A Comparative Analysis of the Effects of Melatonin and Nimodipine on Vasospasm

Objective: In this study, we aimed to compare the efficacy of nimodipine and melatonin in a rat femoral artery vasospasm model. Material and Methods: The rats were divided into five groups as follows: Group I (n=8): control group, Group II (n=8): vasospasm group, Group III (n=8): vasospasm + melatonin group, Group IV (n=8): vasospasm + nimodipine group, Group V (n=8 rats): vasospasm + melatonin + nimodipine group. At the end of the 7th day, the femoral arteries were excised and histopathologically examined under light microscope. Results: Vascular wall of vasospasm group was relatively thicker when compared with the control group. There was a statistically significant difference between the wall thickness of Group II and Group III, IV and V. The maximum reduction of wall thickness was observed in Group V. There was no statistically significant difference in lumen diameter between Group I and Group IV. The lumen diameter was found to be increased in Group III, IV and V when compared with Group II. Conclusion: The development of experimental peripheral vasospasm has been reduced by the melatonin and nimodipine and their effect was increased when they were used concomitantly.


INTRODUCTION
Cerebral vasospasm (CV) is the main cause of morbidity and mortality after subarachnoid hemorrhage (SAH).The etiology is multifactorial and remains obscure [1].Despite experimental and clinical researches, the spasmogenic agents responsible for the development of vasospasm and the mechanisms of action are still poorly understood [2][3][4].Although nimodipine has a limited efficacy in the treatment of CV after SAH, it is being used in routine practice.In several studies, melatonin was reported to be effective against free radicals, which were thought to play a role in the etiopathogenesis of SAH [5][6][7][8].There is no study investigating and comparing the therapeutic effects of these substances on the treatment of CV.
The aim of this study is to investigate the effect of melatonin and nimodipine in CV in the early period after SAH.Furthermore, we aimed to determine which is more effective and whether the combination of these substances improved the efficiency or not.

MATERIALS AND METHODS
The surgical experimental part of this study was performed in Istanbul University, Institute of Experimental Medicine after approved by the ethics committee.Forty male Sprague-Dawley albino rats weighing 220-280 g were used in the study.The "Rat Femoral Artery Vasospasm Model" described by Okada et al. was preferred as vasospasm model [9].The femoral artery vasospasm model was used because the mortality was high in cv models applied in the brain.Okada found similar histopathological findings femoral artery and in the brain vasospasm model.40 rats were seperated randomly into 5 groups each including 8 rats: control, vasospasm, vasospasm+melatonin, vasospasm+nimodipine, vasospasm+nimodipine+melatonine groups.

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All rats were anaesthetized by an injection of ketamine (100 mg/kg, intraperitoneal) and allowed spontaneous breathing.
-Group I (n=8 rats) control group; A 10 mm segment of femoral artery exposed by using a microsurgical technique in the inguinal region and a silastic sheath was wrapped around femoral artery.
-Group II (n=8 rats) vasospasm group; After the same surgical procedure, 0.1 ml of fresh blood derived from ventral tail artery was applied directly to the femoral artery.
-Group III (n=8 rats) vasospasm+ melatonin; vasospasm was induced and melatonin at daily doses of 20 mg/kg was applied for 7 days.
-Group IV (n=8 rats) vasospasm+ nimodipine; vasospasm was induced and nimodipine at daily doses of 0.1 mg/kg was applied for 7 days.
-Group V (n=8 rats): vasospasm was induced and melatonin and nimodipine at daily doses of 20 mg/kg and 0.1 mg/kg concomitantly were applied for 7 days.
At the end of 7 days all rats were anaesthetized and sacrificed.The former incisions were opened, and femoral artery samples dissected.All artery samples fixed in 10% formalin solution for 24 hours and following parafin embedding, tissue sections of 3 μm thickness were obtained.The tissue samples were stained with hematoxylin-eosin (H&E) for histomorphometric analysis and Elastic Van Gieson (EVG) for evaluated internal elastic lamina.The samples from the femoral arteries were measured in 3 different points [10,11].Lumen diameter and wall thickness of femoral arteries were measured using micrometers.

Statistical Analysis
The data were statistically analyzed using the Kruskal-Wallis and the Mann-Whitney U-tests.The value of p< 0.05 was considered as statistically significant.'One-way ANOVA test' was used for the intergroup comparisons of parameters with normal distribution and 'Tukey HSD test' was used to determine the group causing the difference in the study.'Mann Whitney U test' was used for the intergroup comparisons of parameters without normal distribution and 'Kruskal Wallis Test' was used for comparisons of means among more than two groups.The results were evaluated in 95% confidence interval and at a significance level of p<0.05.

Histopathological Findings
All femoral artery samples were examined using the light microscope.The vessels in the group I (control group) had a thin and smooth endothelium, a thin and unfolded internal elastic lamina and smooth muscle cells located concentrically (Figure 1A).There was marked luminal narrowing and an increase in the vessel wall thickness in the group II (vasospasm group).Non-intact endothelium, folding of the internal elastic lamina and vacuolization in muscle layer were determined (Figure 1B).Histopathological findings in each of three groups administered medication were found to be similar.Thin and smooth endothelium, concentrically arrayed smooth muscle cells were observed (Figure 1C, D, E).

Morphometric Analysis
According to the result of analysis, it was concluded that there was a significant difference between the mean vessel wall thicknesses (Table 1).It was seen that the mean vessel wall thickness of the melatonin and nimodipine group was minimum (a decrease by % 50 with respect to group of SAH) and the mean vessel wall thickness of SAH group was maximum.Each of nimodipine and melatonin reduced the wall thickness alone, but melatonin was found to be more effective (a decrease by % 44 with respect to group of SAH) (Figure 2).
When we investigated the mean vessel lumen area, vessel lumen area of vasospasm group was reduced significantly compared to the control group.According to the result of analysis, it was seen that the group administered melatonin had the maximum mean vessel lumen area (Table 2).Vessel lumen area was increased in all of the groups administered treatment compared to vasospasm group, but no statistically significant difference was determined between them (Figure 3).

DISCUSSION
CV begin on the 3rd and 4th days and reaching peak level in the 1st week after SAH and which is severe cause of ischemic neurological deficits [12][13][14][15].Pathophysiology of cerebral vasospasm is complex and multifactorial 16 .Although the specific mechanism of vasospasm has not been determined, neurogenic, metabolic and myogenic theories have been proposed [9,16].
The precense of blood and blood products in cerebrospinal fluid is an etiological factor.There is a correlation between the development CV and increased superoxide anion levels in cerebrospinal fluid after SAH [17].Normally, free radicals or their peroxidation products is neutralized by intrinsic antioxidants such as superoxide dismutase (SOD), catalase, glutathione peroxidase and peroxidase [18,19].Microscopic findings seen in the vessel under the influence of oxidative stress are characterized by increase in vessel wall thickness, luminal narrowing, thickening of internal elastic lamina, deterioration of endothelial cell structure and continuity, vacuoles in smooth muscles, migration of proliferated myointimal cell to the intima, perivascular axonal loss [20,21].Endothelin antagonists, nitric oxide, glutamate antagonists, leukocyte inhibitors, protein kinase inhibitors and many other agents were used for treatment of experimental vasospasm [2,22,23].In this study, we investigated nimodipine currently used for VS treatment and melatonin with known antioxidant efficiency concomitantly and comparing their efficiencies.
Nimodipine has been used in cv treatment after SAH for many years.There is some evidence that voltage-gated calcium channels are involved in vasospasm pathogenesis [4,24.Although it was shown that use of calcium canal blocker after SAH reduced the incidence of cerebral infarction at a rate of 34%, no change was seen in the frequency of CV in the angiographies of the patients using prophylactic nimodipine or symptoms [25,26].It is considered that nimodipine, decreases the risk of the secondary ischemia after SAH, it has a preventive role but there is no evidence that nimodipine decreases the mortality [27][28][29][30].
According to our results, melatonin may reduce vasospasm development and CV related complications.In this study, histopathological and morphometric analysis of rat femoral arteries in experimentally induced vasospasm model supported that melatonin and nimodipine combination therapy may be useful in treating post hemorrhagic vasospasm.When we compared the therapeutic effects of nimodipine and melatonin, the therapeutic effect of melatonin was found to be significantly higher and melatonin and nimodipine were administered concomitantly, their therapeutic effects were increased.

Figure 1 .
Figure 1.(a) Light microscopic view of the control group (Group I; H&E, x200).(b) Light microscopic view of the vasospasm group (Group II; H&E, x200).(c) Light microscopic view of th e group administered melatonin at a dose of 20 mg/kg (Group III, H&E, x200).(d) Light microscopic view of the group administered nimodipine at a dose of 0,1 mg/kg (Group IV, H&E, x200).(e) Light microscopic view of the group administered melatonin at a dose of 20 mg/kg and nimodipine at a dose of 0.1 mg/kg (Group V, H&E, x200).

Figure 2 .Figure 3 .
Figure 2. Comparison of mean vessel wall thickness of the five groups (mm)

Table 1 .
Statistical analysis of the mean vessel wall thickness values of the five groups

Table 2 .
Statistical analysis of the mean vessel lumen area of the five groups SD: Standard deviation