Methodology for the Study of Learning and Memory in Juvenile Rats Using Sevoflurane Anesthesia to Assess Hippocampal p-CREB and c-fos Expression

Summary

This study investigates how sevoflurane impairs memory retention and reduces p-CREB and c-fos expression in hippocampal neurons via the NMDAR/p-CREB/c-fos pathway in juvenile rats. Results show that sevoflurane causes short-term memory deficits and downregulates synaptic plasticity markers, with effects reversible after 3 days, independent of dosing frequency.

Abstract

Studying cognitive impairments during brain development faces challenges due to the dynamic nature of neurodevelopment, methodological constraints, and contextual barriers. This study investigated an integrated methodological approach to study cognitive impairments in the developing brain caused by sevoflurane based on memory and p-CREB and c-fos expression in hippocampal neurons through the NMDAR/p-CREB/c-fos signaling pathway in juvenile rats. Sixty-four 21-day-old SD male rats (body weight <80 g) were divided into five groups: normal (n=8), 60% O₂ (n=16), sham training (n=8), single-dose sevoflurane (n=16), and multiple-dose sevoflurane (n=16). The two sevoflurane groups were sub-grouped into sacrifice 1 day (groups sev1a and sev1b) and 30 days (groups sev30a and sev30b) after training (n=8/subgroup); all other rats were sacrificed 1 h after training. The Y-maze test was used to evaluate learning and memory retention. Immunohistochemistry was used to determine the number of p-CREB- and c-fos-positive neurons in the hippocampus specimens. Compared with the 60% O₂ group (memory retention of 86.33% ± 17.52%), the memory of rats in the sev1a and sev1b groups showed a significant decrease (sev1a: 44.29% ± 11.26%; sev1b: 62.42% ± 7.27%; all P<0.05), while the sev1b and sev30b groups showed no significant differences (sev30a: 84.41% ± 14.15%; sev30b: 85.21% ± 11.61%; all P>0.05). Compared with the 60% O₂ group (c-fos: 92.83 ± 7.88; p-CREB: 72.22 ± 8.89), the expression of p-CREB and c-fos in hippocampal neurons decreased in the sev1a group (c-fos: 23.13 ± 3.28; p-CREB: 22.88 ± 5.18) and the sev30 group (c-fos: 23.22 ± 3.13; p-CREB: 25.58 ± 2.26) (all P<0.05), while the sev30a and sev30b groups showed no significant differences (all P>0.05). Sevoflurane has a short-term negative stimulating effect on the memory maintenance ability of juvenile rats without an impact based on dosing frequency. Sevoflurane decreases the expression of p-CREB and c-fos in hippocampal neurons of juvenile rats.

Introduction

Studying cognitive impairments during brain development faces significant challenges due to the dynamic nature of neurodevelopment, methodological constraints, and contextual barriers. Brain development involves rapid, non-linear changes, complicating the identification of impairments during critical windows^1,2. In low-resource settings, barriers like caregiver literacy, linguistic diversity, and limited access to specialized tools (e.g., MRI) hinder early screening¹. Individual variability in brain maturation makes it difficult to distinguish pathological deviations from typical develo....

Protocol

All protocols in this study were reviewed and approved by the Ethics Committee of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine (approval No. XHEC-C-2016-023-2).

1. Experimental overview of animals and grouping

1. Obtain 64, 21-day-old male SD rats (body weight under 80 g).
2. Divide the rats into five groups: Normal group (n = 8), 60% O₂ group (n = 16), Sham training group (n = 8), Single-dose sevoflurane group (n = 16), and Multiple-dose sevoflurane group (n = 16) (See Table 1).
3. House the rats in an animal facility at 20-24 °C with 50%-60% humi....

Results

Using data from this study as illustrative examples, we have showcased the types of results that can be generated with this methodological framework, including representative immunostaining images as well as behavioral and molecular data from different groups. The emphasis is on presenting typical or expected outcomes produced by this approach, rather than providing an interpretation of their biological significance.

Learning and memory test results of each group of rats
T.......

Discussion

This study investigated how sevoflurane affects memory and modulates p-CREB and c-fos expression in hippocampal neurons through the NMDAR/p-CREB/c-fos signaling pathway in juvenile rats. The results suggest that sevoflurane had a short-term (1 h after training, not after 3 days) negative stimulating effect on the memory maintenance ability of juvenile rats, without impact based on dosing frequency (single dose or seven doses over 7 days). Sevoflurane significantly decreased the expression of p-CREB and c-fos in hippocamp.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Experimental Animals
Sprague-Dawley (SD) Rats	Charles River Laboratories (Beijing, China)	N/A	21-day-old, male
Standard Rat Chow	Keao Xieli Feed (Beijing, China)	N/A	Standard laboratory animal diet
Chemicals and Reagents
Chloral Hydrate	Sinopharm Chemical Reagent (Shanghai, China)	80113818	Anesthetic for terminal perfusion
Citrate Buffer (10x)	ZSGB-BIO (Beijing, China)	ZLI-9064	Antigen retrieval solution, pH 6.0
Ethanol (100%)	Sinopharm Chemical Reagent (Shanghai, China)	100092683	Dehydration agent for histology
Hematoxylin Stain Solution	Baso Diagnostics Inc. (Zhuhai, China)	BA-4041	Counterstain for IHC
Hydrogen Peroxide (H2O2)	Sinopharm Chemical Reagent (Shanghai, China)	10011218	3% solution for blocking endogenous peroxidase
Neutral Balsam	Solarbio (Beijing, China)	G8590	Mounting medium for slides
Nitrogen (N2)	Shanghai Medical Gas Co. (Shanghai, China)	N/A	High purity, for gas mixture
Oxygen (O2)	Shanghai Medical Gas Co. (Shanghai, China)	N/A	Medical grade, for gas mixture
Paraffin	Leica Biosystems (Nussloch, Germany)	39601006	Tissue embedding medium
Paraformaldehyde (PFA)	Sigma-Aldrich (St. Louis, MO, USA)	158127	Fixative for tissue perfusion and fixation
Phosphate-Buffered Saline (PBS)	Gibco (Waltham, MA, USA)	70011044	10x stock solution, pH 7.4
Sevoflurane	AbbVie Inc. (North Chicago, IL, USA)	N/A	Inhalation anesthetic agent
Soda Lime	W. R. Grace and Co. (Columbia, MD, USA)	22-01-0010	CO2 absorbent for inhalation chamber
Sodium Hydroxide (NaOH)	Sinopharm Chemical Reagent (Shanghai, China)	10019818	Used for pH adjustment of PFA solution
Sucrose	Sangon Biotech (Shanghai, China)	A502693	Used for cryoprotection of brain tissue
Xylene	Sinopharm Chemical Reagent (Shanghai, China)	10023418	Clearing agent for histology
Antibodies and Kits
c-Fos Antibody	Abcam (Cambridge, UK)	ab190289	Rabbit anti-mouse, primary antibody
DAB Chromogen Kit	ZSGB-BIO (Beijing, China)	ZLI-9018	Substrate for HRP color development
p-CREB (Ser133) Antibody	Cell Signaling Technology (Danvers, MA, USA)	9198	Rabbit anti-mouse, primary antibody
Polymer HRP Detection Kit	ZSGB-BIO (Beijing, China)	PV-9000	Secondary antibody and detection system for IHC
Equipment
Anesthesia Machine	RWD Life Science (Shenzhen, China)	R500	For controlled gas mixture delivery
Automated Tissue Processor	Leica Biosystems (Nussloch, Germany)	ASP6025	For automated dehydration and clearing
Cryostat/Microtome	Leica Biosystems (Nussloch, Germany)	CM1950	For sectioning of frozen/embedded brain tissue
Microscope System	Olympus (Tokyo, Japan)	BX53	For imaging of IHC slides
Perfusion Pump	Longer Precision Pump Co. (Baoding, China)	BT100-2J	Peristaltic pump for transcardial perfusion
Sevoflurane Vaporizer	RWD Life Science (Shenzhen, China)	R510PS	For precise sevoflurane concentration control
Y-Maze Apparatus	Med Associates Inc. (St. Albans, VT, USA)	MED-VFC-S-Y	With electric grid and lights for active avoidance
Software
GraphPad Prism	GraphPad Software (San Diego, CA, USA)	Version 9	Statistical analysis and data visualization
ImageJ	National Institutes of Health (Bethesda, MD, USA)	Version 1.53	Image analysis software for cell counting
Microsoft Excel	Microsoft Corporation (Redmond, WA, USA)	Office 365	Spreadsheet software for data compilation