysis Values are expressed as mean⫾SEM. Statistical significance was assumed when probability values were ⬍0.05. Repeated measures ANOVA was used to assess the physiological data with the SAS general linear models procedure.16 Linear regression was used to estimate time after occlusion and the rate of [Na⫹] increase, and 95% CIs were calculated.17

Results The physiological data (MBP, PaCO2, PaO2, and pH) from just after the arterial occlusion, at the end of MRI, and just before the CBF study are presented in Table 1 for all of the animals and for the subset in which CBF was determined. There were no differences between the subset in comparison with the remainder of the animals for any of the variables. There were statistically significant differences over time for MBP, PaO2, pH, and [Hb], although none of these differences are important in terms of physiological function. PaCO2 remained stable in these ventilated animals from arterial occlusion to the end of the study, and there were no differences in PaCO2 between the subset and the other animals. This is important because of the CO2 reactivity of CBF18 and its depression in ischemic TABLE 1.

regions.19 The [Na⫹] was 248⫾11 mEq/kg DW in normal brain tissue (n⫽16) and 238⫾15 mEq/kg DW for the subset group (n⫽7) (see Table 3).

Regions at Risk In regions with either low or normal ADC, ADC and CBF did not change between 100 and 450 minutes after arterial occlusion (data not shown). These parameters were used to select those ischemic regions in which [Na⫹] increased. In 84 regions of interest, the relation between CBF versus ADC (Figure 2A) showed a similar pattern as that observed previously10: as CBF decreased, ADC stayed constant until a critical level of CBF of ⬇40 mL 䡠 100 g⫺1 䡠 min⫺1 was reached, after which ADC and CBF are proportional. At-risk regions were chosen as regions with CBF of ⬍40 mL 䡠 100 g⫺1 䡠 min⫺1 and ADC of ⬍520 ␮m2/s. These thresholds were chosen to be ⬇15% above the rat ischemic threshold of 35 mL 䡠 100 g⫺1 䡠 min⫺1,20 and its associated ADC threshold of 460 ␮m2/s.10

Estimation of Time After Onset and the Rate of [Naⴙ] Increase When both CBF and ADC thresholds were used to select at-risk regions in the subset of animals, Ta showed a strong linear correlation with [Na⫹] [R2⫽93%, slope⫽0.93 min/ (mEq/kg DW), P⬍0.0005, Table 2, Figure 2B]. The 95% CIs were 51 at both 180 and 360 minutes after occlusion. With

Physiological Variables MAP, mm Hg

PaCO2, mm Hg

PaO2, mm Hg

pH

[Hb], g/dL

All

112⫾3.0

36.3⫾0.9

164⫾11

7.414⫾0.008

17.8⫾0.3

Subset

114⫾3.5

35.5⫾0.8

138⫾17

7.417⫾0.009

18.9⫾0.2

All

103⫾2.9*

38.4⫾1.9

118⫾5*

7.362⫾0.017*

17.0⫾0.4†

Subset

104⫾2.1†

37.3⫾2.8

118⫾8

7.344⫾0.023*

17.0⫾0.8*

All

105⫾2.4†

35.1⫾0.4

166⫾11

7.400⫾0.010

16.4⫾0.3*

Subset

107⫾3.6

35.1⫾0.4

150⫾21

7.383⫾0.006

16.5⫾0.5*

After MCAO and biCCAO

End of MR

At CBF

MCAO indicates middle cerebral artery occlusion; biCCAO, bilateral common carotid artery occlusion. Values are mean⫾SEM, n⫽16 (7 for the subset group in which CBF was determined). For statistical analysis, see text. *Mean values differ over time from the values at MCAO and biCCAO (P⬍0.01). †Mean values differ over time from the values at MCAO and biCCAO (P⬍0.05).


Wang et al


1389

Figure 2. A, CBF versus ADC in 84 regions of rat brain for the subset of 7 animals in which autoradiographic CBF was determined. The dashed and solid lines show the critical levels of ADC (520 ␮m2/s) and CBF (40 mL 䡠 100 g⫺1 䡠 min⫺1) used to select 19 regions considered to be severely endangered, with [Na⫹] levels that are increasing steadily. The [Na⫹] values of these 19 regions were averaged by animal and plotted versus time after occlusion (B). The regression equation (solid line) and the 95% CIs (dotted lines) demonstrated a strong (R2⫽0.93) linear correlation with a slope of 0.93 min/(mEq/kg DW). The solid vertical line is the CI at 360 minutes.

this example, if a subject arrives at the hospital with an ischemic stroke and has his or her brain tissue [Na⫹] measured with the MRI method,9 the estimated time after onset would have to be ⬍180⫺51⫽129 minutes for tPA to be administrated with 95% confidence that his or her ischemic stroke occurred within the previous 180 minutes. If the subject arrived at 360⫺51⫽309 minutes after onset, pro-UK could be administered with 95% confidence that the insult occurred within 360 minutes. After we used only the ADC threshold to choose regions for analysis for all 16 animals, Ta showed a significant linear relationship with brain tissue [Na⫹] [R2⫽68%, slope⫽0.72 min/(mEq/kg DW), P⬍0.0001, Table 2, Figure 3]. The 95% CIs of Ta at 180 and 360 minutes after occlusion were 52 and 41 minutes. The animal contributing the point in Figure 3 at a [Na⫹] of 641 mEq/g DW at Ta⫽177 minutes had a high [Na⫹] in normal cortex of 337 mEq/kg DW. This value is just ⬎2 SDs above the normal cortex value of 248 mEq/kg DW (248⫹2*44⫽336 mEq/kg DW). If this point is excluded from the linear regression, R2 and the slope approach the result with both ADC and CBF thresholds [R2⫽80%, slope⫽0.77 versus R2⫽93%, slope⫽0.93 minutes/(mEq/kg DW], and the 95% CIs at 180 and 360 minutes become 42 and 39 minutes. The rate of [Na⫹] increase was 0.95 and 1.00 (mEq/kg DW)/min for the data with just ADC criteria and the data with both ADC and CBF criteria, respectively. Note that the units for the rate of Na⫹ increase are the inverse of the slope used to estimate time after onset.

the same strategy could be used to estimate time after onset of ischemic stroke in patients, with several caveats: the time for performing the noninvasive MRI measurement must not be excessive, [Na⫹] must be determined with sufficient accuracy, and this scheme must be validated in the clinical setting of acute stroke. Clearly, minimal variability of the estimate of time is crucial for this scheme to function properly. One factor that affects this variability is the choice of the thresholds of ADC and CBF and whether just ADC or both ADC and CBF are used. These decisions can be based on the fidelity of the regression obtained. The use of a higher threshold might be possible if both ADC and CBF are used to choose the regions that exhibit a steady Na⫹ increase. In initial explorations of which threshold to use, we showed that the R2 and slope stabilized at ⬇0.70 and 0.7 min/(mEq/g DW), respectively, when the threshold was between 480 and 540 ␮m2/s. Thus, the rate of Na⫹ increase in these regions was independent of the ADC threshold level. The higher variability of the time estimate with only the ADC criteria might be reduced by using values of ADC, CBF, and [Na⫹] normalized to the normal cortex as a method of reducing animal-to-animal variability. Another strategy to reduce the variability of the estimate of time after onset would be to increase the number of measurements. The slow process of Na⫹ accumulation in ischemic regions is based on residual blood flow that delivers Na⫹ from plasma and is based primarily on the increase in Na⫹ influx meditated by the stimulation of Na⫹,K⫹-ATPase in ischemic cortex.21 The abluminal location of Na⫹,K⫹-ATPase contributes to the lack of Na⫹ transport from brain to blood.22 Other measurements of brain tissue [Na⫹] in experimental cerebral ischemia at various times after arterial occlusion are presented in Table 3. The rates of Na⫹ increase vary between 0.71 and 1.10 (mEq/kg DW)/min. Our rates are slightly higher than the mean of these estimates: the other studies used larger brain tissue samples, which would possibly include nonischemic tissue with lower, more normal [Na⫹],

Discussion We were able to determine 3 biochemical and physiological variables in multiple brain regions. Using this information, we showed that the time after arterial occlusion in the rat could be estimated within 41 to 52 minutes at the established time limits for the administration of tPA or pro-UK as therapeutic agents for acute ischemic stroke in humans (180 and 360 minutes, respectively). Although we used an experimental model of ischemic stroke in the rat, we suggest that TABLE 2.

Linear Regression Results and Estimates of Time After Arterial Occlusion

Parameters and Thresholds

R2

P

95% CI Time at 180 min, min

95% CI Time at 360 min, min

Linear Regression Equation

ADC ⬍520

68%

⬍0.0001

51

51

Ta⫽⫺127⫹0.72ⴱ[Na⫹]

ADC ⬍520, CBF ⬍40

93%

⬍0.0005

52

41

Ta⫽⫺242⫹0.93ⴱ[Na⫹]

Ta indicates time after occlusion (min); [Na⫹], brain tissue sodium concentration (mEq/kg DW). ADC given in ␮m2/s; CBF given in mL 䡠 100 g⫺1 䡠 min⫺1.


1390

Stroke

June 2000

Figure 3. Forty-nine regions were selected and used to estimate Ta with just the criteria that ADC must be ⬍520 ␮m2/s to select regions from all 16 animals. Each of the data points was composed of 1 to 5 punches from ischemic cortex. The variability of [Na⫹] averaged 13%, expressed as the coefficient of variation. Linear regression and 95% CIs for the time after occlusion, Ta, were determined from the brain tissue [Na⫹]. The 95% CIs at 180 and 360 minutes after occlusion are both 51 minutes. The solid vertical line is the CI at 360 minutes.

lowering the estimate of [Na⫹] in ischemic cortex. Another factor that contributed to our higher rates is our guided sampling procedure. Our sampling was directed at areas with low ADC, minimizing admixture of the ischemic cortex with normal cortex. Using Na⫹ MRI, Thulborn et al9 determined the time course of [Na⫹] in 1 monkey after ischemia caused by an autologous blood clot introduced into the right internal carotid artery. After a delay of ⬇2 hours, Na⫹ increased at a rate of 0.46 (mEq/kg DW)/min in the MCA territory. The delay in the beginning of the Na⫹ increase (⬇2 hours) compared with that obtained through models with direct arterial occlusion, as presented in Table 3, could be because the occlusion was originally incomplete or the clot partially lysed and redistributed downstream. The partial volume effect could have produced lower ischemic cortex [Na⫹] in this MRI study9 and could in part explain their low rate of Na⫹ accumulation in ischemic brain tissue (approximately half the rate we observed). Thulborn et al9 maintain that there is a threshold of brain tissue [Na⫹] above which the restoration of blood flow will no longer be beneficial. This concept would imply that brain tissue Na⫹ is the integral of the time-CBF relationship of cerebral vulnerability proposed by Morawetz et al.23 This TABLE 3.

concept could be applied to individual brain regions with imaging methods. At the time of the transition to irreversible damage at 3 hours usually reported with the direct occlusion models, [Na⫹] is 430 to 455 mEq/kg DW based on the data from this study. The determination of ADC with MRI is well established and is considered a surrogate measure of therapeutic effectiveness in clinical trials.24 However, the MRI methods for determination of CBF25 and especially [Na⫹] are still evolving. Perfusion indices are currently monitored with MRI with spin-tag26,27 and bolus tracking28 –30 methods. The lack of quantification and the inaccuracy at low flows could be overcome with the use of the ratio of ischemic to normal cortex. [Na⫹] determinations with MRI that supply acceptable spatial resolution in humans with imaging times of 10 minutes can be performed in sequence with standard proton imaging for diffusion and perfusion.31 In humans with stroke, these Na⫹ MRI methods have been combined with perfusion and diffusion imaging,9 and quantification has been documented in a rat model.32 At this stage, however, these MRI Na⫹ measurements are not generally available, because they require much attention to MRI hardware and software. The additional time required for Na⫹ imaging would be justified in cases in which the onset of stroke symptoms cannot be determined. This proposed scheme to estimate time after occlusion is a 2-step process. First, the identification of severely endangered tissue with both CBF and ADC or just ADC is needed to choose only the regions in which brain tissue [Na⫹] increases. Both CBF and ADC drop quickly after occlusion and stay relatively constant during the initial hours, so they can be used without regard to the time after occlusion. Second, the pattern of slow and constant change in [Na⫹] is used as a stopwatch that is started at the moment of arterial occlusion. This general scheme could have potential for estimation of the time after ischemic onset in humans because all of these parameters, including ADC, CBF, and [Na⫹], can be estimated with MRI.

Acknowledgments The support of the US Public Health Service (NIH grant NS-30839) is gratefully acknowledged. The statistical services of Kirk Easley

Estimates of the Normal Brain Tissue [Naⴙ] and Rate of [Naⴙ] Increase in Cerebral Ischemia

Reference

Time After Occlusion, h

Normal Brain Tissue [Na⫹], mEq/kg DW

Rate of [Na⫹] Increase, mEq/kg DW/min

Species

Model

Dickenson and Betz (1992)33

Rat

MCAO

4

189

1.10

Schuier and Hossmann (1980)8

Cat

MCAO

4

249

0.81

Young et al (1987)

Rat

MCAO

4

254

0.68

Ito et al (1979)35

Gerbil

CCAO

3

230

0.98

Lo et al (1987)36

208

0.81

䡠䡠䡠 213

0.71

34

Gerbil

CCAO

3

Betz et al (1994)37

Rat

MCAO

6

Warner et al (1987)38

Rat

Normal [Na⫹]

䡠䡠䡠

Mean

224

䡠䡠䡠 0.85

Present study, ADC criteria (n⫽16)

Rat

MCAO and biCCAO

7.5

248

0.95

Present study, ADC and CBF criteria (n⫽7)

Rat

MCAO and biCCAO

7.5

238

1.00

MCAO indicates middle cerebral artery occlusion; CCAO, common carotid artery occlusion; biCCAO, bilateral common carotid artery occlusion.


Wang et al


are greatly appreciated. We thank Keith Thulborn, Fernando Boada, and Thomas Kent for thoughtful criticism and suggestions.

References 1. National Institute of Neurological Disorders and Stroke rt-PA Stroke Study Group. Tissue plasminogen activator for acute ischemic stroke. N Engl J Med. 1995;333:1581–1587. 2. Furlan A, Higashida R, Wechsler L, Gent M, Rowley H, Kase C, Pessin M, Ahuja A, Callahan F, Clark WM, Silver F, Rivera F. Intra-arterial prourokinase for acute ischemic stroke: the PROACT II study: a randomized controlled trial: PROlyse in Acute Cerebral Thromboembolism. JAMA. 1999;282:2003–2011. 3. Del Zoppo GJ, Higashida RT, Furlan AJ, Pessin MS, Rowley HA, Gent M, PROACT Investigators. PROACT: a phase II randomized trial of recombinant pro-urokinase by direct arterial delivery in acute middle cerebral artery stroke. Stroke. 1998;29:4 –11. 4. Clark WM, Wissman S, Albers GW, Jhamandas JH, Madden KP, Hamilton S. Recombinant tissue-type plasminogen activator (Alteplase) for ischemic stroke 3 to 5 hours after symptom onset: the ATLANTIS Study: a randomized controlled trial: Alteplase Thrombolysis for Acute Noninterventional Therapy in Ischemic Stroke. JAMA. 1999;282: 2019 –2026. 5. Wester P, Radberg J, Lundgren B, Peltonen M, The Seek-MedicalAttention-in-Time Study Group. Factors associated with delayed admission to hospital and in-hospital delays in acute stroke and TIA: a prospective, multicenter study. Stroke. 1999;30:40 – 48. 6. Fogelholm R, Murros K, Rissanen A, Ilmavirta M. Factors delaying hospital admission after acute stroke. Stroke. 1996;27:398 – 400. 7. Barsan WG, Brott TG, Broderick JP, Haley EC, Levy DE, Marler JR. Time of hospital presentation in patients with acute stroke. Arch Intern Med. 1993;153:2558 –2561. 8. Schuier FJ, Hossmann KA. Experimental brain infarcts in cats, II: ischemic brain edema. Stroke. 1980;11:593– 601. 9. Thulborn KR, Gindin TS, Davis D, Erb P. Comprehensive MRI protocol for stroke management: tissue sodium concentration as a measure of tissue viability in non-human primate studies and in clinical studies. Radiology. 1999;213:156 –166. 10. Perez-Trepichio AD, Xue M, Ng TC, Majors AW, Furlan AJ, Awad IA, Jones SC. Sensitivity of magnetic resonance diffusion-weighted imaging and regional relationship between the apparent diffusion coefficient and CBF in rat focal cerebral ischemia. Stroke. 1995;26:667– 675. 11. Chen ST, Hsu CY, Hogan EL, Maricq H, Balentine JD. A model of focal ischemic stroke in the rat: reproducible extensive cortical infarction. Stroke. 1986;17:738 –743. 12. Le Bihan D, Breton E, Lallemand D, Grenier P, Cabanis E, Laval-Jeantet M. MR imaging of intravoxel incoherent motions: application to diffusion and perfusion in neurologic disorders. Radiology. 1986;161:401– 407. 13. Reivich M, Jehle J, Sokoloff L, Kety SS. Measurement of regional cerebral blood flow with antipyrine-14C in awake cats. J Appl Physiol. 1969;27:296 –300. 14. Sakurada O, Kennedy C, Jehle J, Brown JD, Carbin GL, Sokoloff L. Measurement of local cerebral blood flow with iodo[14C]antipyrine. Am J Physiol. 1978;234:H59 –H66. 15. Hu W, Kharlamov A, Wang Y, Perez-Trepichio AD, Jones SC. Directed sampling for electrolyte analysis and water content of micro-punch samples shows large differences between normal and ischemic rat brain cortex. Brain Res. In press. 16. SAS Institute. SAS/STAT User’s Guide, Version 6, Volume 2. Cary, NC: SAS Institute, Inc; 1989. 17. Neter J, Wasserman W, Kutner MH. Multiple regression with two independent variables. In: Applied Linear Statistical Models: Regression, Analysis of Variance, and Experimental Designs. Homewood, Ill: RD Irwin; 1985:234 –246.

1391

18. Reivich M. Arterial PCO2 and cerebral hemodynamics. Am J Physiol. 1964;206:25–35. 19. Jones SC, Bose B, Furlan AJ, Friel HT, Easley KA, Meredith MP, Little JR. CO2 reactivity and heterogeneity of cerebral blood flow in ischemic, border zone and normal cortex. Am J Physiol. 1989;257:H473–H482. 20. Perez-Trepichio AD, Furlan AJ, Little JR, Jones SC. Hydroxyethyl starch 200/0.5 reduces infarct volume after embolic stroke in rats. Stroke. 1992; 23:1782–1790. 21. Schielke GP, Moises HC, Betz AL. Blood to brain sodium transport and interstitial fluid potassium concentration during early focal ischemia in the rat. J Cereb Blood Flow Metab. 1991;11:466 – 471. 22. Betz AL, Firth JA, Goldstein GW. Polarity of the blood-brain barrier: distribution of enzymes between the luminal and antiluminal membranes of brain capillary endothelial cells. Brain Res. 1980;192:17–28. 23. Morawetz RB, Crowell RH, DeGirolami U, Marcoux FW, Jones TH, Halsey JH. Regional cerebral blood flow thresholds during cerebral ischemia. Fed Proc. 1979;38:2493–2494. 24. Warach S, Dashe JF, Edelman RR. Clinical outcome in ischemic stroke predicted by early diffusion-weighted and perfusion magnetic resonance imaging: a preliminary analysis. J Cereb Blood Flow Metab. 1996;16: 53–59. 25. Calamante F, Thomas DL, Pell GS, Wiersma J, Turner R. Measuring cerebral blood flow using magnetic resonance imaging techniques. J Cereb Blood Flow Metab. 1999;19:701–735. 26. Williams DS, Detre JA, Leigh JS, Koretsky AP. Magnetic resonance imaging of perfusion using spin inversion of arterial water. Proc Natl Acad Sci U S A. 1992;89:212–216. 27. Detre JA, Zhang W, Roberts DA, Silva AC, Williams DS, Grandis DJ, Koretsky AP, Leigh JS. Tissue specific perfusion imaging using arterial spin labeling. NMR Biomed. 1994;7:75– 82. 28. Villringer A, Rosen BR, Belliveau JW, Ackerman JL, Lauffer RB, Buxton RB, Chao YS, Wedeen VJ, Brady TJ. Dynamic imaging with lanthanide chelates in normal brain: contrast due to magnetic susceptibility effects. Magn Reson Med. 1988;6:164 –174. 29. Rosen BR, Belliveau JW, Vevea JM, Brady TJ. Perfusion imaging with NMR contrast agents. Magn Reson Med. 1990;14:249 –265. 30. Mottet I, Quast MJ, DeWitt DS, Hillman GR, Wei J, Uhrbrock DH, Perez-Polo JR, Kent TA. NG-Nitro-L-arginine methyl ester modifies the input function measured by dynamic susceptibility contrast magnetic resonance imaging. J Cereb Blood Flow Metab. 1997;17:791– 800. 31. Boada FE, Gillen JS, Shen GX, Chang SY, Thulborn KR. Fast three dimensional sodium imaging. Magn Reson Med. 1997;37:706 –715. 32. Christensen JD, Barrere BJ, Boada FE, Vevea JM, Thulborn KR. Quantitative tissue sodium concentration mapping of normal rat brain. Magn Reson Med. 1996;36:83– 89. 33. Dickinson LD, Betz AL. Attenuated development of ischemic brain edema in vasopressin-deficient rats. J Cereb Blood Flow Metab. 1992; 12:681– 690. 34. Young W, Rappaport ZH, Chalif DJ, Flamm ES. Regional brain sodium, potassium, and water changes in the rat middle cerebral artery occlusion model of ischemia. Stroke. 1987;18:751–759. 35. Ito U, Ohno K, Nakamura R, Suganuma F, Inaba Y. Brain edema during ischemia and after restoration of blood flow: measurement of water, sodium, potassium content and plasma protein permeability. Stroke. 1979; 10:542–547. 36. Lo WD, Betz AL, Schielke GP, Hoff JT. Transport of sodium from blood to brain in ischemic brain edema. Stroke. 1987;18:150 –157. 37. Betz AL, Keep RF, Beer ME, Ren X. Blood-brain barrier permeability and brain concentration of sodium, potassium, and chloride during focal ischemia. J Cereb Blood Flow Metab. 1994;14:29 –37. 38. Warner DS, Smith ML, Siesjo BK. Ischemia in normo- and hyperglycemic rats: effects on brain water and electrolytes. Stroke. 1987;18:464 – 471.


1392

Stroke

June 2000

Editorial Comment Of all of the issues that confront clinicians in the quest for improved acute stroke treatment, one of the most vexing has been the time from onset of brain ischemia to the time of treatment initiation. Although results in animal experimental models had long suggested that successful intervention into the process of ischemic stroke was time limited, this fact was not fully appreciated by clinicians until the past 15 years. Undoubtedly, the rapidity with which treatment was brought to bear on patients with ischemic stroke treated with intravenous rtPA greatly influenced the positive outcome from those trials showing benefit, and later delivery likely influenced the neutral outcomes from other studies. One problem that persistently arises in the clinical context of acute stroke treatment is the accurate assignment of the time of onset of ischemia. In most circumstances, the onset of definite focal symptoms (eg, hemiparesis, speech disturbance) is taken as a surrogate for the onset of brain ischemia. Nonspecific symptoms such as headache and general malaise are customarily disregarded in these settings, although there is no guarantee that brain ischemia was not actually occurring during these symptoms. In patients who have onset of symptoms while asleep or those who have unwitnessed onsets but are unable to communicate or are unaware of their deficits, timing the onset becomes problematic, and these patients are usually excluded from treatment. It is now clear

that the presence or absence of early signs of ischemia on initial head CT scans is not a reliable measure of the duration of ischemia, because these signs have now been clearly recognized in patients scanned within 90 minutes of onset, whereas some patients with a much longer durations of symptoms may still have negative scans. In the present article, Wang and colleagues present evidence that in a rat model of focal cerebral ischemia, brain tissue sodium concentrations varied linearly with time in tissues at risk of infarction defined by cerebral blood flow and apparent diffusion coefficient criteria. They suggest that because brain tissue sodium concentrations can be measured in humans with the use of MRI, this work could lead to a method of timing the onset of stroke in patients in whom the clinical information is not adequate. Although considerable work remains to confirm this hypothesis in humans, if the measurements proved to be accurate predictors and the data could be obtained expeditiously, then it might expand the number of patients who could be considered for treatment. Future research in this regard seems warranted. E. Clarke Haley, Jr, MD, Guest Editor Department of Neurology University of Virginia Health System Charlottesville, Virginia
