Influence of sildenafil and donepezil administration on the serum redox balance in experimentally induced lower limb critical ischemia.

INTRODUCTION: Chronic lower limb ischemia (CLLI) leads to endothelial cell dysfunctions and endothelial lesions. The use of substances that release nitric oxide and activate endothelial nitric oxide synthase has proved to be useful in increasing angiogenesis and arteriogenesis under critical ischemia conditions.
OBJECTIVES: To investigate the therapeutic effect of Sildenafil and Donepezil with a vasodilating action in experimentally induced CLLI and on serum redox homeostasis. MATERIAL AND
METHOD: The research was performed in 3 groups of rats (n=10 animals/group) with experimentally induced CLLI: group I - control group; group II - animals treated postoperatively with a therapeutic dose of sildenafil, and group III - animals treated postoperatively with a therapeutic dose of donepezil. Oxidative stress (OS) indicators (malondialdehyde - MDA, protein carbonyls - PC), antioxidant (AO) defense indicators (reduced glutathione - GSH and oxidized glutathione - GSSH), and ceruloplasmin (CP) were determined on days 7, 14, 21 and 30. Statistical processing was performed using the Excel application (Microsoft Office 2007), with the StatsDirect v.2.7.2 software.
RESULTS: Changes in OS were evidenced in all groups on account of a decrease in MDA and PC. The greatest OS decrease in all groups was on day 30. AO defence changes were represented by decreased levels of GSH and GSSG in all groups, at the studied moments. Intracellular AO defense in the cytosol, nucleus and mitochondria was similar in all groups, (decreased GSH, GSSG and GSH/GSSG ratio). We found increased extracellular levels of GSH, GSSG, and CP and increased extracellular GSH/GSSG ratio at level compared to values on day 7.
CONCLUSIONS: 1) The administration of sildenafil (group II) and donepezil (group III) has favorable effects on reducing OS in experimentally induced CLLI. 2) Sildenafil and Donepezil administration stimulates extracellular AO defense on account of CP. 3) Sildenafil and Donepezil administration influences intracellular redox homeostasis on account of the GSH/GSSG couple, the major redox buffer in the body.

Introduction

Many experimental studies have shown that both ischemia and ischemia-reperfusion syndrome are associated with oxidative stress (OS) through the production of reactive oxygen species (ROS) [1,2]. Repeated ischemia and reperfusion episodes may trigger lesions at the level of endothelial cells, mitochondria, muscle fibers, and neuronal axons. These oxidative lesions will initiate chronic changes in the metabolism and structure of muscle fibers, resulting in the loss of their function, which can be entirely attributed to the reduction of blood flow and oxidative lesions [3].

Endothelial cell dysfunction is an early event which will induce lesions in the vascular wall. This will result in the impairment of the capacity of endothelial cells to maintain vascular function and homeostasis [4].

Nitric oxide (NO) is known as a physiologically vasoactive agent, involved in the proliferation and survival of endothelial cells, the increase of their mobility and the activation of mechanisms necessary for angiogenesis [5,6,7]. It plays a role in the regulation of blood pressure, the mediation of vascular permeability, reactive vasodilation, blood flow-dependent dilation and endothelium-dependent relaxing responses to acetylcholine (Ach). It has antiaggregant properties, stimulates endogenous fibrinolysis and maintains the antiinflammatory state of the vascular wall [8].

The use of substances that release NO and activate endothelial nitric oxide synthase (eNOS), such as statins and vascular endothelial growth factor A (VEGF-A), has proved to be useful in increasing angiogenesis and arteriogenesis under critical ischemia conditions [9,10]. However, substances that release nitric oxide may also have negative effects. NO excess associated with the uncoupling of eNOS results in the formation of super-oxide and peroxynitrite radicals, which leads to the extension of the surface of the affected tissues in the ischemia-reperfusion syndrome [11].

NO activates the guanylate cyclase enzyme and increases the concentration of cyclic guanosine mono-phosphate (cGMP), inducing smooth muscle relaxation. Sildenafil is a selective enzyme inhibitor, which enhances the effect of NO through the inhibition of type 5 phosphodiesterase (PDE5), which is responsible for the degradation of cGMP and is mainly found in the smooth muscle of the cavernous body, but also in smooth vascular, visceral and skeletal muscles. Sildenafil treatment may have a protective action in pathological tissue ischemia, through the increase in the tissue level of cGMP and the stimulation of ischemia-induced angiogenesis, without the implication of eNOS [12].

Donepezil, a specific and reversible acetylcholin-esterase inhibitor, induces an increase in Ach levels. Ach causes a reduction of blood pressure and vasodilation through an indirect mechanism: it acts on endothelial cells that release the endothelial relaxing factor NO, through cGMP. Ach is also an activating modulating factor for the increase of Ca2+ concentration, with stimulating effects on eNOS and NO synthesis. On the other hand, a study on the effects of Ach on myocardial cells has shown that Ach prevents hypoxia-induced cell death [13]. However, there are also data demonstrating the negative effects of systemic Ach administration, due to the induction of bronchospasm and mucus hypersecretion at the level of the airways. In cardiovascular patients, Donepezil acts as a therapeutic agent that activates angiogenesis [14]. This is why we used it for testing its effect on rats with experimentally induced chronic lower limb ischemia (CLLI).

Objectives

The objectives of this study were to investigate the therapeutic effect of Sildenafil and Donepezil in experimentally induced CLLI and their influence on serum redox homeostasis.

Material and method

The experimental study was an interventional, prospective, analytical pilot study, with a duration of 30 days. The research was performed in the Experimental Laboratory of the Department of Physiology of the ”Iuliu Haţieganu” University of Medicine and Pharmacy Cluj-Napoca, on white Wistar rats with a weight of 280–300 g, maintained under adequate vivarium conditions; with the approval of the Ethics Board No. 681/18.12.2012. During the experimental research, the legislation in force regarding animal protection was respected; at the end of the experiment, the animals were euthanized.

a) Groups

The groups (n=10 animals/group) were the following:

- group I – control – operated, untreated animals;

- group II – operated animals, treated postoperatively with a therapeutic dose of Sildenafil, 10 mg/kg body weight/day, by oropharyngeal gavage, for 30 days (day 5-day 34); we utilised Sildenafil powder resulting from 100 mg pills dissolved in drinking water, to a final concentration of 40 μg/ml. A resulting dose of 10 mg/kg/day of Sildenafil was achieved, to reach a therapeutic dosing level that would be comparable in man because of the short half life in rats (1 hour in rats versus 4 hour in man) [15];

- group III – operated animals, treated post-operatively with a therapeutic dose of Donepezil, 5 mg/kg/day, by oropharyngeal gavage, for 30 days (day 5-day 34); we utilised Donepezil powder resulting from 10 mg pills dissolved in drinking water, to a final concentration of 50 μg/ml. A resulting dose of 5 mg/kg/day of Donepezil was achieved, to reach a therapeutic dosing level that would be comparable in man. This dose was initially determined to clearly show the expected effects without producing adverse effects in rats [14].

We used Sildenafil (Viagra®, Pfizer 50 mg/compr) and Donepezil (Aricept®, Pfizer 10 mg/tabl).

experimental surgical

The animals were operated in the Experimental Laboratory of the Department of Physiology of the ”Iuliu Haţieganu” University of Medicine and Pharmacy, using a procedure adapted from Colleran et al. and Yang et al. [16,17]. CCLI was induced by the ligation of the common femoral artery, proximally to the origin of the deep femoral artery (day 0), followed by the ligation of the common iliac artery (day 4). The rats were anesthetized with ketamine 100 mg/kg and xylazine 8 mg/kg.

The control of ischemia was performed on day 5 through:

- clinical score (0=normal mobility, 1=pallor, limping, 2=gangrenous tissue limited to less than half of the leg, without lower limb necrosis, 3=gangrenous tissue limited to less than half of the leg, with lower limb necrosis, 4=gangrenous tissue extended to more than half of the leg, 5=extensive necrosis of the lower limb [5], and

- imaging, by Doppler ultrasound.

biochemical

Serum determinations from venous blood collected from the retro-orbital vein were performed in the Laboratory for the Study of Oxidative Stress of the Department of Physiology of the ”Iuliu Haţieganu” University of Medicine and Pharmacy Cluj-Napoca. The selected moments were postoperative days 7, 14, 21, 30.

The following data were measured:

✓ Oxidative stress indicators

- malondialdehyde (MDA) (using the fluorescence method, according to Conti). Concentration values were expressed in nmol/ml, for serum [17].

- protein carbonyls (PC) (using the method of Reznick and Packer). The results were expressed in μmol/ml, for serum [19].

✓ Antioxidant defense indicators

- reduced glutathione (GSH) and oxidized glutathione (GSSG) (using the method of Hu); values were expressed in μmol/l or nmol/ml in the serum [20].

✓ Ceruloplasmin (CP) (using Ravin’s method); values were expressed in mg/100 ml [21].

c) Statistical analysis

Descriptive statistical elements were calculated, and data were presented using centrality, location and distribution indicators.

For testing normal distribution, the Shapiro-Wilk W test was used. For statistical data analysis, in the case of normal distribution data, the (Student) t test was used, variances being tested with the Levene test. In the case of non-uniform distribution values, the non-parametric Mann-Whitney (U) test for two unpaired samples and the Wilcoxon test for two paired samples were used.

The significance threshold for the tests used was α=0.05 (5%): 0.010.05 – statistically insignificant difference.

Statistical processing was carried out with the Excel application (Microsoft Office 2007) and with the StatsDirect v.2.7.2 software.

Results

Comparative statistical analysis by moments between the groups

a. Malondialdehyde

The statistical analysis of MDA values (Table I), considering all studied moments, evidenced highly statistically significant differences between at least two of the studied moments in groups I (p=4.91×10−7), II (p=0.0003) and III (p=7.54×10−6).

Table I

Comparative analysis of MDA values (measured in nmol/ml) at the studied moments and statistical significance.

Group	Moment	Mean	SE	Median	SD	Min.	Max.	Statistical significance (p) between moments
I	Day 7	2.0713	0.1252	1.955	0.3540	1.71	2.82	D7–D14: 0.5618	D14–D30: 0.0002
	Day 14	1.9688	0.0738	1.985	0.2088	1.58	2.27	D7–D21: 0.0002	D21–D30: 0.6342
	Day 21	1.105	0.1178	1.15	0.3333	0.57	1.56	D7–D30: 0.0028
	Day 30	1	0.1720	0.9	0.4866	0.4	1.87	D14–D21: 0.0016
II	Day 7	1.9475	0.0808	1.925	0.2286	1.59	2.35	D7–D14: 0.711	D14–D30: 0.0078
	Day 14	1.9875	0.0569	1.92	0.1609	1.8	2.22	D7–D21: 0.0127	D21–D30: 0.4609
	Day 21	1.195	0.1969	1.21	0.5569	0.32	2.1	D7–D30: 0.0078
	Day 30	1.0213	0.1493	0.865	0.4224	0.58	1.6	D14–D21: 0.0034
III	Day 7	1.6375	0.0745	1.675	0.2108	1.25	1.95	D7–D14: 0.002	D14–D30: 0.0001
	Day 14	2.3788	0.1648	2.23	0.4662	1.65	3.04	D7–D21: 0.2933	D21–D30: 0.0024
	Day 21	1.8025	0.1345	1.7	0.3803	1.33	2.54	D7–D30: 0.0278
	Day 30	1.1588	0.1410	1.26	0.3987	0.53	1.62	D14–D21: 0.0025
Statistical significance (p) between groups		D7 (I–II): 0.4224		D14 (I–II): 0.8436		D21 (I–II): 0.7024		D30 (I–II): 0.9804
		D7 (I–III): 0.0126		D14 (I–III): 0.0465		D21 (I–III): 0.0016		D30 (I–III): 0.488
		D7 (II–III): 0.0136		D14 (II–III): 0.0515		D21 (II–III): 0.0256		D30 (II–III): 0.6454

The statistical analysis of MDA values for paired samples showed:

in group I – highly statistically significant differences between days 7–21 and 14–30 (p<0.001) and very statistically significant differences between days 7–30 and 14–21 (p<0.01);

in group II – statistically significant differences between days 7–21 (p<0.05) and very statistically significant differences between days 7–30, 14–21 and 14–30 (p<0.01);

in group III – highly statistically significant differences between days 14–30 (p<0.001), very statistically significant differences between days 7–14, 14–21 and 21–30 (p<0.01), and statistically significant differences between days 7–30 (p<0.05).

The statistical analysis of MDA values, considering all groups at a certain moment, evidenced statistically significant differences between at least two of the groups on day 7 (p=0.0128) and on day 14 (p=0.0239), and very statistically significant differences between at least two of the groups on day 21 (p=0.0081). On day 30, no statistically significant differences between any of the groups were found (p=0.7241).

The statistical analysis of MDA values for unpaired samples showed:

on day 7 – statistically significant differences between groups I–III and II–III (p<0.05);

on day 14 – statistically significant differences between groups I–III (p<0.05);

on day 21 – very statistically significant differences between groups I–III (p<0.01) and statistically significant differences between groups II–III (p<0.05). On day 30, there were no statistically significant differences between the studied groups.

b. Protein carbonyls

The statistical analysis of PC values (Table II), considering all studied moments, indicated statistically significant differences between at least two of the studied moments in group I (p=0.0114). In groups II and III, there were no statistically significant differences between any of the studied moments (p=0.3348 and p=0.0533, respectively). Although in some cases, considering all studied moments, no statistically significant differences between these could be found, the statistical analysis of PC values for paired samples evidenced:

Table II

Comparative analysis of PC values (measured in nmol/ml) at the studied moments and statistical significance.

Group	Moment	Mean	SE	Median	SD	Min.	Max.	Statistical significance (p) between moments
I	Day 7	1.0125	0.1329	0.9200	0.3758	0.65	1.8	D7–D14: 0.2251	D14–D30: 0.1147
	Day 14	0.8263	0.0439	0.825	0.1242	0.69	1	D7–D21: 0.0357	D21–D30: 0.0093
	Day 21	0.5588	0.0683	0.51	0.1933	0.34	0.89	D7–D30: 0.9847
	Day 30	1.0088	0.1303	1.055	0.3685	0.44	1.5	D14–D21: 0.0106
II	Day 7	0.7488	0.0995	0.72	0.2815	0.43	1.24	D7–D14: 0.0781	D14–D30: 0.4375
	Day 14	1.0275	0.0806	0.935	0.2279	0.81	1.5	D7–D21: 0.9375	D21–D30: 0.25
	Day 21	0.7425	0.1135	0.73	0.3210	0.43	1.21	D7–D30: 0.267
	Day 30	0.9625	0.1449	0.865	0.4099	0.52	1.65	D14–D21: 0.3125
III	Day 7	0.9975	0.1425	0.9	0.4029	0.49	1.86	D7–D14: 0.3041	D14–D30: 0.2421
	Day 14	0.8025	0.0651	0.785	0.1841	0.61	1.1	D7–D21: 0.0845	D21–D30: 0.1068
	Day 21	1.275	0.0925	1.21	0.2616	0.98	1.7	D7–D30: 0.9691
	Day 30	0.9888	0.1395	0.835	0.3947	0.56	1.67	D14–D21: 0.0068
Statistical significance (p) between groups		D7 (I–II): 0.1362		D14 (I–II): 0.0605		D21 (I–II): 0.2328		D30 (I–II): 0.8158
		D7 (I–III): 0.9397		D14 (I–III): 0.7674		D21 (I–III): 3.07 × 10−05		D30 (I–III): 0.918
		D7 (II–III): 0.1759		D14 (II–III): 0.065		D21 (II–III): 0.003		D30 (II–III): 0.898

in group I – statistically significant differences between days 7–21 and 14–21 (p<0.05) and very statistically significant differences between days 21–30 (p<0.01);

in group III – very statistically significant differences between days 14–21 (p<0.01). In group II, no statistically significant differences between the studied days were found.

The statistical analysis of PC values, considering all groups at a certain moment, showed highly statistically significant differences between at least two of the groups on day 21 (p=0.0009). On days 7, 14 and 30, there were no statistically significant differences between any of the groups (p=0.2746, p=0.0833, and p=0.9723, respectively).

The statistical analysis of PC values for unpaired samples evidenced:

on day 21 – highly statistically significant differences between groups I–III (p<0.001) and very statistically significant differences between groups II–III (p<0.01). On days 7, 14 and 30, no statistically significant differences between the studied groups were found.

c. Reduced glutathione

The statistical analysis of GSH values (Table III), considering all studied moments, revealed highly statistically significant differences between at least two of the studied moments in groups I and III (p=0.0004 and p=0.0004, respectively) and very statistically significant differences between at least two of the studied moments in group II (p=0.0081).

Table III

Comparative analysis of GSH values (measured in nmol/ml) at the studied moments and statistical significance.

Group	Moment	Mean	SE	Median	SD	Min.	Max.	Statistical significance (p) between moments
I	Day 7	7.6675	0.3437	7.67	0.9720	6	9.15	D7–D14: 0.1953	D14–D30: 0.6406
	Day 14	6.6263	0.5171	6.355	1.4625	4.99	10	D7–D21: 0.0078	D21–D30: 0.0156
	Day 21	3.0688	0.5184	2.65	1.4663	1.6	6.4	D7–D30: 0.0116
	Day 30	6.0931	0.2689	6.2	0.7607	5	7.3	D14–D21: 0.0156
II	Day 7	7.4175	0.6085	7.4	1.7212	5	11	D7–D14: 0.3092	D14–D30: 0.2457
	Day 14	5.9625	1.0438	5.275	2.9524	2.19	11.35	D7–D21: 0.0095	D21–D30: 0.4865
	Day 21	3.825	0.7088	3.45	2.0048	1.72	7.4	D7–D30: 0.0019
	Day 30	4.5063	0.4091	4.7	1.1571	2.65	5.65	D14–D21: 0.1850
III	Day 7	6.4725	0.2585	6.47	0.7310	5.43	7.58	D7–D14: 1.4 × 10−05	D14–D30: 0.2608
	Day 14	3.4813	0.3210	3.325	0.9080	2.49	5.27	D7–D21: 0.00098	D21–D30: 0.8945
	Day 21	4.075	0.3622	3.95	1.0243	2.75	5.95	D7–D30: 0.02
	Day 30	4.1563	0.7237	3.675	2.0469	2.05	8	D14–D21: 0.1828
Statistical significance (p) between groups		D7 (I–II): 0.7273		D14 (I–II): 0.1949		D21 (I–II): 0.5054		D30 (I–II): 0.0071
		D7 (I–III): 0.0156		D14 (I–III): 0.0003		D21 (I–III): 0.0406		D30 (I–III): 0.0334
		D7 (II–III): 0.1867		D14 (II–III): 0.0527		D21 (II–III): 0.7599		D30 (II–III): 0.6818

The statistical analysis of GSH values for paired samples evidenced:

in group I – very statistically significant differences between days 7–21 (p<0.01) and statistically significant differences between days 7–30, 14–21 and 21–30 (p<0.05);

in group II – very statistically significant differences between days 7–21 and 7–30 (p<0.01);

in group III – highly statistically significant differences between days 7–14, 7–21 (p<0.001) and statistically significant differences between days 7–30 (p<0.05).

The statistical analysis of GSH values, considering all groups at a certain moment, revealed very statistically significant differences between at least two of the groups on day 14 (p=0.0032) and statistically significant differences between at least two of the groups on day 30 (p=0.0295). On days 7 and 21, no statistically significant differences between any of the groups were found (p=0.1418 and p=0.1612, respectively).

Although in some cases, considering all studied groups, there were no statistically significant differences between these, the statistical analysis of GSH values for unpaired samples evidenced:

on day 7 – statistically significant differences between groups I–III (p<0.05);

on day 14 – highly statistically significant differences between groups I–III (p<0.001);

on day 21 – statistically significant differences between groups I–III (p<0.05);

on day 30 – statistically significant differences between groups I–III (p<0.05) and very statistically significant differences between groups I–II (p<0.01).

d. Oxidized glutathione

The statistical analysis of GSSG values (Table IV), considering all studied moments, showed highly statistically significant differences between at least two of the studied moments in group I (p=0.0003). No statistically significant differences were found between the studied moments in groups II and III.

Table IV

Comparative analysis of GSSG values (nmol/ml) at the studied moments and statistical significance.

Group	Moment	Mean	SE	Median	SD	Min.	Max.	Statistical significance (p) between moments
I	Day 7	1.67	0.0609	1.69	0.1724	1.4	1.88	D7–D14: 0.0107	D14–D30: 0.0078
	Day 14	1.3988	0.0551	1.415	0.1558	1.21	1.61	D7–D21: 0.0219	D21–D30: 0.0781
	Day 21	1.325	0.1306	1.3	0.3694	0.6	1.8	D7–D30: 0.0078
	Day 30	0.875	0.0526	0.8	0.1488	0.8	1.2	D14–D21: 0.6721
II	Day 7	1.3325	0.0534	1.32	0.1511	1.12	1.6	D7–D14: 0.0781	D14–D30: 0.2969
	Day 14	1.2188	0.0837	1.16	0.2367	1	1.74	D7–D21: 0.25	D21–D30: 0.5469
	Day 21	1.165	0.1309	1.16	0.3701	0.72	1.6	D7–D30: 0.0228
	Day 30	1.05	0.0732	1	0.2070	0.8	1.4	D14–D21: 0.7422
III	Day 7	1.3125	0.0496	1.27	0.1402	1.18	1.5	D7–D14: 0.7422	D14–D30: 0.3828
	Day 14	1.2225	0.1425	1.245	0.4031	0.5	1.74	D7–D21: 0.1563	D21–D30: 0.2969
	Day 21	1.14	0.07329	1.19	0.2073	0.7	1.36	D7–D30: 0.0781
	Day 30	1	0.1363	0.8	0.3854	0.6	1.6	D14–D21: 0.691
Statistical significance (p) between groups		D7 (I–II): 0.00095		D14 (I–II): 0.0468		D21 (I–II): 0.4884		D30 (I–II): 0.0884
		D7 (I–III): 0.0011		D14 (I–III): 0.2784		D21 (I–III): 0.2424		D30 (I–III): 0.7716
		D7 (II–III): 0.7792		D14 (II–III): 0.9043		D21 (II–III): 0.7052		D30 (II–III): 0.3678

Although in some cases, considering all studied moments, there were no statistically significant differences between these, the statistical analysis of GSSG values for paired samples indicated:

in group I – statistically significant differences between days 7–14 and 7–21 (p<0.05) and very statistically significant differences between days 7–30 and 14–30 (p<0.01);

in group II – statistically significant differences between days 7–30 (p<0.05). In group III, there were no statistically significant differences between the studied days.

The statistical analysis of GSSG values, considering all groups at a certain moment, showed very statistically significant differences between at least two of the groups on day 7 (p=0.0026). On days 14, 21 and 30, there were no statistically significant differences between any of the groups (p=0.2252, p=0.3949, and p=0.2283, respectively).

Although in some cases, considering all studied groups, no statistically significant differences between these could be found, the statistical analysis of GSSG values for unpaired samples evidenced:

on day 7 – highly statistically significant differences between groups I–II (p<0.001) and very statistically significant differences between groups I–III (p<0.01);

on day 14 – statistically significant differences between groups I–II (p<0.05). On days 21 and 30, there were no statistically significant differences between the studied groups.

e. Reduced glutathione/oxidized glutathione ratio

The statistical analysis of GSH/GSSG values (Table V), considering all studied moments, indicated highly statistically significant differences between at least two of the studied moments in group I (p<0.0001). No statistically significant differences were found between any of the studied moments in groups II and III (p=0.4996 and p=0.0541, respectively).

Table V

Comparative analysis of GSH/GSSG values at the studied moments and statistical significance.

Group	Moment	Mean	SE	Median	SD	Min.	Max.	Statistical significance (p) between moments
I	Day 7	4.6463	0.3018	4.555	0.8536	3.75	6.53	D7–D14: 0.1563	D14–D30: 0.0007
	Day 14	3.9913	0.1951	3.935	0.5519	3.19	5.15	D7–D21: 0.0156	D21–D30: 0.0001
	Day 21	2.2575	0.3313	1.99	0.9371	1.29	3.75	D7–D30: 0.0078
	Day 30	7.1288	0.5163	7.37	1.4603	5	9.12	D14–D21: 0.0033
II	Day 7	5.5475	0.3502	5.63	0.9905	3.57	6.87	D7–D14: 0.681	D14–D30: 0.6
	Day 14	5.0988	0.9630	4.785	2.7236	2.02	9.46	D7–D21: 0.1902	D21–D30: 0.7412
	Day 21	3.9675	1.0231	3.14	2.8936	1.08	9.02	D7–D30: 0.0894
	Day 30	4.4663	0.5401	4.3	1.5275	2.12	6.93	D14–D21: 0.4906
III	Day 7	4.9363	0.1478	4.965	0.4180	4.38	5.67	D7–D14: 0.0003	D14–D30: 0.2045
	Day 14	3.1525	0.3532	3.11	0.9989	1.55	4.98	D7–D21: 0.25	D21–D30: 0.4609
	Day 21	3.875	0.7208	3.115	2.0388	2.41	8.5	D7–D30: 0.889
	Day 30	4.7763	1.0281	4.035	2.9079	1.28	10	D14–D21: 0.8438
Statistical significance (p) between groups		D7 (I–II): 0.065		D14 (I–II): 0.2923		D21 (I–II): 0.1505		D30 (I–II): 0.0031
		D7 (I–III): 0.1049		D14 (I–III): 0.0618		D21 (I–III): 0.065		D30 (I–III): 0.0681
		D7 (II–III): 0.1423		D14 (II–III): 0.0902		D21 (II–III): 0.7209		D30 (II–III): 0.7945

Although in some cases, considering all studied moments, no statistically significant differences between these could be evidenced, the statistical analysis of GSH/GSSG values for paired samples showed:

in group I – highly statistically significant differences between days 14–30 and 21–30 (p<0.001), very statistically significant differences between days 7–30 and 14–21 (p<0.01) and statistically significant differences between days 7–21 (p<0.05);

in group III – highly statistically significant differences between days 7–14 (p<0.001). In group II, there were no statistically significant differences between the studied days.

The statistical analysis of GSH/GSSG values, considering all groups at a certain moment, showed statistically significant differences between at least two of the groups on day 7 (p=0.0486) and on day 30 (p=0.0353). On days 7, 14 and 21, no statistically significant differences between any of the groups were found (p=0.0962 and p=0.2128, respectively).

The statistical analysis of GSH/GSSG values for unpaired samples indicated:

on day 30 – very statistically significant differences between groups I–II (p<0.01). On days 14 and 21, no statistically significant differences between the studied groups were found.

Although considering all studied groups on day 7, there should have been statistically significant differences between at least two of these, the statistical analysis of values for unpaired samples did not reveal these statistically significant differences.

f. Ceruloplasmin

The statistical analysis of CP values (Table VI), considering all studied moments, evidenced highly statistically significant differences between at least two of the studied moments in groups I (p=2.96 × 10−7) and II (p=0.0003) and very statistically significant differences between at least two of the studied moments in group III (p=0.0087).

Table VI

Comparative analysis of CP values (measured in mg%) at the studied moments and statistical significance.

Group	Moment	Mean	SE	Median	SD	Min.	Max.	Statistical significance (p) between moments
I	Day 7	50.5175	1.8926	50.52	5.3531	40.4	58.7	D7–D14: 0.8143	D14–D30: 1.26 × 10−05
	Day 14	51.2175	1.7291	51.38	4.8906	44.2	59.1	D7–D21: 0.0009	D21–D30: 0.044
	Day 21	61.0688	1.6769	60.625	4.7430	55.3	68.3	D7–D30: 0.0006
	Day 30	69.0125	2.3784	69.1	6.7272	60.2	77.1	D14–D21: 0.0139
II	Day 7	48.1325	1.2268	47.955	3.4699	43.8	53.9	D7–D14: 0.0027	D14–D30:0.0027
	Day 14	59.4250	3.1918	59.4	9.0279	46.9	73.6	D7–D21: 0.0078	D21–D30: 0.4609
	Day 21	65.7625	3.0306	69	8.5717	54.6	75.4	D7–D30:2.67 × 10−06
	Day 30	69.1688	1.9898	67.425	5.6281	63.4	79.4	D14–D21: 0.0469
III	Day 7	60.5825	2.1894	60.58	6.1926	51.3	68.4	D7–D14: 0.5834	D14–D30:0.0118
	Day 14	57.3675	4.7763	54	13.5093	44.8	81	D7–D21: 0.0214	D21–D30:0.0341
	Day 21	65.1813	2.5784	63.15	7.2929	57.5	78.65	D7–D30: 0.0041
	Day 30	72.6188	1.6954	73.2	4.7954	64.5	78.65	D14–D21: 0.1775
Statistical significance (p) between groups		D7 (I–II): 0.3111		D14 (I–II): 0.045		D21 (I–II): 0.3282		D30 (I–II): 0.9605
		D7 (I–III): 0.0037		D14 (I–III): 0.2568		D21 (I–III): 0.206		D30 (I–III): 0.2388
		D7 (II–III): 0.0004		D14 (II–III): 0.7264		D21 (II–III): 0.9591		D30 (II–III): 0.2081

The statistical analysis of CP values for paired samples showed:

in group I – highly statistically significant differences between days 7–21, 7–30 and 14–30 (p<0.001) and statistically significant differences between days 14–21 and 21–30 (p<0.05);

in group II – highly statistically significant differences between days 7–30 (p<0.001), very statistically significant differences between days 7–14, 7–21 and 14–30 (p<0.01) and statistically significant differences between days 14–21 (p<0.05);

in group III – very statistically significant differences between days 7–30 (p<0.01) and statistically significant differences between days 7–21, 14–30 and 21–30 (p<0.05).

The statistical analysis of CP values, considering all groups at a certain moment, revealed highly statistically significant differences between at least two of the groups on day 7 (p=0.00019). On days 14, 21 and 30, no statistically significant differences between any of the groups were found (p=0.2418, p=0.417, and p=0.3854, respectively).

Although in some cases, considering all studied groups, no statistically significant differences between these could be found, the statistical analysis of CP values for unpaired samples showed:

on day 7 – highly statistically significant differences between groups II–III (p<0.001), very statistically significant differences between groups I–III (p<0.01);

on day 14 – statistically significant differences between groups I–II (p<0.05).

Discussion

The balance between ROS and NO has played an important role in the development of atherosclerosis in patients with peripheral arterial disease.

Cell and gene therapy together with antioxidative pharmacological therapy can be useful in recovering the function of the endothelium and preventing the development of atherosclerosis in patients with critical ischemia [22].

Critical ischemia is characterised by an increased production of all the ROS [23].

In our study we evaluated the effect of the therapy with Sildenafil and Donepezil on the markers of OS and endogeneous AO.

When analysing the groups we noticed the decrease of MDA in the group treated with Donepezil compared to the group that had no treatment and the group with Sildenafil and a significant increase of MDA in the 14th day of treatment. Another marker of OS, GSSG shows a significant decrease in the Sildenafil, Donepezil group compared to the group with ligature and no treatment. Aydin et al. [24] had the same results when administering another vasodilator, levosimendan, which has a different mechanism of action by opening ATP dependent potassium channels (KATP) in the smooth muscle.

According to some theories, Sildenafil is supposed to use the same mechanism of action [25]. However, the decrease in the MDA blood level seen in the Donepezil group can be interpreted through the different mechanism of action: Donepezil lengthens the dilating action of Ach [14] on an intact endothelium; this is true for vessels of neoformation but not for injured vessels.

The administration of PDE5 inhibitor and Sildenafil, decreasing the serum level of GSSG but also decreasing the GSH/GSSG ratio, significantly decreased by the 30th day, in the group with Sildenafil, compared to the group with no treatment.

The 14th day appears to display the highest OS level both in the group with Sildenafil and in the Donepezil therapy group, proved by high levels of stress markers, MDA and GSSG that are decreasing on the 21st day. There are also low levels of AO (GSH) which can be explained by their use in the effort of neutralizing free radicals.

These results are in agreement with the studies of Bivalacqua [26] that demonstrate reduced levels of ROS under the effect of Sildenafil associated with an improvement in endothelial function supported in our study by the increase in e NOS level.

A recent study [27] demonstrates the same effect of reduced oxidative stress by using another PDE5 inhibitor, tadalafil, the effect being reached by lowering MDA level and raising SOD level after 8 weeks of treatment.

Actually PDE5 inhibitors are considered to play an important role in reducing OS in other organs as well. Sildenafil decreases OS and protects the heart in patients with left ventricular hypertrophy and congestive heart failure. Also, at the level of pulmonary artery, Sildenafil diminishes OS, neutralizes the inflammation and remodeling induced by smoking [28]

Our results are in accordance with the literature data regarding the GSH/GSSG couple, which is the most important intracellular redox buffer and the redox indicator of the cellular environment [29,30].

The increase of CP, an extracellular AO, shows the intervention of defence mechanisms in OS, associated with ischemia and reperfusion. The fact that CP increases in all groups and at all times is probably due to its intervention on acute phase reactant in the inflammatory processes of chronic ischemia [31].

Our results show that Sildenafil and Donepezil administration might contribute to the acute increase of blood flow in ischemic tissues, the increase of ischemia-induced angiogenesis and of endothelial cell proliferation in ischemic areas, as previous shown by other studies [32], processes that might also be stimulated by the low ROS production after 14 days. The beneficial effects of ROS occur at low or moderate ROS concentrations [33,34].

The administration of Sildenafil and Donepezil has favorable effects in reducing OS in experimentally induced CLLI.

Sildenafil and Donepezil administration stimulates extracellular AO defence on account of CP.

Sildenafil and Donepezil administration influences intracellular redox homeostasis on account of the GSH/GSSG couple, the major redox buffer in the body.