File size: 39,545 Bytes
8650c17
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
155
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
228
229
230
231
232
233
234
235
236
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
282
283
284
285
286
287
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
331
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
397
398
399
400
401
402
403
404
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
423
424
425
426
427
428
429
430
431
432
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l

i

Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n

r

i

i

l

1 1 / 2 0 / 2 0 2 3

D o w n o a d e d

l

f r o m h t t p : / / j o u r n a s . l

l

w w . c o m / a n e s t h e s a - a n a g e s a

i

l

b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I

f

Neonatal Exposure to Sevoflurane Causes Significant Suppression of Hippocampal Long-Term Potentiation in Postgrowth Rats

Rui Kato, MD, PhD,* Kaori Tachibana, MD, PhD,* Naoki Nishimoto, PhD,† Toshikazu Hashimoto, MD, PhD,* Yosuke Uchida, MD,* Ryoko Ito, MD,* Kenkichi Tsuruga, MD,* Koichi Takita, MD, PhD,* and Yuji Morimoto, MD, PhD*

BACKGROUND: The inhaled anesthetic sevoflurane is commonly used for neonates in the clinical setting. Recent studies have indicated that exposure of neonatal rodents to sevoflu- rane causes acute widespread neurodegeneration and long-lasting neurocognitive dysfunction. Although acute toxic effects of sevoflurane on cellular viability in the hippocampus have been reported in some studies, little is known about the effects of neonatal sevoflurane exposure on long-term hippocampal synaptic plasticity, which has been implicated in the processes of learn- ing and memory formation. Our study is the first to examine the long-term electrophysiological impact of neonatal exposure to a clinically relevant concentration of sevoflurane. METHODS: On postnatal day 7, rats were exposed to sevoflurane (1% or 2% for 2 hours) with oxygen. To eliminate the influence of blood gas abnormalities caused by sevoflurane-induced respiratory suppression, a group of rats were exposed to a high concentration of carbon dioxide (8% for 2 hours) to duplicate respiratory disturbances caused by 2% sevoflurane exposure. RESULTS: Exposure of neonatal rats to 2% sevoflurane for 2 hours caused significant suppres- sion of long-term potentiation (LTP) induction in the postgrowth period. There was no significant difference between the control group and the CO2-exposed group in LTP induction, indicating that sevoflurane-induced LTP suppression was not caused by blood gas abnormalities. CONCLUSION: Our present findings indicate that neonatal exposure to sevoflurane at a higher concentration can cause alterations in the hippocampal synaptic plasticity that persists into adulthood.

(Anesth Analg 2013;117:1429–35)

Neonatal exposure to anesthetics has been shown to

induce acute neuronal cell death and long-lasting behavioral abnormalities in animal models includ- ing primates.1–6 Jevtovic-Todorovic et al.1 reported that isoflurane, individually or in combination with other anes- thetics, caused neurodegeneration, deficits of hippocampal electrophysiological function, and persistent memory and learning impairments in rats. Fredriksson et al.2 reported that neonatal exposure to a combination of thiopental or propofol and ketamine caused a synergistic neurotoxic effect in mice. According to those reports, anesthetics that activate γ-aminobutyric acid-A (GABA) receptors and block N-methyl-d-aspartate (NMDA)-type glutamate receptors have neurotoxic effects in developing animals.6,7

Sevoflurane is one of the most commonly used inhaled anesthetics for neonates in the clinical setting. It activates GABA receptors (GABAR) and blocks NMDA receptors,8,9

From the *Department of Anesthesiology and Critical Care Medicine and †Di- vision of Clinical Trial Management, Center for Translational Research, Hok- kaido University Graduate School of Medicine, Sapporo, Japan.

and several lines of study have shown that it causes neu- rodegeneration in the central nervous system in neona- tal rodents and long-lasting neurocognitive dysfunction, including learning disabilities.3,10–13 The effect of acute anes- thetic toxicity on cellular viability has been well studied; however, the mechanism of long-lasting learning disabili- ties is still uncertain.

Hippocampal synaptic plasticity has been implicated in many learning and memory processes.14–18 Therefore, 1 possible mechanism that underlies the learning disability found after neonatal exposure to sevoflurane is assumed to be impairments in hippocampal synaptic plasticity. In this study, we aimed to characterize the electrophysiologi- cal alterations caused by neonatal sevoflurane exposure by investigating in vivo long-term potentiation (LTP) induc- tion in the hippocampal CA1 region. We also evaluated the effects of hypercapnia and acidosis using a carbon dioxide (CO2) exposure model because respiratory disturbances caused by anesthesia are unavoidable in small animals. The present study is the first to examine the long-term electro- physiological impact of exposing neonatal rats to clinically relevant concentrations of sevoflurane.

Accepted for publication August 2, 2013.

Funding: This work was supported by the Ministry of Education, Science, Sports, and Culture of Japan Society for the Promotion of Science. (Grants-in- Aid for Scientific Research 22591699, 20791060)

The authors declare no conflicts of interest.

Reprints will not be available from the authors.

Address correspondence to Rui Kato, MD, PhD, Department of Anesthesiol- ogy and Critical Care Medicine, Hokkaido University Graduate School of Medicine, Kita-15, Nishi-7, Kita-Ku, Sapporo 060-8638, Japan. Address e-mail to katorui@med.hokudai.ac.jp.

Copyright © 2013 International Anesthesia Research Society DOI: 10.1213/ANE.0b013e3182a8c709

METHODS Animals The experiments were approved by the Committee for Animal Research of the Hokkaido University Graduate School of Medicine. Pregnant Wistar/ST rats were obtained from Shizuoka Laboratory Animal Center (Hamamatsu, Japan). The animals were housed in a room maintained at 22°C to 25°C with a 12-hour light-dark cycle (light from

December 2013 • Volume 117 • Number 6

www.anesthesia-analgesia.org 1429

ReseaRch RepoRt

H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l

i

Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n

r

i

i

l

1 1 / 2 0 / 2 0 2 3

D o w n o a d e d

l

f r o m h t t p : / / j o u r n a s . l

l

w w . c o m / a n e s t h e s a - a n a g e s a

i

l

b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I

f

Neonatal Sevoflurane Exposure Impairs Long-Term Potentiation Induction

06:00 to 18:00) and were given free access to food and water. Rats were handled in accordance with the Guidelines for the Care and Use of Laboratory Animals of the Hokkaido University Graduate School of Medicine.

Gas Exposure At postnatal day 7, male pups were divided into 4 treat- ment groups as follows: animals exposed to (1) 1% sevo- flurane with oxygen (O2), (2) 2% sevoflurane with O2, (3) 8% CO2 with O2 to mimic hypercapnia and acidosis caused by 2% sevoflurane exposure, or (4) 100% O2 as a control. Rat pups were placed in an acrylic chamber and exposed to warmed, humidified gas for 2 hours. The total gas flow was 3 L/min to maintain a stable gas concentration. The concentrations of sevoflurane, CO2, and O2 in the chamber were continuously monitored using an anesthetic gas moni- tor (5250RGM, Datex-Ohmeda GE Healthcare, Chalfont St. Giles, United Kingdom). The gas temperature in the cham- ber was maintained at 27.5°C to 28.6°C, measured using a TR-200 (F.S.T., Foster City, CA), and the rats’ body tempera- tures were maintained at 35.1°C to 36.5°C, measured using a Thermofocus® (Tecnimed, Tokyo, Japan). To avoid litter variability, equal numbers of rat pups from each litter were randomly assigned to the 4 treatment groups. After gas exposure, rat pups were brought up in an austere environ- ment in standardized cages to avoid the influence of envi- ronmental enrichment on later neurocognitive functions.13

Arterial Blood Gas Assessment To assess respiratory disturbances caused by sevoflurane exposure, arterial blood gas assessment was performed as described previously.19,20 At postnatal day 7, rats underwent arterial blood sampling from the left ventricle via transtho- racic puncture under local subcutaneous anesthesia (0.05 mL of 1% lidocaine) at 30, 60, 90, or 120 minutes during 2 hour treatments. Since taking even a single sample of blood could be fatal for the rats, blood sampling was conducted only once for each rat. A small amount of blood (<100 μL) was collected and analyzed immediately using a blood gas analyzer (ABL510, Radiometer Medical, Brønshøj, Denmark) that measured pH, partial pressure of CO2, and O2 (Paco2 and Pao2 in mm Hg, respectively). In all cases, the procedure was completed within 1 minute in the chamber.

Electrophysiological Study Between postnatal days 63 and 70, an electrophysiologi- cal study was performed following our reported method.21 Rats were anesthetized with 1% halothane in a mixture of 21% O2 and 79% nitrogen through a tracheal catheter, and their lungs mechanically ventilated (SN-480–7, Shinano, Tokyo, Japan). They were placed in a stereotaxic apparatus with the bregma and lambda in the same horizontal plane, and their body temperatures were maintained at 37°C ± 0.5°C with a heating pad throughout the recording period. The concentration of halothane and expired CO2 tension was continuously monitored through a tracheal catheter using an anesthetic gas monitor (5250RGM, Datex-Ohmeda GE Healthcare), and the expired CO2 tension was main- tained between 35 and 45 mm Hg. A monopolar recording electrode was inserted into the pyramidal cell body layer

1430

www.anesthesia-analgesia.org

of the hippocampal CA1 region (5.0 mm posterior and 3.0 mm lateral to the bregma and approximately 2.2 mm ven- tral to the dura), to record extracellular population spike amplitude (PSA) (Fig. 1A). A bipolar stimulating electrode was inserted into the ipsilateral Schaffer collaterals (3.0 mm posterior and 1.5 mm lateral to the bregma, and 2.8 mm ventral to the dura) to deliver cathodal stimulus (fre- quency 0.1 Hz, pulse duration 250 μs) (Fig. 1A). A single electrical stimulus evoked action potentials in the Schaffer collaterals, resulting in activation of pyramidal cells of the CA1 region, and extracellular PSA was recorded (MacLab, ADInstruments, Sydney, Australia). We measured the PSA following our previous method.21 Briefly, the PSA was defined as the absolute voltage of a vertical line running from the population spike minimum to its intersection with a line tangential to the population spike onset and popula- tion spike offset. To adjust the test stimulus, changes in PSA caused by varied stimulus intensity were recorded, and the intensity of the test stimulus was then fixed to produce a half-maximal response for each rat. After establishing a sta- ble baseline for 30 minutes, LTP was induced by applying high-frequency stimulation (HFS; 10 trains at 1 Hz each, composed of 8 pulses at 400 Hz), at the same intensity as the test stimulation. Then relative ratios of PSAs before and after the HFS were then plotted every 5 minutes for 60 minutes after HFS. On completion of the experiment, small lesions were made using a direct electric current (5 µA for 30 seconds) at the tips of the recording and stimulating electrodes. The positions of the electrodes were examined histologically (Fig. 1) according to methods in similar pre- vious reports.22,23 Histological confirmation of the locations of the electrodes showed misplacement of the electrodes in 2 rats in each group, so the data obtained from those elec- trodes were excluded from the following assessment. All

Figure 1. Positions of stimulating and recording electrodes (A). The triangle shows the position of the tip of the stimulating electrode, which was located 3 mm posterior and 1.5 mm lateral to the bregma and 2.8 mm under the dura. The circle shows the position of the tip of the recording electrode, which was located 5 mm posterior and 3 mm lateral to the bregma and 2.2 mm under the dura. The locations of the electrodes were examined using a post hoc histological test (B, C). The arrow shows the tip of the stimulating electrode located at the Schaffer collateral (B), and the arrowhead shows the recording electrode located in the pyramidal cell layer of the CA1 region (C).

aNesthesia & aNalgesia

H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l

i

Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n

r

i

i

l

1 1 / 2 0 / 2 0 2 3

D o w n o a d e d

l

f r o m h t t p : / / j o u r n a s . l

l

w w . c o m / a n e s t h e s a - a n a g e s a

i

l

b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I

f

data were collected by investigators who were blinded to treatment assignment.

Statistical Methods All statistical analyses were performed using GraphPad Prism version 5.04 (GraphPad Software, Inc., San Diego, CA). First, we confirmed normal distribution of the obtained data by the Shapiro-Wilk test and a normal-probability plot. Data obtained in arterial blood gas analysis were analyzed using 1-way analysis of variance (ANOVA) followed by Bonferroni multiple comparison test for each time point. In the comparisons of LTP data, we used 2-way repeated-mea- sures ANOVA with treatment and time as factors to assess the interaction, and we used 1-way ANOVA followed by Bonferroni multiple comparison test to evaluate differences among the groups at each time point. All averaged data are presented as mean ± SEM. Probability values (P) of <5% were considered significant.

RESULTS Anesthetic Effect of Neonatal Sevoflurane Exposure Neonatal 2% sevoflurane exposure caused immediate loss of spontaneous movement and righting reflex but pre- served the response to tail pinch. Neonatal 2% sevoflurane also reduced respiratory rate throughout treatment. In the group exposed to 1% sevoflurane, spontaneous movement decreased, but the righting reflex, tail pinch response and frequency of breath were not disturbed. CO2 exposure caused no remarkable change. After treatment, all rats recovered completely.

Arterial Blood Gas Analysis The results from blood gas analysis are summarized in Table 1. In the CO2 and 2% sevoflurane groups, there were significant respiratory disturbances from hypercapnia and respiratory acidosis throughout treatment compared with the control and 1% sevoflurane groups, whereas 1% sevo- flurane exposure caused no significant changes compared with no exposure in the control group. We could not find significant differences in Pao2, Paco2, or pH between the CO2 and 2% sevoflurane-exposed groups, that is, the hyper- capnia and acidosis observed in the 2% sevoflurane group were the same in the CO2 group. Hypoxia was not observed during treatment in any group.

Neonatal Exposure to 2% Sevoflurane Caused Significant Suppression of Hippocampal LTP Induction After the establishment of stable baseline PSAs, the groups (N = 8 for each) were subjected to HFS. PSAs were signifi- cantly augmented in the control and CO2 groups, and LTP was induced as shown in Figure 2A. The maximal responses were 214.1% ± 29.8%, 218.6% ± 25.4%, 170.7% ± 20.1%, and 130.9% ± 10.5% in the control, CO2, 1% sevoflurane, and 2% sevoflurane groups, respectively. Two-way repeated-mea- sures ANOVA revealed significant differences among the treatments (F = 3.90; P = 0.019) but not among time courses (F = 1.86; P = 0.058). There was no interaction among the groups (F = 0.70; P = 0.825). The LTP data between 15 and

December 2013 • Volume 117 • Number 6

60 minutes after HFS were used for this analysis because LTP responses were unstable for 10 minutes after HFS. LTP induction in the 2% sevoflurane group was significantly suppressed compared with the control and CO2 groups at 5, 10, 15, 20, 25, 30, 35, and 40 minutes (Fig. 2B). There were no significant differences among the other groups. However, the statistical power of the analysis between the control and 1% sevoflurane groups might have been insuf- ficient from our sample size. As shown in Table 2, the width of Bonferroni adjusted 95% confidence interval for the mean difference between these groups was greater com- pared with the mean difference at each time point. Further research may be necessary to detect the difference caused by 1% sevoflurane exposure. These results indicated that neonatal 2% sevoflurane exposure caused significant long- lasting suppression of hippocampal LTP induction, but neo- natal 8% CO2 exposure with O2 did not cause any significant electrophysiological changes.

DISCUSSION In this study, we showed that neonatal exposure to 2% sevoflurane caused significant suppression of LTP induc- tion in an in vivo electrophysiological study of adult rats. Hypercapnia and acidosis, which mimicked the sevoflu- rane-induced respiratory disturbances, had no influence on LTP induction.

In the present study, we used 1% halothane to anesthe- tize rats during hippocampal LTP measurement. In vivo electrophysiological study under halothane anesthesia is a well-established procedure in adult rodents.24,25 Halothane anesthesia provides a more stabilized anesthetic depth during experiments compared with the intermittent IV or intraperitoneal injection of anesthetics. Although halothane has been shown to depress CA1 population spikes and LTP induction when applied to hippocampal slices,26 exposure to halothane in vivo does not block the induction of hip- pocampal LTP.27 The discrepancy between these findings in vitro and in vivo could reflect differences in the balance between excitatory and inhibitory circuitry. Thus, halothane has been used successfully for in vivo studies of hippocam- pal synaptic plasticity in rodents, including our previous studies.21,24,25,28,29 To avoid the possible modulation of hippo- campal synaptic activity by halothane-induced physiologi- cal changes (e.g., hypoxia and ischemia), we used intensive respiratory management in our in vivo electrophysiological studies.21,28,29 Respiratory management by tracheal intuba- tion, mechanical ventilation, and monitoring of end-tidal CO2 enabled us to control halothane-induced respiratory depression. Halothane also suppresses circulation; how- ever, our preliminary study showed that 1% halothane had relatively little influence on femoral arterial pressure in adult rats (data not shown). Thus, we assume that halo- thane anesthesia in this in vivo study had minimal electro- physiological and physiological influence on hippocampal synaptic activity.

learn- ing impairments after neonatal exposure to anesthetics remain poorly understood. Although acute widespread cell death has been observed,1,30,31 several studies have shown increased neurogenesis and apparent anatomical recovery

The critical mechanisms that contribute to

www.anesthesia-analgesia.org 1431

H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l

i

Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n

r

i

i

l

1 1 / 2 0 / 2 0 2 3

D o w n o a d e d

l

f r o m h t t p : / / j o u r n a s . l

l

w w . c o m / a n e s t h e s a - a n a g e s a

i

l

b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I

f

Neonatal Sevoflurane Exposure Impairs Long-Term Potentiation Induction

Table 1. Arterial Blood Gas Analysis Group Control

Time (min) 30 60 90 120 30 60 90 120 30 60 90 120 30 60 90 120

CO2

1% Sevoflurane

2% Sevoflurane

N 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5

pH 7.43 ± 0.04 7.36 ± 0.03 7.37 ± 0.02 7.38 ± 0.02 7.18 ± 0.03a 7.16 ± 0.03a 7.14 ± 0.02a 7.20 ± 0.02a 7.41 ± 0.02b 7.40 ± 0.04b 7.36 ± 0.01b 7.34 ± 0.05b 7.18 ± 0.03ac 7.23 ± 0.0ac 7.17 ± 0.03ac 7.18 ± 0.03ac

Paco2 (mm Hg) 37.9 ± 5.4 47.2 ± 7.3 44.0 ± 3.0 46.5 ± 2.6 76.3 ± 4.9a 88.7 ± 5.8a 81.5 ± 5.0a 79.7 ± 6.2a 39.1 ± 2.8b 38.7 ± 3.3b 41.6 ± 1.0b 53.9 ± 5.6b 77.5 ± 5.7ac 79.1 ± 3.8ac 81.7 ± 2.8ac 89.9 ± 5.1ac

Pao2 (mm Hg) 414.0 ± 51.6 414.2 ± 30.5 369.2 ± 53.7 350.6 ± 51.8 296.8 ± 70.9 200.1 ± 24.7 215.2 ± 45.8 237.5 ± 51.2 405.1 ± 77.0 358.4 ± 94.6 360.2 ± 64.4 366.9 ± 66.1 337.6 ± 28.4 226.9 ± 52.8 200.2 ± 28.9 235.4 ± 40.5

Data from arterial blood gas analysis. All groups were composed of 5 rats. Blood samples were collected from the left ventricle every 30 minutes during gas exposure. Statistical analysis revealed that neonatal 2% sevoflurane anesthesia and 8% CO2 exposure caused significant hypercapnia and acidosis in rats. There were no significant differences between the control group and 1% sevoflurane group or the CO2 group and 2% sevoflurane group for pH and Paco2. Hypoxia was not observed in any group. The acidosis and hypercapnia observed in the 2% sevoflurane group were well mimicked by the CO2 group. Data are presented as mean ± SEM. aVersus control group. bVersus CO2 group. cVersus 1% sevoflurane group. P < 0.05.

Figure 2. Neonatal 2% sevoflurane anesthesia suppressed long-term potentiation (LTP) induction in the postgrowth period. Specimen record- ings (A) and time course responses of population spike amplitude (PSA) to 60 minutes after high-frequency stimulation (HFS) (B). Two-way repeated-measures ANOVA revealed that there was no interaction between treatments and time course changes, whereas there were signifi- cant differences between treatments during the period from 15 to 60 minutes after HFS. Statistical analysis revealed that there were signifi- cant differences between the 2% sevoflurane group and the control and CO2 groups at 5, 10, 15, 20, 25, 30, 35, and 40 minutes after HFS. All groups were composed of 8 rats. Data are shown as mean ± SEM. *Versus control group, †Versus CO2 group.

after acute histopathological damage induced by neonatal anesthesia.5,20 In those studies, rat pups that were exposed to anesthesia showed minimal to no histopathological damage as adults. This suggests that the developing brain may have a high capacity for self-repair of morphological

changes. This evidence led us to hypothesize that there might be additional, or even alternative, mechanisms that could mediate anesthetic-induced long-term learning defi- cits and other cognitive dysfunctions besides the immediate morphological changes in the hippocampus.

1432

www.anesthesia-analgesia.org

aNesthesia & aNalgesia

H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l

i

Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n

r

i

i

l

1 1 / 2 0 / 2 0 2 3

D o w n o a d e d

l

f r o m h t t p : / / j o u r n a s . l

l

w w . c o m / a n e s t h e s a - a n a g e s a

i

l

b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I

f

Table 2. Bonferroni Adjusted 95% Confidence Intervals for the Mean Difference Between Treatments in Figure 2B

Control versus CO2

Time points (min)

15 20 25 30 35 40 45 50 55 60

Lower CL (%) −65.09 −61.71 −79.29 −72.26 −69.94 −70.10 −91.01 −86.49 −70.41 −83.53 Control versus 1% sevoflurane

Difference (%) 12.97 11.84 −2.97 9.2 3.92 8.29 −8.37 −2.1 13.92 2.72

Upper CL (%) 91.04 85.4 73.36 90.65 77.78 86.69 74.28 82.29 98.24 88.98

Time points (min)

15 20 25 30 35 40 45 50 55 60

Lower CL (%) −124.87 −122.88 −123.25 −125.13 −115.66 −119.30 −135.91 −131.13 −116.65 −130.26 Control versus 2% sevoflurane

Difference (%) −46.81 −49.33 −46.93 −43.67 −41.81 −40.9 −53.27 −46.74 −32.33 −44.01

Upper CL (%) 31.26 24.22 29.4 37.78 32.05 37.49 29.38 37.66 52 42.24

Time points (min)

15 20 25 30 35 40 45 50 55 60

Difference (%) −90.49 −89.36 −91.08 −90.68 −87.1 −83.76 −89.11 −84.98 −72.2 −88.02

Lower CL (%) −168.55 −162.91 −167.41 −172.13 −160.96 −162.16 −171.76 −169.37 −156.52 −174.27

Upper CL (%) −12.43 −15.80 −14.76 −9.23 −13.24 −5.37 −6.46 −0.59 12.13 −1.76

Bonferroni adjusted 95% confidence interval (95% CI) for the mean difference by the treatment combination between groups in Figure 2B. Mean difference (Difference), lower confidence limits (lower CL), and upper CL between groups at each time point was listed. 95% CI for the mean difference showed that there was a statistical difference in the control group versus 2% Sevoflurane group, and there was no statistical significance difference in the control group versus CO2 group and in the control group versus 1% Sevoflurane group at each time point. However, in the control group versus CO2 group and in the control group versus 1% Sevoflurane group, the statistical power might be insufficient from our sample size, because the width of 95% CI was greater compared with the mean difference between groups. Further, research is required to detect the difference.

One possible reason for anesthetic-induced neurotoxicity is the potential effect of anesthetics on synaptic morphology and function. Previously, we reported that the GABAergic anesthetic pentobarbital, administered at P7, induced long- lasting suppression of hippocampal synaptic plasticity as well as learning disturbances.21 Commonly used anesthet- ics with GABAergic or antiglutamatergic properties such as midazolam, isoflurane, and nitrous oxide, when combined, also suppress hippocampal LTP in adulthood after admin- istration at P7.1 Thus, hippocampal LTP might be associated with the pathophysiology of neonatal anesthetic-related learning deficits remaining in adulthood.

During synaptogenesis, signals via NMDA and/or GABA receptors mediate neuronal survival, migration, and synaptic formation.32,33 It has been reported that abnormal

December 2013 • Volume 117 • Number 6

or excess signals of those receptors may have a long- lasting negative influence over synaptic maturation.34,35 Because sevoflurane has GABAergic and/or antiglutama- tergic properties,8,9 neonatal sevoflurane anesthesia pas- sively modulates those synaptic developmental processes and induces abnormal synaptic formation or ectopic neu- ronal cell distribution that leads to impairment of syn- aptic plasticity such as induction of hippocampal LTP in the CA1 region. We have reported that administration of dexmedetomidine, an α2A adrenergic agonist, causes no impairment in hippocampal synaptic plasticity28 and has no adverse effect on cognitive functions in adulthood.36 The absence of neurotoxicity in rodents treated neonatally with dexmedetomidine, which has no GABAergic or glu- tamatergic properties, may suggest that pharmacological intervention via GABAergic and/or glutamatergic neuro- transmission might play a crucial role in the impairment of hippocampal neural functions, including hippocampal synaptic plasticity. However, further study is needed to clarify the mechanisms underlying sevoflurane-induced LTP impairment shown in this study.

Despite several conflicting reports,1,3,31 it has been well documented that neonatal rodents develop respiratory suppression and subsequent blood gas abnormalities dur- ing anesthetic exposure.21,37,38 Loepke et al.20 pointed out the possible involvement of such blood gas abnormalities in anesthetic-induced neurotoxicity observed in neonatal rodents. Yet the role of blood gas abnormalities in neurode- velopment has remained uncertain. Recently, we attempted to evaluate the influence of blood gas abnormalities in P7 rat pups on their later hippocampal function.29 In that study, P7 rat pups exposed to 13% CO2 under 21% O2 showed blood gas abnormalities along with hypoxia, hypercapnia, and metabolic acidosis, which inhibited hippocampal LTP induction in the postgrowth period.29 Importantly, com- pared with acidosis only, prolonged metabolic acidosis with hypoxia has been thought to carry a higher risk for neuro- nal deficits.39 Furthermore, studies performed in clinical set- tings have demonstrated a correlation between the degree of metabolic acidosis and neonatal neurological outcome.40 This evidence led us to suspect that neither hypercapnia nor low pH, but the coexistence of hypoxia and progres- sive metabolic acidosis during CO2 exposure, could have profound effects on hippocampal synaptic plasticity in later life. In light of this evidence, we used 100% O2 as a carrier gas in the current study. We needed 8% CO2 with 100% O2 to mimic respiratory disturbance and subsequent blood gas abnormalities caused by 2% sevoflurane exposure; this CO2 exposure protocol caused no impairment in later LTP induc- tion, suggesting that respiratory disturbances caused by neonatal exposure to 2% sevoflurane had negligible influ- ence on hippocampal synaptic plasticity in later life. This finding also led us to speculate that hypercapnia and low pH during development, without hypoxia and possible metabolic acidosis, had less effect on hippocampal synaptic plasticity in later life.

In conclusion, our present findings indicate that expo- sure to 2% sevoflurane causes persistent suppression of LTP induction. This persistent change in synaptic plastic- ity after neonatal sevoflurane exposure may be one of the

www.anesthesia-analgesia.org 1433

H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l

i

Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n

r

i

i

l

1 1 / 2 0 / 2 0 2 3

D o w n o a d e d

l

f r o m h t t p : / / j o u r n a s . l

l

w w . c o m / a n e s t h e s a - a n a g e s a

i

l

b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I

f

Neonatal Sevoflurane Exposure Impairs Long-Term Potentiation Induction

mechanisms underlying sevoflurane-induced neurocogni- tive dysfunction. E

DISCLOSURES Name: Rui Kato, MD, PhD. Contribution: This author designed the study, conducted almost all experiments with some other authors, and wrote the manuscript. Attestation: Rui Kato attests to the integrity of the original data and the analysis reported in this manuscript. Yuji Morimoto is the archival author. Name: Kaori Tachibana, MD, PhD. Contribution: This author helped in designing the study and conducting experiments. Name: Naoki Nishimoto, PhD. Contribution: This author helped in statistical analysis. Name: Toshikazu Hashimoto, MD, PhD. Contribution: This author helped in designing the study and preparing the manuscript. Name: Yosuke Uchida, MD. Contribution: This author helped in preparing the manuscript. Name: Ryoko Ito, MD. Contribution: This author helped in electrophysiological tests. Name: Kenkichi Tsuruga, MD Contribution: This author helped in preparing the manuscript. Name: Koichi Takita, MD, PhD. Contribution: This author helped in electrophysiological tests and preparing the manuscript. Name: Yuji Morimoto, MD, PhD. Contribution: This author helped in designing the study and preparing the manuscript. Attestation: Yuji Morimoto is the archival author of this manuscript. This manuscript was handled by: Gregory J. Crosby, MD.

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

2. Fredriksson A, Pontén E, Gordh T, Eriksson P. Neonatal expo- sure to a combination of N-methyl-D-aspartate and gamma- aminobutyric acid type A receptor anesthetic agents potentiates apoptotic neurodegeneration and persistent behavioral deficits. Anesthesiology 2007;107:427–36

3. Satomoto M, Satoh Y, Terui K, Miyao H, Takishima K, Ito M, Imaki J. Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. Anesthesiology 2009;110:628–37

4. 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 neuro- nal cell death in the perinatal rhesus monkey. Toxicol Sci 2007;98:145–58

5. Stratmann G, May LD, Sall JW, Alvi RS, Bell JS, Ormerod BK, Rau V, Hilton JF, Dai R, Lee MT, Visrodia KH, Ku B, Zusmer EJ, Guggenheim J, Firouzian A. Effect of hyper- carbia and isoflurane on brain cell death and neurocogni- tive dysfunction in 7-day-old rats. Anesthesiology 2009;110: 849–61

6. Ikonomidou C, Bosch F, Miksa M, Bittigau P, Vöckler J, Dikranian K, Tenkova TI, Stefovska V, Turski L, Olney JW. Blockade of NMDA receptors and apoptotic neurodegeneration in the developing brain. Science 1999;283:70–4

7. Olney JW, Young C, Wozniak DF, Jevtovic-Todorovic V, Ikonomidou C. Do pediatric drugs cause developing neurons to commit suicide? Trends Pharmacol Sci 2004;25:135–9

1434

www.anesthesia-analgesia.org

8. Hollmann MW, Liu HT, Hoenemann CW, Liu WH, Durieux ME. Modulation of NMDA receptor function by ketamine and magnesium. Part II: interactions with volatile anesthetics. Anesth Analg 2001;92:1182–91

9. Nishikawa K, Harrison NL. The actions of sevoflurane and desflurane on the gamma-aminobutyric acid receptor type A: effects of TM2 mutations in the alpha and beta subunits. Anesthesiology 2003;99:678–84

10. Bercker S, Bert B, Bittigau P, Felderhoff-Müser U, Bührer C, Ikonomidou C, Weise M, Kaisers UX, Kerner T. Neurodegeneration in newborn rats following propofol and sevoflurane anesthesia. Neurotox Res 2009;16:140–7

11. Edwards DA, Shah HP, Cao W, Gravenstein N, Seubert CN, Martynyuk AE. Bumetanide alleviates epileptogenic and neurotoxic effects of sevoflurane in neonatal rat brain. Anesthesiology 2010;112:567–75

12. Kodama M, Satoh Y, Otsubo Y, Araki Y, Yonamine R, Masui K, Kazama T. Neonatal desflurane exposure induces more robust neuroapoptosis than do isoflurane and sevoflurane and impairs working memory. Anesthesiology 2011;115:979–91

13. Shih J, May LD, Gonzalez HE, Lee EW, Alvi RS, Sall JW, Rau V, Bickler PE, Lalchandani GR, Yusupova M, Woodward E, Kang H, Wilk AJ, Carlston CM, Mendoza MV, Guggenheim JN, Schaefer M, Rowe AM, Stratmann G. Delayed environmental enrichment reverses sevoflurane-induced memory impairment in rats. Anesthesiology 2012;116:586–602

14. Morris RG, Garrud P, Rawlins JN, O’Keefe J. Place naviga- tion impaired in rats with hippocampal lesions. Nature 1982;297:681–3

15. Burgess N, Maguire EA, O’Keefe J. The human hippocampus and spatial and episodic memory. Neuron 2002;35:625–41 16. Bliss TV, Collingridge GL. A synaptic model of memory: long- term potentiation in the hippocampus. Nature 1993;361:31–9 17. Gruart A, Muñoz MD, Delgado-García JM. Involvement of the CA3-CA1 synapse in the acquisition of associative learning in behaving mice. J Neurosci 2006;26:1077–87

18. Whitlock JR, Heynen AJ, Shuler MG, Bear MF. Learning induces long-term potentiation in the hippocampus. Science 2006;313:1093–7

19. Lu LX, Yon JH, Carter LB, Jevtovic-Todorovic V. General anes- thesia activates BDNF-dependent neuroapoptosis in the devel- oping rat brain. Apoptosis 2006;11:1603–15

20. Loepke AW, Istaphanous GK, McAuliffe JJ 3rd, Miles L, Hughes EA, McCann JC, Harlow KE, Kurth CD, Williams MT, Vorhees CV, Danzer SC. The effects of neonatal isoflurane exposure in mice on brain cell viability, adult behavior, learning, and mem- ory. Anesth Analg 2009;108:90–104

21. Tachibana K, Hashimoto T, Kato R, Tsuruga K, Ito R, Morimoto Y. Long-lasting effects of neonatal pentobarbital administration on spatial learning and hippocampal synaptic plasticity. Brain Res 2011;1388:69–76

22. Ohashi S, Matsumoto M, Otani H, Mori K, Togashi H, Ueno K, Kaku A, Yoshioka M. Changes in synaptic plastic- ity in the rat hippocampo-medial prefrontal cortex pathway induced by repeated treatments with fluvoxamine. Brain Res 2002;949:131–8

23. Tachibana K, Kato R, Tsuruga K, Takita K, Hashimoto T, Morimoto Y. Altered synaptic transmission in rat anterior cingulate cortex following peripheral nerve injury. Brain Res 2008;1238:53–8

24. Thiels E, Xie X, Yeckel MF, Barrionuevo G, Berger TW. NMDA receptor-dependent LTD in different subfields of hippocampus in vivo and in vitro. Hippocampus 1996;6:43–51

25. Yeckel MF, Berger TW. Spatial distribution of potentiated syn- apses in hippocampus: dependence on cellular mechanisms and network properties. J Neurosci 1998;18:438–50

26. MacIver MB, Tauck DL, Kendig JJ. General anaesthetic modi- fication of synaptic facilitation and long-term potentiation in hippocampus. Br J Anaesth 1989;62:301–10

27. Pearce RA, Stringer JL, Lothman EW. Effect of volatile anes- thetics on synaptic transmission in the rat hippocampus. Anesthesiology 1989;71:591–8

28. Tachibana K, Hashimoto T, Kato R, Uchida Y, Ito R, Takita K, Morimoto Y. Neonatal administration with dexmedetomidine

aNesthesia & aNalgesia

H o 4 X M 0 h C y w C X 1 A W n Y Q p / I l

i

Q H D 3 3 D 0 O d R y 7 T v S F 4 C f 3 V C 4 / O A V p D D a 8 K K G K V 0 Y m y + 7 8 = o n

r

i

i

l

1 1 / 2 0 / 2 0 2 3

D o w n o a d e d

l

f r o m h t t p : / / j o u r n a s . l

l

w w . c o m / a n e s t h e s a - a n a g e s a

i

l

b y B h D M f 5 e P H K a v 1 z E o u m 1 t Q N 4 a + k J L h E Z g b s I

f

does not impair the rat hippocampal synaptic plasticity later in adulthood. Paediatr Anaesth 2012;22:713–9

29. Tachibana K, Hashimoto T, Takita K, Ito R, Kato R, Morimoto Y. Neonatal exposure to high concentration of carbon dioxide produces persistent learning deficits with impaired hippocampal synaptic plasticity. Brain Res 2013; 1507:83–90

30. Istaphanous GK, Howard J, Nan X, Hughes EA, McCann JC, McAuliffe JJ, Danzer SC, Loepke AW. Comparison of the neu- roapoptotic properties of equipotent anesthetic concentrations of desflurane, isoflurane, or sevoflurane in neonatal mice. Anesthesiology 2011;114:578–87

31. Liang G, Ward C, Peng J, Zhao Y, Huang B, Wei H. Isoflurane causes greater neurodegeneration than an equivalent expo- sure of sevoflurane in the developing brain of neonatal mice. Anesthesiology 2010;112:1325–34

32. Komuro H, Rakic P. Modulation of neuronal migration by NMDA receptors. Science 1993;260:95–7

33. Behar TN, Smith SV, Kennedy RT, McKenzie JM, Maric I, Barker JL. GABA(B) receptors mediate motility signals for migrating embryonic cortical cells. Cereb Cortex 2001;11:744–53

34. De Roo M, Klauser P, Briner A, Nikonenko I, Mendez P, Dayer A, Kiss JZ, Muller D, Vutskits L. Anesthetics rapidly promote

December 2013 • Volume 117 • Number 6

synaptogenesis during a critical period of brain development. PLoS One 2009;4:e7043

35. Koyama R, Tao K, Sasaki T, Ichikawa J, Miyamoto D, Muramatsu R, Matsuki N, Ikegaya Y. GABAergic excitation after febrile sei- zures induces ectopic granule cells and adult epilepsy. Nat Med 2012;18:1271–8

36. Sanders RD, Sun P, Patel S, Li M, Maze M, Ma D. Dexmedetomidine provides cortical neuroprotection: impact on anaesthetic-induced neuroapoptosis in the rat developing brain. Acta Anaesthesiol Scand 2010;54:710–6

37. Danneman PJ, Mandrell TD. Evaluation of five agents/meth- ods for anesthesia of neonatal rats. Lab Anim Sci 1997;47:386–95 38. Loepke AW, McCann JC, Kurth CD, McAuliffe JJ. The physi- ologic effects of isoflurane anesthesia in neonatal mice. Anesth Analg 2006;102:75–80

39. Low JA, Galbraith RS, Muir DW, Killen HL, Pater EA, Karchmar EJ. Factors associated with motor and cognitive deficits in chil- dren after intrapartum fetal hypoxia. Am J Obstet Gynecol 1984;148:533–9

40. Hanrahan JD, Cox IJ, Edwards AD, Cowan FM, Sargentoni J, Bell JD, Bryant DJ, Rutherford MA, Azzopardi D. Persistent increases in cerebral lactate concentration after birth asphyxia. Pediatr Res 1998;44:304–11

www.anesthesia-analgesia.org 1435