Anesth Analg 2007;104:898-903
© 2007 International Anesthesia Research Society
doi: 10.1213/01.ane.0000255730.73018.31
NEUROSURGICAL ANESTHESIA
Section Editor:
Adrian W. Gelb
Sevoflurane Affects Neurogenesis After Forebrain Ischemia in Rats
Kristin Engelhard, MD*,
Uta Winkelheide, DVM*,
Christian Werner, MD*,
Julia Kluge, DVD
,
Eva Eberspächer, DVM
,
Regina Hollweck, Dipl Stat
,
Peter Hutzler, PhD||,
Jürgen Winkler, MD¶, and
Eberhard Kochs, MD
From the *Klinik für Anästhesiologie, Johannes Gutenberg-Universität, Mainz; Klinik für Anaesthesiologie, Technische Universität, Munich, Germany; Veterinary Medical Teaching Hospital, UC Davis, California; Institut für Medizinische Statistik und Epidemiologie, Technische Universität, Munich; ||Institut für Pathologie des GSF-Forschungszentrums, Neuherberg, Germany; and ¶Klinik für Neurologie, Universität Regensburg, Regensburg, Germany.
Address correspondence and reprint requests to Kristin Engelhard, MD, Klinik für Anästhesiologie, Johannes Gutenberg-Universität, Langenbeckstr. 1, 55131 Mainz, Germany. Address e-mail to engelhak{at}uni-mainz.de.
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Abstract
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BACKGROUND: The effect of sevoflurane on the neuroregenerative potential after neuronal injury is unclear. We investigated the effect of low and high concentrations of sevoflurane on endogenous neurogenesis after cerebral ischemia.
METHODS: Anesthetized and ventilated rats were randomized to four different treatment groups. Groups 1 and 2: 1.4% sevoflurane; Groups 3 and 4: 2.8% sevoflurane. In Groups 1 and 3, no cerebral ischemia was induced (sham-operated). In Groups 2 and 4, 10 min of forebrain ischemia was induced by bilateral carotid artery occlusion plus hemorrhagic hypotension. Physiological variables were maintained constant. Bromodeoxyuridine was given as a marker of neurogenesis. After 28 days brains were perfused. Histopathological damage of the hippocampus was evaluated in hematoxylin and eosin (HE) stained sections using the HE-index (0 = no damage; 1 = 1%–10% damage; 2 = 11%–50% damage; 3 = 51%–100% damage). Immunohistochemistry was used to detect bromodeoxyuridine-positive neurons. Eight untreated rats were investigated as naive controls (Group 5).
RESULTS: In neither sham-operated group was histopathological damage or change in neurogenesis observed compared to naive controls. In rats anesthetized with 1.4% sevoflurane, cerebral ischemia caused mild neuronal damage (HE-index of 0.64 ± 0.84) and increased neurogenesis by 60% when compared with respective sham-operated animals; with 2.8% sevoflurane, the HE-index was 1.22 ± 1.14, and the number of newly generated neurons increased by 230% when compared with respective sham-operated animals.
CONCLUSION: The present data suggest that high concentrations of sevoflurane stimulate neurogenesis in the dentate gyrus after cerebral ischemia.
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Introduction
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The notion that the adult mammalian central nervous system is incapable of significant self-repair or regeneration was revisited after the detection of self-renewing and multipotent neuronal stem cells. By cell division, stem cells produce progenitor cells which then rapidly generate new neurons, astrocytes, and oligodendrocytes in the adult brain (1). The subventricular zone of the lateral ventricle and the subgranule layer of the dentate gyrus seem to be the most important regenerative centers (2). New cells descending from the subventricular zone migrate into the olfactory bulb via the rostro-migratory stream (3). Cells derived from the subgranular layer of the dentate gyrus move into the granular layer of the dentate gyrus and sprout dendrites to the CA3 region of the hippocampus (4).
Several stimuli can activate the proliferation of progenitor cells such as social contact, exercise, inflammation, or cerebral ischemia (5–7). While some of these proliferated cells undergo apoptotic cell death, others survive. The effects of anesthetics on neurogenesis in the presence and absence of cerebral ischemia is unclear. This study investigates the effect of sevoflurane on the amount of new neurons in the dentate gyrus of rats with or without cerebral ischemia. We hypothesized that sevoflurane would interact, in a dose-dependent fashion, with ischemia to augment neurogenesis.
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METHODS
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Preparation
After approval of the Institutional Animal Care Committee, 40 fasted male Sprague-Dawley rats (370 ± 30 g) were included in the present study. Thirty-two rats were anesthetized in a bell jar saturated with sevoflurane, intubated and mechanically ventilated (Paco2 = 38–42 mm Hg) with 2.0 vol % sevoflurane in oxygen and air (Fio2 = 0.33). Catheters were inserted into the right femoral artery and vein and into the right jugular vein for measurements of mean arterial blood pressure (MAP), blood withdrawal, blood gas analyses, and drug administration. Loose ligatures were placed around the right and left common carotid artery for later clamping. Temperature sensors were inserted into the right temporal muscle and pericranial temperature was maintained constant at 37.5°C using a servo-controlled overhead-heating lamp. Respiratory variables, arterial blood gases, arterial pH, and plasma glucose concentration were monitored and maintained constant during the experiment. Upon completion of the surgical preparation, all incisions were infiltrated with 0.5% bupivacaine (0.1–0.2 mL).
Cerebral Ischemia
The animals were randomly assigned to one of the following treatment groups: Group 1 (n = 8) 1.4 vol % end-tidal sevoflurane concentration, sham-operated animals; Group 2 (n = 8) 1.4 vol % end-tidal sevoflurane concentration, cerebral ischemia; Group 3 (n = 8) 2.8 vol % end-tidal sevoflurane concentration, sham-operated animals; Group 4 (n = 8) 2.8 vol % end-tidal sevoflurane concentration, cerebral ischemia. The concentrations represent the lowest and highest sevoflurane concentration used for neurosurgical patients. Animals of the four groups were ventilated with air and oxygen (Fio2 = 0.33). Additionally, all rats received IV fentanyl (bolus 10 µg/kg, infusion 25 µg · kg–1 · h–1) because 1.4 vol % sevoflurane did not provide adequate analgesia. After randomization to one of the four groups, the sevoflurane concentration was adjusted accordingly, followed by an equilibration period of 30 min. Cerebral ischemia was induced in animals of Groups 2 and 4 by clip-occlusion of both common carotid arteries in combination with hemorrhagic hypotension to a MAP of 40 mm Hg for 10 min. At the end of ischemia, clips were removed and the withdrawn blood was reinfused slowly over 15 min to avoid cerebral hyperemia and systemic hypertension. The physiological variables of animals of the four treatment groups were recorded at four points in time: before hemorrhagic hypotension (baseline), at the end of 10 min of cerebral ischemia (ischemia), 15 min after cerebral ischemia upon reinfusion of the withdrawn blood (reperfusion), and 15 min after the end of reperfusion (recovery). In animals of the sham groups (Groups 1 and 3), physiological variables were recorded at the corresponding time points. At the end of recovery, catheters were removed and the wounds were closed. The total time of sevoflurane administration was 100 min (time points are listed in Table 1). Brains of eight nonanesthetized and nonischemic animals acted as reference to quantify the natural rate of neurogenesis (Group 5, naive).
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Table 1. Time Intervals in Sequence for Each Treatment Procedure. For Sham-Operated Animals, the Cerebral Ischemia was Not Performed, But the Same Time Intervals were Chosen
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Bromodeoxyuridine Labeling and Tissue Preparation
Bromodeoxyuridine (BrdU), a thymidine analog which replaces thymidine in newly synthesized DNA, was used to label endogenous proliferating cells. In animals of the four treatment groups, and in naive animals, BrdU (100 mg/kg) was injected IP for 7 consecutive days starting with the first postischemic day. After 28 days, the animals were deeply anesthetized and transcardially perfused with 100 mL saline 0.9%, followed by 50 mL paraformaldehyde 4% (PFA) in 0.2 M phosphate buffer (pH 7.4). The brains were removed, postfixed for 24 h in PFA-PB and placed for 48 h in 30% sucrose. Sagittal sections (40 µm) of the brain were stored in a cryo-protection solution (glycerol, ethylene glycerol, and 0.1 M phosphate buffer) at –20°C.
Immunohistochemistry
This staining technique was used to count the total amount of BrdU positive cells in the dentate gyrus. To denaturize the DNA, the free-floating brain sections were exposed to 50% formamide and a 2 x saline-sodium citrate buffer at 65°C for 1–2 h. The sections were then rinsed in tris buffered saline (TBS) and incubated in 2 N HCl at 37°C for 30 min, followed by an incubation with 0.1 M boric acid (pH 8.5) at room temperature for 10 min to neutralize the pH. The sections were rinsed again with TBS for 20 min and treated with donkey serum 3% in TBS and TritonX-100 0.1% at room temperature for 30 min. The sections were incubated overnight at 4°C with the primary anti-BrdU antibody (mouse-monoclonal IgG; Roche Molecular Biochemicals, Indianapolis) diluted in TBS with 3% donkey serum and 0.1% TritonX-100 (1:500). After being washed in TBS for 30 min, the sections were incubated in biotinylated secondary donkey anti-mouse antibody (biotin-SP-conjugated, donkey anti-mouse IgG, Jackson ImmunoResearch Laboratories, West Grove) diluted in TBS with 3% donkey serum and 0.1% TritonX-100 (1:500) for 1 h at room temperature. The sections were rinsed in TBS for 30 min and afterwards incubated with an avidin–biotin peroxidase solution for 1 h at room temperature. After another washing period, the sections were incubated in a solution of 20 mg/mL diaminobezidine, H2O2 0.01%, and NiCl2 0.04%. Sections were rinsed in water and then mounted on glass slides. Using a light microscope, the immunohistochemically stained BrdU-positive cells in the subgranular and granular zone of the dentate gyrus were counted in every 10th section. To calculate the total amount of newly generated cells in the dentate gyrus, the results were multiplied by 10. Positive and negative controls were performed to validate the immunohistochemistry staining.
Immunofluorescence-Double Staining
Slices with immunofluorescence-double staining were used to calculate the ration between BrdU+NeuN positive cells and the total amount of BrdU positive cells. All steps for the immunofluorescence-double staining, up to the incubation with the primary antibody, were performed identically to the immunohistochemical staining. The sections were then incubated overnight at 4°C with the primary rat anti-BrdU-antibody (1:500, rat-monoclonal IgG, Oxford Biotechnology, UK) and the mouse anti-NeuN-antibody (1:250, mouse-monoclonal IgG, Chemicon International, Temecula, CA) diluted in TBS with donkey serum 3% and TritonX-100 0.1%. After washing with TBS for 20 min, the sections were incubated in a secondary antibody mix (1:500; fluorescein-conjugated donkey anti-rat IgG, Jackson ImmunoResearch Laboratories, West Grove and rhodamine red-X-conjugated IgG, donkey anti-mouse, Jackson ImmunoResearch Laboratories, West Grove, USA) in TBS with donkey serum 3% and TritonX-100 0.1% for 2 h. Sections were rinsed in TBS and then mounted on glass slides. The analysis of the sections was performed using an Axiovert 200 immunofluorescence microscope (Zeiss GmbH, Göttingen, Germany) combined with an ApoTome (Zeiss GmbH, Göttingen, Germany) and the Axiovision software (Zeiss GmbH, Göttingen, Germany). For each hemisphere, 50 BrdU-positive cells were analyzed for coexpression of BrdU and NeuN to determine the ratio of newly generated neurons (BrdU + NeuN) to the total amount of newborn cells (BrdU). These ratios were then multiplied by the total amount of BrdU-positive cells from the immunohistochemical staining to calculate the absolute number of newborn neurons in the dentate gyrus (7). Positive and negative controls and tests for excluding cross-reactions for the two secondary antibodies were performed.
Histopathological Damage and Volume of the Dentate Gyrus
In brain sections, stained with hematoxylin and eosin (HE), histopathological damage in the hippocampal CA1 and CA3 regions was assessed using light microscopy. The amount of injured neurons (cytoplasmic eosinophilia and pyknotic nuclei) was graded according to the following score (HE-index): 0 = no pathologic change; 1 = 1%–10% of hippocampal neurons show pathologic changes; 2 = 11%–50% of hippocampal neurons show pathologic changes; 3 = more than 50% of hippocampal neurons show pathologic changes. The area of the dentate gyrus was measured in every 10th section using the Image-pro-express software (version 4.5, Media Cybernetics®, Silver Spring). The volume of the dentate gyrus was calculated by multiplying the result with the thickness of one slice (0.04 mm) and by 10 (as only every 10th slice was used).
Statistical Analysis
Continuous variables are presented as mean ± sd. Comparability of treatment groups with respect to physiologic variables were analyzed by Kruskall– Wallis test and in case of significance the bivariate Mann–Whitney test was applied. Wilcoxon’s test was used to detect differences in physiological variables between baseline and ischemic events within each group. Primary end points, such as histopathological damage, total number of newly generated cells or neurons, and the volume of the dentate gyrus were analyzed by Kruskall–Wallis test and post hoc Mann–Whitney test to detect differences among the groups. Bonferroni adjustment was used throughout the analysis to account for multiple testing. Spearman correlation coefficient was calculated to assess the relationship between histopathological damage of the hippocampal CA1 region and the amount of BrdU and NeuN positive cells. All tests were performed two-tailed on a 5% level of significance. Statistical analyses were performed using SPSS 12.0.
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RESULTS
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MAP, arterial blood gas tensions, hemoglobin concentration, and plasma glucose concentration are listed in Table 2. MAP was not different among groups. The arterial oxygen partial pressure was lower in sham-operated animals receiving 2.8 vol % sevoflurane (Group 3) compared to those with 1.4 vol % sevoflurane (Group 1). During ischemia, the Paco2 decreased when compared with baseline in ischemic animals receiving high sevoflurane concentrations (Group 4). In ischemic rats receiving 2.8% sevoflurane (Group 4), the Paco2 was increased during reperfusion and recovery compared to rats receiving 1.4% sevoflurane (Group 2). The hemoglobin concentration and plasma glucose concentration were comparable in animals with cerebral ischemia (Groups 2 and 4).
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Table 2. Mean Arterial Blood Pressure (MAP), Arterial Blood Gas Tensions (Pao2 and Paco2), Hemoglobin Concentration (Hb), and Plasma Glucose Concentration (Gluc) During Baseline, Ischemia, Reperfusion, and Recovery
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Figure 1 shows the amount of newly generated neurons in the dentate gyrus of the brain 28 days after cerebral ischemia. In sham-operated animals (Groups 1 and 3) and in naive rats (Group 5), 1200–1600 new neurons were counted in the dentate gyrus of both hemispheres. After cerebral ischemia, the amount of BrdU-positive neurons increased by 230% in the dentate gyrus of animals treated with 2.8% sevoflurane compared to sham-operated animals (Group 3, statistically significant), and by 160% compared to naive rats (Group 5, not statistically significant).
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Figure 1. Newly generated neurons in the dentate gyrus 28 days after bilateral carotid artery occlusion. Sham = sham-operated animals; isch = animals with cerebral ischemia; *P < 0.05 vs sham-operated animals (data are given as mean ± sd).
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The average volume of the dentate gyrus in one hemisphere for all groups is displayed in Figure 2. The volume of the dentate gyrus was similar for all groups, independent of the sevoflurane concentration or cerebral ischemia.
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Figure 2. Volume of the dentate gyrus in one hemisphere. Sham = sham-operated animals; isch = animals with cerebral ischemia (data are given as mean ± sd).
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Figure 3 shows the histopathological damage in the hippocampal CA1-region 28 days after cerebral ischemia as assessed by HE-index. Sham-operated animals (Groups 1 and 3) and naive rats (Group 5) developed no neuronal injury. After cerebral ischemia, <10% of the hippocampus was injured in animals receiving 1.4% sevoflurane. This difference was not significant when compared with sham-operated (Group 1) or naive animals (Group 5). With 2.8% sevoflurane, the hippocampal injury was significantly higher compared to sham-operated animals (Group 3) and naive rats (Group 5). The CA3-region of the hippocampus was not affected by cerebral ischemia in any group (data not shown).
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Figure 3. Histopathological damage in the CA1-region of the hippocampus assessed by hematoxylin and eosin (HE)-index. Sham = sham-operated animals; isch = animals with cerebral ischemia; 0 = no damage; 1 = damage in 1%–10% of hippocampal neurons; 2 = damage in 11%–50% of hippocampal neurons; 3 = damage in >50% of hippocampal neurons; *P < 0.05 vs sham-operated animals; P < 0.05 vs naive animals (data are given as mean ± sd).
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There was no positive correlation between the amount of newly generated neurons and the histopathological damage (r = 0.269 and P = 0.093).
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DISCUSSION
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The present study shows that neither 1.4 nor 2.8 vol % sevoflurane affected the generation of newborn neurons in the dentate gyrus, when applied for about 100 min in the absence of cerebral ischemia (Fig. 1; Groups 1 and 3 compared to Group 5). Ten minutes of bilateral common carotid occlusion stimulated the generation of new neurons in animals treated with high but not low dose sevoflurane (Group 4 compared to sham-operated animals of Group 3), regardless of the extent of tissue damage. This suggests that neurogenesis requires a combination of stimuli (i.e., deep anesthesia and forebrain ischemia) in this animal model.
In gerbils subjected to bilateral common carotid artery occlusion, the duration of ischemia (varying from 2 to 12 min) was related to the number of new neurons (8). However, neurogenesis was also increased in ischemia-tolerant gerbils in the absence of hippocampal cell death (8). Therefore, it appears that loss of hippocampal neurons is not required for dentate gyrus neurogenesis. Accordingly, there was no positive correlation between the degree of histopathological damage and neurogenesis during the present investigation. This suggests that differences in histopathological damage between the ischemic groups (Groups 2 and 4) did not account for the number of new neurons and, therefore, did not affect the impact of sevoflurane on neurogenesis after cerebral ischemia.
Cerebral ischemia (particularly in the hippocampus) activates progenitor cells in the subgranular zone of the dentate gyrus by stimulation of hippocampal 
-aminobutyric acid (GABA)ergic interneurons (9). GABA has trophic effects on cell proliferation, migration, and neurite outgrowth of progenitor cells (10,11) and stimulation of these receptors using GABAergic drugs (e.g., valporate) increased the number of neurons by activating the proliferation of neuronal progenitors (12). In vitro studies indicate that sevoflurane, at clinical concentrations, activates GABA-receptors (13,14). It is therefore possible that sevoflurane increases the amount of new neurons after cerebral ischemia by activation of GABA receptors.
In the present study, sevoflurane did not affect neurogenesis in sham-operated animals (Groups 1 and 3) 28 days after 100 min of anesthetic exposure. In contrast, experiments in young adult Sprague-Dawley rats, 4 h of 1 MAC isoflurane anesthesia increased the number of newborn cells at 4 days, but not 9 and 16 days after anesthesia (15). This indicates that the activation of neurogenesis by isoflurane was not sustained in the absence of additional stiumuli, while the combination of a volatile anesthetic and cerebral ischemia permanently activated generation of new neurons as observed in the present study. This suggests that anesthetics induce short-term neurogenesis, while sustained effects are available only with additional stimuli.
The ischemia-induced increase of neurogenesis is small when compared with other studies. In the present study, the differentiated long-term surviving (28 days) neurons were observed, whereas other studies investigated the newly proliferated cells after 3–12 days. However, about 80% of the initially proliferated cells disappear within 4 wk, which explains the small increase in new neurons after 28 days (16). The long observation period was chosen because it was expected that after 28 days only those new generated neurons survive, which are integrated into the dentate gyrus. Clinically, only the long-term surviving neurons are of interest.
Histopathological damage was more severe with 2.8 vol % compared to 1.4 vol % sevoflurane (Fig. 3). Yet, the HE-index of 1.22 in rats receiving high sevoflurane concentrations still indicated a modest injury, with only 10% of the hippocampal CA1 region being damaged. Studies using different ischemia models showed sustained neuroprotection with 2.0–2.8 vol % sevoflurane when compared with awake or nitrous oxide/fentanyl-anesthetized control animals after various observation periods (1–28 days) (17–20). However, different concentrations of sevoflurane have never been compared, and no study has investigated the neuroprotective effect of sevoflurane concentrations below 2.0 vol %. It is, therefore, possible that sevoflurane provides more neuroprotection with low compared to high concentrations. A recent study (21) in rats confirmed these assumptions because, in a stroke model of bilateral carotid occlusion increasing concentrations of isoflurane worsen neurological and histopathological damage. This might have been related to the increased direct vasodilatory effect of higher sevoflurane concentrations (22), leading to a redistribution (steal) of cerebral blood flow to nonischemic brain regions with progressive reduction of blood flow in the ischemic territories.
BrdU is an indicator of DNA synthesis, rather than a direct mitotic marker. Its incorporation into a newly synthesized DNA may occur during the S-phase of the cell cycle before cell division as well as during the repair of damaged DNA, which is particularly increased after cerebral ischemia. However, after global ischemia in adult macaques, and after focused beam irradiation in rats, coadministration of Ki-67, which is a nuclear protein expressed in dividing cells for the entire duration of their mitotic processes, showed a similar expression pattern between BrdU and Ki-67 (23,24). Furthermore, after brain ischemia BrdU was not integrated in neurons undergoing DNA damage, as revealed by positive TUNEL staining (25). This suggests that BrdU is a reliable marker of newly generated cells in the brain.
The BrdU clearance time from the brain is 2 h (26). BrdU stains all cells during the S-phase of mitosis, which lasts about 9.5 h in young rats (26). Therefore, a time period of 21 h was covered with one daily BrdU injection, which translates into an 87% detection of the newly generated cells during the present study. To distinguish between neurons and other cells with mitotic potential, a double staining of the BrdU positive cells with a marker for differentiated neurons (NeuN) was performed in the present study. Using the ApoTome® technique, it is possible to identify the colocalization of BrdU and NeuN in the same optical slice of 1 µm. Therefore, labeling of BrdU and NeuN in the same cell reliably identifies a newborn neuron.
In conclusion, histopathological damage was increased with high compared to low sevoflurane concentrations after forebrain ischemia in rats. Likewise, neurogenesis was enhanced with high doses of sevoflurane in the presence, but not the absence, of cerebral ischemia. There was no correlation between the severity of tissue damage and the extent of neurogenesis. This indicates that two triggers, high-dose sevoflurane and ischemic brain damage, are required in this model of cerebral ischemia to foster sustained neurogenesis.
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ACKNOWLEDGMENTS
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The authors thank Doris Droese (Technician, Klinik für Anaesthesiologie, Technische Universität München, Munich, Germany) and Anne Frye (Technician, Klinik für Anaesthesiologie, Technische Universität München, Munich, Germany) for their technical assistance.
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Footnotes
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Accepted for publication November 29, 2006.
Supported by Abbott GmbH, Wiesbaden, Germany and Technische Universität, Munich, Germany.
This paper was presented at the Annual Meeting of the American Society of Anesthesiologists (October 21, 2005) and of the Society of Neurosurgical Anesthesia and Critical Care (October 22, 2005) in Atlanta, USA.
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Abstract
Introduction
