594 Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (7): 594-597

INTRODUCTIONIn patients suffering from subarachnoid hemorrhage(SAH), vasospasm of cerebral arteries is the mostcommon cause of delayed neurological defect.1-3

Vasospasms after subarachnoid hemorrhage depend ona wide variety of causes such as blood, blood products,eicosanoid, endothelin, free radicals, and inflammation.Although various pharmacological drugs have beendeveloped to treat patients with SAH-induced delayedcerebral vasospasm, but in the majority of patients themedications are not satisfactory.4-6

Cobalt nitrate is the inorganic cobalt (II) salt of nitric acid,often with various amounts of water. Cobalt nitrate isgenerally found as a hexahydrate, a red-browndeliquescent salt that is soluble in water and other polarsolvents.7 Cobalt (II) complexes have antimicrobial,anticancer, antiproliferative, and antioxidant activity.8-10

The aim of this study was to determine the effects of

cobalt nitrate on morphometric and histopathologicalchanges in the rat femoral artery wall in an experimentalvasospasm model.

METHODOLOGYThis experimental study was conducted with theapproval of the Ethics Committee Institute forExperimental Medical Research (DETAE), IstanbulFaculty of Medicine, Istanbul University betweenNovember 2009 and September 2010 at the ResearchInstitute, University of Istanbul, Istanbul, Turkey. Twenty-four female Sprague-Dawley rats weighing 180 - 220 gwere used. The animals were fed ad libitum, and a12:12-hour light/dark cycle was maintained.

Rats were separated into three groups: group 1 (n=8,sham-control); group 2 (n=8, vasospasm), and group 3(n=8, vasospasm + cobalt nitrate treated). The group 1rats were delivered 1 cc 0.9% saline. In the group 2 and3 rats, 0.1 cc percutaneous intracardiac blood wascollected and injected into the silastic sheath to generatea peripheral vasospasm model. In addition, the group 3rats were subjected to 0.1 mg/kg/day intraperitonealcobalt nitrate for 7 days.

For the femoral artery vasospasm model, Okada et al.model was preferred.11 Rats were sedated withintraperitoneal 2 mg/kg ketamine HCl (Ketalar vial 50mg/ml, Pfizer). The femoral vascular bundle wasexposed with a longitudinal 2 cm skin incision under asurgical microscope. The femoral artery was dissectedfrom the femoral vein and nerve without being traumatised.

ORIGINAL ARTICLE

Effects of Cobalt Nitrate on Histopathological and MorphometricChanges in Rat Femoral Artery Vasospasm Model

SaygÊn Ucar1, Yasar Dagistan2 and Ali Guler3

ABSTRACTObjective: To determine the effects of cobalt nitrate on the arterial wall in a rat femoral artery vasospasm model.Study Design: Experimental study.Place and Duration of Study: Faculty of Medicine, Istanbul University, between November 2009 and September 2010.Methodology: Twenty-four rats were separated into three groups named group 1 (n=8, sham-control); group 2 (n=8,vasospasm), and group 3 (n=8, vasospasm + cobalt nitrate treated). Group 3 rats were subjected to intraperitonealadministration of 0.1 mg/kg/day cobalt nitrate for 7 days, followed by histological and morphometric analyses. ANOVA andpost hoc analyses were carried out.Results: The mean vascular wall thickness of the group 1, 2 and 3 rats was found to be 133 ±13, 210 ±15 and 160 ±11micron, respectively. The mean vascular lumen diameter of the group 1, 2 and 3 rats was 698 ±20, 240 ±15 and 540 ±22micron, respectively. Arteries in the group 3 had thin smooth endothelium, thin mildly folded internal elastic lamina, andconcentric smooth muscle cells. The mean vascular lumen diameter of the group 3 rats showed a significant decreasecompared to the group 2 rats.Conclusion: Cobalt nitrate may potentially serve as an agent in preventing cerebral vasospasm after a hemorrhagicepisode.

Key Words: Cobalt nitrate. Subarachnoid hemorrhage. Femoral artery vasospasm. Rat.

1 Department of Neurosurgery, Gaziosmanpasa Taksim Trainingand Research Hospital, Istanbul, Turkey.

2 Department of Neurosurgery, Abat Izzet Baysal University,Medical School, Bolu, Turkey.

3 Department of Neurosurgery, Derince Training and ResearchHospital, Kocaeli, Turkey.

Correspondence: Dr. Yasar Dagistan, Assistant Professor,Department of Neurosurgery, Abant Izzet Baysal University,Medical School, Bolu, Turkey.E-mail: dagistanyasar@hotmail.com

Received: June 23, 2014; Accepted: May 31, 2016.

A 1-1.5 cm silastic sheath was wrapped around thefemoral artery and sutured. Autologous cardiac blood ofthe subjects was used as whole blood.

Rats were fed a mouse diet and kept at roomtemperature in separate cages for 7 days. No subjectrequired exclusion due to mortality or disease. At theseventh day, the silastic sheath wrapped around thefemoral artery was accessed by opening the earliersurgical incisions, and the femoral artery was exposedfrom the silastic sheath. In all rats, the femoral arterywas observed to be within the silastic sheath. In allgroups, the right femoral artery was incised 1 - 1.5 cmand removed for histopathological morphometricanalysis.

The extracted femoral artery specimens were imme-diately placed in 10% formalin solution. The tissuespecimens were placed in paraffin blocks, sectioned at5 µm, and stained with hematoxylene and eosine. Thepreparations were examined under 100x, 200x, and400x magnification with an Olympus (Olympus BX7,Japan) microscope, and photographs were taken formorphometric analysis. The vascular wall thickness andlumen area were measured as unit values with theImage J 1.34 program. Measurements were carried outon the photographs taken under 40x magnification. The

specimens were compared in terms of the vascularlumen area and wall thickness in the morphometricanalysis.

SPSS 11.0 software (SPSS Inc., USA) for Windows wasused for the statistical analyses. Data are expressed asmean ±S.D. and analysed for statistical significance byone-way analysis of variance (ANOVA) followed by post-hoc for multiple. Differences between mean values wereconsidered significant when p < 0.05.

RESULTSHistopathologically, the sham-control group rats hadessentially normal femoral artery morphology (Figure1A). In the group 2 rats, the arteries had markednarrowing in the lumen width as well as thickening in thevascular wall, disruption of endothelial integrity, foldingin the internal elastic lamina, and vacuolizations in themuscle layer (Figure 1B). Arteries in the group 3 rats hadthin smooth endothelium, thin and mildly folded internalelastic lamina, and concentric smooth muscle cells(Figure 1C).

After the femoral artery wall thickness and lumen area ofall rats were calculated, the groups were compared. Themean vascular wall thickness of the group 1, 2 and 3 ratswas found to be 133 ±13, 210 ±15 and 160 ±11 micron,

Effects of cobalt nitrate on histopathological and morphometric changes in rat femoral artery vasospasm model

Figure 1(A): Note the thin smooth endotheliumand concentric smooth muscle cells (H&E, 40x)

Figure 1(C): Note the thin, smooth endotheliumand thin internal elastic lamina, and concentricsmooth muscle cells (H&E, 40x)

Figure 1(B): Narrowing in the arterial lumen,thickening in the vascular wall, disruption ofendothelial integrity, and vacuolizations in themuscle layer are seen (H&E, 40x)

Figure 2: (a) Arterial wall thickness and (b) arterial lumen diameter of the femoral artery in the groups 1, 2 and 3.*p < 0.003 and p < 0.001 compared to group 1. † p < 0.001, compared to group 2. Values are mean ± SD.

Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (7): 594-597 595

respectively (Figure 2a). In the group 2 rats, the vascularwall thickness had increased and the lumen diameterhad decreased compared to those of the control groupand the cobalt nitrate treated group (p < 0.003 andp < 0.001, respectively). The mean vascular lumendiameter of the group 1, 2 and 3 rats was 698 ±20,240 ±15 and 540 ±22 micron, respectively (Figure 2b).The mean vascular lumen diameter of the group 3 ratshad increased compared to the group 2 rats (p < 0.001).

DISCUSSIONSpontaneous SAH from the rupture of a cerebralaneurysm is an important clinical problem.12 Afteraneurismal SAH, cerebral vasospasms are still one ofthe most important contributors to morbidity andmortality. It is a common and potentially devastatingcondition that is not completely understood and forwhich the underlying pathological mechanism remainsobscure, despite much research. Additionally, despitethe advances in pharmacological and surgical treatmentof SAH, there is still no exact therapy for cerebralvasospasm.13-15 Recent studies have determined thatinflammation and free oxygen radicals play a key role inthe pathophysiology of cerebral vasospasms.16-18 In thepresent study, the authors demonstrated that cobaltnitrate reduced the development of peripheralvasospasms in a rat model.

Cobalt is an element of biological interest due to itspresence in the active center of cobalamine. Cobaltindirectly regulates the synthesis of DNA, and is involvedin the co-enzyme of vitamin B12.19 Numerous cobaltcomplexes showing antitumor, antiproliferative, anti-microbial, antifungal, antiviral, and antioxidant activityhave been reported in the literature.9,20 Tsiliou et al.explored cobalt (II) complexes with the non-steroidalanti-inflammatory agent tolfenamic acid in the presenceor absence of nitrogen-donor heterocyclic ligands (2,2’-bipyridine, 1,10-phenanthroline, 2,2’-bipyridylamine orpyridine) and synthesized and characterized them withphysicochemical and spectroscopic techniques.19 Theydemonstrated that tolfenamic acid and its cobalt (II)complexes exhibit good binding propensity to human orbovine serum albumin protein, with high bindingconstant values. In a similar study, Dimiza et al.suggested that mefenamic acid and its cobalt (II)complexes exhibit good binding propensity to human orbovine serum albumin protein, with high bindingconstant values.20 Additionally, the antioxidant activity ofthe compounds has been evaluated indicating their highscavenging activity against hydroxyl-free radicals andsuperoxide radicals. Wu et al. investigated thecoordinating ability of various benzimidazole ligands.21

In their study, the synthesis, structure, DNA-bindingproperties, and antioxidant activity of copper (II) andcobalt (II) complexes with bis (N allylbenzimidazol-2-ylmethyl) benzylamine are presented. Both complexes

exhibited potential antioxidant properties in in vitrostudies.

In this study, the arteries in the group 2 rats narrowedsignificantly in the lumen width and thickened in thevascular wall, disruption of endothelial integrity, foldingin the internal elastic lamina, and vacuolization in themuscle layer. Whereas, arteries in the group 3 rats hadthin smooth endothelium, thin mildly folded internalelastic lamina, and concentric smooth muscle cells.In the group 2 rats, the wall thickness increasedsignificantly and the lumen diameter decreasedcompared to the cobalt nitrate treated group.

CONCLUSIONCobalt nitrate histopathologically and morphometricallyprevented the development of peripheral vasospasm inrats. Thus, these findings suggest that cobalt nitrate is apromising therapeutic agent for preventing cerebralvasospasm.

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Saygın Ucar, Yasar Dagistan and Ali Guler

596 Journal of the College of Physicians and Surgeons Pakistan 2016, Vol. 26 (7): 594-597

Effects of cobalt nitrate on histopathological and morphometric changes in rat femoral artery vasospasm model

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