(Stroke. 2000;31:1694.)
© 2000 American Heart Association, Inc.
Original Contributions |
Hypertonic Saline Worsens Infarct Volume After Transient Focal Ischemia in Rats
From the Departments of Anesthesiology/Critical Care Medicine (A.B., I.H., S.J.M., N.J.A., R.C.K., P.D.H., R.J.T.), Neurology (A.B.), and Pathology (B.J.C.), Johns Hopkins University School of Medicine, Baltimore, Md.
Correspondence to Anish Bhardwaj, MD, Neuroscience Critical Care Division, Meyer 8-140, Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287. E-mail abhardwa{at}jhmi.edu
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Background and Purpose—Hypertonic saline (HS) has been advocated as a hyperosmolar agent for the treatment of cerebral edema, especially after traumatic brain injury. We tested the hypothesis that continuous intravenous HS administered during reperfusion from transient focal cerebral ischemia attenuates infarct volume.
Methods—Halothane-anesthetized male Wistar rats were subjected to 2 hours of middle cerebral artery occlusion (MCAO) by the intraluminal occlusion technique. At the onset of reperfusion, rats received a 10-mL/kg intravenous bolus of 0.9% saline (SAL, n=8) or 7.5% SAL (chloride:acetate 50:50, n=8) followed by a continuous infusion for 22 hours. In a second series of experiments, ischemic damage was determined in cohorts treated with equivolumetric 3% saline (n=8) or 20% mannitol (n=8). In a third series, regional cerebral blood flow was measured ([14C]iodoantipyrine autoradiography) at 6 hours of reperfusion in 7.5%-SAL–treated (n=5) or SAL-treated (n=5) animals.
Results—In SAL rats, serum Na+ was 137±3 and 138±2 mEq/L (mean±SEM) at baseline and 22 hours of reperfusion, respectively. In 7.5% SAL, serum Na+ was 136±2 and 154±2 mEq/L at baseline and reperfusion, respectively. Physiological variables and reduction in laser-Doppler signal during MCAO and early reperfusion were not different between the 2 treatment groups. Cortical infarct volume was larger in 7.5%-SAL–treated rats (121±14 mm3; 30±3% of contralateral cortex; P<0.05) than in SAL (64±15 mm3; 16±4% of contralateral cortex). Striatal infarct volume was unchanged by HS therapy. Ipsilateral cortical tissue volume was increased relative to the contralateral side (by 26±5% with SAL; by 41±5% with 7.5% SAL). In contrast, ischemic damage was unaffected by 3%-SAL or 20%-mannitol treatment compared with SAL. Regional cerebral blood flow during reperfusion was heterogeneous in all animals, but there was no evidence of postischemic hypoperfusion or blood flow maldistribution in 7.5%-SAL–treated animals.
Conclusions—These data demonstrate that hypernatremia resulting from postischemic HS infusion worsens cortical infarct volume in transient focal cerebral ischemia. The deleterious effect is not linked to exacerbation of delayed hypoperfusion during early reperfusion (6 hours); however, blood flow defects at later recovery time points remain to be excluded. These results may have implications for HS therapy in clinical ischemic stroke.
Key Words: infarction • ischemia, focal • mannitol • osmolar concentration • reperfusion • saline solution, hypertonic
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Introduction |
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Osmotherapy remains the mainstay of pharmacological intervention in neurological and neurosurgical intensive care for the treatment of cerebral edema and intracranial hypertension of diverse causes. Through the years, a variety of osmotic agents have been used in clinical practice, including mannitol, glycerol, urea, and sorbitol.1 2 3 Mannitol has been the osmotic agent of choice since the 1960s because it ordinarily does not cross the blood-brain barrier (reflection coefficient=0.9).1 Osmotic agents have potent antiedema action, presumably by drawing water from interstitial and intracellular spaces into the intravascular compartment.1 2 3 However, these agents have other nonosmotic cerebral effects, as has been demonstrated in a variety of animal models. For example, mannitol protects the brain by reducing blood viscosity, with consequent improved microvascular cerebral blood flow (CBF),3 4 5 by free radical scavenging properties,6 and by decreasing cerebrospinal fluid formation and reabsorption.7
More recently, hypertonic saline (HS) solutions have received renewed attention and use in clinical practice. Sodium chloride is completely excluded from the intact blood-brain barrier (reflection coefficient=1.0) and is theoretically a better osmotic agent than mannitol.1 Naive animals that are hemodiluted with hypertonic lactated Ringer’s solution (osmolarity of 480 mOsm/L) experience decreased intracranial pressure and total brain water content, along with increased CBF and resulting enhanced oxygen delivery.8 9 10 Furthermore, several case series and randomized clinical trials have demonstrated improved outcome with HS therapy in traumatic brain injury.11 12 13 14 Therefore, it has been hypothesized that HS therapy could be beneficial in cerebral ischemia and stroke.
The purpose of the present study was to test the hypothesis that inducing systemic hypernatremia during reperfusion decreases infarct volume after middle cerebral artery occlusion (MCAO). We also determined whether continuous HS infusion affects regional CBF during reperfusion and whether HS therapy causes histopathological injury independently of ischemia to neurons and glia in the rat.
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Materials and Methods |
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General Preparation and Animal Surgery
The experimental protocol was approved by the Institutional Animal Care and Use Committee and conforms to the National Institutes of Health guidelines for the care and use of animals in research. All techniques are as previously described.15 In brief, adult male Wistar rats (290 to 330 g) were anesthetized with halothane (1.0% to 2.0%) in oxygen-enriched air and allowed to ventilate spontaneously. Under aseptic surgical techniques, the right femoral artery was cannulated to monitor arterial blood pressure and arterial blood gases, and the femoral vein was cannulated for vascular access. After cannulation, both catheters were exteriorized in the posterior mid thorax. Rectal and temporalis muscle temperatures were maintained with a heating lamp throughout the surgical procedures.
Focal Ischemia and Reperfusion
All experiments were performed by a single individual (I.H.).
Cortical perfusion was measured by laser Doppler flowmetry
(LDF) as previously described15 (coordinates: 2 mm
posterior and 6 mm lateral to bregma) (Moor Instruments Ltd, model
MBF3D). To allow continuous monitoring of LDF, the headpiece of the
stereotaxic frame was modified to allow for free rotation
around the longitudinal axis of the rat and was equipped with a snout
mask for spontaneous ventilation and with a holder for the LDF probe.
The probe was positioned over an area devoid of large cortical blood
vessels, and its position was not changed throughout the experiment.
The LDF signal was allowed to stabilize over a 30-minute period before
baseline measurements were obtained. Transient focal ischemia
(2 hours) was produced by MCAO with an intraluminal suture technique as
previously described.15 16 17 At the end of
ischemia, reperfusion was produced by withdrawal of the
intraluminal suture; this was associated with rapid restoration of the
LDF signal. Rats that did not demonstrate a significant reduction of
the LDF signal (
40% of baseline) during MCAO or rapid restoration of
the LDF signal during reperfusion were excluded from the study. LDF
measurements were averaged over 5-minute periods at 5, 15, 30, 60, 90,
and 120 minutes of MCAO and at 15 minutes of reperfusion.
Rats were assigned to 2 treatment groups and received either 0.9% saline (SAL) (308 mOsm/L) or 7.5% SAL (2310 mOsm/L) on reperfusion in a blinded, randomized fashion. An intravenous fluid bolus (10 mL/kg) was administered at the onset of reperfusion, followed by a continuous infusion at 0.5 mL/h until the end of the experiment. In subsequent experiments, the effects of 3% SAL (1027 mOsm/L) and 20% mannitol (1098 mOsm/L) were tested in a blinded, randomized fashion in the same experimental paradigm. Saline infusions were prepared as chloride:acetate (50:50) to avoid hyperchloremic acidosis. Serum Na+, osmolality, hematocrit, arterial blood gases, and glucose were measured at baseline and at 2 and 22 hours of reperfusion. At 15 minutes of reperfusion, rats were allowed to emerge from anesthesia and were provided free access to food and to 0.9% saline rather than free water for oral fluids. At 22 hours of reperfusion, rats were deeply anesthetized with 5% halothane and decapitated. The brain was harvested and sliced into 7 coronal sections 2 mm thick for staining with 1% triphenyltetrazolium chloride in saline at 37°C for 30 minutes as previously described.17 Infarct volume was measured by a blinded observer using digital imaging (Digital Camera 40, Eastman Kodak Co) and image analysis software (SigmaScan Pro, Jandel). The infarcted area was numerically integrated across each section and over the entire ipsilateral hemisphere. Infarct volumes were measured separately in the cerebral cortex and caudoputamen complex and expressed as a percentage of the volume of the ipsilateral side and then corrected for edema as described by Lin et al.18
In another set of blinded, randomized experiments, brain edema was estimated by comparing ratios of wet to dry weight18 in rats treated with either 0.9% or 7.5% SAL and then harvested at 22 hours of reperfusion (n=6 per group). Dry weight of the entire ipsilateral and contralateral hemispheres was determined after the tissue had been heated for 3 days at 100°C in a drying oven. Tissue water content was then calculated as % H2O=(1-dry wt/wet wt)x100%.18
Regional CBF Measurements
CBF was measured in additional cohorts of Wistar rats by
quantitative autoradiography with
[14C]iodoantipyrine
([14C]IAP), as described
previously.15 19 Briefly, rats were instrumented with
femoral venous and arterial catheters, and the MCA was
occluded for 2 hours and treated with continuous 7.5%-SAL (n=5) or SAL
(n=5) infusion with the onset of reperfusion as in the previous
cohorts. At 6 hours of reperfusion, rats were reanesthetized
with halothane; arterial blood pressure and blood gases
were measured. [14C]IAP (40 µCi, New England
Nuclear) in 0.8 mL of isotonic saline was infused
intravenously for 45 seconds. During infusion, fifteen 10-
to 20-µL samples of free-flowing arterial blood from the
femoral artery catheter were collected in heparin-coated sample tubes.
With the filament still in place, the rat was decapitated 45 seconds
after the start of infusion. One postdecapitation arterial
blood sample was also collected. The brain was quickly removed and
frozen at -50°C in 2-methylbutane on dry ice. Each brain was
sectioned by cryostat into coronal sections 20 µm thick at
-20°C and thaw-mounted onto cover glasses. Sections were apposed for
1 week to film (Kodak, Bio-Max MR) with 14C
standards. The concentrations of [14C]IAP in
blood samples were determined by liquid scintillation spectroscopy
(Beckman, model 3801) after decolorization with 0.2 mL of tissue
solubilizer (Soluene-350, Packard Instruments Co).
Autoradiographic images representing 5
different coronal levels (+2.2, +0.2, -1.8, -3.8, and -5.8 mm
from the bregma, 6 to 9 images at each coronal level) were digitized,
and CBF was determined with image analysis software (Inquiry,
Loats Associates). Rates of CBF were calculated by the Kety-Schmidt
modification of the Fick principle as previously
described.15 Standard coefficients for diffusion
equilibrium and tissue:blood partition were used,15 with
the assumption that these variables are unchanged by
ischemia and reperfusion. Theoretical assessment of tracer
techniques suggest that tissue:blood partition coefficients for
diffusible tracers change little with cerebral
ischemia.20
The individual (S.J.M.) performing the CBF data analysis was blinded to the treatment groups. Two methods of analysis were used to determine CBF. First, discrete areas were measured by sampling of 0.08-mm2 squares within those regions most vulnerable to MCA occlusion: frontal and parietal cortex and medial and lateral caudoputamen. Flow rates were then averaged from squares assayed from 6 to 9 consecutive brain slices at each of 3 coronal levels (+2.2, +0.2, and -1.8 mm from the bregma). In the second method, areas for increasing incremental ranges of flow rates were determined by digital image scanning and perimetric measurements for selected slices in the entire ischemic hemisphere. Areas were averaged over 3 images from each brain level (+2.2, +0.2, -1.8, -3.8, and -5.8 mm from the bregma) and then numerically integrated across the 5 coronal levels to obtain an estimate of tissue volume for each incremental range of CBF.
Histopathology
To test whether HS infusion in the absence of MCAO causes
injury, separate naive nonischemic rats were treated with an
intravenous bolus of 10 mL/kg of either 0.9%, 3%, or
7.5% SAL (n=3 per group) followed by a continuous infusion at a rate
of 0.5 mL/h for 4 days. Rats were then killed by decapitation under 5%
halothane anesthesia and perfused with 10% neutral
buffered formalin. The brains were postfixed in formalin and cut
coronally at 2-mm intervals. The coronal slices were embedded in
paraffin, cut at 10 µm, and stained with hematoxylin-eosin
(H-E), H-E with Luxol fast blue counterstain for myelin, and an
antibody to glial fibrillary acidic protein (Dako). Histopathological
studies were interpreted by an individual (B.J.C.) who was blinded to
the 2 treatment groups.
Statistical Analysis
All values are expressed as mean±SEM.
Physiological parameters and mean LDF
measurements among groups were subjected to repeated-measures ANOVA.
Differences in infarct volume, cerebral edema, and
autoradiographic regional CBF were determined by 1-way
ANOVA. Post hoc comparisons were made with the Newman-Keuls test. The
criterion for statistical significance was P<0.05.
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Effect of HS on Infarct Volume
Mean arterial blood pressure, arterial carbon dioxide (PaCO2) and oxygen (PaO2), pH, and temporalis muscle temperatures were within normal physiological ranges in all groups throughout the ischemic insult (Table 1
). No rats died before the end of
the experimental protocol. Two rats treated with 7.5% SAL and 1
treated with SAL were excluded because the residual LDF signal was not
sustained below 40% during 2 hours of MCAO. Eight rats in each
treatment group successfully completed the experimental protocol.
Baseline serum Na+ and osmolality were similar in
all treatment groups. However, serum Na+ and
osmolality were increased at 2 hours (148±0.2 mEq/L; 305±2 mOsm/L)
and 22 hours (154±2 mEq/L; 338±7 mOsm/L) of reperfusion in
7.5%-SAL–treated rats but not in SAL-treated rats (135±1 mEq/L; 293
mOsm/L and 138±1 mEq/L; 301 mOsm/L, respectively) (Table 2). Hemoglobin concentration, hematocrit,
and serum glucose levels were not different among groups. The residual
LDF signal averaged over the 2-hour MCAO period was similar in the
0.9%-saline–treated controls (27±3% of baseline) and the
7.5%-SAL–treated group (20±3%). On withdrawal of the monofilament,
LDF signal returned rapidly to baseline values within 5 minutes in both
groups (Figure 1).
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Triphenyltetrazolium chloride–determined
infarct volume of contralateral cerebral cortex measured at 22 hours of
reperfusion was larger in rats treated with 7.5% SAL (121±14
mm3; 30±3% of contralateral cortex;
P<0.05) than in rats treated with SAL (64±15
mm3; 16±4% of contralateral cortex) (Figure 2). There were no significant differences
among groups for infarct volume in the caudoputamen
complex. Total hemispheric infarct was larger in rats treated with
7.5% SAL (158±17 mm3; 22±2% of
contralateral hemisphere; P<0.05) than in rats treated with
SAL (93±19 mm3; 12±3% of contralateral
hemisphere). Total tissue volume of the ipsilateral cerebral cortex was
increased 26±5% in the SAL group and 41±5% in the 7.5%-SAL group
relative to the contralateral cortex.
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In the 3%-SAL– or 20%-mannitol–treatment groups, there were no
differences in physiological parameters
(Table 1
) or in serum sodium and osmolality (Table 2).
The residual LDF signals during MCAO and early reperfusion in the
3%-SAL group (32±1%) and in the 20%-mannitol group (29±3%) were
similar to that of the SAL group (27±3%). The infarct volumes of the
cortex and caudoputamen in the 3%-SAL group did not differ
from those of the 20%-mannitol or SAL groups (Figure 3).
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Effect of SAL Versus 7.5% SAL on Cerebral Edema After MCAO at
22 Hours
Physiological variables were maintained
within normal values, and there were no differences between treatment
groups before or during MCAO or in early reperfusion. The ipsilateral
LDF signal during MCAO decreased rapidly to 
30% of baseline values
and remained at this level for the duration of occlusion in both
groups. One rat in the 7.5%-SAL group was excluded because the
residual LDF signal during MCAO was not sustained below 40% of
baseline values. Six rats in each treatment group successfully
completed the experimental protocol. Serum Na+
and osmolality in animals treated with SAL were similar between groups
before MCAO. As anticipated, treatment with 7.5% SAL was associated
with an increase in serum Na+ at both 2 hours
(SAL: 136±4 mEq/L; 7.5%-SAL: 144±1 mEq/L, P<0.05) and 22
hours (SAL: 144±2; 7.5%-SAL: 164±3 mEq/L, P<0.05) and in
plasma osmolality at 2 hours (SAL: 298±4 mOsm/L; 7.5%-SAL: 311±4
mOsm/L, P<0.05) and at 22 hours (SAL: 307±2 mOsm/L;
7.5%-SAL: 355±10 mOsm/L, P<0.05). Water content in the
contralateral noninjured hemisphere in the 7.5%-SAL group
(78.3±0.2%) was significantly less (P<0.05) than in the
SAL group (79.0±0.1%) at 22 hours of reperfusion. However, there was
no difference in water content in the injured hemisphere between the
7.5%-SAL group (81.3±0.6%) and the SAL group (81.7±1.0%).
Effects of SAL Versus 7.5% SAL on CBF at 6 Hours of Reperfusion
After MCAO
Physiological variables and baseline serum
Na+ were similar in the SAL- and
7.5%-SAL–treated rats. As expected, serum Na+
levels at 6 hours of reperfusion were 152±2 and 137±1 mEq/L in
7.5%-SAL– and SAL-treated rats, respectively. To examine potential
cortical and subcortical perfusion deficits during reperfusion, both
absolute CBF within the MCA territory and blood flow distribution were
quantified. Absolute CBF in the frontal and parietal cortex at 6 hours
of reperfusion was not different in the SAL and 7.5%-SAL groups
(Figure 4). Blood flow to medial and
lateral sections of the caudoputamen was also similar in
both treatment groups. Furthermore, when brain volume was partitioned
into blood flow increments of 50 mL · 100
g-1 ·
min-1 throughout the
previously ischemic hemisphere, there appeared to be no
difference in blood flow distribution during reperfusion (Figure 5). HS therapy did not alter the
distribution of tissue volume into low-flow zones (ie, <50 mL ·
100 g-1 ·
min-1) compared with SAL.
Scattered hypodensities on the autoradiographs were observed in three
7.5%-SAL–treated rats and 1 SAL-treated rat, but there was no visible
hemorrhage in tissue sections.
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Histopathology
At 24 hours of continuous infusion, serum
Na+ was increased in rats with 7.5% SAL
(155±0.3 mEq/L) compared with SAL (141±1 mEq/L) or 3% SAL (142±1
mEq/L). Hypernatremia was sustained over the 4-day infusion period.
Light microscopy revealed no gross histological
differences among treatment groups. There was no evidence of
hypoxic-ischemic injury to gray or white matter or
macrophage infiltration or myelin pallor or sponginess to
suggest white matter injury or edema. There was no evidence of reactive
gliosis in H-E sections. Immunohistochemistry for glial fibrillary
acidic protein did not reveal reactive astrocytes.
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This study demonstrates 2 important findings. First, administration of intravenous HS initiated at the onset of reperfusion after transient focal ischemia is not efficacious as a cerebral resuscitation treatment in this animal model of focal stroke. HS-induced hypernatremia/hyperosmolality did alter tissue outcome after MCAO, but in an unanticipated manner. We achieved serum Na+ (145 to 155 mEq/L) and serum osmolality (>310 mOsm/L) goals with continuous infusion of 7.5% saline but not with 3% saline. Continuous 7.5% saline, but not 3% saline or mannitol, increased cortical and hemispheric infarct volumes as assessed at 22 hours of reperfusion. Because continuous hypernatremia did not cause histopathological neuronal, glial, or myelin injury in the absence of ischemia, the mechanism by which HS treatment damages brain is most likely linked to an interaction with reperfusion pathophysiology. Second, CBF recovery was robust in both saline- and HS-treated animals at 6 hours of reperfusion. Absolute CBF within the ipsilateral MCA territory or in contralateral nonischemic regions was not altered by ongoing 7.5% saline infusion. Therefore, the mechanism of HS-exacerbated tissue damage is not likely to be gross exacerbation of delayed hypoperfusion during early reperfusion (6 hours); however, blood flow defects at later recovery times remain to be excluded.
The basis for osmotherapy for focal or global cerebral edema of diverse causes can hypothetically be lessened by drawing free water from the interstitial space into the vascular compartment3 in regions of intact blood-brain barrier1 and possibly in regions of defective barrier function.21 HS solutions have been shown to enhance CBF and oxygen delivery in animal models,8 9 10 suggesting beneficial properties by vascular mechanisms. Furthermore, HS and mannitol have been shown to decrease intracranial pressure and improve neurological outcome after neurotrauma. In contrast, hyponatremia accentuates brain damage after traumatic brain injury.11 12 13 14 Thus, HS therapy is increasingly being used as a therapeutic modality in the neurological critical care setting. In our retrospective case series,22 continuous HS therapy improved cerebral edema in neurotrauma and postoperative neurosurgical patients. However, patients with intracerebral hemorrhage and cerebral infarction did not benefit from this therapy, and HS therapy remains unproven in clinical stroke. The present data in animals clearly demonstrate that HS therapy leading to significant hypernatremia can increase tissue damage after vascular occlusion and focal cerebral ischemia.
There are limited data on the histopathological effects on the brain
with the use of HS solutions. Based on published clinical reports,
several complications of HS therapy are possible.2 23
Myelinotoxicity is a well-known complication of rapid correction of
preexisting hyponatremia. Rapidly induced and sustained
severe hypernatremia has also been shown to cause brain myelinolysis in
rats, but a large serum Na+ gradient (
Na)
(39±8 mEq/L) was necessary to demonstrate myelin
injury.24 The Na in our study was only 17 mEq/L.
Maintenance of this Na for 4 days without ischemia
did not cause myelin injury. Furthermore, we did not observe any loss
of neurons, gliosis, or sponginess of white matter indicative of
cerebral edema in our histopathological examination.
Although hypernatremia alone had no apparent deleterious effect on the
brain in this model, treatment with 7.5% SAL during early stroke
recovery exacerbated infarction, and the effect was most prominent in
the cortex. Physiological variables and
intraocclusion LDF reduction were similar among all treatment groups,
so it seems unlikely that ischemic intensity was heightened in
these rats. Although HS solutions have been shown to increase CBF in
the nonischemic cerebral circulation,8 we wished
to exclude grossly enhanced cortical perfusion defects that might be
present during the immediate HS treatment period after MCAO. One
potential cause of abnormal perfusion in HS animals could be
blood-brain barrier opening due to intense endothelial
injury,25 allowing leakage of osmotically active sodium
and water into perivascular tissue elements. The present data
indicate that overall, CBF recovery was not depressed in HS-treated
animals at 6 hours of reperfusion. Furthermore, the volume of
hypoperfused tissue (<50 mL · 100
g-1 ·
min-1) was not
significantly different between the HS and SAL groups. Nevertheless,
some animals in the HS group did have a considerable area of
hypoperfusion at 6 hours, as indicated by the larger SEM relative to
the mean value at CBF <50 mL · 100
g-1 ·
min-1 in Figure 5.
CBF recovery, as measured by [14C]IAP, was
characterized by large intraregional variability in all animals,
regardless of treatment assignment, at 6 hours of reperfusion.
Intrasubject and intragroup blood flow heterogeneity
and patchy CBF recovery patterns have been well characterized by other
investigators with reversible cerebral ischemia in
rat26 and cat.27 Although it may have been
difficult to show small differences in absolute CBF during reperfusion,
the present data clearly do not suggest a gross increase in
postischemic hypoperfusion with HS therapy, at least within
the window of our observations. Differences in CBF distribution may
become more prominent in the later phases of reperfusion in our
experimental paradigm. Nevertheless, non–blood flow–associated
mechanisms should be considered in the surprisingly deleterious
interaction between hypernatremia and reperfusion.
Using wet-to-dry weight comparisons, we examined the possibility that HS therapy paradoxically worsened parenchymal edema rather than dehydrating the injured hemisphere. Cerebral edema was present in all animals at 22 hours of reperfusion, regardless of treatment, because ipsilateral cortical volume increased by 41% and 26% relative to contralateral cortex in the 7.5%-SAL and SAL groups, respectively. Part of this increase in volume can be accounted for by increased brain water. The difference in percent water content between ipsilateral and contralateral hemispheres was 3% in HS-treated animals and 2.7% in the normal saline control group. Consistent with the goal of osmotherapy, we detected lower water content in the noninjured contralateral hemisphere of HS-treated rats and presume that this measurement represents brain regions with intact blood-brain barrier and sodium exclusion. However, there was no evidence that HS therapy worsened (or improved) water accumulation in the injured hemisphere. A greater amount of tissue swelling without a greater increase in water content may imply that the additional tissue volume observed with 7.5% SAL (41% versus 26% in SAL) is due to the greater volume of damaged and infarcted tissue elements rather than more intense edema formation. Alternatively, augmented edema formation by 7.5% SAL in the ischemic core where blood-brain barrier disruption is anticipated may not have been detected by our measurements of water content within the total ipsilateral hemisphere. It is possible that increases in brain water were not large because of dilution of areas of low water content (noninfarcted regions) with those of high water content (infarcted regions). Edema after cerebral ischemia has both vasogenic and cytotoxic components, and HS may have a differential effect over the 22-hour reperfusion period on these 2 components, ie, improving vasogenic but exacerbating cytotoxic edema. A potential for rebound brain uptake of water with HS treatment secondary to increases in idiogenic tissue osmoles also exists. Increased brain osmolality unrelated to tissue electrolyte change or protein extravasation across the blood-brain barrier has also been reported at 3 and 6 hours after MCAO.28
In the present study, the desired end point for serum Na+ was 145 to 155 mmol/L. The rationale for this end point was to achieve a serum osmolality of 310 to 320 mOsm/L, ie, the target for patients with intracranial hypertension and cerebral edema.29 We were able to achieve these goals in our rat model with a continuous infusion of 7.5% SAL. In a further set of experiments, we infused comparable concentrations and equal volumes (10 mL/kg) of 20% mannitol and 3% saline. The goal was to compare the effect of an alternative osmolar agent (mannitol) and a lower level of hypernatremia (3% SAL) with that of 7.5% SAL in our model. Neither agent extended ischemic damage as 7.5% saline therapy did, presumably because increases in osmolality were moderate and mimicked those observed with the control saline treatment. Although evaluation of the potential of mannitol as a stroke resuscitation agent was not a goal of the present study, we noted that 20% mannitol during reperfusion did not improve outcome. Although neuroprotective effects of mannitol have been reported, results have been mixed.30 31 32 33 34 35 It appears that the effects and putative mechanisms of action of mannitol depend on the timing of administration and pathological model studied. Our failure to see a positive effect of mannitol in ischemic stroke may be a result of use of a loading dose followed by a prolonged infusion period (22 hours). Improved outcome through osmotherapy may require administration of large doses of the agent over short durations early in reperfusion, rather than prolonged administration extending beyond the initial period of vasogenic edema. For example, albumin given as a high-dose bolus reduces infarct volume and improves local CBF at 1 hour of reperfusion.36
In conclusion, our data demonstrate that continuous HS therapy worsens infarct volume after transient focal ischemia. We cannot exclude the possibility that HS may have therapeutic efficacy if used in a setting of permanent focal ischemia without reflow, if initiated at a later time than immediately after occlusion, or if given as a single large bolus during early reperfusion rather than as a continuous infusion. However, caution is advised in the use of HS for patients who experience an ischemic insult.
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Acknowledgments |
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This work was supported in part by US Public Health Service National Institutes of Health grants NS-20020 and NS-33668. Dr Bhardwaj was supported in part by a Clinician Scientist Award and Initiation Grant from the American Heart Association. We thank Dr Jeffrey R. Kirsch for his advice and insightful comments throughout the course of this study.
Received February 23, 2000; revision received April 5, 2000; accepted April 26, 2000.
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Editorial Comment
Department of Neurological Surgery University of California, Davis
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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The article by Bhardwaj et al is a thoughtfully executed animal study with some surprising observations concerning hypertonic saline administration during reperfusion after transient focal ischemia. The authors investigated the hypothesis that hypertonic saline administration would improve histological and/or functional outcome after middle cerebral artery occlusion and reperfusion in rats. Their data are negative and in fact demonstrate an exacerbation of the damage after focal ischemia in the hypertonic saline–treated animals.
Negative data are often the subject of even greater scrutiny than those that are positive. However, the methods used by this group are above reproach. The rat filament MCAO model was appropriately chosen, as it is, at the moment, the small-animal model that most closely mimics the clinical scenario of thrombolytic-treated human stroke. Readers not familiar with this model should note that the authors’ use of laser Doppler flowmetry of the ipsilateral hemisphere is an effective method for confirming occlusion of the MCA and for knowing when and if the filament has caused perforation of an intracerebral artery. Studies in which this technique has not been used are more prone to methodological error.
The authors also expressed some surprise over the lack of histological effects of mannitol administration. Indeed, one would expect some benefit of mannitol during ischemia by virtue of its effects on blood viscosity, and such benefits have been shown in animals in terms of outcome and brain water content.R1 R2 R3 An important difference in the current study is the use of mannitol as a continuous infusion, whereas bolus infusion has been the method used in most experimental studies and for patients. Mannitol should not be considered a potent neuroprotective agent during ischemia, and the negative data suggest to me more a reflection of honest data reporting than a problem with the model.
Given that hypertonic saline may exacerbate damage in ischemia/reperfusion, clinicians should avoid such treatment in stroke patients outside the context of a controlled and carefully scrutinized clinical trial.
Received February 23, 2000; revision received April 5, 2000; accepted April 26, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferencesIntroduction References |
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1. Little JR. Modification of acute focal ischemia by treatment with mannitol. Stroke.. 1978;9:4–9.
2. Muizelaar JP, Wei EP, Kontos HA, Becker DP. Mannitol causes compensatory cerebral vasoconstriction and vasodilation in response to blood viscosity changes. J Neurosurg.. 1983;59:822–828.[Medline] [Order article via Infotrieve]
3. Paczynski RP, He YY, Diringer MN, Hsu CY. Multiple-dose mannitol reduces brain water content in a rat model of cortical infarction. Stroke.. 1997;28:1437–1443.
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