(Stroke. 1999;30:1279-1285.)
© 1999 American Heart Association, Inc.
Original Contributions |
From the Departments of Anesthesiology/Critical Care Medicine (T.J.T., A.B., R.J.T., P.D.H.), Neurology (A.B., V.L.D., T.M.D), and Neuroscience (V.L.D., T.M.D.), Johns Hopkins University School of Medicine, Baltimore, Md.
Correspondence to Patricia D. Hurn, PhD, Department of Anesthesiology and Critical Care Medicine, Blalock 1404, Johns Hopkins Hospital, 600 N Wolfe St, Baltimore, MD 21287. E-mail phurn{at}welchlink.welch.jhu.edu
| Abstract |
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MethodsUnder controlled conditions of normoxia, normocarbia, and normothermia, halothane-anesthetized male Wistar rats were subjected to 2 hours of MCAO by the intraluminal occlusion technique in a blinded, randomized experimental trial. Ipsilateral parietal cortical laser-Doppler flowmetry was monitored throughout ischemia. Animals were randomly assigned to 4 pretreatment groups: intravenous FK506 0.3 mg/kg or 1.0 mg/kg, vehicle (cremaphor), or an equivalent volume of saline administered 30 minutes before MCAO. Infarction volume was assessed by a triphenyltetrazolium chloride staining at 22 hours of reperfusion. In separate experiments, microdialysis probes were placed bilaterally into the striatum. Rats were perfused with artificial cerebrospinal fluid containing 3 µmol/L [14C]- L-arginine for 3 hours and then subjected to 2 hours of right MCAO. Intravenous 0.3 mg/kg FK506 or cremaphor was given 30 minutes before right MCAO. Right-left differences between [14C]-L-citrulline in the effluent were assumed to reflect differences in NO production.
ResultsAll values are mean±SE. FK506 at 0.3 mg/kg reduced infarction volume in cortex: 40±12 mm3 compared with saline (109±15 mm3) and cremaphor vehicle (148±23) (P<0.05). Striatal infarction was also reduced by low-dose FK506: 16±4 mm3 versus 36±4 mm3 and 34±4 mm3 in saline and vehicle groups, respectively (P<0.05). High-dose treatment reduced infarction volume in cortex (61±14 mm3, P<0.05 from saline and vehicle groups) and in striatum (22±5 mm3, P<0.05 from saline and vehicle groups). [14C]-L-citrulline recovery via microdialysis was markedly enhanced in ischemic compared with nonischemic striatum. However, ischemia-evoked [14C]-L-citrulline recovery was not different in FK506-treated rats compared with vehicle-treated animals.
ConclusionsThese data demonstrate that FK506 provides robust neuroprotection against transient focal cerebral ischemia in the rat. The mechanism of protection in vivo is not through attenuation of ischemia-evoked NO production during MCAO and early reperfusion.
Key Words: immunosuppressive agents microdialysis nitric oxide stroke rats
| Introduction |
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| Materials and Methods |
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NOS Activity via Microdialysis
Dialysis probes used in these studies were made as described
previously.17 18 Briefly, each was a single hollow
dialysis fiber sealed with epoxy at one end; membrane diameter was
300 µm, with molecular mass cutoff of 5 kDa. Two hollow silica
perfusion tubes were inserted into the dialysis fiber so that their
ends were 3 mm apart. The distance between the tips constitutes
the effective dialyzing area of the cannula.19 Recovery
across the dialysis probe is approximately 15% to 20% in vitro.
In experiments with MCAO accompanied by microdialysis, the animal was initially anesthetized with pentobarbital (60 mg/kg IP), then placed in a Kopf stereotaxic frame for cannula placement in striatum (0.5 mm anterior and 2.5 mm lateral to the bregma; depth, 6 mm from the dura).21 A 2x2-mm area of the skull was removed with a variable-speed drill. A thin layer of bone was left intact and removed with forceps under microscopic observation to minimize trauma to the cortex. Cannulas were advanced to predetermined coordinates with a micromanipulator and fixed in position with dental cement. The animal was then removed from the stereotaxic apparatus and allowed a postsurgical equilibration period during which pentobarbital was discontinued and anesthesia maintained for the remainder of the experiment with 1% to 2% halothane. Starting 1 hour after insertion, the cannulas were perfused at 1 µL/min with artificial cerebrospinal fluid (aCSF): NaCl 131.8, NaHCO3 24.6, CaCl2 2.0, KCl 3.0, MgCl2 0.65, urea 6.7, and dextrose 3.7 mmol/L. The aCSF was filtered, warmed to 37°C, and bubbled with 95% N2/5% CO2 until O2 and CO2 tensions were similar to aCSF and brain tissue.20 Microdialysis effluents were collected over 20-minute periods throughout the ischemic period and compared on a paired basis (right versus left striatum within each animal).
Striatal NO production was measured as previously described,17 18 19 based on modifications of an isotopic conversion assay which quantifies conversion of arginine to equimolar citrulline and NO via NOS.22 Therefore, right-left differences between [14C]-L-citrulline in the effluent presumably reflect differences in local NO production. During continuous infusion of aCSF containing 3 µmol/L [14C]-L-arginine, 20 µL effluent dialysate samples were collected during 20-minute periods and assayed for [14C]-L-citrulline content. Samples were diluted with 200 µL water and poured over 0.5 mL resin columns (AG-50WX8, Na+ form, pH 7.0). Columns were washed with 2 mL buffer containing 30 mmol/L HEPES (pH 5.2), 3 mmol/L EDTA, and 1 mL water. Radioactivity of flow through the column was quantified by liquid scintillation spectroscopy. To determine resin efficiency of arginine trapping, 20 µL aCSF containing 3 µmol/L [14C]-L-arginine (not used for dialysis) was diluted in 200 µL water, poured over a column, and washed as above. Specific activity was corrected for counting efficiency and background activity and expressed as femtomoles per minute of perfusion. As an internal control, 100 µL aCSF not used for dialysis was directly assayed for activity to ensure that consistent concentrations of [14C]-L-arginine were added to the aCSF.
Experimental Protocols
Validation of In Vivo NOS Activity Assay During MCAO
In initial experiments, we evaluated the capability of the
microdialysis loading/sampling method to track changes in NO
production over time during MCAO. Three groups of rats received
initial [14C]-L-arginine
pre-loading via dialysis probe for 3 hours, followed by either MCAO or
sham conditions and measurements of effluents over an additional 3
hours. The [14C]-L-arginine
perfusion was continued throughout the experimental period to assure
adequate cell substrate availability. In 1 group (MCAO, n=8), occlusion
was induced and effluents were collected at 20-minute intervals. In a
second group (sham, n=7), animals were treated with neck surgery
without introduction of the intraluminal suture. A final group received
1 mmol/L L-nitroarginine (L-NNA, n=7) via
microdialysis probe into the right striatum 1 hour before right MCAO.
The inhibitor concentration was selected on the basis of
our previous findings that 1 mmol/L L-NNA reduces NMDA- and
AMPA-stimulated [14C]-L-citrulline
production.17 The right-left effluent
concentrations of
[14C]-L-citrulline were compared on
a paired basis in each animal. Appropriate probe placement relative to
the area of TTC-determined infarction was confirmed in all cases.
FK506 and MCAO
To demonstrate the efficacy of FK506 in reducing
histological injury after MCAO with reperfusion, rats
were assigned in blinded fashion to 1 of 4 intravenous
treatment groups: (1) 0.3 mg/kg FK506 (n=11), (2) 1.0 mg/kg FK506
(n=10), (3) cremaphor (drug vehicle, equivalent volume) (n=10), and (4)
saline (n=11). Dosages were chosen based on efficacy of FK506 in other
ischemic models.1 2 3 4 5 Each infusion was given 30
minutes before MCAO over a 15-minute duration. Preliminary experiments
demonstrated that FK506 at 1 mg/kg or vehicle (n=7 per group) did not
alter mean arterial pressure or LDF immediately on infusion
or for 120 minutes after infusion (data not shown). In a separate
cohort of animals, the effect of FK506 on ischemia-evoked NO
production and on persistent NO production in
reperfusion was evaluated. Rats were randomly assigned to receive
either intravenous infusion of 0.3 mg/kg FK506 (n=5) or
cremaphor vehicle (n=4) 30 minutes before right MCAO. As before, LDF
was evaluated throughout MCAO to determine whether there were
differences in the reduction of LDF signal during MCAO among treatment
groups. Microdialysis effluents were collected over 20-minute intervals
throughout MCAO and 3 hours of reperfusion. All surgical
instrumentation was removed, and the animals recovered. Rats were
killed at 22 hours of reperfusion for confirmation of probe placement
within the area of TTC-determined infarction.
Materials
[14C]-L-Arginine (317
mCi/mmol) was obtained from Amersham, L-NNA from Sigma Chemicals, and
cremaphor from BASF. FK506 was a generous gift from Fujisawa
Pharmaceuticals.
Statistical Analysis
Within each group, the citrulline composition of the
effluent was analyzed by 2-way ANOVA: effluent citrulline from
the 2 striata (ischemic versus nonischemic) were
analyzed as 1 within-subjects factor, and the 20-minute
collections as a second within-subjects factor. If the overall effect
of treatment or treatmentxtime interaction was significant,
comparisons of mean values between the 2 striata at individual time
points were made by orthogonal contrasts. Infarction volume and
physiological data were analyzed by 1-way
ANOVA. A value of P<0.05 was considered significant. Data
are presented as mean±SEM.
| Results |
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Effect of FK506 on Infarction Volume
Physiological variables were held
within normal values, and there were no differences among treatment
groups before MCAO, during MCAO, or during early reperfusion. The
ipsilateral LDF signal during MCAO decreased rapidly to approximately
25% of baseline values and remained at this level for the duration of
occlusion in all groups (Figure 3
). On
reperfusion, LDF returned toward baseline by 15 minutes. If
intraischemic LDF was not reduced to <40% of baseline signal,
the animal was excluded from the study. Three animals were excluded in
this manner in both the cremaphor and saline treatment groups; 2
animals were excluded in each of the FK-506 treatment groups. Averaged
LDF over the ischemic period was not different among groups:
saline, 20±1% of baseline; cremaphor, 27±1%; FK506 0.3 mg/kg,
24±1%; and FK506 1 mg/kg, 20±1%. Cortical infarction volume was
reduced by FK506 at both the 0.3- and 1-mg/kg doses (40±12
mm3 and 61±14 mm3,
respectively) compared with cremaphor vehicle (148±23
mm3, P
0.05) or saline treatment
(109±15 mm3, P
0.05) (Figure 4
). However, infarction size was not
different between drug-treated groups, indicating that both doses
provided equivalent neuroprotection in cortex. In the striatum,
infarction volume was again reduced by both low- and high-dose FK506
treatment (16±4 mm3 and 22±5
mm3, respectively) compared with cremaphor
vehicle (34±4 mm3, P
0.05) or
saline treatment (36±4 mm3,
P
0.05) (Figure 4
).
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Mortality as assessed by survival to 22 hours' reperfusion was as follows for the treatment groups: cremaphor (4/17 animals, or 24%), saline (4/18, 22%), FK506 0.3 mg/kg (5/18, 28%) and FK506 1 mg/kg (5/21, 24%).
Effect of FK506 on NO Production During MCAO and
Reperfusion
Arterial blood pressure, blood gases, and rectal
temperature were within normal physiological range
and not different in FK506- or vehicle-treated animals. As in the
previous protocol, reduction of ischemic LDF was not different
in drug- and vehicle-treated groups. On withdrawal of the monofilament,
LDF restored rapidly to baseline values within 15 minutes in both
groups. As expected, there was a time-dependent increase in labeled
citrulline recovery in the striatum bilaterally in all animals (Figure 5
). FK506 treatment did not significantly
affect labeled citrulline recovery in either ischemic or
nonischemic striatum. Compared with nonischemic
striatum, labeled citrulline recovery increased over the 2 hours of
MCAO and continued to increase throughout reperfusion in both FK506
(Figure 5
, top) or cremaphor vehicletreated (bottom)
animals.
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| Discussion |
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FK506 has been previously shown to have significant neuroprotective properties. In vitro work in primary cortical culture demonstrated that the agent strongly reduces NMDA, but not non-NMDA, neurotoxicity.11 FK506 also proved to be protective in several models of global cerebral ischemia.3 4 5 Further, FK506 0.1 mg/kg reduced tissue infarction after localized, perivascular endothelin administration and consequent loss of regional perfusion, even when administered 60 minutes after the insult.1 Using a standard experimental model of reversible MCAO, we also observed that FK506 pretreatment reduces injury over a range of doses (0.3 to 1 mg/kg).
Because FK506 has numerous mechanisms of action within neurons and inflammatory cells, its protective action is likely multifactorial. FK506 is known to modify immunologic reactions by suppression of interleukin-2 gene expression23 and inhibit protein phosphorylation.10 FK506, complexed with FKBP, inhibits the function of calcineurin thereby inhibiting Ca2+-dependent dephosphorylation and activation of neuronal NOS.10 We hypothesized that FK506 treatment could suppress the activation of neuronal NOS, which ordinarily occurs subsequent to ischemic depolarization, NMDA receptor activation, and resulting rise in intracellular cation flux. If FK506 enhanced NOS phosphorylation in vivo, thus inhibiting the enzyme's catalytic activity during ischemia and early reperfusion, decreased arginine metabolism would be quantifiable as lower labeled citrulline recovery from drug- versus vehicle-treated tissue. However, the rise in striatal citrulline recovery (in femtomoles per minute) during MCAO in untreated animals was also observed with FK506 pretreatment at a dose that reduced striatal infarction. Therefore, the drug had little effect on ischemic NOS catalytic activity, at least within this brain region. It must be emphasized that our dynamic but indirect measurement of NOS activity (arginine-to-citrulline conversion) measures total NOS activity in the dialysate without distinction among contributions from various enzyme isoforms. Because we measured NOS activity during severe focal ischemia up to 6 hours after MCAO, we think it likely that neuronal and endothelial sources contribute most greatly to our signal. The finding that early, ischemia-induced NOS activation is not altered by FK506 treatment is consistent with the observation that the compound protects against NMDA toxicity in cortical neurons cultured from nNOS-deficient mice to the same extent as in wild-type cultures.24
FK506 has also been reported to suppress inducible NOS (iNOS) production in cultured macrophages,25 and ischemia-induced iNOS expression is elevated in reactive astrocytes in some brain regions for days after even brief ischemia.26 Although we detect uniform increases in labeled citrulline recovery within 1 hour of MCAO, relatively little iNOS is elaborated from inflammatory cells by very early reperfusion. Therefore, our measurements of ischemia-evoked NOS production do not address iNOS-mediated mechanisms that may be activated beyond the first 3 hours of reperfusion. Therefore, iNOS could be involved in FK506-induced neuroprotection in the maturing lesion.
Several alternative neuroprotective mechanisms must be considered for the ability of FK506 to salvage tissue during vascular occlusion. Nonimmunosuppressive properties of FK506 may be relevant. FK506 analogs without immunosuppressive activity promote neurite growth and enhance morphological and functional recovery after peripheral nerve injury, as does FK506.27 Glutamate toxicity and Ca2+ influxinitiated intracellular events have been well studied and likely lead to protein kinase redistribution within cell organelles and enhanced tyrosine phosphorylation of synaptic proteins.28 29 Because FK506 inhibits the phosphatase calcineurin, the drug could restore fundamental imbalances in neuronal protein phosphorylation during reperfusion or play a role in Ca2+-triggered apoptosis.30 Intracellular calcium homeostasis may be altered through FK506-sensitive ryanodine and ionositol 1,4,5-triphosphate receptorassociated calcium ion channels within endoplasmic reticulum and cell membranes.31 32 Finally, oxidative stress is known to result in impaired mitochondrial function; the pathophysiology of the injury is thought to involve the formation of inner mitochondrial membrane pores through which protons and possibly calcium ions are released.33 Cyclosporin A, a compound related to FK506, has been shown to prevent or impede pore formation under conditions of oxidative stress accompanied by high-calcium load.34 Thus, the importance of FK506 in protection from free radicalinduced mitochondrial transition states should also be considered.35 36 37 FK506 at doses similar to those of the present study prevents secondary deterioration of mitochondrial function in penumbral areas after MCAO and improves postischemic respiratory rates in vitro.38
As confirmed by postmortem dissection, all microdialysis probe tips were localized in caudate nucleus. The choice of striatum as a measurement site is based on its known vulnerability to ischemia and excitotoxic glutamatergic injury.37 38 Using well-characterized antibodies, we have confirmed an abundance of neuronal and endothelial NOS isoforms in striatum and assume abundant NO synthesis in the area.39 Utilizing microdialysis capture of labeled citrulline, we previously demonstrated17 19 40 that NMDA and AMPA stimulate NO production in rat hippocampus and lamb neocortex. We have now shown that MCAO increases citrulline recovery in striatum and confirmed that recovery is attenuated by the NOS inhibitor L-NNA. Therefore, ischemia-evoked increases in radiolabeled citrulline recovery are assumed to reflect increased NO production. However, it must be emphasized that citrulline recovery is actually an indirect marker of NO production in vivo because of complex compartmental kinetics. For example, the time-dependent increase in labeled citrulline recovery under control conditions likely reflect these kinetics rather than increased NO synthesis over time.17 19 Bilateral microdialysis perfusion was used in a paired experimental design to reduce interanimal variability.
Some differences in absolute levels of basal citrulline recovery
are evident between different groups receiving similar interventions.
Some of this variability may be due to differences in the efficiency of
arginine trapping by the Dowex column. The relative recovery across the
dialysis membrane is dependent on the diffusion coefficient, which
theoretically can be altered by ischemia and intracellular
edema with decreased extracellular space and concentration of an
endogenous substance. However, perfusion with L-NNA caused
rapid and sustained suppression of labeled citrulline recovery during
ischemia in our experimental system (Figure 2
). This
finding argues against the "pooling" of citrulline in the
extracellular space with artifactually concentrated recovery values. We
have quantified the radial spread of labeled arginine from the probe
with autoradiography in nonischemic rat
brain.18 Our results indicated that the labeled compound
spreads at a maximum diameter of 3 mm by 1 hour of perfusion at 1
µL/min. After 5 hours of perfusion, there is no further spread of
label, suggesting that cellular uptake limits diffusion away from the
probe site. Others41 have shown that labeled sucrose
spreads cylindrically over a 1-mm-diameter by 14 minutes of dialysis
perfusion. Finally, although probe insertion can cause disruption of
the blood-brain barrier and an increase in extracellular space
immediately around the probe,41 the volume of tissue
sampled with our technique likely extends well beyond the injured
volume.
In conclusion, this study demonstrates that FK506 pretreatment affords significant neuroprotection in the cortex and the caudoputamen complex in transient focal cerebral ischemia and reperfusion in the rat. Ischemia-evoked NO production during occlusion and acute reperfusion is not attenuated by FK506 at doses that provide neuroprotection. Although this neuroprotection may well involve several mechanisms, the drug does not act via inhibition of acute NO production and consequent early NO-mediated neurotoxicity.
| Acknowledgments |
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| Footnotes |
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Received January 4, 1999; revision received February 22, 1999; accepted March 18, 1999.
| References |
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1-receptor ligand 4-phenyl-1-(4-Phenyl)
piperidine (PPBP) modulates basal and N-methyl-D-aspartate-evoked
nitric oxide production in vivo. Stroke. 1998;29:24042411.Departments of Neurosurgery, Neurology, and Neurological Sciences, Stanford University, Palo Alto, California
| Introduction |
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In addition to the nature of the extremely carefully done and unique observation of the investigation, this study may raise several points that deserve further comment:
(1) The mode of neuroprotection of FK506 in focal cerebral ischemia is likely to be at a cellular level and is unlikely to be related to the changes in cerebral blood flow.
(2) The mechanism underlying the neuroprotection afforded by FK506 on transient focal cerebral ischemia is unknown at present, since it appears that NOS1 inhibition is an unlikely event during the early phase of transient focal cerebral ischemia. It will be of interest to investigate whether this drug will also affect the apoptotic pathway as well as the delayed onset of inducible NOS (NOS2)3 that are known to contribute to the ischemic infarction.
(3) One possible mechanism underlying the neuroprotective action of FK506 and other immunosuppressants, such as cyclosporin A, in brain injuries is the regulation of the mitochondrial permeability transition pore.4 5 In view of recent euphoria about the mitochondrial role as a trigger in necrosis and apoptosis in acute stroke and in neurodegeneration,6 7 pharmacological and therapeutic strategies are warranted to target the mitochondrial bioenergetic function and oxidative stress with immunosuppressants in stroke.
Received January 4, 1999; revision received February 22, 1999; accepted March 18, 1999.
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