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 development^2,3. 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 anesthesia^9,10. 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 infants^13,14, 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 important^19,20. Enhanced NMDAR activity has significant implications for inducing synaptic growth and plasticity^19,20. 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 conclusion^22,23, 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 functions^{11,13,14,23,24}. 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 clearer^25,26,27, 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 brain^28,29. 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.

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% 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 group^33,34.
    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 significance^35,37. 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.

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
The experimental groups were compared with the 60% O₂ group. The memory of rats in the single-dose sevoflurane group (sev1a) and the multiple-dose sevoflurane group (sev1b), i.e., the rats sacrificed 1 h after the test showed a significant decrease (all P < 0.05), while the rats sacrificed 3 days after the test showed no significant differences (all P > 0.05) (Table 2).

Expression of c-fos and p-CREB proteins in the hippocampus of rats
The levels of c-fos in the hippocampal neurons of rats in the normal group were lower than in the other groups, and the p-CREB levels in the hippocampal neurons of rats in the sham training group were lower than in the other groups. Compared with the 60% O₂ group, the expression of p-CREB and c-fos in hippocampal neurons decreased in the sev1a and sev1b groups (i.e., sacrificed 1 h after training), the sham training group, and the normal group (all P < 0.05), while there were no statistically significant differences in the sev30a and sev30b groups (i.e., sacrificed 3 days after training) (Figure 1 and Table 3).

Figure 1: Expression of c-fos and p-CREB proteins in the hippocampus of rats. Please click here to view a larger version of this figure.

Group	Procedure			n
Normal	Expose to 60% air for 2 hours. Adapt to Y-maze for 5 minutes. Perform Y-maze testing 1 day post-exposure. Collect tissue 1 hour after testing.			8
60% O2 Control	Expose to 60% O2 for 2 hours. Perform Y-maze testing at either 1 day or 30 days post-exposure (n=8 each). Collect tissue 1 hour after testing (for 1-day test) or 3 days after testing (for 30-day test).			16
Sham training	Expose to 60% air for 2 hours. Perform Sham Y-maze testing (random stimuli) 1 day post-exposure. Collect tissue 1 hour after testing. Controls for light and electrical stimuli.			8
Single dose sevoflurane	Single 2h inhalation of 60% O2 + 3% sevoflurane. Perform Y-maze testing 1 day post-exposure. Collect tissue 1 hour after testing.  Perform Y-maze testing 30 days post-exposure. Collect tissue 3 days after testing.			16
Multiple doses sevoflurane	Multiple (7 days) 2h daily inhalations of 60% O2 + 3% sevoflurane.Perform Y-maze testing 1 day after last exposure. Collect tissue 1 hour after testing. Perform Y-maze testing 30 days after last exposure. Collect tissue 3 days after testing.			16

Table 1: Experimental rat grouping.

Experimental group	n	Memory retention rate (%)
Sevoflurane single dose sev1a	8	44.29±11.26*
Sevoflurane single dose sev30a	8	84.41±14.15
Sevoflurane multiple sev1b	8	62.42±7.27*
Sevoflurane multiple sev30b	8	85.21±11.61
60% O2 group	8	86.33±17.52
The results are shown as means ± standard error of the mean. *P<0.05 vs. the 60% O2 group.

Table 2: Learning and memory test results of rats in each group.

Group	n	c-fos-positive neurons (n)	p-CREB-positive neurons (n)
Sevoflurane single dose sev1a	8	23.13±3.28*	22.88±5.18*
Sevoflurane single dose sev30a	8	92.32±7.36	72.75±8.24
Sevoflurane multiple sev1b	8	23.22±3.13*	25.58±2.26*
Sevoflurane multiple sev30b	8	91.83±12.12	77.83±8.21
60% O2 group	8	92.83±7.88	72.22±8.89
Sham training	8	44.33±3.12*	7.27±2.75*
Normal	8	9.23±1.28*	7.21±1.12*
The results are shown as means ± standard error of the mean. *P<0.05 vs. the 60% O2 group.

Table 3: Expression results of the c-fos and p-CREB proteins in the hippocampus of rats in each group

Supplementary Figure S1: The rat sevoflurane inhalation anesthesia setup. Please click here to download this File.

Supplementary Figure S2: The Y-maze setup. Please click here to download this File.

Supplementary Figure S3: The surgical process to obtain the hippocampi. Please click here to download this File.

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 hippocampal neurons of juvenile rats 1 h after training.

This study investigated the relationship between sevoflurane and learning and memory retention as a proof of concept for an integrated methodology to study learning and memory. Ca²⁺ can enter the cell through NMDA receptors (NMDARs) or voltage-dependent calcium channels, and subsequently trigger the transcription of various immediate early genes (IEGs), such as c-fos and p-CREB, through multiple signaling pathways¹³. These genes are then translated into corresponding protein products, which act as transcription factors to further regulate the transcription of target genes, thereby participating in the process of synaptic plasticity. CREB is one of the transcription factors whose activity is regulated by phosphorylation. It is localized in the nucleus and expressed in all types of brain cells. Phosphorylation at the Ser-133 residue (p-CREB) serves as a key mediator in initiating the transcription of various downstream genes^38,39. Sevoflurane is a non-competitive antagonist of NMDAR and can bind to the PCP binding site of NMDAR, which may affect the body's learning and memory ability⁴⁰. NMDAR is a subtype of the glutamate receptor, and it mainly plays a key role in inducing and maintaining LTP through cascade reactions within cells after receptor stimulation^19,41. NMDAR can activate various protein kinases and phosphorylate synaptic proteins by regulating intracellular Ca²⁺ and Mg²⁺ concentrations⁴². In the process, CREB can be phosphorylated to generate p-CREB. The transcriptional function of CREB directly affects long-term memory formation and is a very important regulatory factor^43,44. The regulatory gene transcription function of CREB can be significantly demonstrated by binding to CRE, resulting in a 15-20-fold increase in gene transcription levels. Memory formation relies on the induction function of the p-CREB pathway, which enables downstream genes to be expressed rapidly. For example, the early gene c-fos can be significantly upregulated by p-CREB⁴⁵.

Sevoflurane can affect memory in infants^13,14. Traditionally, it was thought that brain hypoxia during general anesthesia was responsible for the cognitive dysfunctions after surgery¹⁰. In the present study, sevoflurane was inhaled without causing brain hypoxia. Therefore, the effects observed here are the effects of sevoflurane, suggesting that it would cause memory dysfunction in infants. On the other hand, the present study suggests that the memory impairment would be short-term and transient, recovering within 3 days after anesthesia, supported by a study in Drosophila⁴⁶. Nevertheless, there are reports of long-term developmental and neurological issues with the use of sevoflurane in infants^23,47. Additional animal studies are necessary to study such effects.

Using the Y-shaped maze experiment, the formation of animal memory can be effectively simulated³⁰. In this experiment, 21-day immature rats were selected mainly because the content of NMDAR in immature brains is richer than in mature brains, and the composition of NMDAR subunits also changes during brain development⁴⁸. The present study found that compared with the 60% O₂ group, the expression of hippocampal p-CREB and c-fos in rats in the sevoflurane group was significantly lower, indicating that sevoflurane can affect the learning and memory function of rats. Those results align with the lower memory retention rates observed with sevoflurane inhalation. Sevoflurane affected the expression of hippocampal c-fos and p-CREB proteins, but there were no significant differences between single and multiple doses, indicating that sevoflurane only has a short-term inhibitory effect on the expression of hippocampal p-CREB and c-fos. Compared with the sham training group and the normal group, there were no significant differences in the expression of p-CREB in the hippocampus of rats between the two groups, while the former showed significantly higher expression of c-fos than the latter. However, at the same time, the expression of p-CREB and c-fos in the 60% O₂ group was higher than in the normal group of the sham training combination. It suggests that the higher expression of p-CREB in the hippocampus was caused by learning and memory training, while the expression of c-fos can be induced by stimuli such as light and electricity. Previous studies reported memory impairments in various animal models after sevoflurane exposure^13,23,40,47, supporting the present study. Still, a study in rats using the Morris water maze (MWM) reported no effects of sevoflurane on memory²². The exact reasons will have to be investigated in the future. Nevertheless, the decreases in p-CREB and c-fos levels after sevoflurane exposure are supported by the literature^49,50,51. Of note, a study showed that resveratrol could reverse, at least in part, the decreases in p-CREB and c-fos observed after sevoflurane exposure in mice⁵⁰. Additional studies are necessary to examine the prevention of POCD after anesthesia.

The hippocampus is a conserved brain structure among mammals⁵². The specific connection between special areas of the brain and learning and memory, especially the high-density neurons present in the hippocampus, indicates a close relationship between learning and memory in the hippocampus in animals^24,53. The present study selected the hippocampus region of rats as the research focus and analyzed their memory activity using stimulus learning. Previous studies showed that the CA1, CA2, CA3, and CA4 regions of the hippocampus, which contain small pyramidal cells, are particularly closely related to spatial cognitive ability and memory activity^54,55. In 1973, Bliss and Lomo⁵⁶ studied the enhancement of synaptic function in the dentate gyrus of the rabbit hippocampus after tetanic stimulation; high-frequency current stimulation of the hippocampus caused rapid synaptic enhancement within s, with a duration of more than 10 h. This long-term enhancement effect, also known as the LTP phenomenon, is a typical feature of learning and memory. The LTP phenomenon of synapses has a significant impact on the information storage process and is a fundamental feature of learning and memory⁵⁷. Previous studies demonstrated that altering the mechanism of synaptic plasticity formation can affect learning and memory. The formation of synaptic plasticity can be observed in the brain regions related to learning and memory, and inducing or enhancing synaptic plasticity in specific environments can also promote or facilitate learning and memory^58,59,60. Still, the exact mechanisms remain poorly understood.

The Y-maze combines electric shock and light signals as stimuli and is highly effective for assessing associative learning and active avoidance memory in animals³⁰. Compared with more complex spatial memory tasks such as the MWM, the Y-maze setup is relatively simple, and the training process (especially for avoidance tasks) can be completed in a short amount of time⁶¹. It is particularly effective for evaluating rapid learning and memory formation. Its high feasibility, combined with standardized voltage and timing parameters, ensures good reproducibility. For preliminary screening studies involving a large number of animals, this method offers relatively high efficiency³⁰. Although it may not comprehensively assess complex spatial navigation abilities like the MWM, nor evaluate recognition memory like the Novel Object Recognition (NOR) task, it provides an effective means to study learning and memory associated with aversive stimuli, which is relevant to the types of stressors that may arise from anesthesia or surgical environments^30,61. The Y-maze active avoidance paradigm is simple to operate, efficient, and capable of rapidly inducing and testing memory, making it especially suitable for observing the immediate impact of learning processes on molecular markers such as immediate early genes (IEGs). In contrast, the MWM focuses on spatial reference memory, NOR emphasizes recognition memory, and fear conditioning mainly evaluates contextual and auditory fear memory^30,61. Our choice of this paradigm was based on the research objective: to investigate the effects of anesthesia on the learning process itself and its induced molecular changes.

Specific parameters for electric shock intensity and signal light duration in a Y-maze experiment can significantly affect experimental outcomes. Shocks ranging from 0.1 mA to 0.7 mA produce varying effects on learning and avoidance behavior⁶². In active avoidance tasks, shock intensities between 0.1 mA and 0.3 mA did not produce statistically significant differences in avoidance rates or escape latency in mice. However, sex-specific interactions were observed, where females showed differential sensitivity to shock intensity⁶². At 0.7 mA, shock effectively measured avoidance learning in rats, with exercised groups showing improved corrective response rates and reduced shock duration. This suggests that while extreme intensities may not alter learning capacity within a moderate range, they must be standardized to ensure reproducibility⁶². Furthermore, moderate light (400 lux) during the Y-maze testing phase enhanced spatial memory retrieval in mice, likely due to optimal stress levels that heighten vigilance without excessive anxiety⁶³. Higher intensities (800-1200 lux) impaired memory retrieval and increased anxiety, as evidenced by reduced exploration in novel arms and elevated stress markers⁶³. Light applied during memory retrieval (testing phase) improved performance, whereas the same intensity during training had no effect. The duration of light exposure (e.g., continuous vs. intermittent) was not explicitly tested, but the timing relative to task phases proved critical⁶³. Those considerations call for reproducible and comparable parameters among studies.

This study introduces a comprehensive methodological framework for examining anesthetic-induced cognitive deficits in developing rats, integrating behavioral testing (Y-maze) with molecular analyses (hippocampal p-CREB and c-fos expression). The protocol's effectiveness and interpretability depend on several key steps, and recognizing possible modifications and troubleshooting procedures can further improve its reliability and reproducibility. A fundamental aspect of this approach is the careful selection of the animal model and age. P21 male Sprague-Dawley rats were selected to represent early adolescence in humans-a phase marked by heightened neuroplasticity and susceptibility^28,29. Using animals of standardized age and weight helps minimize variability, which is essential for both behavioral and molecular investigations. The sevoflurane exposure protocol (3% sevoflurane for 2 h via inhalation) necessitates precise monitoring to maintain a consistent anesthetic depth, thereby avoiding confounding factors that could independently influence neurodevelopment. By comparing single versus multiple exposures, the study addresses a clinically relevant question regarding the effects of varying anesthetic frequencies. Although the Y-maze active avoidance task is straightforward and efficient^30,61, it requires rigorous standardization. Proper habituation of the animals reduces anxiety and enhances task engagement. Critical parameters such as electric shock intensity and the timing/location of the light cue must be finely tuned for the specific age and strain to ensure consistent and reproducible avoidance behavior. Establishing a clear learning criterion is vital for confirming task acquisition before proceeding to memory assessments. The selected testing intervals (1 h and 30 days after training) allow differentiation between immediate and longer-term effects on memory retention. Immunohistochemistry demands careful execution to yield reliable molecular data. Thorough perfusion with PBS followed by fixation with 4% paraformaldehyde is crucial for preserving tissue integrity and antigenicity. Rapid and precise hippocampal dissection minimizes tissue degradation. Antigen retrieval, such as heat-induced epitope retrieval with citrate buffer, must be optimized to ensure antibody accessibility. Validating antibodies for specificity in rat hippocampal tissue, optimizing concentrations, and maintaining consistent incubation conditions are essential for specific staining with minimal background noise. Standardizing imaging parameters (e.g., magnification) and employing unbiased cell counting methods (such as blinded counts in defined, non-overlapping hippocampal regions like CA1, DG, or the entire hippocampus) are critical for quantitative analysis. Potential protocol modifications include adding behavioral tests such as the Morris Water Maze or Novel Object Recognition to evaluate different aspects of memory^30,61. Adjusting sevoflurane concentration, exposure duration, or the developmental stage at exposure could help investigate dose- or age-dependent effects. Expanding molecular analyses to include Western blotting for protein quantification, assessing additional synaptic plasticity markers (e.g., BDNF, Arc), or examining neuroinflammation markers could provide deeper mechanistic insights. Incorporating longer follow-up periods (e.g., several months) would be valuable for evaluating persistent neurocognitive deficits. Common challenges include variability in behavioral outcomes, which can be reduced by maintaining strict environmental controls, consistent animal handling, and optimized stimulus parameters. Issues with immunohistochemical staining, such as weak signals or high background, often stem from suboptimal fixation, inadequate antigen retrieval, or non-optimized antibody concentrations and washing procedures. Troubleshooting should involve systematically evaluating each step, from tissue collection and processing to antibody incubation and detection.

This study had limitations. Of course, rat models had to be used to measure the CREB and c-fos levels in the hippocampus, but whether the results can be translated directly to humans will require additional study. Only two time points (1 h and 3 days) were tested, and the exact pharmacodynamics of sevoflurane on memory retention remains unknown. The relatively small number of animals could influence the results. Future studies should validate the results across different mammalian species and extend the duration of the study to examine the long-term effects of sevoflurane.

In conclusion, the expression of p-CREB and c-fos in rat hippocampal neurons directly relates to learning and memory in juvenile rats. Sevoflurane has a short-term negative stimulating effect on the memory maintenance ability of juvenile rats, and there is no significant impact of the frequency of low-dose sevoflurane anesthesia. Sevoflurane significantly affected the expression of p-CREB and c-fos in hippocampal neurons of juvenile rats.

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