Method Article

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

DOI:

10.3791/68591

⸱

September 19th, 2025

 ,  , 
1Department of Anesthesiology and SICU, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine

In This Article

Summary

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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

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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

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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 windows1,2. 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 development2,3. Conditions like Down syndrome exhibit altered cortical development, but small sample sizes and scarce histological data limit mechanistic insights4. Neurodevelopmental disorders (e.g., ASD, ADHD) often co-occur with anxiety or depression, confounding cognitive assessments5. Severe communication deficits in some populations restrict the use of standardized tests6. Those features lead to several research design issues. Indeed, most studies use cross-sectional designs, failing to model within-individual change over time. There is an overreliance on Western, educated populations ("WEIRD" samples)7. Down syndrome research suffers from small post-mortem brain samples and inadequate age-matched controls4. Ecological momentary assessment tools struggle to account for contextual variables affecting daily cognitive performance5.

The most important functions in the animal and human brain include learning and memory, which are central to human development and life in general8. The brain of infants is in the developmental stage and is vulnerable to external factors like drugs and anesthesia9,10. The damage caused by anesthetics to infants' memory function is a crucial clinical research topic and can even delay optimal treatments for pediatric patients due to the inability to perform anesthesia11. Anesthesia-related cognitive dysfunctions (or postoperative cognitive dysfunction, POCD) may have long-term consequences for infants12. Sevoflurane inhalation can affect memory functions in infants13,14, but traditional anesthesiology believes that brain damage in infants is mainly caused by brain hypoxia during anesthesia, especially in general anesthesia10. Therefore, as long as there is no short-term or long-term hypoxia in the brain, the risk of central nervous system injury in infants should be reduced11. Still, the understanding of POCD remains suboptimal. Infants requiring surgery will require anesthesia, and a better knowledge of the pathways involved could help design anesthesia strategies that would minimize the risk of POCD.

A study involving older adult patients who underwent non-cardiac surgeries revealed that nearly 25% of patients experienced POCD within 1 week after surgery, and the proportion decreased to below 10% after 3 months, while the control group showed POCD rates of 3.4% and 2.8%15. The study also indicated that most POCD cases were reversible, with only a small number of patients experiencing prolonged or permanent POCD15. In the central nervous system, glutamate is the most common excitatory amino acid16. The N-methyl-D-aspartate receptor (NMDAR) is crucial for the induction and maintenance of long-term potentiation (LTP) and long-term depression (LTD)17. Research on mice showed that excitatory synaptic plasticity directly affects brain function development18, with NMDAR activation and calcium influx being particularly important19,20. Enhanced NMDAR activity has significant implications for inducing synaptic growth and plasticity19,20. Therefore, a higher degree of NMDAR blockade by NMDAR antagonists (e.g., sevoflurane) should result in lower learning and memory abilities21. While the effects of sevoflurane on learning and memory in adult brains have not reached a clear conclusion22,23, sevoflurane is commonly used for anesthesia in pediatric surgeries14. As a non-competitive antagonist of NMDAR, sevoflurane can significantly affect the brain's learning and memory functions11,13,14,23,24. Nevertheless, besides NMDAR, other pathways and proteins are involved in learning and memory, and the mechanisms of memory regulation involving phosphorylated-cAMP response element-binding protein (p-CREB) and immediate early gene c-fos are becoming clearer25,26,27, but still require a better understanding for optimal anesthesia delivery in infants and children.

Based on these theoretical frameworks, the present study investigated an integrated methodological approach that could be used 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. The results could help improve our understanding of POCD and memory impairment after general anesthesia. This study used 21-day-old rats because they had just been weaned and had a highly uniform body weight and physiological status, corresponding to early adolescence in humans. Due to their heightened neuroplasticity and vulnerability during this critical developmental period, they can efficiently model the cognitive impairments caused by surgery or anesthesia on the immature brain28,29. This makes them particularly suitable for studying the neurocognitive risks associated with pediatric surgery and exploring potential intervention strategies. The Y-maze is a commonly used method to assess learning and memory30. Immunohistochemistry is commonly used to study neurons and various neuronal proteins.

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Protocol

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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% humidity and natural light.
  4. Provide adequate food and water.
  5. Ensure all protocols are reviewed and approved by the Ethics Committee.
  6. Prepare the inhalation chamber (see Supplementary Figure S1, detailed in section 2.1).
  7. Normal group
    1. Expose the rats to 60% air for 2 h (detailed in section 2.2).
    2. Perform Y-maze testing 1 day post-exposure (detailed in section 4).
    3. Collect tissue after behavioral testing (detailed in section 5).
  8. 60% O2 group
    1. Expose the rats to 60% air for 2 h (detailed in section 2.2).
    2. Perform Y-maze testing 30 days post-exposure (detailed in section 4).
    3. Collect tissue after behavioral testing (detailed in section 5).
  9. Sham training group
    1. Expose the rats to 60% air for 2 h (detailed in section 2.2).
    2. Perform Y-maze testing 1 day post-exposure without structured training (lights and shock zone locations varied randomly), used to control for the effects of light and electric stimuli on learning and memory (detailed in section 4).
    3. Collect tissue after behavioral testing (detailed in section 5).
  10. Single-dose sevoflurane group (sev1a group)
    1. Expose the rats to 60% air + 3% sevoflurane for 2 h (detailed in section 2.2).
    2. Perform Y-maze testing 1 day post-exposure (detailed in section 4).
    3. Collect tissue after behavioral testing (detailed in section 5).
  11. Single-dose sevoflurane group (sev30a group)
    1. Expose the rats to 60% air + 3% sevoflurane for 2 h (detailed in section 2.2).
    2. Perform Y-maze testing 30 days post-exposure (detailed in section 4).
    3. Collect tissue after behavioral testing (detailed in section 5).
  12. Repeated-dose sevoflurane group (sev1b group)
    1. Expose the rats to 60% air + 3% sevoflurane for 2 h per day, for 7 consecutive days (detailed in section 2.2).
    2. Perform Y-maze testing 1 day post-exposure (detailed in section 4).
    3. Collect tissue after behavioral testing (detailed in section 5).
  13. Repeated-dose sevoflurane group (sev30b group)
    1. Expose the rats to 60% air + 3% sevoflurane for 2 h per day, for 7 consecutive days (detailed in section 2.2).
    2. Perform Y-maze testing 30 days post-exposure (detailed in section 4).
    3. Collect tissue after behavioral testing (detailed in section 5).

2. Sevoflurane inhalation

  1. Preparation of the chamber (see Supplementary Figure S1)
    1. Connect the hole on the left of the box to 60% O2/40% N2.
    2. Connect the oxygen supply to the sevoflurane vaporizer.
    3. Connect the hole on the right of the box to the gas analyzer of an anesthesia machine.
    4. Connect the hole on the top of the chamber to the gas waste.
    5. Place lime below the mesh at the bottom of the chamber and add cotton wool.
    6. Place the setup in a fume hood.
    7. Place a heating pad under the chamber.
  2. Inhalation
    1. Turn on the oxygen supply and the sevoflurane vaporizer.
    2. Adjust the oxygen flow to 2 L/min.
    3. When the sevoflurane concentration reaches 3.0%, maintain the flow for 30 min to stabilize the atmosphere in the chamber.
    4. Place a rat on the cotton side of the chamber.
    5. Keep the rat there according to grouping (see step 1.2).
    6. Monitor skin color, respiratory rate, heart rate, and pulse oximetry.
    7. Remove the rat.
    8. Let the rat awaken naturally.
    9. Return the rat to its cage.

3. Experimental reagents and instruments

  1. Reagent preparation
    1. Prepare 250 mL of commercially available sevoflurane for anesthesia administration.
    2. Prepare the p-CREB antibody working solution by diluting the stock antibody 1:350.
    3. Prepare the c-fos antibody working solution by diluting the stock antibody 1:200.
    4. Assemble the kit for immunohistochemical staining.
    5. Prepare 4% paraformaldehyde.
      1. Dissolve 40 g of soluble paraformaldehyde (PFA) in a glass container with 500 mL of high-purity water, continuously heat and stir magnetically to 60-65 °C until it forms a milky suspension.
      2. Add drops of 1.0 mol/L NaOH until the solution becomes clear (pH = 7.0).
      3. Add approximately 500 mL of PBS and thoroughly mix in an ice bath or cold water bath.
      4. Check the pH again, filter, and make up to 1000 mL volume.
      5. Store at 4 °C for future use.
  2. Equipment Setup
    1. Position the Y-maze in the behavioral testing area (See Supplementary Figure S2).
    2. Calibrate the microscope with an integrated imaging system for histological analysis.
  3. Install the microtome with fresh blades for tissue sectioning.
  4. Program the dehydration machine with the standard tissue processing protocol.
  5. Prepare the staining machine with the sequential staining baths according to the machine manufacturer's instructions.
  6. Setup the perfusion apparatus.
    1. Use two plastic-packaged saline bottles, drain the saline from the bottom opening, and fill them with PBS and 4% paraformaldehyde fixative.
    2. Hang the bottles, connect them with infusion sets, and attach a three-way stopcock to the thick needle end of the infusion set.

4. Y-maze and learning/memory testing

  1. Assemble the Y-Maze
    1. Set up the Y-maze with three rectangular arms labeled I, II, and III, connected by a triangular junction (See Supplementary Figure S2).
    2. Place an electric grid with 1 cm spacing on the bottom of the maze.
    3. Attach thin 2 cm copper strips on the maze walls to simulate dangerous areas and install stimulus signal lights.
  2. Define dangerous and safe areas
    1. Electrify regions I, II, and III at 16,000 V/m to indicate dangerous areas (signal light turns on when electrified).
    2. Mark non-electrified regions with a red light to indicate safe areas.
    3. Define the junction between arms as a non-safe area where the rat may receive an electric shock.
  3. Training procedure
    1. At the beginning of the experiment, consider the area where the rat is located as the safe area.
    2. After 2-4 min, apply electricity to the safe area, encouraging the rat to move toward the safe zone.
    3. If the rat moves from the safe area to a dangerous area, it will return to the safe zone under electric shock, displaying a passive avoidance response.
  4. Developing the active avoidance response
    1. After multiple training sessions, turn on the light in the safe area without applying electric shocks.
    2. Observe the rat's movement toward the illuminated safe area, forming a light-dark discrimination conditioned reflex (active avoidance response).
  5. Place the Y-maze in a dimly lit and quiet area to minimize external distractions.
  6. Conduct training daily between 9:00-11:00 AM and 2:00-4:00 PM.
  7. Allow the rats to move freely within the maze for 5 min before the experiment to help them adapt to the maze environment.
  8. Adjust stimulus parameters
    1. Adjust the stimulus voltage (16,000 V/m) during testing to ensure rats escape within 10 s after receiving an electric shock.
    2. Keep the safe area signal light on for 15 s, then turn it off.
    3. Start the next round of shock experiments after a 45 s interval.
  9. Signal light sequence and correct response
    1. Set the sequence of signal light turning on as I → II → III → I, in a cycle.
    2. Define a correct response as the rat escaping from the starting area directly to the illuminated safe zone within 10 s after receiving an electric shock.
  10. Consider the rat has reached the learning standard if it makes the correct escape response in 8 out of 10 consecutive trials.
  11. Record any escape to an arm without light as an incorrect response.
  12. Multiple training and testing sessions
    1. Perform multiple training and testing sessions to evaluate learning and memory.
    2. Record the number of correct and incorrect responses during a fixed number of trials.
    3. Alter the starting area to increase task complexity.
  13. Assess memory retention at intervals (e.g., 24 or 48 h post-training) to evaluate the rats' memory retention capacity.

5. Immunohistochemistry

  1. Anesthetize the rats
    1. Administer an intraperitoneal injection of 10% chloral hydrate (0.4 mL/100 g) 1 h after training to anesthetize the rats. Dispose of chloral hydrate solution according to local regulations.
    2. Perform thoracotomy to fully expose the heart31.
  2. Perfuse anesthetized rats with physiological solution
    1. Insert a needle containing PBS into the left ventricle in the direction of the aorta, secure the needle with hemostatic forceps, and immediately cut open the right auricle of the mouse to release venous blood.
    2. Quickly inject PBS until the effluent is clear and the liver and lungs appear white.
    3. Follow with a rapid and then slow infusion of 500 mL of 10% formalin solution until the animal's body stiffens, achieving formalin fixation.
    4. Dispose of formalin solution according to local regulations.
  3. Remove the brain
    1. Quickly open the skull32.
    2. Remove the whole brain by gently lifting it from the skull and cutting the blood vessels and spinal cord at the base, without damaging the hemispheres (as shown in Supplementary Figure S3).
  4. Dissect the hippocampi
    1. Dissect the bilateral hippocampi.
      1. At the occipital foramen of the mouse, sever the entire brain with scissors.
      2. Carefully insert scissors diagonally into the foramen magnum to cut the parietal bone.
      3. Pry apart both sides of the parietal bone with hemostatic forceps.
      4. Use scissors to cut the optic nerve on one side and probe down to the base of the skull.
    2. Place the hippocampi in 10% formalin solution for 12 h.
    3. Immerse the specimen in formalin containing a gradient of sucrose (10%, 20%, 30%).
    4. Keep overnight at 4°C until the specimen tissue sinks to the bottom.
  5. Embed and section the specimen
    1. Embed the specimen in paraffin. Dispose of formalin solution according to local regulations.
    2. Section the embedded specimen to 4 µm.
  6. Deparaffinize and rehydrate the sections
    1. Soak the sections in 50 mL of xylene three times for deparaffinization, each time for 15 min.
    2. Sequentially immerse in 50 mL of 100%, 90%, 70%, and 50% ethanol for 10 min each.
    3. Rinse three times with 50 mL of 0.01 mM PBS for 3 min each.
  7. Antigen retrieval
    1. Place the sections in 50 mL of citrate buffer (PBS, pH 6.0).
    2. Heat the sections in a stainless steel pressure cooker to 120 °C. Place the sections on a rack, fully immersed in the buffer. Seal the cooker, heat, and maintain under high pressure for 3 min.
    3. Remove the cooker from the heat and allow to cool naturally at room temperature before opening the lid.
    4. Rinse the sections in PBS (pH 7.2) for 2 min, repeating 3 times.
    5. Apply approximately 100-200 µL of 3% H2O2 to cover the sectgion on the slide and incubate at room temperature for 5-10 min to eliminate endogenous peroxidase activity.
    6. Rinse with PBS for 2 min, 3 times.
  8. Primary antibody incubation
    1. Add 200 µL of primary antibodies (diluted 1:150 in 0.01 mol/L PBS) to the sections.
    2. Incubate overnight at 4°C.
  9. Secondary Antibody Incubation
    1. Rinse the sections in PBS for 3 min, 3 times.
    2. Incubate the sections with approximately 100-200 µL of Polymer Helper to cover the section on the slide and incubate at 37 °C for 20 min.
    3. Rinse the sections in 0.1 mol/L PBS for 2 min, 3 times.
    4. Add approximately 100-200 µL of polyperoxidase-anti-mouse/rabbit IgG and incubate at 37°C for 20 min.
    5. Rinse the sections in PBS for 3 min, 3 times.
  10. Color development
    1. Add approximately 100-200 µL of the diaminobenzidine (DAB) solution (enough to cover the section on the slide) for color development.
    2. Let it react for approximately 3 min.
  11. Counterstaining and mounting
    1. Perform counterstaining by adding approximately 100-200 µL of hematoxylin dropwise to completely cover the section on each slide.
  12. Dehydrate and mount the sections
    1. Dehydrate the sections in 50 mL of 50%, 70%, 90%, and 100% ethanol for 10 min each, followed by 50 mL of xylene for 10 min.
    2. Clear and mount the sections using standard procedures.
  13. Microscopic examination
    1. Count the number of p-CREB- and c-fos-positive neurons in the hippocampus of each group33,34.
    2. Examine the sections under a microscope at 400x magnification.
    3. Record the average number of positive neurons in each slice, based on counts from three sections per animal without overlap.
      NOTE: All data were compiled in a computer spreadsheet. All analyses were performed using a statistical software. The results were presented as means ± standard error of the mean (SEM) and analyzed using one-way ANOVA with the SNK-q and Dunnett T3 post hoc tests35. The SNK-q test is commonly used for exploratory studies and is suitable for pairwise comparisons among multiple sample means. It does not rely on predefined hypotheses, making it ideal for comparing all group means36. The Dunnett T3 test is typically used for confirmatory studies with predefined hypotheses. It is suitable for comparing multiple treatment groups with a control group or for comparing means with particular professional significance35,37. When the P-value of the ANOVA test was ≤ 0.05, the pairwise post hoc tests were performed to determine which groups were significantly different from the others. Two-sided P-values < 0.05 were considered statistically significant.

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Results

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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...

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Discussion

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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...

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Disclosures

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The authors report there are no competing interests to declare.

Acknowledgements

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Ye Jiang and Guohui Li designed the study. Ye Jiang and Lai Jiang collected and analyzed the data and drafted the manuscript. Ye Jiang and Guohui Li revised the manuscript. All authors reviewed and approved the final version of the manuscript for submission. This work is supported by grants to Guohui Li from Shanghai Pujiang Programme (grant no. 23PJD057).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Experimental Animals
Sprague-Dawley (SD) RatsCharles River Laboratories (Beijing, China)N/A21-day-old, male
Standard Rat ChowKeao Xieli Feed (Beijing, China)N/AStandard laboratory animal diet
Chemicals and Reagents
Chloral HydrateSinopharm Chemical Reagent (Shanghai, China)80113818Anesthetic for terminal perfusion
Citrate Buffer (10x)ZSGB-BIO (Beijing, China)ZLI-9064Antigen retrieval solution, pH 6.0
Ethanol (100%)Sinopharm Chemical Reagent (Shanghai, China)100092683Dehydration agent for histology
Hematoxylin Stain SolutionBaso Diagnostics Inc. (Zhuhai, China)BA-4041Counterstain for IHC
Hydrogen Peroxide (H2O2)Sinopharm Chemical Reagent (Shanghai, China)100112183% solution for blocking endogenous peroxidase
Neutral BalsamSolarbio (Beijing, China)G8590Mounting medium for slides
Nitrogen (N2)Shanghai Medical Gas Co. (Shanghai, China)N/AHigh purity, for gas mixture
Oxygen (O2)Shanghai Medical Gas Co. (Shanghai, China)N/AMedical grade, for gas mixture
ParaffinLeica Biosystems (Nussloch, Germany)39601006Tissue embedding medium
Paraformaldehyde (PFA)Sigma-Aldrich (St. Louis, MO, USA)158127Fixative for tissue perfusion and fixation
Phosphate-Buffered Saline (PBS)Gibco (Waltham, MA, USA)7001104410x stock solution, pH 7.4
SevofluraneAbbVie Inc. (North Chicago, IL, USA)N/AInhalation anesthetic agent
Soda LimeW. R. Grace and Co. (Columbia, MD, USA)22-01-0010CO2 absorbent for inhalation chamber
Sodium Hydroxide (NaOH)Sinopharm Chemical Reagent (Shanghai, China)10019818Used for pH adjustment of PFA solution
SucroseSangon Biotech (Shanghai, China)A502693Used for cryoprotection of brain tissue
XyleneSinopharm Chemical Reagent (Shanghai, China)10023418Clearing agent for histology
Antibodies and Kits
c-Fos AntibodyAbcam (Cambridge, UK)ab190289Rabbit anti-mouse, primary antibody
DAB Chromogen KitZSGB-BIO (Beijing, China)ZLI-9018Substrate for HRP color development
p-CREB (Ser133) AntibodyCell Signaling Technology (Danvers, MA, USA)9198Rabbit anti-mouse, primary antibody
Polymer HRP Detection KitZSGB-BIO (Beijing, China)PV-9000Secondary antibody and detection system for IHC
Equipment
Anesthesia MachineRWD Life Science (Shenzhen, China)R500For controlled gas mixture delivery
Automated Tissue ProcessorLeica Biosystems (Nussloch, Germany)ASP6025For automated dehydration and clearing
Cryostat/MicrotomeLeica Biosystems (Nussloch, Germany)CM1950For sectioning of frozen/embedded brain tissue
Microscope SystemOlympus (Tokyo, Japan)BX53For imaging of IHC slides
Perfusion PumpLonger Precision Pump Co. (Baoding, China)BT100-2JPeristaltic pump for transcardial perfusion
Sevoflurane VaporizerRWD Life Science (Shenzhen, China)R510PSFor precise sevoflurane concentration control
Y-Maze ApparatusMed Associates Inc. (St. Albans, VT, USA)MED-VFC-S-YWith electric grid and lights for active avoidance
Software
GraphPad PrismGraphPad Software (San Diego, CA, USA)Version 9Statistical analysis and data visualization
ImageJNational Institutes of Health (Bethesda, MD, USA)Version 1.53Image analysis software for cell counting
Microsoft ExcelMicrosoft Corporation (Redmond, WA, USA)Office 365Spreadsheet software for data compilation

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Sevoflurane AnesthesiaJuvenile RatsLearning And MemoryHippocampal Neuronsp CREB Expressionc fos ExpressionY Maze TestImmunohistochemistryNMDAR SignalingCognitive Impairment

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