- Email: [email protected]
Effect of L-Arginine Therapy on Vasospasm: Experimental Study in Rats
Original Article
Effect of L-Arginine Therapy on Vasospasm: Experimental Study in Rats Ezgi Akar1, Selin Tural Emon1, Serap Uslu2, Metin Orakdogen3, Hakan Somay3
OBJECTIVE: Cerebral vasospasm occurring after subarachnoid hemorrhage is a serious cause of morbidity. Cerebral vasospasmLrelated studies aim to prevent complications after subarachnoid hemorrhage. Nitric oxide affects brain blood flow and local vascular hemodynamics. L-arginine is used in the synthesis of nitric oxide, and hence we have investigated the efficacy of L-arginine treatment by using femoral artery vasospasm model.
vasospasm in rats. Therefore we think that L-arginine therapy can be used in the prevention and treatment of cerebral vasospasm after subarachnoid hemorrhage.
METHODS: Twenty-four male Sprague-Dawley rats have been divided into 3 groups as vasospasm, vasospasm D Larginine, and control. In this study, we have preferred the “Rat Femoral Artery Vasospasm Model” described by Okada et al. Rats in the vasospasm D L-arginine group were given 300 mg/kg L-arginine for 7 days. At the end of the study, all samples of rat femoral arteries have been dissected and examined microscopically for histopathologic analysis. Statistical analysis was performed using Kruskal-Wallis and Mann-Whitney U tests, and P < 0.05 value was considered statistically significant.
ubarachnoid hemorrhage (SAH) is the determination of blood between pia mater and arachnoid layer. The most frequent cause of spontaneously developed SAH is aneurysmal rupture. Cerebral vasospasm (CV) is the narrowing of the large arteries of the basis cranii induced by blood products and other chemical substances.1 The etiology of cerebral vasospasm, which is highly associated with neurologic deterioration in cases with subarachnoid hemorrhage, is still unclear.2 Degradation of erythrocytes, hypotension, spasmogenic agents, arachidonic acid, serotonin, thromboxane A2, histamine, thrombin, plasmin, angiotensin, potassium, and inflammation are possible factors responsible for the etiology of CV.1 CV-related studies aim to prevent complications after SAH. Nitric oxide (NO) is a gaseous signaling molecule and has an effect on brain blood flow and local vascular hemodynamics. Thus in recent years, many studies have focused on the role of NO in the CV developing secondary to aneurysmal SAH and the role of NO donors and reactive nitrogen products has been the focus of many researchers.3 For NO synthesis, 1 arginine, 2 oxygen molecules, and 1.5 NADP molecules are required and this is done with an NO synthase enzyme.4 Since L-arginine is detected to be used in the synthesis of NO, it may also be an agent that can be used in the prevention and treatment of CV.
-
-
RESULTS: L-arginine treatment reduced the morphometric changes such as irregularity of the elastic lamina, disruption of the endothelial cells, vacuolization, and hemorrhages that are caused by vasospasm. When the wall thickness and lumen diameter measurements were evaluated statistically, significant improvement was observed in the vasospasm D L-arginine group compared with the vasospasm group (P < 0.01).
-
CONCLUSIONS: In our study, the use of L-arginine, as a nitric oxide substrate, improved the experimental
-
Key words Cerebral vasospasm - L-arginine - Nitric oxide - Subarachnoid hemorrhage - Vasospasm -
Abbreviations and Acronyms CV: Cerebral vasospasm NO: Nitric oxide SAH: Subarachnoid hemorrhage
WORLD NEUROSURGERY -: e1-e4, - 2019
INTRODUCTION
S
From the Departments of 1Neurosurgery, Haydarpasa Numune Training and Research Hospital; 2Histology and Embryology, Istanbul Medeniyet University; and 3Neurosurgery, Medicana Kadıkoy Hospital, Istanbul, Turkey To whom correspondence should be addressed: Ezgi Akar, M.D. [E-mail: [email protected]] Citation: World Neurosurg. (2019). https://doi.org/10.1016/j.wneu.2019.08.119 Journal homepage: www.journals.elsevier.com/world-neurosurgery Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2019 Elsevier Inc. All rights reserved.
www.journals.elsevier.com/world-neurosurgery
e1
ORIGINAL ARTICLE EZGI AKAR ET AL.
EFFECT OF L-ARGININE ON VASOSPASM
The aim of this study is to investigate the effect of L-arginine therapy on the rat femoral artery in a vasospasm model. MATERIAL AND METHODS Consent has been given for the use of laboratory animals by Marmara University, Research Ethics Commission. Animal care has been given in compliance with the international laws regarding the use of laboratory animals. The study has included 24 male Sprague-Dawley rats weighing between 220 and 280 gm. The “Rat Femoral Artery Vasospasm Model” described by Okada et al5 was the preferred vasospasm model. Anesthesia was performed using an intraperitoneal injection of ketamine (100 mg/kg). The termination point of the experiment has been determined on the seventh day, when the vasospasm has been maximum. Twenty-four rats were divided randomly into 3 groups: 1. Group 1 (n ¼ 8, control group): A 10-mm segment of femoral artery has been exposed by using a microsurgical technique in the inguinal region, and a Silastic (Dow Corning Corp., Midland, Michigan, USA) sheath was wrapped around the femoral artery without applying vasospasm or any other treatment.5 2. Group 2 (n ¼ 8, vasospasm group): After the same surgical procedure, 0.1 mL of fresh blood derived from ventral tail artery has been applied around the femoral artery without any treatment.5 3. Group 3 (n ¼ 8, vasospasmþ L-arginine group): Vasospasm was induced, and L-arginine was applied intraperitoneally at daily doses of 300 mg/kg for 7 days. At the end of 7 days, all animals were reanesthetized using intraperitoneal ketamine and sacrificed. The former incision was opened, and femoral arteries were dissected. After fixation (10% neutral buffered formalin), the tissue blocks have been dehydrated through graded ethanol (80%, 95%, and 100% sequentially); sterilized in xylene; and embedded in paraffin wax. Tissue sections of 5 mm have been stained with hematoxylin-eosin for
Figure 1. (A) Control group light microscopic findings: thin and regular endothelium, unfolded internal elastic lamina. (B) Vasospasm group light microscopic findings: luminal narrowing, increased wall thickness, nonintact endothelium, curling of the internal elastic lamina. (C)
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histologic and histomorphometric analyses. Histomorphometry, lumen diameter, and wall thickness of femoral arteries have been performed after the images have been transferred from a light microscope (Olympus BX53, Tokyo, Japan) via camera (Olympus DP21) into a computer. All measurements were made with a semiautomatic image analysis system (The University of Texas Health Science Center at San Antonio image tool). The histomorphometric measurements were carried out from 4 different points by a blinded histologist. Statistical Analysis The Kruskal-Wallis and Mann-Whitney U-tests were used for statistical evaluation. The Mann-Whitney U test was preferred for the intergroup comparisons of parameters without normal distribution, and the Kruskal-Wallis test was preferred for comparing more than 2 groups with each other. For all groups alike, maximum, minimum, mean, and standard deviation values were detected and the values of P < 0.05 were considered statistically significant. RESULTS Histopathologic Examination In the control group, histopathologic examination of the femoral artery was revealed with thin and smooth endothelium. Unfolded internal elastic lamina and smooth muscle cells were located concentrically, and the lumen was clearly observed (Figure 1A). In the vasospasm group, marked luminal narrowing and an increase in the vessel wall thickness were observed. Nonintact endothelium, curling of the internal elastic lamina, and vacuolization in smooth muscle layer were observed (see Figure 1B). The L-arginine group’s vascular structure was similar to the control group. Thin and smooth endothelium and concentrically arrayed smooth muscle cells were observed (see Figure 1C). Morphometric Analysis Mean thickness of wall and lumen diameter in all groups have been measured and statistically analyzed as mean and standard deviation (Table 1). The mean wall thickness has been found to be 149.3, 192.3, and 116.4 in Groups 1, 2, and 3, respectively. We observed statistically significant mean wall thickness differences
Vasospasm þ L-arginine group light microscopic findings: thin and smooth endothelium, concentrically arrayed smooth muscle cells. Hematoxylin-eosin, 200. Thin arrow, endothelial layer; thick arrow, disruption of endothelial integrity; *, tunica muscularis bleeding.
WORLD NEUROSURGERY, https://doi.org/10.1016/j.wneu.2019.08.119
ORIGINAL ARTICLE EZGI AKAR ET AL.
EFFECT OF L-ARGININE ON VASOSPASM
Table 1. Vascular Wall Thickness and Lumen Diameter Measurements of All Groups Vascular Wall Thickness and Lumen Diameter of Groups 1. Control
2. Vasospasm
3. Vasospasm D Arginine
P*
Pairwise Comparison; Py
118e179.3
170e212.7
53.7e162.5
0.0004
(Groups 1 and 2) 0.00062 (Groups 2 and 3) 0.000155 (Groups 1e3) 0.13
0.04
(Groups 1 and 2) 0.57 (Groups 2 and 3) 0.01 (Groups 1e3) 0.08
Wall thickness MinMax Median Mean SD
151
195
135.8
149.3
192.3
116.4
19
13.9
43.7
131e269
56e338
184e600
Lumen diameter MinMax Median Mean SD
205
164.7
342
201.5
168
365
55
101
165
SD, standard deviation. *Kruskal-Wallis Test. yMann-Whitney U Test.
between the groups (P < 0.05). The mean wall thickness values of the vasospasm group were significantly higher than the control and vasospasm þ L-arginine groups (P < 0.05). We did not observe any significant statistical difference between mean wall thickness of control and vasospasm þ L-arginine groups (P < 0.05). The mean lumen diameter was 201.5, 167.9, and 365 in Groups 1, 2, and 3, and a statistically significant difference was found among the groups (P < 0.05). The mean lumen diameter of the vasospasm group was significantly lower than the other groups, and the vasospasm þ L-arginine group’s mean lumen diameter values were significantly higher than the control group (P < 0.05). DISCUSSION Cerebral vasospasm caused by cerebral ischemia causes morbidity and mortality after SAH.6 Cerebral vasospasm began on the third and fourth days, which reached the peak level in the first week after the SAH, presenting itself with delayed ischemic neurologic deficits.1,4 Despite much work being done for a long time on the pathogenesis of SAH-induced vasospasm, it is still a matter of debate and an adequate treatment is not available yet.7,8 The etiopathogenesis of vasospasm is multifactorial, and neurogenic factors, free radical species, and metabolic and myogenic damages are possible effective mechanisms. The presence of blood in the cerebrospinal fluid and the amount of blood, as well as free oxygen products from erythrocytes, are the main causes of post-SAH cerebral vasospasm. Also, oxidative stress resulting from free radical reactions and inflammation are among the possible factors for the etiopathogenesis of vasospasm.2,7,9,10 Many different animal models are known to form a cerebral arterial vasospasm model. Okada et al5 described experimental vasospasm in the rat femoral artery. They found that histopathologic changes in the rat femoral arteries exposed to
WORLD NEUROSURGERY -: e1-e4, - 2019
periadventitial blood were similar to those of cerebral arterial vasospasm after SAH. This method has been preferred by many researchers because of its ease of application and low mortality and morbidity rates and similar findings with cerebral vessels. However, this being the case, in order to have more significant results, working on different models with which we can evaluate the cerebral arteries will increase the credibility of the findings. Many different pharmacologic agents have been tried to prevent and treat CV in both clinical practice and experimental studies; free radical scavengers, vasodilators agents, glutamate antagonists, leukocyte inhibitors, and protein kinase inhibitors are some of these agents.11,12 Although many agents are found useful in the experimental setting, CV is still a difficult condition to prevent and treat. One encouraging mechanism to prevent complications after SAH-related vasospasm involves enhancement of NO signaling in the cerebral vasculature.3 NO is a signaling molecule critical for retaining blood vessel patency and promoting local hemodynamics in cerebrum.10 NO promotes cerebral blood flow in resting mode and during synaptic activity and metabolic activity such as hypercapnia conditions.13 NO activity and its effects on cerebral blood flow are determined by NO signaling and affect NO synthesis. This being the case, many experimental studies determine that NO and NO donors have a protective effect against vasospasm and improve neurologic outcomes after SAH.10 In our study, mean wall thickness and lumen diameter measurements of L-arginine group showed statistically significant improvement compared with vasospasm group. Light microscopic findings of L-arginine group were similar to the control group, and nearly normal microvascular structure was seen. Thus L-arginine played a protective role against the effects of vasospasm. Pluta14 showed that NO is protective against vasospasm and can prevent angiographic vasospasm. Some agents of NO donor, such as nitroglycerin, have been also considered to possibly contribute to clinical
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ORIGINAL ARTICLE EZGI AKAR ET AL.
EFFECT OF L-ARGININE ON VASOSPASM
improvement in SAH. The hemoglobin that emerges after SAH acts as a NO scavenger, and NO synthesis is rapidly upregulated by free radical products after aneurysmal SAH.13 Thus many researchers have studied on the role of NO and NO donors on the development and prevention of cerebral vasospasm after SAH. Different materials have been used in previous studies as NO donor, such as NONOates, S-nitrosothiols, nitroglycerin, peroxynitrite, and sodium nitrite.15,16 Intravascular administration of L-arginine, as a NO substrate, has been shown to cause vasodilatation and increase cerebral blood flow; however, no finding has been found to show that it prevents cerebral vasospasm. This effect of L arginine is thought to exist with an increase in NO synthesis by upregulation of eNOS or iNOS in macrophages, reactive astrocytes, and smooth-muscle cells activated by hemoglobin products after SAH.17 L-arginine and superoxide dismutase have been shown to be effective in preventing cerebral vasospasm by intracisternal use in dogs.18 We have studied the efficiency of L-arginine peripheral administration in an experimental vasospasm model formed in the rat femoral artery. Because L-arginine is an NO donor, our study indicates that it can be used in the treatment and prevention of vasospasm after SAH. In our study, in the L-arginine group, vascular wall thickness and lumen diameter measurements showed statistically significant improvement compared with the vasospasm group. As a result of
REFERENCES 1. Chehrazi BB, Giri S, Joy RM. Prostaglandins and vasoactive amines in cerebral vasospasm after aneurysmal subarachnoid hemorrhage. Stroke. 1989;20:217-224. 2. Baggott CD, Aagaard-Kienitz B. Cerebral vasospasm. Neurosurg Clin N Am. 2014;25:497-528. 3. Förstermann U, Münzel T. Endothelial nitric oxide synthase in vascular disease. Circulation. 2006;13: 1708-1714. 4. Nathan C, Xie Q-W. Nitric oxide synthase: roles, tolls, and controls. Cell. 1994;78:915-918. 5. Okada T, Harada T, Bark DH, Mayberg MR. A rat femoral artery model for vasospasm. Neurosurgery. 1990;27:349-356. 6. Kılıç T, Konya D. Temel Nöros¸irurji. Ankara: Turkish Neurosurgical Society; 2005:191-192. 7. Hakan T, Berkman MZ, Ersoy T, Karatas¸ I, San T, Arbak S. Antiinflammatory effect of meloxicam on experimental vasospasm in the rat femoral artery. J Clin Neurosci. 2008;15:55-59. 8. Ayer RE, Zhang JH. Oxidative stress in subarachnoid haemorrhage: significance in acute brain injury and vasospasm. Acta Neurochirurg. 2008;104:33-41.
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histopathologic and morphometric analysis of femoral artery, we observed that L-arginine has a vasospasm healing effect. Previous studies have shown that NO has a therapeutic efficacy in cerebral vasospasm and is important in brain blood supply.3,4,14 The data we obtained in our study once again showed the effectiveness of L-arginine and NO in arterial vasospasm in rats. We think that the effectiveness of L-arginine in experimental femoral arterial vasospasm is used in NO synthesis and indirectly increases NO. However, our study has some limitations. Firstly, histopathologic findings of femoral artery vasospasm are similar to vasospasm of cerebral blood vessels but may not always be identical. Furthermore, in order to use L-arginine in the prevention and treatment of VS, these findings must be supported by different experimental models and clinical studies in which cerebral vessels can be evaluated. CONCLUSION According to our results, L-arginine reduced vasospasm development in rat femoral artery vasospasm model. We suggest that the beneficial effect of L-arginine is that it contributes to NO synthesis and thus vasodilatation. These results may be promising in the prevention of cerebral vasospasm after subarachnoid hemorrhage. However, the support of this view with human studies will increase its reliability in clinical use.
9. Pyne-Geithman GJ, Caudell DN, Prakash P, Clark JH. Glutathione peroxidase and subarachnoid hemorrhage: implications for the role of oxidative stress in cerebral vasospasm. Neurol Res. 2009;31:195-199.
16. Wolf EW, Banerjee A, Soble-Smith J, Dohan FC Jr, White RP, Robertson JT. Reversal of cerebral vasospasm using an intrathecally administered nitric oxide donor. J Neurosurg. 1998;89:279-288.
10. Megyesi JF, Vollrath B, Cook DA, Findlay JM. In vivo animal models of cerebral vasospasm: a review. Neurosurgery. 2000;46:448-461.
17. Suzuki S, Kassell NF, Lee KS. Hemin activation of an inducible isoform of nitric oxide synthase in vascular smooth-muscle cells. J Neurosurg. 1995;83: 862-866.
11. Karaoglan A, Akdemir O, Barut S, et al. The effects of resveratrol on vasospasm after experimental subarachnoidal hemorrhage in rats. Surg Neurol. 2008;70:337-343.
18. Kajita Y, Suzuki Y, Oyama H, et al. Combined effect of L-arginine and superoxide dismutase on the spastic basilar artery after subarachnoid hemorrhage in dogs. J Neurosurg. 1994;80:476-483.
12. Munakata A, Ohkuma H, Nakano T, Shimamura N, Asano K, Naraoka M. Effect of a free radical scavenger, edaravone, in the treatment of patients with aneurysmal subarachnoid hemorrhage. Neurosurgery. 2009;64:423-428.
Conflict of interest statement: The authors declare that the article content was composed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
13. Edvinsson L, Krause DN. Cerebral Blood Flow and Metabolism. 2nd ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2002. 14. Pluta RM. Delayed cerebral vasospasm and nitric oxide: review, new hypothesis, and proposed treatment. Pharmacol Ther. 2005;105:23-56. 15. Tierney TS, Pradilla G, Wang PP, Clatterbuck RE, Tamargo RJ. Intracranial delivery of the nitric oxide donor diethylenetriamine/nitric oxide from a controlled-release polymeter: toxicity in cynomolgus monkeys. Neurosurgery. 2006;58:952-960.
Received 6 June 2019; accepted 16 August 2019 Citation: World Neurosurg. (2019). https://doi.org/10.1016/j.wneu.2019.08.119 Journal homepage: www.journals.elsevier.com/worldneurosurgery Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2019 Elsevier Inc. All rights reserved.
WORLD NEUROSURGERY, https://doi.org/10.1016/j.wneu.2019.08.119
Effect of L-Arginine Therapy on Vasospasm: Experimental Study in Rats Ezgi Akar1, Selin Tural Emon1, Serap Uslu2, Metin Orakdogen3, Hakan Somay3
OBJECTIVE: Cerebral vasospasm occurring after subarachnoid hemorrhage is a serious cause of morbidity. Cerebral vasospasmLrelated studies aim to prevent complications after subarachnoid hemorrhage. Nitric oxide affects brain blood flow and local vascular hemodynamics. L-arginine is used in the synthesis of nitric oxide, and hence we have investigated the efficacy of L-arginine treatment by using femoral artery vasospasm model.
vasospasm in rats. Therefore we think that L-arginine therapy can be used in the prevention and treatment of cerebral vasospasm after subarachnoid hemorrhage.
METHODS: Twenty-four male Sprague-Dawley rats have been divided into 3 groups as vasospasm, vasospasm D Larginine, and control. In this study, we have preferred the “Rat Femoral Artery Vasospasm Model” described by Okada et al. Rats in the vasospasm D L-arginine group were given 300 mg/kg L-arginine for 7 days. At the end of the study, all samples of rat femoral arteries have been dissected and examined microscopically for histopathologic analysis. Statistical analysis was performed using Kruskal-Wallis and Mann-Whitney U tests, and P < 0.05 value was considered statistically significant.
ubarachnoid hemorrhage (SAH) is the determination of blood between pia mater and arachnoid layer. The most frequent cause of spontaneously developed SAH is aneurysmal rupture. Cerebral vasospasm (CV) is the narrowing of the large arteries of the basis cranii induced by blood products and other chemical substances.1 The etiology of cerebral vasospasm, which is highly associated with neurologic deterioration in cases with subarachnoid hemorrhage, is still unclear.2 Degradation of erythrocytes, hypotension, spasmogenic agents, arachidonic acid, serotonin, thromboxane A2, histamine, thrombin, plasmin, angiotensin, potassium, and inflammation are possible factors responsible for the etiology of CV.1 CV-related studies aim to prevent complications after SAH. Nitric oxide (NO) is a gaseous signaling molecule and has an effect on brain blood flow and local vascular hemodynamics. Thus in recent years, many studies have focused on the role of NO in the CV developing secondary to aneurysmal SAH and the role of NO donors and reactive nitrogen products has been the focus of many researchers.3 For NO synthesis, 1 arginine, 2 oxygen molecules, and 1.5 NADP molecules are required and this is done with an NO synthase enzyme.4 Since L-arginine is detected to be used in the synthesis of NO, it may also be an agent that can be used in the prevention and treatment of CV.
-
-
RESULTS: L-arginine treatment reduced the morphometric changes such as irregularity of the elastic lamina, disruption of the endothelial cells, vacuolization, and hemorrhages that are caused by vasospasm. When the wall thickness and lumen diameter measurements were evaluated statistically, significant improvement was observed in the vasospasm D L-arginine group compared with the vasospasm group (P < 0.01).
-
CONCLUSIONS: In our study, the use of L-arginine, as a nitric oxide substrate, improved the experimental
-
Key words Cerebral vasospasm - L-arginine - Nitric oxide - Subarachnoid hemorrhage - Vasospasm -
Abbreviations and Acronyms CV: Cerebral vasospasm NO: Nitric oxide SAH: Subarachnoid hemorrhage
WORLD NEUROSURGERY -: e1-e4, - 2019
INTRODUCTION
S
From the Departments of 1Neurosurgery, Haydarpasa Numune Training and Research Hospital; 2Histology and Embryology, Istanbul Medeniyet University; and 3Neurosurgery, Medicana Kadıkoy Hospital, Istanbul, Turkey To whom correspondence should be addressed: Ezgi Akar, M.D. [E-mail: [email protected]] Citation: World Neurosurg. (2019). https://doi.org/10.1016/j.wneu.2019.08.119 Journal homepage: www.journals.elsevier.com/world-neurosurgery Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2019 Elsevier Inc. All rights reserved.
www.journals.elsevier.com/world-neurosurgery
e1
ORIGINAL ARTICLE EZGI AKAR ET AL.
EFFECT OF L-ARGININE ON VASOSPASM
The aim of this study is to investigate the effect of L-arginine therapy on the rat femoral artery in a vasospasm model. MATERIAL AND METHODS Consent has been given for the use of laboratory animals by Marmara University, Research Ethics Commission. Animal care has been given in compliance with the international laws regarding the use of laboratory animals. The study has included 24 male Sprague-Dawley rats weighing between 220 and 280 gm. The “Rat Femoral Artery Vasospasm Model” described by Okada et al5 was the preferred vasospasm model. Anesthesia was performed using an intraperitoneal injection of ketamine (100 mg/kg). The termination point of the experiment has been determined on the seventh day, when the vasospasm has been maximum. Twenty-four rats were divided randomly into 3 groups: 1. Group 1 (n ¼ 8, control group): A 10-mm segment of femoral artery has been exposed by using a microsurgical technique in the inguinal region, and a Silastic (Dow Corning Corp., Midland, Michigan, USA) sheath was wrapped around the femoral artery without applying vasospasm or any other treatment.5 2. Group 2 (n ¼ 8, vasospasm group): After the same surgical procedure, 0.1 mL of fresh blood derived from ventral tail artery has been applied around the femoral artery without any treatment.5 3. Group 3 (n ¼ 8, vasospasmþ L-arginine group): Vasospasm was induced, and L-arginine was applied intraperitoneally at daily doses of 300 mg/kg for 7 days. At the end of 7 days, all animals were reanesthetized using intraperitoneal ketamine and sacrificed. The former incision was opened, and femoral arteries were dissected. After fixation (10% neutral buffered formalin), the tissue blocks have been dehydrated through graded ethanol (80%, 95%, and 100% sequentially); sterilized in xylene; and embedded in paraffin wax. Tissue sections of 5 mm have been stained with hematoxylin-eosin for
Figure 1. (A) Control group light microscopic findings: thin and regular endothelium, unfolded internal elastic lamina. (B) Vasospasm group light microscopic findings: luminal narrowing, increased wall thickness, nonintact endothelium, curling of the internal elastic lamina. (C)
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histologic and histomorphometric analyses. Histomorphometry, lumen diameter, and wall thickness of femoral arteries have been performed after the images have been transferred from a light microscope (Olympus BX53, Tokyo, Japan) via camera (Olympus DP21) into a computer. All measurements were made with a semiautomatic image analysis system (The University of Texas Health Science Center at San Antonio image tool). The histomorphometric measurements were carried out from 4 different points by a blinded histologist. Statistical Analysis The Kruskal-Wallis and Mann-Whitney U-tests were used for statistical evaluation. The Mann-Whitney U test was preferred for the intergroup comparisons of parameters without normal distribution, and the Kruskal-Wallis test was preferred for comparing more than 2 groups with each other. For all groups alike, maximum, minimum, mean, and standard deviation values were detected and the values of P < 0.05 were considered statistically significant. RESULTS Histopathologic Examination In the control group, histopathologic examination of the femoral artery was revealed with thin and smooth endothelium. Unfolded internal elastic lamina and smooth muscle cells were located concentrically, and the lumen was clearly observed (Figure 1A). In the vasospasm group, marked luminal narrowing and an increase in the vessel wall thickness were observed. Nonintact endothelium, curling of the internal elastic lamina, and vacuolization in smooth muscle layer were observed (see Figure 1B). The L-arginine group’s vascular structure was similar to the control group. Thin and smooth endothelium and concentrically arrayed smooth muscle cells were observed (see Figure 1C). Morphometric Analysis Mean thickness of wall and lumen diameter in all groups have been measured and statistically analyzed as mean and standard deviation (Table 1). The mean wall thickness has been found to be 149.3, 192.3, and 116.4 in Groups 1, 2, and 3, respectively. We observed statistically significant mean wall thickness differences
Vasospasm þ L-arginine group light microscopic findings: thin and smooth endothelium, concentrically arrayed smooth muscle cells. Hematoxylin-eosin, 200. Thin arrow, endothelial layer; thick arrow, disruption of endothelial integrity; *, tunica muscularis bleeding.
WORLD NEUROSURGERY, https://doi.org/10.1016/j.wneu.2019.08.119
ORIGINAL ARTICLE EZGI AKAR ET AL.
EFFECT OF L-ARGININE ON VASOSPASM
Table 1. Vascular Wall Thickness and Lumen Diameter Measurements of All Groups Vascular Wall Thickness and Lumen Diameter of Groups 1. Control
2. Vasospasm
3. Vasospasm D Arginine
P*
Pairwise Comparison; Py
118e179.3
170e212.7
53.7e162.5
0.0004
(Groups 1 and 2) 0.00062 (Groups 2 and 3) 0.000155 (Groups 1e3) 0.13
0.04
(Groups 1 and 2) 0.57 (Groups 2 and 3) 0.01 (Groups 1e3) 0.08
Wall thickness MinMax Median Mean SD
151
195
135.8
149.3
192.3
116.4
19
13.9
43.7
131e269
56e338
184e600
Lumen diameter MinMax Median Mean SD
205
164.7
342
201.5
168
365
55
101
165
SD, standard deviation. *Kruskal-Wallis Test. yMann-Whitney U Test.
between the groups (P < 0.05). The mean wall thickness values of the vasospasm group were significantly higher than the control and vasospasm þ L-arginine groups (P < 0.05). We did not observe any significant statistical difference between mean wall thickness of control and vasospasm þ L-arginine groups (P < 0.05). The mean lumen diameter was 201.5, 167.9, and 365 in Groups 1, 2, and 3, and a statistically significant difference was found among the groups (P < 0.05). The mean lumen diameter of the vasospasm group was significantly lower than the other groups, and the vasospasm þ L-arginine group’s mean lumen diameter values were significantly higher than the control group (P < 0.05). DISCUSSION Cerebral vasospasm caused by cerebral ischemia causes morbidity and mortality after SAH.6 Cerebral vasospasm began on the third and fourth days, which reached the peak level in the first week after the SAH, presenting itself with delayed ischemic neurologic deficits.1,4 Despite much work being done for a long time on the pathogenesis of SAH-induced vasospasm, it is still a matter of debate and an adequate treatment is not available yet.7,8 The etiopathogenesis of vasospasm is multifactorial, and neurogenic factors, free radical species, and metabolic and myogenic damages are possible effective mechanisms. The presence of blood in the cerebrospinal fluid and the amount of blood, as well as free oxygen products from erythrocytes, are the main causes of post-SAH cerebral vasospasm. Also, oxidative stress resulting from free radical reactions and inflammation are among the possible factors for the etiopathogenesis of vasospasm.2,7,9,10 Many different animal models are known to form a cerebral arterial vasospasm model. Okada et al5 described experimental vasospasm in the rat femoral artery. They found that histopathologic changes in the rat femoral arteries exposed to
WORLD NEUROSURGERY -: e1-e4, - 2019
periadventitial blood were similar to those of cerebral arterial vasospasm after SAH. This method has been preferred by many researchers because of its ease of application and low mortality and morbidity rates and similar findings with cerebral vessels. However, this being the case, in order to have more significant results, working on different models with which we can evaluate the cerebral arteries will increase the credibility of the findings. Many different pharmacologic agents have been tried to prevent and treat CV in both clinical practice and experimental studies; free radical scavengers, vasodilators agents, glutamate antagonists, leukocyte inhibitors, and protein kinase inhibitors are some of these agents.11,12 Although many agents are found useful in the experimental setting, CV is still a difficult condition to prevent and treat. One encouraging mechanism to prevent complications after SAH-related vasospasm involves enhancement of NO signaling in the cerebral vasculature.3 NO is a signaling molecule critical for retaining blood vessel patency and promoting local hemodynamics in cerebrum.10 NO promotes cerebral blood flow in resting mode and during synaptic activity and metabolic activity such as hypercapnia conditions.13 NO activity and its effects on cerebral blood flow are determined by NO signaling and affect NO synthesis. This being the case, many experimental studies determine that NO and NO donors have a protective effect against vasospasm and improve neurologic outcomes after SAH.10 In our study, mean wall thickness and lumen diameter measurements of L-arginine group showed statistically significant improvement compared with vasospasm group. Light microscopic findings of L-arginine group were similar to the control group, and nearly normal microvascular structure was seen. Thus L-arginine played a protective role against the effects of vasospasm. Pluta14 showed that NO is protective against vasospasm and can prevent angiographic vasospasm. Some agents of NO donor, such as nitroglycerin, have been also considered to possibly contribute to clinical
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ORIGINAL ARTICLE EZGI AKAR ET AL.
EFFECT OF L-ARGININE ON VASOSPASM
improvement in SAH. The hemoglobin that emerges after SAH acts as a NO scavenger, and NO synthesis is rapidly upregulated by free radical products after aneurysmal SAH.13 Thus many researchers have studied on the role of NO and NO donors on the development and prevention of cerebral vasospasm after SAH. Different materials have been used in previous studies as NO donor, such as NONOates, S-nitrosothiols, nitroglycerin, peroxynitrite, and sodium nitrite.15,16 Intravascular administration of L-arginine, as a NO substrate, has been shown to cause vasodilatation and increase cerebral blood flow; however, no finding has been found to show that it prevents cerebral vasospasm. This effect of L arginine is thought to exist with an increase in NO synthesis by upregulation of eNOS or iNOS in macrophages, reactive astrocytes, and smooth-muscle cells activated by hemoglobin products after SAH.17 L-arginine and superoxide dismutase have been shown to be effective in preventing cerebral vasospasm by intracisternal use in dogs.18 We have studied the efficiency of L-arginine peripheral administration in an experimental vasospasm model formed in the rat femoral artery. Because L-arginine is an NO donor, our study indicates that it can be used in the treatment and prevention of vasospasm after SAH. In our study, in the L-arginine group, vascular wall thickness and lumen diameter measurements showed statistically significant improvement compared with the vasospasm group. As a result of
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histopathologic and morphometric analysis of femoral artery, we observed that L-arginine has a vasospasm healing effect. Previous studies have shown that NO has a therapeutic efficacy in cerebral vasospasm and is important in brain blood supply.3,4,14 The data we obtained in our study once again showed the effectiveness of L-arginine and NO in arterial vasospasm in rats. We think that the effectiveness of L-arginine in experimental femoral arterial vasospasm is used in NO synthesis and indirectly increases NO. However, our study has some limitations. Firstly, histopathologic findings of femoral artery vasospasm are similar to vasospasm of cerebral blood vessels but may not always be identical. Furthermore, in order to use L-arginine in the prevention and treatment of VS, these findings must be supported by different experimental models and clinical studies in which cerebral vessels can be evaluated. CONCLUSION According to our results, L-arginine reduced vasospasm development in rat femoral artery vasospasm model. We suggest that the beneficial effect of L-arginine is that it contributes to NO synthesis and thus vasodilatation. These results may be promising in the prevention of cerebral vasospasm after subarachnoid hemorrhage. However, the support of this view with human studies will increase its reliability in clinical use.
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18. Kajita Y, Suzuki Y, Oyama H, et al. Combined effect of L-arginine and superoxide dismutase on the spastic basilar artery after subarachnoid hemorrhage in dogs. J Neurosurg. 1994;80:476-483.
12. Munakata A, Ohkuma H, Nakano T, Shimamura N, Asano K, Naraoka M. Effect of a free radical scavenger, edaravone, in the treatment of patients with aneurysmal subarachnoid hemorrhage. Neurosurgery. 2009;64:423-428.
Conflict of interest statement: The authors declare that the article content was composed in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
13. Edvinsson L, Krause DN. Cerebral Blood Flow and Metabolism. 2nd ed. Philadelphia, PA: Lippincott Williams & Wilkins; 2002. 14. Pluta RM. Delayed cerebral vasospasm and nitric oxide: review, new hypothesis, and proposed treatment. Pharmacol Ther. 2005;105:23-56. 15. Tierney TS, Pradilla G, Wang PP, Clatterbuck RE, Tamargo RJ. Intracranial delivery of the nitric oxide donor diethylenetriamine/nitric oxide from a controlled-release polymeter: toxicity in cynomolgus monkeys. Neurosurgery. 2006;58:952-960.
Received 6 June 2019; accepted 16 August 2019 Citation: World Neurosurg. (2019). https://doi.org/10.1016/j.wneu.2019.08.119 Journal homepage: www.journals.elsevier.com/worldneurosurgery Available online: www.sciencedirect.com 1878-8750/$ - see front matter ª 2019 Elsevier Inc. All rights reserved.
WORLD NEUROSURGERY, https://doi.org/10.1016/j.wneu.2019.08.119