- Email: [email protected]
Inflammatory response to nitrous oxide in the central nervous system
BR A IN RE S EA RCH 1 2 46 ( 20 0 8 ) 8 8 –95
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Inflammatory response to nitrous oxide in the central nervous system Jens Lehmberg a,b,⁎, Maximilian Waldner b , Alexander Baethmann b , Eberhard Uhl b,c a
Department of Neurosurgery, Technical University, Munich, Germany Institute for Surgical Research, Ludwig-Maximilians-University, Munich, Germany c Department of Neurosurgery, LKH Klagenfurt, Austria b
A R T I C LE I N FO
AB S T R A C T
Article history:
Nitrous oxide is a widely used anesthetic gas. The aim of this study was to investigate the
Accepted 16 September 2008
effect of this agent on inflammatory side effects in the brain. The cerebral microcirculation
Available online 7 October 2008
of Mongolian gerbils was investigated by fluorescent intravital microscopy for up to 7 h after induction of anesthesia. Anesthesia was induced and maintained with isoflurane or
Keywords:
halothane alone or in combination with nitrous oxide (70%). The number of leukocytes that
Nitrous oxide
were rolling along and firmly adherent to the endothelial wall of cerebral venules was
Leukocyte
significantly elevated in animals anesthetized with nitrous oxide in combination with
Microcirculation
isoflurane and halothane compared to isoflurane and halothane alone. A significantly
Inflammation
increased number of neutrophil granulocytes invading the brain parenchyma in histological slices from animals treated with the combination of isoflurane or halothane and nitrous oxide compared to controls treated with isoflurane or halothane alone was observed. Our data show that prolonged anesthesia with nitrous oxide induces inflammation of the cerebral microcirculation and brain. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Nitrous oxide (N2O; laughing gas) has been widely used in clinical practice for decades because its effective analgesic properties are achieved at concentrations below those required for general anesthesia. These analgesic effects, coupled with a rapid onset and short duration of action, have made nitrous oxide the oldest inhalational anesthetic in clinical anesthesia and analgesia. Because of its low potency, it is used in combination with other anesthetics. After decades of the use of nitrous oxide, the first reports have emerged describing the cellular mechanisms of the analgesic effects of nitrous
oxide and revealed its function as an NMDA (N-methyl-Daspartate) glutamate receptor antagonist (Mennerick et al., 1998). Nitrous oxide is known to be a potent cerebrovasodilator that increases the total cerebral blood flow and alters the distribution of the regional cerebral blood flow towards higher flows in subcortical areas of the brain (Reinstrup et al., 1997). The cerebral blood volume was not altered in this study, raising questions about the finding of increased intracranial pressure after administration of nitrous oxide (Moss and McDowall, 1979; Reinstrup et al., 2001). By inactivating methionine synthase, nitrous oxide can induce bone marrow depression with
⁎ Corresponding author. Department of Neurosurgery, Ismaninger Strasse 22, 81675 Munich, Germany. Fax: +49 89 4140 4889. E-mail address: [email protected] (J. Lehmberg). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.09.064
89
BR A IN RE S E A RCH 1 2 46 ( 20 0 8 ) 8 8 –9 5
Fig. 1 – Mean arterial blood pressure (A) and concentration of isoflurane/halothane (B). Mean arterial blood pressure [mm Hg] did not differ between groups and remained constant throughout the experiment (A). The anesthetic/analgesic effect of nitrous oxide added to the gas mixture was not sufficient to reduce the need for isoflurane or halothane (B).
peripheral cytopenia like that associated with B12 deficiency, but only after prolonged administration (Lassen et al., 1956). In addition to this hematotoxic effect, a neurotoxic effect of
nitrous oxide has been described. Human subjects exposed to nitrous oxide, either professionally or through abuse, developed sensorimotor polyneuropathy and myelopathy (Layzer, 1978). As an NMDA receptor antagonist, nitrous oxide can be neuroprotective or neurotoxic. Short-term exposure to nitrous oxide causes a rapidly reversible vacuole reaction in neurons, whereas prolonged exposure causes neuronal cell death (Jevtovic-Todorovic et al., 2003a). Early exposure of the developing mammalian brain to nitrous oxide causes apoptotic neurodegeneration and learning deficits (Jevtovic-Todorovic et al., 2003b). On the other hand, nitrous oxide administered in subanesthetic doses after experimental stroke in rodents has been shown to reduce neuronal cell death (David et al., 2003). Nitrous oxide also interferes with the immune system, and infections following anesthesia are attributed to this effect. Although controversial, in vitro as well as in vivo studies have revealed decreased neutrophil motility and chemotaxis as well as impairment of the leukocyte oxidative response in response to nitrous oxide (Frohlich et al, 1998b; Hill et al., 1978; Kripke et al., 1987; Moudgil et al., 1984; Nunn and O'Morain, 1982; Welch, 1984; Welch and Zaccari, 1982). Furthermore, changes in the expression of endogenous sugar receptors on polymorphonuclear leukocytes after anesthesia with nitrous oxide and the impact of nitrous oxide on the impairment of intracellular signaling in leukocytes indicate that it interferes with the leukocyte adhesion– activation cascade (Bardosi et al., 1992; Frohlich et al., 1998a). The aim of the present study was to investigate the influence of nitrous oxide on leukocyte–endothelium interactions in cerebral microvessels and the subsequent leukocyte invasion of the brain parenchyma, in order to detect any effect that this agent might have on an inflammatory response in the brain. Due to the weak anesthetic properties of nitrous oxide, it was administered in combination with the inhalative anesthetics halothane and isoflurane. Whereas halothane is rarely administered to humans in western countries but frequently used in animal research, isoflurane is widely used in the clinical practice.
Table 1 – Blood gases obtained during the surgical preparation, during the experiment, and at the end of the experiment Parameter pH
pCO2 [mm Hg]
pO2 [mm Hg]
BE [mmol/l]
Group
During preparation
During experiment
Isoflurane Isoflurane + N2O Halothane Halothane + N2O Isoflurane Isoflurane + N2O Halothane Halothane + N2O Isoflurane Isoflurane + N2O Halothane Halothane + N2O Isoflurane Isoflurane + N2O Halothane Halothane + N2O
7.44 ± 0.04 7.46 ± 0.06 7.41 ± 0.04 7.42 ± 0.03 37 ± 3 32 ± 5 42 ± 5 40 ± 5 131 ± 8 129 ± 9 127 ± 37 115 ± 14 0.5 ± 1.8 −0.5 ± 1.4 1.3 ± 1.5 1.0 ± 2.1
7.37 ± 0.03 7.37 ± 0.03 7.33 ± 0.03 7.31 ± 0.04 33 ± 3 34 ± 5 36 ± 3 37 ± 5 139 ± 6 134 ± 8 121 ± 19 111 ± 24 −5.9 ± 2.0 −5.3 ± 1.9 −6.4 ± 1.5 −7.0 ± 3.4
End of experiment 7.29 ± 0.04 7.28 ± 0.04 7.25 ± 0.05 7.23 ± 0.06 36 ± 4 36 ± 7 35 ± 2 38 ± 4 119 ± 29 136 ± 14 93 ± 34 101 ± 23 − 8.6 ± 1.2 − 9.7 ± 2.3 −10.9 ± 1.8 −11.0 ± 2.6
90
2.
BR A IN RE S EA RCH 1 2 46 ( 20 0 8 ) 8 8 –95
Results
Mean arterial blood pressure [mm Hg] did not differ between groups and remained constant throughout the experiment (Fig. 1A). As shown in Fig. 1B, the anesthetic/analgesic effect of the nitrous oxide added to the gas mixture was not sufficient to reduce the need for isoflurane or halothane. Measurement of blood gases showed a reduction of the pH as well as base excess in the course of the experiment, without differences among the four experimental groups (Table 1). The pCO2 in the four groups was in the normal range and did not differ significantly among groups, and there was no change over the course of the experiment. Measurement of the pO2 revealed excellent oxygenation in all animals throughout the experiment (Table 1). In all groups the number of rolling leukocytes [n × 100μm− 1 × min− 1] increased continuously over the course of the experiment. At 5 h of observation, 6.6 ± 2.8 and 3.6 ± 5.6 rolling leukocytes were found in the animals anesthetized with isoflurane and with halothane, respectively. The addition of nitrous oxide to isoflurane or halothane significantly (P < 0.05) increased the number of rolling leukocytes to 29.0 ± 16.6 and 19.1 ± 21.0, respectively (Fig. 2A). Firm adherence of leukocytes increased significantly over the course of the experiment in animals receiving additional nitrous oxide. Isoflurane or halothane alone had no effect. The total number of adherent leukocytes [n × 100 μm− 1 × min− 1] increased from 0 ± 0 at the beginning of the experiment to 1.7 ± 1.5 at 5 h in animals with isoflurane/nitrous oxide and to 1.4 ± 2.0 at 5 h (P < 0.05) in animals receiving halothane/nitrous oxide (Fig. 2B). The blood count revealed an increase in the number of leukocytes over the course of the experiment. 5.3 ± 1.4, 6.9 ± 2.3, 7.6 ± 2.4, and 7.3 ± 2.4 leukocytes × 103/μl were found at the beginning of the experiment in animals anesthetized with isoflurane, isoflurane/nitrous oxide, halothane, and halothane/nitrous oxide, respectively, whereas 12.2 ± 3.5, 14.7 ± 4.7, 14.2 ± 2.3, and 11.2 ± 6.6, respectively, were found at the end of the experiment. In animals anesthetized with isoflurane and halothane alone and with isoflurane/nitrous oxide but not in animals with halothane/nitrous oxide this increase was significant (P < 0.05). There was no correlation between the leukocyte count and the number of rolling leukocytes. The blood counts of erythrocytes and thrombocytes did not differ between the groups, nor did they change over the course of the experiment (Table 2). Examples of the intravital microscopic images are given in Figs. 3A and B. The diameters of pial arterioles (range 23–85 μm) and pial venules (range 16–82 μm), as well as the capillary density, did not change throughout the experiment (Figs. 4A–C). The AVTT, a measure of local microvascular perfusion, was 0.81 ± 0.28 and 0.71 ± 0.34, respectively, in animals that received isoflurane or halothane alone at the beginning of the measurements. The addition of nitrous oxide to isoflurane or halothane, did not alter the respective AVTTs, which were 0.76 ± 0.43 and 0.66 ± 0.21. A trend towards prolonged AVTT in animals treated with isoflurane or isoflurane/nitrous oxide was observed when they were compared with animals anesthetized with halothane or halothane/nitrous oxide, but the difference was not statistically significant (Fig. 4D). Opening of the blood-brain
Fig. 2 – Number of rolling (A) and firm adherent (B) leukocytes at the endothelium of cerebral venules. Narcosis with isoflurane or halothane alone or in combination with nitrous oxide was induced 2 h prior to the first intravital microscopic imaging of the gerbils' brain surface. A steady increase in the number of rolling leukocytes was observed during the 300 min observation period. Addition of nitrous oxide to isoflurane or halothane significantly increased the number of rolling leukocytes at the endothelium. A significant increase in the number of firmly adherent leukocytes was observed in animals anesthetized with isoflurane or halothane in combination with nitrous oxide. Firm adherence did not significantly increase over time in animals treated with isoflurane or halothane alone. Comparison of the animals treated with monotherapy vs. combination therapy with nitrous oxide revealed no significant difference.*P < 0.05, isoflurane or halothane as monotherapy vs. isoflurane or halothane in combination with nitrous oxide. †P < 0.05, the following measurements vs. baseline at 0 min. Mean ± SD, n = 8 per group.
barrier was not observed in either group at the end of the experiment. The number of esterase positive leukocytes in coronal slices of the brain obtained 7 h after induction of anesthesia with isoflurane or halothane in combination with nitrous oxide (40.1 ± 22.8 and 67.4 ± 35.6, respectively) was significantly increased relative to controls treated with isoflurane or halothane alone (17.1 ± 13.9 and 25.1 ± 14.6, respectively; P < 0.05,
BR A IN RE S E A RCH 1 2 46 ( 20 0 8 ) 8 8 –9 5
Table 2 – Blood count during the preparation and at the end of the experiment Parameter WBC [n × 103/μl]
RBC [n × 106/μl]
Plt [n × 103/μl]
a
Group
During preparation
End of experiment
Isoflurane Isoflurane + N2O Halothane Halothane + N2O Isoflurane Isoflurane + N2O Halothane Halothane + N2O Isoflurane Isoflurane + N2O Halothane Halothane + N2O
5.3 ± 1.4 6.9 ± 2.3 7.6 ± 2.4 7.3 ± 2.4 6.1 ± 0.8 6.5 ± 0.7 6.7 ± 1.0 6.3 ± 0.6 368 ± 284 428 ± 268 432 ± 187 390 ± 215
12.2 ± 3.5a 14.7 ± 4.7a 14.1 ± 2.3a 11.2 ± 6.6 6.1 ± 0.7 5.9 ± 0.5 5.9 ± 0.7 5.7 ± 1.2 308 ± 146 371 ± 139 297 ± 74 313 ± 119
P < 0.05, Mann–Whitney rank-sum test.
91
endothelium interactions. Therefore, it can be hypothesized that leukocyte adhesion to the venular endothelium was induced by the functional activation of selectins and integrins as well as their counter receptors, glycoproteins and the members of IgG superfamily, respectively (Springer, 1990). Whether this activation was a direct effect of nitrous oxide on the adhesion molecules or a secondary one due to accumulation of different mediators or chemokines such as bradykinin, platelet-activating factor, endothelin, tumor necrosis factor alpha, interleukins, and others cannot be answered by the present experiments. Changes in the expression of endogenous sugar receptors on polymorphonuclear leukocytes after anesthesia with nitrous oxide and the effect of nitrous oxide on the impairment of intracellular signaling in leukocytes indicate that nitrous oxide interferes with the leukocyte adhesion– activation cascade. The blood counts done during sample preparation and at the end of the experiment showed an increase in the leukocyte count over the course of the experiment. In
Fig. 5). An example of two esterase positive leukocytes is given in Fig. 6.
3.
Discussion
Nitrous oxide has been widely used as an anesthetic in humans since the middle of the 19th century. Its short-acting analgesic properties reduce the need for the more potent anesthetic with which it is combined. Nitrous oxide has a longstanding safety record and is generally considered to be a relatively safe anesthetic. Nevertheless, several adverse effects, including megaloblastic anemia, homocysteinemia and its possible risk for atherosclerosis, thrombosis, cognitive dysfunction, neurotoxicity, possible teratogenicity, increased intracranial pressure and cerebral blood flow, expansion of air spaces and hypoxia, post-operative nausea and vomiting, and possible immunosuppression are known. The experiments presented here indicate that an inflammatory response is initiated in the cerebral microcirculation after exposure to nitrous oxide, as revealed by increased numbers of rolling and adherent leukocytes. This effect was not limited to the surface of the cortex, but was also observed in histological specimens of deeper brain sections. Leukocytes were also seen extravascularly in the brain parenchyma, indicating that nitrous oxide initiates more than the first steps of an inflammatory response. The trend toward a slightly lower cerebral blood flow, measured by calculation of the AVTT, in animals anesthetized with isoflurane compared to halothane, is a characteristic of these anesthetics that has been described in other species (Boarini et al., 1984; Hansen et al., 1988; Reinstrup et al., 1995). In contrast to other workers (Drummond et al., 1987; Hansen et al., 1989), we found no differences in cerebral blood flow when nitrous oxide was added to isoflurane or halothane. We also found no differences among the four narcotic regimens in other microvascular parameters, such as the arteriolar and venular diameters and the capillary density. Furthermore, we found that the extent of leukocyte–endothelium interactions and the cerebral blood flow were independent, thus excluding a major influence of mechanical forces on the evolution of leukocyte–
Fig. 3 – In vivo fluorescence microphotograph of the brain surface demonstrating pial venules at the beginning of the experiment (A) and 7 h after induction of narcosis with isoflurane and nitrous oxide (B). Leukocytes were stained in vivo by intravenous injection of rhodamine 6G. An increased number of rolling and firmly adherent leukocytes was observed after prolonged application of nitrous oxide. Bar = 50 μm.
92
BR A IN RE S EA RCH 1 2 46 ( 20 0 8 ) 8 8 –95
Fig. 4 – Microvascular parameters. The diameter of pial arterioles (A) and venules (B), the capillary density of the brain surface (C), and the arterio-venous transit time (AVTT; D) are shown. The cerebral microvascular blood flow in Mongolian gerbils was measured by calculation of the AVTT. The AVTT was slightly prolonged (i.e. the cerebral blood flow was lower) in animals anesthetized with isoflurane alone or in combination with nitrous oxide compared to animals anesthetized with halothane alone or in combination with nitrous oxide. Addition of nitrous oxide to either of the other two anesthetics had no influence on cerebral blood flow. Mean ± SD, n = 8 per group.
animals anesthetized with isoflurane and halothane alone and with isoflurane combined with nitrous oxide, but not in animals with halothane combined with nitrous oxide, this rise was statistically significant. The increase in the leukocyte count might have been due to the minor trauma of catheter placement and preparation of the closed cranial window, or to the narcosis (Khan et al., 1995). The most interesting and important finding is that the degree of leukocyte activation as measured by the total leukocyte count was divergent from the extent of leukocyte adhesion in the brain. In the histological slices, increased numbers of neutrophil granulocytes was found in the animals anesthetized with the combination of nitrous oxide with halothane or isoflurane, demonstrating that the subsequent steps of leukocyte extravasation were not interrupted. While initial research focused on the role of leukocytes in host defense and tissue repair, evidence emerged implicating leukocytes as mediators of tissue damage in different diseases including, among others, arteriosclerosis, ischemia/ reperfusion and trauma (Hartl et al., 1996). For example, in several studies attenuation of brain damage after cerebral ischemia was observed after anti-leukocyte intervention,
using leukocyte depletion, antibodies directed against adhesion molecules or knockout mice deficient in an adhesion molecule (Bednar et al., 1991; Connolly et al., 1997; Zhang et al., 1995). The finding of increased leukocyte invasion of the brain parenchyma after anesthesia with nitrous oxide suggests the possibility that these activated leukocytes may damage intact parenchyma by releasing proteolytic enzymes and oxidative radicals. The post-surgical mental disturbances often referred to as toxic psychosis (i.e., delirium, confusion, disorientation, and memory impairment) may be due not only to sudden disturbances (i.e., blood loss, fluid and nutritional imbalance, post-operative pain, and fever), but also to the inflammatory response after exposure to nitrous oxide. In conclusion, nitrous oxide in combination with isoflurane or halothane increased leukocyte recruitment, a hallmark of the inflammatory response. The enhanced leukocyte adhesion to the endothelium of pail venules was not due to altered blood rheology. This justifies the hypothesis of a direct or secondary action of nitrous oxide on cell adhesion molecules. Further investigation is needed to determine whether the invasion of the brain parenchyma by leukocytes
BR A IN RE S E A RCH 1 2 46 ( 20 0 8 ) 8 8 –9 5
Fig. 5 – Neutrophil granulocytes were stained with a naphthol AS_D chloroacetate esterase stain. The number of esterase positive cells was counted in coronal slices of the brains of gerbils anesthetized for a total of 7 h with isoflurane or halothane alone or in combination with nitrous oxide. Addition of nitrous oxide to the anesthetic gases caused a significant increase in the number of neutrophil granulocytes in the brain slices. *P < 0.05, isoflurane or halothane as monotherapy vs. isoflurane or halothane in combination with nitrous oxide. Box and whisker plot representing the median, 25th/75th percentile, and the 5th/95th percentile, n = 8 per group.
can cause disturbances of brain tissue or function and is therefore a harmful side effect of anesthesia with nitrous oxide.
4.
Experimental procedure
The study used 32 Mongolian gerbils (body weight 60 to 75 g, age 3 to 5 months). The animals had free access to tap water and pellet food and were kept on a 12 h dark/light cycle; room temperature was 22 °C. The experiments were conducted according to institutional guidelines and approved by the State Government of Bavaria. The animals were randomized to four experimental groups (n = 8 in each group) receiving isoflurane, halothane, or one of these anesthetics in combination with nitrous oxide. In our study, nitrous oxide was given in a concentration of 70% with the intent to use the maximal dose that could be given without using hyperbaric pressure. The addition of more potent anesthetics was also necessary, because the minimal active concentration (MAC) for nitrous oxide in rodents is higher than that for humans (Mahmoudi et al., 1989). Taking the different MAC for rodents and humans in consideration, the concentration of 70% in gerbils might be comparable to 50% in humans. The concentration of 50% is a widely used concentration for general anesthesia in humans. The desired concentration in every animal was 1.0 MAC, the concentration was continuously controlled by monitoring the blood pressure and the frequency of breathing, and at intervals by a tail-clamp stimulus. The concentration of isoflurane or halothane was individually adjusted for each animal. At the beginning of the narcosis (time period 30 min), the animals were given con-
93
centrations of isoflurane and halothane that have been described in the literature as appropriate for mice (1.4% for isoflurane and 1.0% for halothane) (Deady et al., 1981). Thereafter, concentrations of isoflurane and halothane where individually adapted for every animal. As shown in Fig. 1B, the differences in the individual concentrations for every animal were in narrow margins. The oxygen content of the inspired gas was 30%. The gas mixture was given via a face mask at a rate of 1 l/min. The surgical preparation, intravital microscopy workstation, and technique of intravital microscopy have been described in detail previously and thus are only briefly summarized here (Uhl et al., 2000). Body temperature was maintained at 37.0 °C using a feedback controlled heating pad. Polyethylene catheters (Portex, Hythe, UK) were inserted into the tail artery for continuous blood pressure monitoring and into the femoral vein for injection of fluorescent dyes. The skull was then fixed in a stereotactic frame (Stoelting Co., Wood Dale, IL, USA) and a rectangular 4 × 4 mm cranial window was prepared over the left parietal hemisphere with the dura mater left intact. The intact dura mater ensures a physiological milieu of the electrolytes and gases of the cerebrospinal fluid and consequently the brain surface and its vessels. During intravital microscopy the dura mater was rinsed continuously with physiological saline at 37.0 °C. The duration of the preparation was fixed at 2 h. Thereafter, five intravital microscopical measurements were performed, at hourly intervals. Enhancement of microvessels was achieved by an intravenous injection of a 0.2 ml bolus of 65 μM fluorescein isothiocyanate-labeled dextran (FITC-dextran; molecular weight: 150,000; Sigma, Taufkirchen, Germany) before the first measurement. Leukocytes were stained in vivo before each measurement by intravenous injection of 0.05 ml of a 200 mM solution of rhodamine 6G (Merck, Darmstadt, Germany). The
Fig. 6 – Histological section of the gerbil brain showing deep brain parenchyma with two venules. Two leukocytes (arrows) can be seen beyond the vascular wall starting to invaded the parenchyma. The brain was harvested 7 h after induction of narcosis with isoflurane and nitrous oxide. Staining with naphthol AS-D chloroacetate-esterase for leukocytes and hematoxylin and eosin. Bar = 20 μm.
94
BR A IN RE S EA RCH 1 2 46 ( 20 0 8 ) 8 8 –95
intravital fluorescence microscope (Leitz, Wetzlar, Germany) was equipped with a 75 W xenon bulb and N2 and L3 Ploemopak filter blocks for epiillumination. The microcirculatory parameters were analyzed using a 25× salt water immersion objective. To test the integrity of the blood-brain barrier at the end of the experiment, 0.2 ml of a 2.7 mM sodium fluorescein solution (Sigma) was injected intravenously. For the measurement of the arterio-venous transit time (AVTT), a bolus of rhodamine 6G (0.05 ml) was given intravenously. A computercontrolled microscope X–Y stage was used for repeated analysis of identical regions of interest, comprising at least 2 capillary beds, 2 arterioles, and 4 venules. Images were recorded by a SIT-video camera (C 2400, Hamamatsu Photonics, Herrsching, Germany) and stored on S-VHS videotapes. Arteriolar and venular diameters [μm], the number of rolling and adherent leukocytes in postcapillary venules [n × 100 μm− 1 × min− 1], the capillary density [cm− 1], and the integrity of the blood-brain barrier [yes/no] were measured using a computerassisted microcirculation analysis system (CapImage; Ingenieurbüro Dr. Zeintl, Heidelberg, Germany). The white blood cells were classified according to their interaction with the venular endothelium as rolling or adherent leukocytes as described previously (Uhl et al., 2000). AVTT was measured using an image-analysis system (IBAS 2.0, Kontron, Eching, Germany). For analysis of AVTT, 22 images over a period of 6 s were digitized. Means of grey tones in an area of 40 × 40 pixels in the arteriole and the venule were calculated in each image. The calculation of the mean AVTT was performed with the difference integral method as suggested by Rovainen et al. (Rovainen et al., 1993). After intravital microscopy, the animals were sacrificed and their brains were harvested for histological evaluation. Transcardial perfusion was performed with a phosphate-buffered paraformaldehyde solution (4%) after flushing the circulation with physiological saline for 60 s at a pressure of 100 cm H2O. Brains were removed and stored in paraformaldehyde solution for at least 24 h until further processing. The brains were then dehydrated in ethanol and embedded in paraffin. Coronal slices of 5 μm thickness were cut 1.7 mm caudal and 0.5 mm rostral to the bregma. The specific staining for neutrophil granulocytes was performed with a naphthol AS-D chloroacetateesterase stain (Li et al., 1973; Yam et al., 1971). The number [n] of esterase positive leukocytes in brain tissue was counted in each slice by an investigator blinded to the treatment group. Statistical analysis was performed using Sigma Stat 2.0 (SPSS Science, Chicago, IL, U.S.A.). The Kruskal–Wallis oneway analysis of variance on ranks followed by the Dunnett test was used for analyzing differences between control and treatment groups. The Friedman repeated-measures analysis of variance on ranks followed by Dunnett's method were used to detect differences within each group. The Wilcoxon signedrank test was used to compare numbers of WBCs counted during preparation and at the end of the experiment. Searches for correlation were performed with the regression tool in SigmaPlot 2000 (SPSS Science, Chicago, IL, U.S.A.). All data are presented as mean ± SD, except data presented as box and whisker plots, where the median, the 25th/75th percentiles, and the 5th/95th percentiles are given. A statistically significant difference was assumed at P < 0.05.
Acknowledgment We gratefully acknowledge the excellent technical assistance of Miss V. Bischoff. REFERENCES
Bardosi, L., Bardosi, A., Gabius, H.J., 1992. Changes of expression of endogenous sugar receptors by polymorphonuclear leukocytes after prolonged anaesthesia and surgery. Can. J. Anaesth. 39, 143–150. Bednar, M.M., Raymond, S., McAuliffe, T., Lodge, P.A., Gross, C.E., 1991. The role of neutrophils and platelets in a rabbit model of thromboembolic stroke. Stroke 22, 44–50. Boarini, D.J., Kassell, N.F., Coester, H.C., Butler, M., Sokoll, M.D., 1984. Comparison of systemic and cerebrovascular effects of isoflurane and halothane. Neurosurgery 15, 400–409. Connolly Jr., E.S., Winfree, C.J., Prestigiacomo, C.J., Kim, S.C., Choudhri, T.F., Hoh, B.L., Naka, Y., Solomon, R.A., Pinsky, D.J., 1997. Exacerbation of cerebral injury in mice that express the P-selectin gene: identification of P-selectin blockade as a new target for the treatment of stroke. Circ. Res. 81, 304–310. David, H.N., Leveille, F., Chazalviel, L., MacKenzie, E.T., Buisson, A., Lemaire, M., Abraini, J.H., 2003. Reduction of ischemic brain damage by nitrous oxide and xenon. J. Cereb. Blood Flow Metab. 23, 1168–1173. Deady, J.E., Koblin, D.D., Eger, E.I., Heavner, J.E., D'Aoust, B., 1981. Anesthetic potencies and the unitary theory of narcosis. Anesth. Analg. 60, 380–384. Drummond, J.C., Scheller, M.S., Todd, M.M., 1987. The effect of nitrous oxide on cortical cerebral blood flow during anesthesia with halothane and isoflurane, with and without morphine, in the rabbit. Anesth. Analg. 66, 1083–1089. Frohlich, D., Rothe, G., Schmitz, G., Taeger, K., 1998a. Nitrous oxide impairs the signaling of neutrophils downstream of receptors. Toxicol. Lett. 100–101, 121–127. Frohlich, D., Rothe, G., Wittmann, S., Schmitz, G., Schmid, P., Taeger, K., Hobbhahn, J., 1998b. Nitrous oxide impairs the neutrophil oxidative response. Anesthesiology 88, 1281–1290. Hansen, T.D., Warner, D.S., Todd, M.M., Vust, L.J., Trawick, D.C., 1988. Distribution of cerebral blood flow during halothane versus isoflurane anesthesia in rats. Anesthesiology 69, 332–337. Hansen, T.D., Warner, D.S., Todd, M.M., Vust, L.J., 1989. Effects of nitrous oxide and volatile anaesthetics on cerebral blood flow. Br. J. Anaesth. 63, 290–295. Hartl, R., Schurer, L., Schmid-Schonbein, G.W., del Zoppo, G.J., 1996. Experimental antileukocyte interventions in cerebral ischemia. J. Cereb. Blood Flow Metab. 16, 1108–1119. Hill, G.E., English, J.B., Stanley, T.H., Kawamura, R., Loeser, E.A., Hill, H.R., 1978. Nitrous oxide and neutrophil chemotaxis in man. Br. J. Anaesth. 50, 555–558. Jevtovic-Todorovic, V., Beals, J., Benshoff, N., Olney, J.W., 2003a. Prolonged exposure to inhalational anesthetic nitrous oxide kills neurons in adult rat brain. Neuroscience 122, 609–616. Jevtovic-Todorovic, V., Hartman, R.E., Izumi, Y., Benshoff, N.D., Dikranian, K., Zorumski, C.F., Olney, J.W., Wozniak, D.F., 2003b. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J. Neurosci. 23, 876–882. Khan, F.A., Kamal, R.S., Mithani, C.H., Khurshid, M., 1995. Effect of general anaesthesia and surgery on neutrophil function. Anaesthesia 50, 769–775. Kripke, B.J., Kupferman, A., Luu, K.C., 1987. Suppression of chemotaxis to corneal inflammation by nitrous oxide.
BR A IN RE S E A RCH 1 2 46 ( 20 0 8 ) 8 8 –9 5
Zhonghua Min Guo. Wei Sheng Wu Ji. Mian. Yi. Xue. Za Zhi. 20, 302–310. Lassen, H.C.A., Henriksen, E., Neukirch, F., Kristensen, H.S., 1956. Treatment of tetanus. Severe bone-marrow depression after prolonged nitrous oxide anaesthesia. Lancet 527–530. Layzer, R.B., 1978. Myeloneuropathy after prolonged exposure to nitrous oxide. Lancet 2, 1227–1230. Li, C.Y., Lam, K.W., Yam, L.T., 1973. Esterases in human leukocytes. J. Histochem. Cytochem. 21, 1–12. Mahmoudi, N.W., Cole, D.J., Shapiro, H.M., 1989. Insufficient anesthetic potency of nitrous oxide in the rat. Anesthesiology 70, 345–349. Mennerick, S., Jevtovic-Todorovic, V., Todorovic, S.M., Shen, W., Olney, J.W., Zorumski, C.F., 1998. Effect of nitrous oxide on excitatory and inhibitory synaptic transmission in hippocampal cultures. J. Neurosci. 18, 9716–9726. Moss, E., McDowall, D.G., 1979. I.C.P. increases with 50% nitrous oxide in oxygen in severe head injuries during controlled ventilation. Br. J. Anaesth. 51, 757–761. Moudgil, G.C., Gordon, J., Forrest, J.B., 1984. Comparative effects of volatile anaesthetic agents and nitrous oxide on human leucocyte chemotaxis in vitro. Can. Anaesth. Soc. J. 31, 631–637. Nunn, J.F., O'Morain, C., 1982. Nitrous oxide decreases motility of human neutrophils in vitro. Anesthesiology 56, 45–48. Reinstrup, P., Ryding, E., Algotsson, L., Messeter, K., Asgeirsson, B., Uski, T., 1995. Distribution of cerebral blood flow during anesthesia with isoflurane or halothane in humans. Anesthesiology 82, 359–366. Reinstrup, P., Ryding, E., Algotsson, L., Berntman, L., Uski, T., 1997. Regional cerebral blood flow (SPECT) during anaesthesia with
95
isoflurane and nitrous oxide in humans. Br. J. Anaesth. 78, 407–411. Reinstrup, P., Ryding, E., Ohlsson, T., Dahm, P.L., Uski, T., 2001. Cerebral blood volume (CBV) in humans during normo- and hypocapnia: influence of nitrous oxide (N(2)O). Anesthesiology 95, 1079–1082. Rovainen, C.M., Woolsey, T.A., Blocher, N.C., Wang, D.B., Robinson, O.F., 1993. Blood flow in single surface arterioles and venules on the mouse somatosensory cortex measured with videomicroscopy, fluorescent dextrans, nonoccluding fluorescent beads, and computer-assisted image analysis. J. Cereb. Blood Flow Metab 13, 359–371. Springer, T.A., 1990. Adhesion receptors of the immune system. Nature 346, 425–434. Uhl, E., Beck, J., Stummer, W., Lehmberg, J., Baethmann, A., 2000. Leukocyte–endothelium interactions in pial venules during the early and late reperfusion period after global cerebral ischemia in gerbils. J. Cereb. Blood Flow Metab. 20, 979–987. Welch, W.D., 1984. Effect of enflurane, isoflurane, and nitrous oxide on the microbicidal activity of human polymorphonuclear leukocytes. Anesthesiology 61, 188–192. Welch, W.D., Zaccari, J., 1982. Effect of halothane and N2O on the oxidative activity of human neutrophils. Anesthesiology 57, 172–176. Yam, L.T., Li, C.Y., Crosby, W.H., 1971. Cytochemical identification of monocytes and granulocytes. Am. J. Clin. Pathol. 55, 283–290. Zhang, Z.G., Chopp, M., Tang, W.X., Jiang, N., Zhang, R.L., 1995. Postischemic treatment (2–4 h) with anti-CD11b and anti-CD18 monoclonal antibodies are neuroprotective after transient (2 h) focal cerebral ischemia in the rat. Brain Res. 698, 79–85.
a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m
w w w. e l s e v i e r. c o m / l o c a t e / b r a i n r e s
Research Report
Inflammatory response to nitrous oxide in the central nervous system Jens Lehmberg a,b,⁎, Maximilian Waldner b , Alexander Baethmann b , Eberhard Uhl b,c a
Department of Neurosurgery, Technical University, Munich, Germany Institute for Surgical Research, Ludwig-Maximilians-University, Munich, Germany c Department of Neurosurgery, LKH Klagenfurt, Austria b
A R T I C LE I N FO
AB S T R A C T
Article history:
Nitrous oxide is a widely used anesthetic gas. The aim of this study was to investigate the
Accepted 16 September 2008
effect of this agent on inflammatory side effects in the brain. The cerebral microcirculation
Available online 7 October 2008
of Mongolian gerbils was investigated by fluorescent intravital microscopy for up to 7 h after induction of anesthesia. Anesthesia was induced and maintained with isoflurane or
Keywords:
halothane alone or in combination with nitrous oxide (70%). The number of leukocytes that
Nitrous oxide
were rolling along and firmly adherent to the endothelial wall of cerebral venules was
Leukocyte
significantly elevated in animals anesthetized with nitrous oxide in combination with
Microcirculation
isoflurane and halothane compared to isoflurane and halothane alone. A significantly
Inflammation
increased number of neutrophil granulocytes invading the brain parenchyma in histological slices from animals treated with the combination of isoflurane or halothane and nitrous oxide compared to controls treated with isoflurane or halothane alone was observed. Our data show that prolonged anesthesia with nitrous oxide induces inflammation of the cerebral microcirculation and brain. © 2008 Elsevier B.V. All rights reserved.
1.
Introduction
Nitrous oxide (N2O; laughing gas) has been widely used in clinical practice for decades because its effective analgesic properties are achieved at concentrations below those required for general anesthesia. These analgesic effects, coupled with a rapid onset and short duration of action, have made nitrous oxide the oldest inhalational anesthetic in clinical anesthesia and analgesia. Because of its low potency, it is used in combination with other anesthetics. After decades of the use of nitrous oxide, the first reports have emerged describing the cellular mechanisms of the analgesic effects of nitrous
oxide and revealed its function as an NMDA (N-methyl-Daspartate) glutamate receptor antagonist (Mennerick et al., 1998). Nitrous oxide is known to be a potent cerebrovasodilator that increases the total cerebral blood flow and alters the distribution of the regional cerebral blood flow towards higher flows in subcortical areas of the brain (Reinstrup et al., 1997). The cerebral blood volume was not altered in this study, raising questions about the finding of increased intracranial pressure after administration of nitrous oxide (Moss and McDowall, 1979; Reinstrup et al., 2001). By inactivating methionine synthase, nitrous oxide can induce bone marrow depression with
⁎ Corresponding author. Department of Neurosurgery, Ismaninger Strasse 22, 81675 Munich, Germany. Fax: +49 89 4140 4889. E-mail address: [email protected] (J. Lehmberg). 0006-8993/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2008.09.064
89
BR A IN RE S E A RCH 1 2 46 ( 20 0 8 ) 8 8 –9 5
Fig. 1 – Mean arterial blood pressure (A) and concentration of isoflurane/halothane (B). Mean arterial blood pressure [mm Hg] did not differ between groups and remained constant throughout the experiment (A). The anesthetic/analgesic effect of nitrous oxide added to the gas mixture was not sufficient to reduce the need for isoflurane or halothane (B).
peripheral cytopenia like that associated with B12 deficiency, but only after prolonged administration (Lassen et al., 1956). In addition to this hematotoxic effect, a neurotoxic effect of
nitrous oxide has been described. Human subjects exposed to nitrous oxide, either professionally or through abuse, developed sensorimotor polyneuropathy and myelopathy (Layzer, 1978). As an NMDA receptor antagonist, nitrous oxide can be neuroprotective or neurotoxic. Short-term exposure to nitrous oxide causes a rapidly reversible vacuole reaction in neurons, whereas prolonged exposure causes neuronal cell death (Jevtovic-Todorovic et al., 2003a). Early exposure of the developing mammalian brain to nitrous oxide causes apoptotic neurodegeneration and learning deficits (Jevtovic-Todorovic et al., 2003b). On the other hand, nitrous oxide administered in subanesthetic doses after experimental stroke in rodents has been shown to reduce neuronal cell death (David et al., 2003). Nitrous oxide also interferes with the immune system, and infections following anesthesia are attributed to this effect. Although controversial, in vitro as well as in vivo studies have revealed decreased neutrophil motility and chemotaxis as well as impairment of the leukocyte oxidative response in response to nitrous oxide (Frohlich et al, 1998b; Hill et al., 1978; Kripke et al., 1987; Moudgil et al., 1984; Nunn and O'Morain, 1982; Welch, 1984; Welch and Zaccari, 1982). Furthermore, changes in the expression of endogenous sugar receptors on polymorphonuclear leukocytes after anesthesia with nitrous oxide and the impact of nitrous oxide on the impairment of intracellular signaling in leukocytes indicate that it interferes with the leukocyte adhesion– activation cascade (Bardosi et al., 1992; Frohlich et al., 1998a). The aim of the present study was to investigate the influence of nitrous oxide on leukocyte–endothelium interactions in cerebral microvessels and the subsequent leukocyte invasion of the brain parenchyma, in order to detect any effect that this agent might have on an inflammatory response in the brain. Due to the weak anesthetic properties of nitrous oxide, it was administered in combination with the inhalative anesthetics halothane and isoflurane. Whereas halothane is rarely administered to humans in western countries but frequently used in animal research, isoflurane is widely used in the clinical practice.
Table 1 – Blood gases obtained during the surgical preparation, during the experiment, and at the end of the experiment Parameter pH
pCO2 [mm Hg]
pO2 [mm Hg]
BE [mmol/l]
Group
During preparation
During experiment
Isoflurane Isoflurane + N2O Halothane Halothane + N2O Isoflurane Isoflurane + N2O Halothane Halothane + N2O Isoflurane Isoflurane + N2O Halothane Halothane + N2O Isoflurane Isoflurane + N2O Halothane Halothane + N2O
7.44 ± 0.04 7.46 ± 0.06 7.41 ± 0.04 7.42 ± 0.03 37 ± 3 32 ± 5 42 ± 5 40 ± 5 131 ± 8 129 ± 9 127 ± 37 115 ± 14 0.5 ± 1.8 −0.5 ± 1.4 1.3 ± 1.5 1.0 ± 2.1
7.37 ± 0.03 7.37 ± 0.03 7.33 ± 0.03 7.31 ± 0.04 33 ± 3 34 ± 5 36 ± 3 37 ± 5 139 ± 6 134 ± 8 121 ± 19 111 ± 24 −5.9 ± 2.0 −5.3 ± 1.9 −6.4 ± 1.5 −7.0 ± 3.4
End of experiment 7.29 ± 0.04 7.28 ± 0.04 7.25 ± 0.05 7.23 ± 0.06 36 ± 4 36 ± 7 35 ± 2 38 ± 4 119 ± 29 136 ± 14 93 ± 34 101 ± 23 − 8.6 ± 1.2 − 9.7 ± 2.3 −10.9 ± 1.8 −11.0 ± 2.6
90
2.
BR A IN RE S EA RCH 1 2 46 ( 20 0 8 ) 8 8 –95
Results
Mean arterial blood pressure [mm Hg] did not differ between groups and remained constant throughout the experiment (Fig. 1A). As shown in Fig. 1B, the anesthetic/analgesic effect of the nitrous oxide added to the gas mixture was not sufficient to reduce the need for isoflurane or halothane. Measurement of blood gases showed a reduction of the pH as well as base excess in the course of the experiment, without differences among the four experimental groups (Table 1). The pCO2 in the four groups was in the normal range and did not differ significantly among groups, and there was no change over the course of the experiment. Measurement of the pO2 revealed excellent oxygenation in all animals throughout the experiment (Table 1). In all groups the number of rolling leukocytes [n × 100μm− 1 × min− 1] increased continuously over the course of the experiment. At 5 h of observation, 6.6 ± 2.8 and 3.6 ± 5.6 rolling leukocytes were found in the animals anesthetized with isoflurane and with halothane, respectively. The addition of nitrous oxide to isoflurane or halothane significantly (P < 0.05) increased the number of rolling leukocytes to 29.0 ± 16.6 and 19.1 ± 21.0, respectively (Fig. 2A). Firm adherence of leukocytes increased significantly over the course of the experiment in animals receiving additional nitrous oxide. Isoflurane or halothane alone had no effect. The total number of adherent leukocytes [n × 100 μm− 1 × min− 1] increased from 0 ± 0 at the beginning of the experiment to 1.7 ± 1.5 at 5 h in animals with isoflurane/nitrous oxide and to 1.4 ± 2.0 at 5 h (P < 0.05) in animals receiving halothane/nitrous oxide (Fig. 2B). The blood count revealed an increase in the number of leukocytes over the course of the experiment. 5.3 ± 1.4, 6.9 ± 2.3, 7.6 ± 2.4, and 7.3 ± 2.4 leukocytes × 103/μl were found at the beginning of the experiment in animals anesthetized with isoflurane, isoflurane/nitrous oxide, halothane, and halothane/nitrous oxide, respectively, whereas 12.2 ± 3.5, 14.7 ± 4.7, 14.2 ± 2.3, and 11.2 ± 6.6, respectively, were found at the end of the experiment. In animals anesthetized with isoflurane and halothane alone and with isoflurane/nitrous oxide but not in animals with halothane/nitrous oxide this increase was significant (P < 0.05). There was no correlation between the leukocyte count and the number of rolling leukocytes. The blood counts of erythrocytes and thrombocytes did not differ between the groups, nor did they change over the course of the experiment (Table 2). Examples of the intravital microscopic images are given in Figs. 3A and B. The diameters of pial arterioles (range 23–85 μm) and pial venules (range 16–82 μm), as well as the capillary density, did not change throughout the experiment (Figs. 4A–C). The AVTT, a measure of local microvascular perfusion, was 0.81 ± 0.28 and 0.71 ± 0.34, respectively, in animals that received isoflurane or halothane alone at the beginning of the measurements. The addition of nitrous oxide to isoflurane or halothane, did not alter the respective AVTTs, which were 0.76 ± 0.43 and 0.66 ± 0.21. A trend towards prolonged AVTT in animals treated with isoflurane or isoflurane/nitrous oxide was observed when they were compared with animals anesthetized with halothane or halothane/nitrous oxide, but the difference was not statistically significant (Fig. 4D). Opening of the blood-brain
Fig. 2 – Number of rolling (A) and firm adherent (B) leukocytes at the endothelium of cerebral venules. Narcosis with isoflurane or halothane alone or in combination with nitrous oxide was induced 2 h prior to the first intravital microscopic imaging of the gerbils' brain surface. A steady increase in the number of rolling leukocytes was observed during the 300 min observation period. Addition of nitrous oxide to isoflurane or halothane significantly increased the number of rolling leukocytes at the endothelium. A significant increase in the number of firmly adherent leukocytes was observed in animals anesthetized with isoflurane or halothane in combination with nitrous oxide. Firm adherence did not significantly increase over time in animals treated with isoflurane or halothane alone. Comparison of the animals treated with monotherapy vs. combination therapy with nitrous oxide revealed no significant difference.*P < 0.05, isoflurane or halothane as monotherapy vs. isoflurane or halothane in combination with nitrous oxide. †P < 0.05, the following measurements vs. baseline at 0 min. Mean ± SD, n = 8 per group.
barrier was not observed in either group at the end of the experiment. The number of esterase positive leukocytes in coronal slices of the brain obtained 7 h after induction of anesthesia with isoflurane or halothane in combination with nitrous oxide (40.1 ± 22.8 and 67.4 ± 35.6, respectively) was significantly increased relative to controls treated with isoflurane or halothane alone (17.1 ± 13.9 and 25.1 ± 14.6, respectively; P < 0.05,
BR A IN RE S E A RCH 1 2 46 ( 20 0 8 ) 8 8 –9 5
Table 2 – Blood count during the preparation and at the end of the experiment Parameter WBC [n × 103/μl]
RBC [n × 106/μl]
Plt [n × 103/μl]
a
Group
During preparation
End of experiment
Isoflurane Isoflurane + N2O Halothane Halothane + N2O Isoflurane Isoflurane + N2O Halothane Halothane + N2O Isoflurane Isoflurane + N2O Halothane Halothane + N2O
5.3 ± 1.4 6.9 ± 2.3 7.6 ± 2.4 7.3 ± 2.4 6.1 ± 0.8 6.5 ± 0.7 6.7 ± 1.0 6.3 ± 0.6 368 ± 284 428 ± 268 432 ± 187 390 ± 215
12.2 ± 3.5a 14.7 ± 4.7a 14.1 ± 2.3a 11.2 ± 6.6 6.1 ± 0.7 5.9 ± 0.5 5.9 ± 0.7 5.7 ± 1.2 308 ± 146 371 ± 139 297 ± 74 313 ± 119
P < 0.05, Mann–Whitney rank-sum test.
91
endothelium interactions. Therefore, it can be hypothesized that leukocyte adhesion to the venular endothelium was induced by the functional activation of selectins and integrins as well as their counter receptors, glycoproteins and the members of IgG superfamily, respectively (Springer, 1990). Whether this activation was a direct effect of nitrous oxide on the adhesion molecules or a secondary one due to accumulation of different mediators or chemokines such as bradykinin, platelet-activating factor, endothelin, tumor necrosis factor alpha, interleukins, and others cannot be answered by the present experiments. Changes in the expression of endogenous sugar receptors on polymorphonuclear leukocytes after anesthesia with nitrous oxide and the effect of nitrous oxide on the impairment of intracellular signaling in leukocytes indicate that nitrous oxide interferes with the leukocyte adhesion– activation cascade. The blood counts done during sample preparation and at the end of the experiment showed an increase in the leukocyte count over the course of the experiment. In
Fig. 5). An example of two esterase positive leukocytes is given in Fig. 6.
3.
Discussion
Nitrous oxide has been widely used as an anesthetic in humans since the middle of the 19th century. Its short-acting analgesic properties reduce the need for the more potent anesthetic with which it is combined. Nitrous oxide has a longstanding safety record and is generally considered to be a relatively safe anesthetic. Nevertheless, several adverse effects, including megaloblastic anemia, homocysteinemia and its possible risk for atherosclerosis, thrombosis, cognitive dysfunction, neurotoxicity, possible teratogenicity, increased intracranial pressure and cerebral blood flow, expansion of air spaces and hypoxia, post-operative nausea and vomiting, and possible immunosuppression are known. The experiments presented here indicate that an inflammatory response is initiated in the cerebral microcirculation after exposure to nitrous oxide, as revealed by increased numbers of rolling and adherent leukocytes. This effect was not limited to the surface of the cortex, but was also observed in histological specimens of deeper brain sections. Leukocytes were also seen extravascularly in the brain parenchyma, indicating that nitrous oxide initiates more than the first steps of an inflammatory response. The trend toward a slightly lower cerebral blood flow, measured by calculation of the AVTT, in animals anesthetized with isoflurane compared to halothane, is a characteristic of these anesthetics that has been described in other species (Boarini et al., 1984; Hansen et al., 1988; Reinstrup et al., 1995). In contrast to other workers (Drummond et al., 1987; Hansen et al., 1989), we found no differences in cerebral blood flow when nitrous oxide was added to isoflurane or halothane. We also found no differences among the four narcotic regimens in other microvascular parameters, such as the arteriolar and venular diameters and the capillary density. Furthermore, we found that the extent of leukocyte–endothelium interactions and the cerebral blood flow were independent, thus excluding a major influence of mechanical forces on the evolution of leukocyte–
Fig. 3 – In vivo fluorescence microphotograph of the brain surface demonstrating pial venules at the beginning of the experiment (A) and 7 h after induction of narcosis with isoflurane and nitrous oxide (B). Leukocytes were stained in vivo by intravenous injection of rhodamine 6G. An increased number of rolling and firmly adherent leukocytes was observed after prolonged application of nitrous oxide. Bar = 50 μm.
92
BR A IN RE S EA RCH 1 2 46 ( 20 0 8 ) 8 8 –95
Fig. 4 – Microvascular parameters. The diameter of pial arterioles (A) and venules (B), the capillary density of the brain surface (C), and the arterio-venous transit time (AVTT; D) are shown. The cerebral microvascular blood flow in Mongolian gerbils was measured by calculation of the AVTT. The AVTT was slightly prolonged (i.e. the cerebral blood flow was lower) in animals anesthetized with isoflurane alone or in combination with nitrous oxide compared to animals anesthetized with halothane alone or in combination with nitrous oxide. Addition of nitrous oxide to either of the other two anesthetics had no influence on cerebral blood flow. Mean ± SD, n = 8 per group.
animals anesthetized with isoflurane and halothane alone and with isoflurane combined with nitrous oxide, but not in animals with halothane combined with nitrous oxide, this rise was statistically significant. The increase in the leukocyte count might have been due to the minor trauma of catheter placement and preparation of the closed cranial window, or to the narcosis (Khan et al., 1995). The most interesting and important finding is that the degree of leukocyte activation as measured by the total leukocyte count was divergent from the extent of leukocyte adhesion in the brain. In the histological slices, increased numbers of neutrophil granulocytes was found in the animals anesthetized with the combination of nitrous oxide with halothane or isoflurane, demonstrating that the subsequent steps of leukocyte extravasation were not interrupted. While initial research focused on the role of leukocytes in host defense and tissue repair, evidence emerged implicating leukocytes as mediators of tissue damage in different diseases including, among others, arteriosclerosis, ischemia/ reperfusion and trauma (Hartl et al., 1996). For example, in several studies attenuation of brain damage after cerebral ischemia was observed after anti-leukocyte intervention,
using leukocyte depletion, antibodies directed against adhesion molecules or knockout mice deficient in an adhesion molecule (Bednar et al., 1991; Connolly et al., 1997; Zhang et al., 1995). The finding of increased leukocyte invasion of the brain parenchyma after anesthesia with nitrous oxide suggests the possibility that these activated leukocytes may damage intact parenchyma by releasing proteolytic enzymes and oxidative radicals. The post-surgical mental disturbances often referred to as toxic psychosis (i.e., delirium, confusion, disorientation, and memory impairment) may be due not only to sudden disturbances (i.e., blood loss, fluid and nutritional imbalance, post-operative pain, and fever), but also to the inflammatory response after exposure to nitrous oxide. In conclusion, nitrous oxide in combination with isoflurane or halothane increased leukocyte recruitment, a hallmark of the inflammatory response. The enhanced leukocyte adhesion to the endothelium of pail venules was not due to altered blood rheology. This justifies the hypothesis of a direct or secondary action of nitrous oxide on cell adhesion molecules. Further investigation is needed to determine whether the invasion of the brain parenchyma by leukocytes
BR A IN RE S E A RCH 1 2 46 ( 20 0 8 ) 8 8 –9 5
Fig. 5 – Neutrophil granulocytes were stained with a naphthol AS_D chloroacetate esterase stain. The number of esterase positive cells was counted in coronal slices of the brains of gerbils anesthetized for a total of 7 h with isoflurane or halothane alone or in combination with nitrous oxide. Addition of nitrous oxide to the anesthetic gases caused a significant increase in the number of neutrophil granulocytes in the brain slices. *P < 0.05, isoflurane or halothane as monotherapy vs. isoflurane or halothane in combination with nitrous oxide. Box and whisker plot representing the median, 25th/75th percentile, and the 5th/95th percentile, n = 8 per group.
can cause disturbances of brain tissue or function and is therefore a harmful side effect of anesthesia with nitrous oxide.
4.
Experimental procedure
The study used 32 Mongolian gerbils (body weight 60 to 75 g, age 3 to 5 months). The animals had free access to tap water and pellet food and were kept on a 12 h dark/light cycle; room temperature was 22 °C. The experiments were conducted according to institutional guidelines and approved by the State Government of Bavaria. The animals were randomized to four experimental groups (n = 8 in each group) receiving isoflurane, halothane, or one of these anesthetics in combination with nitrous oxide. In our study, nitrous oxide was given in a concentration of 70% with the intent to use the maximal dose that could be given without using hyperbaric pressure. The addition of more potent anesthetics was also necessary, because the minimal active concentration (MAC) for nitrous oxide in rodents is higher than that for humans (Mahmoudi et al., 1989). Taking the different MAC for rodents and humans in consideration, the concentration of 70% in gerbils might be comparable to 50% in humans. The concentration of 50% is a widely used concentration for general anesthesia in humans. The desired concentration in every animal was 1.0 MAC, the concentration was continuously controlled by monitoring the blood pressure and the frequency of breathing, and at intervals by a tail-clamp stimulus. The concentration of isoflurane or halothane was individually adjusted for each animal. At the beginning of the narcosis (time period 30 min), the animals were given con-
93
centrations of isoflurane and halothane that have been described in the literature as appropriate for mice (1.4% for isoflurane and 1.0% for halothane) (Deady et al., 1981). Thereafter, concentrations of isoflurane and halothane where individually adapted for every animal. As shown in Fig. 1B, the differences in the individual concentrations for every animal were in narrow margins. The oxygen content of the inspired gas was 30%. The gas mixture was given via a face mask at a rate of 1 l/min. The surgical preparation, intravital microscopy workstation, and technique of intravital microscopy have been described in detail previously and thus are only briefly summarized here (Uhl et al., 2000). Body temperature was maintained at 37.0 °C using a feedback controlled heating pad. Polyethylene catheters (Portex, Hythe, UK) were inserted into the tail artery for continuous blood pressure monitoring and into the femoral vein for injection of fluorescent dyes. The skull was then fixed in a stereotactic frame (Stoelting Co., Wood Dale, IL, USA) and a rectangular 4 × 4 mm cranial window was prepared over the left parietal hemisphere with the dura mater left intact. The intact dura mater ensures a physiological milieu of the electrolytes and gases of the cerebrospinal fluid and consequently the brain surface and its vessels. During intravital microscopy the dura mater was rinsed continuously with physiological saline at 37.0 °C. The duration of the preparation was fixed at 2 h. Thereafter, five intravital microscopical measurements were performed, at hourly intervals. Enhancement of microvessels was achieved by an intravenous injection of a 0.2 ml bolus of 65 μM fluorescein isothiocyanate-labeled dextran (FITC-dextran; molecular weight: 150,000; Sigma, Taufkirchen, Germany) before the first measurement. Leukocytes were stained in vivo before each measurement by intravenous injection of 0.05 ml of a 200 mM solution of rhodamine 6G (Merck, Darmstadt, Germany). The
Fig. 6 – Histological section of the gerbil brain showing deep brain parenchyma with two venules. Two leukocytes (arrows) can be seen beyond the vascular wall starting to invaded the parenchyma. The brain was harvested 7 h after induction of narcosis with isoflurane and nitrous oxide. Staining with naphthol AS-D chloroacetate-esterase for leukocytes and hematoxylin and eosin. Bar = 20 μm.
94
BR A IN RE S EA RCH 1 2 46 ( 20 0 8 ) 8 8 –95
intravital fluorescence microscope (Leitz, Wetzlar, Germany) was equipped with a 75 W xenon bulb and N2 and L3 Ploemopak filter blocks for epiillumination. The microcirculatory parameters were analyzed using a 25× salt water immersion objective. To test the integrity of the blood-brain barrier at the end of the experiment, 0.2 ml of a 2.7 mM sodium fluorescein solution (Sigma) was injected intravenously. For the measurement of the arterio-venous transit time (AVTT), a bolus of rhodamine 6G (0.05 ml) was given intravenously. A computercontrolled microscope X–Y stage was used for repeated analysis of identical regions of interest, comprising at least 2 capillary beds, 2 arterioles, and 4 venules. Images were recorded by a SIT-video camera (C 2400, Hamamatsu Photonics, Herrsching, Germany) and stored on S-VHS videotapes. Arteriolar and venular diameters [μm], the number of rolling and adherent leukocytes in postcapillary venules [n × 100 μm− 1 × min− 1], the capillary density [cm− 1], and the integrity of the blood-brain barrier [yes/no] were measured using a computerassisted microcirculation analysis system (CapImage; Ingenieurbüro Dr. Zeintl, Heidelberg, Germany). The white blood cells were classified according to their interaction with the venular endothelium as rolling or adherent leukocytes as described previously (Uhl et al., 2000). AVTT was measured using an image-analysis system (IBAS 2.0, Kontron, Eching, Germany). For analysis of AVTT, 22 images over a period of 6 s were digitized. Means of grey tones in an area of 40 × 40 pixels in the arteriole and the venule were calculated in each image. The calculation of the mean AVTT was performed with the difference integral method as suggested by Rovainen et al. (Rovainen et al., 1993). After intravital microscopy, the animals were sacrificed and their brains were harvested for histological evaluation. Transcardial perfusion was performed with a phosphate-buffered paraformaldehyde solution (4%) after flushing the circulation with physiological saline for 60 s at a pressure of 100 cm H2O. Brains were removed and stored in paraformaldehyde solution for at least 24 h until further processing. The brains were then dehydrated in ethanol and embedded in paraffin. Coronal slices of 5 μm thickness were cut 1.7 mm caudal and 0.5 mm rostral to the bregma. The specific staining for neutrophil granulocytes was performed with a naphthol AS-D chloroacetateesterase stain (Li et al., 1973; Yam et al., 1971). The number [n] of esterase positive leukocytes in brain tissue was counted in each slice by an investigator blinded to the treatment group. Statistical analysis was performed using Sigma Stat 2.0 (SPSS Science, Chicago, IL, U.S.A.). The Kruskal–Wallis oneway analysis of variance on ranks followed by the Dunnett test was used for analyzing differences between control and treatment groups. The Friedman repeated-measures analysis of variance on ranks followed by Dunnett's method were used to detect differences within each group. The Wilcoxon signedrank test was used to compare numbers of WBCs counted during preparation and at the end of the experiment. Searches for correlation were performed with the regression tool in SigmaPlot 2000 (SPSS Science, Chicago, IL, U.S.A.). All data are presented as mean ± SD, except data presented as box and whisker plots, where the median, the 25th/75th percentiles, and the 5th/95th percentiles are given. A statistically significant difference was assumed at P < 0.05.
Acknowledgment We gratefully acknowledge the excellent technical assistance of Miss V. Bischoff. REFERENCES
Bardosi, L., Bardosi, A., Gabius, H.J., 1992. Changes of expression of endogenous sugar receptors by polymorphonuclear leukocytes after prolonged anaesthesia and surgery. Can. J. Anaesth. 39, 143–150. Bednar, M.M., Raymond, S., McAuliffe, T., Lodge, P.A., Gross, C.E., 1991. The role of neutrophils and platelets in a rabbit model of thromboembolic stroke. Stroke 22, 44–50. Boarini, D.J., Kassell, N.F., Coester, H.C., Butler, M., Sokoll, M.D., 1984. Comparison of systemic and cerebrovascular effects of isoflurane and halothane. Neurosurgery 15, 400–409. Connolly Jr., E.S., Winfree, C.J., Prestigiacomo, C.J., Kim, S.C., Choudhri, T.F., Hoh, B.L., Naka, Y., Solomon, R.A., Pinsky, D.J., 1997. Exacerbation of cerebral injury in mice that express the P-selectin gene: identification of P-selectin blockade as a new target for the treatment of stroke. Circ. Res. 81, 304–310. David, H.N., Leveille, F., Chazalviel, L., MacKenzie, E.T., Buisson, A., Lemaire, M., Abraini, J.H., 2003. Reduction of ischemic brain damage by nitrous oxide and xenon. J. Cereb. Blood Flow Metab. 23, 1168–1173. Deady, J.E., Koblin, D.D., Eger, E.I., Heavner, J.E., D'Aoust, B., 1981. Anesthetic potencies and the unitary theory of narcosis. Anesth. Analg. 60, 380–384. Drummond, J.C., Scheller, M.S., Todd, M.M., 1987. The effect of nitrous oxide on cortical cerebral blood flow during anesthesia with halothane and isoflurane, with and without morphine, in the rabbit. Anesth. Analg. 66, 1083–1089. Frohlich, D., Rothe, G., Schmitz, G., Taeger, K., 1998a. Nitrous oxide impairs the signaling of neutrophils downstream of receptors. Toxicol. Lett. 100–101, 121–127. Frohlich, D., Rothe, G., Wittmann, S., Schmitz, G., Schmid, P., Taeger, K., Hobbhahn, J., 1998b. Nitrous oxide impairs the neutrophil oxidative response. Anesthesiology 88, 1281–1290. Hansen, T.D., Warner, D.S., Todd, M.M., Vust, L.J., Trawick, D.C., 1988. Distribution of cerebral blood flow during halothane versus isoflurane anesthesia in rats. Anesthesiology 69, 332–337. Hansen, T.D., Warner, D.S., Todd, M.M., Vust, L.J., 1989. Effects of nitrous oxide and volatile anaesthetics on cerebral blood flow. Br. J. Anaesth. 63, 290–295. Hartl, R., Schurer, L., Schmid-Schonbein, G.W., del Zoppo, G.J., 1996. Experimental antileukocyte interventions in cerebral ischemia. J. Cereb. Blood Flow Metab. 16, 1108–1119. Hill, G.E., English, J.B., Stanley, T.H., Kawamura, R., Loeser, E.A., Hill, H.R., 1978. Nitrous oxide and neutrophil chemotaxis in man. Br. J. Anaesth. 50, 555–558. Jevtovic-Todorovic, V., Beals, J., Benshoff, N., Olney, J.W., 2003a. Prolonged exposure to inhalational anesthetic nitrous oxide kills neurons in adult rat brain. Neuroscience 122, 609–616. Jevtovic-Todorovic, V., Hartman, R.E., Izumi, Y., Benshoff, N.D., Dikranian, K., Zorumski, C.F., Olney, J.W., Wozniak, D.F., 2003b. Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J. Neurosci. 23, 876–882. Khan, F.A., Kamal, R.S., Mithani, C.H., Khurshid, M., 1995. Effect of general anaesthesia and surgery on neutrophil function. Anaesthesia 50, 769–775. Kripke, B.J., Kupferman, A., Luu, K.C., 1987. Suppression of chemotaxis to corneal inflammation by nitrous oxide.
BR A IN RE S E A RCH 1 2 46 ( 20 0 8 ) 8 8 –9 5
Zhonghua Min Guo. Wei Sheng Wu Ji. Mian. Yi. Xue. Za Zhi. 20, 302–310. Lassen, H.C.A., Henriksen, E., Neukirch, F., Kristensen, H.S., 1956. Treatment of tetanus. Severe bone-marrow depression after prolonged nitrous oxide anaesthesia. Lancet 527–530. Layzer, R.B., 1978. Myeloneuropathy after prolonged exposure to nitrous oxide. Lancet 2, 1227–1230. Li, C.Y., Lam, K.W., Yam, L.T., 1973. Esterases in human leukocytes. J. Histochem. Cytochem. 21, 1–12. Mahmoudi, N.W., Cole, D.J., Shapiro, H.M., 1989. Insufficient anesthetic potency of nitrous oxide in the rat. Anesthesiology 70, 345–349. Mennerick, S., Jevtovic-Todorovic, V., Todorovic, S.M., Shen, W., Olney, J.W., Zorumski, C.F., 1998. Effect of nitrous oxide on excitatory and inhibitory synaptic transmission in hippocampal cultures. J. Neurosci. 18, 9716–9726. Moss, E., McDowall, D.G., 1979. I.C.P. increases with 50% nitrous oxide in oxygen in severe head injuries during controlled ventilation. Br. J. Anaesth. 51, 757–761. Moudgil, G.C., Gordon, J., Forrest, J.B., 1984. Comparative effects of volatile anaesthetic agents and nitrous oxide on human leucocyte chemotaxis in vitro. Can. Anaesth. Soc. J. 31, 631–637. Nunn, J.F., O'Morain, C., 1982. Nitrous oxide decreases motility of human neutrophils in vitro. Anesthesiology 56, 45–48. Reinstrup, P., Ryding, E., Algotsson, L., Messeter, K., Asgeirsson, B., Uski, T., 1995. Distribution of cerebral blood flow during anesthesia with isoflurane or halothane in humans. Anesthesiology 82, 359–366. Reinstrup, P., Ryding, E., Algotsson, L., Berntman, L., Uski, T., 1997. Regional cerebral blood flow (SPECT) during anaesthesia with
95
isoflurane and nitrous oxide in humans. Br. J. Anaesth. 78, 407–411. Reinstrup, P., Ryding, E., Ohlsson, T., Dahm, P.L., Uski, T., 2001. Cerebral blood volume (CBV) in humans during normo- and hypocapnia: influence of nitrous oxide (N(2)O). Anesthesiology 95, 1079–1082. Rovainen, C.M., Woolsey, T.A., Blocher, N.C., Wang, D.B., Robinson, O.F., 1993. Blood flow in single surface arterioles and venules on the mouse somatosensory cortex measured with videomicroscopy, fluorescent dextrans, nonoccluding fluorescent beads, and computer-assisted image analysis. J. Cereb. Blood Flow Metab 13, 359–371. Springer, T.A., 1990. Adhesion receptors of the immune system. Nature 346, 425–434. Uhl, E., Beck, J., Stummer, W., Lehmberg, J., Baethmann, A., 2000. Leukocyte–endothelium interactions in pial venules during the early and late reperfusion period after global cerebral ischemia in gerbils. J. Cereb. Blood Flow Metab. 20, 979–987. Welch, W.D., 1984. Effect of enflurane, isoflurane, and nitrous oxide on the microbicidal activity of human polymorphonuclear leukocytes. Anesthesiology 61, 188–192. Welch, W.D., Zaccari, J., 1982. Effect of halothane and N2O on the oxidative activity of human neutrophils. Anesthesiology 57, 172–176. Yam, L.T., Li, C.Y., Crosby, W.H., 1971. Cytochemical identification of monocytes and granulocytes. Am. J. Clin. Pathol. 55, 283–290. Zhang, Z.G., Chopp, M., Tang, W.X., Jiang, N., Zhang, R.L., 1995. Postischemic treatment (2–4 h) with anti-CD11b and anti-CD18 monoclonal antibodies are neuroprotective after transient (2 h) focal cerebral ischemia in the rat. Brain Res. 698, 79–85.