Nylon Monofilament for Intraluminal Middle Cerebral Artery Occlusion in Rats
Background and Purpose In rat middle cerebral artery (MCA) occlusion models with an intraluminal nylon monofilament, lesion volume and its reproducibility vary among laboratories. These variations may be caused by differences in the properties of nylon monofilaments. We performed the present study to compare the effects of two different types of 4-0 nylon monofilament on neurological and morphological outcomes in this model.
Methods We randomly and blindly used two types of 4-0 nylon monofilament (Ethilon, n=15, and Nitcho, n=15) to permanently occlude the ostium of the right MCA intraluminally in rats. Neurological outcome and lesion size were compared 24 hours after MCA occlusion between the two groups. The diameter, tensile strength, and extensibility of each filament were measured.
Results Neurological outcome was not different between the groups. The mortality within 24 hours was 13% (2/15) in the Ethilon group and 0% in the Nitcho group. Total lesion volume in the former was 295.9±97.2 mm3 (mean±SD), significantly larger and more reproducible than that in the latter (190.3±144.7 mm3, P=.026). The Ethilon filament had a significantly larger diameter (Ethilon, 0.193±0.001 mm; Nitcho, 0.180±0.001 mm; P<.0001), poorer tensile strength, and better extensibility than the other.
Conclusions Exact diameter and quality are not always the same among nylon monofilaments, even if they meet the standard for the designation 4-0. The present study indicates that slight differences of filament characteristics significantly affect lesion volume and its reproducibility. This result may explain some conflicting observations in this MCA occlusion model.
The intraluminal filament MCA occlusion model in rats introduced by Koizumi et al1 2 and modified by Zea Longa et al3 has gained increasing acceptance for studies of cerebral ischemic pathophysiology and therapeutic intervention.4 5 6 7 8 9 10 11 12 13 In this model, however, lesion size, rates of subarachnoid hemorrhage due to vessel perforation, and early mortality within 24 to 48 hours are not necessarily similar from one laboratory to another.
Recently, Laing et al14 compared the effectiveness and reliability of the method of Koizumi et al, which uses a 4-0 nylon monofilament coated with silicone, with those of the method of Zea Longa et al, which uses a blunt, noncoated filament. By measuring CBF after MCA occlusion, they observed a higher residual CBF in the area supplied by the occluded MCA with a relatively large variation (95% confidence interval, 28.3 to 33.7 mL/100 g per minute) in the latter compared with the former group (9.9 to 11.5 mL/100 g per minute). Vessel perforation occurred more frequently with the blunt (10/52) than the coated filament. The authors concluded that the method of Koizumi et al was more reliable than the method of Zea Longa et al. Other investigators, however, criticized the conclusion.15 16 Their discussions were focused on silicone coating, 3-0 or 4-0 filament size, length of the filament insertion, body weight of the animal, and other end-point measurements. The exact diameter and quality of the filament, however, may not be completely the same, even when a surgical nylon monofilament labeled 4-0 is used. The diameter of a 4-0 nylon monofilament ranges from 0.150 to 0.199 mm, according to the USP XXII standard. Quality may also vary from one 4-0 filament to another.
We performed the present study to compare the effects of two different types of non–silicone-coated, 4-0 surgical nylon monofilament with a different diameter and quality on neurological and morphological outcomes.
Materials and Methods
We used 30 male Sprague-Dawley rats weighing 276 to 346 g. All procedures were performed in accordance with institutional guidelines. The rats were fasted overnight before the experiment but allowed free access to water. An intraluminal occluder was made of 4-0 surgical nylon monofilament, either “Ethilon” (Ethicon Co, Inc) or “Nitcho” (Nitcho Kogyo Co, Inc). The tips of the occluders were rounded by flame heating but not coated with silicone, as in the method of Zea Longa et al.
Animals were initially anesthetized with 400 mg/kg IP chloral hydrate, and the anesthetic state was maintained with 0.5% to 2.0% halothane delivered in an air-oxygen mixture gas through a face mask. A polyethylene catheter (PE-50) with a PE-10 tip was introduced into the left femoral artery for continuous monitoring of arterial blood pressure and sampling of blood for analysis of blood gases and plasma glucose concentration before and 15 and 60 minutes after MCA occlusion. The rat’s body and head temperatures were monitored with a rectal probe and with a needle-type thermocouple probe (PTW-100A, PTN-800, Unique Medical Co, Inc), which were inserted between the right temporalis muscle and the lateral aspect of the skull, respectively. They were maintained at 37°C with a heat lamp and a heating pad during the operation.
The ostium of the right MCA in each rat was occluded intraluminally with a method described in detail previously.8 Briefly, an occluder was introduced through the right CCA into the internal carotid artery, then advanced approximately 17 mm intracranially from the CCA bifurcation. The animals were randomly assigned to either the Ethicon filament (Ethilon group, n=15) or the Nitcho filament (Nitcho group, n=15) immediately before the filament was inserted into the CCA. The type of filament was not known by the examiners (Y.K. and K.M.) throughout the experiment and data collection.
Sixty minutes after MCA occlusion, the femoral arterial catheter was removed and the artery was ligated to prevent bleeding. The animals were permitted to recover from the anesthesia at room temperature.
Neurological evaluation was performed 24 hours after the induction of ischemia and scored on a 6-point scale, as described previously,11 which was modified from the scale proposed by Zea Longa et al.3 The animals were then anesthetized with 300 mg/kg IP chloral hydrate and decapitated. The brains were quickly removed and inspected to confirm the position of the intraluminal occluder and the absence of subarachnoid hemorrhage and/or arterial penetration with the occluder. The brains were sectioned coronally with a tissue chopper (McIlwain Tissue Chopper, Mickle Laboratory Engineering Co, Inc) at 1-mm intervals, incubated for 30 minutes in a 2% solution of TTC at 37°C for vital staining,17 and fixed by immersion in 10% phosphate-buffered formalin solution. The 13 brain sections per animal, stained with TTC, were recorded with a 3-CCD color video camera (KY-M380, Nippon Victor Co, Inc) on an operating microscope 48 hours after the formalin fixation for measurement of lesion areas. Areas not stained red with TTC, which were considered lesioned, were calculated by the video image analyzing system (NIH Image, version 1.52). The total lesion volume (in cubic millimeters) was calculated by the use of numerical integration of the TTC-pale areas for all of the sections per animal and the distances between them.
The diameter and tensile strength of each type of filament were measured with a dial gauge (n=15) and by the knot-pull (n=5) and straight-pull (n=5) methods, according to the method described in the Sutures Diameter and Tensile Strength section in the USP XXII. Extensibility was also determined as percent elongation of a 50-mm filament section when 1 kilogram-force pulled the filament (n=5).
An unpaired t test was used to assess the significance of differences in physiological variables and lesion volume between the groups and in data from the measurements of filaments. A two-factor, repeated-measures ANOVA was performed to assess the significance of differences due to the type of filament, brain sections, and their interactions with measured areas of TTC-determined lesion. The Mann-Whitney U test was used for comparison of the neurological grade score. All values presented are mean±SD. A two-tailed value of P<.05 was considered significant.
The results of measurements of the filaments are shown in the Table⇓. Although both filaments met the USP standard for a 4-0 surgical nylon monofilament, the Ethilon filament was larger in diameter, had less tensile strength, and was more elastic than the other; the differences were statistically significant (P<.0001 for each comparison).
There were no statistically significant differences in arterial pH, Po2, and Pco2, plasma glucose concentration, or mean arterial blood pressure between the groups throughout the experiment (data not shown). Two rats in the Ethilon group died 18 and 20 hours after MCA occlusion, respectively (mortality, 13%) and were scored as 5 on the neurological grade scale, then immediately subjected to TTC staining. All rats in the Nitcho group survived for 24 hours after MCA occlusion. No subarachnoid hemorrhage was observed in any animals. The neurological grades at 24 hours were 2.6±1.4 in the Ethilon group and 2.1±1.0 in the Nitcho group (P=NS).
The total TTC lesion volume in the Ethilon group was 295.9±97.2 mm3, significantly larger than that in the Nitcho group (190.3±144.7 mm3, P=.026). As depicted in the Figure⇓, the TTC lesion area on each coronal slice was larger in the Ethilon group than in the Nitcho group (F[1, 28]=5.501, P=.026). The coefficient of variation of the TTC lesion volume was smaller in the Ethilon group (33%) than in the Nitcho group (76%), suggesting better reproducibility of lesion volume in the Ethilon group than in the Nitcho group.
The present results obtained from the Ethilon group support our previous observations8 11 and the claims by Garcia15 and Holland et al16 that an extensive and reproducible lesion with a low rate of subarachnoid hemorrhage (vessel perforation) results from MCA occlusion when an intraluminal nylon monofilament without silicone coating is used. Animals in the Nitcho group had a smaller and more variable lesion volume. The neurological grade scores were not sensitive enough to detect differences between the two groups.
We evaluated animals neurologically and morphologically 24 hours after MCA occlusion to avoid a decrease in the number of experimental animals because of increasing mortality later than 24 hours, which was observed in our pilot study (data not shown). Several studies that used similar models reported that mortality within the initial 48 hours after occlusion was extremely high.1 4 An infarcted lesion matures fully at 72 hours in a similar model, and TTC-pale areas represent mitochondrial dysfunction rather than neuronal necrosis.13 17 The present method and time point of morphological evaluation are unlikely to significantly affect the results because TTC-pale areas correspond fairly well with those determined histologically,12 17 and the lesion size at 24 hours has been reported to be approximately 80% that at 72 hours.13
No previous reports using this MCA occlusion model have provided details concerning the diameter and quality of the filament. The present study indicates that even slight differences in the diameter and quality of a filament significantly affect lesion volume. A filament with larger diameter is likely to cause a more complete vessel occlusion, lower residual CBF, and more reproducible lesion volume. The progression from ischemic injury to pannecrosis that occurs in the rat brain after MCA occlusion is partly attributed to a worsening of the blood circulation through the microvessels, resulting from the influx of leukocytes and platelets, and the structural changes of astrocytes and endothelial cells.18 19 20 Stimuli to the endothelial cells with the filament tip may cause platelet aggregation and thrombus formation in this model.1 The two kinds of filament may differently affect these processes, although we did not examine their effects on microcirculation, microvessels, and platelets.
No subarachnoid hemorrhage (vessel perforation) was observed in either of the groups of the present study. Subarachnoid hemorrhage (vessel perforation) seems to result from excessive insertion pressure of the intraluminal filament.15
In the intraluminal filament MCA occlusion model, several modifications have been proposed, including the use of a 3-0 monofilament with or without its tip tapered and rounded with fine sandpaper7 or a 4-0 monofilament coated with silicone (mentioned above), the method of inserting the filament into the blood vessel, and the strain and body weight of the rat.1 11 These variables should be standardized to avoid inconsistent results among laboratories and to establish the universal validity of this unique MCA occlusion model.
Selected Abbreviations and Acronyms
|CBF||=||cerebral blood flow|
|CCA||=||common carotid artery|
|MCA||=||middle cerebral artery|
|USP||=||United States Pharmacopeia|
This study was supported in part by Special Coordination Funds for Promoting Science and Technology (Encouragement System of COE) from the Science and Technology Agency of Japan, Research Grants for Cardiovascular Diseases 5A-5 and 6A-1 from the Ministry of Health and Welfare of Japan, and a research grant from the Japan Cardiovascular Research Foundation. The authors thank Dr Marc Fisher (University of Massachusetts Medical School, Worcester) for reviewing the manuscript. We also thank Johnson & Johnson Medical Co, Inc (Tokyo, Japan) for measuring the diameter, tensile strength, and extensibility of both filaments.
Reprint requests to Kazuo Minematsu, MD, Cerebrovascular Division, Department of Medicine, National Cardiovascular Center, 5-7-1 Fujishirodai, Suita, Osaka 565, Japan.
- Received November 14, 1994.
- Revision received May 12, 1995.
- Accepted May 25, 1995.
- Copyright © 1995 by American Heart Association
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