brain research 1622 (2015) 279–291

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Research Report

Phosphodiesterase-4 inhibitors ameliorates cognitive deficits in deoxycorticosterone acetate induced hypertensive rats via cAMP/CREB signaling system S. Sugin Lal Jabarisa, Haridass Sumathya, Ramesh Girishb,a, Shridhar Narayananc, Mani Sugumara, Chidambaram Saravana Babua,n, Sadagopan Thanikachalama, Mohan Thanikachalamd a

Centre for Toxicology and Developmental Research (CEFT), Sri Ramachandra University (SRU), Chennai 600116, Tamil Nadu, India b Department of Nephrology, Sri Ramachandra University, Chennai 600116, Tamil Nadu, India c Foundation for Neglected Disease Research, Sri Krishnadevaraya Research Centre, Bangalore 562 157, India d Tufts University School of Medicine, Boston, MA 02111, USA

art i cle i nfo

ab st rac t

Article history:

Phosphodiesterase-4 (PDE-4) inhibitors promote memory by blocking the degradation of

Accepted 3 July 2015

cAMP. Existing evidence also shows that neuronal survival and plasticity are dependent on

Available online 11 July 2015

the phosphorylation of cAMP-response element-binding protein. In this regard, PDE-4

Keywords:

inhibitors have also been shown to reverse pharmacologically and genetically induced

Hypertension

memory impairment in animal models. In the present study, the authors examined the

Cognition

effect of both rolipram and roflumilast (PDE-4 inhibitors) on the impairment of learning

Rolipram

and memory observed in hypertensive rats. Deoxycorticosterone acetate (DOCA) salt

Roflumilast

hypertensive model was used to induce learning and memory deficits. The mRNA

pCREB

expression of different PDE-4 subtypes along with the protein levels of pCREB and BDNF

BDNF

in the hippocampus was quantified. Systolic blood pressure was significantly increased in DOCA salt hypertensive rats when compared to sham operated rats. This effect was reversed by clonidine, an α2 receptor agonist, while PDE-4 inhibitors did not. PDE-4 inhibitors significantly improved the time-induced memory deficits in object recognition task (ORT). In DOCA salt hypertensive rats, the gene expression of PDE-4B and PDE-4D was significantly increased. Furthermore, both pCREB and BDNF showed decreased levels of expression in hypertensive rats in comparison to sham operated rats. Repeated administration of PDE-4 inhibitors significantly decreased both PDE-4B and PDE-4D with an increase in the expression of pCREB and BDNF in hypersensitive rats. Also, rolipram, roflumilast and roflumilast N-oxide showed a linear increase in the plasma and brain

n

Corresponding author. E-mail address: [email protected] (C. Saravana Babu).

http://dx.doi.org/10.1016/j.brainres.2015.07.003 0006-8993/& 2015 Elsevier B.V. All rights reserved.

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concentrations after ORT. Our present findings suggested that PDE-4 inhibitors ameliorate hypertension-induced learning impairment via cAMP/CREB signaling that regulates BDNF expression downstream in the rat hippocampus. & 2015 Elsevier B.V. All rights reserved.

1.

Introduction

Phosphodiesterases (PDEs) play a vital role in the hydrolysis of cAMP/cGMP (Conti et al., 2003; Houslay, 2001). Currently, more than 50 isoforms have been identified (Houslay, 2001; Rose et al., 2005; Reneerkens et al., 2009). The PDE-4 gene family members are distributed throughout the brain and expressed in various neurons. PDE-4 C is exclusively found in the olfactory bulb (Perez-Torres et al., 2000; Bian et al., 2004). Inhibition of PDE-4 increases the intracellular cAMP levels in the cerebral region. This results in the subsequent phosphorylation of cAMP response element-binding protein (CREB) which in turn regulates the transcription of genes, important for the mediation of memory enhancement and synaptic plasticity (Frey et al., 1993; Bailey et al., 1996; Barad et al., 1998). Rolipram, a first-generation cAMP-specific PDE-4 inhibitor, has been shown to improve memory deficits in various studies (Zhang et al., 2013). Rolipram ameliorated the working memory deficits in rats (Zhang and O'Donnell, 2000; Zhang et al., 2000) and reversed recognition memory impairment upon sub-chronic treatment in rats or in CBPþ/  mice (Rutten et al., 2008a; Bourtchouladze et al., 2003). In addition, treatment with rolipram improved the neuronal cell death caused by transient ischemia (Block et al., 1997) and also microsphere embolism induced defects in memory function and neurogenesis in the hippocampus associated with cAMP/PKA/CREB signal transition system (Nagakura et al., 2002). Hypertension (HT) is also related to behavioral, electrophysiological and neurotransmitter associated alterations (Hacioglu et al., 2003). Many pre-clinical models of hypertension have been shown to induce deficits in learning and memory. (Wyss et al., 1992; Meneses et al., 1996; Meneses and Hong, 1998; Hirawa et al., 1999; Wyss et al., 2000; Hacioglu et al., 2003). We have previously reported that PDE-4 inhibitors improved the memory performance of rodents in an object recognition task and retention latency in elevated plus maze in experimentally induced hypertension (two-kidney one-clip) model (Jabaris et al., 2015).

On this basis, the present study was undertaken to evaluate the effects of PDE-4 inhibitors, rolipram and roflumilast in DOCA salt-induced hypertensive rat model and explore the molecular mechanisms of cAMP/PKA/CREB signal transduction systems, post treatment in the rat hippocampus.

2.

Results

2.1.

Effect of PDE-4 inhibitors on blood pressure

Fig. 1 The mean systolic blood pressure in male Wistar rats weighing 120–140 g was approximately 109.772.8 mm Hg prior to uninephrectomy. This increased to 136.872 mm Hg at the end of the third week and further increased to 197.875.8 mm Hg, at the end of 14th week after uninephrectomy. Systolic blood pressure (SBP) and responses to systemic administration of rolipram and roflumilast, selective PDE-4 inhibitors were examined under the conscious state to determine the influence of PDE-4 on blood pressure in rats. As shown in Fig. 2A, PDE-4 inhibitors did not cause a reduction in systolic blood pressure in DOCA salt-induced hypertensive rats, indicating that PDE-4 inhibitors do not have an effect on the blood pressure. Clonidine caused statistically significant reductions in SBP at the dose of 25 μg/kg in rats. The blood pressure recordings on days 1, 5 and 9 were 169.774.6, 127.572.1, and 107.672.8 mm Hg respectively. No significant change in SBP was observed during the experimental periods in the vehicle-treated rats. Fig. 2B–D shows the mean value of the weight of right kidneys, weight of heart and heart index (heart weight normalized for body weight). The right kidneys weight was significantly increased in vehicle treated group when compared to sham operated group (2.0170.09 Vs 1.2870.07 g; Po0.001). As anticipated, vehicle controls showed significant increase in heart weight (1.3870.04 Vs 1.0270.04 g; Po0.001) and heart index (4.3870.21 Vs 2.5970.21; Po0.001) when compared to the normotensive group. Both the PDE-4 inhibitors caused no significant differences in heart weight and heart index, compared to the hypertensive control rats.

Fig. 1 – Schedule of drug treatment and experiment orders. Vehicle (0.5% CMC; 10 ml/kg, p.o.), clonidine (25 lg/kg, p.o.), rolipram (0.03, 0.1, 0.3 mg/kg, i.p.) and roflumilast (0.1, 0.3, 1 mg/kg, p.o.). On day 8, habituation (H) of the ORT was performed, followed by acquisition (A) trial the next day and 24 h after interval discrimination (D) trial.

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225

1.5

Sham + Vehicle (10ml/kg.,p.o)

DOCA + Rolipram (0.03mg/kg.,i.p)

DOCA + Vehicle (10ml/kg.,p.o)

DOCA + Rolipram (0.1mg/kg.,i.p)

DOCA + Clonidine (25μg/kg.,p.o)

DOCA + Rolipram (0.3mg/kg.,i.p)

DOCA + Roflumilast (0.3mg/kg.,p.o)

DOCA + Roflumilast (0.1mg/kg.,p.o)

***

DOCA + Roflumilast (1mg/kg.,p.o)

175

Heart.wt(gm)

200

SBP (mm Hg)

###

***

150

***

125 100

1.0

0.5

***

75 9

1

0.

Sh a Ve m hi C lo cle ni di ne

3

0.0

3

5 Treatment

0. 1

1

1

14

0.

12

0.

10

After surgery (wks)

03

6 8

0.

Basal Before surgery

(mg/kg) Roflumilast

Rolipram

2.5

5.0

###

###

4.5

2.0 Right Kidney wt (gm)

Heart index(mg/g)

4.0

**

3.5 3.0 2.5 2.0

1.5

1.0

1.5 0.5

1.0 0.5

Roflumilast

1

3 0.

1 0.

0. 3

1 0.

03 0.

am Sh

1

0. 3

1 0.

0. 3

1 0.

03 0.

am Ve hi cl C e lo ni di ne

Sh

(mg/kg) Rolipram

Ve hi c C lo le ni di ne

0.0

0.0

(mg/kg) Rolipram

Roflumilast

Fig. 2 – Effect of PDE-4 inhibitors on SBP, kidney and heart weight and heart index in hypertensive rats. Data were expressed as mean7S.E.M. nnPo0.01, nnnPo0.001 (one-way ANOVA followed by Dunnett's post hoc test) in comparison with vehicletreated group; ###Po0.001 Vs sham group.

Clonidine, however, caused a significant reduction in heart weight and the heart index (Po0.001; Po0.01).

2.2.

Object recognition task

The effect of repeated doses of PDE-4 inhibitors in hypertension-induced deficits on cognitive performance was investigated by performing the object recognition

task. During the acquisition trial (T1), sham, vehicle and clonidine treated rats displayed a similar level of exploration of the objects. However, PDE-4 inhibitors treated rats displayed less of exploration of the objects when compared to the sham and vehicle treated groups, but not to the significant level. Following 24 h delay, rats with repeated doses of PDE-4 inhibitors significantly increased the time spent in exploring the novel object in comparison

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T1 Exploration Time (sec)

12.5

10.0

7.5

5.0

2.5

1

3

1

0.

3

0.

0.

0.

1

Sh a Ve m hi c C lo le ni di ne 0. 03

0.0 (mg/kg) Roflumilast

Rolipram

1.0

***

***

20

*

*

*

**

**

10

*** *

* 0.4

0.2

0

***

**

0.6

##

Roflumilast

1

3

1

0.

0.

0. 3

0. 1

03 0.

am

hi

c C lo le ni di ne

(mg/kg) Rolipram

Ve

1

0. 3

1 0.

0. 3

1 0.

03 0.

Sh

a Ve m hi C lo cle ni di ne

0.0

Sh

Time exploring object (sec)

***

Discrimination Index

0.8 Novel object

Familiar object

30

(mg/kg) Rolipram

Roflumilast

Fig. 3 – Effect of PDE-4 inhibitors on Object Recognition Task (ORT) in hypertensive rats. (A) Discrimination index. (B) Time exploring the familiar and the novel objects. Data were expressed as mean7S.E.M. (n ¼6–7 per group); nPo0.05, nnPo0.01, nnn Po0.001 Vs vehicle control; ## Po0.01 Vs sham group. to the familiar object, thereby showing an increase in memory retention (Fig. 3A–C). However, the recognition task results revealed that both the vehicle and centrally acting anti-hypertensive drug (clonidine) treated animals displayed less exploration of novel object.

2.3. Rolipram, roflumilast and roflumilast N-oxide concentrations in plasma and brain The exposure levels of rolipram, roflumilast and roflumilast N-oxide (active metabolite of roflumilast) in plasma and brain

samples are represented in Fig. 4A–C. Rolipram-treated animals showed dose-dependent increase levels of rolipram in plasma and brain, when the final dose was administrated 45 min before sampling. The rolipram concentrations were 137.27714.87, 270.91735.83 and 697.27716.22 nM in plasma and 216.37719.72, 524.55720.59 and 1315.73750.19 nM in brain at 0.03, 0.1 and 0.3 mg/kg, i.p., respectively. Also, roflumilast treated animals (at 0.1, 0.3 and 1 mg/kg, p.o.) showed dose-dependent increase levels of roflumilast and its active metabolite in plasma as well as brain whose concentrations were 10.4871.51, 11.4771.38, 19.2172.12 nM

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brain research 1622 (2015) 279–291

2500 Plasma Brain

Con(nM)

2000 1500 1000 500

0. 3

0.

0.

03

1

0

Rolipram (mg/kg) 80

250 Roflumilast N-oxide Con (nM)

Plasma Brain

40

20

Brain

200 150 100 50

0.

Roflumilast (mg/kg)

3 0.

03

3 0.

1 0.

03 0.

1

0

0

0.

Con (nM)

60

Plasma

Roflumilast (mg/kg)

4000 3500

1

3 0.

0. 1

0. 3

0. 1

3000

03

Fig. 5 shows plasma corticosterone concentration in DOCA salt-induced hypertension rats treated with vehicle, clonidine and PDE-4 inhibitors. There was no significant increase in the plasma corticosterone levels of the vehicle-treated group when compared to the sham operated group (4155.67161.6 Vs 3827734.5 pg/ml). However, high doses of rolipram and roflumilast treatment groups significantly increased the plasma corticosterone levels (Po0.01 and Po0.001), when compared to vehicle treated animals.

4500

0.

Corticosterone concentration in rats

**

5000

a Ve m hi cl C lo e ni di ne

2.4.

***

5500

Sh

and 13.6470.67, 89.24710.55, 118.80710.43 nM in plasma; 7.4170.88, 22.1672.61, 45.6474.68 nM and 6.9270.98, 18.6172.33, 58.6878.85 nM in brain, respectively, when the final dose was administrated 1 h before sampling.

Plasma Corticosterone (pg/ml)

Fig. 4 – Plasma and brain concentrations of rolipram, roflumilast and roflumilast N-oxide. (A) Rolipram, (B) Roflumilast, (C) Roflumilast N-oxide. Data were expressed as mean7S.E.M. (n¼ 4 per dose).

(m g/kg) Rolipram

Roflum ilast

Fig. 5 – Effect of PDE-4 inhibitors on plasma corticosterone levels in hypertensive rats. Data were expressed as mean7S.E.M. (n ¼ 5 per group); nnPo0.01, nnnPo0.001 Vs vehicle control.

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1.25

PDE4A / β -actin

1.00

0.75

0.50

0.25

1

1

0. 3

3

0.

0.

1 0.

03 0.

Sh

a Ve m h C icl lo e ni di ne

0.00 (mg/kg) Roflumilast

Rolipram

2.50 2.75 2.50

2.25

###

2.25

1.75 1.50

1.75

**

*** ***

1.25

***

***

1.00

***

PDE4D / β -actin

PDE4B / β -actin

2.00

##

2.00

** **

1.50

***

1.25

*** *** ***

1.00 0.75

0.75 0.50

0.50

0.25

0.25

(mg/kg) Roflumilast

1

3 0.

1

3 0.

0.

1 0.

03 0.

Sh

1

1

0. 3

0.

3 0.

1 0.

Sh a Ve m hi C lo cle ni di ne 0. 03

Rolipram

a Ve m hi C lo cle ni di ne

0.00

0.00

(mg/kg) Rolipram

Roflumilast

Fig. 6 – Effect of PDE-4 inhibitors on the expression of PDE-4 A, PDE-4B, and PDE-4D mRNA in hypertensive rat hippocampus tissues. Levels of mRNA were quantified by densitometry and are shown in the bar graph. Data were expressed as mean7S.E. M. (n ¼3 per group); nnP o0.01, nnnPo0.001 Vs vehicle control; ##Po0.05, ###Po0.001 Vs sham group.

2.5.

Determination of PDE-4A, B and D mRNA expression

PDE-4A, B and D mRNA expression in the hippocampus, was examined by RT-PCR, following the behavioral test to verify the effects of PDE-4 inhibitors on hypertension-induced changes in PDE-4 subtypes in the hippocampus. In the case of PDE-4B and D, DOCA salt-induced hypertension group showed significantly increased expression of PDE-4B (Po0.001), and PDE-4D (Po0.01); this expression was downregulated by PDE-4 inhibitors in a dose-dependent manner, compared to those treated with the vehicle. No significant changes in expression of PDE-4 subtypes were observed in clonidine treated rats. In contrast, PDE-4A mRNA expression

was not alter by hypertension and treatments as well (Fig. 6A–D).

2.6. Effect of hypertension on pCREB levels in hippocampus The authors examined pCREB expression in the hippocampus using the same rats, which were decapitated after the behavioral task to determine if hypertension had an effect on cAMP dependent phosphorylation of CREB (pCREB) activity and whether cAMP/CREB signaling contributed to the effect of rolipram and roflumilast on reversing hypertension-induced cognitive deficits. The vehicle treated group showed a

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significant decrease in pCREB levels when compared to the sham operated group; pCREB was found to be significantly increased by rolipram at doses of 0.03, 0.1 and 0.3 mg/kg, i.p. 55 kD 36 kD

43 kD

2.7. Effect of PDE-4 inhibitors on mRNA and protein levels of BDNF in hypertensive rats

200

***

150

*** ***

100

** *

*

## 50

1

3 0.

0. 1

3

1

0.

0.

03 0.

Sh am Ve hi cl C e lo ni di ne

0 (mg/kg) Rolipram

Roflumilast

Fig. 7 – Effect of PDE-4 inhibitors on the expression of pCREB in hypertensive rat hippocampus tissues. Levels of proteins from all the groups were quantified by densitometry and were normalized with β-actin and are shown in the bar graph. Data were expressed as mean7S.E.M. (n¼ 3–4 per group); nPo0.05, nnPo0.01, nnnPo0.001 Vs vehicle control; ## Po0.01 Vs sham group.

2.5

BDNF

There is evidence that phosphorylated CREB dimer initiates transcription and regulation of important gene products such as Brain Derived Neurotrophic Factor (BDNF). The authors determined BDNF expression, both at the RNA level and at the protein level in the hippocampal region to verify the activation of CREB signaling cascade following the effect of PDE-4 inhibitors on hypertension. As shown in Fig. 8A and B, the vehicle-treated group significantly decreased the mRNA levels of BDNF in hippocampus when compared to the sham operated animals. Interestingly, repeated doses of PDE-4 inhibitors showed a dosedependent increase in BDNF expression in PDE-4 inhibitors treated groups when compared to those treated with vehicle. Furthermore, BDNF protein levels were also decreased in the hippocampus tissues of DOCA salt-induced hypertensive rats in comparison to the sham operated group. Importantly, the levels of BDNF were also observed to increase in a dose dependent manner in rolipram and roflumilast administered rats. One-way ANOVA revealed no significant changes in BDNF levels in the hippocampus of the clonidine treated rats.

3.

In the present study, the authors used the DOCA salt-induced hypertension model, a mineralocorticoid-based model of

17 kD 10 kD

14 kD

200

β-actin

***

2.0

*** *** ***

1.5

*** 1.0

*

#

***

150

BDNF (% of vehicle)

***

100

0.0

*

* ##

50

0.5

***

**

Sh

(mg/kg) Rolipram

Roflumilast

1

3 0.

3

0. 1

1

0.

03

0.

1

3 0.

1 0.

0. 3

0. 1

03 0.

Sh

am Ve hi c C lo le ni di ne

0 0.

BDNF/ β -actin

Discussion

am Ve hi cl C e lo ni di ne

pCREB (% of vehicle)

and roflumilast (0.1, 0.3 and 1 mg/kg, p.o.) in dose-dependent manner. On the contrary, no significant changes in level of pCREB was observed in the clonidine treated group of rats (Fig. 7).

(mg/kg) Rolipram

Roflumilast

Fig. 8 – Effect of PDE-4 inhibitors on the expression of mRNA and protein levels of BDNF in hypertensive rat hippocampus tissues. Levels of proteins from all the groups were quantified by densitometry and were normalized with β-actin and are shown in the bar graph. Data were expressed as mean7S.E.M. (n¼ 3–4 per group); nPo0.05, nnPo0.01, nnnPo0.001 Vs vehicle control; #Po0.05, ##Po0.01 Vs sham group.

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brain research 1622 (2015) 279–291

hypertension in rats which mimics "essential" hypertension in the clinical setting (Stewart, 1999; Hacioglu et al., 2003). The SBP, right kidney weight, heart weight and the heart index in DOCA salt-induced hypertensive rats were found to be significantly increased when compared to the sham operated group. These results were found to be in agreement with earlier studies that show significantly higher blood pressure, right kidney weight and heart weight in DOCA salt-induced hypertensive rats as compared to the sham operated rats (Hacioglu et al., 2003). Hypertension is one of the most critical risk factors for cognitive impairment and the renin angiotensin aldosterone system (RAAS) is a key player in hypertension, both in a blood pressure- dependent and- independent manner (Yagi et al., 2011; Yagi et al., 2013). The functional mechanisms of hypertension increasing the risk of cognitive impairment remain ambiguous. The putative factors that contribute to cognitive impairment and memory deterioration include oxidative stress, inflammation, platelet aggregation, vasoconstriction, amyloid plaques and apoptotic neuronal death (Hardy and Selkoe, 2002; Iyer et al., 2010; Yagi et al., 2011; Carnevale et al., 2012). Besides, few cohort studies have also revealed a positive association between hypertension and cognitive decline (Launer et al., 1995; Skoog et al., 1996; Kilander et al., 1998). In the present study, it was also found that DOCA salt-induced hypertension caused deficits of learning and recognition memory in ORT accompanied by over expression of PDE-4 subtypes and down-regulation of phosphorylation of cAMP response element-binding protein in the hippocampus. cAMP specific phosphodiesterase inhibitor such as rolipram induced behavioral sedation produced by the elevation of brain cAMP availability (Griebel et al., 1991; Smith, 1990), given that rolipram and roflumilast at the doses we used in the present study did not alter significantly in the acquisition trial (T1) in ORT. In the present study, the lack of the behavioral sedative property of rolipram and roflumilast may be attributed to the fact that animals were administered with rolipram and roflumilast, 45 min and 1 h respectively, prior to ORT, and could possibly attenuate the CNS related side-effects such as sedation during the task. It has been well established that selective PDE-4 inhibitor, rolipram alone enhances memory in animal models (Barad et al., 1998; Bourtchouladze et al., 2003; Rutten et al., 2006; Vecsey et al., 2009; Wang et al., 2012) with memory deficits resulting from normal aging, sleep deprivation, genetically, chemically and Aβ- induced cognition deficits. In the present study, the effect of hypertension-induced cognition deficits was reversed by repeated doses of rolipram and secondgeneration PDE-4 inhibitor, roflumilast (Davis et al., 2009), in ORT without influencing the SBP. Recent reports suggest that roflumilast treatment reduced pulmonary hypertension induced by hypoxia and monocrotaline (MCT), but did not affect systemic arterial pressure (SAP) in rats (Izikki et al., 2009). Also, in human studies, SAP was unaffected by roflumilast treatment (Louw et al., 2007). On the other hand, an increase in cAMP levels inhibited the pro-inflammatory and apoptotic responses and provided an anti-inflammatory milieu (Dastidar et al., 2009; Naderi et al., 2009; Hoyle, 2010; Kim et al., 2010). So, it could be concluded that inhibition of PDE-4 reversed the hypertension-induced cognitive deficits independent of blood pressure. This might be attributed to

the antioxidant and anti-inflammatory properties of PDE-4 inhibitors (Barnette et al., 1996; Aoki et al., 2000; Guabiraba et al., 2010). In addition, the plasma and brain concentrations of rolipram, roflumilast and roflumilast N-oxide were also found to be linear. Hypertension induces damage to localized brain areas such as hippocampus and cortex (Poulet et al., 2006; Sabbatini et al., 2002), and it has been previously demonstrated that pretreatment with clonidine significantly alleviated the drug-induced memory deficits but not hippocampal damage-induced memory deficits (Bardgett et al., 2008). This provides supporting evidence to the findings of the present study and suggested that clonidine could not reverse the memory deficits induced due to hippocampal damage. In the present study, clonidine treatment showed no improvement in the deficits of memory function induced by hypertension; but it significantly decreased SBP throughout the treatment period and, as a result showed significant activity on heart weight and heart index. These results were consistent with the findings reported by the authors in our earlier study using the two-kidney one-clip (2k-1c) hypertension model (Jabaris et al., 2015). In the present study, we found that repeated administration of high doses of PDE-4 inhibitors caused a significant increase in plasma corticosterone levels in DOCA salt-induced hypertensive rats, but had no effect on the corticosterone levels at lower doses. These results were supported by findings from previous studies, in which inhibition of PDE-4 caused an increase in plasma corticosterone levels in rats (Kumari et al., 1997; Kung et al., 2000; Jabaris et al., 2015). Contrasting reports from previous studies demonstrated that exogenous administration of corticosterone and elevation of the hormone in circulation following stress exposure reduced hippocampal synaptic potentiation and hippocampus-specific learning and memory (Kim et al., 2001; Alfarez et al., 2002; Woodson et al., 2003). However, in the present study, it was found that the elevation of corticosterone due to the administration of PDE-4 inhibitors did not affect the reversal of cognition deficit, as shown by the results of behavioral task. At this juncture, further investigation is needed to explore the individual role of corticosterone in hypertension-induced cognition deficit. Further, the role of PDE-4 inhibition in long-term potentiation (LTP) and memory needs to be explored. The authors also investigated the PDE-4 subtypes such as PDE-4 A, B and D (Houslay and Milligan, 1997), which are distributed throughout the brain (Perez-Torres et al., 2000; Bian et al., 2004), Specifically, PDE-4 A is highly expressed in rat hippocampus CA1 sub-region (Zhang et al., 2004); PDE-4B is predominantly present in the amygdala, hypothalamus, striatum, frontal cortex and olfactory bulb as well as hippocampus (Dlaboga et al., 2006; Zhang et al., 2008); PDE-4 C is minimally expressed in the CNS (Zhang et al., 2004); PDE-4D is expressed in hippocampal CA1 region (Zhang et al., 2002; Rutten et al., 2008b). All these subtypes are selective for cAMP (Rutter et al., 2014). In the present study, the authors have demonstrated the subtype – specific inhibitory properties of rolipram and roflumilast on PDE-4, of which PDE-4B and PDE4D were significantly inhibited. This was in agreement with earlier findings in which GEBR-7b, a novel PDE-4D selective inhibitor, increased the hippocampal cAMP level as well as object memory performance (Bruno et al., 2011). Till date, only a few studies have shown that PDE-4B might be involved

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in depression, schizophrenia as well as anxiety (Millar et al., 2005; Zhang et al., 2008). Clonidine treatment showed similar effects as that of the vehicle group. While PDE-4 A subtype was not unaltered in both the hypertension group as well as the PDE-4 treatment. These results are also consistent with our recent findings in which rolipram and roflumilast attenuated the memory impairment induced by 2k-1c hypertension model accompanied by increased mRNA expression of PDE-4 subtypes such as PDE-4B and D (Jabaris et al., 2015). Therefore, the present study provides further evidence that PDE-4B and D play a prominent role in mediating the memory deficit processes induced by hypertension. The cAMP-responsive element-binding protein is a nuclear protein, which modulates the transcription of genes with cAMP-responsive elements. An increase in the concentration of cAMP activates the phosphorylation of CREB (pCREB). Activation of this transcription factor (CREB) is a component of intracellular signaling events that regulate a several range of biological functions like memory (Silva et al., 1998). PDE-4 is an enzyme which regulates the intracellular cAMP concentration in various types of cell. Inhibition of PDE-4 by rolipram increases cAMP and activates the phosphorylation of CREB and thereby facilitates induction of hippocampal LTP and enhances memory (Barad et al., 1998; Li et al., 2011). Here, the authors have demonstrated that pCREB levels are decreased in the hippocampus associated with cognitive deficits in hypertensive rats. Repeated treatment with rolipram and roflumilast reversed hypertension-induced decrease in pCREB in hippocampal region, but clonidine did not. These results suggested that inhibition of PDE-4 reverses hypertension-induced cognitive deficits via increased expression of pCREB, suggesting an important role of cAMP/CREB signaling in the mediation of the memory-enhancing effect of PDE-4 inhibitors in the animal model of hypertension induced cognitive impairment. BDNF is widely expressed in the CNS and its expression is down-regulated in various pathological conditions (Durany et al., 2000; Hock et al., 2000; Zuccato, 2001). Previous studies have shown that the activation of the cAMP plays a major role in the regulation of BDNF mRNA expression (Condorelli et al., 1994). Further, chronic administration of rolipram increased the level of BDNF mRNA in hippocampus (Nibuya et al., 1996). Also, the levels of BDNF mRNA were reported to be decreased in the hippocampal region in acute and chronic stress animal models (Smith et al., 1995). Here, the authors have demonstrated that hypertensive rats produced deficits of learning and memory accompanied by decreased BDNF mRNA and protein expression in the hippocampus. These effects of hypertension were reversed by repeated doses of rolipram as well as roflumilast, which increased the BDNF expression in the hippocampus, but not with clonidine. These results were supported by findings from previous studies, in which BDNF-induced hippocampal LTP in-vivo has been shown to up-regulate Arc expression, in parallel with CREB activation (Ying et al., 2002). Taken together, the findings of the present study support the possibility that activation of the cAMP/CREB system and increased expression of BDNF contribute to the neural adaptations that underlie the action of cognitive improvement induced by hypertension.

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In summary, the authors have provided a real-time demonstration of reversal effect by PDE-4 inhibitors such as rolipram and roflumilast on memory deficits caused by hypertension. We have also emphasized on the finding that inhibition of PDE-4 reversed hypertension-induced memory deficits and it is mediated by cAMP/CREB signaling involving BDNF. The findings of our earlier studies also suggest that PDE-4 inhibitors can arguably serve as potential therapeutic agents for cognitive disorders caused by hypertension.

4.

Experimental procedure

4.1.

Animals

Adult male Wistar rats were obtained from Central Animal Facility, Sri Ramachandra University, Chennai, India. Animals were housed in groups (n¼ 4/cage) on soft bedding with food and water available ad libitum, in a temperature-controlled environment with a light-dark cycle of 12:12 h. All animals were allowed to habituate to the housing facilities for at least 1 week prior to surgery. Guidelines of “Guide for the Care and Use of Laboratory Animals” (Institute of Laboratory Animal Resources, National Academic Press 1996; NIH publication number #85-23, revised 1996) were strictly followed throughout the study. All experimental procedures were approved by the Institutional Animal Ethical Committee (IAEC), Sri Ramachandra University, constituted as per the directions of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), India (IAEC NO: IAEC/ XXXV111/SRU/340/2014).

4.2.

Drugs

Rolipram and clonidine hydrochloride were purchased from Tokyo Chemical Industry Co., Ltd, Japan. DOCA salt was purchased from Sigma (St. Louis, MO, USA). Roflumilast was kindly gifted by Matrix Laboratories, Hyderabad, India.

4.3. Deoxycorticosterone acetate (DOCA) salt-induced hypertension in rats Prior to the surgical procedure, systolic blood pressure (SBP) was measured by tail-cuff method (model MC 4000; Hatteras Instruments, Cary, NC, USA). DOCA salt-induced HT was performed as per the method described previously with minor modifications (Brown et al., 1999). In brief, rats weighing 120–140 g at the time of surgery were anaesthetized by an intra- peritoneal (i.p.) injection of ketamine (75 mg/kg) and midazolam (1 mg/kg) mixture. A small incision was made and the left kidney was removed after the ligation of left renal artery, vein and ureter, and then the animals were returned to their home cages. Sham animals were operated as follows: an incision was made, then the left kidney was exposed and the incision was sutured. After uninephrectomy, postoperative care was given to the rats for 1 week. Then the rats were maintained on drinking water containing 0.9% NaCl and subcutaneous injection of DOCA (15 mg/kg) twice a week for 14 weeks except the sham operated group. SBP was measured 3 weeks after uninephrectomy for 14 weeks.

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Animals were selected based on SBP (Z 175 mm Hg); selected animals were then stratified into groups based on their mean SBP so that the mean baseline did not differ between the groups.

4.4.

Behavioral task

To examine the effect of PDE-4 inhibitors on DOCA saltinduced hypertension associated with memory deficits, nine groups of six to seven rats each were tested for memory task. Vehicle (0.5% CMC) or clonidine (25 mg/kg, p.o.), rolipram (0.03, 0.1, 0.3 mg/kg, i.p. dissolved in normal saline containing 5% DMSO), roflumilast (0.1, 0.3, and 1 mg/kg, p.o.) were given once per day for 10 consecutive days. Clonidine, an α2 receptor agonist and centrally acting anti-hypertensive drug was selected to evaluate whether anti-hypertensive treatment alone would attenuate the cognitive impairments associated with HT in the therapeutic model (Total no. ¼ 62). The behavioral test was performed 1 h following the treatment except in the rolipram group (45 min after dosing) as per the schedule (Fig. 1).

4.4.1.

Object recognition task

This recognition task was performed as described previously (Prickaerts et al., 2002). During habituation, the rats were allowed to explore the empty apparatus for 15 min (8th day of treatment). During the acquisition trial (T1), the apparatus contained two identical objects. A rat was taken from its home cage and placed into the apparatus, central from the two objects, facing the wall in front of the observer and the time spent actively exploring (exploration was defined as the animal sniffing, or touching it) the objects during 3 min. After T1, the rat was transferred to its home cage. Twenty-four hours later (10th day of treatment), the rat was again placed into the apparatus for the discrimination trial (T2). Now, the exploration arena contained two different objects, a familiar object and a novel object. The time spent exploring the two objects during T1 and T2 was recorded and discrimination index (DI) was calculated, as per the following formula. DI¼RI/(Time spent in exploring novel objectþTime spent in exploring familiar object) Recognition Index (RI)¼Time spent in exploring novel object Time spent in exploring familiar object.

4.5. Rolipram, roflumilast and roflumilast N-oxide concentrations in plasma and brain Five minutes after ORT (10th day of experiment), rats were anaesthetized using isoflurane, and blood was collected retro-orbitally. After blood sampling, rats were euthanized by over dose of isoflurane and the brain was removed. Blood was centrifuged at 3000  g for 10 min at 4 1C. Plasma and brain were frozen at 80 1C until compound level determination took place. The concentrations of roflumilast and roflumilast N-oxide in plasma and brain were measured using a gradient LC/MS/MS procedure as described previously (Thappali et al., 2012). For rolipram, 200 ml plasma/brain homogenate (80:20 acetonitrile and water) were vortexed with 200 ml of acetonitrile for 60 sec. Then, the samples were centrifuged at 10000 g for 15 min, 50 ml of supernatant was

injected into the chromatographic system. The HPLC (Shimadzu Ltd., Japan) system consisted of a LC-20 AD binary pump, auto injector (SIL-20 AC HT), column oven (CTO-10 AS VP) and PDA detector (SPD-M20A). The wavelength of the detector was set at 230 nm. Detector output was quantified on Lab Solutions platform. Separation was carried out on an Enable C18G column 250  4.6 mm) using a mixture of 0.5% formic acid and ACN (60:40) as a mobile phase, at a flow rate of 1 ml/min. Total analysis time was 15 min.

4.6.

Corticosterone analysis

The plasma concentration of corticosterone was quantified using enzyme-linked immunosorbent assay (Cayman Chemical, Ann Arbor, MI, USA).

4.7.

Reverse transcriptase-PCR

Reverse transcriptase-PCR (RT-PCR) was performed to determine the mRNA expression of PDE-4 A, B and D in the rat hippocampus. Briefly, total RNA was extracted from hippocampus using TRIzol Reagent (Sigma). After homogenization, the tubes were incubated for 10 min and centrifuged at 100 g for 5 min. Then, 200 μl of chloroform was added to the supernatant, allowed to incubate for 5 min at room temperature and centrifuged at 12000 g for 20 min. The supernatant was transferred to the new centrifuge tube to which 500 μl of isopropyl alcohol was added to precipitate the total RNA and centrifuged at 12000 g for 15 min following the incubation period of 10 min. The supernatant was decanted carefully and the pellet was washed three times with 75% ethanol and centrifuged at 12000 g for 15 min and the pellet was dried and resuspended in 20 μl of RNase free water. The concentration of RNA was determined using the nanodrop spectrophotometer and RNA samples were stored at 80 1C until use. Two hundred nanogram of RNA were used for RT-PCR according to the manufacturer's instructions (Genet Bio, Korea). Isolated RNA was allowed to undergo reverse transcription and polymerization reaction to synthesize cDNA using PCR master cycler gradient. The following steps were performed for each PCR reaction: 42 1C for 30 sec, 94 1C for 5 min (1 cycle); 94 1C for 1 min, β-actin (55.4 1C), PDE-4 A (71.0 1C), PDE-4B (59.7 1C), PDE-4D (64.0 1C) and BDNF (60.2 1C) and 72 1C for 1 min (with 35 cycles); and a final extension phase at 72 1C for 5 min. The concentration of Table 1 – Primer sequences for reverse transcriptase – PCR. Name

Primer

Sequence

PDE-4A

Upstream Downstream Upstream Downstream Upstream Downstream Upstream Downstream Upstream Downstream

50 -gcttgaacaccaacgtcccacggt-30 50 -gctgaggttctggaagatgtcgcag-30 50 -atgacccagataagtggagtgaag-30 50 -tcaggctgaaccaggtctgcccag-30 50 -atggcctccaacaagttcaagagg-30 50 -gctggctttcctcttctgctacagcc-30 50 -agcctcctctgctctttctgctgga-3’ 50 -cttttgtctatgcccctgcagcctt-3’ 50 -tcatgaagtgtgacgttgacatccgt-30 50 -cctagaagcatttgcggtgcacgatg-30

PDE-4B PDE-4D BDNF β Actin

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cDNA was analyzed using the nanodrop spectrophotometer. The RT-PCR products were loaded in 1% agarose gel and electrophoresed at 40 V for 30 min and bands obtained were quantified using gel documentation (Vilber, France). The primers are listed in Table 1

4.8.

Blotting analysis

Hippocampal samples were homogenized in ice-cold RIPA buffer containing 0.1% phenyl methyl sulfonyl fluoride (PMSF) and assessed for expressions of phosphorylation of CREB (pCREB) and BDNF. After lysis in ice, samples were centrifuged at 10000  g for 30 min at 4 1C. After determining the protein concentration (Bradford, 1976), equal amount of protein (75 mg for pCREB and 25 mg for BDNF) from each samples was separated by electrophoresis in 12% polyacrylamide gels (100 V for 2 h), prior to the transfer to PVDF membranes for pCREB and nitrocellulose membrane for BDNF (25 V for 3 h). Nonspecific bindings were blocked with Tris- buffer saline 0.02 M TBS containing 5% BSA for pCREB and 5% nonfat milk for BDNF with 0.1% Tween-20, for 1 h at room temperature. Membranes were then incubated with rabbit anti-pCREB (Ser133), (1:500; Santa Cruz, CA, USA), BDNF (1:200; Santa Cruz, CA, USA), β-actin (1:100; Santa Cruz, CA, USA) at 4 1C overnight. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary Ab (1:2000, Product No, A4919; Sigma, USA) for 2 h at room temperature. Blots were visualized using a DAB system (Genei, Bangalore, India). The relative expression levels of pCREB and BDNF were determined by densitometry using Image J (Image Processing & Analysis in Java) software.

4.9.

Statistical analysis

Data are presented as mean7S.E.M. Statistical analysis was performed using GraphPad Prism Software (Version 5; Inc.). The difference between time spent exploring the novel object versus familiar object during discrimination trial was calculated for each group and the level of significance was analyzed using two-sided Student's t test. For other parameters, the effect of sham Vs vehicle was analyzed using a two-sided Student’s t test and each treatment group Vs vehicle group was analyzed using one-way analysis of variance (ANOVA) followed by Dunnet’s post hoc test. The level of significance was set at Po0.05.

Conflict of interest statement The authors declare no conflict of interest.

Acknowledgment The authors express their sincere thanks to Dr. S. P. Thyagarajan, Professor of Eminence & Dean (Research), SRU for his advice and support. Authors extend their thanks to all technical and non-technical staff of CEFT, SRU for their help during the conduct of the experiments.

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