Ro 20-1724 Ameliorates Learning Deficit and Long-Term Memory Impairment Secondary to Repeated Ketamine Anesthesia in Young Rats

doi:10.4236/nm.2013.43025

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Neuroscience & Medicine, 2013, 4, 155-160

http://dx.doi.org/10.4236/nm.2013.43025 Published Online September 2013 (http://www.scirp.org/journal/nm)

155

Ro 20-1724 Ameliorates Learning Deficit and Long-Term

Memory Impairment Secondary to Repeated Ketamine

Anesthesia in Young Rats

Sheng Peng1, Hai Yan Sun2, Gong-Jian Liu3, Xia Yang4, George Mychaskiw II5*

1Department of Anesthesiology, Soochow University, Wuxi, China; 2Department of Anesthesiology, Traditional Chinese Medicine

Hospital of Zhang Jiang, China; 3Department of Anesthesiology, Xuzhou Medical College, Xuzhou, China; 4Department of Anesthe-

siology, Tengzhou Central People’s Hospital, Tengzhou, China; 5Department of Anesthesiology, Nemours Children’s Hospital, Or-

lando, USA.

Email: *[email protected]

Received May 20th, 2013; revised June 25th, 2013; accepted July 4th, 2013

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

To investigate effects and possible mechanism of Ro 20-1724, a PDE4 inhibitor, on long-time learning and memory

ability following repeated ketamine exposure in immature rats. Methods: Sixty 21-day-old SD rats were randomly di-

vided into five groups (n = 12): C: Normal control group, S: Saline control group, K: Ketamine, K + Ro: Ketamine +

Ro 20-1724, K + E: Ketamine + ethanol vehicle. Ro 20-1724 (0.5 mg·kg−1) or its vehicle (ethanol) was administered in-

traperitoneally 30 minutes after ketamine anesthesia (70 mg·kg−1), daily for seven days. Nine weeks after birth, the

Morris water maze was used to test the ability of learning and spatial localization memory on the rats. Following be-

havioral testing, animals’ hippocampi were removed for Western blot and electron microscopic examination. Results:

In the Morris water maze test, compared with controls, the escape latency in groups exposed to ketamine or ketamine

plus the ethanol vehicle were significantly prolonged (P < 0. 05), the ability of spatial localization was lower (P < 0.05),

and the expression of p-CREB in the hippocampus was also decreased (P < 0.05), while there was no significant differ-

ence between control groups and animals treated with Ro 20-1724 following ketamine exposure (P > 0.05). Electron

microscopy demonstrated degenerative changes in hippocampal neurons of animals repetitively exposed to 70 mg·kg−1

Ketamine, which was ameliorated by Ro 20-1724 (0.5 mg·kg−1). Conclusion: The PDE-4 inhibitor Ro 20-1724 (0.5

mg·kg−1) reverses cognitive deficits associated with repeated ketamine exposure in young rats, most likely via increased

cAMP/CREB signaling in the hippocampus and preservation of hippocampal neuronal ultrastructure.

Keywords: Research; Animal; Anesthesia; Neurodegeneration; Phsophodisterase Inhibitors; Memory

1. Introduction

Ketamine is an N-methyl-D-aspartate receptor (NMDA)

antagonist, widely used in anesthesia. Pediatric burn pa-

tients who require multiple escharotomy procedures and

dressing changes frequently undergo numerous and re-

peated ketmaine anesthetics as part of their medical care.

Exposure of the developing brain to NMDA receptor

antagonists, including ketamine, is associated with wide-

spread neurodegeneration [1]. Previous studies show that

ketamine, either following single or multiple exposures,

may lead to cognitive impairment, including learning and

memory ability in humans [1,2] and rodents [3-5]. Whe-

ther anesthetic exposure in childhood, particularly in hu-

mans, results in long-term learning disability and cogni-

tive dysfunction is a matter of widespread debate. Most

animal studies focus on recent cognitive function, with

there being fewer studies of the long-term effects of an-

esthetic exposure in the literature. Therefore, this rat mo-

del was designed to examine long-term learning and me-

mory behavior after repeated ketamine anesthesia in young

rats.

Ro 20-1724 is a specific phosphodiesterase-4 (PDE-4)

inhibitor. Recent studies show PDE-4 inhibitors can ame-

liorate cognitive dysfunction caused by sleep deprivation

[6], Aβ protein exposure, neuroinflammation [7], in-

tracerebroventricular streptozotocin injection [8], and

*Corresponding author.

Ro 20-1724 Ameliorates Learning Deficit and Long-Term Memory Impairment

Secondary to Repeated Ketamine Anesthesia in Young Rats

156

neural ischemia [9]. This study examines whether Ro

20-1724 decreases long-term cognitive dysfunction fol-

lowing multiple ketamine exposures.

2. Materials and Methods

2.1. Animals

This study was approved by the Animal Use and Care

Committee (AUCC) of Suzhou University. Sixty Spra-

gue-Dawley rats, aged 21 days and weighing 60.0 ± 5.0

grams, were provided by the Laboratory Animal Center of

Xuzhou Medical College. The housing and treatment of

the animals was in accordance with institutional guide-

lines and approved by the AUCC. The mice were ran-

domly divided into five groups of 12 individuals: a normal

control group (C group), a 0.9% saline exposure group (S

group), a ketamine exposure group (K group), a ketamine

+ Ro 20-1724 group (K + Ro group), and ketamine +

vehicle group (K + E group) (the Ro 20-1724 is supplied

in an ethanol vehicle). There were six individuals per

group. Group K was anesthetized with 70 mg/kg intrap-

eritoneal (ip) ketamine. Group K+R received 0.5 mg/kg

Ro 20-1724 (Sigma, USA) ip 30 minutes following the

ketamine. Group K+E received an equal volume of the

ethanol vehicle instead of Ro 20-1724. Group S received

saline only and group C received no treatment. The in-

jections were administrated once daily for seven con-

secutive days. Following the series of injections, all ani-

mals were housed and fed under stamdard conditions until

8 weeks of age. On the first day of the ninth week, the

Morris water maze was used to test cognitive ability,

including a four-day consecutive place navigation test to

evaluate learning function and a one-day spatial probe test

to evaluate memory function. After completion of be-

havioral testing, the animals were sacrificed, their brains

dissected and hippocampi rapidly removed for measure-

ment of expression of phosphorylated cyclic AMP re-

sponse element binding protein (p-CREB) by Western

blot and examination of neuronal ultrastructure by trans-

mission electron microscopy (TEM).

2.2. Morris Water Maze Testing

The Morris water maze (MWM) is a widely used tool to

assess hippocampal-dependent learning and memory [10].

Evaluation of learning and memory was performed using

the Morris water maze in a quiet, low-light environment at

the same time every afternoon. The MWM consists on a

black circular pool (120 cm diameter, 60 cm high) filled

with water (30 cm depth) at 25˚C ± 1˚C and virtually

divided into four equivalent quadrants. An escape plat-

form (15 cm diameter, 30 cm high) was submerged 1.5 cm

below the water surface and placed in the middle of one of

the quadrants equidistant from the sidewall and the center

of the pool.

1) Place navigation test: The Place navigation test was

performed as previously described elsewhere [11].

Briefly, the animal was put into the MWM and forced

to swim. When the animal finds the underwater plat-

form, it gets a short break; if it is unable to climb onto

the platform within 120 seconds, the animal is manu-

ally placed on the site and allowed to stay for 30 sec-

onds. In this study, the place navigation test was re-

peated over a period of four days. The decrease in la-

tency from being placed in the MWM to finding the

platform is a measurement of the animal’s learning

ability.

2) Spatial probe test: The spatial probe test was pre-

formed as previously described elsewhere [12]. Briefly,

once the animal has been trained to find the platform in

the MWM, the platform is removed. The animal then

tried to located the platform persistently based on

memory acquired during training. The frequency of

passing the platform’s previous locating (FPP) is re-

flective of the animal’s memory ability.

2.3. Content of p-CREB Protein in Hippocampus

by Western Blot

After the behavior test, all animals were anesthetized by ip

injection of chloral hydrate (0.4 ml/100 g), the thorax was

opened and heart exposed. A tube was them placed

through the left ventricle into the aorta and 200 ml of

saline was rapidly injected. 400 ml of freshly prepared 4%

paraformaldehyde in PBS buffer solution was then per-

fused at 4˚C.

The animals were then decapitated, the entire brain was

removed and hippocampus quickly separated. In six

animals from each group, a lysis buffer was added (4μl:1

mg) and the specimen was homogenized and centerfuged,

with supernatant collected. p-CREB protein content was

detected by bicinchonininc acid (BCA) assay. A 10%

sodium dodecyl sulfate-polyacrylamide gel electrophore-

sis (SDS-PAGE) was prepared according to the relative

molecular mass of the target protein. The measured sam-

ple size was 70 μg. After concentration and electropho-

retic separation process, the target protein was trans-

membrane separated for 53 minutes.

Western-blot (WB) hybridization: A 10 ml washing

buffer solution containing 3% fetal bovine serum (BSA)

was used to prepare the sample, gently shaking for 4 hours

at room temperature. The first antibody of the CREB

polyclonal (1:1000, Cell Signaling, USA) was added and

the hybridization reaction proceeded at 4˚C overnight.

The membrane was then washed three times, five minutes

per wash, and rat anti-rabbit IgG secondary antibodies

(1:1000, Beyotime Company, China) were added, gently

shaking for 4 hours at room temperature. The membrane

was again washed three times for 5 minutes per wash and

Ro 20-1724 Ameliorates Learning Deficit and Long-Term Memory Impairment

Secondary to Repeated Ketamine Anesthesia in Young Rats

157

3. Results

the reaction was observed 15 minutes after adding col-

orimetric solution. The resulting color bands were then

scanned and images saved. The expression of CREB

protein was determined by semi-quantitative analysis of

the gray scale ratio, using Image J analysis software.

3.1. Place Navigation Test

During the four-day process, escape latency gradually

shortened in all animals and each animal could find the

underwater platform in the end. The escape latency was

significantly prolonged in the ketamine group (K), com-

pared with either normal controls (C) or animals that

received only saline (S). In the animals treated with Ro

20-1724 (K + R) the escape latency was significantly

shorter than group K on the first day during the process

(P < 0.05), and the difference continued to increase on

days 2, 3 and 4 (P < 0.01). Both the saline and ketamine

plus ethanol vehicle (K + E) groups demonstrated no

effects, as there was no difference between either the C

and S groups or the K and K+E groups, respectively (P >

0.05) (Figure 1).

2.4. Electron Microscopy of Hippocampal

Neurons

In one animal from each group, after the brain was re-

moved, the hippocampus was double fixed with glu-

taraldehyde-osmium tetroxide, epoxy-embedded, and

cut into ultrathin sections. The hippocampal neurons

were then observed by transmission electron microscopy

(×10,000).

2.5. Statistical Analysis

SPSS16.0 Software for was used for statistical analysis.

Data are expressed as mean ± standard deviation. Single

factor analysis of variance (One-way ANOVA) was used

for intergroup comparison. The least significant differ-

ence (LSD) test was analyzed between any two groups.

A p value < 0.05 was considered to be statistically sig-

nificant.

3.2. Spatial Probe Test

The frequency of passing the platform (FPP) was sig-

nificantly reduced in the K group, compared with C

group (P < 0.05). Animals treated with Ro 20-1724

demonstrated significant improvement over the K group

(Figures 2(a) and (b)).

Figure 1. After seven consecutive days of anesthesia using ketamine on 21-day-old rats, the place navigation test was per-

formed within the Morris water maze at 9 weeks of age. Over the learning course of four days, the animals that received

ketamine anesthesia (K), demonstrated longer escape latency in learning the procedure compared with normal controls (P <

0.01) Animals treated with Ro 20-1724 (K + R), showed significantly shorter escape latency compared to the K group (P <

0.01 or P < 0.05). Both saline and the ethanol vehicle had no effect.

Ro 20-1724 Ameliorates Learning Deficit and Long-Term Memory Impairment

Secondary to Repeated Ketamine Anesthesia in Young Rats

158

(a) (b)

Figure 2. After four-days of training, all animals performed the spatial probe memory test. Frequency of passing the plat-

form (FPP) was recorded to measure memory ability. The results shows significant reduction in the K group, compared with

C group (P < 0.05); and after treatment by Ro 20-1724, FPP was significant reversed. There are no differences between

groups C and S or K and K + E, respectively.

3.3. Expression of p-CREB Protein in

Hippocampus

Expression of p-CREB protein determined by Western

blot was significantly reduced in the K group (P < 0.05)

and the decrease was reversed by Ro 20-1724 admini-

stration (P < 0.05). There were no significant differences

between C group and S groups, or between the K and K

+ E groups (P > 0.05) (Figure 3).

3.4. Electron Microscopy

The ultrastructure of hippocampal neurons in the CA1

area was observed by electron microscopy. In group C and

group S, large amounts of rough endoplasmic reticulum in

hippocampal neuronal cytoplasm can be clearly observed.

Ribosomes are well distributed, nuclear membrane integ‐

rity is preserved, and clear structure of neuritis can be seen.

In group K and group K + E, hippocampal neurons

demonstrated dramatic cell body swelling, the nuclear

membrane appeared fractured, there was a lower density

of ribosomes and degranulation of rough endoplasmic

reticulum. In group K + Ro, hippocampal neurons showed

only slight swelling, a slightly lower density of ribosomes

and the nuclear membrane basic structure was maintained

intact (Figure 4).

4. Discussion

Previous studies have shown that ketamine exposure in

the immature brain can lead to cognitive dysfunction [1-

5]. However, these studies are primarily concerned with

short-term function and long-term studies in children are

rare. Although much of the literature has focused on the

developing brain, even in the adult, chronic ketamine

Figure 3. Expression of p-CREB protein was significantly

reduced in the K group (P < 0.05) and the decrease was

reversed by Ro 20-1724 administration (P < 0.05).

Figure 4. 10,000× EM of neurons. Arrows indicate nuclear

membrane structure, which was maintained by administra-

tion of Ro 20-1724.

exposure (20 mg/kg/day × 14 daily injections) may lead

to permanent cognitive change [13]. In neonatal rhesus

monkeys, even one ketamine exposure for 24 hours, may

cause long-lasting cognitive deficits [14]. The potential

for ketamine anesthesia in childhood to affect cognitive

function in adolescence has not been well studied.

This study confirmed that repeated ketamine anesthe-

Ro 20-1724 Ameliorates Learning Deficit and Long-Term Memory Impairment

Secondary to Repeated Ketamine Anesthesia in Young Rats

159

sia led to long-term learning and memory dysfunction, as

measured by MWM testing. Escape latency represents

the average time from the animal being placed into the

pool to find the underwater platform. In the group ex-

posed to ketamine, escape latency was significantly pro-

longed, suggesting that the learning deficits resulting

from repeated ketamine anesthesia may last until adoles-

cence. However, in animals that received Ro 20-1724

thirty minutes following each ketamine exposure, the

increase in the escape latency was significantly rescued,

especially on day 4, when it was only about half that of

the K group and close to normal, suggesting that Ro

20-1724 could ameliorate the learning deficits secondary

to repeated ketamine exposure. These results parallel

measurement of memory in the place probe test.

It appears, therefore, that repeated ketamine exposure

in minor rats can result in learning and memory impair-

ment that persists into adolescence, but the molecular

events involved in this cognitive decline are poorly un-

derstood. Phosphodiesterase-4 (PDE4) is a specific in-

tracellular high-affinity cAMP hydrolytic enzyme. PDE4

and its inhibitor can affect physiological function by

regulating the intracellular cAMP levels. As a second

messenger of signal transduction pathways in learning

and memory [15-17], cAMP plays a pivotal role [18,19].

cAMP activates downstream protein kinase A (PKA) and

protein kinase C (PKC) subunits entry to the nucleus,

induces phosphorylation of specific serine stump site in

cAMP response element binding protein (CREB) and

activates CREB [20,21]. Phosphorylated CREB (p-

CREB) binds specifically to the cAMP response element

(CRE) which, with cAMP regulates target genes in De-

oxyribonucleic acid (DNA), starts downstream gene tran-

scription and generates proteins believed necessary for

learning and memory. PDE-4 can directly regulate the

level of intracellular cAMP levels by hydrolysis, de-

crease phosphorylation of CREB, reduce activation and

down-regulate relative gene expression that is controlled

by CREB. This leads to proteins that participate in learn-

ing and memory not being synthesized, resulting in long-

term learning and memory function impairment [22].

There are many members of the PDE-4 family. The most

frequently studied drug is rolipram, which is developed

as a potential treatment for depression and Parkinson’s

Disease. However, it was not introduced into clinical

practice due to adverse effects of nausea and vomiting

[23]. Therefore, we chose Ro 20-1724 as a research tool.

In our study, p-CREB expression in the hippocampus

was analyzed by Western blot. In the ketamine group,

p-CREB was significantly reduced after repeated expo-

sure, however, when treated by Ro 20-1724, not only did

behavior tasks of both learning and memory improve-

ment, but also p-CREB expression in hippocampus was

preserved. This suggests that Ro 20-1724 alleviates the

impairment of learning and memory function caused by

ketamine through enhancement of p-CREB expression in

the hippocampus. Our study also demonstrated preserva-

tion of neuronal ultrastructure in the hippocampus on

electron microscopy when animals exposed to ketamine

were treated with Ro 20-1724. Yu et al. found that Ro

20-1724 can not only reverse ketamine-induced cognitive

dysfunction, but can also shorten duration of anesthesia

[24].

In summary, this study shows Ro 20-1724 can reverse

the long-term learning and memory impairment caused

by repeated ketamine exposure, possibly through enhanc-

ed p-CREB expression in the hippocampus. Specific

PDE4 inhibitors, such as Ro 20-1724, may be promising

agents for clinical applications, including the treatment

of post-anesthesia cognitive dysfunction and further in-

vestigation is warranted.

5. Acknowledgements

This study was approved by the Animal Use and Care

Committee (AUCC) of Suzhou University. This work

was supported by The National Natural Science Founda-

tion of China (Grant: 81000469) and Scientific Founda-

tion from Health Office of Jiangsu province (H201070).

The authors have no financial conflicts of interest.

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