Edited by: Yun-Qing Li, The Fourth Military Medical University, China
Reviewed by: Bing Lang, University of Aberdeen, UK; Riyi Shi, Purdue University, USA
*Correspondence: Shengxi Wu
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Sevoflurane is widely used in adult and pediatric patients during clinical surgeries. Although studies have shown that exposure to sevoflurane impairs solfactory memory after an operation, the neuropathological changes underlying this effect are not clear. This study detected the effect of sevoflurane exposure on the development of calcium-binding proteins-expressing interneurons in the main olfactory bulb (MOB). We exposed neonatal mice to 2% sevoflurane at two different developmental time points and found that exposing mice to sevoflurane at postnatal day (PD) 7 significantly decreased the expression of GAD67 and parvalbumin (PV) in the olfactory bulb (OB) but did not alter the expression of calretinin (CR) or calbindin D28k (CB). The number and dendritic morphology of PV-expressing interneurons in the MOB were impaired by exposure to sevoflurane at PD7. However, exposure to sevoflurane at PD10 had no effect on calcium-binding protein expression or the number and dendritic morphology of PV-expressing interneurons in the MOB. These results suggest that exposing neonatal mice to sevoflurane during a critical period of olfactory development affects the development of PV-expressing interneurons in the MOB.
Inhalation anesthetics are widely used in adult and pediatric patients during surgeries, and sevoflurane is one of the most frequently used inhalation anesthetics in infants and children because it has a low blood/gas ratio, low pungency and a rapid onset and recovery (Sakai et al.,
The olfactory system consists of the olfactory epithelium, the main olfactory bulb (MOB) and the olfactory cortex. The MOB is an important part of the olfactory system for olfactory functions. It has high plasticity because its neural network can be modified even by simple stimulation during an olfactory experience, such as exposure to an odorant (Buonviso et al.,
Interneurons in the MOB use γ-aminobutyric acid (GABA) as their main neurotransmitter (Groh et al.,
C57BL/6 mice were purchased from the Experimental Animal Center of the Fourth Military Medical University and individually housed with free access to food and water in a temperature- and humidity-controlled environment with a 12:12 h light/dark cycle. When the neonatal mice reached PD7 or PD10, the treatments were performed. All experimental procedures received prior approval from the Animal Use and Care Committee for Research and Education of the Fourth Military Medical University (Xi’an, China). Every effort was taken to minimize animal suffering and to reduce the number of animals used. A total of 12 liters containing 102 male offspring were used in this study.
When the pups reached PD7 or PD10, they were randomly divided into a sevoflurane-treated group and an air-treated control group. Mice in the sevoflurane-treated group were placed in a plastic container and continuously exposed to 2% sevoflurane for 6 h using air as a carrier with a gas flow of 2 liters/min. During sevoflurane exposure, the container was heated to 38°C. The control animals were exposed to air without sevoflurane. After 6 h of treatment, the pups were placed back into their maternal cages.
For nissl staining, mice at PD14, PD28 and PD42 in each group were deeply anesthetized with sodium pentobarbital (50 mg/kg) and then perfused with 20 mL 0.01 M phosphate-buffered saline (PBS, pH = 7.4), followed by 100 mL 4% paraformaldehyde in 0.1 M phosphate buffer solution (PB, pH = 7.4). Then the Olfactory Bulbs (OB) were removed and post-fixed in the same fixative for 3 h and then cryoprotected for 24 h at 4°C in 0.1 M PB containing 30% sucrose. Coronal sections (30 μm) were cut in a freezing microtome (Leica CM1800, Heidelberg, Germany) at −20°C and collected in 0.01 M PBS. For staining, the sections were mounted on gelatin coated glass slides. When dried, the sections were defatted in 75% ethanol at 37°C overnight. Then the sections were stained for 10 min in 0.1% cresyl violet solution at RT, after rinsing with water, sections were incubated with 70% ethanol (3 s), 80% ethanol (3 s), 90% ethanol (3 s), 95% ethanol (3 s), absolute ethanol I (3 s) and absolute ethanol II (5 min) and then with xylene I (10 min) and xylene II (30 min). Sections were observed under an optical microscope after mounting with permount.
When pups reached their ages, they were sacrificed, and the olfactory were rapidly removed. The tissue samples were homogenized using an ultrasonic wave (10 s, 3 times) in RIPA lysis buffer, which contained a cocktail of proteinase and phosphatase inhibitors (Roche). After centrifugation at 12,000 rpm for 15 min at 4°C, the protein-containing supernatants were collected. The protein concentrations were determined with a BCA-based kit (Pierce). Lysate samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis; then, the proteins were transferred onto polyvinylidenedifluoride (PVDF) membranes (Bio-Rad). After blocked with 5% defatted milk in Tween/Tris-buffered saline (TBST) for 1 h at room temperature, the membranes were incubated with the primary antibody at 4°C overnight. The following primary antibodies were used: mouse monoclonal GAD67 (Chemicon® 1:5000), mouse monoclonal CB (Sigma-Aldrich 1:1000), goat polyclonal CR (Abcam 1:2000), rabbit polyclonal PV (Abcam 1:1000) and mouse polyclonal β-actin (Sigma-Aldrich 1:5000). After washing in TBST, the membranes were incubated in a secondary antibody for 2 h at room temperature. All of the blots were detected by an enhanced chemiluminescence (ECL) detection system (Advansta). The scanned images were quantified with ImageJ (version 1.47) Software.
The mice atPD42 in each group were deeply anesthetized with a lethal dose of sodium pentobarbital (50 mg/kg of body weight) and then perfused with 0.01 M PBS (pH 7.4), followed by a 4% phosphate-buffered formalin in a 0.1 M phosphate buffer solution (pH 7.4). Coronal olfactory sections (30 μm) were cut in a freezing microtome (Leica CM1800, Heidelberg, Germany) at −20°C and collected in 0.01 M PBS. During the staining, the cryostated sections were washed in PBS three times, and following blocking in 10% normal donkey serum in PBS, the sections were incubated with primary antibodies overnight at 4°C. The following antibodies were used: mouse monoclonal CB (Sigma-Aldrich 1:500), goat polyclonal CR (Abcam 1:1000), and rabbit polyclonal PV (Abcam 1:500). After rinsing in PBS, the slides were incubated with secondary antibodies conjugated with Alexa Fluor 488 (Invitrogen/Life Technologies 1:500) for 2 h at room temperature and counterstained with 100 ng/ml DAPI. After rinsing in PBS, the sections were mounted on gelatin-coated glass slides and cover slipped in Flouromount G. The sections were observed and captured with a confocal laser scanning microscope (Olympus FV1000, Japan). Imaging-Pro-Plus (LEIKA DMLB) was used to perform quantitative analysis of the positive immunostained cells numbers. Every fourth coronal section through the OB was collected, and a total of five sections from each mouse were used for quantification. The number of immunostained cells in each field was counted at a higher magnification (200×). Three random fields were captured in each section, and the mean number of immunostained neurons per view in the three views was included as the data for each section. The final average number of immunostained neurons per vision in all sections was included as the data for each sample.
All data were presented as Mean ± SD. The statistical analysis was performed with GraphPad Prism 5.0 Software (GraphPad Software) and comparisons of the means of two groups were performed using the Student
To explore the effect of sevoflurane exposure on the overall morphology of the MOB, Nissl staining was used to show the structure of the MOB. A distinct laminar organization can be seen in the MOB, with intact glomeruli and clear layers. Following sevoflurane exposure at PD7 and PD10, the organization of the MOB was the same as that in control mice (Figure
To assess the effect of sevoflurane exposure at PD7 on interneurons in the OB at different developmental stages, we first analyzed GAD67 expression in the OB in the sevoflurane-treated mice and control mice at PD14, 28 and 42 using western blot analysis. Compared to the control group, the expression of GAD67 was significantly decreased at PD14, 28 and 42 in the OB in the sevoflurane-treated mice. The significance of this difference was decreased as the age of the mice increased (Figures
To determine whether the observed molecular changes represented alterations at the cellular level, immunofluorescence staining was performed to display calcium-binding proteins in immunoreactive (ir) interneurons. The pattern of distribution for PV-ir, CR-ir and CB-ir interneurons were not qualitatively different between sevoflurane-treated mice and control mice. The soma of the PV-ir interneurons were mainly located in the EPL and few PV-ir cells located in ML and IPL. The CB-ir interneurons were periglomerular interneurons, and the CR-ir interneurons were observed in all layers of the MOB, with the highest packing density in the GL (Figures
To further evaluate the effect of sevoflurane on neuronal morphologies, immunofluorescence staining was used to display the dendritic arbor architecture of these interneurons in control and sevoflurane-exposed mice at PD42. The results showed that the PV-ir interneurons in the MOB in control mice were multipolar neurons with extensive and clear dendrite complexities with visible secondary and tertiary dendrites (Figures
To further explore the effect of sevoflurane exposure on phases of OB interneuron development, mice were subjected to sevoflurane exposure at PD10, and the expression of GAD67 and calcium-binding proteinswas detected using western blot analysis. At PD14, 28 and 42, there were no significant differences in the expression of GAD67, PV, CB, and CR in the OB between control mice and sevoflurane-exposed mice (Figure
To further confirm the effects of sevoflurane exposure at PD10 on PV-expressing interneurons at the cellular level, immunofluorescence staining was used to detect the numbers and morphologies of PV-ir interneurons in the MOB of control and sevoflurane-treated mice at PD42. No difference was observed in the number of PV-ir interneurons not only in EPL but also in the ML and IPL of the MOB between control mice and sevoflurane-treated mice (Figures
In the present study, by exposing neonatal mice to sevoflurane, we explored the effects of sevoflurane on interneurons in the OB of developing mice. First, we found that the laminar organization of the MOB was not adversely affected by sevoflurane exposure at either PD7 or PD10. Second, sevoflurane exposure at PD7 affected the survival and dendritic development of PV-expressing interneurons in the MOB. Finally, sevoflurane exposure at PD10 had no effect on calcium-binding protein-expressing interneurons in the MOB.
Sevoflurane is one of the most commonly used anesthetics in neonatal and pediatric patients (Lerman et al.,
A previous report demonstrated that olfactory acuity was intact in patients after anesthesia with sevoflurane, whereas olfactory memory was impaired (Kostopanagiotou et al.,
In humans and rodents, postnatal development is also an important period for the precise formation of neuronal circuits in the central nervous system (Petit et al.,
Interneurons in the OB are a heterogeneous population that is produced beginning in embryogenesis and continuing through adulthood. The development of different calcium-binding protein-expressing neurons occurs in a spatially and temporally regulated manner (Stenman et al.,
In conclusion, this study is the first to describe the effect of sevoflurane exposure at two different developmental time points on the development of interneurons in the MOB. The results suggest that in neonatal mice, exposure to 2% sevoflurane of at PD7 can affect the survival and dendritic development of PV-expressing interneurons in the MOB, but it does not affect CB- and CR-expressing interneurons in the MOB. These findings may lay a morphological foundation for studies aimed at determining the effects of sevoflurane exposure on olfactory functions.
JY designed the experiments and drafted the manuscript. JC and GC participated in the study design and coordination. JY performed the experiments on immunofluorescence and western blotting and analyzed these data with TS. RL performed experiment on animal treatment with anesthesia. TL performed experiment on Nissl staining. SL and SW provided the financial and administrative support for this project. All authors read and approved the final manuscript.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The handling Editor declared a shared affiliation, though no other collaboration, with several of the authors JC, GC, RL, TL and SW and states that the process nevertheless met the standards of a fair and objective review.
This work was supported by grants from the National Natural Science Foundation of China (81371404 and 81571243) and Innovation Teams Project in Priority Areas Accredited by the Ministry of Science and Technology of the People’s Republic of China (2014RA4029).
calbindinD28k
calretinin
chemiluminescence
external plexiform layer
γ-aminobutyric acid
glomerular cell layer
glomerular layer
main olfactory bulb
postnatal days
parvalbumin
polyvinylidenedifluoride.