20230808-AI coding-1st round/1043 – Gao 2021.txt

PMID: 34645094 DOI: 10.31083/j.jin2003065
2. Methods
2.1 Animals
Six adult female Sprague-Dawley (SD) rats, weighing 180–220 g, were raised with free diet and water intake in polypropylene cages for 7 days. Then the female SD rats were mated with male SD rats with sexual experience at 7:00 PM after adaptive feeding. Vaginal smears were performed the next morning and pregnancy day 0, G0, was defined by sperm detection. The pregnant rats were randomly divided into two groups: a control group (control, n = 3) and a sevoflurane group (SeV, n = 3). The six female SD rats were raised to G14.5 (middle pregnancy).

2.2 Anesthesia
On pregnancy day 14.5, the rats allocated to sevoflurane exposure were put inside a 30 cm 
×
 20 cm 
×
 120 cm box. A mixture of oxygen and sevoflurane (2 L/min with 4% sevoflurane) was delivered through an inlet port connected to a vaporizer, while a gas analyzer installed on a second port allowed monitoring of anesthetic gas concentration. Pregnant rats are more sensitive to sevoflurane and a minimum alveolar concentration (MAC) of 2.4% in healthy adult rats [27], so the concentration of sevoflurane (3%) is equivalent to 1.3 MAC to maintain a surgical level of anesthesia. The rats in the control group inhaled oxygen (2 L/min). However, limited to anesthesia machine conditions that it is not completely airtight, the inhalation concentration of sevoflurane should be 4% in order to reach 1.8 MAC to maintain a surgical level of anesthesia in the SeV group. The inhalation time is 3 hours in the SeV group. During the procedure, the skin color of the rats’ mouths, noses, limbs, and respiratory amplitudes and frequencies were observed to avoid hypoxia respiratory depression. After anesthesia, the rats were sent back to their cages after the righting reflex was recovered. After sevoflurane anesthetization, an arterial blood gas analysis was performed to assess gas exchange and glycemic status in female rats. The site of blood sampling was left heart artery. If there was a significant derangement, e.g., severe hypoxemia, these female rats were no longer involved in the follow-up experiments. No female rats were excluded in the study. Then the rat offspring were reared and delivered naturally.

2.3 Tissue section preparation
Three offspring were randomly selected from each group with one offspring/dam. The ex vivo brain samples of the offspring rats were harvested at the day 30 of postpartum (P30) for histology, immunostaining and Golgi staining. Rats in both the control and SeV groups were executed and perfused through the left ventricle with precooling saline followed by 4% paraformaldehyde in 0.01 M phosphate buffered saline (PBS) pH 7.35. The brain tissue of the rats was taken and post-fixed for 24 hours for paraffin and frozen sections.

To analyze the status of the NRG1–ErbB4 pathway in the interneurons, the expression levels of NRG1 and ErbB4 in LII and III of the ECT were examined via immunohistochemistry. The NRG1–ErbB4 pathway plays an important role in the genesis, migration, differentiation, maturation, and neurotransmitter synthesis of GABAergic interneurons. We assumed that the number of GABAergic interneurons the LII/LIII ECT of offspring in could be affected by alterations of the NRG1–ErbB4 pathway. Therefore, we label GABAergic interneurons with PV to represent PV interneurons. We also used glutamic acid decarboxylase 67 (GAD67) to label GABAergic interneuron (the key enzyme of GABA neurotransmitter synthesis) positive cells to represent the total GABAergic interneurons in the LII/LIII ECT. The expression levels of PV and GAD67 in LII and LIII of the ECT were examined via immunohistochemistry. That is, Interneurons were identified by immunoreactivity to PV and GAD67. To investigate whether NRG1–ErbB4 pathway changes in offspring after prenatal sevoflurane exposure affect the formation of subunits during maturation, we detected NMDA receptor subunit 2A (NR2A) and NMDA receptor subunit 2B (NR2B) by immunofluorescence. The expression levels of NR2A and NR2B in LII and LIII of the ECT were examined via immunohistochemistry to analyze the status of NMDA receptors in the ECT. There is a fixed pattern of neurite growth in the developing brain. We assumed that prenatal sevoflurane exposure could affect the inherent growth pattern of dendrites and dendritic spines in pyramidal neurons through NRG1–ErbB4 alterations. Therefore, we used Golgi silver staining to investigate the length of dendrites and the number of branches and dendritic spines. Golgi staining was performed to analyze the total dendrite length, number of dendritic branches, spatial distribution of dendrites, and density of dendritic spines in the pyramidal neurons in the ECT.

2.4 Histology and immunohistochemistry
The coronal sections of the brain were deparaffinized, rehydrated, and immersed in 3% H
2
O
2
 at room temperature for 30 min. Antigens were retrieved in a 0.01 mol/L citric buffer (pH 6.0) at 97 
∘
C for 15 min. The coronal sections were cooled down for 1 h before being blocked by 10% bovine serum albumin (BSA) solution. Staining with diluted primary antibodies was conducted at 4 
∘
C overnight (for at least 18 h). The primary antibodies included rabbit anti rat NRG-1 (1:1000, Cat. No Ab191139, Abcam, Cambridge, UK), rabbit anti rat ErbB4 (1:250, Cat. No Sc-283, Santa Cruz, Dallas, Texas, USA), rabbit anti rat NR2A (1:1000, Cat. No cell signaling technology, Massachusetts, USA), rabbit anti rat NR2B (1:1000, Cat. No 06-600, Millipore, Massachusetts, USA), mice anti rat PV (1:1000, Cat. No #2886709, Millipore), and rat anti rat GAD67 (1:2500, Cat. No. MAB5406, Millipore, Massachusetts, USA). After being washed by 0.1% PBST for three times (5 min), the sections were stained with diluted second antibodies at room temperature for 2 h and kept in a dark place. After washed by 0.1% PBST for three times (5 min), the sections were counterstained with hematoxylin, dehydrated with ethanol and mounted with coverslips. Then the expression levels of NRG1, ErbB4, PV, GAD67, NR2, A and NR2B were examined using a fluorescence microscope (Leica DM6000B, Germany). The results were shown as positive cells/sections. In each rat, we randomly select 5–6 coronal sections to count the cells to avoid error resulting from the section status. The brain area sections (ECT) we selected for immunohistochemical section is fixed. There is an inward concave angle under the area of the ECT, which is used to locate the central cortex and reduce the error. The size of the ECT in this part of the rat brain is relatively fixed, so the randomly selected sections can be regarded as roughly the same size which is comparable.

2.5 Golgi stain
150 
μ
m-thick frozen brain sections were obtained from control and SeV rats. Golgi–Cox staining was performed using the FD Rapid Golgi stain kit (Cat. NO. PK401, FD NeuroTechnologies, Inc. Columbia, USA) according to the manufacturer’s protocols. Ten well-individualized pyramidal neurons in LII and LIII of the ECT were randomly selected from each rat. Sequential optical sections of 1392 
×
 1040 pixels were taken at 1.5 
μ
m intervals along the z-axis (Leica, DMi8 + DFC7000J, Germany). The Imaris software (BitPlane AG, Zurich, Switzerland) was used for tridimensional reconstruction. The total dendrite length, number of dendritic branches, and spatial distribution of dendrites in the pyramidal neurons of the ECT were estimated using Sholl analysis [28]. To measure the density of dendritic spines, a straight dendrite was scanned on the z-axis using a 100 
×
 objective microscope. A 40-
μ
m long dendrite was randomly intercepted with image J 1.46r (National institute of health, Bethesda, Maryland, USA). The number of synaptic spines was counted and the density of synaptic spines (spines/10 
μ
m) was calculated. At least 10 terminal dendrites were selected for each sample.

2.6 Statistical analysis
All the data was expressed as mean 
±
 standard deviation. JMP software version 16.0 (SAS Institute, Cary, NC, USA) was used for statistical processing. All parameters were tested for normal distribution using the Kolmogorov-Smirnov test. Two independent-sample t tests were conducted used to compare the parameters differences between the control and sevoflurane groups, including NRG1, ErbB4, PV, GAD67, NR2B and NR2A. Dendrites were analyzed with Kruskal-Wallis test (Sholl analysis) and Steel Dwass post hoc test using JMP software version 16.0 (SAS Institute, Cary, NC, USA) [28]. It was considered that a difference was statistically significant when P 
<
 0.05. GraphPad Prism 5.0 (GraphPad Software, San Diego, CA, USA) software was used to make drawings.