& the Role of Nitric Oxide in Resolution of Vasospasam Corresponding with Cerebral Vasospasms after Subarachnoid Haemo- Rrhage: Animal Model Kemal Dizdarević: the Role of Nitric Oxide in Resolution of Vasospasam Corresponding with Cerebral Vasospasms after Subarachnoid Haemorrhage: Animal Model

Intracranial aneurysmal rupture is the common cause of spontaneous subarachnoid haemorrhage (SAH). Th is haemorrhage is typically diff use and located in extracerebral subarachnoid space in which main cerebral arterial branches are situated. Th e intimate and long-term contact of arterial wall and blood products in the closed space causes the cerebral vasospasm as a serious and frequent complication of SAH. It is connected with signifi cant morbidity and mortality due to developing of focal cerebral ischaemia and subsequently cerebral infarction. Th e aim of our experimental research was to create the animal model of vasospasm using the femoral artery due to examination of reduced basic dilator activity cause in arterial wall after SAH. Th e important characteristic of major cerebral arteries is their localization in the closed subarachnoid space which enables their to have long-term contact with blood products after haemorrhage. Th irty six femoral arteries (FA) of eighteen female rats weighing about  g were used. In vivo, femoral arteries are microsurgically prepared in both inguinal regions in all rats. Eighteen arteries were encompassed by polytetrafl uoroethylene (PTFE) material forming closed tube and autolo-gous blood was injected in the tube around the arterial wall. Additional eighteen arteries, as a control group, were also put in PTFE tube but without exposing to the blood. All rats are left to live for eight days. Afterwards, rats were sacrifi ced and their arteries were in vitro examined including an isometric tension measurement and histo-logical changes analysis. Th e tension was measured during application of vasoconstrictors and vasodilatators (ni-tric oxide, NO). FA exposed to periadventitial blood exhibit hyper reactivity to constrictors (KCl, phenylephrine, acetylcholine) compared to control group. It was also found that NO donor (sodium nitroprusside) diminished arterial spasm induced by blood and vasoconstrictors. In conclusion, FA can be used as a model for vasospasm correlating with cerebral vasospasm after SAH and therefore this model can be utilized in future experiments assessing cerebral vasospasm. Th e reduced basic dilator activity of spastic femoral artery is caused by an absence of gaseous messenger NO next to the arteries but not by diminished response vasculature to NO. Absence of NO after SAH probably causes the reduced basic dilator activity of cerebral arteries as well. Th e guanylate-cyclase level in the arterial wall is consequently reduced after SAH primary due to absence of NO but not due to direct reduction of enzyme activities caused …


Introduction
Cerebral vasospasm is a devastating complication of spontaneous subarachnoid hemorrhage (SAH).The presence of blood products in subarachnoid space cause an imbalance between intrinsic constrictor and dilator mechanisms in cerebral arteries with consecutive increased constrictor mechanism as a crucial contribution for onset and development of vasospasm (,,).Clinical signifi cant and diagnostic measurable vasospasm occurs on approximately day  post-SAH, although acute vasospasm is also stressed in novel reports.Recently, nitric oxide is recognized as an important factor in cerebral dilatation, but its exact role in the pathophysiological processes after SAH is not completely elucidated (,,) .Th e key question associated with post-SAH vasospasm is whether increased basic constrictor activity in arterial wall is caused by reduction of nitric oxide (NO) or by insufficient artery response to NO i.e. by changes of mural arterial enzyme systems in which guanylate (guanylyl)-cyclase plays the most important role (, ) If the problem is reduction of NO, the simple NO addition could resolve it.In the case of an impaired vasodilator response of vasculature to NO, the real problem is a reduced capacity of soluble guanylate-cyclase to generate cGMP, which is direct vasodilator (, ).Our objectives were to create experimental, animal model of delayed vasospasm using rat femoral artery and to examine the cause of reduced basic dilator activity after SAH with the help of this model.

Materials and Methods
Two hypotheses were put forward concerning this research: .A rat femoral artery can be an appropriate model for vasospasm correlating with post SAH spasm in the setting of closed system which implies the longterm contact of blood products with arterial wall and when microsurgical dissection is performed.
.An impairment of basic vasodilator activity is determined by diminished production of NO (or NO inactivation) but not primary by reduced effectiveness of guanylate-cyclase, enzyme activated by NO.Eighteen female Wistar rats weighing - g were used.Th e thirty-six rat femoral arteries with outer diameter of , mm (Figure .) were in vivo dissected by utilizing microsurgical technique and Zeiss operative microscope.Rats are anesthetized with intraperitoneal Nembutal ( mg/kg) and allowed to breathe spontaneously.Autologous periadventitial blood was applied to eighteen femoral arteries and vessels were encompassed by the polytetrafl uoroethylene (PTFE) material forming closed tube.Additional eighteen arteries, as a control group, were also put in the PTFE tube but without exposing to the blood.Both groups of arteries in all rats were prepared and left for eight days in vivo (Figure .).Afterwards all animals were sacrifi ced and arteries were processed for in vitro examination including an isometric tension measurement and histological changes analysis.Th e tension was measured in both groups of arteries during application of vasoconstrictors (KCl, phenylephrine, and acetylcholine) and vasodilator -NO.This model corresponds with post -SAH situation of cerebral arteries.Th e most intensive rat vessels spastic reaction can be noted eight days after exposing arteries to the blood so this period was a right moment for sacrifi cing animals.Femoral arteries, exposed to the blood and its products during eight days, were excised from the rats as an  mm long segment.In vitro this segment was cut in two ring segments of  mm length.One segment was sent to the histological analysis.Th rough lumen of another one, two thin wires were introduced and metal triangles were formed from these wires with the help Nitric oxide donor was sodium-nitroprusside/ NaFe(CN)NO/.The concentration of  mmol/ dm donor was added to solution.Before donor NO adding the reaction of arteries was examined by using the KCl, phenilephrine and acetylcholine.We used KCL in concentration of  mmol/dm  .Transducer had a system for load adjustment.The loading was made on arterial wall with the help of thread and metal triangle.It caused the extending of arterial wall and its tunica media.After bringing the artery from in vivo to in vitro conditions, artery was primary put in Krebs' solution (water bath) during  min to be adjusted to new conditions.Afterwards, the test length/tension was made.Firstly, artery exposed to blood was examined on the following way:


-Increased the tension on the arterial wall by adjusting the load on the transducer up to  gram, and artery was left with that load for  minutes.-Additional load, up to  grams, on the arterial wall was made.It was waited for additional  minutes.
-Additional load up to  grams.Additional  minutes waiting -Adding the ,  ml KCl solution, in the concentration of  mol, to the organ chamber which has been fi lled out with  ml of Krebs' solution.On this way we get the adequate concentration of KCl at the organ chamber ( mmol/ dm KCl).
-Washing out and waiting for additional  minutes -Adding of phenylephrine in increasing concentration from -, - to - mmol/dm -Washing out and leaving the artery at the water bath waiting for additional  minutes -Adding the acetylcholine in the concentration of - mmol/dm -Washing out and waiting for  minutes.
-Repetition of whole procedure after primary adding of NO.Th e same artery was included in the same protocol but with one important diff erence.Before adding the abovementioned constrictors, artery was treated by sodium nitroprusside (NO donor) in the concentration of  mmol/ dm.In fact, NO was added at the chamber with spastic artery and whole procedure was repeated with KCl, phenylephrine, and acetylcholine.
-Assessment of control group of arteries with the same protocol but without adding NO.Th e reason for this approach is our intention to compare reaction on the constrictors of two groups of arteries: control group and group of arteries exposed to blood products without NO addition Th e group of arteries exposed to blood products and with NO addition should have showed whether NO has an ability to modulate response of arteries in the case when their basic isometric tension are increased.Shortly, we measured the response of arteries on constrictors and that response was recorded on millimeter papers by utilizing the isometric transducer.Transducer was converted the induced forces from the arterial wall in the electrical signal.Fifty minutes after arterial excision, its connection to transducer and fi xation to the wall of bath, the addition of abovementioned substances was followed according to described protocol.Th e response was recorded on millimeter papers.Th e  mm segment of each artery was sent to the histological examination.

Results
Th ree groups of arteries were created by  examined arteries.Th e fi rst group of arteries included spastic ones e.g.those arteries exposed only to blood (spastic).Th e second group is the fi rst group which was primary exposed to blood and secondly to the NO donor (spastic + NO).Th ird group is the control group e. g arteries which were not exposed to blood and NO.Tables , , and  show an arterial response to the constrictors (KCl, phenylephrin, acetylcholine) in the terms of millimeters.For example, the control group showed the minimal response to KCl (in the range  to  mm) but spastic group showed extensive response ( to  mm).Th e response was proportional to the tension increase in the arterial wall which means to the strength of contraction (level of spasm) (, ).
To additionally confirm our results from other point of view, ten spastic arteries were randomized after histological confirmation of morphological changes corresponding with spasm.The Figure  illustrate the descent of line on the millimeter papers after adding of NO donor.This means that isometric tension in the arterial wall is decreased.Tension is converted in the electrical signal which is recorded on the papers.The height of one segment of paper is  mm.
Th ere was a statistically signifi cant diff erence in descending line on millimeter paper after adding of NO donor meaning there is a signifi cant diff erence in arterial dilatation i.e. p-value < , and X = ,.(Table  and ).Histological analysis showed that corrugation of the elastic laminas and endothelial cells changes are constantly present histological fi ndings in spastic arteries (Figure ).

Discussion
A original rat model for vasospasm which correlates with cerebral vasospasm after SAH has been developed (, ).The peripheral artery () was microsurgically exposed to autologous blood and situated in closed system in vivo imitating the cerebral artery in subarachnoid space.Th e contractile responses on KCl, phenylephrine and acetylcholine indicated that arteries exposed to periadventitial blood (spastic arteries) exhibit hyper-reactivity compared to control group of arteries.It was proved that NO modulates spastic arteries reactivity on vasoconstrictor substances.It was also found that previously induced vasospasm can be clearly diminished if sodium nitroprusside (NO donor) is used  ).Additionally, in vitro experiment was also included ten randomized arteries, previously exposed to periadventitial blood in which histological changes corresponding with morphological signs of spasm had already been found.Th ese arteries were added NO donor (sodium nitroprusside).A decrease of isometric tension in arterial wall was noted in the form of descending line on millimeter paper.Th e tension decrease was a sign of arterial dilatation and a confi rmation that even spastic arteries can respond to NO. Th is was additional argument for hypothesis that post SAH problem is not insuffi cient artery response to NO due to changes of mural arterial enzyme guanylate (guanylyl)-cyclase which generates cGMP, direct vasodilator.Th e real problem is reduction of NO itself so the simple NO addition could be a solution.
Histological analysis helped us to notice morphological changes in the walls of spastic arteries and to confi rm quantitative diff erence between inner and outer diameter of arteries exposed to blood and those not exposed to blood.The adventitia was not measured because this measurement is uncertain ().Our animal model of vasospasm with peripheral artery could correspond to other animal model of vasospasm after SAH in which cerebral arteries were used.Our model is simple and can be used easily.Also, the exposing of rat femoral artery is standard part of microsurgical training for neurosurgeons, plastic and vascular surgeons.

Conclusion
. Femoral artery can be used as a model for vasospasm correlating with cerebral vasospasm after SAH and therefore this model can be utilized in future experiments assessing cerebral vasospasm..Th e reduced basic dilator activity of spastic femoral artery is caused by an absence of gaseous messenger NO next to the arteries but not by diminished vasculature response to NO. .Absence of NO after SAH causes the reduced basic dilator activity of cerebral arteries as well..Th e cGMP level in the arterial wall is probably reduced after SAH primary due to absence of NO but not due to direct guanylate-cyclase inactivation caused by factors generated from the process of blood degradation inside the subarachnoid space.
BOSNIAN JOURNAL OF BASIC MEDICAL SCIENCES 2008; 8 (2): 179-182 KEMAL DIZDAREVIĆ: THE ROLE OF NITRIC OXIDE IN RESOLUTION OF VASOSPASAM CORRESPONDING WITH CEREBRAL VASOSPASMS AFTER SUBARACHNOID HAEMORRHAGE: ANIMAL MODEL of microscope and microinstruments.The metal triangles were connected with nonelastic threads.Arterial rings, prepared in accordance with above-mentioned principles, were brought to the water bath with organ chamber and Krebs's solution due to isometric tension recording.One part of thread was tied to the transducer and another one was fi xed in the bath (Figure ).Vasoconstrictors were added to the both groups of arteries, in the organ chamber, owing to registration of arterial reactivity differences.NO donor (sodium nitroprusside) is also added due to examination of NO modulatory influence to spastic arteries and appraisal of vascular relaxation.Water bath was filled out with distillated water with constant temperature of   C. The organ chamber with  ml Krebs' solution was situated inside the central part of water bath.Krebs' solution was aerated ( O i  CO) under pressure just before the experiment during one and a half hour.Th e transducer was designed for measuring the forces inside the tunica media of artery.Artery was in isometric tension due to induced vasospasm developed by infl uence of blood breakdown products.Th e function of transducer was consistent with the fact that produced forces, brought to carbon fi bers, are converted to proportional electrical signal.Th is signal was visualized and recorded on the millimeter paper by the writer machine.Namely, the writer machine was connected to transducer so arterial tonus with changes in arterial wall was recorded directly.Isometric transducer measures the changes in force in the setting of constant length.We used isometric transducer ( model) which was suggested for the forces of - grams.It is adequate for no sensitive organs or for the small force.Arteries and tracheal rings are the most common organs for examination with this transducer.Krebs' solution was consisted of KHPO , mmol/ dm; KCl , mmol/ dm; CaCl , mmol/ dm; MgSO , mmol/ dm  ; NaHCO , mmol/ dm; NaCl , mmol/ dm  ; Dextrose , mmol/ dm  .
KEMAL DIZDAREVIĆ: THE ROLE OF NITRIC OXIDE IN RESOLUTION OF VASOSPASAM CORRESPONDING WITH CEREBRAL VASOSPASMS AFTER SUBARACHNOID HAEMORRHAGE: ANIMAL MODEL

TABLE 1 .
Arterial response to KCl in the millimeters.

TABLE 3 .
Arterial response to acetylcholine in the millimeters.

TABLE 4 .
Descent of line in millimeters as a response of the spastic arteries to NO addition.

TABLE 5 .
Number of arteries with regard to descent of line in millimeters.KEMAL DIZDAREVIĆ: THE ROLE OF NITRIC OXIDE IN RESOLUTION OF VASOSPASAM CORRESPONDING WITH CEREBRAL VASOSPASMS AFTER SUBARACHNOID HAEMORRHAGE: ANIMAL MODEL around spastic artery.Th e nitric oxide increases the ba- sic dilator activities of arterial wall(, , ).Th e crucial reason for spasm developing after SAH is absence of NO, not absence of vasculature response to nitric oxide.Absence of NO changes basic tonus of arterial wall which becomes hyper-reactive to constrictors.Th e autologous periadventitial blood application inside closed PTFE tube containing femoral artery is truly imitation of SAH situation in human, so our model of vasospasm has involved the same mechanisms found in cerebral vasospasm after SAH (, , , 