| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Laboratory for Research in Neonatal Physiology, Departments of Physiology and Pediatrics, and Vascular Biology Center, University of Tennessee Health Science Center, Memphis, Tennessee 38163
Submitted 28 January 2003 ; accepted in final form 8 May 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
cerebral circulation; seizure; bicuculline; carbon monoxide; tin protoporphyrin; vascular injury; vascular reactivity; pial arterioles; cranial window; newborn piglets
Mechanisms by which seizures increase blood flow to the brain may involve heme oxygenase (HO). HO inhibitors greatly reduced cerebral hyperemia caused by seizures in rats and newborn pigs (24, 30). HO, a rate-limiting enzyme in heme degradation to biliverdin and carbon monoxide (CO), is highly expressed in the brain and in cortical microvessels (18, 20). CO is a potent vasodilator in the cerebral microcirculation of newborn pigs (15, 18). HO isoforms include inducible HO-1 (heat shock protein 32) and constitutive HO-2 (20). HO-1 is rapidly upregulated in response to stress and growth factors (20). Under basal conditions, high expression and activity of constitutive HO-2 is found in the brain tissue and in cortical microvessels (18, 20, 27).
Glutamate is a major excitatory neurotransmitter in the brain. Excessive
neuronal release of glutamate, imbalance between inhibitory and excitatory
neurotransmitters, and decreased reuptake result in the accumulation of
extracellular glutamate in the brain during seizures
(22). Glutamate is a
vasodilator in the cerebral circulation
(5,
32). Dilator effects of
glutamate may involve a CO-mediated mechanism. In the cerebral
microcirculation of newborn pigs, glutamate stimulates CO production and
causes CO-dependent vasodilation. CO production by cerebral microvessels and
cerebral microvascular endothelial cells from newborn pigs is stimulated by
glutamate via an ionotropic glutamate receptor-mediated mechanism
(26,
32). Isolated cerebral
arterioles respond to glutamate by endothelium-dependent vasodilation, which
can be blocked by HO inhibitors
(15). In vivo, activation of
N-methyl-D-aspartate (NMDA) and
-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate
receptors results in CO-dependent dilation of pial arterioles
(32). These data indicate that
activation of HO in the cerebral microcirculation via a glutamate
receptor-mediated mechanism may contribute to the increased cerebral blood
flow response to seizures. HO activation during seizures may protect against
excitotoxic injury in the cerebral microcirculation. The products of the HO
pathway, CO biliverdin, and its end metabolite, bilirubin, have protective
effects against cell injury and oxidative stress
(9,
10,
20,
35).
We hypothesized that seizures cause severe impairment of cerebral vascular functions when HO enzymatic activity is inhibited. In a newborn pig model of bicuculline-induced seizures in vivo, we addressed the following questions: 1) do seizures cause HO activation in the brain? 2) do seizures cause cerebral vascular dysfunction during the recovery period? and 3) is HO activation important in preventing cerebral vascular dysfunction? To assess HO activity/expression in the brain before and during seizures, we measured the CO concentration in cortical periarachnoid cerebrospinal fluid (CSF) and expression of the HO-1 and HO-2 isoforms in cerebral microvessels. With the use of cranial window techniques, we examined cerebral vascular reactivity to endothelium-dependent (hypercapnia and bradykinin) and endothelium-independent (isoproterenol and sodium nitroprusside) stimuli during the recovery from seizures in control animals and in animals with inhibited HO activity. To inhibit HO activity in the brain, we used a synthetic nonmetabolized heme analog, tin protoporphyrin (SnPP), a potent competitive inhibitor of HO, which binds to the active site of the enzyme (1). In newborn animals, systemically administered SnPP (7–30 mg/kg iv) effectively inhibits HO activity in all organs, including the brain (11, 21).
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
Catheter and cranial window placement. Newborn pigs (1–5
days old, 1.5–2.5 kg) were initially anesthetized with ketamine
hydrochloride (33 mg/kg im) and acepromazine (3.3 mg/kg im) and maintained on
-chloralose (30).
Although ketamine is a NMDA antagonist, it was used only for initial
anesthesia. Because the effects of ketamine-acepromazine anesthesia are
transient (piglets regain consciousness in 15–20 min), the residual
effects of ketamine on cerebral vascular responses to glutamate receptor
agonists are negligent in our experimental model
(5,
32). The catheters were placed
into a femoral vein for infusion of 5% dextrose saline (4 ml ·
kg–1 · h–1) and
SnPP (3 mg/kg when indicated). Another catheter was inserted into the
abdominal aorta via a femoral artery for recording blood pressure and for
sampling of blood for pH and gases. The animals were ventilated with room air.
Before seizure induction, arterial blood gases and pH were maintained as
follows: arterial PO2 (PaO2)
90 mmHg, arterial PCO2
(PaCO2) 40 mmHg, and pH 7.45. Mean arterial
blood pressure (MABP) was within a range of 60–80 mmHg. Body temperature
was maintained at 37–38°C with a servo-controlled heating pad. The
animals were equipped with a stainless steel cranial window that allowed
1) measurement of pial arteriolar diameter with a videomicrometer
coupled to a television camera, 2) delivery of drugs directly to the
brain surface through the ports of the window, and 3) cortical
periarachnoid CSF collection. The space under the window was filled with
artificial CSF (aCSF), which contained (in mM) 3.0 KCl, 1.5 MgC2,
1.5 CaCl2, 132 NaCl, 6.6 urea, 3.7 dextrose, and 24.6
NaHCO3, equilibrated with 6% CO2-6% O2-88%
N2 to pH 7.3–7.35 at 37°C. To detect cerebral production
of CO, cortical periarachnoid CSF (500 µl) was sampled from under the
window.
Seizure induction. Seizures were induced by administration of bicuculline (3 mg/kg iv) (6, 30, 31). Bicuculline, a GABA receptor blocker, induces spontaneous seizures by disruption of the normal balance between excitatory and inhibitory neurotransmitters. Shortly before the experiment, bicuculline was dissolved in 0.1 N HCl (3 mg/ml), neutralized with 1 N NaOH to pH 4–5, and diluted with 5 ml saline. Bicuculline-induced seizures are characterized by abnormal electrocortical activity, increased intracranial pressure, and increased heart rate (HR) sustained for over a 1-h period (6, 31). Animals were paralyzed with pancuronium bromide (0.2 mg/kg iv). Pancuromium attenuates the increase in MABP and stabilizes arterial blood gases and pH without affecting the cerebral blood flow response to seizures (31). Additional injections of pancuronium (0.1 ml/kg) were given if visible evidences of seizures were seen. To inhibit HO activity, SnPP (3 mg/kg) was administered 30 min before seizure induction. Although different metalloporphyrins (ZnPP, CrMP, and SnPP) effectively inhibit HO activity and HO-mediated cerebral vascular responses in vivo and in vitro (17, 18, 26, 27, 32), we selected SnPP because it was more effective in inhibiting cerebral dilation to seizures and was better tolerated by animals upon systemic administration (30). SnPP was dissolved in 10% ethanolamine (stock solution, 15 mg/ml) and kept protected from light. Before use, SnPP was diluted with saline (1:30) and administered intravenously by a 0.45-µm Millipore filter-equipped syringe. Pial arteriolar diameter, HR, and MABP were constantly monitored for 2 h after seizure induction. Blood gases were measured every 20 min, and adjustments in ventilation were made to assure that blood gases remained within physiological ranges. Cortical periarachnoid CSF (500 µl) was sampled from under the window every 10 min for detection of cerebral production of CO. Two hours after the bicuculline injection, cerebral vascular reactivity to endothelium-dependent and -independent stimuli was tested.
Vascular reactivity. To test vascular reactivity, we used endothelium-dependent dilator stimuli, hypercapnia and bradykinin, and endothelium-independent vasodilators, isoproterenol and sodium nitroprusside. In addition, the responses of pial arterioles to the topical HO substrate, heme [as heme-L-lysinate (HLL)], were tested to probe for functional activity of HO in the cerebral microcirculation. Three pial arterioles (60–80 µm) in each animal were selected for observation. To determine the control diameter values, arterioles were measured over a 10-min period during basal conditions. Hypercapnia (PaCO2, 84 ± 5 mmHg, PaO2, 100 ± 8 mmHg, pH 7.00 ± 0.05) was induced for 10 min by ventilating piglets with a mixture of 10% CO2 and 21% O2 in 69% N2. At the end of the hypercapnia period, the ventilation gas was returned to air, and the cerebral surface was flushed with aCSF for 20 min to allow arterial blood gases, pH, and pial arteriolar diameter to return to basal values. Bradykinin at progressively increasing concentrations (10–6 and 10–5 M) was applied to the cerebral surface, and changes in pial arteriolar diameter were recorded. After the cerebral surface was flushed with aCSF for 20 min, pial arteriolar diameter returned to the basal level, and isoproterenol (10–6 and 10–5 M) was applied. The brain surface was flushed with aCSF, and HLL at increasing concentrations (10–8, 10–7, and 10–6 M) was applied. HLL solution in aCSF was protected from light at all times, and the cranial window was illuminated only shortly for the pial arteriolar diameter measurements. Pial arteriolar diameter measurements in response to topical compounds were taken three times over a 10-min period after the application of each concentration. The stable diameter achieved between 5 and 10 min was taken as the response. At the end of the experiment, the brain was rapidly removed, and cerebral microvessels were isolated to detect HO protein expression.
Experimental groups. To investigate the effects of seizures on cerebral vascular reactivity, vascular responses to hypercapnia, bradykinin, isoproterenol, sodium nitroprusside, and HLL were tested in 1) intact animals (group I, n = 12); 2) animals during the seizure recovery period (2 h after bicuculline injection, saline control-seizure, group II, n = 7); and 3) SnPP-pretreated animals during the seizure recovery period (2 h after bicuculline injection, SnPP-seizure, group III, n = 7). To evaluate SnPP selectivity, we investigated the acute (30 min) and prolonged (2 h) effects of SnPP (3 mg/kg iv by single injection) on cerebral vascular reactivity to the above stimuli (SnPP control, group IV, n = 9; and SnPP control, group V, n = 9, respectively).
Detection of CO in cortical periarachnoid CSF by gas chromatography/mass spectrometry. To collect CSF, 1 ml of aCSF was slowly infused through the port of the cranial window onto the brain surface, and drops of CSF from the opposite port were collected. The CSF samples were transferred to amber vials (2 ml), and the total volume was adjusted to 1.7 ml with aCSF. The internal standard (5 µl) of a saturated solution of heavy 13C16O (isotopic purity >99%, 1 mM, Isotech; Miamisburg, OH) was injected into the bottom of the vial (26). The vials were sealed with Teflon-lined caps, and the samples were left overnight at room temperature. The samples were placed in hot water (75°C) for 30 min. The headspace gas mixture was analyzed by a Hewlett-Packard 5970 ion detector interfaced to a Hewlett-Packard 5890A gas chromatograph. The separation of CO was carried out on a Varian-5A Molesieve capillary column (30 m, 0.32 mm internal diameter, Varian) with a linear temperature gradient from 35 to 65°C. Helium was the carrier gas at a column head pressure of 4.0 psi. Aliquots (100 µl) of the headspace gas were injected with a gas-tight syringe into the injector (120°C). Ions at a mass-to-charge ratio (m/z) of 28 and 29, corresponding to 12C16O and 13C16O, respectively, were recorded via selective ion monitoring. The amount of CO was calculated from the ratio of the peak areas of m/z 28 and m/z 29.
Isolation of cerebral microvessels and Western immunoblotting. Cerebral microvessels (60–300 µm) were isolated from the brain cortex by differential filtration of the tissue homogenate through nylon 300- and 60-µm mesh screens (27). Microvessels were homogenized in 1 ml of 10 mM Tris-1% SDS, pH 7.4, containing 1 mM sodium orthovanadate, and heated at 100°C for 5 min. Proteins (50 µg/lane) were separated by 9% SDS-PAGE, transferred to nitrocellulose membranes, and blocked with 5% BSA-0.1% Tween 20. The membranes were probed with polyclonal anti-human HO-2 (1:5,000 dilution, SPA 897 from StressGen; Victoria, Canada) or polyclonal anti-human HO-1 (1:5,000 dilution, SPA 895 from StressGen), followed by peroxidase-conjugated donkey anti-rabbit IgG (dilution 1:10,000, Jackson Immunoresearch; West Grove, PA). As positive controls, we used recombinant rat HO-1 and human HO-2 proteins (StressGen). For quantification purposes, the membranes were reprobed with monoclonal antibodies against actin (dilution 1:10,000, Chemicon International; Temecula, CA), followed by peroxidase-conjugated donkey anti-mouse IgG (Jackson Immunoresearch). Bands were visualized with the Renaissance chemiluminescence kit (NEN Life Science Products; Boston, MA) and quantified by digital densitometry using NIH Image 1.63.
Statistical analysis. Values are presented as means ± SE of absolute values or as a percentage of control. ANOVA with repeated measures and Fisher's protected least-significant-difference test were used to confirm differences among and then between groups, respectively. A level of P < 0.05 was considered significant in all statistical tests.
Materials. HLL was prepared using methods described previously (17). SnPP was purchased from Porphyrin Products (Logan, UT). Pancuronium bromide was from Astra Pharmaceutical Products (Westborough, MA). Bicuculline, bradykinin, isoproterenol, and sodium nitroprusside were from Sigma (St. Louis, MO).
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
Time-dependent cerebral vascular effects of SnPP on HO-mediated responses. To characterize the functional expression of HO in the cerebral microcirculation and to confirm that systemic SnPP does inhibit HO in the cerebral microcirculation, we studied the responses of pial arterioles to HLL, a HO substrate (Fig. 1). In intact piglets, HLL (10–8–10–6 M) caused dose-dependent vasodilation of pial arterioles (maximal dilation, 38 ± 5%, was achieved at 10–7 M). In animals pretreated with SnPP (3 mg/kg iv) for 30 min, responses to HLL were greatly reduced (maximal dilation, 12 ± 1%). However, the dilation of pial arterioles to HLL was restored within 2 h of SnPP administration (maximal dilation, 36 ± 3%). These data indicate that upon systemic administration, SnPP rapidly but reversibly inhibits HO in the cerebral circulation by 70–80%, as assessed by responses of pial arterioles to the HO substrate.
|
Effects of SnPP on cerebral vascular responses to hypercapnia, isoproterenol, sodium nitroprusside, and bradykinin. To address the specificity of SnPP in inhibiting HO-mediated cerebral vascular responses, we investigated the effects of the inhibitor on dilation of pial arterioles to endothelium-dependent and -independent vasodilator stimuli. In piglets pretreated with SnPP (3 mg/kg) for 30 min, dilator responses of cerebral arterioles to hypercapnia (PaCO2, 84 ± 5 mmHg; PaO2, 110 ± 8 mmHg, pH 7.02 ± 0.03) and isoproterenol (10–5 M) were reduced by 20–30%; the inhibition was completely reversed in 2 h (Fig. 2). SnPP did not inhibit cerebral vasodilation to sodium nitroprusside (10–6 M), as evaluated 30 min or 2 h after the inhibitor administration (Fig. 2). Low effectiveness or inability of SnPP to inhibit responses of pial arterioles to hypercapnia, isoproterenol, and sodium nitroprusside indicates that the HO inhibitor has very little nonspecific effect in the cerebral circulation.
|
In contrast to the results with hypercapnia, isoproterenol, and sodium nitroprusside, in piglets pretreated with SnPP for 30 min, dilation of pial arterioles to the endothelium-dependent dilator bradykinin (10–6–10–5 M) was greatly inhibited (70–80% inhibition; Fig. 3). These results indicate that HO/CO may contribute to cerebral vasodilation in response to bradykinin. The inhibitory effect of SnPP on the dilator responses to bradykinin is reversible, as is the case with the heme substrate. By 2 h after SnPP administration, the vasodilation to bradykinin returned near to the control level (Fig. 3).
|
Effects of seizures on the cerebral and systemic circulation. Bicuculline (3 mg/kg iv) caused immediate and sustained dilation of pial arterioles. Maximal vasodilation (>1.8-fold above the baseline) was observed during the first 2–5 min of seizures (Fig. 4). In animals with intact HO activity, dilation of pial arterioles (1.4- to 1.6-fold above the basal diameter) was sustained for 2 h after seizure induction (Fig. 4). However, when HO activity was inhibited at the seizure onset, time-dependent cerebral vasodilation to seizures was greatly reduced. In SnPP-treated animals, initial dilation (1.5-fold above the baseline in 2–10 min of seizure induction) was followed by a rapid return of pial arteriolar diameter to near the basal level in 1 h (Fig. 4).
|
We investigated whether SnPP affects systemic cardiovascular effects of
seizures. Seizures caused immediate and sustained increase in HR (from
120 to 200 beats/min; Fig.
5A) and a transient moderate increase in MABP (from
60–70 to 75–80 mmHg in 2–10 min;
Fig. 5B) in both
control and SnPP-treated groups of piglets. Arterial blood gases were
constantly monitored and maintained at physiological levels for the duration
of seizures in control and SnPP-treated piglets: PaCO2,
30–40 mmHg (Fig.
5C); and PaO2, 95 ± 5 mmHg.
Bicuculline administration resulted in an immediate drop in blood pH from 7.34
± 0.01 to 7.24 ± 0.01, indicating a moderate shift toward
metabolic acidosis during seizures that was slightly more accentuated in
SnPP-treated group of piglets (Fig.
5D). Overall, SnPP administration did not cause any major
changes in systemic responses to seizures.
|
Effects of seizures on the CO level in cortical periarachnoid CSF. We detected CO in cortical periarachnoid CSF by gas chromatography/mass spectrography. In control animals during resting conditions, the CO level was 88 ± 20 pmol/ml CSF (n = 25); therefore, the CO concentration in cortical periarachnoid CSF corresponded to 9 ± 2 x 10–8 M. Injection of pancuronium bromide (0.2 mg/kg) did not alter the CO level. Seizures caused an immediate and sustained increase of CO in cortical periarachnoid CSF. The maximal increase in CO concentration (20- to 30-fold over the baseline) was observed in the first 10 min after seizure induction (Fig. 6). The CO production was maintained at the increased level (5- to 10-fold above the baseline) for the first 1 h of seizures. By 2 h after seizure induction, CO returned to the basal level (Fig. 6). Overall, the time-dependent dynamics of CO concentration in cortical periarachnoid CSF followed the changes in pial arteriolar diameter during seizures. We observed a good correlation between the increase in pial arteriolar diameter and the CO level in CSF (correlation coefficient = 0.63, P < 0.05; Fig. 7). SnPP (3 mg/kg iv) in 30 min decreased the basal cortical CSF CO level by 30–50% (114 ± 26 and 61 ± 12 pmol/ml before and after SnPP injection, n = 15), indicating inhibition of the HO enzymatic activity in the brain. In SnPP-treated piglets, a significant increase in CO (4-fold above the baseline) was observed only during first 10 min of seizures (Fig. 6). At all time points during active seizures (1-h period), the CO level in CSF was significantly lower in SnPP-treated piglets than in the untreated animals (Fig. 6).
|
|
HO-1 and HO-2 expression in cerebral microvessels. We compared HO-1 and HO-2 protein expression in cerebral microvessels from intact animals and from animals recovering from seizures (4 h after bicuculline administration). As revealed by Western immunoblotting, cerebral microvessels of intact piglets express HO-2 but not HO-1, consistent with our previous observations (27). HO-2 expression in microvessels was not altered by seizures (Fig. 8). HO-1 was not immunodetectable in cerebral microvessels isolated 4 h after seizure induction (Fig. 8). These data indicate that seizures do not result in an immediate induction of HO-1 and HO-2 proteins in cerebral microvessels.
|
Acute effects of seizures on cerebral vascular reactivity during the recovery period. We investigated whether seizures cause acute impairment of cerebral vascular functions in HO-deficient animals. Cerebral vascular responsiveness to endothelium-dependent (hypercapnia and bradykinin) and endothelium-independent (isoproterenol and sodium nitroprusside) stimuli was tested during the seizure recovery period (2 h after bicuculline injection). Although the electric activity in the brain subsides by 1 h after bicuculline injection (5, 31), the recovery period is characterized by sustained pial arteriolar dilation (Fig. 4), increased HR (Fig. 5A), and moderate metabolic acidosis (Fig. 5D). As the above data in intact animals demonstrate, HO/CO contributes to cerebral vascular responses to HLL and bradykinin but not to hypercapnia, isoproterenol, or nitroprusside. First, we tested whether seizures alter CO-independent cerebral vascular responses. In saline control animals (HO active), the responsiveness of cerebral arterioles to hypercapnia, isoproterenol, and sodium nitroprusside was completely preserved during the seizure recovery period (Fig. 9). However, in SnPP-treated animals (HO inhibited at the onset of seizures), cerebral vascular responses to hypercapnia, isoproterenol, and sodium nitroprusside were greatly impaired during the recovery (50–80% inhibition; Fig. 9). Second, we tested whether seizures reduce cerebral vascular responsiveness to CO-dependent stimuli, HLL and bradykinin. In saline control animals (active HO), dilator responses to HLL (10–8–10–6 M) and bradykinin (10–6–10–5 M) were not altered by seizures (Fig. 10). However, when HO activity was inhibited at the onset of seizures, the recovery period was characterized by great reduction of cerebral vasodilation to HLL and bradykinin (60–80% inhibition; Fig. 10). This is not due to the direct inhibitory effects of the HO inhibitor on CO-mediated responses because the effects of SnPP were completely reversed in 2 h of the administration (Figs. 1 and 3). Overall, in animals with intact HO activity, seizures did not cause acute effects on cerebral vascular reactivity as tested during the recovery period. However, inhibition of HO activity in cerebral microcirculation at the onset of seizures resulted in a great impairment of cerebral vascular responsiveness to endothelium-dependent and -independent stimuli, including CO-dependent and -independent vasodilators.
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
Seizures cause an immediate increase in cerebral blood flow to match metabolic demands of the hyperactive neurons (2, 4, 6, 28, 31, 37). There are multiple and, possibly, synergistic mechanisms by which seizures induce cerebral hyperemia. Nitric oxide (NO) synthase plays an essential role in seizure-induced cerebral hyperemia in rabbits (13) and in rats (23), and NO may act as an endogenous anticonvulsant (37). Products of arachidonic acid metabolism via the phospholipase A2/cyclooxygenase pathways are important in seizure-induced increased blood flow in newborn piglets (6) and in prevention of neurotoxicity (14). HO, which catalyzes heme conversion to CO, a gaseous vasodilator, and biliverdin/bilirubin, potent antioxidants, contributes to the cerebral hyperemia response to seizures in rats (24) and in newborn piglets (31).
Bicuculline-induced seizures in newborn pigs are associated with rapid and sustained HO activation in the brain. During basal conditions, the CO level in cortical CSF was 9 ± 2 x 10–8 M. At this concentration range, CO has vasodilator effects in pressurized cerebral arterioles (15) and in the cerebral circulation of newborn pigs in vivo (18). In the brain, constitutive HO-2 is highly expressed in neurons (9, 10, 18) and in non-neuronal cells, including endothelial and smooth muscle cells from cerebral microvessels (18, 27). HO-1 can be induced by stress factors, including oxidative stress (12, 20). Therefore, multiple sources in the brain can potentially contribute to CO production in response to seizures. The HO/CO system in the brain is activated at the onset of seizures, and the activation persists for the duration of seizures. The onset of seizures (2–10 min) is characterized by a >20-fold increase in the CO level and maximal cerebral vasodilation. HO activation (5- to 10-fold) is sustained for the duration of seizures (1 h in our experimental model) (31). During the recovery period (2 h after seizure induction), CO returns to the basal level. Overall, CO dynamics in cortical CSF follow time-dependent dilation of pial arterioles, suggesting a possibility of cause-effect relationships between CO and cerebral hyperemia during sustained seizures.
Systemically administered SnPP (3 mg/kg) effectively and selectively
inhibits HO activity in the brain without changing any systemic parameters
(blood pressure and HR, among others). Systemic SnPP reduced the basal CO
level in cortical CSF and nearly completely blocked CO-mediated cerebral
vasodilation to heme. SnPP did not alter pial arteriolar dilation to sodium
nitroprusside and only slightly (
30%) decreased responses to hypercapnia
and isoproterenol, indicating the specificity of the inhibitor toward
HO-mediated cerebral vascular responses. When HO activity was inhibited, the
CO increase and the cerebral dilation to seizures were greatly reduced.
Inhibitory effects of systemic SnPP in the cerebral circulation are time
dependent and reversible. Pial arteriolar dilation to heme was blocked in 30
min of SnPP administration, whereas by 2 h the inhibition was completely
reversed. Heavy metal protoporphyrins are competitive HO inhibitors
(1,
19–21)
and can be displaced from the active site by the substrate. Systemically
administered SnPP is accumulated mainly in the liver and kidneys, causing
prolonged (up to 7 days) inhibition of HO, whereas only a small amount of SnPP
is retained by the brain (1,
19,
20). It appears that SnPP
clearance from the systemic circulation removes its inhibitory effects on HO
in the brain. It is possible that systemic SnPP may primarily target
endothelial HO in the cerebral microcirculation. Our preliminary data show
that SnPP did not produce substantial reduction of neuronal excitation and did
not affect the duration of seizures as detected by electroencephalography
(unpublished observations).
The excitatory neurotransmitter glutamate is a major excitotoxic factor that contributes to seizures and seizure-related brain injury. Accumulation of extracellular glutamate in the brain during seizures has been reported in patients (34, 38) and in animal models (25, 33). In a model of bicuculline-induced seizures, blocking inhibitory GABA receptors on glutamatergic neurons enhances glutamate release and activates excitatory amino acid receptors on nerve terminals. Excessive release of glutamate during seizures is a central event in recruiting neurons into epileptic discharges, and glutamate receptors play a crucial role in epilepsy (16, 34). Glutamate transporter deficiency is also related to epilepsy and brain injury (36). Another excitatory amino acid, aspartate, also contributes to seizures (25). We (26) have previously demonstrated that glutamate and aspartate target cerebral microvessels via a glutamate receptor-mediated mechanism. Ionotropic glutamate receptors (NMDA, AMPA, and kainate type) in the cerebral microvasculature are functionally coupled to HO, so that their stimulation rapidly increases CO production (26) and causes endothelium-dependent CO-mediated vasodilation (15). Therefore, excitatory neurotransmitters may directly target the cerebral microvasculature and stimulate CO production in the cerebral circulation during sustained seizures.
What is the mechanism by which CO production by the brain is rapidly stimulated by seizures? We found neither HO-1 induction nor changes in HO-2 expression in cerebral microvessels 4 h after seizure onset. The rapid increase in CO in the brain (10–20 min) indicates rapid activation of the preexisting constitutive isoform (HO-2). Mechanisms of HO-2 activation at posttranslational level may include protein phosphorylation (10, 17). In cerebral microvessels, a protein tyrosine kinase-mediated pathway is involved in rapid stimulation of CO production by glutamate (17). Excitatory neurotransmitters excessively released during seizures may directly target cerebral microvessels, activate vascular HO, and increase production of endogenous CO, which increases cerebral blood flow. Although it is a strong possibility that activation of HO in neuronal populations also contributes to the CO increase in cortical periarachnoid CSF, experimental data connecting neuronal HO activity with seizures are not currently available.
We investigated whether activation of HO/CO system in the brain can protect
against seizure-induced cerebral vascular injury. As functional probes for
cerebral vascular injury, we tested the reactivity of pial arterioles to
physiologically relevant endothelium-dependent and -independent stimuli during
the recovery period (2 h after seizure induction). In newborn pigs, cerebral
vasodilation to hypercapnia and bradykinin required the presence of intact
endothelium (15,
39). Cerebral dilator
responses to isoproterenol, a 
-adrenoreceptor agonist, and sodium
nitroprusside, a NO donor, result from direct stimulation of vascular smooth
muscle via a cAMP- or cGMP-dependent mechanisms, respectively. In animals with
active HO, cerebral vascular responsiveness to endothelium-dependent and
-independent stimuli, including CO-mediated vasodilators (heme and
bradykinin), was completely preserved during the seizure recovery period.
Other authors (8) also did not
find changes in cerebral vascular reactivity to hypercapnia, topical
nitroprusside, or adenosine after bicuculline-induced seizures in newborn
pigs. Therefore, during the postictal period, cerebral vascular functions are
completely preserved in newborn pigs with functionally active HO. In contrast,
when HO activity was inhibited at the onset of seizures, cerebral vascular
responsiveness to all examined dilators was greatly reduced during the
postictal period, thus reflecting vascular injury in the cerebral circulation.
Blood-brain barrier integrity is severely affected by seizures
(3,
7), indicating that the
endothelium is a likely target for excitotoxic injury. However, in animals
with inhibited HO activity, seizure-induced cerebral vascular dysfunction goes
beyond endothelial injury and also involves vascular smooth muscle, including
both cAMP- and cGMP-dependent signaling pathways. Changes in cerebral vascular
reactivity may contribute to brain injury after neonatal seizures.
What is a possible mechanism of HO protection against the seizures-induced vascular injury? Although metabolic acidosis was more accentuated in SnPP-versus saline-treated piglets during the recovery period, the pH values (pH 7.20 ± 0.01 and 7.24 ± 0.01, respectively) remained in physiological range for human infants and newborn piglets. Furthermore, vascular reactivity in the saline-treated piglets during the recovery was not altered compared with that of intact piglets, despite the more significant differences in pH values (pH 7.24 ± 0.01 and 7.31 ± 0.02, respectively). Therefore, it is highly unlikely that the severe cerebral vascular dysfunction observed during the seizure recovery period is due to pH variations.
Because cerebral metabolism increases markedly during seizures and HO inhibition blunts the increase in cerebral blood flow, the result is relative ischemia, which could produce vascular injury. Furthermore, HO activation is not limited to production of vasodilator CO. The products of the HO pathway have protective effects against cell injury and oxidative stress (9, 10, 20, 29, 35). HO is a major antioxidant system, leading to a decrease in heme, a prooxidant molecule, and an increase in biliverdin and bilirubin, potent antioxidants (9, 20, 29, 35). In neurons, HO is essential in protection against glutamate-induced apoptosis and oxidative stress (9, 10). However, molecular mechanisms involved in HO protection against stress-induced cell injury and death are not completely understood.
All together, our data indicate that HO in the brain is absolutely essential in protection from seizure-induced cerebral vascular injury.
|
DISCLOSURES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONDISCLOSURESREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  X. Qin, H. Kwansa, E. Bucci, S. Dore, D. Boehning, D. Shugar, and R. C. Koehler Role of heme oxygenase-2 in pial arteriolar response to acetylcholine in mice with and without transfusion of cell-free hemoglobin polymers Am J Physiol Regulatory Integrative Comp Physiol, August 1, 2008; 295(2): R498 - R504. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Kanu and C. W. Leffler Carbon monoxide and Ca2+-activated K+ channels in cerebral arteriolar responses to glutamate and hypoxia in newborn pigs Am J Physiol Heart Circ Physiol, November 1, 2007; 293(5): H3193 - H3200. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Zimmermann, C. W. Leffler, D. Tcheranova, A. L. Fedinec, and H. Parfenova Cerebroprotective effects of the CO-releasing molecule CORM-A1 against seizure-induced neonatal vascular injury Am J Physiol Heart Circ Physiol, October 1, 2007; 293(4): H2501 - H2507. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. W. Leffler, H. Parfenova, A. L. Fedinec, S. Basuroy, and D. Tcheranova Contributions of astrocytes and CO to pial arteriolar dilation to glutamate in newborn pigs Am J Physiol Heart Circ Physiol, December 1, 2006; 291(6): H2897 - H2904. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Basuroy, S. Bhattacharya, D. Tcheranova, Y. Qu, R. F. Regan, C. W. Leffler, and H. Parfenova HO-2 provides endogenous protection against oxidative stress and apoptosis caused by TNF-{alpha} in cerebral vascular endothelial cells Am J Physiol Cell Physiol, November 1, 2006; 291(5): C897 - C908. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Kanu, J. Whitfield, and C. W. Leffler Carbon monoxide contributes to hypotension-induced cerebrovascular vasodilation in piglets Am J Physiol Heart Circ Physiol, November 1, 2006; 291(5): H2409 - H2414. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Parfenova, S. Basuroy, S. Bhattacharya, D. Tcheranova, Y. Qu, R. F. Regan, and C. W. Leffler Glutamate induces oxidative stress and apoptosis in cerebral vascular endothelial cells: contributions of HO-1 and HO-2 to cytoprotection Am J Physiol Cell Physiol, May 1, 2006; 290(5): C1399 - C1410. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. W. Leffler, H. Parfenova, J. H. Jaggar, and R. Wang Carbon monoxide and hydrogen sulfide: gaseous messengers in cerebrovascular circulation J Appl Physiol, March 1, 2006; 100(3): 1065 - 1076. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Parfenova, P. Carratu, D. |
P
< 0.05 compared with the corresponding time point in the saline control
animals.
