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Background: Our previous research showed that 4 h of maternal anesthesia with isoflurane during early gestation in pregnant rats leads to a deficit in spatial memory of adult male offspring. Because spatial memory is predominantly a hippocampally-mediated task, we asked the question if early gestational exposure to isoflurane affects development of the hippocampus in the offspring. Findings: Previously behaviorally characterized adult male rats that were exposed to isoflurane during second trimester were sacrificed at 4 months of age (N = 10 and 13, control and isoflurane groups, respectively) for quantita- tive histology of hippocampal subregions. Sections were stained with cresyl violet and the total number of cells in the granular layer of the dentate gyrus and the pyramidal cell layer in the CA1 region were determined by a blinded observer using unbiased stereological principles and the optical fractionator method. Data were analyzed using Student’s t test; P < 0.05 was accorded statistical significance. Stereological examination revealed 9% fewer cells in the granular layer of the dentate gyrus of isoflurane-exposed adult rats compared to controls (1,002,122 ± 84,870 vs. 1,091,829 ± 65,791, respectively; Mean ± S.D, *P = 0.01). In contrast, there were no changes in the cell number in the CA1 region, nor were there changes in the volumes of both regions. Conclusions: Our results show that maternal isoflurane anesthesia in rodents causes region-specific cell loss in the hippocampus of adult male offspring. These changes may, in part, account for the behavioral deficits reported in adult rats exposed to isoflurane in utero. Keywords: Anesthesia during pregnancy, Anesthetic neurotoxicity, Maternal anesthesia, Spatial working memory, Stereology, Hippocampus, Dentate gyrus, CA1 region  is propelled by gamma-amino butyric acid (GABA) Main text and glutamate [7–9] precisely the same neurotransmit- Introduction ter mechanisms by which anesthetic agents exert their Despite robust evidence for anesthesia-induced neuro- effects [ 10]. Thus, clinical necessity and practice may toxicity in postnatal rodent models [1–4] not much is inadvertently place the fetal brain at risk by disrupting known about the effects of maternal anesthesia on the the trophic milieu that propels early neurodevelopment fetal brain. This is significant because most non-obstet - . We previously showed that a single 4 h exposure to ric surgeries and fetal intervention procedures that are isoflurane during mid-gestation impairs acquisition of performed during the second trimester require mater- spatial memory in the adult male offspring [ 12]. This sug - nal anesthesia . Furthermore, the robust neuronal gested that prolonged and unphysiological stimulation of proliferation and migration observed during this period these neurotransmitter mechanisms can be detrimental to fetal brain development. Because acquisition of spatial *Correspondence: email@example.com memory is reliant on the structural integrity of the hip- Present Address: Department of Anesthesiology, Washington University pocampus , we hypothesized that early gestational School of Medicine, St. Louis, MO, USA Full list of author information is available at the end of the article © The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Palanisamy et al. Behav Brain Funct (2017) 13:14 Page 2 of 5 exposure to isoflurane during maternal anesthesia will (Fig. 1A–C), were delineated using a stereotaxic rat brain derail normal development of the hippocampal forma- atlas. Approximately 72 sections/brain that included tion. Therefore, in the present study, we assessed the the regions of interest were collected, stained with 0.1% changes in neuronal number in the pyramidal cell layer aqueous cresyl violet (Sigma, St. Louis, MO), dehydrated of CA1 region and the granule cell layer of the dentate in ethanol, cleared in xylene and cover-slipped with Per- gyrus of the behaviorally dysfunctional adult male rats mount mounting medium (Fisher Scientific, PA, USA). that were exposed to isoflurane in utero. Stereological quantitation Materials and methods Every 12th section of the series was chosen for ste- Experiments were conducted on timed-pregnant reological analysis because we established that every Sprague–Dawley rats (Charles River Laboratories, Inc, 12th section, when compared to every 8th or 10th sec- Wilmington, MA) on embryonic day 14 (E 14), a period tion, produced reliable estimates of cell numbers in the that corresponds to the second trimester of human preg- dentate gyrus as well as the CA1 area. Moreover, this nancy , and their male offspring as described previ - approach has been validated by an elegant study done ously . We chose 2–3 dams/treatment condition previously to study anesthetic neurotoxicity in post- based on results from our previous study that indicated natal rodents . The first section of the series was absence of maternal–fetal interaction during statisti- chosen at random, based on the roll of a die. The stere - cal analyses. Briefly, animals randomized to anesthesia ology equipment consisted of a light microscope (Nikon received 1.4% isoflurane in 100% oxygen for 4 h whereas E2000) connected to a CCD camera (Microfire; Optron - control animals received 100% oxygen for 4 h in identical ics, CA, USA), motorized X–Y stage (Ludl Electronics anesthetizing chambers. The animals breathed sponta - Products, NY, USA), z-axis indicator (MT12 microca- neously and the isoflurane concentration was measured tor; Heidenhain, Traunreut, Germany) and a computer continuously with an agent analyzer (Datex, Tewksbury, running Stereo Investigator software (Micro- Bright- MA; Ohmeda, Madison, WI). During isoflurane anes - field, Inc., VT, USA). Briefly, outlines of the dentate thesia, maternal blood pressure was measured non-inva- gyrus and the CA1 were drawn at 2× magnification in sively every hour, rectal temperature was maintained at 37 ° ± 0.5 °C with heating pads, and venous blood gas and plasma glucose were measured at the end of the anesthetic. Following anesthesia, the anesthetized and control dams recovered in 100% oxygen for 20 min fol- lowing return of righting reflex. Maternal weight was monitored daily until delivery of the pups on gestational day 22 . All experiments had the approval of the Insti- tutional Animal Care and Use Committee of Longwood Medical Area (Boston, MA, USA), and appropriate care was taken to minimize the number of animals used and their suffering. Preparation for stereology Following behavioral characterization, male rat offspring (N = 10 and 13 in control and anesthesia groups, respec- tively) were anesthetized and perfused intracardially using 0.9% saline followed by a fixative containing 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) on postnatal day 120. The brains were removed and post- fixed in the same fixative at 4 °C overnight and cryopro - Fig. 1 Sample microphotographs to illustrate the regions investi- tected in 30% sucrose at 4 °C overnight until sectioning. gated. A A 2× image of a Nissl-stained section from a control rat Coronal sections were cut at 50-μm thicknesses using demonstrating the CA1 area and the dentate gyrus in the right hip- pocampus. The arrow indicates the point of intersection to determine a Leica 1800 CM cryostat at – 20 °C (Leica Microsys- the CA1 area. B A 63x image of a Nissl-stained section from a control tems Inc., IL, USA). Every section that contained the rat showing pyramidal cell bodies in the stratum pyramidale of the hippocampus was collected in a rostral-to-caudal CA1 area, and C A 63x image of a Nissl-stained section from a control serial order in phosphate buffer (0.1 M, pH 7.2) and rat showing a densely packed granule cell layer of the dentate gyrus. the regions of interest, i.e., CA1 and the dentate gyrus Scale bars as indicated Palanisamy et al. Behav Brain Funct (2017) 13:14 Page 3 of 5 the right hippocampus from each section. Systematic Results random sampling was performed by randomly translat- Isoflurane anesthesia was well tolerated by the dams with ing a 110 × 250 μm grid onto the section of interest. A no evidence for physiologic perturbance. There were no 35 × 35 × 10 μm optical dissector was created at each differences in the male litter size (6 ± 2.8 vs. 5.3 ± 1.2; sampling site using a 63× objective (oil; numerical aper- P = 0.72) nor were there any differences in the weights ture, 1.3). The average mounted thickness was 15-μm of the adult male offspring (370 ± 29 vs. 370 ± 19 g; with a guard zone of 2-μm. Neurons were identified by P = 0.92) between control and isoflurane groups, respec - a clear nuclear profile containing a nucleolus and Nissl tively. Stereological quantification revealed no dif - substance in the cell body, as the focus went through the ferences in pyramidal cell number of the CA1 region section from one grid to the other. Cell counts were lim- between control and gestationally isoflurane-exposed ited to the pyramidal cell layer in the CA1 region and the groups (545,375 ± 51,272 vs. 556,493 ± 47,726, respec- granule cell layer (GCL) of the dentate gyrus. The soft - tively; P = 0.60) (Fig. 2a). However, adult male rats ware package converted the counts into total numbers of exposed to isoflurane during early gestation showed a cells, which were subsequently multiplied by 2 to obtain significant decrease in the number of cells in the granule estimates of cell numbers for both hemispheres. The total cell layer of the dentate gyrus compared to control rats volumes of the GCL of the dentate gyrus and the CA1 (1,002,122 ± 84,870 vs. 1,091,829 ± 65,791, *P = 0.01) pyramidal cell layer in the right hippocampus were esti- (Fig. 2b). mated in the same sections used to determine the total There were no differences in the total volumes of the neuronal count using the Cavalieri principle and the Ste- CA1 pyramidal layer and the dentate gyrus granule cell reo Investigator software. Briefly, the layers of interest layer (in mm ) between the control and the isoflurane- were outlined in each section at 4× magnification and an exposed groups (1.275 ± 0.10 vs. 1.316 ± 0.05 and unbiased point counting method was employed. The area 2.016 ± 0.20 vs. 1.958 ± 0.12; P = 0.20 and 0.40, respec- of the grid associated with each point on the section, A/P, tively) (Fig. 3a, b). was 50 µm , and a value of 7 was assigned as the shape factor. The volume was calculated using the formula Discussion V = ∑P X A/P X T, where ∑P is the sum of the cross- Our results indicate that even a single exposure to a clini- sectional area of the points inside the outlined borders, T cally relevant concentration of isoflurane during early is the cut thickness of the Section (50-μm), and A/P the gestation causes region-specific hippocampal cell loss area associated with each point (50 µm ). The Gunder - that lasts into adulthood. More specifically, in utero iso - son coefficient of error (CE), a measure of the quality of flurane exposure, at a period equivalent to the human the estimates, was < 10% in the stereology runs for both second trimester, causes a persistent decrease in the cell numbers and volumes. Data were analyzed using Stu- granule cell number of the dentate gyri of adult male rats. dent’s t-test and expressed as mean ± S.D; P < 0.05 was This may, in part, explain the impairment of spatial mem - accorded statistical significance. ory acquisition seen in these rats . Fig. 2 Gestational exposure to isoflurane resulted in region-specific cell loss in the adult hippocampus. No changes were detected in the pyramidal layer of the CA1 area of adult male rats following isoflurane exposure in utero (N = 13) compared to control rats (N = 10) (a). However, isoflurane exposure at E14 (N = 13) was associated with a significant decrease in the number of cells in the granule cell layer of the dentate gyrus (*P = 0.01) compared to control rats (N = 10) (b). Data were analyzed using Student’s t-test and expressed as mean ± S.D Palanisamy et al. Behav Brain Funct (2017) 13:14 Page 4 of 5 Fig. 3 Comparison of the total volumes of the CA1 pyramidal cell layer and the granule cell layer of the dentate gyrus between control and gesta- tionally isoflurane-exposed groups. No significant differences were detected in either of the volumes between the two groups (P = 0.20 and 0.40, a, b, respectively). Data were analyzed using Student’s t-test and expressed as mean ± S.D The results are, perhaps, not surprising considering is that we did not analyze the brains of female offspring. that both GABA and glutamatergic transmission, the Though ideal, we limited our analysis to the hippocampi principal mechanisms of anesthetic action, is primarily from behaviorally characterized male offspring to corre - involved in early neurodevelopment. It is, therefore, con- late morphological changes with behavior. The timeline ceivable that an acute modulation of such transmission of the previous study predates the NIH policy on sex as a as would occur with in utero anesthetic exposure could biological variable, and unfortunately, we did not process leave a lasting neurodevelopmental imprint. Though our the brains of the female offspring at that time. study did not investigate the mechanisms responsible for In summary, our results indicate that gestational the reduction in the granule cell number of the dentate exposure to isoflurane causes granule cell loss in the gyrus, evidence from other studies suggest a myriad of dentate gyrus of adult male rats suggesting that this possibilities. For example, isoflurane is known to cause may, in part, be responsible for the deficits in spatial a dose-dependent decrease in the renewal potential of working memory seen in the male offspring of exposed neural stem/progenitor cells (NPCs) in vitro , as well dams. In a broader sense, our study suggests that anes- as decreased progenitor proliferation in vivo [15, 17]. thetic agents have the potential to disrupt orderly devel- Furthermore, isoflurane, when administered alone or in opment of the brain and warrants inclusion in the list combination during the critical brain growth spurt, is of various environmental and pharmacological agents known to cause extensive apoptotic neurodegeneration that are known to affect normal brain development. . At present, the literature on anesthetic-mediated cell This is reinforced by the latest Drug Safety Communi - death during in utero brain development is conflicting. A cation from the Food and Drug Administration (FDA) study in fetal guinea pigs clearly demonstrated caspase-3 warning that repeated or lengthy use of general anes- activation and neurodegeneration after 4 h of 0.55% iso- thetic or sedative drugs during the third trimester or flurane anesthesia , whereas a similar study done in the first 3 years of life may have consequences for early fetal rats on embryonic day 21 (E 21) with 6 h of 1.3% iso- brain development (https://www.fda.gov/Drugs/Drug- flurane showed a decrease in apoptosis in the hippocam - Safety/ucm532356.htm). Further studies, therefore, are pal CA1 region at 2 h following anesthesia . These required to determine the causative mechanisms and to differences can be partly reconciled by species differences identify anesthetic agents that are safer for obstetric and in mammalian neural development. For example, the fetal perinatal use. guinea pig brain is almost fully developed before birth, unlike rats, and hence, anesthetic exposure occurred at Abbreviations the time of synaptogenesis when the developing brain is GABA: gamma amino-butyric acid; CA1: cornu ammonis area 1; E 14: most vulnerable. This incongruence, nevertheless, intro - embryonic day 14; NPC: neural stem/progenitor cell; FDA: Food and Drug Administration. duces an element of caution in interpreting anesthetic neurotoxicity studies from disparate animal models. It is Authors’ contributions also unclear why isoflurane exposure during 2nd trimes - AP conceived of the study, performed rat anesthesia, stereological quantifica- tion, and collection and analysis of data. GC participated in perfusion, harvest- ter equivalent (E 14) causes a differential, region-specific ing of the adult rat brains, and interpretation of data. 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Neonatal exposure to sevoflurane induces abnormal social behaviors and deficits in fear conditioning in mice. Anesthesiology. 2009;110(3):628–37. Submit your next manuscript to BioMed Central and we will help you at every step: • We accept pre-submission inquiries • Our selector tool helps you to ﬁnd the most relevant journal • We provide round the clock customer support • Convenient online submission • Thorough peer review • Inclusion in PubMed and all major indexing services • Maximum visibility for your research Submit your manuscript at www.biomedcentral.com/submit
Behavioral and Brain Functions – Springer Journals
Published: Dec 26, 2017
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