# Effects of Atipamezole Preconditioning on Cognitive Function and Protein Expression in Related Brain Regions of Aged Rats after General Anesthesia

Authors: Kebing Huang; Xiong Wang
Journal: Disease Markers
 (2022)

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## Abstract

To explore the possible mechanism of atipamezole in improving cognitive function after general anesthesia in aged rats, forty-five aged SD rats were separated into control, model, and atipamezole groups. Rats in the model group were anesthetized by intraperitoneal injection of 75 mg/kg ketamine plus 5 mg/kg midazolam. Results showed that the escape incubation period of the atipamezole group versus model group on the 2nd, 3rd, and 4th days was shortened, residence time of platform quadrant was prolonged on the 5th day, and number of times of crossing platform quadrant was increased. Compared with the control group, the residence time in the central region of the model group was shortened on the 1st, 2nd, and 3rd days. Atipamezole group’s central residence time was prolonged on the 1st, 2nd, and 3rd days compared to the model group. Concentrations of IL-1, IL-6, and TNF-α in the hippocampus of the atipamezole group decreased significantly compared to the model group. The expressions of p-CREB and c-fos proteins in the prefrontal cortex and nucleus accumbens of rats in the atimezazole group were higher than those in the model group. In conclusion, atipamezole preconditioning can reduce cognitive dysfunction in aged rats after general anesthesia, and its mechanism may be related to inhibiting hippocampal inflammatory reaction and improving protein expression levels of p-CREB and c-fos in related brain regions of aged rats.


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## Body

## 1. Introduction

Postoperative cognitive dysfunction (POCD) is a central nervous system complication after surgery and anesthesia, and its incidence rate is relatively high in elderly patients [1]. However, mechanism of POCD is still unclear. Previous studies have shown that general anesthesia drugs, as one of the influencing factors of postoperative cognitive dysfunction, can have a broad impact on cognitive function in elderly patients [2, 3].Studies have confirmed [4] that anesthesia induces cognitive dysfunction in aged animals, and its mechanism is related to excessive release of hippocampal proinflammatory factors. However, excessive inflammatory reaction in the hippocampus is one of the important causes of cognitive dysfunction. Release of neuronal inflammatory factors (TNF-α, IL-1β, etc.) can lead to cognitive decline. Release of inflammatory factors can inhibit long-term potentiation of the hippocampus [5].Studies have shown [6] that cyclic AMP response element binding protein (CREB) is a transcription factor in long-term memory, and phosphorylation is its activated form (p-CREB). The increase of p-CREB is beneficial to the enhancement of long-term memory in hippocampus. P-CREB also participates in hippocampal neurogenesis and is important in adult hippocampal neurogenesis and spatial memory.Studies have shown that POCD [7, 8] is related to oxidative stress response and decreased expression level of p-CREB in related brain regions. c-fos protein is the most common transcription factor in the Fos family used to study animal drug dependence [9]. Studies have confirmed [10] that general anesthetic drugs can affect the CREB protein expression in relevant brain regions of experimental animals.Atimezole is aα2-adrenoceptor antagonist with imidazole structure, which can rapidly reverse the sedative and anesthetic effects induced by α2-adrenoceptor agonists [11]. Clinically, atipamezole is often used to antagonize the anesthetic effect of 2-adrenaline receptor agonists (especially dexmedetomidine) and can also effectively relieve adverse reactions caused by medetomidine-midazolam-ketamine combined anesthesia [12], the current study aimed to investigate effect of atipamezole on learning and memory ability and expression of p-CREB and c-fos protein in related brain tissues after general anesthesia in aged rats and to explore the possible mechanism of atipamezole in improving cognitive function after general anesthesia in aged rats.


## 2. Materials and Methods

### 2.1. Laboratory Animals and Groups

Forty-five healthy male aged SD rats, aged 19-20 months, weighed568.12±21.63g. Rats were separated into the control group (n=15), model group (n=15), and atipamezole group (n=15).


### 2.2. Preparation of the Animal Model

The rats in the model group were anesthetized by intraperitoneal injection of 75 mg/kg ketamine (Fujian Gutian, China) plus 5 mg/kg midazolam (Jiangsu Hengrui, Chian). After satisfactory anesthesia, the rats were fixed in the conventional supine position, the abdominal skin was disinfected, and a longitudinal incision of about 3 cm was made along the abdominal midline 0.5 cm below the rib. Abdominal exploration was carried out every 5 min, and the liver, stomach, small intestine, and large intestine were explored in turn. The total operation time was 20 min. After the exploration, the abdominal cavity was closed. The rats in the atipamezole group were given intraperitoneal injection of 0.5 mg/kg atipamezole, and anesthesia and exploratory laparotomy were performed 60 min later. Method was the same as that of the model group. The control group was not given any treatment.


### 2.3. Morris Water Maze (MWM)

After anesthesia, MWM was carried out at the same time every morning, including (1) positioning navigation experiment: the mice were gently put into the pool with their heads facing the pool wall in the order of quadrants 1 to 4, and the time required for the rats to find this platform from each water entry point within 60s was recorded, i.e., to escape incubation period. The averages of the escape incubation period in 4 quadrants of rats were recorded on the 1st, 2nd, 3rd, and 4th days, respectively. (2) Spatial probe test for rats: platform was removed on the morning of the 5th day, and the mice were put into the pool from the 3rd quadrant. The residence time of the platform quadrant and number of times of crossing platform quadrant were recorded within 60 s.


### 2.4. Open Field Test

After anesthesia, open field tests were conducted at the same time every afternoon. After testing one mouse each time, thoroughly clean the open field box to avoid interference. The rats were tested continuously for 3 days, and the residence time in the central region of the rats was recorded on the 1st, 2nd, and 3rd days, respectively.


### 2.5. Determination of IL-1, IL-6, and TNF-α Concentrations in the Hippocampus

After the behavioral test, that is, the rats were anesthetized with ether immediately on the 5th day after anesthesia, the heads were decapitated and craniotomy was carried out quickly, the hippocampus tissue was completely taken out, weighed, and homogenized at 4°C, and the supernatant was centrifuged. IL-1, IL-6, and TNF-α indexes in the hippocampus were determined by ELISA kits (Beyotime, China).


### 2.6. Detect the Expression of P-CREB and c-fos in the Relevant Brain Regions by Western Blot

After the behavioral test, that is, the rats were anesthetized with ether immediately on the 5th day after anesthesia, and the craniotomy was carried out quickly. The tissues of relevant brain regions of the rats, including hippocampus, prefrontal cortex, and nucleus accumbens, were completely removed. 100 mg of brain tissue was weighed, and nucleoprotein was extracted by nucleoprotein and cytoplasmic protein extraction kit. 50μg of that nucleoprotein was sample and subjected to polyacrylamide gel protein electrophoresis. The proteins were transferred onto a PVDF membrane (Millipore, USA) and incubated at room temperature for 1 h in a blocking solution made of 5 g skim milk powder, 0.2 ml 0.05% Tween 20, and phosphate buffer solution buffer. The primary antibodies against p-CREB and c-fos (Abcam, USA) and the secondary antibody labeled with horseradish peroxidase (ProteinTech, China) were added at a ratio of 1 : 1000. The protein expression was detected by the chemiluminescence method, and β-actin was used as internal reference by secondary hybridization.


### 2.7. Statistical Analysis

SPSS 25.0 software was used for statistical analysis, and the measurement data were expressed by (x¯±s). Variance analysis with LSD t-test was used. P<0.05 was statistically significant.


## 2.1. Laboratory Animals and Groups

Forty-five healthy male aged SD rats, aged 19-20 months, weighed568.12±21.63g. Rats were separated into the control group (n=15), model group (n=15), and atipamezole group (n=15).


## 2.2. Preparation of the Animal Model

The rats in the model group were anesthetized by intraperitoneal injection of 75 mg/kg ketamine (Fujian Gutian, China) plus 5 mg/kg midazolam (Jiangsu Hengrui, Chian). After satisfactory anesthesia, the rats were fixed in the conventional supine position, the abdominal skin was disinfected, and a longitudinal incision of about 3 cm was made along the abdominal midline 0.5 cm below the rib. Abdominal exploration was carried out every 5 min, and the liver, stomach, small intestine, and large intestine were explored in turn. The total operation time was 20 min. After the exploration, the abdominal cavity was closed. The rats in the atipamezole group were given intraperitoneal injection of 0.5 mg/kg atipamezole, and anesthesia and exploratory laparotomy were performed 60 min later. Method was the same as that of the model group. The control group was not given any treatment.


## 2.3. Morris Water Maze (MWM)

After anesthesia, MWM was carried out at the same time every morning, including (1) positioning navigation experiment: the mice were gently put into the pool with their heads facing the pool wall in the order of quadrants 1 to 4, and the time required for the rats to find this platform from each water entry point within 60s was recorded, i.e., to escape incubation period. The averages of the escape incubation period in 4 quadrants of rats were recorded on the 1st, 2nd, 3rd, and 4th days, respectively. (2) Spatial probe test for rats: platform was removed on the morning of the 5th day, and the mice were put into the pool from the 3rd quadrant. The residence time of the platform quadrant and number of times of crossing platform quadrant were recorded within 60 s.


## 2.4. Open Field Test

After anesthesia, open field tests were conducted at the same time every afternoon. After testing one mouse each time, thoroughly clean the open field box to avoid interference. The rats were tested continuously for 3 days, and the residence time in the central region of the rats was recorded on the 1st, 2nd, and 3rd days, respectively.


## 2.5. Determination of IL-1, IL-6, and TNF-α Concentrations in the Hippocampus

After the behavioral test, that is, the rats were anesthetized with ether immediately on the 5th day after anesthesia, the heads were decapitated and craniotomy was carried out quickly, the hippocampus tissue was completely taken out, weighed, and homogenized at 4°C, and the supernatant was centrifuged. IL-1, IL-6, and TNF-α indexes in the hippocampus were determined by ELISA kits (Beyotime, China).


## 2.6. Detect the Expression of P-CREB and c-fos in the Relevant Brain Regions by Western Blot

After the behavioral test, that is, the rats were anesthetized with ether immediately on the 5th day after anesthesia, and the craniotomy was carried out quickly. The tissues of relevant brain regions of the rats, including hippocampus, prefrontal cortex, and nucleus accumbens, were completely removed. 100 mg of brain tissue was weighed, and nucleoprotein was extracted by nucleoprotein and cytoplasmic protein extraction kit. 50μg of that nucleoprotein was sample and subjected to polyacrylamide gel protein electrophoresis. The proteins were transferred onto a PVDF membrane (Millipore, USA) and incubated at room temperature for 1 h in a blocking solution made of 5 g skim milk powder, 0.2 ml 0.05% Tween 20, and phosphate buffer solution buffer. The primary antibodies against p-CREB and c-fos (Abcam, USA) and the secondary antibody labeled with horseradish peroxidase (ProteinTech, China) were added at a ratio of 1 : 1000. The protein expression was detected by the chemiluminescence method, and β-actin was used as internal reference by secondary hybridization.


## 2.7. Statistical Analysis

SPSS 25.0 software was used for statistical analysis, and the measurement data were expressed by (x¯±s). Variance analysis with LSD t-test was used. P<0.05 was statistically significant.


## 3. Results

### 3.1. The Escape Incubation Period between Different Groups

The escape incubation period of the model group was prolonged on the 2nd, 3rd, and 4th days (P<0.05), the time spent in the platform quadrant was shortened on the 5th day, and the number of crossing the platform quadrant was decreased (P<0.05). Compared with the model group, the escape incubation period on the 2nd, 3rd, and 4th days in the atipamazole group was shortened (P<0.05), the stay time in the platform quadrant on the 5th day was significantly prolonged, and the number of times of crossing the platform was significantly prolonged. Quadrants were increased (P<0.05, Table 1).Table 1
Comparison of MWM results.
GroupnEscape incubation period (s)Residence time in platform quadrant (s)Number of times of crossing platform quadrant (frequency)On the 1st dayOn the 2nd dayOn the 3rd dayOn the 4th dayControl group1546.56±3.5134.57±2.8421.61±1.9314.58±1.0431.32±3.4714.25±1.31Model group1556.85±4.6945.08±4.22a24.15±2.84a19.43±1.55a17.82±2.63a6.14±0.92aAtipamezole group1549.23±3.0437.95±3.16b22.68±1.45b15.19±1.55b32.58±3.06b15.03±1.45bCompared with the control group,aP<0.05; compared with the model group, bP<0.05. The same below.


### 3.2. The Residence Time in Central Area

Residence time in central region of the model group was shortened on the 1st, 2nd, and 3rd days compared to the control group (P<0.05). The residence time of the atipamezole group was prolonged on the 1st, 2nd, and 3rd days (P<0.05) compared to the model group (Table 2), indicating that atipamezole increased residence time in central area.Table 2
Comparison of residence time in central area.
GroupnOn the 1st dayOn the 2nd dayOn the 3rd dayControl group1570.75±8.1872.94±9.3268.53 ± 10.16Model group1541.43±6.05a36.37±3.66a38.15±5.47aAtipamezole group1575.58±9.67b76.92±8.41b69.81±9.29b


### 3.3. The Levels of Serum Markers in the Hippocampus

Concentrations of IL-1, IL-6, and TNF-α in the hippocampus of the model group increased compared to the control group (P<0.05). In contrast, levels of IL-1, IL-6, and TNF-α in the hippocampus of the atipamezole group significantly decreased (P<0.05, Table 3). The results indicated atipamezole decreased levels of serum makers compared to the model group.Table 3
Comparison of serum markers in the hippocampus.
GroupnTNF-αIL-6IL-1Control group154.83±0.323.04±0.284.21±0.41Model group159.57±0.91a6.15±0.43a11.87±0.66aAtipamezole group155.15±0.26b4.78±0.55b5.53±0.37b


### 3.4. The Expressions of P-CREB and c-fos in the Hippocampus

As shown in Figure1, expression levels of p-CREB and c-fos in hippocampus of rats in model group versus control group decreased (P<0.05). However, the expression of p-CREB and c-fos in the atipamezole group significantly increased compared to the model group (P<0.05).Figure 1
Comparison of expression levels of p-CREB and c-fos proteins in the hippocampus of rats in each group (n=15).


### 3.5. The Expressions of P-CREB and c-fos in the Prefrontal Cortex

Similar to expression pattern observed in the hippocampus, the protein expression levels of p-CREB and c-fos in the prefrontal cortex in the model group decreased compared to the control group (P<0.05). The atipamezole increased p-CREB and c-fos expressions in the prefrontal cortex compared to the model group (P<0.05, Figure 2).Figure 2
Comparison of expression levels of p-CREB and c-fos proteins in the prefrontal cortex of rats in each group (n=15).


### 3.6. The Expressions of P-CREB and c-fos in Nucleus Accumbens

As shown in Figure3, protein expression levels of p-CREB and c-fos in nucleus accumbens of rats in the model group decreased significantly (P<0.05). The expression of p-CREB and c-fos in nucleus accumbens of the atipamezole group increased compared to the model group (P<0.05).Figure 3
Comparison of expression levels of p-CREB and c-fos proteins in nucleus accumbens of rats in each group (n=15).


## 3.1. The Escape Incubation Period between Different Groups

The escape incubation period of the model group was prolonged on the 2nd, 3rd, and 4th days (P<0.05), the time spent in the platform quadrant was shortened on the 5th day, and the number of crossing the platform quadrant was decreased (P<0.05). Compared with the model group, the escape incubation period on the 2nd, 3rd, and 4th days in the atipamazole group was shortened (P<0.05), the stay time in the platform quadrant on the 5th day was significantly prolonged, and the number of times of crossing the platform was significantly prolonged. Quadrants were increased (P<0.05, Table 1).Table 1
Comparison of MWM results.
GroupnEscape incubation period (s)Residence time in platform quadrant (s)Number of times of crossing platform quadrant (frequency)On the 1st dayOn the 2nd dayOn the 3rd dayOn the 4th dayControl group1546.56±3.5134.57±2.8421.61±1.9314.58±1.0431.32±3.4714.25±1.31Model group1556.85±4.6945.08±4.22a24.15±2.84a19.43±1.55a17.82±2.63a6.14±0.92aAtipamezole group1549.23±3.0437.95±3.16b22.68±1.45b15.19±1.55b32.58±3.06b15.03±1.45bCompared with the control group,aP<0.05; compared with the model group, bP<0.05. The same below.


## 3.2. The Residence Time in Central Area

Residence time in central region of the model group was shortened on the 1st, 2nd, and 3rd days compared to the control group (P<0.05). The residence time of the atipamezole group was prolonged on the 1st, 2nd, and 3rd days (P<0.05) compared to the model group (Table 2), indicating that atipamezole increased residence time in central area.Table 2
Comparison of residence time in central area.
GroupnOn the 1st dayOn the 2nd dayOn the 3rd dayControl group1570.75±8.1872.94±9.3268.53 ± 10.16Model group1541.43±6.05a36.37±3.66a38.15±5.47aAtipamezole group1575.58±9.67b76.92±8.41b69.81±9.29b


## 3.3. The Levels of Serum Markers in the Hippocampus

Concentrations of IL-1, IL-6, and TNF-α in the hippocampus of the model group increased compared to the control group (P<0.05). In contrast, levels of IL-1, IL-6, and TNF-α in the hippocampus of the atipamezole group significantly decreased (P<0.05, Table 3). The results indicated atipamezole decreased levels of serum makers compared to the model group.Table 3
Comparison of serum markers in the hippocampus.
GroupnTNF-αIL-6IL-1Control group154.83±0.323.04±0.284.21±0.41Model group159.57±0.91a6.15±0.43a11.87±0.66aAtipamezole group155.15±0.26b4.78±0.55b5.53±0.37b


## 3.4. The Expressions of P-CREB and c-fos in the Hippocampus

As shown in Figure1, expression levels of p-CREB and c-fos in hippocampus of rats in model group versus control group decreased (P<0.05). However, the expression of p-CREB and c-fos in the atipamezole group significantly increased compared to the model group (P<0.05).Figure 1
Comparison of expression levels of p-CREB and c-fos proteins in the hippocampus of rats in each group (n=15).


## 3.5. The Expressions of P-CREB and c-fos in the Prefrontal Cortex

Similar to expression pattern observed in the hippocampus, the protein expression levels of p-CREB and c-fos in the prefrontal cortex in the model group decreased compared to the control group (P<0.05). The atipamezole increased p-CREB and c-fos expressions in the prefrontal cortex compared to the model group (P<0.05, Figure 2).Figure 2
Comparison of expression levels of p-CREB and c-fos proteins in the prefrontal cortex of rats in each group (n=15).


## 3.6. The Expressions of P-CREB and c-fos in Nucleus Accumbens

As shown in Figure3, protein expression levels of p-CREB and c-fos in nucleus accumbens of rats in the model group decreased significantly (P<0.05). The expression of p-CREB and c-fos in nucleus accumbens of the atipamezole group increased compared to the model group (P<0.05).Figure 3
Comparison of expression levels of p-CREB and c-fos proteins in nucleus accumbens of rats in each group (n=15).


## 4. Discussion

HR, SpO2, blood gas, and other physiological indexes of rats were monitored and controlled during anesthesia to eliminate the interference of ischemia and hypoxia on the results in this study. During the whole anesthesia process, the skin color of rat nose, lip, and toe was kept ruddy. Morris water maze is a simple experimental method applied to the study of brain learning and memory mechanism. It is especially sensitive to the structural damage of hippocampal region of animals and can objectively reflect the cognitive level of experimental animals [13]. Open field experiment is a classical experimental model to evaluate animal behavior, which mainly reflects the autonomous behavior and inquiry behavior of animals in the new environment [14]. The results of Morris water maze and open field experiments in this study show that atipamezole can reduce the learning, exploration, and memory disorders caused by isoflurane anesthesia in elderly rats.Systemic inflammatory response and central nervous system inflammatory response may be the key factor leading to POCD [15]. Surgery can activate specific steady-state reactions and trigger cascade effects through various inflammatory mediators. Research shows that [16, 17] oxidative free radicals, IL-1, IL-6, TNF-α, and other cytokines directly penetrate blood-brain barrier through active transport mechanism or indirectly through vagus nerve stimulation, bind their own receptors in the central nervous system, and activate microglia and vascular endothelial cells. They can induce neuroinflammatory response and cause a series of effects on the central nervous system. Trauma, surgery, and anesthesia can stimulate the expression of IL-1, IL-6, and TNF-α. Results of this experiment also showed that the concentrations of IL-1, IL-6, and TNF-α produced in the hippocampus of rats in the atipamezole group were lower than those in the model group. It is suggested that atipamezole can inhibit the release of related proinflammatory factors, thus alleviating neurocognitive dysfunction related to neuroinflammatory response.CREB plays a role in promoting neuron regeneration, survival, and repair after stress injury of the central nervous system [18]. P-CREB binds to the cAMP response element sequence in the target gene promoter to induce upregulation of the immediate early gene expression. c-fos and c-jun are central events that produce a series of neurophysiological function-dependent changes. c-fos is the most common transcription factor in the Fos family used to study drug dependence in animals. Studies have found that [19, 20] general anesthetic drugs ketamine and propofol can affect the CREB protein expression in relevant brain regions of experimental animals. The results of this study showed that the protein expression levels of p-CREB and c-fos in the hippocampus, prefrontal cortex, and nucleus accumbens of atipamezole group rats increased versus model group. It is suggested that atipamezole pretreatment can significantly improve the expression of p-CREB and c-fos proteins in brain regions related to behavioral memory after general anesthesia in aged rats.


## 5. Conclusion

Atipamezole preconditioning can reduce cognitive dysfunction in aged rats after general anesthesia, and its mechanism may be related to inhibiting hippocampal inflammatory reaction and improving protein expression levels of p-CREB and c-fos in related brain regions of aged rats.



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