(Stroke. 1995;26:305-311.)
© 1995 American Heart Association, Inc.
Articles |
N-Acetylcysteine Enhances Hippocampal Neuronal Survival After Transient Forebrain Ischemia in Rats
From the Department of Clinical Neuroscience, Program in Neurosurgery, Rhode Island Hospital/Brown University, Providence, RI.
Correspondence to Neville W. Knuckey, 110 Lockwood St, Providence, RI 02903.
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Abstract |
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Background and Purpose Free radical scavengers enhance neuronal survival in some models of transient forebrain ischemia. Recent experiments have suggested that N-acetylcysteine prevents cellular injury after a reperfusion injury. No information is available regarding the neuroprotective potential of the free radical scavenger N-acetylcysteine after transient forebrain ischemia. In this study we evaluated the potential of N-acetylcysteine to improve hippocampal neuronal survival after transient forebrain ischemia in the rat.
Methods In series A and B, ventilated, paralyzed, normothermic rats had 10 minutes of transient forebrain ischemia induced by bilateral carotid occlusion with hypotension induced by blood withdrawal (mean arterial blood pressure, 45 mm Hg). In series A, animals were administered N-acetylcysteine (163 mg/kg) 30 minutes and 5 minutes before transient forebrain ischemia. In series B, N-acetylcysteine (326 mg/kg) was administered 15 minutes after transient forebrain ischemia. In series C, N-acetylcysteine (326 mg/kg) was administered 15 minutes after transient forebrain ischemia in animals with a mean arterial blood pressure of 30 mm Hg during transient forebrain ischemia. All series had normal control, sham, and vehicle treatment groups. In all series, the rats were allowed to recover and were killed at 7 days after ischemia. The effect of forebrain ischemia was assessed by evaluating the number of viable neurons at bregma sections -3.3, -3.8, and -4.3 of the CA1 region of the hippocampus.
Results The results demonstrated no physiological difference among the various treatment groups. There were no differences in the number of viable neurons between the transient forebrain ischemia with no treatment group and the vehicle (saline)-treated transient forebrain ischemic groups. Animals pretreated with N-acetylcysteine (mean number of neurons, 84±6) had a significant increase (P<.05) in neuronal survival compared with vehicle-treated animals (mean number of neurons, 43±4). Animals posttreated with N-acetylcysteine (mean number of neurons, 89±9) had a significant increase in neuronal survival compared with vehicle-treated animals (mean number of neurons, 7±1). However, N-acetylcysteine protection was only partial at 45 mm Hg and did not improve neuronal survival (mean number of neurons, 22±3) in animals with a more severe ischemic insult (mean arterial blood pressure, 30 mm Hg during transient forebrain ischemia) compared with vehicle-treated animals (mean number of neurons, 10±1).
Conclusions N-Acetylcysteine partially improved neuronal survival when administered before or after ischemia following transient cerebral ischemia (mean arterial blood pressure, 45 mm Hg) but not with a more severe ischemic insult of 10 minutes of transient cerebral ischemia with a mean arterial blood pressure of 30 mm Hg.
Key Words: acetylcysteine • cerebral ischemia, transient • free radicals • hippocampus • rats
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Introduction |
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Transient forebrain ischemia secondary to systemic hypotension is common after head injury, cardiac arrest, and shock. Thirty percent of head-injured patients have an episode of significant systemic hypotension.1 The clinical importance of the systemic hypotension is manifested by multiple memory deficits in these patients compared with head-injured patients with no systemic hypotension.1 The memory deficits result from the neuronal damage to the CA1 region of the hippocampus.2 3 The CA1 human hippocampal damage is reproducible in rat animal models of transient forebrain ischemia, which makes these models suitable to investigate potential treatment options. Treatment modalities that enhance neuronal survival after transient forebrain ischemia could significantly improve patient outcome.
The mechanisms contributing to hippocampal neuronal death have implicated increases in factors such as intracellular calcium,4 excitatory amino acid release,5 6 calcium-sensitive proteases,7 lipid peroxidation,8 and free radicals.9 10 11 Despite the multiplicity of factors, there is considerable interaction among them: a common pathway in neuronal damage may be the production of free radicals.12 13 For example, Pellegrini-Giampietro et al14 illustrated that the production and release of glutamate are enhanced by free radicals. Furthermore, the neurotoxic nature of calcium-sensitive proteases and lipid peroxidation is related to the production of free radicals.15 While free radical ions have been theorized for many years to precipitate neuronal death, the increased production of free radicals in vivo recently has been demonstrated after cerebral ischemia.16 In vitro experiments have likewise revealed the importance of free radicals by demonstrating the neurotoxic nature of these ions.9 17 Hence, free radical ions may be important in the pathogenesis of neuronal death after transient cerebral ischemia.
Prevention of free radical production and damage enhances neuronal survival in some animal models of ischemia. Enhancement of the brain's endogenous scavenging system by the superoxide dismutase/catalase system has improved neuronal survival after focal ischemia,18 but results have been variable after transient forebrain ischemia. Superoxide dismutase/catalase improved neuronal survival in the gerbil19 20 but not in the dog.21 Glutathione peroxidase is an alternative but little explored endogenous scavenger system. Ebselen, which enhances glutathione peroxidase activity, decreases cortical infarct size after focal ischemia.22 An alternative compound, N-acetylcysteine (NAC), also enhances the endogenous glutathione scavenging system.23 NAC decreases free radical–induced cellular damage after various injury models such as head injury,24 endotoxin-induced lung damage,15 25 isolated liver reperfusion injury,26 and decreased cardiac rhythm disorders following cardiac ischemia and reperfusion.27 Since free radicals appear important in neuronal death, we evaluated the potential of NAC to enhance CA1 hippocampal neuronal survival after transient forebrain ischemia.
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Materials and Methods |
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Surgical Preparation
This study was approved by the Rhode Island Hospital Institutional Animal Welfare Committee. Adult male Sprague-Dawley rats (Charles River, Mass) weighing 250 to 350 g were fasted overnight but allowed water ad libitum. Anesthesia was induced with 3% halothane/27% O2/balanced NO2; once the animal was sedated the halothane was reduced to 1.5%. Initially, heparin-filled (heparin 50 µL/mL) polyethylene catheters (PE-50) were placed in each femoral artery and one femoral vein. Through a longitudinal cervical skin incision, a ligature was looped around both carotid arteries. The animal was endotracheally intubated, paralyzed (pancuronium 0.02 mg/kg), supplemented as required, and ventilated on a Harvard rodent ventilator (Harvard Apparatus) with 1.5% halothane/27% O2/balanced NO2. The rat was placed in a stereotaxic frame, and thermostatic probes (Tele-Thermometer, Yellow Scientific Instrument) were placed in the temporalis muscle and rectum. Brain temperature was maintained at 37.0±0.5°C. A bipolar electroencephalogram (EEG) was recorded by one reference central scalp needle electrode and two active lateral electrodes, which were interfaced with a DAM 50 differential amplifier (World Precision Instruments). The EEG and blood pressure were recorded with a MacLab (Analog Digital Instruments) data acquisition system. The animal was measured for brain temperature, blood pressure, arterial blood gases, and blood glucose (Life Scan, Johnson and Johnson). The physiological parameters were recorded for 15 minutes before the induction of transient forebrain ischemia.
Transient forebrain ischemia was induced by occluding both common carotid arteries, and arterial blood was withdrawn to maintain a mean arterial blood pressure (MABP) of 45 mm Hg or 30 mm Hg. Transient forebrain ischemia was recorded from the time the EEG became isoelectric and was maintained for 10 minutes. At the completion of transient forebrain ischemia, the carotid clamps were removed and the warmed blood reinfused. Fifteen minutes after transient forebrain ischemia, blood gases and blood glucose were measured, following which the arterial and venous lines were removed and wounds sutured. The animals were allowed to recover, extubated, and returned to their cages and allowed free access to food and water until they were killed.
Experimental Protocols
We explored the potential neuroprotective role of NAC (Fisher
Scientific) in three experimental series (A through C) of animals. In
series A, a group of animals was evaluated to assess the potential of
pretreatment with NAC to enhance neuronal survival followed by
transient forebrain ischemia, which was induced with an MABP of 45
mm Hg and an isoelectric EEG maintained for 10 minutes. The animals
were randomly assigned to one of four treatment groups: group 1,
control animals, no surgical procedure; group 2, sham animals in which
transient forebrain ischemia was induced but no other treatment was
administered; group 3, vehicle animals with saline (0.9% NaCl, 1.9
mL/kg IP) administered at 30 minutes and 5 minutes before transient
forebrain ischemia; and group 4, treatment animals with NAC (163 mg/kg
IP) administered 30 minutes and 5 minutes before transient forebrain
ischemia. In series B, after treatment we evaluated the administration
of NAC 15 minutes after transient forebrain ischemia, which was induced
with an MABP of 45 mm Hg. Groups 1 and 2 were the same as above. Group
5 consisted of vehicle animals with saline (1.9 mL/kg IP) administered
15 minutes after transient forebrain ischemia, and group 6 consisted of
treatment animals with NAC (326 mg/kg IP) administered 15 minutes after
transient forebrain ischemia. In series C we evaluated NAC after a more
severe ischemic insult by inducing transient forebrain ischemia with
carotid occlusion and an MABP of 30 mm Hg. This more severe ischemic
insult has been assessed by Gionet at al,28 who
demonstrated a more severe reduction in hippocampal blood flow and more
severe histological damage compared with the standard model of
hypotension and carotid occlusion. Group 7 consisted of a vehicle group
with saline (1.9 mL/kg IP) administered 15 minutes after forebrain
ischemia. Group 8 consisted of a treatment group with NAC (326 mg/kg
IP) administered 15 minutes after transient forebrain ischemia with an
MABP of 30 mm Hg.
Histological Analysis
At 7 days after transient forebrain ischemia, the animals were
anesthetized with pentobarbital (100 mg/kg) and transcardially perfused
with 0.05 mol/L phosphate-buffered saline (200 mL) followed by 200 mL
of 10% neutral-buffered formalin. The brains were removed, postfixed
in neutral-buffered formalin overnight at 4°C, and paraffin embedded.
The brains were sectioned at a thickness of 8 µm according to Paxinos
and Watson29 from bregma -3.3, -3.8, and -4.3 and were
stained with cresyl violet. Since the CA1 region of the hippocampus is
the brain region most vulnerable to ischemia, only the CA1 region was
assessed in this study. The medial, intermediate, and lateral segments
of the hippocampal CA1 region per 1000-µm lengths from bregma -3.3,
-3.8, and -4.3 were counted for viable cells with use of the criteria
of Brierley.30 The counter was blind to each treatment
group.
Data Analysis
Data in text and figures are mean±SEM values of the different
regions of the hippocampus of each group. Data analysis was by
ANOVA, followed by post hoc Bonferroni/Dunn. Significance was assumed
at P<.05. Physiological data were analyzed with the
Bonferroni/Dunn test.
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Results |
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Physiological values for experimental series A through C are summarized in Table 1
. All groups were normothermic,
with a preoperative MABP of 95 to 132 mm Hg,
PCO2 of 34 to 42 mm Hg,
PO2 of 98 to 134 mm Hg, arterial pH of 7.30
to 7.49, and blood glucose of 3.80 to 4.90 mmol/L. There were no
significant intergroup differences within the physiological variables,
and NAC did not affect arterial blood pressure.
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We initially explored the effect of the administration of NAC 30
minutes and 5 minutes before transient forebrain ischemia, using the
dose regimen of Ellis et al.24 Fig 1 shows
the results of series A (pretreatment with NAC) experiments, and the
hippocampal morphology is illustrated in Fig 2. Animals
pretreated with NAC (group 4; n=7) had a mean viable neuronal count of
84±6, which was significantly different (P<.05) from
vehicle-treated animals (group 3; n=5) with a mean viable neuronal
count of 43±4. Intrarater reliability was analyzed with the two-tailed
paired t test, and the difference was 8%, which was not
significant (P>.05).
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We next determined whether postadministration of NAC, 15 minutes after
transient forebrain ischemia, improved neuronal survival. Fig 3 shows the results of series B experiments, with
hippocampal morphology demonstrated in Fig 2
. Animals posttreated with
NAC (group 6; n=5) had a significant increase in viable neurons (number
of neurons, 89±9) compared with vehicle-treated animals (n=4) (number
of neurons, 7±1). While neuronal survival was significantly improved,
its improvement was only to 37% that of normal nonischemic animals.
Since posttreatment with NAC improved neuronal survival, we performed
experiments with a more severe ischemic insult (Fig 3). In this group
of animals, series C (posttreatment with NAC and MABP=30 mm Hg), we
found no significant neuronal protection in the animals treated with
NAC (group 8; n=5), with 22±3 neurons per 1000 µm compared with the
vehicle-treated animals (n=4) (number of neurons, 10±1).
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We also analyzed whether there was any regionality to the
neuroprotection of the postadministration/preadministration of
NAC. The results in Table 2 demonstrate no significant
differences in neuronal survival at the three bregma sections -3.3,
-3.8, and -4.3. Similarly, we found no significant difference between
the left and right hippocampi at bregma -3.8 or within the lateral,
intermediate, and medial segments of the CA1 region of the hippocampus
at bregma -3.8 with respect to NAC-related neuroprotection.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transient forebrain ischemia results in death of the neurons in the CA1 region of the hippocampus. In this study we demonstrated that NAC partially improved neuronal survival within the CA1 region of the hippocampus after transient forebrain ischemia. After 10 minutes of transient forebrain ischemia at an MABP of 45 mm Hg, the administration of NAC before or after ischemia significantly improved hippocampal neuronal survival compared with saline-infused ischemic animals. However, NAC administered after a more severe ischemic insult (MABP, 30 mm Hg) did not significantly improve CA1 hippocampal neuronal survival.
Although this study was not designed to address the mechanisms of neuronal death or the mechanisms of action of NAC, there is considerable evidence in the literature to suggest that free radicals are important in neuronal death and that these radicals are scavenged by NAC or its metabolites. Multiple free radicals are generated after transient forebrain ischemia.31 Indirect evidence of free radicals was demonstrated by a decrease in the brain's level of free radical scavengers such as glutathione and ascorbic acid.32 Direct evidence of free radicals was demonstrated by the spin-trapping technique of ischemic brain perfusate.16 The superoxide ion is the initial free radical generated after transient forebrain ischemia.33 34 35 The superoxide ion is produced by the metabolism of arachidonic acid by cyclooxygenase and lipoxygenase17 and by the metabolism of hypoxanthine to xanthine after the activation of xanthine dehydrogenase to xanthine oxidase.36 The superoxide ion is rapidly metabolized by superoxide dismutase to H2O2.9 Furthermore, the superoxide ion exists in equilibrium with the hydroperoxyl radical, which is favored in the acidic brain environment after transient forebrain ischemia.
The mechanism by which NAC improves neuronal survival in our model is unknown, but the literature suggests that the scavenging of free radicals is the most likely mechanism of action. A potentially important direct function of NAC is scavenging free radicals that are generated during transient ischemia. In vitro experiments have demonstrated that NAC, a thiol-containing compound, scavenges the hydroxyl radical22 that is generated after forebrain ischemia. NAC also has indirect free radical scavenging potential because NAC is deacetylated to cysteine (a thiol reducing agent), which supports glutathione biosynthesis.21 Glutathione is an important natural brain free radical scavenger that is depleted after transient forebrain ischemia.32 The increased systemic production of glutathione may replenish tissue supplies of glutathione.37
While the scavenging of free radicals is the most likely mechanism of action of NAC, the literature suggests several other possibilities, which may account for its neuroprotective action. Ischemic-reperfusion injury has a deleterious effect on the microvascular and endothelial function38 39 that may be ameliorated by NAC. The disturbance of endothelial cells and the accumulation of neutrophils after ischemia results in stimulation of nitric oxide synthase and generation of nitric oxide.40 The nitric oxide reacts with the superoxide ion, with the production of peroxynitrate.41 The increased production of nitric oxide appears to be detrimental for neuronal survival because inhibition of nitric oxide synthase decreases cortical infarction.42 Ellis et al24 demonstrated that administration of NAC either before injury or after injury restores cerebrovascular reactivity following concussive brain injury. NAC improves endothelial function and inhibits the endothelial/neutrophil interaction and platelet-derived factor activation, both of which will precipitate further free radical formation after recirculation ischemia.15 Our study has demonstrated that NAC partially improved neuronal survival, suggesting that free radicals are potentially involved in neuronal death, but our study was not designed to determine its mechanism of action. Further experimentation is required to determine which potential action of NAC is the most critical in our model of transient forebrain ischemia.
The enhancement of neuronal survival by free radical scavengers has
been evaluated in many animal models and with different drugs. The
results have been variable and, similar to our results, have not
demonstrated total neuron protection. Uyama et al20 used
the brain's natural defense mechanisms to demonstrate the
neuroprotective effect of human recombinant superoxide dismutase by
decreasing CA1 hippocampal neuronal loss in gerbils. However, complete
global ischemia in the dog model revealed no improvement in
neurological outcome after administration of superoxide dismutase and
catalase.21 An alternative approach is the synthetic 21
amino acids (U74006F, tirilazad mesylate), which have had variable
success in preventing neuronal damage after transient global ischemia.
Hall43 reported the efficacy of U74006F in decreasing
hippocampal damage in the gerbil but not the rat carotid hypotension or
four-vessel occlusion model of transient global ischemia. Our results
have demonstrated that NAC partially improves neuronal survival at the
administered dosage. While our results suggest that free radicals are
potentially involved in neuronal death, we found only 37% neuronal
survival compared with nonischemic animals. The results suggest that
other mechanisms are also important in neuronal death. For example, the
-amino-3-hydroxy-5-methyl-4-isoxasole proprionic acid (AMPA)
receptor inhibitor NBQX
[2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)quinoxaline] results in
70% neuronal preservation,44 and the protease inhibitor
leupeptin results in 80% neuronal preservation.45
Although caution must be exercised in comparing results in different
animals with different routes of drug administration, the results taken
together suggest that neuronal death is multifactorial and suggest that
a combination of different drugs may be required to obtain optimal
neuronal survival. The dose used in this study was the maximum
previously reported dose that has been demonstrated in other
reperfusion models to ameliorate cellular injury. This study does not
address the possibility that higher doses, different dose scheduling,
or different routes of administration of NAC may improve the degree of
neuronal protection.
In summary, we demonstrated that NAC partially improved hippocampal neuronal survival after transient forebrain ischemia. Our results suggest that free radicals are involved in the pathogenesis of neuronal death after ischemia, but since the protection is only partial our results also suggest that free radicals are part of a complex interaction of neurotoxic factors that induce neuronal death after transient forebrain ischemia.
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Acknowledgments |
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The authors appreciate the assistance of Anthony Spangerberger for photography, Carole Thompson for graphs, and Rhode Island Hospital for research funds.
Received June 1, 1994; revision received August 26, 1994; accepted October 21, 1994.
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