The phosphodiesterase-4 inhibitor Rolipram promotes cognitive function recovery in prenatal Escherichia coli infected offspring

Tao Zhu, Tianming Yuan, Huimin Yu, Weizhong Gu, Xi Chen & Peifang Jiang

To cite this article: Tao Zhu, Tianming Yuan, Huimin Yu, Weizhong Gu, Xi Chen & Peifang Jiang (2018): The phosphodiesterase-4 inhibitor Rolipram promotes cognitive function recovery in prenatal Escherichia coli infected offspring, The Journal of Maternal-Fetal & Neonatal Medicine, DOI: 10.1080/14767058.2018.1542682
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The phosphodiesterase-4 inhibitorroliprampromotescognitive function recoveryin prenatal escherichia coli infected offspring

Tao Zhua, Tianming Yuanb, Huimin Yub, Weizhong Guc, Xi Chenc,
Peifang Jiangb, d,*

aDepartment of Critical Care Medicine, Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, Hangzhou 310016, China
bDepartment of Neonatology, Children’s Hospital, Zhejiang University School of Medicine,
Hangzhou 310052, China
cCentral Laboratory, Children’s Hospital, Zhejiang University School of Medicine, Hangzhou 310052, China
dDepartments of Neurology, Children’s Hospital, Zhejiang University School of Medicine, Hangzhou 310052, China

*Correspondence:Peifang Jiang, M.D., Ph.D.Departments of Neurology, Children’sHospital, Zhejiang University School of Medicine,Hangzhou 310052, China. Tel: +86 571 86670900. Fax:
+8657186658672.E-mailaddress:[email protected] (P. Jiang).

Objective.Preterm infants are especially vulnerable to intrauterine infection-induced brain injury, which is closely relevant with cognitive deficits and cerebral palsy. Rolipram, a phosphodiesterase-4 inhibitor, can improve cognition in rodents. However, the underlying roles and mechanisms are not well investigated.
Methods.In thepresent study, we used intrauterineescherichia coli(E. coli) infectedmodel.
E. coli was inoculated into pregnant rats’ uterine cervix at embryonic day 15 (E15)whilethe control group wasgivennormal saline.Rolipram wasadministered by intraperitoneal(i.p.) injectiononce daily from postnatal day (P) 1 to 7.Morris water mazetest was used for cognitive behavior test.Hippocampalneural stem/precursor cells (NSPCs) proliferation and neuronal

differentiationwerestudied by immunofluorescentstaining. The expressions ofp-CREB, p-Akt, TrkB and BDNF were estimated by Western-blot analysis.
Results.The data showed that rolipram could ameliorate cognitive deficits and enhance NSPCs proliferation and neuronal differentiation in intrauterine infected offspring. Additionally, rolipram could significantly increase p-CREB/CREB, p-Akt/Akt, TrkB and BDNF levels.
Conclusions.These results suggested that rolipram might play a neuroprotective role to promote cognitive function recovery after intrauterine infection. And hippocampal NSPCs proliferation and neuronal differentiation might be enhanced via CREB/Akt/BDNF signal transduction.

Keywords:Intrauterine infection,cognitive deficit,rolipram,NSPCs proliferation, neuronal differentiation

Abbreviations:BrdU = 5-bromodeoxyuridine; CREB = cAMP response element-binding protein; DMSO = dimethyl sulfoxide; E. coli = escherichia coli; E = embryonic day; i.p. =
intraperitoneal;MWM = Morris water maze; NSPCs = neural stem/precursor cells; P = postnatal day; PBS = phosphate-buffered saline; PDEs = phosphodiesterases; PLC = phospholipase C; PTB = preterm birth; PVDF = polyvinylidenedifluoride

Increasing evidence supports the facts that intrauterine infection can cause preterm infants brain injury[1], which is associated with cognitive disability, mood and behavior disorders, physical dysfunction, cerebral palsy and even death [2, 3]. In most countries, many interventions have been introduced in clinical practice and shown to be effective, such as hypothermia for newborns with hypoxic ischemic encephalopathy (HIE) and Magnesium sulphate as neuroprophylaxisfor preterm infants. Nevertheless, owing to some potential limitations of
these therapeutic methods, the later development ofneuropsychological diseasesinduced by preterm infant brain injuries still remains high incidence rate.

(PDEs) play a vital role in the hydrolysis of cAMP/cGMP [4]. Inhibition of PDEs can increase the intracellular cAMP and subsequent phosphorylation of cAMP response element-binding protein (CREB) levels in the cerebral region [5]. The increased CREB is important for synaptic plasticity regulation and memory enhancement [6-8]. PDEs inhibitor has been proposed as a

therapeutic method for Alzheimer’s disease, ischemia, depression and anxiety [9-11], which is owing to increased neurogenesis. Rolipram, a PDE-4 inhibitor, also can produce antidepressant- like effects owing to increased CREB [6]. Neurons can modify their synaptic strength through activation of specific gene BDNF. Studies have identified that close relationship may reside in BDNF and CREB activation, which plays an important role in long-term memory formation [12] [13]. However, the effect and molecular mechanism of rolipram on intrauterine infection induced brain injury have not been demonstrated.

on the above, we used intrauterineE. coli infected neonatalrat modelto evaluate theeffectofrolipramon cognitive function recovery, NSPCs proliferation and neuronal differentiation, expressions of p-CREB/CREB, p-Akt/Akt, TrkB and BDNF. The possible molecular mechanisms of rolipramon cognitive function recovery and hippocampalneurogenesis following intrauterine infectionwere expected to be found.

Materials and Methods
Pregnant rats (E12) were obtained from the Experimental Animal Center of Zhejiang Medical Academy of Science. Animals were housed in groups with available water and food adlibitum, in a temperature (22±1°C) and humidity (50±10%)-controlled environment with a light-dark cycle of 12:12h. They were allowed to acclimate to our animal facility prior to experimental manipulation. All procedures were approved by Zhejiang University Animal Care Committee in accordance with Laboratory Animal Care Principles (NIH publication 80-23, revised 1996).

Experimental protocols
Intrauterine E. coli infected model was successfully establishedby endocervical injection of 0.4 ml of E. coli suspension (2.5×108–4.0×108 colony-forming units per mL) in E15 pregnant rats [14, 15]. In this study, the female pups were removed at weaning. The litters of pups (n=75) were arbitrarily divided into three groups: (1) Control+saline group (n=25), normal pups received 0.9% saline containing 1% dimethyl sulfoxide (DMSO) by intraperitoneal (i.p.) injection; (2) E. coli+saline group (n=25), E. coli infected pups received i.p. injection of 0.9% saline containing 1% DMSO; (3) E. coli+rolipram group (n=25), E. coli infected pups received i.p. injection of rolipram. Thirty male pups were randomly divided into three groups (10 pups/group)at postnatal 28 days (P28) for testing in the Morris Water Maze.At postnatal7 days (P7), five

pups in each group were killed and cell proliferation was evaluated. Neuronal differentiation analysis was performed at P28 from five pups in each group. Five pups were killed at P28 and their hippocampus tissues were immediately dissected and stored at −80 °C for Western blot study.

Administration of drugs
(1) Rolipram (PDE4 inhibitor, 10 mM, Sigma) dissolved in saline containing 1% DMSO (Sigma) was administered i.p. injection (1 mg/kg) once daily from P1 to P7.
(2) 5-bromodeoxyuridine BrdU (10 μg/μL in saline, pH 7.0, Sigma, 50 mg/kg) was given i.p. injection twice daily (at 12-hr intervals) from P5 to P7, and rats were killed at P7 to evaluate cell proliferation. Same dosage of BrdU was given from P1 to P7, and rats were killed at P28 to evaluate neuronal differentiation [16].

Morris water maze (MWM) test
The equipment of MWM consisted of a pool (150 cm in diameter and 60 cm in height) which was filled with water to 45 cm depth. Water temperature was maintained at 23 ± 1°C. The pool was divided into I, II, III, and IV equal quadrants. A black circular platform (12 cm in diameter) was located in the quadrant III center which is supposed to be the target quadrant. The platform submerged 2 cm below the water surface. Rat climbed onto the platform in order to escape from water. When the rat stayed on it for more than 3 s, the camera over the pool would automatically stop recording (Huaibei Biological Equipment Co., Ltd, China).

navigation and spatial probe tests were operated in the MWM test. During the place navigation, rats were subjected to training trials for 5 days. Briefly, the location of the platform remained constant and rats were allowed to swim for 120 s or until they located the platform. Rats that failed to locate the target within 120 s were guided manually to the platform. They were remained for
10 s before returning to their home cage. There was a 5 min interval among trials and the training procedure was repeated over the next 4 days. The camera automatically records the trail and the escape latency. During the spatial probe, the platform was removed from the tank. Rats were released into water from the starting point which was in the opposite of the target quadrant and allowed to swim for 120 s individually. The rats’ target crossings and residence time in the target

quadrant which the platform had previously been located were recorded to evaluate learning and memory abilities.

Tissue fixation, immunohistochemistry and immunofluorescent staining
Under anesthesia of sodium pentobarbital (40 mg/kg, i.p.), rats were perfused transcardially with 0.9% saline, followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) (pH 7.4). The brains were removed and post-fixed in 4% paraformaldehyde at 22±1°C for
2 days. After formalin-fixation and paraffin-embedding, brain tissue was performed for the immunohistochemical staining. In each case, 5-μm thick serial paraffin sections spanning the whole hippocampus were obtained on silanized slides. After being further processing, the slides were incubated overnight at 4°C with primary antibody: mouse monoclonal anti-BrdU (1:50; Santa Cruz Biotechnology, Santa Cruz, CA). Using the MCID system, the BrdU-labeled cells were examined at a magnification of 20 and counted. For immunofluorescent staining, sections were incubated with mouse monoclonal anti-Nestin (1:250; Millipore), mouse monoclonal anti-NeuN (1:100; Millipore) and rat monoclonal anti-BrdU (1:40; Abcam). FITC conjugated goat anti-
rabbit (1:100; Zymed), goat anti-mouse (1:100; Zymed) or TRITC conjugated goat anti-rat (1:100; Zymed) antibodies were used. The sections were counterstained with DAPI. Immunoactivity was recorded under a Zeiss confocal microscope.

Cell counting and quantization
Cell counting and quantization were conducted as described previously [17]. Observers were blind to individual treatment status of the animals. Counts were averaged and normalized by measuring the linear distance (in mm) of the hippocampal DG region for each section. The number of
BrdU-positive cells (red stained) and BrdU/Nestin or BrdU/NeuN double-positive cells (yellow after merge) were counted in the DG. The percentage of BrdU/Nestin and BrdU/NeuN double- positive cells over the total number of BrdU-positive cells in the DG was estimated and used as a parameter to evaluate new neural stem cells and new neurons.

Western blot analysis
Proteins (50 μg) of each sample were loaded into 8-10% SDS polyacrylamide gel for electrophoresis. Polyvinylidene difluoride (PVDF) membranes (Amersham) with transferred

proteins were blocked in 5% milk powder in TBS-T (20 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20) for 2 hr. The primary antibodies such as BDNF (1: 1000, Abcam), TrkB (1:1000, Abcam), phospho-Akt (1:2000, Cell Signaling), total Akt (1:1000, Cell Signaling, US), phospho- CREB (1:1000, Cell Signaling), total CREB (1:1000, Cell Signaling) andβ-actin (1:5000,
Sigma-Aldrich) were used. After incubation at 4°C overnight, membranes were washed. Then peroxidase-labeled secondary antibody was incubated for 2 hr. Enhanced chemiluminescence (ECL Plus Detection Kit, Beyotime) was used to detect bands. Blots were incubated in the stripping buffer for 30 min at 50–55°C. The level of β-actin was used for loading control.
Densitometric analysis of immunoreactivity for each protein was conducted using Image Pro Plus software.

Statistical analysis
All data are presented as the means ± SEM, and analyzed by One-way ANOVA followed by Tukey’s post hoc comparisons. The average escape latency was calculated and evaluated by repeated measures ANCOVA followed by Tukey’s post hoc comparisons. P<0.05 was considered statistically significant. Results Rolipram promoted cognitive function recovery in prenatally infected offspring In the place navigation trial, a noticeable difference was found in the average latency amonggroups (group: F(2,27)=18.72, P<0.01; day: F(4,108)=349.58, P<0.01; day*group: F(8,108)=6.22, P<0.01). Data indicated that the E. coli+saline group had longer escape latency than the Control +saline group (P<0.01). After administration of rolipram, the escape latency was markedly reduced in the E. coli+rolipram group (P<0.01) (Fig.1). In the spatial probe test, we found that the E. coli+rolipram group had more platform crossing movement than the E. coli+saline group (Control+saline: 6.60±0.48,E. coli+saline: 4.30±0.52,E. coli+rolipram: 6.20±0.42, F(2,27)=6.78, P<0.01) (Fig.2). Similarly, the E. coli+rolipram group spent more time in the target quadrant than the E. coli+saline group (Control+saline: 48.36±4.22, E. coli+saline: 36.31±3.33,E. coli+rolipram: 49.90±1.85, F(2,27)=5.14, P<0.01) (Fig.3). Roliprampromoted neuronal proliferation in prenatally infected offspring Quantitative analysis of BrdU-positive cells revealed that there was a significant increase in the E. coli+rolipram group. (Control+saline: 2980.00±213.07,E. coli+saline: 4792.00±224.11,E. coli +rolipram: 5632.00±189.69, F(2,12)=41.88, P<0.01) (Fig.4). To verify the increased proliferative cells had the property of NSPs, we used BrdU/Nestindouble-positive cells to represent NSPs. The ratio of BrdU/Nestin double-positive cells was not significantly different in three groups (F(2,12)=3.73, P>0.05). But a remarkable increase of the absolute number of BrdU/Nestin double- positive cells was observed in the E. coli+rolipram group (Control+saline: 987.53±97.23,E. coli
+saline: 1637.88±69.86,E. coli+rolipram: 2113.38±153.06, F(2,12)=25.38, P<0.01) (Fig.5). Rolipram enhanced neuronal differentiation in hippocampus We used BrdU/NeuN double-positivecells to represent mature neurons. Our data showed that the ratio of BrdU/NeuN double-positive cells was not significantly different in three groups (F(2,12)=1.49, P>0.05). The absolute number of BrdU/NeuNdouble-positive cells was significantly reduced in the E. coli+saline group. After rolipram treatment, the absolute number of BrdU/ NeuNdouble-positive cells was specifically increased (Control+saline: 884.95±35.27, E. coli+saline: 725.79±38.33, E. coli+rolipram: 1084.73±60.83; F(2,12)=15.13, P<0.01) (Fig.6). Rolipram increased p-CREB, p-Akt, TrkB and BDNF levels in hippocampus The total CREB levels showed no significant difference between groups (F(2,12)=1.43, P>0.05). A noticeable decrease of p-CREB/CREB was found in the E. coli+saline group. After treatment of rolipram, p-CREB/CREB was significantly increased (Control+saline: 0.75±0.04,E. coli+saline: 0.64±0.03, E. coli+rolipram: 1.06±0.08, F(2,12)=15.02, P<0.01) (Fig. 7A). Furthermore, we studied the possible CREB regulatory mechanism and several of its upstream or downstream targets. The similar increased tendency was observed with p-Akt/Akt (Control+saline: 0.71±0.01,E. coli +saline: 0.70±0.01,E. coli+rolipram: 0.89±0.02, F(2,12)=42.03, P<0.01) (Fig. 7B), TrkB (Control +saline: 0.85±0.02,E. coli+saline: 0.94±0.03,E. coli+rolipram: 1.12±0.04, F(2,12)=20.06, P<0.01) (Fig. 7C) and BDNF levels (Control+saline: 0.95±0.05,E. coli+saline: 0.97±0.04,E. coli+rolipram: 1.18±0.03, F(2,12)=9.98, P<0.01) (Fig. 7D) after administration of rolipram. Discussion Preterm birth (PTB) is the primary cause of neonatal mortality, and surviving prematureinfants are at great risk for neuropsychological diseases.Intrauterine infection is an important etiological factor that drives PTB, whichcan cause brain injury.Brain injury in thepremature neonate is linked with the later development of physical dysfunction,dysgnosia, secondary epilepsy and so on[18, 19].Studies of brain injury in thepremature neonatehave been proceeded, but the method toamelioratechildren’scognitive deficitsowing to premature neonate’sbrain injuryinduced by intrauterine infectionremains to be explored. has been identified to persist in the sub-ventricular zone (SVZ) and sub-granular zone (SGZ) of the hippocampal dentate gyrus (DG) in postnatal and adult mammalian brain. But endogenous neurogenesis has not been found in the brain white matter. Intrauterine infection usually induces brain injury in the preterm brain, which mostly affects the brain’s white matter, while cortex and subcortical grey matter involvement are less common. In cerebral ischemic injuries models, studies have verified that hippocampal endogenous neurogenesis can potentially contribute to the regeneration of injured nerves. So, hippocampal endogenous neurogenesis was studied in patients with intrauterine infection with an attempt to identify the possibility of its potential contribution in repairing injured nerve cells. is highly sensitive to trauma, acute seizures, hypoxia-ischemia and infection [20-24]. Hippocampal pyramidal neurons are especially vulnerable to various brain injuries in terms of cognitive deficits [25]. Once hippocampus is damaged, the progression and severity of cognitive dysfunction will be further underpined. [26, 27]. However, secondary neurogenesis activated by various brain injuries is important for cognitive function recovery such as memory consolidation and pattern separation [27-31]. Many positive or negative regulators have been reported to modulate hippocampal endogenous neurogenesis [32, 33]. Currently, therapeutic strategies of cognitive deficits have been tried, but limited progress has been obtained. We suggested that NSPCs recruitment, activation and survival may be helpful for promoting cognitive function recovery in prenatal E. coli infected offspring. have showed that a prompt inflammatory cascade is initiated at early stageof infection,which is toeliminate the pathogen. Subsequently,brain damagecharacterized as neuronsapoptosisoccurred.In recent years, however, there has been an increasing recognition thatinflammation is a double-edged sword.Following theproinflammatory mediators released, many damaged and dead neuronswere cleared. Along with the inflammation progression, repairprocess characterized withhippocampalneurogenesisensues.In our study, we foundasignificant increaseof BrdU-positive cells appearedaroundhippocampus in the prenatal E. coli infected offspring. It was demonstrated that intrauterine inflammationparticipated insecondarycell proliferation right afterearlyinflammatory reaction. Furthermore, these proliferative cells showed NSPCs property. Thereafter prominent increased BrdU/NeuN double-positive cells were found in the cell proliferation regions. We presumed that the new born neurons exerted a positive effect in brain self-repair and functional recovery process following intrauterine E. coli infection. However, little improvement of cognitive function was obtained in spite of increased cell proliferation and new born neurons. The prenatally infected offspring showed noticeable cognitive deficits (e.g. longer escape latency and less time stayed in the target quadrant). Based on the above, we deemed that the neurogenesis was tenuous and transient. The new born neurons were immature and usually had incomplete function. Within weeks, the early abundant proliferative premature neurons did not accomplish survival and differentiation. Most of these premature neurons died through apoptosis and the cognitive function recovery was damaged. So, activating the NSPCs proliferation and promoting mature neurons differentiation hold therapeutic promise on cognitive deficits. inhibitor rolipram can increase cAMP and activate CREB phosphorylation, which can promote neurogenesis and represent a kind of nervous structural plasticity. Newborn mature neurons were electrically active and could make connections to the hippocampal CA3 field with the administration of rolipram [34]. Thereby the increased compensatory hippocampal neurogenesis could facilitate hippocampal LTP induction, which favors to enhance learning and memory. Our study showed the similar result that the rolipram-treated pups had noticeable cognitive function improvement (e.g. decreased escape latency and increased time stayed in the target quadrant) in prenatal E. coli infected offspring. It suggested that rolipram could promote premature neurons survival and differentiation. The enhanced hippocampal neurogenesis induced by rolipram is believed to contribute to ameliorate cognitive dysfunction induced by intrauterine infection. the cAMP pathway, there are numerous pathways can activate CREB [35-37], such as protein kinase pathways, phospholipase C (PLC)-PKC signaling, Ras/ERK/RSK2 and PI3K/Akt pathways. However, some of the literature considered that there is a cross-talk between the cAMP and PI3K pathways. Studies indicated cAMP can either stimulate or inhibit Akt activity [38]. The activation of the cAMP plays a major role in the regulation of BDNF mRNA expression [39, 40]. Chronic administration of PDE4 inhibitor can increase BDNF mRNA in hippocampus [41]. BDNF, a cognate ligand for TrkB receptor, promotes the activation of PI3K and activate downstream molecule Akt [42]. In our study, the p-CREB/CREB, p-Akt/Akt, TrkB and BDNF protein levels were markedly increased in the rolipram-treated pups. It suggested that phosphorylation of CREB can stimulate Akt activity, and phosphorylation of Akt can subsequently regulate BDNF expression. The activated CREB/Akt/BDNF signaling pathway might participate the neurons proliferation and survival following intrauterine infection. is one of the major phosphodiesterase isoenzymes expressed in the central nervous system, and therefore nausea and emesis are common side effects. Other side effects include fatigue, headache, dyspepsia, diarrhea, gastroenteritis and nasopharyngitis [43]. Rolipram was the first selective PDE4 inhibitor investigated, and it has been studied as an antidepressant several years. In recent years, advancements in PDE4-targeted therapies have shown promise for treating some autoimmune diseases, Alzheimer’s disease, Multiple sclerosis etc. Despite the fact that it has anti-inflammatory and neurogenetic properties, rolipram has not yet been approved for clinical use in intrauterine infected-brain injured patients due to its unacceptable threshold of nausea and emesis triggered in patients. Conclusion Our data suggested that rolipram could ameliorate cognitive deficits and promote hippocampal neurogenesis probably mediated by CREB/Akt/BDNF signaling pathway after intrauterine infection. Further investigation is needed to demonstrate by which way rolipram promotes hippocampal neurogenesis after blocking of PI3K/Akt signaling, and to elucidate the exact molecular mechanisms of CREB/Akt/BDNF signaling pathway in vitro. 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Figure legends Fig.1. Changes of the offspring’s escape latency after rolipram treatment. Compared with the Control+saline group, the average incubation period of seeking the platform was longer in the E. coli+saline groups (P<0.05). After rolipram treatment, the escape latency was significantly shorten in the E. coli+rolipram group (P<0.05). Values represent mean±SEM (n=10 per group). *P<0.05 vs. Control+saline group, ▲P<0.05 vs. E. coli+saline group. Fig.2. Changes of the offspring’s target crossings after rolipram treatment. The target crossing in the E. coli+saline groups was less than that in the Control+saline group (P<0.01). After rolipram treatment, the target crossing was significantly increased. More target crossings was seen in the E. coli+rolipram group than the E. coli+saline group (P<0.05). Values represent mean±SEM (n=10 per group). *P<0.05. Fig.3. Changes of the offspring’s residence time after rolipram treatment. Time in the target quadrant in the E. coli+saline groups was less than that in the Control+saline group (P<0.05). After rolipram treatment, the residence time was significantly increased in the E. coli+rolipram group. Values represent mean±SEM (n=10 per group). *P<0.05. Fig.4. Cell proliferation after rolipram treatment. A-C, The cells with BrdU (brown stained) that clearly localized to the nucleus were counted as BrdU-labeled cells. Histological results (20×) illustrate the increased number of BrdU-labeled cells in the E. coli+rolipram group. sgz, subgranular zone; gcl, granule cell layer. Scale bars=200μm. D, Quantitative data are expressed as total number of dentate gyrus BrdU-labeled cells. Values represent mean±SEM, *P<0.05, n=5. Fig.5.NSPCs proliferation after rolipram treatment.A-C,Confocal images of double-stained cells for BrdU (red) and Nestin (green) illustrates co-localization in the DG in representative sections from rats. Nuclei are counterstained with DAPI (blue). Scale bars=100μm. A1-C4, Representative images of BrdU/Nestin double-stained cells divided into single color images. Scale bars=20μm. D, Quantification of the total number of BrdU+/Nestin+ cells in the DG. A remarkable increase of BrdU+/Nestin+ cells was observed in the E. coli+rolipram group (P<0.01). Values represent mean ±SEM, *P<0.05, n=5. Fig. 6.Neuronal differentiation after rolipram treatment.A1-C4, Confocal image of double-stained cells for BrdU (red) and NeuN (green) illustrates co-localization in the DG in representative sections from rats. Nuclei are counterstained with DAPI (blue). Scale bars=100μm. D1-F4, Representative images of BrdU/NeuN double-stained cells divided into single color images. Scale bars=20μm. G, BrdU/NeuN double-stained cells are shown in x-y orthogonal planes and z-sectioning at 0.5μm intervals (right) to confirm overlap of the two immunoreactions. Scale bars=20μm. H, Quantification of the total number of BrdU+/NeuN+ cells in the DG. Values are represented as mean±SEM, *P < 0.05, n=5. Fig.7. Changes of p-CREB/CREB, p-Akt/Akt, TrkB and BDNF protein expressions after rolipram treatment.A-D, Representative autoradiographs of p-CREB/CREB, p-Akt/Akt, TrkB and BDNF expression are shown. Intensities of p-CREB/CREB (A1), p-Akt/Akt (B1), TrkB (C1) and BDNF
(D1) protein bands were quantified by densitometry analysis, respectively. Significant increase of p-CREB/CREB (P<0.01), p-Akt/Akt (P<0.01), TrkB (P<0.01) and BDNF (P<0.01) were observed in the E. coli+rolipram group. Values are represented as mean±SEM, *P<0.05, n=5.