(Stroke. 2000;31:1990.)
© 2000 American Heart Association, Inc.
Original Contributions |
Superoxide Generation Links Nociceptin/Orphanin FQ (NOC/oFQ) Release to Impaired N-Methyl-D-Aspartate Cerebrovasodilation After Brain Injury
From the Departments of Anesthesia and Pharmacology, University of Pennsylvania, Philadelphia, Pa.
Correspondence to William M. Armstead, PhD, Department of Anesthesia, University of Pennsylvania, 3400 Spruce St, Philadelphia, PA 19104. E-mail armsteaw{at}mail.med.upenn.edu
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Abstract |
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Background and Purpose—Although activation of the N-methyl-D-aspartate (NMDA) receptor is thought to contribute to altered cerebrovascular regulation after traumatic brain injury, the effects of such injury on the vascular response to NMDA itself has been less well appreciated. The newly described opioid nociceptin/orphanin FQ (NOC/oFQ) elicits pial artery dilation, at least in part, in a prostaglandin-dependent manner and is released into cerebrospinal fluid after fluid percussion brain injury (FPI). Generation of superoxide anion (O2-) occurs after FPI, and a byproduct of cyclooxygenase metabolism is the generation of O2-. This study was designed to determine whether NOC/oFQ generates O2-, which in turn could link NOC/oFQ release to impaired NMDA-induced pial artery dilation after FPI.
Methods—Injury of moderate severity (1.9 to 2.1 atm) was produced by the lateral FPI technique in anesthetized newborn pigs equipped with a closed cranial window. Superoxide dismutase–inhibitable nitroblue tetrazolium (NBT) reduction was determined as an index of O2- generation.
Results—Under non–brain injury conditions, topical NOC/oFQ (10-10 mol/L, the concentration present in cerebrospinal fluid after FPI) increased superoxide dismutase–inhibitable NBT reduction from 1±1 to 20±3 pmol/mm2 but had no effect itself on pial artery diameter. Indomethacin (5 mg/kg IV) blunted such NBT reduction (1±1 to 6±2 pmol/mm2), whereas the NOC/oFQ receptor antagonist [F/G] NOC/oFQ (1-13) NH2 (10-6 mol/L) blocked NBT reduction. [F/G] NOC/oFQ (1-13) NH2 and indomethacin also blunted the NBT reduction observed after FPI (1±1 to 15±1 versus 1±1 to 4±1 versus 1±1 to 4±1 pmol/mm2 for sham, NOC/oFQ antagonist, and indomethacin-treated animals, respectively). NMDA (10-8 and 10-6 mol/L)–induced pial artery dilation was reversed to vasoconstriction after FPI, and [F/G] NOC/oFQ (1-13) NH2 attenuated such vasoconstriction (sham 9±1% and 16±1% versus FPI -7±1% and -12±1% versus FPI–[F/G] NOC/oFQ (1-13) NH2–pretreated animals -2±1% and -3±1%). Indomethacin and the free radical scavengers polyethylene glycol superoxide dismutase and catalase also partially restored NMDA-induced vasodilation.
Conclusions—These data show that NOC/oFQ, in concentrations present in cerebrospinal fluid after FPI, increased O2- production in a cyclooxygenase-dependent manner and contributes to such production after FPI. These data show that NOC/oFQ contributes to impaired NMDA-induced pial artery dilation after FPI. Therefore, these data suggest that cyclooxygenase-dependent O2- generation links NOC/oFQ release to impaired NMDA-induced cerebrovasodilation after brain injury.
Key Words: cerebral circulation • excitatory amino acids • newborn • opioids • oxygen free radicals • pigs
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Introduction |
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Traumatic brain injury is a leading cause of morbidity and mortality in children.1 Decreased cerebral blood flow has been described in children after brain injury and may contribute to the severity of sequelae.2 Fluid percussion injury (FPI) in animals has been suggested to model human concussive trauma.3 In the newborn pig, FPI results in pial artery vasoconstriction and reductions in cerebral blood flow within 10 minutes of injury.4 Additionally, neurohumoral control of the cerebral circulation is altered after brain injury.5 6 Because the oxygen free radical scavengers polyethylene glycol superoxide dismutase (PEG-SOD) and catalase have been observed to partially restore impaired vasodilator responses to several stimuli after FPI,7 oxygen free radicals such as superoxide anion (O2-) appear to be involved in FPI-associated hemodynamic sequelae. Additionally, cortical periarachnoid cerebrospinal fluid (CSF) opioid concentrations have been shown to increase after FPI in newborn pigs and appear to contribute to altered cerebral hemodynamics after the insult.8 Opioids themselves are thought to be important contributors to cerebrovascular regulation in the newborn.9
Glutamate is an important excitatory amino acid transmitter in the
brain. It can bind to any of 3 different inotropic receptor
subtypes named after specific synthetic analogues:
N-methyl-D-aspartate (NMDA),
kainate, and AMPA (
-amino-3-hydroxy-5-methyl-4-isoxazole
priopionic acid). Activation of NMDA receptors has been observed to
elicit cerebrovascular dilation and may represent one of the
mechanisms for the coupling of local cerebral metabolism to
blood flow.10 NMDA-induced pial artery dilation has
been observed to be attenuated after hypoxia and
ischemia/reperfusion in the piglet.11 12 13
Mechanisms for such altered dilation to NMDA after such an insult have
been less well characterized. Additionally, although activation of the
NMDA receptor is thought to contribute to altered cerebrovascular
regulation after traumatic brain injury,14 the effects of
such injury on the vascular action of NMDA have been less well
appreciated.
During the last 5 years, several groups have isolated and cloned a new
G protein–coupled receptor that showed high homology with opioid
receptors.15 16 17 The peptide ligand for this receptor does
not bind to classic opioid receptors (µ, 
, and 
) and was named
orphanin FQ by Reinscheid et al18 because its sequence
begins with phenylalanine (F) and ends with a glutamine (Q). The same
peptide was called nociceptin by Meunier et al19 because
it increased the reactivity to pain in animals in contrast to the
analgesic effects of opioid drugs. Recently, nociceptin/orphanin FQ
(NOC/oFQ) has been observed to elicit pial artery vasodilation in the
newborn pig at least in part by a prostaglandin-dependent
mechanism.20 21 However, little is known about the role of
NOC/oFQ in the physiological or
pathophysiological control of cerebral
hemodynamics. Although somewhat
controversial,22 23 the identification of an NOC/oFQ
receptor antagonist, [F/G] NOC/oFQ (1-13)
NH2, and its demonstrated selectivity for NOC/oFQ
in the piglet cerebral circulation20 have resulted in the
development of an avenue for the characterization of the functional
significance of this newly described opioid. Recent studies have shown
that the CSF concentration of NOC/oFQ is elevated after FPI (W.M.A.,
unpublished observations, 2000). Interestingly, it has also been
observed that NOC/oFQ can both inhibit the release of glutamate from
rat cerebrocortical slices and inhibit glutamatergic transmission in
the rat spinal cord, as well as have its own signaling modulated by
NMDA.24 25 26 Finally, because a byproduct of
cyclooxygenase metabolism is the
generation of O2-, and NOC/oFQ
elicits dilation in a prostaglandin-dependent manner, it is
uncertain whether NOC/oFQ will cause release of
O2-.
The present study, therefore, was designed to (1) determine whether NOC/oFQ, in a concentration present in CSF after FPI, increased superoxide dismutase (SOD)–inhibitable nitroblue tetrazolium (NBT) reduction, an index of O2- production; (2) determine whether such NOC/oFQ-mediated NBT reduction was dependent on the cyclooxygenase pathway; (3) determine whether NOC/oFQ and the cyclooxygenase pathway contribute to O2- production after FPI; and (4) characterize the relationship between NOC/oFQ, the cyclooxygenase pathway, and O2- generation in determining the effects of FPI on NMDA-induced pial artery dilation.
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Materials and Methods |
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Newborn (1 to 5 days old; weight 1.3 to 2.1 kg) pigs of either sex were used in these experiments. All protocols were approved by the Institutional Animal Care and Use Committee. Animals were sedated with isoflurane (1 to 2 minimum alveolar concentration). Anesthesia was maintained with
-chloralose (30 to 50
mg/kg, supplemented with 5 mg ·
kg-1 ·
h-1 IV). A catheter was
inserted into a femoral artery to monitor blood pressure and to sample
for blood gas tensions and pH. Drugs to maintain anesthesia
were administered through a second catheter placed in a femoral vein.
The trachea was cannulated, and the animals were mechanically
ventilated with room air. A heating pad was used to maintain the
animals at 37°C to 39°C. A cranial window was placed in the parietal skull of these anesthetized animals. This window consisted of 3 parts: a stainless steel ring, a circular glass coverslip, and 3 ports consisting of 17-gauge hypodermic needles attached to 3 precut holes in the stainless steel ring. For placement, the dura was cut and retracted over the cut bone edge. The cranial window was placed in the opening and cemented in place with dental acrylic. The volume under the window was filled with a solution, similar to CSF, of the following composition (in mmol/L): 3.0 KCl, 1.5 MgC12, 1.5 CaCI2, 132 NaCl, 6.6 urea, 3.7 dextrose, and 24.6 NaHCO3. This artificial CSF was warmed to 37°C and had the following chemistry: pH 7.33, PCO2 46 mm Hg, and PO2 43 mm Hg, which was similar to that of endogenous CSF. Pial arterial vessels were observed with a dissecting microscope, a television camera mounted on the microscope, and a video output screen. Vascular diameter was measured with a video microscaler.
Methods for brain FPI have been described previously.27 A device designed by the Medical College of Virginia was used. A small opening was made in the parietal skull contralateral to the cranial window. A metal shaft was sealed into the opening on top of intact dura. This shaft was connected to the transducer housing, which was in turn connected to the fluid percussion device. The device itself consisted of an acrylic plastic cylindrical reservoir 60 cm long, 4.5 cm in diameter, and 0.5 cm thick. One end of the device was connected to the transducer housing, whereas the other end had an acrylic plastic piston mounted on O rings. The exposed end of the piston was covered with a rubber pad. The entire system was filled with 0.9% saline. The percussion device was supported by 2 brackets mounted on a platform. FPI was induced by striking the piston with a 4.8-kg pendulum. The intensity of the blow (usually 1.9 to 2.3 atm with a constant duration of 19 to 23 ms) was controlled by varying the height from which the pendulum was allowed to fall. The pressure pulse of the blow was recorded on a storage oscilloscope triggered photoelectrically by the fall of the pendulum. The amplitude of the pressure pulse was used to determine the intensity of the injury.
Protocol
Two types of pial arterial vessels, small arteries
(resting diameter 120 to 160 µm) and arterioles (resting
diameter 50 to 70 µm), were examined to determine whether
segmental differences in the effects of FPI on NMDA and glutamate pial
dilation could be identified. Pial arterial vessel diameter
was determined every minute for a l-minute exposure period after
infusion onto the exposed parietal cortex of artificial CSF before NMDA
and after the topical application of NMDA. Typically, 2 to 3 mL of CSF
was flushed through the window over a 30-second period, and excess CSF
was allowed to run off through one of the needle ports. For
sample collection, 300 µL of the total cranial window volume of 500
µL was collected by slowly infusing CSF into one side of the window
and allowing the CSF to drip freely into a collection tube on the
opposite side.
Eleven major types of experiments were performed: (1) generation of O2- with NOC/oFQ (n=7); (2) generation of O2- with NOC/oFQ in the presence of indomethacin (n=7); (3) generation of O2- with NOC/oFQ in the presence of the NOC/oFQ receptor antagonist [F/G] NOC/oFQ (1-13) NH2 (n=7); (4) generation of O2- with FPI (n=7); (5) generation of O2- with FPI in indomethacin-pretreated animals (n=7); (6) generation of O2- with FPI in [F/G] NOC/oFQ (1-13) NH2–pretreated animals (n=7); (7) vascular responses to agonists in the absence of FPI (sham control) (n=7); (8) vascular responses to agonists after FPI (n=7); (9) vascular responses to agonists after FPI in indomethacin-pretreated animals (n=7); (10) vascular responses to agonists after FPI in [F/G] NOC/oFQ (1-13) NH2–pretreated animals (n=7); and (11) vascular responses with FPI in PEG-SOD and catalase (SODCAT)–pretreated animals (n=7).
In the first 3 series of experiments designed to investigate generation of O2-, NOC/oFQ (10-10 mol/L, Phoenix Pharmaceuticals, Inc) was applied to the cerebral cortex for 20 minutes in either the absence or presence of indomethacin (5 mg/kg IV) or [F/G] NOC/oFQ (1-13) NH2 (10-6 mol/L, Phoenix). In the next 3 series of experiments, generation of O2- 1 hour after FPI was investigated in the absence and presence of indomethacin or [F/G] NOC/oFQ (1-13) NH2. In these experiments, indomethacin or [F/G] NOC/oFQ (1-13) NH2 was administered 20 minutes before FPI. The NOC/oFQ antagonist was kept in constant contact with the cerebral cortex for the duration of the experiment. Because the technique for measurement of O2- generation (see below) involves placement of detection solutions on the cerebral cortex for 20 minutes, such measurement in fact reflects O2- generation during the first 20-minute period 1 hour after FPI.
In the vascular experiments, responses of arterial vessels to NMDA and glutamate (10-8 or 10-6 mol/L, Sigma) were obtained before and 1 hour after FPI either in the absence or presence of indomethacin, [F/G] NOC/oFQ (1-13) NH2, and SODCAT (1000 U/kg and 10 000 U/kg of PEGSOD and catalase, respectively).
O-2 Analysis
SOD-inhibitable NBT reduction was determined as an index of
O2- generation, as previously
described.28 29 Such reduction was determined by placing
NBT (Sigma, 2.4 mmol/L) dissolved in artificial CSF under 1 window
and NBT (2.5 mmol/L) and SOD (Sigma, 60 U/mL) in artificial CSF
under the other window 1 hour after FPI. Because such solutions
remained on the surface for 20 minutes, data are quantified as
picomoles of NBT reduced for 20 minutes. Two windows were placed
contralateral to the adapter for induction of FPI for these
experiments.
NBT is water soluble and forms a yellow solution that is converted to nitroblue formazan, an insoluble purple precipitate, in the presence of reducing agents, eg, O2-. The SOD-inhibitable NBT reduction was determined by the difference in the quantities of nitroblue formazan precipitated on the brain surface under the 2 windows. Although NBT can be reduced by a variety of agents, SOD provides specificity for the assay. Slices of the brain surface 1 mm thick under each cranial window were obtained. The slices were minced and homogenized in 1N NaOH and 0.1% sodium dodecyl sulfate solution. The supernatant was discarded, and the pellet was resuspended in 3 mL of pyridine. The formazan was dissolved in the pyridine during heating at 80°C for 1 hour. Particulate matter was removed by a second centrifugation at 10 000g for 10 minutes. The concentration of nitroblue formazan in the supernatant was then determined spectrophotometrically at 515 nm. The nitroblue formazan on the side with NBT alone was analyzed against the background of the SOD-treated side. Freshly prepared calibration solutions were used with each set of samples and treated identically to the samples. Recovery of NBT averaged 88±4%.
NOC/oFQ Analysis
The CSF samples that were collected were acidified, rapidly
frozen, and stored at -20°C. Radioimmunoassay kits for NOC/o FQ are
commercially available (Phoenix). The radioimmunoassay uses
simultaneous addition of sample, rabbit anti-NOC/oFQ
antibody, and the 125I-labeled derivative of
NOC/oFQ. After an overnight incubation at 4°C, free NOC/oFQ was
separated from NOC/oFQ bound to antibody by the addition of goat
anti-rabbit IgG serum and normal rabbit serum. After being
centrifuged at 760g for 10 minutes, the supernatant
was decanted and the pellet counted with a gamma scintillation counter.
All samples and standards were assayed in duplicate. Data are
calculated as %B/Bo versus concentration, where
%B/Bo=[(average cpm of sample-average cpm of
nonspecific binding tube)/Bo]x100 and
Bo=(average cpm of total binding tube-average
cpm of nonspecific binding tube), where cpm is counts per
minute.
Statistical Analysis
Pial arteriolar diameter, systemic arterial
pressure, amount of NBT reduced, and NOC/oFQ levels were
analyzed by ANOVA for repeated measures or t test
where appropriate. If the value was significant, the data were then
analyzed by Fisher’s protected least significant difference
test. An -level of P<0.05 was considered significant in
all statistical tests. Values are represented as mean±SE
of the absolute values or percent changes from control values.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Influence of FPI on CSF NOC/oFQ Concentration
Experiments were initially designed to characterize the influence of FPI on CSF NOC/oFQ concentration. Cortical periarachnoid CSF NOC/oFQ was elevated from 70±3 to 444±56 pg/mL within 1 hour of FPI (n=7, 2.0±0.1 atm). On a molar basis, CSF NOC/oFQ was
10-11 mol/L under
resting sham control conditions and
10-10 mol/L at 1 hour
after FPI.
Role of the Cyclooxygenase Pathway in
NOC/oFQ-Induced O2- Generation During
Non–Brain Injury and Brain Injury Conditions
Topical application of NOC/oFQ
(10-10 mol/L, the
concentration present in CSF after FPI) to the cerebral cortical
surface of non–brain injured animals increased SOD-inhibitable NBT
reduction (Figure 1A
). Such NBT reduction
by NOC/oFQ was blunted by indomethacin (5 mg/kg IV) and
blocked by the NOC/oFQ receptor antagonist [F/G] NOC/oFQ
(1-13) NH2
(10-6 mol/L) (Figure 1A). Under brain injury conditions, SOD-inhibitable NBT
reduction was increased 1 hour after FPI (Figure 1B). Such
enhanced NBT reduction after FPI was blunted by both
indomethacin and [F/G] NOC/oFQ (1-13)
NH2 (Figure 1B).
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P<0.05 vs absence
of indomethacin or [F/G] NOC/oFQ (1-13)
NH2.
Role of NOC/oFQ, the Cyclooxygenase Pathway,
and O2- Generation in Impaired Excitatory
Amino Acid–Induced Pial Artery Dilation After Brain Injury
NMDA and glutamate
(10-8 and
10-6 mol/L) elicited
reproducible pial small-artery (120 to 160 µm) and arteriole (50
to 70 µm) vasodilation in sham control animals (data not shown).
However, NMDA- and glutamate-induced vasodilation was reversed to
vasoconstriction within 1 hour after FPI (2.0±0.1 atm) (Figures 2 and 3).
Such postinsult excitatory amino acid–induced vasoconstriction was
attenuated by [F/G] NOC/oFQ (1-13) NH2
(10-6 mol/L) (Figures 2 and 3). Both indomethacin and SODCAT
administration reversed that postinsult excitatory amino acid
vasoconstriction back to vasodilation, although responses were only
partially restored to control value (Figures 2 and 3).
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Effect of Indomethacin [F/G] NOC/oFQ (1-13)
NH2, SODCAT, and NOC/oFQ on Pial Artery Diameter
Indomethacin produced pial artery vasoconstriction
(143±5 versus 129±5 µm), whereas [F/G] NOC/oFQ (1-13)
NH2, SODCAT, and NOC/oFQ
(10-10 mol/L) had no
effect on pial artery diameter.
Blood Chemistry and Injury Intensity Level
The arterial blood gas and pH for the piglets at the
beginning and end of the experiments were no different between all the
experimental groups (eg, 7.45±0.02, 34±3, and 93±6 versus
7.44±0.02, 33±3, and 91±5 mm Hg for pH,
PO2, and
PO2). The injury intensity level was
2.0±0.1 atm.
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Discussion |
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Results of the present study show that under non–brain injury conditions, topical administration of NOC/oFQ, in a concentration observed in CSF after FPI, results in increased SOD-inhibitable NBT reduction by newborn pig brain, indicating that O2- was generated. Because indomethacin blunted such elevation in SOD-inhibitable NBT reduction by NOC/oFQ, these data indicate that activation of cyclooxygenase contributes to O2- generation by this opioid. Moreover, the putative NOC/oFQ antagonist, [F/G] NOC/oFQ (1-13) NH2, blocked such NBT reduction, indicating that NOC/oFQ generates O2- in a selective manner. Additionally, both indomethacin and [F/G] NOC/oFQ (1-13) NH2 blunted brain injury–induced elevated SOD-inhibitable NBT reduction. Previously, FPI has been observed to be associated with generation of O2- on the piglet cerebral cortical surface.28 In those studies, it was also observed that FPI caused the release of endothelin-1 (ET-1) into CSF, which in turn contributed to the generation of O2- after injury through activation of protein kinase C.28 29 Results of the present study extend the latter observations in that, taken as a whole, such results suggest that NOC/oFQ also contributes to the generation of O2- after injury through activation of cyclooxygenase. Because the concentration of NOC/oFQ observed in CSF after FPI (10-10 mol/L) did not have any effect on pial artery diameter, such O2- generation by NOC/oFQ appears independent of vascular contributory effect. However, concerns related to the accuracy of the NBT assay have been raised recently.30
The cerebrovascular consequences of free radical production are not fully understood. However, there is a significant amount of evidence that supports a role of oxygen radicals in brain injury. For example, brain injury in cats has been reported to cause the generation of superoxide for at least 1 hour after injury.31 In that study, the sustained dilation and abnormal responsiveness of pial arterioles observed after injury could be reversed by treatment with the free radical scavengers SOD and catalase.31 Oxygen radicals also have been shown to increase blood-brain barrier permeability,32 produce ultrastructural changes in pial vessel endothelium,32 and cause abnormal arteriolar reactivity.33 In addition, oxygen radical scavengers have been shown to improve vascular function and blood flow during focal ischemia in rats, which may account for the observed reductions in infarct size.34 Intracellular generation of superoxide or other species could alter structure and/or production of nucleotides, second messengers, receptors, and membranes, and the movement of superoxide out of the cell through anion channels could result in high concentrations of activated oxygen species at cell surfaces, including endothelium. Such oxygen species are thought to antagonize NO function and to contribute to altered cerebral hemodynamics after FPI in the piglet, because free radical scavengers partially restored decreased CSF cGMP concentration and decreased responses to NO-dependent dilator stimuli such as opioids.7
The role of the systemic pressor response after FPI in altered adult cerebral hemodynamics has been investigated. For example, it was hypothesized that acute elevations of blood pressure after injury in the adult result in the release and metabolism of arachidonic acid, which would generate oxygen free radicals, causing cerebral functional abnormalities.27 31 32 35 However, in contrast to studies performed in adult and juvenile animals, there was no acute elevation in blood pressure after FPI in the newborn pig.8 Because the elevation in systemic blood pressure was thought to be an absolute requirement for cerebral generation of free radicals after injury,27 31 32 35 the observed decrease in blood pressure was perplexing initially. More recent studies, however, have shown that the peptide ET-1 is released after FPI in the piglet.28 Topical administration of ET-1 in the same concentration observed after FPI resulted in the generation of substantial amounts of superoxide on the cerebral cortical surface.28 These results, therefore, link the cerebral release of the peptide to superoxide generation after FPI in the piglet. Interestingly, decreased opioid-induced dilation and associated CSF cGMP release after FPI were partially restored in animals pretreated with the ET-l antagonist BQ 123.28 These data, then, suggest that ET-1 contributes to altered cerebral hemodynamics after FPI at least in part through elevated superoxide production.
Because it had been observed previously that NOC/oFQ interacts with NMDA and glutamate in studies unrelated to vascular activity,24 25 26 additional studies were designed to investigate the relationship between NOC/oFQ, O2-, the cyclooxygenase pathway, and excitatory amino acid–induced vascular activity after FPI. Results of those studies show that NMDA- and glutamate-induced pial artery dilation was reversed to vasoconstriction after FPI. Such postinsult excitatory amino acid–induced vasoconstriction was attenuated by [F/G] NOC/oFQ (1-13) NH2, which indicates NOC/oFQ involvement in such altered vascular activity. However, both indomethacin and SODCAT administration reversed the postinsult excitatory amino acid vasoconstriction back to vasodilation, although responses were only partially restored to control values. Taken together, these data suggest that cyclooxygenase-dependent O2- generation links NOC/oFQ release to impaired NMDA- and glutamate-induced pial artery dilation after brain injury. However, because both indomethacin and SODCAT restored such excitatory amino acid dilation to a greater extent than [F/G] NOC/oFQ (1-13) NH2, those data further suggest that other yet to be determined factors also contribute to activation of cyclooxygenase, subsequent O2- generation, and final impairment of excitatory amino acid–induced vasodilation after FPI.
Global cerebral ischemia in a piglet model has previously been observed to result in attenuated pial artery dilation to NMDA.12 Results of the present study extend those of others in that the present study shows that glutamate- as well as NMDA-induced pial artery dilation is altered in a model of injury distinct from previously published reports. Additionally, others had not noted a reversal of NMDA-induced dilation to vasoconstriction after global cerebral ischemia.12
The mechanism by which NMDA-induced pial artery dilation is altered after global cerebral ischemia/reperfusion or combined hypoxia/ischemia/reperfusion is unclear at this time. Recent work by others suggests a role for oxygen free radicals and protein synthesis.11 12 36 In that proposed scenario, increased cyclooxygenase synthesis might account for the previously observed role for oxygen free radicals in ischemia/reperfusion-associated cerebrovascular derangement.36 Alternatively, the observed beneficial action of protein synthase inhibitors might relate to the blocking of the production of an unidentified regulatory protein that is rapidly overexpressed after ischemia.36 Interestingly, adenosine, which is released during hypoxia, has been observed to inhibit NMDA-induced pial artery dilation when coadministered with this excitatory amino acid,13 very similarly to that observed with NOC/oFQ. In those studies, it was suggested that adenosine might reduce calcium entry into nerve cells and activation of nitric oxide synthase by promoting hyperpolarization or by blocking N- and Q-type channels.13 It was further suggested that adenosine might reduce presynaptic glutamate release and thus suppress autoamplification of glutamate effects.13 Equally interesting, then, is the observation that NOC/oFQ can both inhibit the release of glutamate from rat cerebrocortical slices and inhibit glutamatergic transmission in the rat spinal cord as well as have its own signaling modulated by NMDA.24 25 26 More distal mechanisms by which NOC/oFQ-induced O2- generation might alter NMDA-induced pial artery dilation as observed in the present study are currently uncertain.
The experimental design of the present study did not allow for the identification of the cellular site of origin for NOC/oFQ detected in cortical periarachnoid CSF. Potential cellular sites of origin include neurons, glia, vascular smooth muscle, and endothelial cells.
Although glutamate is an excitatory neurotransmitter thought to be a predominant contributor to neurotoxicity associated with traumatic brain injury,14 little attention has been paid to the functional implications of vascular abnormalities to NMDA and glutamate after such an insult. In the present study, endogenous NOC/oFQ could either function to limit vascular responses to abnormally high glutamate levels after FPI or, alternatively, exacerbate them. It is speculated that the latter is more plausible. Recent data show that at higher concentrations than that studied presently, NOC/oFQ-induced vasodilation is reversed to vasoconstriction after FPI (W.M.A., unpublished observations, 2000). The preadministration of the NOC/oFQ antagonist [F/G] NOC/oFQ (1-13) NH2 attenuated reductions in cerebral blood flow observed after FPI, thereby acting in a neuroprotective or vasoprotective manner (unpublished observations). Therefore, it is hypothesized that the abnormal vascular responses to glutamate and NMDA are deleterious and that FPI-accentuated release of NOC/oFQ contributes to impaired cerebral hemodynamics via modulation of vasodilation by excitatory neurotransmitters.
Opioids are important contributors to the regulation of the piglet cerebral circulation,9 including brain injury.8 Results of the present study extend such studies by characterizing the contribution of the newly described opioid NOC/oFQ to altered cerebrovascular regulation observed after FPI.
In conclusion, results of the present study show that NOC/oFQ, in concentrations present in CSF after FPI, increased O2- production in a cyclooxygenase-dependent manner and contributes to such production after FPI. These data also show that NOC/oFQ contributes to impaired NMDA- and glutamate-induced pial artery dilation after FPI. These data suggest, therefore, that cyclooxygenase-dependent O2- generation links NOC/oFQ release to impaired NMDA-induced cerebrovasodilation after brain injury.
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Acknowledgments |
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This research was supported by grants from the National Institutes of Health, the American Heart Association–Pennsylvania/Delware Affiliate, and the University of Pennsylvania Research Foundation.
Received February 22, 2000; revision received May 15, 2000; accepted May 18, 2000.
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Editorial Comment
Medical College of Virginia Virginia Commonwealth University Richmond, Virginia
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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It is well established that cerebral vascular reactivity is altered after experimental traumatic brain injury. Many abnormalities have been described. These include alterations in autoregulation, abnormal responses to changes in CO2 tension, and altered responses to endothelium-dependent vasodilators. Most of these abnormalities have been attributed to generation of reactive oxygen species.
In the preceding article, Kulkarni and Armstead showed that the vasodilation in response to NMDA is also abnormal after traumatic brain injury. These investigators traced this abnormality to the generation of superoxide as a result of release of the opioid nociceptin/orphanin FQ. This peptide causes vasodilation in part by generating superoxide as a by-product of increased cyclooxygenase production. These observations extend our knowledge of the mechanisms by which traumatic brain injury affects the function of the microcirculation in the brain.
Received February 22, 2000; revision received May 15, 2000; accepted May 18, 2000.
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