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From the *Klinik für Anästhesiolgie Klinikum der Johannes Gutenberg-Universität, Mainz, Germany; University of California Anesthesia/Critical Patient Care Veterinary Medical Teaching Hospital, Davis, California; Klinik für Anaesthesiologie Technischen Universität München, Klinikum rechts der Isar, and Institut für Pathologie der GSF, Munich, Germany.
Address correspondence and reprint requests to Monika Pape, MD, Klinik für Anästhesiolgie Klinikum der Johannes Gutenberg-Universität, Langenbeckstraße 1, 55131 Mainz, Germany. Address e-mail to pape{at}uni-mainz.de.
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Abstract |
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 Top Abstract IntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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Introduction |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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METHODS |
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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After surgical preparation, the administration of isoflurane was terminated and animals were randomly assigned to one of the following treatment groups: Group 1 (control, n = 32): fentanyl IV (bolus: 10 µg/kg, infusion: 25 µg · kg–1 · h–1) and N2O/O2 (Fio2 = 0.33); Group 2 (sevoflurane, n = 32): 2.0 vol% sevoflurane in air/O2 (Fio2 = 0.33). After an equilibration period of 52 min cerebral ischemia was induced by hemorrhagic hypotension (mean arterial blood pressure, 40 mm Hg) and temporary clip occlusion of the right common carotid artery. After 45 min, reperfusion was initiated by release of the clip and subsequent reinfusion of shed blood over 15 min. Arterial pH was maintained at physiological levels by IV infusion of bicarbonate. Vecuronium was continuously infused IV (0.1 mg/kg/min) to maintain neuromuscular blockade. Physiological variables were recorded at baseline, 45 min of cerebral ischemia (ischemia), 15 min on reinfusion of shed blood (reperfusion), and 45 min after reperfusion (recovery).
Animals were randomly assigned to one of four different postischemic observation periods of 1, 3, 7, and 28 days. After this time, animals were killed by decapitation during deep halothane anesthesia. Brains were immediately removed, frozen at –70°C, and cut into 7-µm slices. Brains of 8 untreated naive animals were identically prepared for assessment of the normal concentration of the investigated proteins (Group 3, non-ischemic rats).
Two 7-µm coronal tissue sections of each brain (bregma –2.3 mm) were stained with hematoxylin and eosin (HE) and sections were examined using light microscopy by a blinded investigator. In the ischemic hemisphere of each section the percentage of eosinophilic neurons of the hippocampal regions CA1–3 on total hippocampal neurons of these regions was calculated. The hippocampus of the non-ischemic hemisphere was also investigated for eosinophilic neurons, but the total number of neurons was not counted. In the case of severe histopathological damage (e.g., formation of scars after 28 days) hippocampal damage was defined as 100%.
Brain slices were fixed with 100% ice-cold ethanol and washed with phosphate-buffered saline (PBS) containing 0.1% Tween® 20 (PBST). Subsequently, sections were incubated in blocking buffer (10% fetal calve serum in PBST) for 60 min to prevent unspecific binding of the antibodies. Thereafter, slices were incubated with the primary antibody (rabbit polyclonal antibodies; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h using antisera specific for Bax (I-19 and P-19, 1:80 each), Bcl-2 (
C21, 1:60), Mdm-2 (C18, 1:60), and p53 (FL-393, 1:80). Sections were then washed with PBST and incubated for 1 h with the secondary antibody (Alexa Fluor®488 goat anti-rabbit IgG antibody, Molecular Probes, Leiden, Netherlands, 1:800) and washed with PBST. After staining, sections were covered with mounting medium (Vectashield®H-1000; Vectorlabs, Burlingame, CA) and cover slips, and stored at 4°C. With every staining, negative controls were performed by omitting the primary antibody to detect nonspecific fluorescence. Tissue sections of rat liver were used as a positive control as the proteins Bax, Bcl-2, Mdm-2 and p53 are constantly expressed in this tissue (5). Within the next 24 h, immunofluorescence intensity of the proteins (2 slices per protein) was recorded in the CA1–CA3 regions of hippocampus of the ischemic and non-ischemic hemisphere (4 images per hemisphere) using a confocal laser scanning microscope (LSM 410, Carl Zeiss, Jena, Germany). Constant intensity of the laser light was controlled using a powermeter. With the KS400-software (Carl Zeiss Vision, Jena, Germany) images were evaluated marking the hippocampal neurons of the CA1–CA3 regions. Within these marked neurons the mean intensity of immunofluorescence (gray levels) was determined, which is proportional to the concentration of the fluorescence marker and therefore proportional to the mean protein-concentration. From the signal of the marked neurons the background intensity was substracted.
Frozen brain sections were fixed in 4% paraformaldehyde and washed with PBS. To block endogenous peroxidase, sections were immersed in 3% H2O2 in methanol and washed in PBS. To prevent unspecific binding of the antibodies, sections were incubated with blocking solution (Dako Protein Block Serum-Free, Hamburg, Germany). Sequential staining of each primary antibody was performed for double immunostaining. The active form of caspase-3 was detected with purified rabbit anti-active caspase-3 monoclonal antibody (Clone C92-605, BD Biosciences, Franklin Lakes, NJ, 1:200), which was incubated for 50 min. After washing in PBS, sections were incubated for 60 min with the secondary antibody (Universal-LSAB TM Kit, DAKO, Hamburg, Germany). Brain sections were then incubated with streptavidin conjugated horseradish peroxidase (Universal-LSAB TM Kit, DAK) and washed again in PBS. Chromogen substrate (DAKO) was incubated, until sufficient staining intensity was achieved. Peroxidase reaction was stopped with several PBS washes. Sections were then incubated with a purified monoclonal mouse anti-neuron-specific nuclear protein antibody (NeuN; Chemicon International, Temecula, CA, 1:500) for neuronal staining for 60 min. After rinsing, sections were incubated with a biotinylated horse anti-mouse antibody (Vector Laboratories, 1:200) for 60 min, followed by incubation with an alkaline phosphatase streptavidin as a ready-to-use-solution (Vector Laboratories) and then by reaction with red chromagen (Vektor Red Alkaline Phosphatase Substrate Kit I, Vektor Laboratories). Sections were dehydrated through graded ethanol, cleared in a xylene substitute (Roti®-Histol, Carl Roth GmbH & CO, Karlsruhe, Deutschland), mounted in Roti®-Histokitt (Carl Roth GmbH & CO), and coverslipped. Sections without primary antibodies were similarly processed to control for unspecific binding of the secondary antibodies. Naive rat brain was used as a negative control. Cells that were double positive for activated caspase-3 and NeuN were counted in the CA1–3 regions of the hippocampus. Their number was compared to the total amount of neurons of the CA1–3 regions.
Continuous variables are presented as mean ± sd. Inferential statistics between factors were assessed by analysis of variance models in the context of general linear model. Post hoc Student's t-test using Bonferroni adjustment was performed for bivariate comparison. Greenhouse-Geisser correction was applied if sphericity assumption was not met. The following models were performed: physiological variables were analyzed via a full factorial repeated-measures model using 4 repetitions (baseline, ischemia, reperfusion, and recovery) as within-subject factor and day (4 factors) and intervention group (2 factors) as between-subject factors. The concentration of apoptosis-related proteins (Bax, Bcl-2, Mdm-2, and p53) was recorded on ischemic and non-ischemic hemisphere. Full factorial repeated measures model was applied with within-subject factor hemisphere and between-subject factors group and day. The factor group was extended to naive rats (n = 8, untreated, 1 time point only) without cerebral ischemia to account for apoptotic cells resulting from ischemia. For the investigation of HE and activated caspase-3 a univariate analysis of variance model was conducted. Group (naive, control, and sevoflurane) and day (1,3,7,28) were treated as between-subject factors. Statistical analyses were performed using SPSS 11.5 (SPSS, Chicago, IL). All tests were performed two sided on a 5% level of significance. P values <0.001 were recorded as <0.001.
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RESULTS |
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During cerebral ischemia, red blood cell flow velocity (CBF) was reduced by 50% in the ischemic hemisphere and by 25% of baseline in the contralateral hemisphere. CBF returned to baseline levels after reperfusion (Fig. 1).
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In sevoflurane-anesthetized rats, no eosinophilic neurons were detected. In contrast, eosinophilic neurons were present in the ischemic hippocampus of control animals during the entire observation period (percentage of eosinophilic neurons 27% ± 43% day 1, 32% ± 34% day 3, 38% ± 45% day 7, 9% ± 16% day 28). On day 3, the difference between the groups was statistically significant (Fig. 2). The non-ischemic hippocampus of all groups did not reveal any histopathological neuronal damage.
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On days 1 and 3, the concentration of the pro-apoptotic protein Bax was larger in both hemispheres of control animals compared with the sevoflurane group (P < 0.001), whereas the anti-apoptotic protein Bcl-2 was decreased (P < 0.014, day 1, P < 0.001, day 3) in the ischemic hemisphere of control animals. In the non-ischemic hemisphere of control animals, Bcl-2 was smaller on days 1 (P < 0.063) and 3 (P < 0.001) (Fig. 3).
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Sevoflurane anesthesia was associated with larger concentrations of the anti-apoptotic Mdm-2 in the ischemic hemisphere on day 1 (P < 0.013) and 3 (P < 0.009) and on days 1 (P < 0.014), 3 (P < 0.010), and 7 (P < 0.005) in the non-ischemic hemisphere compared with control animals (Fig. 3). P53 concentration did not differ between the groups on days 1, 7, and 28 but was increased in the ischemic hippocampus of sevoflurane-treated animals on day 3.
Activated caspase-3 was present in neurons of both groups without significant differences between the groups (Fig. 4). However, the overall treatment effect of sevoflurane on activated caspase-3 over the time was significant (P < 0.029). In the non-ischemic hemisphere of both groups and in naive animals, no localized activated caspase-3 immunoreactivity was present.
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DISCUSSION |
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There are two major mechanisms of ischemic neuronal death: necrosis and apoptosis (7). Necrosis, as a passive form of cell death, is the main mechanism of neuronal death resulting from acute cerebral injury, such as brain infarction or acute circulatory arrest leading to insufficient oxygen supply, followed by breakdown of aerobic metabolism and depletion of adenosine triphosphate (8). Morphologically, necrosis is characterized by cell swelling, nuclear pyknosis, karyolysis, increased cytoplasmic eosinophilia, and inflammatory response (9). Sevoflurane completely prevented the eosinophilic damage (detected by HE staining) of the brain seen in the ischemic hippocampus of control animals over 28 days. As an anionic dye, eosin stains acidophilic agents in damaged neurons that occur during ischemia in the context of a mismatch between oxygen demand and supply. Therefore, eosinophilic staining of neurons is a valid indicator of the extent of damage caused by cerebral ischemia, and eosinophilic neurons are considered moribund, if not already dead (10,11). In rats subjected to cerebral ischemia sevoflurane reduced histopathological damage and improved neurologic outcome (1,2). These neuroprotective effects of sevoflurane have been attributed to a reduction of cerebral metabolism and, thereby, a re-balancing of the relation between energy requirement and supply during cerebral ischemia. Furthermore, sevoflurane reduces the release of cerebral catecholamines and excitotoxic neurotransmitters such as glutamate, minimizing deleterious stimulation of the neurons (12). Sevoflurane inhibited neuronal eosinophilic damage for 28 days after incomplete hemispheric cerebral ischemia. In contrast, in a rat focal ischemia model isoflurane did not lead to sustained neuroprotection after 7 days (13). This suggests that the severity of focal ischemia is beyond the anesthetic potential to block necrosis. However, anesthetics do provide sustained neuroprotection in the presence of less severe low-flow ischemic states, as observed in the present study (14). Isoflurane anesthesia before an ischemic insult can increase the tolerance towards middle cerebral artery occlusion in rats. This effect is described as preconditioning (15). As isoflurane was given for 1 hour during the preparation period, preconditioning with isoflurane cannot be excluded in the present study. However, all treatment groups received isoflurane and, therefore, an overall effect of preconditioning should have had no influence on the comparability of both groups.
In contrast to necrosis, apoptosis is an active and complex process triggered by protein-regulated programmed cell death, which typically shows shrinkage of the cell body and cytoplasm with membrane blebbing, followed by nuclear chromatin condensation (9). There are several proteins regulating and executing apoptosis. Bax, a 21 kD protein, is a promoter of apoptosis, which is counteracted by the anti-apoptotic protein Bcl-2 (16). Upregulation of Bax and downregulation of Bcl-2 are governed by p53, which is activated by oxidative modification and fragmentation of DNA, e.g., in the sequel of oxidative stress after ischemia and reperfusion. Moreover, it was demonstrated in M1-myeloid leukemia cells that p53 also regulates the transcription of the anti-apoptotic protein Mdm-2 (17). The activity of p53 protein and the transcription of the Mdm-2 gene are regulated by a mutual feedback loop (18). The homologous proteins Bax and Bcl-2 are located in the outer and inner mitochondrial membrane (19). Over-expression of Bcl-2 prevents ischemic neuronal damage. Cell protective properties of Bcl-2 are related to (1) regulation of Ca2+ transport across mitochondrial membranes (19), (2) protection against breaches in the membrane of the mitochondria (16) and (3) control of antioxidant pathways reducing oxidative stress on the membranes (20). The influence of Bax and Bcl-2 on the regulation of apoptosis also depends on their potential to build dimers, whereby the probability of formation of Bax-homodimers, Bcl-2-homodimers or Bax-Bcl-2-heterodimers depends on the relative quantity of the two proteins (16). When Bax-homodimers represent the dominating protein cluster, the death repressor activity of Bcl-2 can be counteracted and apoptotic cell death is accelerated (7), whereas excessive formation of Bcl-2-homodimers provides protection and stress-resistance of cells (21).
The present data show a significant increase in the concentration of the anti-apoptotic protein Bcl-2 in both hemispheres of sevoflurane-anesthetized animals. In a focal ischemia model over-expression of Bcl-2 reduced apoptotic cell death (22). This suggests that the upregulation of Bcl-2 relates to the use of sevoflurane and its neuroprotective effect. Analogously, the concentration of the pro-apoptotic protein Bax was significantly less in sevoflurane-treated animals compared with the control Group 1 and 3 days after the insult. The increased Bax concentration as found in the control group is consistent with the findings in another focal ischemia model in rat brains (7).
In contrast to studies in rats subjected to focal cerebral ischemia, this experiment did not show overexpression of p53 (23). However, anesthesia with sevoflurane caused an increase of the anti-apoptotic protein Mdm-2 in the ischemic hemisphere and in the non-ischemic hemisphere compared with controls. Likewise, Mdm-2 was also increased with peri-ischemic administration of the alpha2-agonist, dexmedetomidine, at 4 hours from ischemia and reperfusion (24). In the ischemic hemisphere of spontaneously hypertensive rats, Mdm-2-expression was maximally increased 24 hours after a 90-minute middle cerebral artery occlusion, which was interpreted as a component of a repair response in injured neurons (23). This repair response may be amplified from the treatment with sevoflurane by the significant increase of Mdm-2.
Interactions of Bax-homodimers with the mitochondrial membrane elicit the release of cytochrome c, which activates cytosolic pro-caspases, inducing apoptotic cell death. Apoptosomes, consisting of cytochrome c, apoptotic protease activating factor-1 and caspase-9 activate caspase-3, which degrades DNA and key substrates essential for cellular integrity resulting in the formation of apoptotic bodies (25).
The sustained presence of activated caspase-3 reflects the execution of the apoptotic program in damaged neurons. In a rat model of focal cerebral ischemia, the percentage of apoptotic cells in the infarction zone amounted to 5.4% (26). During the present study, 2.2% of all neurons of the ischemic hippocampus of control animals were positive for activated caspase-3. This finding may be related to differences in injury severity (incomplete cerebral ischemia versus focal ischemia), which is reflected by the different percentages of necrotic cells (49.9%, focal ischemia versus 26.5% in the present study). Sevoflurane anesthesia significantly reduces apoptotic cell death, as reflected by the small presence of activated caspase-3 in the ischemic hippocampus. No evidence for activation of caspase-3 was detected in the non-ischemic hemisphere and in the brain of naive rats.
In this model of incomplete cerebral ischemia with reperfusion, changes in the concentrations of apoptosis-regulating proteins are similar in the hippocampus of both hemispheres. This may be attributable to a decrease in CBF in the non-ischemic hemisphere of 25% (24) caused by the hemorrhagic hypotension during ischemia. Although the extent of CBF-reduction is above the threshold to induce neuronal necrosis in the non-ischemic hemisphere (27), it apparently induces apoptotic signals in a vulnerable brain region such as the hippocampus.
The intra-ischemic plasma glucose concentration was smaller in control animals compared with sevoflurane-anesthetized rats. This may represent a confounding factor because hyperglycemia as well as hypoglycemia may worsen outcome from cerebral ischemia. However, studies in laboratory animals have shown that neuronal injury occurs only at a plasma glucose concentration <40 mg/dL (28). Likewise, electroencephalographic examination did not reveal seizure activity at any time and the smaller plasma glucose concentration occurred during but not after cerebral ischemia. This suggests that hypoglycemic injury is not a major factor determining outcome during these experiments. Arterial blood pressure was lower during baseline and recovery in animals anesthetized with sevoflurane. This may have contributed to secondary brain damage. However, the sevofluraneanesthetized rats showed no necrosis and more anti-apoptotic promoters. It is, therefore, unlikely that the differences in arterial blood pressure contributed to differences seen in outcome between the two groups.
In summary, this is the first report of complete and sustained inhibition of neuronal damage after incomplete cerebral ischemia with reperfusion in rats anesthetized with sevoflurane. Moreover, sevoflurane seems to promote anti-apoptotic pathways after cerebral ischemia. Although the caspase-3 dependent apoptotic pathway appears to be of minor importance in this model, sevoflurane reduced the activation of caspase-3. These data indicate the presence of neuroprotection by sevoflurane.
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Footnotes |
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Supported, in part, by a grant from Else Kröner-Fresenius-Stiftung, Bad Homburg v.d. Höhe, Germany and by a grant from Abbott GmbH, Wiesbaden, Germany.
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TopAbstractIntroductionMETHODSRESULTSDISCUSSIONREFERENCES |
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