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Research Report
Minocycline mitigates isoflurane-induced cognitive
impairment in aged rats
Shi-Yong Li
a
, Li-Xia Xia
b
, Yi-Lin Zhao
a
, Liu Yang
a
, Ye-Lin Chen
a
, Jin-Tao Wang
a
,
Ai-Lin Luo
a,
a
Department of Anesthesiology, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, 1095 JieFang
Avenue, Wuhan, Hubei 430030, China
b
Operating Room, Tongji Hospital of Tongji Medical College, Huazhong University of Science and Technology, 1095 JieFang Avenue, Wuhan,
Hubei 430030, China
article info
Article history:
Accepted 6 December 2012
Available online 25 December 2012
Keywords:
Minocycline
Isoflurane
Postoperative cognitive dysfunction
TNF-a
abstract
Postoperative cognitive dysfunction (POCD) is a severe neurological sequela that occurs in
individuals who have undergone anesthesia and surgery, especially in the geriatric surgical
population. Although it is known that isoflurane exposure impairs cognitive function in
aged rodents, there are few clinical interventions for the prophylaxis and treatment of this
disorder. Minocycline, a derivative of tetracycline, produces neuroprotection from several
neurodegenerative diseases. Therefore, we set out to investigate the effects of minocycline
pretreatment on isoflurane-induced cognitive impairment in aged rats. We found that
pretreatment with minocycline remarkably alleviated isoflurane-induced cognitive
dysfunction and inhibited the isoflurane-induced over expression of TNF-a, IL-1b, and
IL-6, possibly by inhibiting the degradation of IkBa. In addition, minocycline downregulated
the isoflurane-induced increase in the protein levels of cleaved caspase 3 and bax, and
upregulated the bcl-2 protein level. These findings highlight the beneficial role of
minocycline in preventing isoflurane-induced cognitive impairment and suggested that
minocycline can be used as a clinical treatment to mitigate the cognitive impairment
induced by isoflurane in elderly patients.
&2013 Elsevier B.V. All rights reserved.
1. Introduction
Postoperative cognitive dysfunction (POCD) is characterized by
a continuous deterioration of cognitive performance after
anesthesia and surgery, as evaluated by preoperative and post-
operative cognitive testing (Moller et al., 1998;Rasmussen,
1998). It is reported that at the time of discharge from the
hospital, 41.4% of elderly patients (60 years or older) were
subjected to POCD after non-cardiac surgery (Monk et al.,
2008;Steinmetz et al., 2009). Although perioperative morbidity
and mortality have been dramatically reduced over the past
decades, little progress has been made in alleviating the
prevalence of POCD, which imposes a serious burden on quality
of life, as well as on healthcare costs.
0006-8993/$ - see front matter &2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.brainres.2012.12.025
Abbreviations: AD, Alzheimer’s disease; BBB, blood brain barrier; IL, interleukin; IkB, inhibitor of nuclear factor of kappa light
polypeptide gene enhancer in B-cells; NF-kB, nuclear factor kappa-light-chain-enhancer of activated B-cells; POCD, postoperative
cognitive dysfunction; TNF, tumor necrosis factor
Corresponding author. Fax: þ86 27 83665480.
E-mail address: ailinluo@yahoo.cn (A.-L. Luo).
brain research 1496 (2013) 84–93
Although the etiology of POCD remains elusive, factors
such as advanced age and duration of anesthesia have been
considered as major risk factors (Moller et al., 1998;Monk
et al., 2008;Ramaiah and Lam, 2009). Some studies suggest
that there is no difference in the incidence of POCD attributed
to general anesthesia vs. regional anesthesia (Newman et al.,
2007). Nevertheless, exposure to general anesthetics remains
a cardinal cause of POCD (Lin et al., 2012;Mawhinney et al.,
2012;Su et al., 2011;Wan et al., 2007). The exact role of
general anesthetics in POCD is yet to be fully elucidated, but
extensive information gained over the past decade indicates
that the excessive release of proinflammatory cytokines,
including tumor necrosis factor (TNF)-a, interleukin (IL)-1b
and IL-6, is involved in cognitive impairment after surgery
and anesthesia (Cibelli et al., 2010;Lin et al., 2012;Lucas et al.,
2006;Terrando et al., 2010;Wan et al., 2010,2007;Wu et al.,
2012). The degradation of the inhibitory nuclear factor kappa
light polypeptide gene enhancer in B-cells (IkB)ais associated
with neuroinflammation including nuclear factor of kappa
light polypeptide gene enhancer in B-cells (NF-kB) activation
and proinflammatory cytokines production (Nikodemova
et al., 2006). Furthermore, overproduction of TNF-a, IL-band
IL-6 can impair neuronal cells function including impairment
of synaptic plasticity, apoptosis (Lynch and Lynch, 2002;
Shaftel et al., 2008;Sheng et al., 2005;Wei et al., 2011).
A burgeoning series of studies demonstrate that isoflurane-
induced hippocampal neuronal apoptosis is associated with
the hippocampus-dependent memory deficit (Lin and Zuo,
2011;Xie et al., 2006;Zhang et al., 2012). Our previous results
also demonstrated that isoflurane exposure induces neuronal
apoptosis (Zhao et al., 2011a,2011b) and increases the
secretion of proinflammatory cytokines in the hippocampus
of neonatal rats (Shi et al., 2010). Theoretically, drugs with
anti-neuroinflammatory and anti-neuroapoptotic properties
could be effective, at least in part, inpreventing the cognitive
dysfunction that occurs after general anesthesia.
Minocycline is a tetracycline derivative that easily crosses
the blood brain barrier (BBB) (Arvin et al., 2002;Yrjanheikki
et al., 1999). Accumulating evidence suggests that the neuro-
protective effect of minocycline is mainly caused by the
inhibition of inflammation and neuroapoptosis (Choi et al.,
2007;Lucas et al., 2006;Tikka et al., 2001). An increasing
number of studies have revealed that minocycline improves
cognitive function in neurodegenerative diseases such as
Alzheimer’s disease, Huntington’s disease and Parkinson’s
disease (Choi et al., 2007;Thomas et al., 2003;Tikka et al.,
2001). Hence, we hypothesize that pretreatment with mino-
cycline could alleviate the cognitive impairment that is
triggered by isoflurane in aged rats.
2. Results
2.1. Isoflurane anesthesia did not induce circulatory or
respiratory distress
To exclude the possibility that isoflurane depressed the
circulatory and respiratory systems, mean blood pressure
and heart rate were continuously monitored and were
recorded hourly. As shown in Fig. 1, isoflurane anesthesia
reduced mean blood pressure (Fig. 1A) and heart rate (Fig. 1B),
but the difference was not statistically significant (P¼0.45).
To determine if isoflurane anesthesia caused hypoxia, arterial
blood was drawn by cardiac puncture and arterial gas
analysis was performed immediately after the anesthesia
ended. As shown in Table 1, PaCO
2
,PaO
2
, pH, blood glucose
and arterial oxygen saturation (SaO
2
) did not change
significantly.
2.2. Minocycline pretreatment improved cognitive
function after isoflurane exposure
To determine the effect of minocycline on cognitive function
after isoflurane anesthesia, the Morris water maze (MWM)
was used to assess learning and memory. As shown in
Fig. 2A, both the repeated factor (training days) and the
over-group factor significantly affected the latency of the rats
to locate the platform. However, no interactive effect between
Fig. 1 – Isoflurane anesthesia had no significant effect on
mean blood pressure and heart rate. (A) Mean blood
pressure was relatively stable in both the isoflurane
untreated group and the isoflurane treated group. (B) Heart
rate did not change significantly in isoflurane untreated rats
or isoflurane treated rats. The isoflurane untreated group
included the CON and MINO groups, and the isoflurane
treated group comprised the ISO and MINOþISO groups.
The results are presented as the mean7SEM (n¼30).
brain research 1496 (2013) 84–93 85
group and training days was found (P¼0.68). On the third and
fourth training days, rats in the minocyclineþisofluranegroup
(MINOþISO) spent less time locating the platform (Day 3,
Po0.001; Day 4, P¼0.007, MINOþISO vs. ISO). There was no
significant difference in the latency between the control
(CON) and MINO groups (P¼0.72).
In the probe trial, the percentage time of rats in the
MINOþISO group spent in the target quadrant was much
greater than the rats in the ISO group (Po0.001) (Fig. 2B).
There was no significant difference between the MINOþISO
and CON groups (P¼0.55).
2.3. Minocycline reduced the isoflurane-induced
upregulation of inflammatory cytokine levels in the
hippocampus
The levels of TNF-a, IL-1band IL-6 detected at a series of time
points after anesthesia were respectively shown in Fig. 3A, B
and C. After isoflurane exposure the expression of TNF-a,
IL-1band IL-6 increased significantly immediately, peaked 3 h
after isoflurane exposure; and the level of IL-1b(Fig. 3B) and
IL-6 (Fig. 3C) persisted till 6 h after anesthesia, then fell to
baseline levels 12 h after anesthesia; but the increase of
TNF-a(Fig. 3A) lasted to 12 h and fell to baseline 24 h after
anesthesia. When rats were pretreated with minocycline, the
elevated levels of TNF-a, IL-1band IL-6 in the hippocampus
were reversed. Sole minocycline treatment did not change
the expression of TNF-a, IL-1band IL-6 at all detected time
points. To investigate the mechanism involved in the
suppression of proinflammatory cytokines by minocycline,
the level of IkBaprotein was detected by western blot. As
shown in Fig. 3D, isoflurane markedly decreased the expres-
sion of IkBafrom the end of anesthesia to 6 h after anesthesia
(0 h: Po0.001; 3 h: Po0.001; 6 h: P¼0.002), which was reversed
by treatment with MINO. Sole intraperitoneal injection of
minocycline did not affect the protein levels of IkBaat any
time point after anesthesia.
2.4. Minocycline decreased the levels of TNF-amRNA and
protein in vitro
To determine whether minocycline could inhibit the secretion
of neuronal TNF-athat was induced by isoflurane, we assayed
both the mRNA and protein levels of TNF-ain hippocampal
neurons in vitro. In line with previous studies, isoflurane
increased TNF-aexpression at both the mRNA and protein
levels (Po0.05). Pretreatment with minocycline 30 min before
isoflurane exposure resulted in the downregulation of TNF-a
mRNA (Fig. 4A) and TNF-aprotein (Fig. 4B) (Po0.05, MINOþISO
vs. ISO). To further explore the mechanisms involved in the
inhibition of TNF-aexpression, we determined the protein level
of IkBa.AsshowninFig. 4C,minocyclineincreasedthelevel
of IkBaprotein, which was reduced by isoflurane (Po0.001,
MINOþISO vs. ISO).
Table 1 – Effect of isoflurane exposure on physiological parameters of arterial blood gas analysis.
ABG CON ISO MINOþISO MINO
pH 7.3170.05 7.3270.06 7.3570.06 7.3670.04
PaCO
2
(mm Hg) 36.274.3 36.172.7 39.273.6 40.772.1
PaO
2
(mm Hg) 108714 106713 10179 10578
Glucose (mmol/l) 4.570.4 4.470.7 3.970.7 4.470.5
SaO
2
(%) 99719770.6 9870.9 9970.8
pH, PaCO
2
,PaO
2
, glucose and SaO
2
levels did not differ significantly among the four groups. The results are presented as
the mean7SEM (n¼5).
Fig. 2 – Minocycline pretreatment mitigated the isoflurane-
induced spatial memory impairment. (A) The effect of
minocycline pretreatment on average latency to reach the
platform in the spatial acquisition trials. (B) The effect of
minocycline pretreatment on the percentage of time spent
in the target quadrant. The results are presented as the
mean7SEM (n¼10).
Po0.05 MINOþISO vs. ISO;
#
Po0.05
ISO vs. CON.
brain research 1496 (2013) 84–9386
2.5. Minocycline decreased isoflurane-mediated neuronal
apoptosis in the hippocampi of aged rats
To evaluate the effect of minocycline on isoflurane-induced
neuroapoptosis in the hippocampus, we set out to examine
the activity of caspase 3 and the protein expression of cleaved
caspase 3. As shown in Fig. 5, isoflurane activated caspase 3
(Fig. 5A, B) (caspase 3 activity: Po0.001; cleaved caspase 3:
Po0.001). To further investigate the mechanisms involved in
the anti-neuroapoptotic properties of minocycline, we revealed
the expression of bax and bcl-2 by western blot. It was observed
that isoflurane upregulated the expression of bax (Fig. 5C)
(P¼0.006) and reduced the expression of bcl-2 (Fig. 5D)
(P¼0.004). However, pretreatment with minocycline inhibited
the activation of caspase 3 (caspase 3 activity: Po0.001; cleaved
caspase 3: Po0.001), decreased the expression of bax (Po0.001),
and increased the expression of bcl-2 (P¼0.027) as compared to
the ISO group.
3. Discussion
Although the neurobiological basis of POCD remains unclear, it
is has been determined that POCD is age-dependent by clinical
trials (Monk et al., 2008) and experimental models (Mawhinney
et al., 2012;Stratmann et al., 2009;Su et al., 2011;Zhang et al.,
2012). In addition, a recent meta-analysis revealed that general
anesthesia is a possible cause of POCD (Mason et al., 2010),
despite previous reports which suggest that the incidence of
POCD in general anesthesia compared to regional anesthesia
is not significantly different (Rasmussen et al., 2003;Williams-
Russo et al., 1995). As the global population ages, the number
of elderly patients who are subjected to anesthesia and
surgery is increasing. We therefore sought to investigate the
effect of minocycline on isoflurane-induced cognitive disorder.
In present study, we found that pretreatment with minocy-
cline alleviated cognitive impairment in aged rats exposed to
1.4% isoflurane for 6 h. The protective role of minocycline was
associated with significant suppression of the excessive
release of proinflammatory cytokines and a marked reduction
of neuroapoptosis in the hippocampus. No significant changes
of basic physiological parameters were observed in all groups,
which implying that the side effect of isoflurane on the
circulatory and respiratory systems did not significantly influ-
ence the results gained in present study, although heart rate
and mean blood pressure were slightly inhibited.
It has been demonstrated that hippocampal neuroinflam-
mation triggered by increases in TNF-aand IL-1bunderlies
Fig. 3 – The effect of minocycline on the expression of TNF-a, IL-1b, IL-6 and IjBain hippocampus. (A)–(C) respectively
showed that minocycline decreased the expression of TNF-a, IL-1band IL-6 after isoflurane exposure in aged rats. (D)
Quantification of western blot results showed that minocycline increased the expression of IjBaafter isoflurane exposure in
aged rats. The results are presented as the mean7SEM (n¼5).
Po0.05 MINOþISO vs. ISO;
#
Po0.05 ISO vs. CON.
brain research 1496 (2013) 84–93 87
cognitive deficits, while the blockade of TNF-aand IL-1b
improves cognitive function (Bluthe et al., 1995;Cibelli
et al., 2010;Kent et al., 1992;Terrando et al., 2010). Consistent
with previous studies (Vizcaychipi et al., 2011;Wu et al.,
2012), we found that isoflurane elevated the levels of TNF-a,
IL-1band IL-6 in vivo and resulted in the induction of
neuroinflammatory processes. However, an elaborate
research demonstrates isoflurane does not induce increase
of IL-1band IL-6 in the hippocampi of nearly three-month-old
mice (Cibelli et al., 2010). Several other studies using different
protocols of anesthesia also display that anesthesia does not
change the level of proinflammatory cytokines (Rosczyk
et al., 2008;Wan et al., 2007). To our knowledge, the different
age of used animal might be the principal reason as it is
reported that increasing age is an independent risk factor of
POCD (Monk et al., 2008). Furthermore, the isoflurane con-
centration and anesthesia duration should be taken into
account.
Previously, surgery-induced secretion of proinflammatory
cytokines were glial-derived (Rosczyk et al., 2008;Terrando
et al., 2010;Wan et al., 2007), but in our results showed
isoflurane-modulated TNF-aincrease derived from neuron.
Two other studies support the result that production of TNF-a
induced by neurotoxins is from neurons (Takahashi et al.,
2008;Wu et al., 2012). This inconsistency is probably due to
that surgery-stimulated cytokines originated from peripheral
immune system and then activate the neuroinflammatory
response in hippocampus (Terrando et al., 2010), while
isoflurane can readily spread to the brain and directly target
the neurons. It seems that the neuroflammatory response is
activated by sole anesthesia or surgery in different pathways.
Of note, proinflammatory cytokine likely have bidirec-
tional effect on brain functions in different contexts (Liu
et al., 2011;Pickering et al., 2005;Wan et al., 2007;Wei et al.,
2011). However, it is confirmed that increased IL-1bin
hippocampus is contributed to age-related impairment in
long-term potentiation and age-related neuronal apoptosis
(Lynch and Lynch, 2002). Although the relationship between
isoflurane-induced neuroinflammation and cognitive disor-
der is undetermined, studies reveal that proinflammatory
cytokines are involved in the pathogenesis of neurodegen-
erative diseases such as AD (Akiyama et al., 2000;
Eikelenboom et al., 2002;Rozemuller et al., 2012), which is
exacerbated after isoflurane anesthesia (Wei and Xie, 2009;
Xie et al., 2007). Taken together, the isoflurane-induced
upregulation of TNF-a, IL-1band IL-6 and the resulting
inflammatory response may interrupt cognitive function in
aged rats.
A recent study verifies that the mitochondrial pathway of
neuroapoptosis was responsible for isoflurane-induced cog-
nitive dysfunction in aged mice (Zhang et al., 2012). A number
of studies revealed that clinically relevant concentration of
isoflurane activates mitochondrial apoptotic pathway and
finally increases the levels of cleaved caspase 3 (the active
form of caspase) in neurons or in neuroglial lines transfected
with amyloid precursor protein (Xie et al., 2006,2007;Zhang
Fig. 4 – The effect of minocycline on TNF-aproduction in hippocampal neurons. (A) Pretreatment with minocycline led to a
decline in TNF-amRNA levels that were upregulated by isoflurane exposure. (B) Pretreatment with minocycline led to a
decrease in the levels of TNF-aprotein that were upregulated by isoflurane exposure. (C) Pretreatment with minocycline
increased the level of IjBaprotein that was reduced by isoflurane exposure. The results are presented as the mean7SEM
(n¼5).
Po0.05 MINOþISO vs. ISO;
#
Po0.05 ISO vs. CON.
brain research 1496 (2013) 84–9388
et al., 2012). In mitochondrial apoptotic pathway, the pro-
apoptotic and anti-apoptotic members of Bcl family initiate
the dysfunction of mitochondrial membrane (Gross et al.,
1999). In present study, we found that isoflurane activated
caspase 3, upregulated bax and downregulated bcl-2.
However, neuroleptic general anesthesia (fentanyl–droperi-
dol) decreases the ratio of bcl-2: bax while does not activate
caspase 3 in adult rats (Wan et al., 2007). This difference
may be owing to using different aging rats and different
anesthetics.
It is noteworthy that proinflammatory cytokines can induce
neuronal apoptosis. It is reported TNF-amodulates caspase
3-dependent and caspase 3-independent apoptosis in neural
cells (Alvarez et al., 2011;Huang et al., 2005). In the process of
normal aging, increased level of IL-1band IL-1 receptor is
accompanied by elevated activity of caspase 3 in hippocampus
(Lynch and Lynch, 2002). Coupled with the results that isoflurane
can activate caspase 3, the question comes out whether neu-
roinflammation and neuroapoptosis are two coinstantaneously
distinguished mechanisms or the former is initial while the
latter is secondary. This is also the caveat of the present study.
Further work need to be done to establish the relevance between
isoflurane-induced neuroinflammation and neuroapoptosis.
Suppressing the inflammatory response by minocycline
inhibits glial activation and therefore improves cognitive
function (Fan et al., 2007;Krady et al., 2005;Seabrook et al.,
2006;Yrjanheikki et al., 1999). As aforementioned, our study
and other study demonstrate that isoflurane-induced upre-
gulation of TNF-ais neuron-derived. Thus, we investigate
whether minocycline can inhibit isoflurane-induced increase
of proinflammatory cytokines originating from neurons.
Interestingly, we found that pretreatment with minocycline
inhibited the neuronal secretion of TNF-ain vivo and in vitro.
The mechanism of minocycline on the regulation of
TNF-aproduction seems to be associated with inhibiting the
degradation of IkBa. The degradation of IkBais pivotal for the
release of NF-kB. Of interest, minocycline exerts its inhibitory
role on NF-kB transcriptional activity by attenuating the degra-
dation of IkBain microglia (Nikodemova et al., 2006). In the
present study, we showed that minocycline restored the
isoflurane-induced downregulation of IkBain aged rats. Similar
changes were observed in cultured neurons.
Minocycline suppresses the mitochondrial apoptotic path-
way by inhibiting the loss of the mitochondrial membrane
potential, thus reducing the cytoplasmic level of cytochrome
C in animal models of Huntington’s disease (Wang et al.,
Fig. 5 – Minocycline alleviated neuroapoptosis in the hippocampus after isoflurane anesthesia. (A) The activity of caspase 3
was suppressed by minocycline pretreatment. The relative levels of cleaved caspase 3 (B), bax (C) and bcl-2 (D) in hippocampi
of aged rats, normalized to the internal reference b-actin. The upper immunoblots are representative of cleaved caspase 3,
bax, and bcl-2. The results are presented as the mean7SEM (n¼10).
Po0.05 MINOþISO vs. ISO;
#
Po0.05 ISO vs. CON. clvd
casp3 is an abbreviation of cleaved caspase 3.
brain research 1496 (2013) 84–93 89
2003). It is noted that minocycline induces bcl-2 accumula-
tion in neuronal mitochondria, in which bcl-2 antagonizes
pro-apoptotic molecules such as bax (Wang et al., 2003; Wang
et al., 2004; Zhang et al., 2012). According to these results, it is
indicated that the inhibition of hippocampal neuroapoptosis
may be another mechanism involved in the neuroprotective
properties of minocycline against isoflurane insult in
aged rats.
In conclusion, minocycline alleviated the impairment in
cognitive function that was caused by clinical concentrations
of isoflurane in aged rats. Minocycline was involved in the
suppression of the neuroinflammatory response and apopto-
sis in the hippocampus. Considering the inexpensive cost,
the good safety record and the high BBB permeability,
minocycline is a potential candidate for the clinical prophy-
laxis and treatment of POCD.
4. Experimental procedures
4.1. Animals and drug administration
All animal experiments were approved by the Ethical Com-
mittee on Animal Experimentation of Tongji Medical College,
Huazhong University of Science and Technology, China.
The present study used 20-month-old male Sprague Dawley
rats, weighing 350–400 g, which were purchased from the
Center of Experimental Animal (Tongji Medical College) and
raised under standard laboratory conditions (room tempera-
ture: 22721C, relative humidity: 6075%,12 h light/dark cycle).
Standard rodent food and water were available ad libitum.
Rats were randomly assigned into four groups: control
(CON), isoflurane (ISO), minocyclineþisoflurane (MINOþISO)
and minocycline (MINO) (n¼40 rats per group). Rats in the
MINO and MINOþISO groups received a dose of 50 mg/kg
minocycline (Sigma, St. Louis, MO) by intraperitoneal injec-
tion 12 h before exposure to isoflurane anesthesia. Rats in the
CON and ISO groups were intraperitoneally injected with an
identical volume of saline. Rats in the CON and MINO groups
were exposed to vehicle gas (30% oxygen and 70% nitrogen)
for 6 h, and rats in the ISO and MINOþISO groups were
exposed to 1.4% isoflurane for 6 h.
Anesthetic exposure was performed in anesthesia-
induction chambers kept in a homoeothermic incubator to
maintain the environmental temperature at 37 1C Rats were
exposed to 1.4% isoflurane for 6 h through a calibrated
isoflurane vaporizer, using 30% oxygen and 70% nitrogen as
the vehicle gas, or just to the vehicle gas at the same flow rate
(Su et al., 2011). Two liters of total gas flow were used to
ascertain a steady state of anesthetic gas and prevent the
accumulation of expired carbon dioxide in the chamber.
An infrared probe (OhmedaS/5 Compact, Datex-Ohmeda,
Louisville, CO) was adopted to continuously monitor the
concentrations of oxygen, carbon dioxide and isoflurane in
the exhalant gas. Rats were visually inspected for respiratory
effort and skin color.
At the end of anesthesia, five rats in each group were
randomized to dynamically assess the expression of TNF-a,
IL-6 and IL-1b(0 h, 3 h, 6 h, 12 h and 24 h after isoflurane
exposure). Arterial gas analysis was performed in the rats
used to detect proinflammtory cytokines 0 h after anesthesia.
Half of the hippocampus was used for the detection of TNF-a,
IL-6 and IL-1bby ELISA and the other half was used for
extraction of total protein to detect the expression of IkBa.
Two weeks after isoflurane exposure, rats underwent
MWM testing to examine their spatial memory abilities. After
MWM testing, the rats were decapitated and the hippocampi
were dissociated for evaluation of caspase 3 activity and the
protein levels of cleaved caspase 3, bax, and bcl-2.
4.2. Primary neuron culture and drug administration
Pregnant rats were purchased from the Center of Experimen-
tal Animal of Tongji Medical College. Rats with a gestation
stage of Day 18 were killed with carbon dioxide and a
Cesarean section was performed. The hippocampi were
dissected from the embryonic brains for neuron culture.
Primary culture of the hippocampal neurons was performed
as described in our previous studies (Xiang et al., 2009;Zhao
et al., 2011a,2011b). Ten days after the harvest, the neurons
were divided into four groups: CON, ISO, MINOþISO and
MINO. Each group contained ten dishes, five for mRNA
extraction and five for the detection of IkBaprotein.
The minocycline pretreatment protocol was described in
previous studies (Choi et al., 2007;Gonzalez et al., 2007).
Briefly, minocycline (final concentration of 10 mM) was added
to neurons in the MINOþISO and MINO groups 30 min prior
to isoflurane or vehicle gas exposure. The TNF-aprotein level
in the media was assessed by a TNF-aELISA kit (BioSource
International Inc., Camarillo, CA).
4.3. Arterial gas analysis and hemodynamic monitoring
Mean blood pressure and heart rate were dynamically mon-
itored during anesthesia using a noninvasive blood pressure
meter (BP-98 A, Softron, Beijing, China). Five rats in each
group were used for arterial gas analysis with an
ABL-800FLEX analyzer (Radiometer, Denmark). At the end of
anesthesia, arterial blood was drawn by cardiac puncture for
arterial blood gas analysis (Jevtovic-Todorovic et al., 2003).
4.4. Morris water maze test
The MWM trials were performed as previously described
(D’Hooge and De Deyn, 2001;Vorhees and Williams, 2006).
The apparatus, consisting of a circular pool (120 cm diameter
and 50 cm high) containing a hidden platform in a dimly lit
room, was employed to train and test the learning and memory
of rats. The rats were trained to identify the location of the
hidden platform using only distal extra-maze cues attached to
the walls of the room. The pool was divided into four equal
quadrants and was filled with a water and carbonic ink mixture
to a height of 1.5 cm above the top of the black 15-cm-diameter
platform. The pool was kept at 20 1C. A video camera was
mounted above the pool to track the rats. The experiment was
recorded and analyzed using a camera connected to a video
recorder and the EthoVision tracking system (Noldus Informa-
tion Technology, Wageningen, the Netherlands).
On the 15th day following isoflurane exposure, the MWM
training and testing began. Testing lasted for five days.
brain research 1496 (2013) 84–9390
The spatial acquisition trial was performed on the first four
days, beginning at 9:00 A.M. every day. At the beginning of
each trial, the rat was placed into the water, facing the wall of
the pool, into one of the three quadrants that did not contain
the platform.
Each rat was given 60 s to search and mount the platform,
and could remain on the platform for 15 s after it was located.
Then, the rat was sent back to its home cages, wiped dry with
a towel, and warmed with a heating lamp. Rats that failed to
find the platform in 60 s were manually guided to the plat-
form and remained on the platform for 15 s before being
returned to cages. The time spent on searching and mounting
the platform (latency) was calculated.
A probe trial, in which the platform was removed, was
given to assess reference memory on the fifth day. Rats were
randomly placed into a quadrant that did not contain
the platform and were allowed to swim freely for 60 s.
The percentage of time spent in the target quadrant was
considered an indicator of memory performance.
4.5. Western blot analysis
The dissected rat hippocampi were homogenized in RIPA
buffer (150 mM sodium chloride, Triton X-100, 0.5% sodium
deoxycholate, 0.1% sodium dodecyl sulfate,50 mM Tris, pH
8.0) containing protease inhibitors (1 mg/ml antipine, 5 mg/
ml pepstatin, 1 mg/ml leupeptin, 1 mg/ml aprotinin) (Beyo-
time Institute Biotechnology, Haimen, China) and phospha-
tase inhibitors (1 mM NaF, 0.4 mM Na
3
VO
4
, 0.5 mM okadaic
acid). After centrifugation at 12,000 g/min for 10 min, the
supernatant was harvested. The protein samples were dena-
tured with 5 loading buffer in boiling water for 5 min. The
samples (75 mg) were separated on 12% sodium dodecyl
sulfate polyacrylamide gels using electrophoresis and were
transferred to 0.22 mm nitrocellulose membranes (Millipore,
Billerica, MA). The membranes were blocked with 5% milk in
Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h at
room temperature and then incubated at 4 1C with the
appropriate primary antibody. The primary antibodies were:
rabbit anti-cleaved caspase 3 (1:1000, Cell Signaling Technol-
ogy, Beverly, MA), rabbit anti-IkBa(1:1000, Cell Signaling
Technology), mouse anti-bcl-2, anti-bax (1:1000, BD
bioscience, MA) or mouse anti-bactin (1:5000, Sigma, St.
Louis, MO). After incubation in the primary antibodies, the
membranes were rinsed and then incubated for 2 h at room
temperature with horseradish peroxidase (HRP)-conjugated
secondary antibody (anti-mouse or anti-rabbit (1:7500,
Abcam, Cambridge, MA)). Following the species-appropriate
HRP-conjugated secondary incubation, detection was per-
formed using SuperSignal
s
West Pico (ThermoScientific,
Rockford, IL) and the blots were photographed using Image
Lab
TM
Software Systems (BIO-RAD, Hercules, CA). Software
Image Lab 3.0 (BIO-RAD) was used to analyze the relative
intensity of the bands.
4.6. Assay of caspase 3 activity
Caspase 3 activity was assayed by the Caspase 3 Colorimetric
Assay Kit (R&D Systems
s
, Minneapolis, MN). The procedure
was performed following the supplier’s instructions.
4.7. Quantification of TNF-a, IL-6 and IL-1bwith enzyme-
linked immunosorbent assay (ELISA)
The protein levels of TNF-a, IL-6 and IL-1bin hippocampal
tissues and the protein level of TNF-ain the media of
cultured hippocampal neurons were determined by commer-
cially available ELISA kits (BioSource International Inc,
Camarillo, CA) following the protocols provided by manufac-
turer. All samples were assayed in duplicate. The readings
were normalized to the amount of standard protein.
4.8. RNA isolation and quantitative real-time polymerase
chain reaction
Total RNA extraction of hippocampal neurons and the reverse
transcription procedure were performed as previously
described with minor modifications (Zhao et al., 2011a,
2011b). In brief, the total RNA of neurons was extracted with
TRIzol
s
(Invitrogen, Carlsbad, CA) according to the manufac-
turer’s instructions. One microgram of total RNA was reverse
transcribed using random primers and Superscript II Reverse
Transcriptase (Takara, Shiga, Japan) in a 20 ml reaction sys-
tem. Specific primers for TNF-a(forward:50-AACTGGCAGAG-
GAGGCG-30; and reverse: 50-CAGAAGAGCGT GGTGGC-30), and
for the endogenous control GAPDH (forward: 50-GGCACAGT-
CAAGGCTGAGAATG-30; and reverse: 50-ATGGTGGTGAA-
GACGCCAGTA-30) were designed and synthesized by Takara
(Takara BioTechnology (Dalian), China). ABI Stepone version
2.0 (Applied Biosystems, Foster City, CA) was used to conduct
the qRT-PCR using the Power SYBR
TM
Green PCRMaster Mix
(Takara). Each sample was run in triplicate. Five independent
polymerase chain reaction amplification experiments were
performed for each sample. Data were analyzed using
Sequence Detection Software version 3.0 (Applied Biosys-
tems). Relative quantification was performed by means of
the 2
DDCt
method (Livak and Schmittgen, 2001;Schmittgen
and Livak, 2008). Data are expressed as fold changes normal-
ized to control groups.
4.9. Statistical analysis
All data were presented and graphed as the mean7SEM.
The Statistical Package for the Social Sciences 16.0 software
was used for the statistical analyses. Data acquired from the
detection of protein and mRNA were analyzed with an
analysis of variance (ANOVA), followed by a least square
difference (LSD) multiple comparison test. Data collected
from the spatial acquisition trials were analyzed using a
repeated measures ANOVA (the different treatments were
the between groups factors and time was the repeated
measures factor), followed by a post-hoc test to compare four
groups. Differences were deemed statistically significant if
Po0.05.
Acknowledgments
The present work was supported by a grant from the National
Natural Science Foundation of China (Nos. 30772086,
30901390, 81271233, and 81200880).
brain research 1496 (2013) 84–93 91
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