Perinatal stroke is a common human disease. Neonatal brains are immature and engaged in active synaptogenesis. Preconditioning adult rats with the volatile anesthetic isoflurane induces neuroprotection. Whether isoflurane preconditioning induces neuroprotection in neonates is not known.
Seven-day-old Sprague-Dawley rats had left common carotid arterial ligation followed by hypoxia with 8% oxygen for 1, 2, or 2.5 h at 37 degrees C. Isoflurane preconditioning with 1 or 1.5% isoflurane for 30 min was performed at 24 h before the brain hypoxia/ischemia. The inducible nitric oxide synthase inhibitor aminoguanidine (200 mg/kg, intraperitoneally) was administered 30 min before the isoflurane pretreatment. The weight ratio of left to right cerebral hemispheres at 7 days after the brain hypoxia/ischemia was calculated. The mortality during the period from cerebral hypoxia/ischemia to 7 days afterwards was monitored. In another experiment, 6-day-old rats were exposed to 1.5% isoflurane for 30 min. The cerebral hemispheres were removed at various time points for Western analysis of inducible nitric oxide synthase.
The mortality was about 40% in neonates with brain hypoxia/ischemia for 2 h or 2.5 h and was not altered by isoflurane preconditioning. The weight ratio of left/right cerebral hemispheres in the survivors was 0.99 +/- 0.02, 0.65 +/- 0.19, and 0.86 +/- 0.15 (n = 7-18) for the rats in control, brain hypoxia/ischemia for 2.5 h, and isoflurane preconditioning plus brain hypoxia/ischemia for 2.5 h groups, respectively (P < 0.05 for the comparisons between control versus brain hypoxia/ischemia and brain hypoxia/ischemia versus isoflurane preconditioning plus brain hypoxia/ischemia). This isoflurane preconditioning-induced neuroprotection was abolished by aminoguanidine (the weight ratio was 0.61 +/- 0.18, n = 12). Isoflurane induced a time-dependent increase in the inducible nitric oxide synthase proteins.
Isoflurane preconditioning induces neuroprotection in neonatal rats. This neuroprotection is inducible nitric oxide synthase-dependent.
PERINATAL stroke is a cerebrovascular event that results in brain ischemia during the period from 28 weeks gestation to 1 week of age in human.1It is estimated that perinatal stroke occurs in 1 of 4,000 births, a rate that is four to five times higher than the rate of childhood stroke.1The National Hospital Discharge Survey has shown that the in-hospital mortality rate for neonatal stroke was 2.67 of 100,000 live births in the United States from 1980 to 1998.1In the survivors, long-term follow-up showed that ∼40% were neurologically normal and ∼60% were neurologically or cognitively abnormal presenting delayed mental and motor development (88%), epilepsy (50%), spastic hemiparesis (88%), and major cognitive deficits (88%).1–3Many interventions, such as induction of hypothermia and use of glutamate receptor antagonists, have been explored for potential neuroprotection against ischemia.4,5However, clinically practical methods to reduce ischemic brain injury have not been well established yet.
It has been demonstrated that pretreatment of various organs, such as brain and heart, with brief episodes of sublethal ischemia induces ischemic tolerance. This phenomenon is called ischemic preconditioning.6,7Two phases of ischemic tolerance have been described: the acute phase is observed within minutes and disappears 2–3 h later and the delayed phase develops hours after the preconditioning stimulus and lasts for several days to weeks.6,7Recently, multiple studies have shown that pretreatment of adult rats with volatile anesthetics such as isoflurane and halothane also induces acute and delayed phases of ischemic tolerance in the brains.8–10However, it is not known whether isoflurane induces preconditioning effects on neonatal brains. Unlike adult brains, neonatal brains are immature and engaged in active synaptogenesis.11Neonatal brains are more vulnerable to glutamate-mediated excitotoxicity than adult brains, possibly as a result of the differences in the subunit composition of N -methyl-d-aspartate receptors (one type of glutamate receptors).12In addition, unlike in the case of adult stroke that is usually caused by single-vessel occlusion from emboli or thrombi, perinatal stroke is commonly caused by brain ischemia superimposed on severe systemic hypoxemia resulting from failure of ventilation or oxygen supply.13Thus, in this study we investigated whether isoflurane preconditioning could induce neuroprotection in neonatal rats after brain asphyxia. In addition, inducible nitric oxide synthase has been shown to play an important role in cardioprotection or neuroprotection induced by many preconditioning stimuli.9,14We hypothesize that inducible nitric oxide synthase is important for isoflurane preconditioning-induced neuroprotection in neonatal rats.
Materials and Methods
The animal protocol was approved by the institutional Animal Care and Use Committee of the University of Virginia (Charlottesville, VA). All animal experiments were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All reagents unless specified below were obtained from Sigma Chemical (St. Louis, MO).
Neonatal Cerebral Hypoxia/Ischemia Model
Cerebral hypoxia/ischemia was induced as previously described with minor modifications.15Briefly, 7-day-old male and female Sprague-Dawley rats were anesthetized by isoflurane in 30% O2-70% N2and their left common carotid arteries were permanently ligated with a double 7–0 surgical silk (this took about 5–7 min per rat). Isoflurane anesthesia was stopped and the rats were returned to their cages with the mothers for 3 h. The neonates were then placed in a chamber containing humidified 8% O2-92% N2for 1, 2, or 2.5 h at 37°C (air temperature in the chamber was continuously monitored and maintained at 37°C). After the hypoxic periods animals were exposed to room air for 15 min before being returned to their cages.
Isoflurane Preconditioning and Study Groups
Isoflurane preconditioning was performed by placing 6-day-old rats in a chamber containing 1% or 1.5% isoflurane carried by 30% O2-70% N2for 30 min at 24 h before the cerebral hypoxia/ischemia. Neonates were studied under the following conditions (n = 14–32 for each experimental condition; table 1):
2) cerebral hypoxia/ischemia for 1 h, 2 h, or 2.5 h,
3) preconditioning with 1.5% isoflurane and then cerebral hypoxia/ischemia 1 h, 2 h, or 2.5 h,
4) preconditioning with 1% isoflurane and then cerebral hypoxia/ischemia for 2.5 h,
5) aminoguanidine (200 mg/kg, intraperitoneally), which at this regimen has been shown to inhibit inducible nitric oxide synthase (iNOS) activity in rat brain,16administered 24 h before the cerebral hypoxia/ischemia for 2.5 h, and
6) aminoguanidine administered 30 min before the isoflurane (1.5%) preconditioning followed by cerebral hypoxia/ischemia for 2.5 h.
Neonates from the same mother were assigned to several experimental conditions. While some neonates were preconditioned with isoflurane, the others from the same mother were placed in a chamber containing 30% O2-70% N2but without isoflurane for 30 min.
Arterial Blood Gas Determination
To determine the adequacy of ventilation during isoflurane preconditioning, arterial blood was taken from three groups of 6-day-old rats (n = 6 for each group): 1) control, 2) 1% isoflurane, and 3) 1.5% isoflurane. About 200 μl of blood per rat were taken via left ventricular puncture at the end of the 30-min exposure to isoflurane or at 20 min after local infiltration with 1% lidocaine at the puncture site (control animals). The samples were immediately analyzed by an IRMA blood analyzer (Diametrics Medical Inc., St Paul, MN).
Brain Injury/Loss Quantification, Mortality and Body Weight Monitoring
Seven days after the cerebral hypoxia/ischemia, rats were euthanized by isoflurane and their brains were removed. The hindbrain was resected from the cerebral hemispheres and the two hemispheres were then separated and weighed. The weight ratio of left to right hemispheres was calculated. In addition, death during the period from cerebral hypoxia/ischemia to 7 days afterwards was recorded and mortality was calculated. Neonatal body weights were measured just before and 7 days after the cerebral hypoxia/ischemia.
Brain histopathological evaluation was performed in rats at 8 days after the exposure to 1.5% isoflurane for 30 min (n = 6, no brain hypoxia/ischemia was induced on these rats) or at 7 days after brain hypoxia/ischemia for 2.5 h (two groups, hypoxia/ischemia only and 1.5% isoflurane preconditioning plus hypoxia/ischemia, were studied, n = 3 for each group) (time control rats were used for comparison, n = 6). For this purpose, rats were euthanized by isoflurane and perfused with 30 ml of saline. They were then perfused transcardiacally with 30 ml of 4% phosphate-buffered paraformaldehyde. Brains were removed and stored in the fixative for 4 h at room temperature. Four-μm thick cryostat coronal sections at approximately 3.3 mm caudal to bregma were obtained and subjected to Nissl staining. These sections were examined by an observer blinded to the group assignment of the sections. Neuronal density in the retrosplenial granular cortex and the ventral posteromedial thalamic nucleus of the time control rats and the rats at 8 days after isoflurane pretreatment was determined as follows. A reticle (∼0.034 mm2) was used to count cells in the same size area. Nissl staining positive cells were counted in the area. Three determinations, each on different locations in these two brain regions, were performed and averaged to yield a single number (density of the neurons) for each brain region of individual rat.
Western Blot Analysis
Six-day-old neonates were exposed to 1.5% isoflurane in 30% O2-70% N2for 30 min. The cerebral hemispheres were removed at 1 h, 3 h, 6 h, and 24 h after the isoflurane exposure (n = 6 for each time point), and homogenized in ice-cold 1% sodium dodecyl sulfate with a glass homogenizer. The lysate was centrifuged at 13,000g at 4°C for 30 min. The supernatant was stored at −70°C for Western blot. The protein concentrations of the supernatants were determined by the Lowry assay using a protein assay kit from Sigma Chemical. Equal protein samples (60 μg per lane) were separated by 8% sodium dodecyl sulfate-polyacrylamide gels and then electrotransferred onto nitrocellulose membranes (Bio-Rad, Hercules, CA). The primary antibody was a rabbit polyclonal antibody raised against mouse iNOS protein with cross-reactivity to the corresponding rat proteins from Santa Cruz (Santa Cruz, CA). Protein bands were visualized by the enhanced chemiluminescence detection method with reagents from Amersham Pharmacia Biotech (Piscataway, NJ). The protein band volumes were quantified by a densitometry with ImageQuant 5.0 Windows NT software (Molecular Dynamics, Sunnyvale, CA). The volumes of iNOS protein bands were normalized to those of β-actin to control for errors in protein sample loading and transferring during the Western blot analysis. The results in the groups after the isoflurane exposure were then normalized to those of control animals that were exposed to 30% O2-70% N2for 30 min at 10 h before the brain samples were collected.
Data are presented as means ± SD. Brain weight, brain weight ratio, arterial blood gas results, and Western blot data of the different study groups were compared by one-way analysis of variance followed by the Student-Newman-Keuls method after confirmation of a normal distribution of the data or by Kruskal-Wallis test followed by Mann–Whitney rank sum test when the data were not normally distributed. The comparison between left and right hemispheric weights of the same group of rats and the difference of neuronal density between control and isoflurane-pretreated rats was tested by Student t test. The mortality among groups was analyzed by z testing. The comparison of body weight among groups was performed by analysis of variance for repeated measures followed by the Student-Newman-Keuls method. A P < 0.05 was considered significant.
General characteristics of various study groups are presented in table 1. The mortality was approximately 40% in the groups of neonates that had brain hypoxia/ischemia for 2 h or 2.5 h and was not significantly altered by isoflurane preconditioning. Neonates with brain hypoxia/ischemia for 1 h had a mortality rate of 14%, which is similar to that (17%) reported in a previous study also using brain hypoxia/ischemia for 1 h.17Most of deaths (77 of 80 deaths) occurred during the period of brain hypoxia/ischemia. The body weight of 7-day-old neonates (just before the brain hypoxia/ischemia) and 14-day-old neonates (7 days after the brain hypoxia/ischemia) among all study groups including control rats was not different (table 1).
The results of arterial blood gases at the end of isoflurane pretreatment and the results from control animals are presented in table 2. Isoflurane (1% or 1.5%) exposure for 30 min did not significantly change the pH, Pco2, and Po2of arterial blood compared with results of control rats.
Brain hypoxia/ischemia dose-dependently caused brain loss/damage assessed at 7 days after the insult (fig. 1and table 3). Brain hypoxia/ischemia for 2 h or 2.5 h induced a significant brain loss/damage compared with control (weight ratio of left/right cerebral hemisphere was 0.99 ± 0.02, 0.70 ± 0.20, and 0.65 ± 0.19, respectively, for the control rats and rats with brain hypoxia/ischemia for 2 h and 2.5 h, n = 7–18, P < 0.05). This brain loss induced by either 2 h or 2.5 h of brain hypoxia/ischemia was significantly decreased by preconditioning with 1.5% isoflurane (weight ratio was 0.90 ± 0.12 and 0.86 ± 0.15, respectively, in rats with isoflurane preconditioning plus brain hypoxia/ischemia for 2 h and 2.5 h, P < 0.05 compared with the results of the corresponding brain hypoxia/ischemia only, n = 9–18, fig. 1and table 3). Although aminoguanidine did not affect the ischemia-induced brain loss/damage, aminoguanidine abolished isoflurane preconditioning-induced neuroprotection (figs. 2, 3and table 4).
Histopathological studies showed that all three rats subjected to brain hypoxia/ischemia for 2.5 h had brain infarction presented as empty space in the brain sections (fig. 4). One of three rats that were preconditioned with 1.5% isoflurane for 30 min before the brain hypoxia/ischemia had brain infarction. The overall histologic structure of the ischemic hemisphere in the other two rats with isoflurane preconditioning was similar to that of control rats (fig. 4).
Isoflurane induced a time-dependent increase in the iNOS protein expression in the cerebrum. This increase peaked at about 6 h after the isoflurane exposure (fig. 5).
There were no significant differences in neuronal density and morphology in the retrosplenial granular cortex and the ventral posteromedial thalamic nucleus between the time control rats and the rats at 8 days after isoflurane pretreatment (fig. 6).
Our current study has the following major findings: 1) preconditioning neonatal rats with 1.5% isoflurane induced a delayed phase of ischemic tolerance in the brain because there is less brain loss/damage in the ischemic cerebral hemisphere of rats pretreated with isoflurane before brain hypoxia/ischemia than that in rats with brain hypoxia/ischemia only; 2) isoflurane induced a time-dependent increase in iNOS expression in neonatal brains, and 3) iNOS may be a mediator for the isoflurane preconditioning-induced neuroprotection.
To induce brain ischemia in the neonatal rats, we performed unilateral common carotid arterial ligation followed by exposure to 8% oxygen for various durations on 7-day-old rats. This is a commonly used animal model to simulate human perinatal stroke.18Postnatal day 7 in the rat is a period of rapid growth and synaptogenesis (the period spans from 1 or 2 days before birth to about 2 weeks after birth in rats and spans from the third trimester to several years after birth in human), and the maturation of the brain in the 7-day-old rat is similar to that of the human newborn brain.11,19In addition, as the left hemisphere is more often affected than the right one in humans,1we performed left common carotid arterial ligation in our study.
To quantify ischemia-induced brain damage, we used the weight ratio of left/right cerebral hemispheres. This parameter has been used for this purpose in previous studies.15,20Brain weight reduction was associated with neuronal loss in postnatal day 12 rats that were exposed to ethanol at postnatal day 7.21In addition, brain weight ratio has been found to be as sensitive as and to be correlated with histopathological and biochemical (activity of choline acetyltransferase, a neuronal enzyme) data to quantitate brain injury 5 days after an unilateral injection of N -methyl-d-aspartate to striatum of 7-day-old rats.22Our results showed that the ischemia of left hemisphere significantly reduced the weight ratio of left/right hemispheres and this ischemia-induced brain loss was decreased by preconditioning neonates with 1.5% isoflurane. In addition, brain histopathological changes induced by brain hypoxia/ischemia were reduced by isoflurane preconditioning. These results suggest that isoflurane preconditioning induces neuroprotection in the neonates. However, two important factors need to be considered here in this regard. First, during the 30 min of isoflurane pretreatment, we did not intubate and mechanically ventilate the neonates because of the technique difficulty. Because isoflurane can inhibit respiration, neonates may have CO2retention and hypoxemia during the pretreatment. For this reason, we measured the arterial blood gases at the end of isoflurane pretreatment, which should represent the worst situation. Our results showed that there was no difference in pH, Paco2, or Po2between control and isoflurane-treated rats. In addition, no rats had hypoxemia (Pao2< 60 mm Hg) under anesthesia with 1.5% isoflurane. Thus, it is unlikely that the mild CO2retention, acidemia, or hypoxemia during the isoflurane pretreatment contributes to the final isoflurane preconditioning effects. The second important factor that deserved attention is blood pressure. It is possible that isoflurane pretreatment caused hypotension that induced preconditioning effects. However, a previous study showed that the degree of hypotension associated with the application of 1.4% isoflurane did not induce neuroprotection in adult rats9(of note, there may be differences in physiologic and biochemical responses to hypotension between adult and neonatal rats). Thus, the neuroprotection induced by 1.5% isoflurane may be mainly caused by the pharmacological effects of isoflurane rather than the effects secondary to the disturbance of physiologic parameters in the presence of isoflurane.
We have also monitored the mortality and body weight gain in the neonates. We used these two parameters to measure the well being of the rats in each study group. The fact that the body weight gain in the survived neonates is similar among all groups suggests that this parameter is not a sensitive measure to reflect the degree of brain injury after the brain ischemia. Our results showed that preconditioning neonates with 1.5% isoflurane did not reduce the mortality induced by brain hypoxia/ischemia, suggesting that isoflurane preconditioning did not increase the general tolerance of the neonates to the brain ischemia and hypoxemia used in this study. Although multiple factors such as individual tolerance and health may contribute to death, the severity of brain injury after the brain hypoxia/ischemia may be an important factor to determine the fate of the rats (rats with severe brain injury may die and rats with less severe brain injury may survive). Thus, our results showing that isoflurane preconditioning induced neuroprotection in the survivors and did not affect mortality induced by the brain hypoxia/ischemia suggest that the presence and benefit of the neuroprotection induced by isoflurane preconditioning depend on the severity of brain injury.
Previous studies have shown that nitric oxide plays a critical role in ischemic or hypoxic preconditioning-induced neuroprotection.7Although nitric oxide synthesized by different types of nitric oxide synthases (NOS; iNOS, endothelial NOS, or neuronal NOS) has been implicated in the neuroprotection induced by various preconditioning stimuli,7a recent paper suggests that iNOS may be important to mediate volatile anesthetic preconditioning-induced neuroprotection in adult rats.9It has been generally accepted that the development of delayed phase of neuroprotection requires new protein synthesis.7Previously we showed isoflurane induced increased iNOS expression that resulted in a higher amount of nitric oxide release in cultured macrophages.23In this study, we showed that isoflurane induced a time-dependent increase in iNOS protein expression in the neonatal cerebrum and that the iNOS inhibitor aminoguanidine abolished the isoflurane preconditioning-induced neuroprotection. These results suggest that iNOS plays an important role in the delayed phase of ischemic tolerance induced by isoflurane preconditioning in the neonates. Although a big increase of iNOS expression is often associated with inflammation and may be harmful in most of those cases,24multiple studies have shown that a relatively small increase of iNOS expression may be beneficial and plays a critical role in cardioprotection and neuroprotection induced by many preconditioning stimuli.9,14The downstream events of iNOS to induce protection as shown in the previous studies include activation of protein kinase C and mitogen-activated protein kinases that then induce synthesis of multiple protective proteins such as heat shock proteins and manganese superoxide dismutase.7,25In addition, protein kinase C can also activate adenosine triphosphate sensitive potassium channels, an important effector for cardioprotection induced by many preconditioning stimuli.25Thus, further studies are needed to elucidate what the downstream events of iNOS are to mediate the isoflurane preconditioning-induced neuroprotection. Further studies are also needed to determine whether endothelial NOS or neuronal NOS plays a role in the isoflurane preconditioning-induced neuroprotection.
Although isoflurane has been used clinically for all ages of patients, a recent study showed that 7-day-old rats anesthetized by isoflurane for 6 h had widespread apoptotic neuronal death.26To determine whether isoflurane pretreatment that was used in this study induced neuronal loss, we compared the density and morphology of neurons in the retrosplenial granular cortex and the ventral posteromedial thalamic nucleus, two brain regions sensitive to isoflurane in the previous study,26in isoflurane-treated and control rats. Our results showed that there was no significant reduction of neuronal density in these brain regions of rats at 8 days after the isoflurane exposure compared with controls (cumulative neuronal loss over the 8 days would have been expected if the isoflurane pretreatment induced neuronal apoptosis). These results suggest that our isoflurane pretreatment did not induce neuronal loss/neurodegeneration observed in the previous study.26The reasons for this discrepancy are not known. It may be that 30 min of isoflurane exposure is not long enough to cause neuronal loss/neurodegeneration as 6 h of isoflurane exposure did. Alternatively, other factors such as changes in blood glucose concentration and body temperature that are often associated with prolonged isoflurane anesthesia may also contribute to the different results.
In summary, our data demonstrate that preconditioning neonatal rats with 1.5% isoflurane reduces brain hypoxia/ischemia-induced brain loss/injury in the survivors. This isoflurane preconditioning-induced neuroprotection is iNOS-mediated.