Psychopharmacology

, Volume 223, Issue 3, pp 307–317

Defensive effect of natrium diethyldithiocarbamate trihydrate (NDDCT) and lisinopril in DOCA–salt hypertension-induced vascular dementia in rats

Authors

  • Bhupesh Sharma
    • Pharmacology Division, Department of Pharmaceutical Sciences and Drug Research, Faculty of MedicinePunjabi University
    • Pharmacology Division, Department of Pharmaceutical Sciences and Drug Research, Faculty of MedicinePunjabi University
Original Investigation

DOI: 10.1007/s00213-012-2718-9

Cite this article as:
Sharma, B. & Singh, N. Psychopharmacology (2012) 223: 307. doi:10.1007/s00213-012-2718-9
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Abstract

Rationale

Vascular dementia and hypertension are increasing day by day, with a high degree of co-occurrence. Tremendous amount of research work is required so that new pharmacological agents may be identified for their appropriate therapeutic utility to combat different dementing disorders.

Objectives

This study investigates the effect of natrium diethyldithiocarbamate trihydrate (NDDCT), a nuclear factor kappa-B (NF-κB) inhibitor, as well as lisinopril, an angiotensin converting enzyme (ACE) inhibitor, on deoxycorticosterone acetate (DOCA) hypertension-induced vascular dementia in rats.

Methods

DOCA was used to induce hypertension and associated vascular dementia. Morris water maze (MWM) was used for testing learning and memory. Endothelial function was assessed by acetylcholine-induced endothelium-dependent relaxation of aortic strips. Different biochemical estimations were used to assess oxidative stress (aortic superoxide anion, serum and brain thiobarbituric acid reactive species, and brain glutathione), nitric oxide levels (serum nitrite/nitrate), and cholinergic activity (brain acetyl cholinesterase activity).

Results

DOCA treatment significantly raised the mean arterial blood pressure of rats, and these hypertensive rats performed poorly on MWM, reflecting impairment of learning and memory. DOCA treatment also impaired vascular endothelial function and different biochemical parameters. Treatments of NDDCT as well as lisinopril significantly attenuated DOCA hypertension-induced impairment of learning and memory, endothelial dysfunction, and changes in various biochemical levels.

Conclusions

DOCA–salt hypertension induces vascular dementia in rats. NF-κB as well as ACE inhibitors may be considered as potential pharmacological agents for the management of hypertension-induced vascular dementia.

Keywords

Nuclear factor kappa-BAngiotensin converting enzymeEndothelial dysfunctionMorris water mazeAlzheimer’s diseaseOxidative stress

Introduction

Cardiovascular risk factors, including hypertension have been linked to subsequent incidence, onset, and progression rate of dementia of vascular origin and other etiology like Alzheimer’s disease (AD) (Monsuez et al. 2011). Vascular dementia (VaD) represents the second most common cause of dementia after AD in the elderly and is referred as the “silent epidemic of the twenty-first century” (Battistin and Cagnin 2010). Several epidemiological studies reported associations of hypertension, diabetes, obesity, and inflammation with VaD and in some cases, AD (Moretti et al. 2011). Optimal treatment of cardiovascular risk factors prevents and slows down age-related cognitive disorders (Monsuez et al. 2011). Further, it has been suggested that dementia prevention can become effective without delay if the vascular components of dementia are aggressively targeted through the treatment of vascular risk factors such as hypertension (Wehling and Groth 2011).

Hypertension has been found to increase the probability of endothelial dysfunction and VaD (Lorenza et al. 2011; Yang et al. 2011). Endothelial dysfunction is a hallmark of, and plays a pivotal role in the pathogenesis of cardiometabolic diseases, including type II diabetes, obesity, and hypertension (Zhang et al. 2011). Integrity of the vascular system is essential for the efficient functioning of the brain. Aging-related structural and functional disturbances in the macro- or microcirculation of the brain make it vulnerable to cognitive dysfunction, leading to dementing illness (Kalaria 2010). In previous reports from our lab, we have shown that endothelial dysfunction results in vascular dementia in different metabolic-disordered conditions (Koladiya et al. 2008; Sharma and Singh 2010, 2011).

The nuclear factor kappa-B (NF-κB) family of transcription factors regulates the induction and resolution of inflammation. NF-κB is thought to promote cardiovascular disorder including endothelial dysfunction through its pro-inflammatory, pro-adhesion, and prooxidant gene transcription (Anthony et al. 2009). It has also been suggested that NF-κB is involved in amyloid beta-42-induced neuroal cell death and subsequent memory impairment (Kim et al. 2009). In aging-induced dementia, there is an activation of NF-κB which results in the upregulation of genes of pro-inflammatory enzymes (Kim et al. 2010). It has previously been reported that NF-κB plays an important role in learning and memory (Meffert et al. 2003). NF-κB has been reported to impair spatial learning in mice (Fridmacher et al. 2003) and reduce long-term potentiation and long-term depression (Kaltschmidt et al. 2006). NF-κB has reported to reduce protein kinase A activity and thus regulate PKA/CREB signaling, which is an essential pathway for learning and memory (Kandel 2001). Furthermore, NF-κB has also been reported as an important regulator of neuronal morphology and shapes brain structures that are important for learning and memory (Kaltschmidt and Kaltschmidt 2009). Overexpression of NF-κB has also been reported to cause defects in novel task acquisition and reduced short-term memory (Denis-Donini et al. 2008). NF-κB has been suggested to be involved in consolidation of memory (Levenson et al. 2004). It interferes with the activity of glutamatergic as well as GABAergic neurons which are responsible for learning and memory (Kaltschmidt and Kaltschmidt 2009). NF-κB inhibition has shown enhanced spatial learning memory and LTP (Kaltschmidt and Kaltschmidt 2009). However, the modulation of NF-κB in dementia of vascular origin due to hypertension is still not explored.

Angiotensin converting enzyme (ACE) inhibitors decrease blood pressure and systemic vascular resistance by blocking the formation of angiotensin II, the endogenous pressor substance of the renin–angiotensin cascade. While these agents have been used primarily in the treatment of hypertension and congestive heart failure, there are few pre-clinical and clinical studies in which ACE inhibitors have been shown to reduce the incidence of dementia or slow down the rate of cognitive decline in patients with hypertension (Sink et al. 2009; Yamada et al. 2010). Moreover, utility of ACE inhibitors in hypertension-induced vascular dementia is still unknown.

In the light of the one mentioned above, the present study has been undertaken to investigate the potential of NF-κB inhibitor, natrium diethyldithiocarbamate trihydrate (Spiller et al. 2011) as well as ACE inhibitor, lisinopril in deoxycorticosterone acetate (DOCA) hypertension-induced vascular dementia in rats. Donepezil has been used as positive control in this study.

Material and methods

Animals

Albino Wistar rats, weighing 200–250 g, were employed in the present study and were housed in animal house with free access to water and standard laboratory pellet chow diet (Kisan Feeds Ltd, Mumbai, India). The animals were exposed to12-h light/dark cycle. The experiments were conducted between 900 and 1800 hours in a semi sound-proof laboratory. The animals were acclimatized to the laboratory condition 5 days prior to behavioral study. The protocol of the study was duly approved by the Institutional Animal Ethics Committee, and care of the animals was taken as per the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests, Government of India (Reg. No. 107/1999/CPCSEA).

Drugs and chemicals

All the drug solutions were freshly prepared before use. Lisinopril was obtained as ex gratia from Cipla. Natrium diethyldithiocarbamate trihydrate (NDDCT), deoxycorticosterone acetate (DOCA), and 1,1,3,3 tetra methoxy propane were purchased from Sigma-Aldrich (St. Louis, MO, USA). 5,5′-Dithiobis (2-nitro benzoic acid), bovine serum albumin, glutathione (GSH) reduced standard, and nitroblue tetrazolium were purchased from Sisco Research Laboratories Pvt Ltd., Mumbai, India. Thiobarbituric acid was purchased from Loba Chemie, Mumbai. NDDCT and donepezil were dissolved in saline and administered intraperitoneally. Lisinopril was suspended in 1 % w/v of sodium carboxy methyl cellulose (CMC). DOCA was dissolved in Arachis oil and administered subcutaneously. Lisinopril and CMC were administered orally with the help of an oral tube (canulla). All the drug solutions were freshly prepared before use.

Deoxycorticosterone acetate hypertension-induced vascular dementia

Rats were administered with DOCA (20 mg/kg, s.c. twice weekly) for 90 days, and the drinking water was replaced with solution of 1 % sodium chloride (NaCl) and 0.2 % potassium chloride (KCl), to produce hypertension (Bockman et al. 1992). The animals were used on the 86th day for the behavioral and other assessment. Mean arterial blood pressure (MABP) was measured by BIOPAC MP100, using AcqKnowledge 3.8.2. analysis system.

Assessment of learning and memory by Morris water maze

Morris water maze (Morris 1984; Parle and Singh 2004; Sharma and Singh 2010) is one of the most commonly used animal models to test memory. Morris water maze (MWM) procedure was based on the principle where animal was placed in a large pool of water divided into four equal quadrants, as animal dislikes swimming, its tendency to escape was accomplished by finding a hidden escape platform. Each animal was subjected to four consecutive training trials (with an inter gap of 5 min) each day for four consecutive days in search for a hidden platform. The day 4 escape latency time (ELT) to locate the hidden platform in water maze was taken as the index of acquisition or learning. On the fifth day, the hidden platform was removed. Each animal was allowed to explore the pool for 120 s. The mean time spent in all the quadrants in search of the hidden platform was noted. The mean time spent by the animal in the target quadrant was taken as the index of retrieval or memory.

Assessment of vascular endothelial function using isolated rat aortic ring preparation

Rats were decapitated and the thoracic aorta was removed, cut into a ring of 4 to 5 mm width, and mounted in organ bath containing Krebs–Henseleit bubbled with carbonated oxygen (95 % O2:5 % CO2), and maintained at 37.8°C. The preparation was allowed to equilibrate for 90 min under 1.5-g tension. The isometric contractions were recorded with a force–displacement transducer (Ft-2147) connected to Physiograph (INCO, Ambala, India). The preparation was primed with 80 mmol L−1 KCl to check its functional integrity and to improve its contractility. The cumulative dose–responses of acetylcholine (ACh; 10−8 to 10−4 mol L−1) or sodium nitroprusside (SNP; 10−8 to 10−4 mol L−1) were recorded in phenylephrine (3 × 10−6 mol L−1)-precontracted preparations (Koladiya et al. 2008; Sharma and Singh 2010; Sharma and Singh 2011). The intimal layer of aortic ring was rubbed gently with a moistened filter paper for 30 s to obtain endothelium-free preparations. Loss of ACh (1 × 10−6 mol L−1)-induced relaxation confirmed the absence of vascular endothelium (Koladiya et al. 2008; Sharma and Singh 2010; Sharma and Singh 2011).

Biochemical parameters

Collection of sample

Blood samples for biochemical estimation were collected by retro-orbital bleeding. The blood was kept at room temperature for 30 min and then centrifuged at 4,000 rpm for 15 min to separate the serum.

After last retro-orbital bleeding, animals were killed by cervical dislocation; thoracic aorta and brain tissue were carefully removed. Thoracic aorta was used for endothelium-dependent and endothelium-independent relaxation as well as for the estimation of superoxide anion, whereas brain tissue was subjected to various biochemical estimations. The removed brains were homogenized in phosphate buffer (pH 7.4, 10 % w/v) using Teflon homogenizer and centrifuged at 3,000 rpm for 15 min to obtain the clear supernatant. Serum and clear supernatant were then used for different biochemical estimations.

Estimation of serum glucose levels

The glucose levels were estimated spectrophotometerically (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at 505 nm by glucose oxidase peroxidase method using a commercially available kit (Reckon diagnostics Pvt. Ltd. Vadodra, India).

Estimation of serum nitrite concentration

Serum nitrite concentration was serum nitrite was measured spectophotometrically (DU 640B Spectrophotometer, Beckman Coulter Inc., CA, USA) at 545 nm, using the method of Sastry et al. (2002; Sharma and Singh 2010).

Estimation of aortic production of super oxide anion

The superoxide anion was determined spectrophotometrically (DU 640B Spectrophotometer, Beckman Coulter, Inc.) at 540 nm using the method of Wang et al. (1998; Sharma and Singh 2010).

Estimation of brain acetyl cholinesterase activity

The whole brain acetyl cholinesterase (AChE) activity was measured spectrophotometerically (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at 420 nm by the method of Ellman et al. (1961; Sharma and Singh 2010).

Estimation of thiobarbituric acid reactive substances

The brain/serum thiobarbituric acid reactive substances (TBARS) was measured spectrophotometrically (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at 532 nm using the method of Ohkawa et al. (1979; Sharma and Singh 2010).

Estimation of reduced glutathione

The reduced GSH content in the brain was estimated spectrophotometrically (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at 412 nm using the method of Beutler et al. (1963; Sharma and Singh 2010).

Estimation of brain total protein

The brain total protein was determined spectrophotometrically (DU 640B spectrophotometer, Beckman Coulter Inc., CA, USA) at 750 nm using the method of Lowry’s et al. (1951; Sharma and Singh 2010).

Experimental procedure

In total, 15 groups have been employed in the present study and each group consisted of eight male Wistar rats. Based on our pilot studies and some literature, we selected two best effective doses of NDDCT (5 and 10 mg kg−1, i.p.). Similarly, various doses of lisinopril have been used in various conditions starting from as low as 0.1, 1, 75 mg/kg, and so on. We have used the doses of lisinopril (10 and 15 mg kg−1, p.o.) on the bases of pilot studies and published research reports.

In the present study, hypertension and subsequent VaD in rats were induced by administering DOCA and salt solution for 90 days (85 + 5 days during MWM exposure). We carried out some pilot studies and did measure the blood pressure at different time intervals and found that after DOCA–salt treatment a stable hypertension is induced in around 50 days (2 months approximately). This is also supported by the published reports suggested that DOCA induces hypertension in 45–90 days (Sahan-Firat et al. 2010; Khazaei et al. 2012). Thus we have used the total of 90 days protocol, which includes 50 days for establishment of stable hypertension. Our pilot studies also suggest that significant observable learning and memory impairment occurs in approximately 3 months period after administration of DOCA and salt solution. Further, to observe the effects of the therapeutics on hypertension-induced VaD, both the drugs were administered for the last 40 days viz day 51 to day 90 (35 + 5 days during MWM exposure) in DOCA-treated animals. The idea was to target hypertension-induced memory deficits (vascular dementia) hence this particular period of drug treatment was selected.

Group I—control group

Animals were exposed to Morris water maze for acquisition trial from day 1 to day 4 and retrieval trial on day 5.

Group II—vehicle control group (0.9 % saline)

Animals were administered saline (10 ml kg−1 i.p., daily) for 35 days followed by exposure to Morris water maze. The treatment was continued during acquisition (from the 36th to 39th day) and retrieval trials (on the 40th day) on Morris water maze.

Group III—vehicle control group (CMC)

Animals were administered with CMC (10 ml kg−1 i.p., daily) for 35 days; the rest of the procedure was same as described in group II.

Group IV—vehicle control group (Arachis oil)

Animals were administered with Arachis oil (maximum 2.5 ml kg−1 s.c., twice weekly), for 85 days followed by exposure to Morris water maze. The treatment was continued during acquisition (from the 86th to 89th day) and retrieval trials (on the 90th day) on Morris water maze.

Group V—DOCA treatment group

Animals were administered with DOCA (20 mg kg−1 s.c., twice weekly), for 85 days followed by exposure to Morris water maze. The treatment was continued during acquisition (from the 86th to 89th day) and retrieval trials (on the 90th day) on Morris water maze. Drinking water of these animals was replaced with the solution of 1 % NaCl and 0.2 % KCl.

Group VI—natrium diethyldithiocarbamate trihydrate (5 mg kg−1 day−1) per se

Animals were administered with NDDCT (5 mg kg−1 i.p., daily) for 35 days; the rest of the procedure was the same as described in group II.

Group VII—NDDCT (10 mg kg−1 day−1) per se

Animals were administered with NDDCT (10 mg kg−1 i.p., daily) for 35 days; the rest of the procedure was the same as described in group II.

Group VIII—DOCA and NDDCT (5 mg kg−1 day−1)

NDDCT (5 mg kg−1 i.p., daily) was administered to the DOCA-treated rats, starting from the 51st day of DOCA treatment followed by exposure to Morris water maze on the 86th day of DOCA administration. The treatment was continued during acquisition (from the 86th to 89th day) and retrieval trials (on the 90th day) on Morris water maze.

Group IX—DOCA and NDDCT (10 mg kg−1 day−1)

NDDCT (10 mg kg−1 i.p., daily) was administered to the DOCA-treated rats; the rest of the procedure was the same as described in group VIII.

Group X—lisinopril (10 mg kg−1 day−1) per se

Animals were administered with lisinopril (10 mg kg−1 p.o., daily) for 35 days; the rest of the procedure was the same as described in group II.

Group XI—lisinopril (15 mg kg−1 day−1) per se

Animals were administered with lisinopril (15 mg kg−1 p.o., daily) for 35 days; the rest of the procedure was the same as described in group II.

Group XII—DOCA and lisinopril (10 mg kg−1 day−1)

Lisinopril (10 mg kg−1 p.o., daily) was administered to the DOCA-treated rats, starting from the 51st day of DOCA treatment followed by exposure to Morris water maze on the 86th day of DOCA administration. The treatment was continued during acquisition (from the 86th to 89th day) and retrieval trials (on the 90th day) on Morris water maze.

Group XIII—DOCA and lisinopril (15 mg kg−1 day−1)

Lisinopril (15 mg kg−1 p.o., daily) was administered to the DOCA-treated rats; the rest of the procedure was the same as described in group XII.

Group XIV—donepezil (0. 5 mg kg−1 day−1) per se

Animals were administered with donepezil (0.5 mg kg−1 i.p., daily) for 35 days; the rest of the procedure was the same as described in group II.

Group XV—DOCA and donepezil

Donepezil (0.5 mg kg−1 i.p., daily) was administered to the DOCA (20 mg kg−1 s.c., twice weekly)-treated rats, starting from the 51st day of DOCA treatment followed by exposure to Morris water maze on the 86th day of DOCA administration. The treatment was continued during acquisition (from the 86th to 89th day) and retrieval trials (on the 90th day) on Morris water maze.

Statistical analysis

All results were expressed as mean ± S.E.M. Data for isolated aortic ring preparation were statistically analyzed using repeated measures of analysis of variance (ANOVA) followed by Newman–Keuls test. All other results were analyzed using one-way ANOVA followed by Tukey’s multiple range test. P < 0.05 was considered to be statistically significant.

Results

Effect on escape latency time and time spent in target quadrant, using Morris water maze

Control rats showed a downward trend in their ELT. There was a significant fall in day 4 ELT, when compared to day 1 ELT of these rats (Table 1), reflecting normal learning ability. Further, on day 5 a significant rise in time spent in target quadrant (TSTQ) was observed, when compared to time spent in other quadrants (Fig. 1), reflecting normal retrieval as well. Administration of 0.9 % saline water (10 ml kg−1 i.p., 40 days)/CMC (10 ml kg−1 p.o., 40 days)/Arachis oil (maximum 2.5 ml kg−1 s.c., twice weekly for 90 days) did not show any significant effect on ELT and TSTQ as compared to the control rats. Administration of NDDCT (5 mg kg−1 i.p./10 mg kg−1 i.p., 40 days)/lisinopril (10 mg kg−1 p.o./15 mg kg−1 p.o., 40 days)/donepezil (0.5 mg kg−1 i.p., 40 days) did not show any significant per se effect on ELT and TSTQ (Table 1 and Fig. 1). However, DOCA (20 mg kg−1 s.c., twice weekly for 90 days)-treated rats showed a significant increase in day 4 ELT (89th day of DOCA treatment), when compared to day 4 ELT of the control animals (Table 1), indicating impairment of acquisition. Further, DOCA administration also produced a significant decrease in day 5 TSTQ (90th day of DOCA treatment), when compared day 5 TSTQ of the control animals (Fig. 1), indicating impairment of memory as well.
Table 1

Effect of various agents, on mean arterial blood pressure (MABP) and day 4 escape latency time (ELT) of animals, using Morris water maze

Group

Name of the group

Mean arterial blood pressure (mmHg)

Escape latency time (in seconds)

Basal

Final

Day 1

Day 4

I

Control

88.1 ± 2.3

92.2 ± 2.9

99.2 ± 2.3

42.2 ± 2.6*

II

Vehicle control (0.9 % NaCl—10 ml kg−1)

90.3 ± 3.2

91.1 ± 2.7

101.5 ± 3.2

48.2 ± 4.3*

III

Vehicle control (CMC—10 ml kg−1)

89.5 ± 2.8

90.6 ± 2.5

103.4 ± 2.7

49.6 ± 3.6*

IV

Vehicle control (Arachis oil—2.5 ml kg−1)

87.7 ± 3.3

89.1 ± 3.1

97.4 ± 3.5

44.2 ± 2.8*

V

DOCA (20 ml kg−1)

90.7 ± 2.4

165.6 ± 4.2**

106.3 ± 2.8

82.2 ± 3.6***

VI

NDDCT (5 ml kg−1) per se

87.1 ± 2.9

90.5 ± 2.3

98.2 ± 4.1

47.3 ± 3.2*

VII

NDDCT (10 ml kg−1) per se

88.3 ± 3.6

91.3 ± 2.1

100.4 ± 3.2

50.1 ± 2.3*

VIII

DOCA and NDDCT (5 ml kg−1)

87.3 ± 3.1

157.2 ± 3.1**

102.3 ± 4.2

60.4 ± 2.9*, ****

IX

DOCA and NDDCT (10 ml kg−1)

89.4 ± 2.4

160.3 ± 4.1**

106.3 ± 4.2

54.5 ± 3.8*, ****

X

Lisinopril (10 ml kg−1) per se

89.1 ± 3.2

87.1 ± 2.6

101.2 ± 3.8

48.1 ± 2.4*

XI

Lisinopril (15 ml kg−1) per se

91.3 ± 1.9

90.3 ± 3.4

102.1 ± 2.8

45.1 ± 3.6*

XII

DOCA and lisinopril (10 ml kg−1)

90.3 ± 2.7

118.2 ± 5.6*****

105.5 ± 4.1

64.7 ± 2.8*, ****

XIII

DOCA and lisinopril (15 ml kg−1)

86.6 ± 2.8

108.1 ± 5.2*****

102.1 ± 3.2

53.1 ± 4.3*, ****

XIV

Donepezil (0.5 ml kg−1) per se

88.4 ± 3.3

92.2 ± 2.9

103.5 ± 2.7

44.2 ± 3.6*

XV

DOCA and donepezil (0. 5 ml kg−1)

91.5 ± 3.4

161.5 ± 3.3**

112.1 ± 3.2

53.1 ± 4.4*, ****

Results are mean ± standard error of means; one-way ANOVA followed by Tukey’s multiple range test (n = 8)

NaCl sodium chloride, CMC sodium carboxymethylcellulose, DOCA deoxycorticosterone acetate, NDDCT natrium diethyldithiocarbamate trihydrate

*P < 0.001 versus day 1 ELT in respective group (day 4 ELT—F (14, 105) = 9.048); **P < 0.001 versus MABP of control group (MABP—F (14, 105) = 82.338); ***P < 0.001 versus day 4 ELT of control group; ****P < 0.001 versus day 4 ELT of DOCA-treated group (day 4 ELT—F (14, 105) = 9.048);*****P < 0.001 versus MABP of DOCA-treated group (MABP—F (14, 105) = 82.338);

https://static-content.springer.com/image/art%3A10.1007%2Fs00213-012-2718-9/MediaObjects/213_2012_2718_Fig1_HTML.gif
Fig. 1

Effect on time spent in target quadrant (TSTQ) of animals using Morris water maze (n = 8). Results are mean ± standard error of means; one-way ANOVA followed by Tukey’s multiple range test. a P < 0.001 versus mean time spent in other quadrants in control; b P < 0.001 versus mean time spent in target quadrant in control group; c P < 0.001 versus mean time spent in target quadrant in DOCA-treated group, F (14, 105) = 20.896. NaCl sodium chloride, CMC sodium carboxymethylcellulose, DOCA deoxycorticosterone acetate, NDDCT natrium diethyldithiocarbamate trihydrate

Administration of NDDCT (5 mg kg−1 i.p./10 mg kg−1 i.p., 40 days)/lisinopril (10 mg kg−1 p.o./15 mg kg−1 p.o., 40 days)/donepezil (0.5 mg kg−1 i.p., 40 days) significantly prevented DOCA-induced rise in day 4 ELT, indicating reversal of DOCA-induced impairment of acquisition (Table 1). Further, treatment of these drugs also attenuated DOCA-induced decrease in day 5 TSTQ in a significant manner, indicating reversal of DOCA-induced impairment of memory (Fig. 1).

Effect on endothelium-dependent and endothelium-independent relaxation

ACh and SNP in a dose-dependent manner produced endothelium-dependent and endothelium-independent relaxation in phenylephrine (3 × 10−6 M)-precontracted isolated rat aortic ring preparation. DOCA administration significantly attenuated acetylcholine-induced endothelium-dependent relaxation (Fig. 2); however, it did not affect SNP-induced endothelium-independent relaxation (Fig. 3). Treatment of NDDCT (5 mg kg−1 i.p./10 mg kg−1 i.p., 40 days)/lisinopril (10 mg kg−1 p.o./15 mg kg−1 p.o., 40 days)/donepezil (0.5 mg kg−1 i.p., 40 days) significantly abolished the effect of DOCA on endothelial-dependent relaxation. Further, NDDCT (5 mg kg−1 i.p./10 mg kg−1 i.p., 40 days)/lisinopril (10 mg kg−1 p.o./15 mg kg−1 p.o., 40 days)/donepezil (0.5 mg kg−1 i.p., 40 days) did not show any per se effect on endothelium-dependent relaxation.
https://static-content.springer.com/image/art%3A10.1007%2Fs00213-012-2718-9/MediaObjects/213_2012_2718_Fig2_HTML.gif
Fig. 2

Effect on acetylcholine-induced endothelium-dependent relaxation using aortic ring preparation (n = 8). Responses are expressed as percentage of precontraction induced by 3 × 10−6 M phenylephrine. Results are mean ± standard error of means; repeated measure ANOVA followed by Newman–Keuls test. a P < 0.05 versus control; b P < 0.05 versus DOCA-treated group. Ach acetylcholine, DOCA deoxycorticosterone acetate, NDDCT natrium diethyldithiocarbamate trihydrate

https://static-content.springer.com/image/art%3A10.1007%2Fs00213-012-2718-9/MediaObjects/213_2012_2718_Fig3_HTML.gif
Fig. 3

Effect of various treatments on sodium nitroprusside-induced endothelium-independent relaxation using aortic ring preparation. Responses are expressed as percentage of precontraction induced by 3 × 10−6 M phenylephrine. (n = 8) Results are mean ± standard error of means; repeated measures of ANOVA followed by Newman–Keuls test. SNP sodium nitroprusside, DOCA deoxycorticosterone acetate, NDDCT natrium diethyldithiocarbamate trihydrate

Effect on mean arterial blood pressure

Administration of DOCA produced a significant increase in MABP as compared to the control rats. Treatment with NDDCT (5 mg kg−1 i.p./10 mg kg−1 i.p., 40 days)/donepezil (0.5 mg kg−1 i.p., 40 days) did not shown any significant change in DOCA-induced increase in MABP (Table 1). Lisinopril (10 mg kg−1 p.o./15 mg kg−1 p.o., 40 days) has significantly reduced the DOCA-induced increase in MABP. Further, NDDCT (5 mg kg−1 i.p./10 mg kg−1 i.p., 40 days)/lisinopril (10 mg kg−1 p.o./15 mg kg−1 p.o., 40 days)/donepezil (0.5 mg kg−1 i.p., 40 days) did not show any significant per se effect on MABP of the animals (Table 1).

Effect on serum nitrite level

Administration of DOCA produced a significant decrease in serum nitrite, when compared to the control rats. Treatment with NDDCT (5 mg kg−1 i.p./10 mg kg−1 i.p., 40 days)/lisinopril (10 mg kg−1 p.o./15 mg kg−1 p.o., 40 days)/donepezil (0.5 mg kg−1 i.p., 40 days) prevented DOCA-induced decrease in serum nitrite level in a significant manner (Table 2). Further, NDDCT (5 mg kg−1 i.p./10 mg kg−1 i.p., 40 days)/lisinopril (10 mg kg−1 p.o./15 mg kg−1 p.o., 40 days)/donepezil (0.5 mg kg−1 i.p., 40 days) did not show any significant per se effect on serum nitrite level (Table 2).
Table 2

Effect of various agents on oxidative stress (superoxide anion, TBARS, and GSH) and brain acetyl cholinesterase (AChE) activity and serum nitrite/nitrate levels of animals

Group

Name of the Group

Serum nitrite/nitrate (μM/L)

Serum TBARS (μM/L)

Aortic superoxide anion (reduced NBT—pM/min/mg)

Brain AchE activity (μM of ACh hydrolyzed/min/mg protein)

Brain TBARS (nM/mg protein)

Brain GSH (μM/mg of protein)

I

Control

12.8 ± 1.2

3.8 ± 0.6

3.7 ± 0.4

3.1 ± 0.5

4.2 ± 0.7

18.2 ± 2.2

II

Vehicle control (0.9 % NaCl—10 ml kg−1)

11.2 ± 1.1

3.7 ± 0.4

3.6 ± 0.5

3.3 ± 0.7

4.1 ± 0.6

17.3 ± 2.1

III

Vehicle control (CMC—10 ml kg−1)

12.2 ± 1.1

3.8 ± 0.4

3.5 ± 0.4

3.0 ± 0.4

3.9 ± 0.9

18.4 ± 2.2

IV

Vehicle control (Arachis oil—2.5 ml kg−1)

11.6 ± 1.0

3.6 ± 0.3

3.3 ± 0.7

2.9 ± 0.9

4.5 ± 0.5

17.5 ± 2.1

V

DOCA (20 ml kg−1)

6.5 ± 0.6*

7.3 ± 1.1**

6.9 ± 0.7***

11.6 ± 1.7****

11.2 ± 1.2*****

7.2 ± 0.9

VI

NDDCT (5 ml kg−1) per se

11.4 ± 1.0

3.6 ± 0.5

3.1 ± 0.5

3.5 ± 0.6

3.9 ± 0.9

17.8 ± 2.1

VII

NDDCT (10 ml kg−1) per se

12.3 ± 1.1

3.9 ± 0.5

3.2 ± 0.8

3.6 ± 0.4

4.7 ± 0.6

18.3 ± 2.2

VIII

DOCA and NDDCT (5 ml kg−1)

8.9 ± 0.8******

5.4 ± 1.3*******

5.9 ± 0.4********

7.1 ± 1.9*********

7.2 ± 1.8**********

10.3 ± 1.2

IX

DOCA and NDDCT (10 ml kg−1)

9.7 ± 0.9******

6.1 ± 2.1*******

4.6 ± 0.8********

6.4 ± 2.3*********

5.9 ± 2.6**********

11.5 ± 1.4

X

Lisinopril (10 ml kg−1) per se

11.7 ± 1.1

3.3 ± 0.4

3.4 ± 0.3

3.2 ± 0.7

4.3 ± 0.8

18.7 ± 2.2

XI

Lisinopril (15 ml kg−1) per se

11.9 ± 1.1

3.4 ± 0.5

3.1 ± 0.9

3.5 ± 0.5

4.5 ± 0.9

18.3 ± 2.2

XII

DOCA and lisinopril (10 ml kg−1)

9.1 ± 0.8******

6.4 ± 1.2*******

5.5 ± 0.7********

8.6 ± 1.3*********

8.8 ± 1.6**********

13.2 ± 1.6

XIII

DOCA and lisinopril (15 ml kg−1)

10.2 ± 0.9******

5.1 ± 1.6*******

5.1 ± 0.5********

7.7 ± 1.7*********

7.5 ± 1.5**********

11.5 ± 1.4

XIV

Donepezil (0.5 ml kg−1)per se

12.1 ± 1.1

3.7 ± 0.4

3.9 ± 0.7

3.4 ± 0.6

4.2 ± 0.7

17.5 ± 2.1

XV

DOCA and donepezil (0. 5 ml kg−1)

9.9 ± 0.9******

6.1 ± 0.7*******

6.0 ± 0.3********

4.5 ± 0.9*********

7.6 ± 0.8**********

13.7 ± 1.6

Results are mean ± standard error of means; one-way ANOVA followed by Tukey’s multiple range test (n = 8). Brain GSH—F (14, 105) = 3.898

TBARS thiobarbituric acid reactive species, GSH reduced form of glutathione, NaCl sodium chloride, CMC sodium carboxymethylcellulose, DOCA deoxycorticosterone acetate, NDDCT natrium diethyldithiocarbamate trihydrate

*P < 0.001 versus control group (serum nitrite/nitrate—F (14, 105) = 3.06); **P < 0.05 versus control group (serum TBARS—F (14, 105) = 1.920); ***P < 0.001 versus control group (aortic superoxide anion—F (14, 105) = 4.302); ****P < 0.001 versus control group (brain AChE activity—F (14, 105) = 5.098); *****P < 0.001 versus control group (brain TBARS—F (14, 105) = 3.341); ******P < 0.001 versus DOCA-treated group (serum nitrite/nitrate—F (14, 105) = 3.06); *******P < 0.05 versus DOCA-treated group (serum TBARS—F (14, 105) = 1.920); ********P < 0.001 versus DOCA-treated group (aortic superoxide anion—F (14, 105) = 4.302); *********P < 0.001 versus DOCA-treated group (brain AChE activity—F (14, 105) = 5.098); **********P < 0.001 versus DOCA-treated group (brain TBARS—F (14, 105) = 3.341)

Effect on brain acetyl cholinesterase activity

Administration of DOCA produced a significant increase in brain AChE activity, when compared to the control rats. Treatment with NDDCT (5 mg kg−1 i.p./10 mg kg−1 i.p., 40 days)/lisinopril (10 mg kg−1 p.o./15 mg kg−1 p.o., 40 days)/donepezil (0.5 mg kg−1 i.p., 40 days) significantly prevented DOCA-induced rise in brain AChE activity. Further, NDDCT (5 mg kg−1 i.p./10 mg kg−1 i.p., 40 days)/lisinopril (10 mg kg−1 p.o./15 mg kg−1 p.o., 40 days)/donepezil (0.5 mg kg−1 i.p., 40 days) did not show any significant per se effect on brain AChE activity (Table 2).

Effect on oxidative stress levels

Administration of DOCA produced a significant increase in aortic superoxide anion level/brain and serum TBARS (Table 2) and a significant decrease in the brain levels of reduced form of GSH (Table 2), when compared to the control rats, hence reflecting induction of oxidative stress. Treatment with NDDCT (5 mg kg−1 i.p./10 mg kg−1 i.p., 40 days)/lisinopril (10 mg kg−1 p.o./15 mg kg−1 p.o., 40 days)/donepezil (0.5 mg kg−1 i.p., 40 days) significantly prevented DOCA-induced oxidative stress. Further, NDDCT (5 mg kg−1 i.p./10 mg kg−1 i.p., 40 days)/lisinopril (10 mg kg−1 p.o./15 mg kg−1 p.o., 40 days)/donepezil (0.5 mg kg−1 i.p., 40 days) did not show any significant per se effect on oxidative stress level (Table 2).

Discussion and conclusions

Administration of DOCA (20 mg kg−1 s.c., twice weekly for 90 days) in the present investigation has resulted in marked hypertension and associated vascular endothelial dysfunction and memory deficit along with alterations in various biochemical parameters of rats. DOCA hypertensive rats are widely used for the assessment of hypertensive condition and its secondary complications including vascular endothelial dysfunction (Bockman et al. 1992; Kandlikar and Fink 2011; Ocaranza et al. 2011).

DOCA hypertensive rats performed poorly on MWM test, indicating impairment in their learning abilities and memory capacities. Furthermore, a significant rise in brain AChE activity, brain and serum TBARS, aortic superoxide anion along with a fall in brain GSH, and serum nitrite/nitrate levels was also noted. Morris water maze was employed in the present study being one of the most widely used and accepted model to test learning and memory of rodents (Morris 1984; Sharma et al. 2008a and b; Sharma and Singh 2010, 2011).

Chronic administration of DOCA, in our study, produced significant degree of vascular endothelial dysfunction reflected by impairment of acetylcholine-induced endothelial-dependent relaxation of aortic strips and reduction in serum nitrite/nitrate levels. Moreover, a marked level of oxidative stress (increase in aortic superoxide anion in thoracic aorta and serum TBARS) in DOCA-treated rats was also noted. DOCA in earlier studies has been demonstrated to induce endothelial dysfunction, so our findings are in line with previous findings (Sahan-Firat et al. 2010; Szasz and Watts 2010). Further, DOCA has also been documented to enhance the production of free radicals with subsequent increase in oxidative stress (Szasz et al. 2010; Borde et al. 2011). DOCA-induced rise in superoxide anion in aortic strip of present study is a reflection of oxidative stress and probably is one of the major contributing factors in DOCA-induced endothelial dysfunction. Moreover in our earlier studies, we have demonstrated that vascular endothelial dysfunction in addition to impairment of memory and oxidative stress produces rise in brain AChE activity (Koladiya et al. 2008, 2009; Sharma and Singh 2010, 2011). Therefore, the observed DOCA-induced vascular dementia may be due to increase in oxidative stress levels both peripherally as well as centrally, impairment of endothelial function, and increase in brain acetylcholinesterase activity.

Treatments of NDDCT (a NF-κB inhibitor)/lisinopril (an ACE inhibitor)/donepezil (an acetylcholinesterase inhibitor) to DOCA hypertensive rats attenuated endothelial dysfunction, learning and memory impairment, and various biochemical parameters. Lisinopril in addition to the above effects also significantly reduced mean arterial blood pressure of DOCA hypertensive animals; however, no such effect was noted in case of NDDCT and donepezil.

Few earlier studies have documented that DOCA-induced hypertension is involved in the activation of NF-κB (Jadhav et al. 2008, 2009). The NF-κB family of transcription factors regulates the induction and resolution of inflammation. Two main pathways, classical and alternative, control the nuclear translocation of NF-κB. Classical NF-κB activation is usually a rapid and transient response to a wide range of stimuli whose main effector is RelA/p50. The alternative NF-κB pathway is a more delayed response to a smaller range of stimuli resulting in DNA binding of RelB/p52 complexes. Additional complexity in this system involves the posttranslational modification of NF-κB proteins and an ever-increasing range of co-activators, co-repressors, and NF-κB complex proteins (Sanz et al. 2010). NF-κB activation a central event of inflammation has been considered as a common feature of many neurodegenerative diseases including Huntington, Parkinson, stroke, and AD (Granic et al. 2009). Brains of AD patients have shown enhanced NF-κB activity, which has been found predominantly in neurons and glial cells in amyloid beta (Aβ) plaque surrounding areas. Further Aβ and/or a secreted form of Aβ precursor proteins are reported to induce an upregulation of NF-κB activity, and that pathological hallmarks of AD (Aβ and hyperphosphorylated tau) are capable of inducing NF-κB activation (Granic et al. 2009). NF-κB has also been reported to increase acetylcholinesterase in the brains of rats with cognitive deficits (Kuhad et al. 2009). Furthermore, the redox-sensitive transcription factor, NF-κB, has been implicated in oxidative stress also (Muhammad et al. 2011). In a recent study, activation of NF-κB has been demonstrated to induce endothelial dysfunction (Jablonski et al. 2011). Therefore, the observed beneficial effect of NDDCT in DOCA hypertension-induced vascular dementia may primarily be attributed to its NF-κB inhibitory activity, anti-acetylcholinesterase, antioxidant, and enhancement of endothelial function.

While ACE inhibitors have been used primarily in the treatment of hypertension and congestive heart failure, there are some clinical studies in which ACE inhibitors have been shown to reduce the incidence of dementia or slow down the rate of cognitive decline in patients with hypertension (Yasar et al. 2008; Sink et al. 2009). It has been reported that treatment with centrally active ACE inhibitors like lisinopril, but not non-centrally active ACE inhibitors, could slow down the rate of cognitive decline in mild-to-moderate Alzheimer’s disease patients with hypertension (Ohrui et al. 2004; Yamada et al. 2010). However, the mechanisms responsible for the pro-cognitive effect of this class of drugs have not yet been elucidated (Ohrui et al. 2004; Yamada et al. 2010). Few reports have shown the activation of renin–angiotensin system in the brains of patients with Alzheimer’s disease (Miners et al. 2008, 2009), and increased ACE activity has been found to be positively correlated with the amyloid beta load in patients with dementia of AD (Miners et al. 2008, 2009; Savaskan et al. 2001; Barnes et al. 1991).

ACE inhibitors in addition to their potential antihypertensive action, in recent studies, have been shown to improve endothelial dysfunction (Silva et al. 2011). ACE inhibitors have been reported to unregulated eNOS protein expression and activity that results in enhanced levels nitric oxide levels (Silva et al. 2011). It has been suggested that these drugs lower angiotensin II formation and increase bradykinin levels, which in turn, stimulate receptors on endothelial cells causing the release of vasodilators (viz nitric oxide, prostacyclin, and endothelium-derived hyperpolarizing factor) along with significant reduction in oxidative stress and vasoconstriction (Silva et al. 2011). Recently, it has been reported that ACE inhibitor (perindopril) decreased activity and mRNA expression of AChE and ACE in dementia. Moreover, perindopril per se has shown no effect on AChE activity and expression. However, perindopril per se significantly reduced ACE activity but increased mRNA expression of ACE in rat brain (Tota et al. 2012). Thus reduction of brain AChE activity by lisinopril is in line with the reports of Tota et al. (2012). Hence, in the present study, beneficial effect of lisinopril in DOCA hypertension-induced vascular dementia appears to be attributed to its multiple actions viz anti-acetylcholinesterase activity, antioxidant action, enhancement of endothelial function, and off course anti-ACE activity. However, antihypertensive effect of lisinopril cannot also be ignored at this point.

Acetylcholinesterase inhibitors are the main class of drugs which are frequently used for the management of memory deficits. In our previous reports, we have demonstrated that donepezil in addition to its usefulness in dementia of AD (Sharma et al. 2008a, b) also exert beneficial effect in different animal models of vascular dementia (Koladiya et al. 2008, 2009; Sharma and Singh 2010, 2011). Donepezil is already in clinical use for management of dementia of various etiologies and hence it served as positive standard in this study.

Hence, depending on the results of this study and above discussions, it is concluded that experimental hypertension by DOCA–salt administration has induced endothelial dysfunction and subsequent vascular dementia. Treatments of NDDCT (a NF-κB inhibitor)/lisinopril (an ACE inhibitor)/donepezil (an acetylcholinesterase inhibitor) have ameliorated hypertension-induced vascular dementia in rats by virtue of their hypertension-dependent and hypertension-independent actions. Nevertheless, further studies are required to find the full potential and exact mechanism of these pharmacological agents for the management of hypertension-associated vascular dementia.

Acknowledgments

The authors are thankful to the Department of Pharmaceutical Sciences and Drug Research, Faculty of Medicine, Punjabi University, Patiala, Punjab, India for providing all the necessary facilities and funding to conduct this research. We are also thankful to Mr. A.S. Jaggi, Assistant Prof. in Pharmacology for his valuable suggestions.

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© Springer-Verlag 2012