Experimental microaneurysms in rats: I. Model for induction

Aneurysm

Experimental Microaneurysms in Rats: I. Model for Induction Nilton Eduardo Guerreiro, M.D., Ph.D.,* Benedicto Oscar Colli, M.D., Ph.D.,† Carlos Gilberto Carlotti, Jr., M.D., Ph.D.† and Leila Chimelli, M.D., Ph.D.‡ *Department of Surgery, Division of Neurosurgery, Marı´lia School of Medicine, Marı´lia, Sa ˜ o Paulo, Brazil; †Department of Surgery, Division of Neurosurgery, Ribeira ˜ o Preto´ Medical School, University of Sa ˜ o Paulo, Ribeira ˜ o Preto, Sa ˜ o Paulo, Brazil; ‡Department of Pathology, School of Medicine, Federal University of Rio de Janeiro, Rio de Janeiro, Brazil

Guerreiro NE, Colli BO, Carlotti CG, Chimelli L. Experimental microaneurysms in rats: I. model for induction. Surg Neurol 2004; 62:406 – 412.

KEY WORDS

Experimental microaneurysms, mechanical trauma, aorta bifurcation, rats.

BACKGROUND

Small aneurysms (lesser than 2 mm) in humans called sessile, baby aneurysms, or microaneurysms, generally are not able to be clipped or to be coil-packed through endovascular route. Among the modalities of treatment that have been used for treating microaneurysms, bipolar coagulation and wrapping of the lesion are outstanding. Nevertheless, demonstration of the efficacy of these treatments is difficult because most reported experimental models for inducting aneurysms are complex and difficult to be reproduced. This study aimed to develop a simple and reproducible model for inducing microaneurysms. METHODS

Microaneurysms were induced using a mechanical lesion of the bifurcation of the aorta in 72 rats. Three groups of 10 animals were sacrificed 7, 14, and 21 days after the lesion and 2 groups (35 and 7 animals) after 30 days. The aortic bifurcation was macro/microscopically analyzed in the first 4 groups and a resistance test was applied in the fifth group. RESULTS

Microaneurysms occurred in 77.8% of cases. Microscopically, degenerative changes were observed in the intima, media, and adventitia and in the internal elastic lamina. The bursting pressure ranged from 368 to 1,472 mm Hg during the resistance test in the fifth group. CONCLUSIONS

The presented model of experimental microaneurysm induction is simple, reproducible and gives a high rate of positivity. © 2004 Elsevier Inc. All rights reserved.

mall aneurysms (lesser than 2 mm) in humans are called sessile, baby aneurysms, or microaneurysms [9,25,28]. Frequently, they cannot be clipped or coil-packed through an endovascular route. Several modalities of treatment have been used for treating microaneurysms. Special attention has been directed to bipolar coagulation [19,28] and wrapping of the lesion with some material aiming to increase the resistance of the aneurysm wall [5,12,15,22,26,28]. Nevertheless, the treatment of intracranial microaneurysms remains controversial [20,18,28]. Better understanding of the efficacy of the treatments for these lesions requires reproducible experimental models. However, most experimental aneurysm models reported in the literature are complex and difficult to be reproduced. Simpler models of microaneurysm induction can be performed using direct mechanical lesion of the vessel wall. Nevertheless, the obtained results using these methods are not constant. The object of this study was to develop a simple and reproducible method for induction of experimental microaneurysm in rats.

S

Materials and Methods Study performed at the Laboratory of Experimental Surgery, Department of Surgery, Ribeira˜o Preto Medical School, University of Sa˜o Paulo, Ribeira˜o Preto, Sao Paulo, Brazil, as part of the requirements for Master of Science Thesis (N.E.G.). Address reprint requests to: Benedicto Oscar Colli, M.D., Departamento de Cirurgia–HCFMRP, Campus Universita´rio USP, 14048-900 Ribeira˜o Preto, Sao Paulo, Brazil. Received July 21, 2003; accepted January 13, 2004. 0090-3019/04/$–see front matter doi:10.1016/j.surneu.2004.01.023

The experiments were performed using 72 male Wistar rats, each weighing between 280 and 480 g. Experimental protocols were designed conforms to the NIH guidelines for the care and use of animals in research (1985 Guide for the Care and Use of Lab© 2004 Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010 –1710

Microaneurysms: Experimental Induction

oratory Animals, NIH Publication 85–23). All animals were submitted to the proposed method for induction of microaneurysms, and they were sequentially distributed into 5 groups: Group 1, with 35 rats; Groups 2, 3, and 4 with 10 rats each, and, Group 5 with 7 rats. The animals of Groups 1 and 5 were killed 1 month and the animals of Groups 2, 3, and 4 were killed, respectively, 21, 14, and 7 days after aneurysm induction. The distal abdominal aorta and the proximal iliac arteries from animals of Groups 1 to 4 were removed for histopathological analysis, and in the animals of Group 5 these vessels were submitted to a resistance test. INDUCTION OF MICROANEURYSMS All animals were anesthetized by sulfuric ether inhalation. Arterial blood pressure was monitored through a PE 10 catheter inserted in the caudal artery and kept between 90 and 100 mm Hg [2]. The temperature remained around 36.5°C. during the experiment. Under the surgical microscope, the aortic bifurcation was approached through a midline abdominal incision, and the vessels were isolated 5-mm proximal, and 5-mm distal to the bifurcation. The mechanical lesion was performed grasping and compressing one-third of the circumference at the aortic bifurcation with a number 3 jeweler’s forceps. The site of pinching was the ventral wall of the aortic bifurcation, just above the origin of the inferior mesenteric artery. The intensity of compression was enough for causing local changes in the color of the vessel. This occurs because of erythrocyte infiltration and outpouching in the vessel. Eventually, some local bleeding transudation occurred in the site of the lesion, and it was stopped using gentle cottonoid compression. The abdominal incision was sutured in a unique layer using 4 – 0 mono-nylon running suture, and the animals were kept in cages with food and water ad libitum until the date of sacrifice. According to the group distribution, the animals were anesthetized, the site of the lesion was inspected, and the animals were killed with massive sulfuric ether inhalation. The arterial segments containing the aneurysms were removed and submitted to morphologic studies. RESISTANCE TEST OF THE MICROANEURYSMS The resistance test was performed aiming to prove if the site of the mechanical lesion became a week point in the vessel in 6 animals that presented macroscopic microaneurysm. One month after the induction of the microaneurysms, the animals of Group 5 were anesthetized, and the resistance test

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was performed according the technique reported by Dias et al [4] (1995). After inspection and exposition of the bifurcation of the abdominal aortas (approximately 5 mm of the aorta and 5 mm of the iliac arteries), the animals were killed. The aorta was catheterized just below the emergency of the renal arteries, with an 18-gauge polyethylene catheter whose tip was positioned at the aortic bifurcation. The catheter was attached to the vessel with a 4 – 0 cotton suture, the proximal iliac arteries were occluded with 4 – 0 cotton sutures, and the proximal extremity of the catheter was connected to a short arm of a Y-polyethylene infusion system. The other short arm of the Y was connected to a manometer ranging from 0 to 2,576 mm Hg, and the long arm was connected to an air compressor system (Figure 1). The site of the aneurysm was covered with saline solution. After that, air was allowed to enter slowly (50 mL/min) through the infusion system, until aneurysm rupture verified through air bubbling out the vessel in the saline solution. The bursting pressure was considered as the highest pressure observed in the manometer. MORPHOLOGIC ANALYSIS macroscopic analysis. It was based on the evidence of outpouching (microaneurysm) of the site of the lesion under the surgical microscope, in anesthetized animals. The microaneurysms were classified as: a) absent, b) small, and c) large. The microaneurysms were considered small or large when they were respectively lesser or greater than half the vessel wall diameter at the site of the lesion. After removal, arterial samples were fixed in 10% neutral buffered formalin during 48 hours and included in paraffin blocks in its anatomic form (Y-shape). Serial 5-mm slices were obtained perpendicular to the long axis of the vessels, and the slices were stained using Hematoxilin & Eosin, Masson and Verhoeff techniques. The slices were observed under the optic microscope looking for inflammatory cells, bleeding, fibrosis, thickening/thinning, and interruption/ disorganization of the layers of the arterial wall.

histopathological analysis.

Results MACROSCOPIC ANALYSIS The macroscopic findings in the animals of Groups 1 to 5 are presented in Table 1. Microaneurysm formation was observed to occur in the ventral wall of the aortic bifurcation, just above the origin of the inferior mesenteric artery. The outpouching reached from one-third to the entire anterior wall

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Surg Neurol 409 2004;62:406 – 412

Upper Left–Device used for performing resistance test of the arterial wall. (A) Arm of the Y to be connected to the compressed air network; (B) Arm of the Y to be connected to the arterial segment to be tested; and (C) Arm of the Y to be connected to the manometer. Upper Right—(D) photomicrograph of the aortic bifurcation of one animal of group 1 (30 days survival) showing a large microaneurysm occupying the entire extension of the anterior wall of the bifurcation. Center—Photomicrographs of the aorta of one normal animal showing the intima, the media, and the adventitia. (E) 100X, H&E; (F) highlight of the internal elastic lamina (100X, Verhoeff); Lower—Photomicrographs of the aorta of one animal of group 2 (21 days survival), showing a slight focal thinning and outpouching of the vessel wall (microaneurysm) (arrows). (G) Disorganization of the adventitia and media (40X Verhoeff) (H) interruption of the endothelium and internal elastic lamina (arrows) (100X, Verhoeff). 4™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™™

1

(Figure 2). Considering small and large macroscopic outpouchings, microaneurysms were detected in 77.8% of the 72 animals analyzed in our study, most of them of small size. HISTOPATHOLOGICAL ANALYSIS The main histopathological findings observed in the animals of Groups 1 to 4 are presented in Table 2 and, except for the outpouching, they were similar in animals with and without macroscopic outpouchings. Microscopic analysis of the vessels of 65 animals showed microaneurysm formation in 93.8% of the animals (34 animals of Group 1, 9 of Group 2, 8 of Group 3, and 10 of Group 4 presented microscopic outpouching or depression on the site of trauma, and in some cases both alterations coexisted). One animal of Group 2 and another of Group 3 were excluded because of technical problems. Areas of focal thinning and/or thickening occurred in all layers of the vessel wall, varying from disorganization to complete interruption of the layer (Table 2; Figure 3). Infiltration of lymphomononuclear inflammatory cells and sparse erythrocytes, hemosiderin and fibroblast proliferation were found in the media and in the adventitia layers in most cases of Groups 1 and 2. Interruptions of the internal elastic lamina were observed in all but 2 animals (in 1 animal it was normal and in another it was disorganized). Erythrocytes, fibrin and polymorphonuclear inflammatory cells were found in all layers of the arterial wall in some animals of Group 3, and similar findings were observed in Group 4, except for the

1

higher intensity response.

of

the

acute

inflammatory

RESISTANCE TEST OF MICROANEURYMS The vessels of all 6 animals of Group 5 submitted to resistance tests suffered rupture under pressures of 1,472 mm Hg in 2 animals, and of 957 mm Hg, 770 mm Hg, 736 mm Hg, and 368 mm Hg in the 4 remaining animals.

Discussion Several experimental models have been developed for attempting a better understanding of the pathophysiology and treatment of intracranial aneurysms. These models can be grouped in 2 categories: 1) direct trauma on the arterial wall, and 2) simulation of etiologic factors theoretically involved in the pathogenesis of this disease, as hemodynamic stress. Ideal experimental models should be practical and try to simulate some clinical conditions, have a high success rate and be reproducible. These models should also be developed in resistant animals, easy to be obtained, handled, and kept in laboratories. Because of this, we chose the rat for development of the proposed model for induction of microaneurysms. The human extracranial arteries, the cerebral arteries, are constituted of 3 layers: an outer adventitia composed of loosely woven collagen; a media

Macroscopic Evidence of Microaneurysms Induced in the Abdominal Aortic Bifurcation in the Animals of Groups 1 to 5

MICROANEURYSMS GROUPS

ABSENT

SMALL

LARGE

POSITIVITY

TOTAL

1 2 3 4 5 Total

6 0 5 4 1 16 (22.2%)

24 3 3 5 4 39 (54.2%)

5 7 2 1 2 17 (23.6%)

29 (82.9%) 10 (100%) 5 (50%) 6 (60%) 6 (85.7%) 56 (77.8%)

35 10 10 10 7 72

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Histopathological Analysis of the Aortic Bifurcation in the Animals of Groups 1 to 4

HISTOPATHOLOGICAL FINDINGS GROUPS/ARTERIAL LAYERS 1 Intima Internal Elastic Media Adventitia 2 Intima Internal Elastic Media Adventitia 3 Intima Internal Elastic Media Adventitia 4 Intima Internal Elastic Media Adventitia

Lamina

Lamina

Lamina

Lamina

THINNING

THICKENING

THINNING/ THICKENING

DISORGANIZATION

FIBROSIS

LAYER INTERRUPTION

5 — 5 22 — — — 9 2 — — 5 10 — — 10

8 — 5 4 — — — — 2 — 2 2 — — — —

13 — 17 — 9 — 9 — 3 — 5 1 0 — 10 —

34 — 32 33 9 — 9 9 9 — 0 9 10 — 10 —

34 — 33 24 9 — 9 9 6 — 8 9 3 — 10 5

19 32 31 24 9 9 9 6 9 9 9 9 10 10 10 10

of smooth muscle; and an inner layer, the intima, composed of internal elastic lamina, a thin collagen layer and the endothelium [23]. Nevertheless, the cerebral arteries differ from the extracranial arteries of a similar size because their media and intima are thinner and they do not have an external elastic lamina. In addition, adventitias of major cerebral arteries are surrounded by cerebrospinal fluid instead of surrounding tissue. Despite the histology of the rat arteries not being similar to the human intracranial arteries, there are many experimental studies for induction of aneurysms reported in the literature using this animal as the subject [1,7,8,11,13,20,22,29]. The microscopic examination of saccular intracranial aneurysms in humans shows that the media layer and the internal elastic lamina of the artery disappear at the aneurysm neck, although some small patches of fragmented elastic may extend for a short distance into the aneurysm sac [10]. The elastic and muscle of the media are replaced by fibrous tissue and the thickness may vary greatly; thickened walls are more commonly seen in large aneurysms [21]. Aneurysms typically arise at branch points, bifurcations and fenestrations of cerebral vessels [21]. Ferguson [6] experimentally demonstrated that hemodynamic stress resulting from the impingement of axial blood flow at the point of bifurcation is much greater than that in the main arterial trunk or branches. The impact and sudden deflection of the central stream at the apex transmits a pulsatile impulse to the bifurcation. These forces may cause

degeneration of the internal elastic lamina, accounting for the preferential occurrence of aneurysm formation at the sites of maximal hemodynamic stress. Once an outpouching has occurred, turbulent flow starts at this site and contributes to its enlargement and rupture [3,6]. This sequence of events is confirmed by experimental models of intracranial aneurysms [1,7,14,16]. Traumatic destruction of the internal elastic lamina and media layers is the premise for some experimental models of aneurysms reported in the literature [13,18,20,27,30]. This was also the basis for the development of the method now proposed. In an attempt to get experimental traumatic microaneurysms we tried to reproduce 2 of the simpler models reported in the literature. One of them uses intramural injection of calcium gluconate in vessels of dogs for destroying the media layer [30]. The other consists in removing fragments of the internal elastic lamina and of the media layer of the cervical carotid bifurcation of dogs [27]. We did not get constant results using these models, and the shape of obtained aneurysms was not similar to a microaneurysm. The attempt of compressing the arterial wall with a jeweler’s forceps for interrupting the internal elastic lamina and media layers allowing an aneurysm formation was supported by the study of Maxwell et al [17], which observed aneurysm dilatation in 1 in 5 arteries after adventitia removal and grasping with a mosquito forceps, clearly proving the correlation between arterial wall trauma and aneurysm formation. The method proposed now is based in the direct

Microaneurysms: Experimental Induction

lesion of the arterial wall layers, mainly rupture of the internal elastic lamina and of the media layer on the rat aortic bifurcation. Generally, immediately after the trauma, an outpouching was observed in the vessel wall at the site of compression. Sometimes, a small bleeding occurred that was easily stopped with gentle cottonoid compression, as reported by Maxwell et al [17]. The compression cannot be very intense because of the risk of complete rupture of the arterial wall. The immediate local outpouching of the vessel causes local turbulence of the blood flow easing the aneurysm formation, as proposed by Ferguson and co-workers [3,6]. The histopathological examination of the aortic bifurcation in our study showed that at 1 month after the arterial trauma, the adventitia frequently presented focal areas of thinning and eventually of thickening. The media layer presented areas of thinning and/or thickening, and all layers, especially the internal elastic lamina, had focal areas of interruptions that were substituted by fibrous tissue. The decision for waiting one month as the longer time to perform the microscopic analysis of the vessels in this study was because the rate of healing in rats is very fast. That time is enough for microaneurysm formation and complete healing of the vessel and adjacent structures. In addition, the persistence of the turbulence at the vessel bifurcation for longer time could lead to the formation of berry aneurysms, although we have not tested this hypothesis. In our study, microaneurysm formation was observed to occur in the ventral wall of the aortic bifurcation. The outpouching reached from onethird to the entire anterior wall, most of them of small size. Considering small and large macroscopic outpouchings, microaneurysms were detected in 76.9% of the 65 animals analyzed. Animals with macroscopic outpouching generally had this find confirmed by microscopic analysis. Nevertheless, some of these animals presented invagination or depression or coexistence of outpouching and depression of the arterial wall at the microscopic analysis. This apparent contradictory find is a technical artifact of the fixation process that provokes inversion of the outpouching [14]. Microscopic alterations of different severity of the arterial wall were the rule in the animals of all experimental groups, mainly in the medial layer and in the internal elastic lamina. Great amounts of erythrocytes and polymorphonuclear and mononuclear inflammatory cells and fibrin were found in the animals of the groups with shorter duration. These elements were decreasing with time and were changed by a progressive fibroblastic proliferation. The high fre-

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quency of microaneurysms in animals with lesions of the internal elastic lamina and of the media layer supports the theory that these lesions are involved in the aneurysm formation [13,14,17,20,24]. A study concerning experimental aneurysm induction using a mechanical lesion of the arterial wall [20] was the basis for the development of the experimental model used in this study. The resistance test was performed in our study aiming to prove if the site of the mechanical lesion became a weak point in the vessel wall. This was well demonstrated when compared with tests performed in a previous study [4] with normal and hypertensive arteries, which supported intra-arterial pressure of 1,440 mm Hg (the superior limit of the manometer) without rupture. Similar results were also obtained in experimental aneurysms inducted by arteriotomy closed with laser [20]. Based on the lesions observed in the arterial wall (lesions of internal elastic membrane and of the media layer) and on the morphology of the aneurysm, the proposed model can be considered similar to human microaneurysms. The morphology of the microaneurysm was always constant and similar to human microaneurysms (lesser than 2 mm and sessile aneurysms [9,25,28]). Additionally, the site of microaneurysm formation was just in the anterior wall of the aortic bifurcation, as can be observed on many middle cerebral artery bifurcation microaneurysms. Although the proposed model cannot be completely transposed to human microaneurysms, it produces similar lesions in the arterial wall (media layer and internal elastic lamina). It also causes an outpouching in the arterial wall that leads to turbulence related to hemodynamic stress [3,6] creating a weak point in the arterial wall.

Conclusions The proposed method for induction of microaneurysm in the abdominal aorta of rats is simple to be performed using nonsophisticated material or equipment. Also, it allows a high rate of macroscopic (77,8%) and microscopic (93.8%) microaneurysm formation, which can easily be reproduced. REFERENCES 1. Ammirati M, Ciric I, Rabin E. Induction of experimental aneurysms on the rat common carotid artery using a microsurgical CO2 laser. Microsurgery 1988;9: 78 –81. 2. Carlotti CG Jr, Colli BO, Kazuo JY. Avaliac¸a˜o da isquemia cerebral pela respirac¸a˜o mitocondrial: modelo experimental. Arg Neuropsiguiatr 2001:365–71.

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t does not matter how slowly you go so long as you do not stop.

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—Confucius (551 BC— 479 BC) Chinese philosopher and reformer