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.
Received May 20th, 2013; revised June 25th, 2013; accepted July 4th, 2013
Copyright © 2013 Sheng Peng et al. This is an open access article distributed under the Creative Commons Attribution License,
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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