Anesthesiology 2009; 110:1077– 85

Copyright © 2009, the American Society of Anesthesiologists, Inc. Lippincott Williams & Wilkins, Inc.

Dexmedetomidine Attenuates Isoflurane-induced Neurocognitive Impairment in Neonatal Rats Robert D. Sanders, B.Sc., M.B.B.S., F.R.C.A.,* Jing Xu, M.D.,† Yi Shu, B.Sc.,‡ Adam Januszewski, B.Sc., M.B.B.S.,§ Sunil Halder, B.Sc., M.B.B.S.,(cid:1) Antonio Fidalgo, M.Sc.,‡ Pamela Sun, B.Sc.,# Mahmuda Hossain, Ph.D.,** Daqing Ma, M.D., Ph.D.,†† Mervyn Maze, M.B., Ch.B., F.R.C.P., F.R.C.A., F.Med.Sci.‡‡

Background: Neuroapoptosis is induced by the administra- tion of anesthetic agents to the young. As (cid:1)2 adrenoceptor signaling plays a trophic role during development and is neu- roprotective in several settings of neuronal injury, the authors investigated whether dexmedetomidine could provide func- tional protection against isoflurane-induced injury.

Methods: Isoflurane-induced injury was provoked in organo- typic hippocampal slice cultures in vitro or in vivo in postnatal day 7 rats by a 6-h exposure to 0.75% isoflurane with or without dexmedetomidine. In vivo, the (cid:1)2 adrenoceptor antagonist ati- pamezole was used to identify if dexmedetomidine neuropro- tection involved (cid:1)2 adrenoceptor activation. The (cid:2)-amino-bu- tyric-acid type A antagonist, gabazine, was also added to the organotypic hippocampal slice cultures in the presence of isoflurane. Apoptosis was assessed using cleaved caspase-3 im- munohistochemistry. Cognitive function was assessed in vivo on postnatal day 40 using fear conditioning.

Results: In vivo dexmedetomidine dose-dependently pre- vented isoflurane-induced injury in the hippocampus, thala- mus, and cortex; this neuroprotection was attenuated by treat- ment with atipamezole. Although anesthetic treatment did not affect the acquisition of short-term memory, isoflurane did induce long-term memory impairment. This neurocognitive deficit was prevented by administration of dexmedetomidine, which also inhibited isoflurane-induced caspase-3 expression in organotypic hippocampal slice cultures in vitro; however, gabazine did not modify this neuroapoptosis.

Conclusion: Dexmedetomidine attenuates isoflurane-induced injury in the developing brain, providing neurocognitive pro- tection. Isoflurane-induced injury in vitro appears to be inde- pendent of activation of the (cid:2)-amino-butyric-acid type A recep- tor. If isoflurane-induced neuroapoptosis proves to be a clinical problem, administration of dexmedetomidine may be an im- portant adjunct to prevent isoflurane-induced neurotoxicity.

ANESTHESIA has recently been associated with wide- spread apoptotic neurodegeneration in the neonatal rat brain with persistent functional neurocognitive impair-

ment, exemplified by impaired memory formation.1– 4 This discovery has led to concern about the possible detrimental effects of anesthesia and sedation in the pediatric population. The observed apoptotic neurode- generation mimics the neuronal injury of fetal alcohol syndrome5 and is thought to be secondary to impaired neurotransmission during a critical period of synapto- genesis that triggers so-called neuronal suicide. Indeed, there is significant evidence that preventing synaptic neurotransmission causes deleterious long-term central nervous system changes,6 with synaptic neurotransmis- sion critical to avoid synaptic pruning and apoptosis of activity-deprived neurons.7,8

Generically, anesthetic agents are thought to inhibit synaptic neurotransmission by potentiating (cid:1)-amino- butyric-acid type A (GABAA) receptors, inhibiting gluta- mate N-methyl-D-aspartate (NMDA) channels or activat- ing two-pore potassium channels.9 The net result leads to cellular hyperpolarization and a reduction in neuronal activity. However, during development, this artificial silenc- ing of synapses is thought to induce an apoptotic cascade via disruption of the action of trophic factors, notably brain-derived neurotrophic factor,2,3 phosphorylated ex- tracellular signal-regulated protein kinase 1 and 2 (pERK),2 and phosphorylated-cyclic-adenosine monophosphate (AMP) response element binding protein with subse- quent stimulation of the intrinsic apoptotic cascade.4,10 The intrinsic cascade results in cytochrome C release and Bax signaling to activate the caspase enzymes that provoke cell death by apoptosis.4,10,11 Subsequently, ex- trinsic apoptotic signaling may also be activated.10 These toxic effects have now been established after as little as 60 min of below 1 minimum alveolar concentration of isoflurane in the 7-day-old rat12; thus, a relationship be- tween anesthesia, neuroapoptosis and cognitive dys- function has been established.

Academic Clinical Fellow, ‡ Doctoral Student, § House Officer, # Medical Student, ** Research Technician, †† Senior Lecturer, ‡‡ Sir Ivan Magill Professor of Anaesthesia, Department of Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, United Kingdom; (cid:1) Honorary Research Fellow, Imperial College London, and Specialty Trainee, Department of Anaesthetics, Reading General Hospital, Reading, United Kingdom; † Attending Physician, Department of Anesthesiology, Gongli Hospital, Pudong, Shanghai, China. Professor Maze has been a consultant for Abbott Laboratories, Abbott Park, Illinois, to facilitate registration of dexmedetomidine in the United States. Received from the Department of Anaesthetics, Pain Medicine and Intensive Care, Imperial College London, United Kingdom. Submitted for publication January 23, 2008. Accepted December 2, 2008. Supported by Chelsea and Westminster Healthcare NHS Trust, London, United Kingdom, and the Westmin- ster Medical School Research Trust, London, United Kingdom.

Address correspondence to Professor Maze: Magill Department of Anaesthet- ics, Intensive Care and Pain Medicine, Imperial College London, Chelsea and Westminster Hospital, 369 Fulham Road, London SW10 9NH. m.maze@ imperial.ac.uk. Information on purchasing reprints may be found at www. anesthesiology.org or on the masthead page at the beginning of this issue. ANESTHESIOLOGY’s articles are made freely accessible to all readers, for personal use only, 6 months from the cover date of the issue.

The NMDA antagonist ketamine (20 mg kg(cid:1)1 and above) and the GABAergic agonist midazolam (9 mg kg(cid:1)1) both induce apoptotic neurodegeneration in in- fant mice13 despite having different mechanisms of an- esthetic action. This has significant implications for pedi- atric anesthesia as these drugs are used for premedication, sedation or analgesia in several clinical settings. Further- more, as these agents have differing mechanisms of anes- thetic action, yet induce this neuroapoptosis, it has been argued that it is the anesthetic state that produces the injury.11 To date, only one exception to this rule has been identified, the noble anesthetic gas xenon, which prevented isoflurane-induced toxicity.4 However, xenon

Anesthesiology, V 110, No 5, May 2009

1077

1078

is currently not widely available; therefore, we have been seeking to identify alternative methods to amelio- rate this toxicity.

Early in life, (cid:2)2 adrenoceptors are thought to play a trophic role in central nervous system signaling,14,15 with endogenous norepinephrine activating cellular survival mechanisms such as the Ras-Raf-pERK pathway.16,17 Acti- vation of this Ras-Raf-pERK pathway has been associated with neuroprotection against the apoptosis induced by NMDA antagonists in the young.2 Dexmedetomidine also increases the expression of the antiapoptotic proteins mdm2 and bcl-2 in a model of adult ischemic cerebral injury18; in vitro, it has been shown to upregulate brain- derived neurotrophic factor, phosphorylated-cyclic-AMP response element binding protein, and pERK signal- ing.16,19,20 However, it is not known whether modifica- tion of these proteins represents a true antiapoptotic effect of dexmedetomidine or whether these findings were merely a correlate of increased cellular survival. Herein, we show that dexmedetomidine protects against anesthetic-induced apoptosis in vivo and in vitro, indi- cating that it does possess antiapoptotic qualities. Im- portantly, we again establish that isoflurane injury provokes a long-term neurocognitive deficit and then demonstrate that this functional deficit can be atten- uated by dexmedetomidine.

Materials and Methods

The study protocol was approved by the Home Office (London, United Kingdom) and conforms to the United Kingdom Animals (Scientific Procedures) Act of 1986.

In Vitro Experiments Organotypic hippocampal slices were derived from postnatal day 8 or 9 C57Bl/6 mice pups (Harlan Labora- tories, Huntingdon, United Kingdom) and cultured by the interface method21,22 with some modifications. In brief, the brain was quickly dissected and placed in ice-cooled (4°C) dissection solution. All stages of slice preparation were performed under sterile and ice-cooled conditions. Excess tissue (including the cerebellum, ol- factory bulbs, and meninges) was removed, and the brain was cut into 400-(cid:3)m sagittal slices using a McIl- lwain Tissue Chopper (Mickle Laboratory, Cambridge, United Kingdom). Under a dissecting microscope and avoiding contact with the hippocampus, the slices were separated using fine forceps. Slices containing the intact hippocampus were selected and positioned onto 30-mm- diameter semiporous cell culture inserts (five slices per insert) (Falcon; Becton Dickinson Labware, Millipore, Bedford, MA) and placed in a six-well tissue culture tray (Multiwell; Falcon, Becton Dickinson Labware). Eagle minimum essential medium enhanced with heat-inacti- vated horse serum (1.5 ml) was then transferred to each well.

Anesthesiology, V 110, No 5, May 2009

SANDERS ET AL.

The slices were incubated for 24 h in humidified air at 37°C, enriched with 5% carbon dioxide. The culture medium was replaced the next day with fresh, temper- ature-equilibrated medium before exposure to gas treat- ments. The groups of slices (n (cid:2) 15 per group) were assigned to control (air (cid:3) 5% carbon dioxide), dexme- detomidine 1 (cid:3)M, gabazine 50 (cid:3)M, 0.75% isoflurane, 0.75% isoflurane (cid:3) dexmedetomidine 1 (cid:3)M, and 0.75% isoflurane (cid:3) gabazine 50 (cid:3)M.

All subsequent gas exposure occurred in a specially con- structed exposure chamber as previously described.23 The gases, warmed by a water bath, were delivered in the headspace above the slices by a standard anesthetic machine at 2–3 l/min, and concentrations were moni- tored with an S/5 spirometry module (Datex-Ohmeda, Bradford, United Kingdom). After 3– 4 min of gas flow, the chambers were sealed and placed in a 37°C incuba- tor for 6 h (Galaxy R Carbon Dioxide Chamber; Wolf Laboratories, Pocklington, York, United Kingdom). After exposure, the slices were returned to the incubator for a further 12 h of culture to allow for suitable caspase-3 expression and then fixed overnight in 4% paraformal- dehyde and subsequently immersed in 30% sucrose for a further 24 h at 4°C before slicing with a cryostat.

In Vivo Experiments Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane in 25% oxygen or air in a temperature-controlled chamber (n (cid:2) 6 per group). Three doses of saline or dexmedetomidine (1, 10, or 25 (cid:3)g/kg) were administered by intraperitoneal injection over the 6-h exposure (at 0, 2, and 4 h). One group received 0.75% isoflurane, 25 (cid:3)g/kg dexmedetomidine, and 500 (cid:3)g/kg nonselective (cid:2)2 adrenoceptor antagonist atipamezole in 3 doses over the 6-h exposure (n (cid:2) 4 per group). An additional three doses of 75 (cid:3)g/kg dexme- detomidine in air were given to establish at extreme doses of dexmedetomidine whether apoptosis could be induced (n (cid:2) 6 per group).

The animals were sacrificed (with 100 mg/kg sodium pentobarbital by intraperitoneal injection) at the end of gas exposure and perfused transcardially with heparin- ized saline followed by 4% paraformaldehyde in 0.1 M buffer. After removal of the brain and storage overnight at 4°C in paraformaldehyde, it was transferred to 30% sucrose solution with phosphate buffer and 1% sodium azide and kept at 4°C until the brains were sectioned and stained immunohistochemically for caspase-3.

Immunohistochemistry For the in vitro experiments, the slices were sectioned at 25-(cid:3)m intervals using a cryostat, and the inner sec- tions were mounted onto Super Plus-coated glass slides (VWR International, Lutterworth, United Kingdom). The sections were allowed to dry at 37°C for 24 h and then immunostained while adherent to the slides. Concerning

DEXMEDETOMIDINE INHIBITS ISOFLURANE-INDUCED INJURY

the in vivo experiments, the brain was sliced at 30-(cid:3)m intervals beginning at (cid:1)3.6 mm from the bregma, the sections were then transferred to a six-well plate con- taining phosphate-buffered saline. Sections were dried at 37°C for 24 h and then immunostained while adherent to the slides, before preincubation with hydrogen 0.3% peroxidase in methanol for 30 min and then rinsed in phosphate-buffered saline. The sections were then incu- bated overnight at 4°C with rabbit anti-cleaved caspase-3 (1:2,500; New England Biolab, Hitchin, United King- dom) and then washed three times in phosphate-buff- ered saline with 3% Triton at room temperature. Biotin- ylated secondary antibodies (1:200; Sigma, St. Louis, MO) and the avidin-biotin-peroxidase complex (Vector Laboratories, Orton Southgate, Peterborough, United Kingdom) were applied. The sections were again washed in phosphate-buffered saline before incubating with 0.02% 3,3=-diaminobenzidine with nickel ammo- nium sulfate in 0.003% hydrogen peroxide (DAB kit, Vector Laboratories). The sections were dehydrated through a gradient of ethanol solutions (70 –100%) and then mounted (floating section) and covered with a cover slip.

Neurocognitive Evaluation Seven-day-old Sprague-Dawley rat pups were exposed to 6 h of 0.75% isoflurane in 25% oxygen or air in a temperature-controlled chamber (n (cid:2) 6 per group). Three doses of saline or 25 (cid:3)g/kg dexmedetomidine were administered by intraperitoneal injection over the 6-h exposure (at 0, 2, and 4 h). The animals were al- lowed to mature until postnatal day 40 and then tested for hippocampal-dependent memory and learning func- tion in a previously reported contextual fear-condition- ing behavioral paradigm24 in which the rats were taken from the vivarium in the behavioral room on the first test day and allowed to sit undisturbed in their homecage for 10 min. Once placed in the conditioning chamber, the rats were allowed 198 s of exploration.

The conditioning chamber was cubic (30 cm (cid:4) 24 cm (cid:4) 21 cm; Med Associates, Inc., St. Albans, VT) and had a white opaque back wall, aluminum sidewalls, and a clear polycarbonate front door. The conditioning box had a removable grid floor and waste pan. Between each rat, the box was cleaned with an almond-scented solution and dried thoroughly. The grid floor contained 36 stain- less steel rods (diameter, 3 mm) spaced 8 mm center to center. When placed in the chamber, the grid floor made contact with a circuit board through which a scrambled shock was delivered. During training and context test- ing, a standard HEPA filter provided background white noise of 65 db.

Afterwards, all animals received 6 cycles of 214 s of trace fear conditioning. The tone was presented for 16 s (2 kHz) followed by a trace interval of 18 s and subse- quent foot shock (2 s, 0.85 mA). The rats were removed

from the conditioning chamber 198 s after the last shock and returned to their home cage. The total time of the acquisition phase was 26 min. Acquisition time was defined as the time spent immobile after a shock divided by the intertrial interval. On the next day, trained rats were exposed to the same acquisition environment but received neither tone nor shock for 8 min (context test). The percentage of time an animal froze during the 8-min observation periods was calculated as the number of observations judged to be freezing divided by the total number of observations in 8 min (i.e., 60 observations). Freezing time was assessed using VideoFreeze software (Med Associates Inc., Burlington, VT); therefore, the assessment can be considered objective. The percentage of freezing time (context results) and the area under curve were derived from plots between the percentage freezing time and trial time in the tone test and were used for statistical comparison (mean (cid:5) SD, n (cid:2) 6 per group).

Statistical Analyses The number of caspase-3–positive neurons in the cor- tex, thalamus, and hippocampus in each brain slice were counted by an observer blinded to the experimental protocol. Four brain slices were counted per animal. The immunohistochemical and behavioral data are presented as mean (cid:5) SD. Statistical analyses was performed by ANOVA followed by post hoc Newman Keuls testing using the INSTAT (London, United Kingdom) program. P (cid:6) 0.05 was set as significant.

Results

All animals survived the in vivo experiments. Isoflu- rane induced neuroapoptosis throughout the cortex, thalamus, and hippocampus reflected by the increase in the number of caspase-3–positive cells observed (fig. 1, A–C). Isoflurane (0.75% (cid:3) saline) increased caspase-3 expression relative to air ((cid:3) saline) controls in the cor- tex from 44 (cid:5) 7 to 270 (cid:5) 34 cells (P (cid:6) 0.05; fig. 1D), in the hippocampus from 8 (cid:5) 3 to 80 (cid:5) 11 cells (P (cid:6) 0.05; fig. 1E), and in the thalamus from 4 (cid:5) 2 to 62 (cid:5) 15 cells (P (cid:6) 0.05; fig. 1F). In contrast dexmedetomidine in the presence of air did not increase cellular caspase-3 ex- pression relative to controls (fig. 1, E–F).

Dexmedetomidine (1–25 (cid:3)g/kg) provided dose-dependent neuroprotection reducing isoflurane-induced caspase-3 ex- pression in the cortex (186 (cid:5) 23 to 129 (cid:5) 29 cells; P (cid:6) 0.05) relative to isoflurane (270 (cid:5) 34 cells), hippocam- pus (28 (cid:5) 11 to 15 (cid:5) 5 cells; P (cid:6) 0.05) relative to isoflurane (80 (cid:5) 11 cells), and thalamus (21 (cid:5) 6 to 9 (cid:5) 4 cells; P (cid:6) 0.05) relative to isoflurane (62 (cid:5) 15 cells). The addition of 25 (cid:3)g/kg dexmedetomidine provided the most potent protection that was significantly better than 1 or 10 (cid:3)g/kg dexmedetomidine (P (cid:6) 0.05) in each

Anesthesiology, V 110, No 5, May 2009

1079

1080

brain area. In the hippocampus and thalamus, but not the cortex, 25 (cid:3)g/kg dexmedetomidine reduced the injury induced by isoflurane to baseline (P (cid:7) 0.05 vs. Air (cid:3) Saline). Reversal of dexmedetomidine neuroprotection by the (cid:2)2 adrenoceptor antagonist, atipamezole, in the hippocampus (P (cid:6) 0.05; fig. 1E), thalamus, and cortex (nonsignificant) indicates that this effect is at least partly mediated by (cid:2)2 adrenoceptors in these regions.

Consistent with previous data,4 6 hours of 0.75% isoflu- rane also induced neuroapoptosis in organotypic hip- pocampal slice cultures, increasing caspase-3 expression by 44% (control 18 (cid:5) 6 vs. isoflurane 26 (cid:5) 11 cells; P (cid:6) 0.01; fig. 2, A and B). This effect was reversed by addi- tion of 1 (cid:3)M dexmedetomidine (20 (cid:5) 7 cells; P (cid:6) 0.05, fig. 2C), reducing caspase-3 expression to within 10% of controls. As reported previously,25 gabazine itself was nontoxic (showing caspase-3 expression 92% of air-sa- line treated controls; P (cid:7) 0.05); it did not attenuate isoflurane-induced apoptosis (P (cid:7) 0.05 vs.. isoflurane; fig. 2, D and E).

At postnatal day 40, neonatal treatment with isoflu- induced

rane-saline, but not air-dexmedetomidine,

Anesthesiology, V 110, No 5, May 2009

SANDERS ET AL.

Fig. 1. Dexmedetomidine (Dex) inhibits isoflurane-induced neuroapoptosis in vivo. Seven-day-old rats were exposed to air or isoflurane (0.75%) for 6 h with injections of saline or intraperitoneal dexmedetomidine given three times at 0, 2, and 4 h. (A) Photomicrograph of a cor- tical section from an animal exposed to air and three doses of saline over 6 h stained immunohistochemically for caspase-3. (B) A similar photomicrograph of a cortical section from an animal exposed to isoflu- rane and three doses of saline over 6 h. (C) Photomicrograph of a cortical section from an animal exposed to isoflurane and three doses of 1 (cid:3)g/kg dexmedetomi- dine over 6 h stained immunohisto- chemically for caspase-3. (D) Histogram showing the number of caspase-3–posi- tive cortical neurons against interven- tion. (E) Histogram showing the number of caspase-3–positive hippocampal neu- rons against intervention. (F) Histogram showing the number of caspase-3–posi- tive thalamic neurons against interven- tion. The interventions include: Air (cid:4) Sa- line intraperitoneal (Air/Sal), Air (cid:4) Dex 25 (cid:3)g/kg (Air/Dex25), Air (cid:4) Dex 75 (cid:3)g/kg (Iso/Dex75), Isoflurane (cid:4) saline (Iso/Sal), Iso (cid:4) Dex 1 (cid:3)g/kg (Iso/Dex1), Iso (cid:4) Dex 10 (cid:3)g/kg (Iso/Dex10), Iso (cid:4) Dex 25 (cid:3)g/kg (Iso/Dex25), Iso (cid:4) Dex 25 (cid:3)g/kg (cid:4) Atipamezole 500 (cid:3)g/kg (Iso/ Dex25/Atp); n (cid:5) 4 – 6 per group. * (cid:5) P < 0.05 versus Air (cid:4) Sal; ** (cid:5) P < 0.001 versus Air (cid:4) Sal; # (cid:5) P < 0.05 versus Iso (cid:4) Sal; (cid:4) (cid:5) P < 0.01 versus Iso (cid:4) Sal; ˆ (cid:5) P < 0.001 versus Iso (cid:4) Sal; § (cid:5) P < 0.05 versus Iso (cid:4) Dex 25; ˜ (cid:5) P < 0.05 versus Iso (cid:4) Dex1 or Iso (cid:4) Dex10.

neurocognitive impairment as assessed by context fear conditioning (a marker of long-term memory); however, none of the groups exhibited any deficit in the acquisition phase (indicating no deficit in short- term memory; fig. 3A). The percentage freezing time in the contextual fear-conditioning experiment was 48 (cid:5) 5% in controls (air-saline), 45 (cid:5) 11% with air- dexmedetomidine–treated animals, and 29 (cid:5) 7% with isoflurane-saline–treated animals (fig. 3B). Dexmedeto- midine ameliorated the neurocognitive impairment in- duced by isoflurane; percentage freezing time 46 (cid:5) 9% with isoflurane-dexmedetomidine–treated animals.

Discussion

Isoflurane induced widespread cerebral neuroapopto- sis in neonatal rat pups with subsequent long-term neu- rocognitive impairment of the animals. As the injury occurred in the neonatal period and animal training and testing followed this injury, this indicates impairment in learning and memory consistent with a significant hip-

DEXMEDETOMIDINE INHIBITS ISOFLURANE-INDUCED INJURY

isoflurane treatment. Importantly, in contrast to isoflu- rane (and other agents such as midazolam and ket- amine13), dexmedetomidine itself lacks neurotoxicity even at extremely high doses such as 75 (cid:3)g/kg dexme- detomidine (a dose that is 75 times the ED50 for hypno- sis27). Dexmedetomidine also did not induce neurocog- nitive impairment at the clinically relevant dose of 25 (cid:3)g/kg. Although dexmedetomidine could also attenuate isoflurane-induced neuroapoptosis in organotypic hip- pocampal slice cultures, gabazine did not reverse this effect, suggesting that isoflurane’s neuroapoptotic effect is not mediated by GABAA receptors.

Caveats These results indicate that dexmedetomidine can in- hibit neuroapoptosis provoked by isoflurane in vitro and in vivo; however, several caveats need to be raised before further interpretation of our data. Previous re- ports have shown that the apoptosis involved neurons, and the injured cells in our study morphologically ap- pear to be neurons. Therefore we assume the dying cells are neurons; this is supported by our data showing a neurocognitive deficit induced by isoflurane. In addition, our marker of apoptosis and cell death, caspase-3 expres- sion, has been previously validated in this model of anesthetic-injury.1– 4 Although we (and others1) have correlated the apoptosis observed with the neurocogni- tive deficits induced by isoflurane in neonatal rats, we still cannot exclude that other mechanisms (such as effects on neurogenesis or synaptic function) do not contribute to the pathogenesis or the protection af- forded by dexmedetomidine.

Fig. 2. Dexmedetomidine (Dex) inhibits isoflurane-induced neuroapoptosis in vitro. C57Bl/6 mice pup organotypic hip- pocampal cultures were exposed to (A) air (cid:4) 5% carbon dioxide (control), (B) isoflurane 0.75% (Iso), (C) isoflurane 0.75% (cid:4) Dex 1 (cid:3)M (Iso (cid:4) Dex), and (D) isoflurane (cid:4) gabazine 50 (cid:3)M (Iso (cid:4) Gab) for 6 h and then stained for caspase-3 using immuno- histochemistry. Quantified data are presented in section E. * (cid:5) P < 0.01 versus Control; ** (cid:5) P < 0.001 versus Control; # (cid:5) P < 0.05 versus Iso and P < 0.01 versus Iso (cid:4) Gab.

pocampal lesion.24,26 These data support previous ex- periments showing a significant hippocampal injury af- ter anesthetic treatment.1 Dexmedetomidine provided neuroprotection against isoflurane-induced neuroapop- tosis in a dose-dependent manner, acting via activation of (cid:2)2 adrenoceptors (as atipamezole reversed dexme- detomidine’s neuroprotective effect). Crucially, dexme- detomidine prevented the neurocognitive sequelae of

Despite data showing that the hypnotic, analgesic, and neuroprotective effects of dexmedetomidine primarily relate to activation of the (cid:2)2A adrenoceptor,28,29 atipam- ezole significantly inhibited dexmedetomidine’s neuro- protective effect only in the hippocampus. Although there was a trend to a reversal in effect in the thalamus and cortex, atipamezole did not significantly alter dexmedetomidine protection in these regions. This may indicate alternate receptor targets in these regions, such as imidazoline receptors,30 but we suspect a type II error may also account for these findings.

Minimal disturbances in arterial blood gases1,3 have been reported in previous studies; however, the poten- tial to induce hypoglycemia in these animals during the anesthetic period is of concern,31 but this has also been shown not to occur.12 Indeed, it is possible that the addition of dexmedetomidine to isoflurane could exac- erbate both the cardiovascular and respiratory depres- sion of the anesthetic state; however, it is also conceiv- able that high doses of dexmedetomidine may have increased either blood pressure (via activation of (cid:2)2B adrenoceptors) or glucose (via (cid:2)2A adrenoceptors). Therefore, we also conducted an in vitro experiment to control for the potential confounding effects of hypoxia,

Anesthesiology, V 110, No 5, May 2009

1081

1082

glucose, and temperature dysregulation using the orga- notypic hippocampal slice culture model. We employed the latter experimental paradigm because synapses re- main intact, which is imperative because inhibition of synaptic neurotransmission is hypothesized as critical to the injury. It is known that isoflurane is directly neurotoxic in the organotypic hippocampal slice culture model.4,32 We used a single dose of dexmedetomidine (1 (cid:3)M) in our organotypic hippocampal slice culture studies and did not corroborate the extensive dose response curve that was obtained in vivo because the aim in this experiment was to identify whether dexmedetomidine was acting via a direct or physiologic mechanism. These data suggest that dexmedetomidine can prevent the isoflurane injury by direct action within the central nervous system.

It should also be noted that dexmedetomidine, al- though neuroprotective, does not entirely reverse the isoflurane injury in the cortex (despite prominent neu- roprotection of the thalamus and hippocampus being observed). However, our neurocognitive tests did not uncover an isoflurane-associated deficit in memory ac- quisition that typically depends on a functional prefron- tal cortex.33 Therefore, despite significant apoptosis in the cortex, our study suggests the cortex is not function- ally impaired after isoflurane treatment (0.75% for 6 h),

Anesthesiology, V 110, No 5, May 2009

SANDERS ET AL.

Fig. 3. Cognitive function assessed by trace fear conditioning. Seven-day-old Sprague-Dawley rat pups were exposed with air or 0.75% isoflurane (Iso) in ox- ygen with or without saline or dexme- detomidine (Dex) treatment for 6 h. They were allowed to live up to 40 days and then tested for hippocampal-dependent memory and learning function. (A) The plot of the mean percentage of freezing time of acquisition against six test trials of trace fear conditioning (day 1). (B) The mean of the percentage of freezing time (context results) obtained from trace fear conditioning (day 2). Mean (cid:6) SD (n (cid:5) 6); * (cid:5) P < 0.05 versus other groups.

but we do suggest that the effects of other doses of isoflurane still require investigation. In monkeys, ket- amine injury is primarily involved the cortex rather than subcortical structures34; it is possible that, if observed in humans, cortical apoptosis induced by anesthetics may be the predominant injury. Further tests of cortex-based neurocognitive function in rodents and primates should be conducted before this injury is dismissed.

A final difficulty plaguing all preclinical studies is the ability to extrapolate across species; in this regard, differ- ential interspecies vulnerability to isoflurane injury may be apparent. Indeed, recent data have suggested that monkey brains may be less vulnerable to ketamine injury than ro- dent brains.34 However, isoflurane may be more potent at inducing apoptosis than ketamine, especially because the injury is apparent after subanesthetic isoflurane concentra- tions lasting only 1 h.12 Whether anesthetic-induced neu- rotoxicity is a clinical problem requires further investiga- tion, including studies involving monkeys and ultimately humans; while we await these answers, we need to strive to obtain a safe anesthetic therapy.

Mechanism of Dexmedetomidine Neuroprotection In these studies, we have explored whether dexme- detomidine is antiapoptotic (as suggested, but not di-

DEXMEDETOMIDINE INHIBITS ISOFLURANE-INDUCED INJURY

rectly investigated by many preclinical studies). We pro- pose that administration of an (cid:2)2 adrenoceptor agonist during the critical phase of synaptogenesis activates the endogenous postsynaptic norepinephrine-mediated tro- phic system, which couples to a pERK-Bcl-2 pathway to produce its antiapoptotic effect.19,20,21 Further studies will probe the involvement of this pathway in vivo. Despite our data showing a role for (cid:2)2 adrenoceptor activation in dexmedetomidine neuroprotection, other potential receptor subtypes, such as the imidazoline re- ceptors, can upregulate pERK and are activated by dexmedetomidine,30 thus providing an alternative mech- anism for dexmedetomidine’s neuroprotective effect.

Mechanism of Isoflurane Neurotoxicity Interestingly, gabazine could not reverse the isoflu- rane-induced neurodegeneration despite the hypothesis that, by potentiating GABAA receptor activity, isoflurane inhibits neurotransmission detrimentally during the crit- ical period of synaptogenesis.11 Thus, GABAA receptor activation may not be critical for isoflurane-induced neu- rotoxicity, although it is of interest that GABAA receptor antagonists can attenuate isoflurane’s neuroprotective effect.25,35 While GABAA receptor activation remains an important target for anesthesia, especially for intrave- nous anesthetics such as propofol, its role in haloge- nated volatile anesthesia is less clear.36,37 Therefore, it would appear that activation of the GABAA receptor is important for isoflurane neuroprotection but not neces- sarily critical for toxicity. Whether GABAA receptor antag- onism can attenuate propofol-induced neurotoxicity38 will be of interest because it reverses the propofol anesthetic state.39 However, GABAA receptors are not involved in the neurotoxicity; therefore, it may be possible to design a safe anesthetic agent for use in the young. Interest- ingly, a difference in the ability of sevoflurane and isoflu- rane to induce apoptosis has also been observed previ- ously,40 although preliminary evidence suggests that sevoflurane, similar to isoflurane, also induces neuro- apoptosis in the neonatal rat brain.41

Another receptor target that may be responsible for the isoflurane injury is the NMDA receptor, which plays a critical role in neurodevelopment.8 Each of the neuro- apoptotic-inducing anesthetics, including isoflurane, ket- amine, and MK-801, inhibit the NMDA receptor subtype of the glutamate receptor.1–5,10 –13 An exception to this rule is xenon, another NMDA receptor antagonist, which produces protection against isoflurane-induced injury rather than neuroapoptosis in the neonatal rat brain.4 We consider it likely that xenon exerts an antiapoptotic effect independent of its action at the NMDA receptor. It is of interest that both (cid:2)2 adrenoceptor agonists and xenon can attenuate the injury produced by NMDA an- tagonists in the adult brain42,43; therefore, despite differ- ences in the morphology of the adult and neonatal tox- icity, we cannot discount overlapping mechanisms of

injury. In addition, we have not as yet evaluated whether a neuroprotective cocktail of xenon and dexmedetomi- dine can be employed to further reduce isoflurane tox- icity because they provide synergistic protection against neonatal hypoxic-ischemic injury.44

Neurocognitive Effects Our results from our fear conditioning paradigm sup- port the previous reports of neurocognitive impairment in adult rats after neonatal anesthesia.1,45 Fear condition- ing consists of placing a rat or a mouse in a chamber and giving one or more mild electric foot shocks. After a shock, the animal becomes immobile (freezing), a natu- ral response to fear that can be used as an indication of memory formation. Complex neuronal circuitry involv- ing the frontal cortex, hippocampus, periaqueductal gray, and rostral ventral medulla underlie the acquisition and retention of fear conditioning.24 Notably, a damaged hippocampus is unable to process the incoming stimuli producing a memory deficit.26

Interestingly, all groups displayed normal learning as- the animals sessed by the acquisition of memory; showed increasing levels of freezing across the training tones, with freezing levels post-shock on sixth pairing approximately 70%. This indicates a normal short-term memory, a function predominantly involving the pre- frontal cortex.31 In the context assessment, 24 h after acquisition training, animals exposed to isoflurane and saline displayed less freezing when compared to naive controls, indicating a neurocognitive deficit. Thus isoflu- rane-treated animals showed an abnormal response to contextual fear conditioning, indicating a severe hip- pocampal lesion24,26 consistent with previous reports.1 However, our experiments employed a much lower dose of anesthetic than in the previous studies (0.75% isoflurane vs. 0.75% isoflurane plus 75% nitrous oxide and 9 mg/kg midazolam). Even with subanesthetic dos- ing, the potential for functional neurocognitive deficit is apparent. In contrast, dexmedetomidine alone did not induce any memory deficit. Furthermore, the addition of dexmedetomidine to isoflurane reversed the neurocog- nitive compromise induced by isoflurane. This is of crit- ical importance because dexmedetomidine is the first agent to be shown to reverse the neurocognitive dys- function provoked by isoflurane.

Dexmedetomidine is widely available and has an ex- panding role in pediatric clinical practice; therefore, if anesthetic-induced neurodegeneration is proven to be a clinical problem, we may already have available a thera- peutic intervention that can be employed in this setting where necessary. In situations where dexmedetomidine is not available, another (cid:2)2 agonist, clonidine, could be a candidate, although further studies are warranted because, although atipamezole significantly reversed dexmedetomi- dine’s neuroprotective effect in the hippocampus, the pro- tection afforded in the thalamus and cortex were not sig-

Anesthesiology, V 110, No 5, May 2009

1083

1084

nificantly attenuated; we cannot be sure that all (cid:2)2 adrenoceptor agonists will afford this protection.

Clinical Implications Clinically, no information has detailed the extent of anesthetic-induced neonatal neurodegeneration in hu- mans. In terms of neurodevelopment, a 7-day-old rat pup represents the peak of the synaptogenic period, but this period extends from birth to up to 2–3 yr in humans; therefore, the window of vulnerability may be greater in humans.46,47 One cannot advocate withholding anesthe- sia or analgesia during early human life on the basis of these findings because of the harm that this can do.47–50 However, if anesthetic-induced neurodegeneration is re- vealed as a clinical problem for pediatric anesthesia, administration of an (cid:2)2 adrenoceptor agonist during the anesthesia maybe prudent. Thus, this study has uncov- ered a plausible and promising novel application of a widely available class of drugs that may significantly affect the safety of clinical practice.

The use of (cid:2)2 adrenoceptor agonists in pediatric prac- tice is expanding as a result of their potent sedative/ hypnotic qualities, analgesic action, potential organ-pro- tective effects, reduction in postoperative nausea and vomiting and delirium, and relative lack of respiratory side effects.51,52 Their use in neonatal practice requires evaluation based on these factors.51 In the future, their organ-protective, including neuroprotective, effects may be of importance to the provision of safe, balanced pediatric anesthesia.47

References

1. Jevtovic-Todorovic V, Hartman RE, Izumi Y, Benshoff ND, Dikranian K, Zorumski CF, Olney JW, Wozniak DF: Early exposure to common anesthetic agents causes widespread neurodegeneration in the developing rat brain and persistent learning deficits. J Neurosci 2003; 23:876–82

2. Hansen HH, Briem T, Dzietko M, Sifringer M, Voss A, Rzeski W, Zdzisinska B, Thor F, Heumann R, Stepulak A, Bittigau P, Ikonomidou C: Mechanisms leading to disseminated apoptosis following NMDA receptor blockade in the developing rat brain. Neurobiol Dis 2004; 16:440–53

3. Lu LX, Yon JH, Carter LB, Jevtovic-Todorovic V: General anesthesia activates BDNF-dependent neuroapoptosis in the developing rat brain. Apoptosis 2006; 11:1603–15

4. Ma D, Williamson P, Januszewski A, Nogaro MC, Hossain M, Ong LP, Shu Y, Franks NP, Maze M: Xenon mitigates isoflurane-induced neuronal apoptosis in the developing rodent brain. ANESTHESIOLOGY 2007; 106:746–53

5. Ikonomidou C, Bittigau P, Ishimaru MJ, Wozniak DF, Koch C, Genz K, Price MT, Stefovska V, Horster F, Tenkova T, Dikranian K, Olney JW: Ethanol-induced apoptotic neurodegeneration and fetal alcohol syndrome. Science 2000; 287: 1056–60

6. Jain N, Florence SL, Qi HX, Kaas JH: Growth of new brainstem connections in adult monkeys with massive sensory loss. Proc Natl Acad Sci U S A. 2000; 97:5546–50

7. Hestrin S: Developmental regulation of NMDA receptor-mediated synaptic

currents at a central synapse. Nature 1992; 357:686–9

8. Hardingham GE, Bading H: The Yin and Yang of NMDA receptor signaling.

Trends Neurosci 2003; 26:81–9

9. Franks NP, Lieb WR: Molecular and cellular mechanisms of general anaes-

thesia. Nature 1994; 367:607–14.

10. Yon JH, Daniel-Johnson J, Carter LB, Jevtovic-Todorovic V: Anesthesia induces neuronal cell death in the developing rat brain via the intrinsic and extrinsic apoptotic pathways. Neuroscience 2005; 135:815–27

11. Olney JW, Young C, Wozniak DF, Ikonomidou C, Jevtovic-Todorovic V: Anesthesia-induced developmental neuroapoptosis. Does it happen in humans? ANESTHESIOLOGY 2004; 101:273–5

Anesthesiology, V 110, No 5, May 2009

SANDERS ET AL.

12. Johnson SA, Young C, Olney JW: Isoflurane-induced neuroapoptosis in the developing brain of nonhypoglycemic mice. J Neurosurg Anesthesiol 2008; 20:21–8

13. Young C, Jevtovic-Todorovic V, Qin YQ, Tenkova T, Wang H, Labruyere J, Olney JW: Potential of ketamine and midazolam, individually or in combination, to induce apoptotic neurodegeneration in the infant mouse brain. Br J Pharmacol 2005; 146:189–97

14. Winzer-Serhan UH, Leslie FM: Expression of alpha2A adrenoceptors during

rat neocortical development. J Neurobiol 1999; 38:259–69

15. Song ZM, Abou-Zeid O, Fang YY: Alpha2a adrenoceptors regulate phos- phorylation of microtubule-associated protein-2 in cultured cortical neurons. Neuroscience 2004; 123:405–18

16. Wang Q, Lu R, Zhao J, Limbird LE: Arrestin serves as a molecular switch, linking endogenous alpha2-adrenergic receptor to SRC-dependent, but not SRC- independent, ERK activation. J Biol Chem 2006; 281:25948–55

17. Philipp M, Brede ME, Hadamek K, Gessler M, Lohse MJ, Hein L: Placental alpha(2)-adrenoceptors control vascular development at the interface between mother and embryo. Nat Genet 2002; 31:311–5

18. Engelhard K, Werner C, Eberspa¨cher E, Bachl M, Blobner M, Hildt E, Hutzler P, Kochs E: The effect of the alpha 2-agonist dexmedetomidine and the N-methyl-D-aspartate antagonist S((cid:3))-ketamine on the expression of apoptosis- regulating proteins after incomplete cerebral ischemia and reperfusion in rats. Anesth Analg 2003; 96:524–31

19. Dahmani S, Paris A, Jannier V, Hein L, Rouelle D, Scholz J, Gressens P, Desmonts JM, Mantz J: Dexmedetomidine increases hippocampal phosphory- lated extracellular signal-regulated protein kinase 1 and 2 content by an alpha 2-adrenoceptor-independent mechanism: Evidence for the involvement of imida- zoline I1 receptors. ANESTHESIOLOGY 2008; 108:457–66

20. Dahmani S, Rouelle D, Gressens P, Mantz J: Effect of dexmedetomidine on brain derived neurotrophic factor expression in rat hippocampal slices (ab- stract). ANESTHESIOLOGY 2006; 105:A681

21. Gahwiler BH: Organotypic monolayer cultures of nervous tissue. J Neuro-

sci Methods 1981; 4:329–42

22. Stoppini L, Buchs PA, Muller D: A simple method for organotypic cultures

of nervous tissue. J Neurosci Methods 1991; 37:173–82

23. Wilhelm S, Ma D, Maze M, Franks NP: Effects of xenon on in vitro and in

vivo models of neuronal injury. ANESTHESIOLOGY 2002; 96:1485–91

24. Quinn JJ, Loya F, Ma QD, Fanselow MS: Dorsal hippocampus NMDA receptors differentially mediate trace and contextual fear conditioning. Hip- pocampus 2005; 15:665–74

25. Ma D, Hossain M, Rajakumaraswamy N, Franks NP, Maze M: Combination of xenon and isoflurane produces a synergistic protective effect against oxygen- glucose deprivation injury in a neuronal-glial co-culture model. ANESTHESIOLOGY 2003; 99:748–51

26. Fanselow MS: Contextual fear, gestalt memories, and the hippocampus.

Behavioural Brain Res 2000; 110:73–81

27. Sanders RD, Giombini M, Ma D, Ohashi Y, Hossain M, Fujinaga M, Maze M: Dexmedetomidine exerts dose-dependent age-independent antinociception but age-dependent hypnosis in Fischer rats. Anesth Analg 2003; 100:1295–302

28. Lakhlani PP, MacMillan LB, Guo TZ, McCool BA, Lovinger DM, Maze M, Limbird LE: Substitution of a mutant alpha2a-adrenergic receptor via “hit and run” gene targeting reveals the role of this subtype in sedative, analgesic, and anesthetic-sparing responses in vivo. Proc Natl Acad Sci U S A 1997; 94:9950–5 29. Ma D, Hossain M, Rajakumaraswamy N, Arshad M, Sanders RD, Franks NP, Maze M: Dexmedetomidine produces its neuroprotective effect via the (cid:2)2A- adrenoceptor subtype. Eur J Pharmacol 2004; 502:87–97

30. Dahmani S, Paris A, Hein L, Gressens P, Mantz J: Dexmedetomidine activates ERK 1 & 2 via a non-(cid:2)2 adrenoceptors-mediated pathway (abstract). ANESTHESIOLOGY 2007:A12

31. Loepke AW, McCann JC, Kurth CD, McAuliffe JJ: The physiologic effects

of isoflurane anesthesia in neonatal mice. Anesth Analg 2006; 102:75–80

32. Wise-Faberowski L, Zhang H, Ing R, Pearlstein RD, Warner DS: Isoflurane- induced neuronal degeneration: An evaluation in organotypic hippocampal slice cultures. Anesth Analg 2005; 101:651–7

33. Blum S, Hebert AE, Dash PK: A role for the prefrontal cortex in recall of

recent and remote memories. Neuroreport 2005; 17:341–4

34. Slikker W Jr, Zou X, Hotchkiss CE, Divine RL, Sadovova N, Twaddle NC, Doerge DR, Scallet AC, Patterson TA, Hanig JP, Paule MG, Wang C: Ketamine- induced neuronal cell death in the perinatal rhesus monkey. Toxicol Sci 2007; 98:145–58

35. Elsersy H, Mixco J, Sheng H, Pearlstein RD, Warner DS: Selective gamma- aminobutyric acid type A receptor antagonism reverses isoflurane ischemic neuroprotection. ANESTHESIOLOGY 2006; 105:81–90

36. Jurd R, Arras M, Lambert S, Drexler B, Siegwart R, Crestani F, Zaugg M, Vogt KE, Ledermann B, Antkowiak B, Rudolph U: General anesthetic actions in vivo strongly attenuated by a point mutation in the GABA(A) receptor beta3 subunit. FASEB J 2003; 17:250–2

37. Sonner JM, Zhang Y, Stabernack C, Abaigar W, Xing Y, Laster MJ: GABA(A) receptor blockade antagonizes the immobilizing action of propofol but not ketamine or isoflurane in a dose-related manner. Anesth Analg 2003; 96:706–12 38. Cattano D, Young C, Olney J: Subanesthetic doses of propofol induce neuroapoptosis in the infant mouse brain (abstract). ANESTHESIOLOGY 2007; 106: A1984

DEXMEDETOMIDINE INHIBITS ISOFLURANE-INDUCED INJURY

39. Nelson LE, Lu J, Guo T, Saper CB, Franks NP, Maze M: The sedative component of anesthesia is mediated by GABA(A) receptors in an endogenous sleep pathway. Nat Neurosci 2002; 5:979–84

combination of N-methyl-D-Aspartate and gamma-aminobutyric acid type A re- ceptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioural deficits. ANESTHESIOLOGY 2007; 107:427–36

40. Wei H, Kang B, Wei W, Liang G, Meng QC, Li Y, Eckenhoff RG: Isoflurane and sevoflurane affect cell survival and BCL-2/BAX ratio differently. Brain Res 2005; 1037:139–47

41. Satomoto M, Satoh Y, Terui K, Imaki J: Inhaled sevoflurane induces apoptosis in the developing brain and behavioural abnormality in mice (abstract). ANESTHESIOLOGY 2008; 109:A26

42. Marrs W, Kuperman J, Avedian T, Roth RH, Jentsch JD: Alpha-2 adreno- ceptor activation inhibits phencyclidine-induced deficits of spatial working mem- ory in rats. Neuropsychopharmacology 2005; 30:1500–10

46. Dobbing J, Sands J: Comparative aspects of the brain growth spurt. Early

Hum Dev 1979; 3:79–83

47. Sanders RD, Ma D, Brooks P, Maze M: Balancing paediatric anaesthesia: Preclinical insights into analgesia, hypnosis, neuroprotection and neurotoxicity. Br J Anaesth 2008; 101:597–609

48. Anand KJS, Hickey PR: Pain and its effects in the human neonate and fetus.

N Engl J Med 1987; 317:1321–29

49. Porter FL, Grunau RE, Anand KJ: Long-term effects of pain in infants. J Dev

43. Nagata A, Nakao Si S, Nishizawa N, Masuzawa M, Inada T, Murao K, Miyamoto E, Shingu K: Xenon inhibits but N(2)O enhances ketamine-induced c-Fos expression in the rat posterior cingulate and retrosplenial cortices. Anesth Analg 2001; 92:362–8

44. Rajakumaraswamy N, Ma D, Hossain M, Sanders RD, Franks NP, Maze M: Neuroprotective interaction produced by xenon and dexmedetomidine on in vitro and in vivo neuronal injury models. Neurosci Lett 2006; 409:128–33

45. Fredriksson A, Ponten E, Gordh T, Eriksson P: Neonatal exposure to a

Behav Pediatr 1999; 20:253–61

50. Taddio A, Katz J, Ilersich AL, Koren G: Effect of neonatal circumcision on pain response during subsequent routine vaccination. Lancet 1997; 349:599–603 51. Sanders RD, Maze M: Alpha2-adrenoceptor agonists. Curr Opin Investig Drugs 2007; 8:25–33

52. Bergendahl H, Lonnqvist PA, Eksborg S: Clonidine: An alternative to benzodiazepines for premedication in children. Curr Opin Anaesthesiol 2005; 18:608–13

Anesthesiology, V 110, No 5, May 2009

1085