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Nylon filament coated with paraffin for intraluminal permanent middle cerebral artery occlusion in rats
Neuroscience Letters 519 (2012) 42–46
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Nylon filament coated with paraffin for intraluminal permanent middle cerebral artery occlusion in rats Xia-lin Zuo 1 , Ping Wu 1 , Ai-min Ji ∗ Center for Drug Research and Development, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, PR China
h i g h l i g h t s
In this study we tested the effects of paraffin-coated nylon filament for rat MCAO model. Rats models of MCAO with paraffin-coated nylon filament (cMCAO) have larger infarct areas. Rats models in cMCAO group have lower mortality. The success rate in cMCAO group is 100% (n = 20). The paraffin-coated nylon filament was applicable to making MCAO model.
a r t i c l e
i n f o
Article history: Received 5 January 2012 Received in revised form 2 May 2012 Accepted 3 May 2012 Keywords: Nylon filament Middle cerebral artery occlusion Rats
a b s t r a c t A variety of intraluminal nylon filament has been used in rat middle cerebral artery occlusion (MCAO) models. However the lesion extent and its reproducibility vary among laboratories. The properties of nylon filament play a part of reasons for these variations. In the present study, we used paraffin-coated nylon filament for rat MCAO model, tested the effects and advanced improvement for making the rat MCAO. Forty male Sprague-Dawley (SD) rats were randomized into two groups, MCAO with traditional uncoated nylon filament (uMCAO) and MCAO with paraffin-coated nylon filament (cMCAO), three rats as normal group and sham group respectively. Assessment included mortality rates, model success rates, neurological deficit evaluation, and infarct volume. The study showed two rats died in uMCAO group, no rat died in cMCAO group within the 12 h. The model success rate of uMCAO was 100%, while the uMCAO group was 55% (n = 20, two died within 12 h, seven rats were excluded as the brain slices showed no TTC staining due to subarachanoid hemorrhage). Neurological evaluation demonstrated group cMCAO had more worse neurological outcomes than group uMCAO, and the difference was statistically signification (p < 0.05). TTC staining cMCAO group had significantly larger infarct volumes than uMCAO group, and also showed statistically significant difference (p < 0.05). The result demonstrated that the paraffincoated nylon filament intraluminal occlusion provide better occlusion of middle cerebral artery than the uncoated nylon filament, improve the consistent of model, and raise the success rate to reduce the number of experimental animals. These positive results are much encouraging and interesting. © 2012 Elsevier Ireland Ltd. All rights reserved.
Stroke as one of the leading causes of death and adult disability in the worldwide, it has produced significant family and society burdens [1]. However, treatment options to date are very limited. To meet the need for validating the novel therapeutic approaches and understanding the physiopathology of the ischemic brain injury, experimental strokes were must used research tools. There are many kinds of experimental strokes, however, the most frequently used is cerebral ischemia model due to its a closer approximation
∗ Corresponding author. Tel.: +86 02061643500; fax: +86 02084300639. E-mail address: [email protected] (A.-m. Ji). 1 These authors contributed equally to this work and should be considered co-first authors. 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.05.017
to clinical ischemic stroke. The animal species for experimental stroke is usually limited to laboratory species, rat and mice are most commonly used as they are relatively inexpensive and their cranial circulatory anatomy is similar to humans [15]. In the methods of cerebral ischemic stroke, intraluminal occluding the middle cerebral artery (MCA) is the most frequently used; the vessel can be occluded either permanently or transiently, and damage results from both ischemia and reperfusion [5,6,8,12,14]. However, the properties of filaments, including diameter, flexibility, surface finish and tip end smoothness affect the results of model, especially the smoothness of tip end is critical for the model making due to insert blood vessel and leading the filament pass through the blood vessel, a little bit of tip end unevenness increase the risk of damage the blood wall causing hemorrhage. Researchers had
X.-l. Zuo et al. / Neuroscience Letters 519 (2012) 42–46
awarded this issue long time ago, various methods had been taken to improve it, such as heating the tip of nylon filament to make it blunted, silicone or glue coated the filament [2,4,7]. Coated filament showed improved results as the experimental animals with larger infarct areas/low mortality rate than uncoated nylon filament [2,11]. Coated nylon filament have been used in MCAO related studies, however, the coated materials vary in different laboratories and no detail description the preparation methods were introduced. For this reason, a detailed study of the coated nylon filament for MCAO is much needed. Paraffin is high carbon mineral grease, with the advantage of strong inertia, good stability and biocompatibility, covered on the nylon filament surface easily and adhere well; simple preparation is practical that can be applied to in other laboratories. In this article, we investigated the paraffin-coated nylon filament for permanent MCAO model in rats, compared the results with the traditional uncoated nylon filament.
1. Materials and methods 1.1. Animals Adult male Sprague-Dawley rats, 10–12 W age, weight 250–280 g, were purchased from animal center of Southern Medical University, Guangzhou, China (No. 0080980). Rats were house in cages maintained in a regulated environment (12 h light/dark cycle), supplied with food and water without restriction.
1.2. Preparation of paraffin-coated nylon filament Nylon filament (diameter, 0.26 mm) was cut into 70 mm long under the microscope. The nylon filament was inserted into polyethylene tubing (inner diameter = 0.28 mm) at the length of 8 mm, the tubing was filled with melting paraffin for 10 s, then the tubing was removed after the paraffin coagulated. Put the paraffincoated filament in the bechtop irradiation with UV light for 30 min before using. To maintain consistency, it is recommended that one person prepare all of the paraffin-coated nylon filaments.
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1.3. Permanent middle cerebral artery occlusion (pMCAO) Middle cerebral artery occlusion (MCAO) was induced as described in previous studies. In brief, rats were anesthetized with chloral hydrate (300 mg/kg), maintained the body temperature at 37 ◦ C during surgery procedures until the rats awoke from surgery by a heating lamp. Disinfected the skin and surrounding fur with 70% ethyl alcohol, after a midline neck incision, pulled apart the soft tissues and exposed the left common carotid artery (CCA), gently separated it from the vagal nerves, the left external carotid artery (ECA) and the left internal carotid artery (ICA) were carefully dissected free from the surrounding nerves, then ligated the CCA and ECA with 3-0 silk thread. A small hole was cut in the CCA before it bifurcates to the ECA and the ICA [13,16]. The paraffin-coated nylon filament or uncoated nylon filament (Fig. 2B) was then introduced into the ICA to ∼17 mm until resistance was felt, lodged the filament in the ACA and blocked blood flow to the MCA (Fig. 1B), then incision was sutured. The animal was kept warm at 37 ◦ C with a heating lamp until came awake. In sham operations group, the filament is introduced into the ICA about 10 mm (Fig. 1C), the subsequent operation is identical to the experiment group. After 6 h, evaluating the neurological status as described by Longa et al. [10], then the rats were killed at 12 h after permanent MCAO and taken the brain tissue as quickly as possible. 1.4. Evaluate the neurological deficit scores Neurological deficits evaluations were carried out at 6 h after MAC occlusion by an observer masked to the identity of experimental groups using the following criteria as described by Longa et al. [10]: 0, no neurologic deficit or normal function; 1, left Horner s syndrome, 2, failure to extend right forepaw fully, 3, turning to right and 4, circling to right or absence of spontaneous motor activity. Hence, the higher the score the poor the neurological function is. 1.5. Triphenyltetrazolium chloride staining Animals were reanesthetized with chloral hydrate (300 mg/kg) and decapitated after 12 h of modeling. The rat brains were
Fig. 1. (A) Schematic illustration of the cerebral artery in the rats. Anterior cerebral artery (ACA), middle cerebral artery (MCA), posterior cerebral artery (PCA), internal carotid artery (ICA), external carotid artery (ECA), common carotid artery (CCA). (B) Schematic illustration of MCAO by nylon filament (black) is shown. (C) Schematic illustration of the sham operations.
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Fig. 2. Microscope observation the tip of nylon filament (40×). Traditional uncoated nylon filament (A); paraffin-coated nylon filament (B).
quickly removed and was frozen at −30 ◦ C for 10 min, dissected into 5 coronal slices and immediately incubated in 2% 2,3,5triphenyltetrazolium chloride (TTC) at 37 ◦ C for 10 min as described as previous reported [9]. The TTC stained viable brain tissue was dark red, whereas infracted tissue was unstained. After TTC staining, the slices were fixed in 4% paraformaldehyde. The border between infarcted and noninfarcted tissue was outlined and analysis with the Image J software, and the area of infarction was measured by subtracting the area of the nonischemic ipsilateral hemisphere from that of the contralateral side. Infarct area were calculated and expressed as a percentage of infarct area to total hemispheric area for each slice. The unsuccessful rat models had to be excluded in this study. 1.6. Statistical analysis The data were expressed as mean ± standard deviation (SD). Statistical analysis of two groups of MCAO (uncoated filament or coated filament method) was performed with variance analysis, and p < 0.05 was considered statistically significant. 2. Results 2.1. Preparation of paraffin-coated nylon filament Nylon filament was processed by coating paraffin at the tip of nylon filament. Under the microscope, the tip of uncoated nylon filaments were sharp (Fig. 2A) that can easy damage artery wall, however, the tip of paraffin-coated nylon filament (Fig. 2B) were blunted and with smoother surface.
Fig. 3. Neurological deficit scores of each studied group. The group of cMCAO had worse neurological outcomes than the group of uMCAO. There was significant difference in MCAO with paraffin-coated nylon filament group (cMCAO) and MCAO with traditional uncoated nylon filament (uMCAO) (p < 0.05). Data are mean ± SD (n = 20). *p < 0.05.
2.2. Mortality of pMCAO In uMCAO group, there were two rats died 10 h later of modeling, none of the rats died within the 12 h in cMCAO group. Autopsy found the reason for the died rats were intracranial hemorrhage and edema. The overall mortality was 10% in uMCAO group.
score of cMCAO group was 3.5 ± 0.7 (n = 20) versus uMCAO group was 2.63 ± 0.67 (n = 20). cMCAO group had worse neurological deficits than group uMCAO, there was a statistically significant difference between the two groups (p = 0.01) (Fig. 3).
2.3. Success rate of model In uMCAO group, there were two rats died of intracranial hemorrhage and edema in 10 h after modeling, and seven rats were excluded from the success rate account as these rats were found intracranial hemorrhage when sacrificed at 12 h, even though these rats showed neurological deficit. The success rate of uMCAO group in this study is 55.0% (n = 20). In cMCAO group, there was no rat died after modeling and no rat suffered intracranial hemorrhage, the success rate of model is 100% (n = 20). 2.4. Neurological deficits Neurologic examinations were performed at 6 h after MCA occlusion. Neurological evaluation demonstrated the neurological
2.5. TTC staining Rats were sacrificed 12 h after permanent MCAO and assessed the infarct areas with TTC staining. The result showed, in the uMCAO group, 11 rats brain had infarcts areas (two death, and the brain of seven rats suffered intracranial hemorrhage without showing TTC staining), whereas in the cMCAO group, all rats had infarcts (n = 20). cMCAO group had larger infarct areas than uMCAO group (24.28 ± 11.31% vs. 11.57 ± 5.56%, respectively) (Fig. 4). The mean of infarct areas was 209.97% larger compared with uMCAO group, and there was a statistically significant difference between the two groups (p = 0.015).
X.-l. Zuo et al. / Neuroscience Letters 519 (2012) 42–46
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Fig. 4. Representative TTC staining brain samples 12 h after doing different treatments. MCAO with paraffin-coated nylon filament (cMCAO), MCAO with traditional uncoated nylon filament (uMCAO), sham operation (sham) and normal group are show. (B) Infraction area (%) was shown for rats MCAO model with traditional uncoated nylon filament and paraffin-coated nylon filament. Data are mean ± SD. *p < 0.05.
3. Discussion In previous experiments, we also found the success rate of using uncoated nylon filament for MCAO model was rather low, accordance with the previous research that the success rate of middle cerebral artery model with uncoated filament in rats was less than 60% [3]. Thus, we tried to do some improvements, blunted the tip or paraffin-coated nylon for filament respectively, the results showed the paraffin-coated nylon has much more promising improvements than tip blunted method, which was in line with the previous research by Longa et al. [10], their study showed tip blunted method without making significant improvement, the MCAO model was still relatively low reproducibility and success rate was only 56%. A previous study demonstrated the silicone coated-nylon filament showed better results [11]. Hence, doing a systematic research to standardize the process of paraffin-coated nylon filament for MCAO model is much interesting. In our present study, we chose paraffin as coating material. Paraffin belongs to high carbon mineral grease, a mixture of alkanes that falls within the 20 ≤ n ≤ 40 range; enter the liquid phase past approximately 37 ◦ C and solid at the room temperate quickly. In addition, paraffin with good lubricity, chemical stability, biocompatibility, and covered on the nylon filament surface easily and stick well. Simple preparing method is practical for popularity. We systematically studied the paraffin-coated filament in the permanent MCAO model. The reason we chose permanent MCAO model is because the inducing damage only by the occlusion effects, exclude the reperfusion induced damage by temporary occlusion method. TTC-defined core expands to involve most of the cortical tissue supplied by the MCAO, this occurs quite rapidly and is complete (by TTC) within 6–12 h after stroke onset. We chose 6 h after the modeling to evaluate the acute phase damage effects by assessing the neurological defects. Certainly by 12 h, all of the territories supplied by the MCA is irreversibly damaged, TTC staining can show the infract areas to reflect the finally occlusion effects. Our study results demonstrated that the mortality of cMCAO group within 12 h was 0% (0/20), while in the uMCAO group was 10% (2/20), the reason for the died rats were intracranial hemorrhage and edema, this suggested the paraffin coated-nylon filament with good biocompatibility for blood vessel. The model success rate of uMCAO
was 100%, while the uMCAO group was 55% (n = 20, two died within 12 h, seven rats were excluded as the brain slices showed no TTC staining that means they were not true sense of ischemia stroke model). Neurological defects evaluation after 6 h of modeling, the cMCAO group have more severe defects than uMCAO group and showed statistically significant between two groups (p = 0.01). The most encouraging in our study was we found in cMCAO group, TTC staining showed lager infract area, the mean infarct areas was 209.97% than group uMCAO. In uMCAO group, seven rats suffered intracranial hemorrhage when decapitated at 12 h, and TTC staining showed no infarct areas of these rat brains, although these rats showed neurological defects, so these rats should be exclude from cerebral ischemia study to avoid false positive results. Also, we added an additional experiment in this study. The results showed the mortality of this model (cMCAO) within 12–24 h was 12.5% (2/16); 50% within 24–48 h (8/16); 37.5% within 48–84 h((6/16)). 12.5% animals model were survival less than 24 h and more than 87.5% animals model were survival at 24 h or more longer, such survival time and mortality would be enough to test novel therapies. In summary, we conclude that the paraffin-coated nylon filament for intraluminal permanent middle cerebral artery occlusion provide better occlusion than the traditional uncoated nylon filament in the aspects of model success rate, unify infarct area and neurological defects, by virtue of thickness, blunt tip and smooth surface. Presently, there is limited published data to support the use of one type of nylon filament to the exclusion of the other. In this study, we demonstrated that paraffin-coated filament for intraluminal permanent middle cerebral artery occlusion is worth popularity. Acknowledgements Authors acknowledge the support given by the National Science and Technology Major Project (Project No. 2009ZX09103605); Joint Project of the Chinese Academy of Sciences and the Guangdong Province Guangdong Science and Technology plan Project (Project No. 2009B091300129); and the Science and Technology Development project of Guangzhou (Project No. 2010UI-E00531-7).
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References [1] American Heart Association, Heart disease and stroke statistics, 2012 update, Circulation 125 (2012) e2–e220. [2] J. Bouley, M. Fisher, N. Henninger, Comparison between coated vs. uncoated suture middle cerebral artery occlusion in the rat as assessed by perfusion/diffusion weighted imaging, Neuroscience Letters 412 (2007) 185–190. [3] L. Belayev, O.F. Alonso, R. Busto, W.Z. Zhao, M.D. Ginsberg, Middle cerebral artery occlusion in the rat by intraluminal suture, Stroke 27 (1996) 1616–1623. [4] P.A. Barber, L. Hoyte, D. Kirk, T. Foniok, A. Buchan, U. Tuor, Early T1- and T2weighted MRI signatures of transient and permanent middle cerebral artery occlusion in a murine stroke model studied at 9.4T, Neuroscience Letters 388 (2005) 54–59. [5] S.T. Carmichael, Rodent models of focal stroke: size, mechanism, and purpose, NeuroRx 2 (3) (2005) 396–409. [6] T. Gerriets, E. Stolz, M. Walberer, C. Muller, C. Rottger, A. Kluge A, M. Kaps, M. Fisher, G. Bachmann, Complications and pitfalls in rat stroke models for middle cerebral artery occlusion: a comparison between the suture and the macrosphere model using magnetic resonance angiography, Stroke 35 (2004) 2372–2377. [7] Y.J. Guan, Y.T. Wang, F.L. Yuan, H.Y. Lu, Y.Q. Ren, T.Q. Xiao, K.M. Chen, D.A. Greenberg, K.L. Jin, G.Y. Yang, Effect of suture properties on stability of middle cerebral artery occlusion evaluated by synchrotron radiation angiography, Stroke 17 (2011) 111–117. [8] J. Koizumi, Y. Yoshida, T. Nakazawa, G. Ooneda, Experimental studies of ischemic brain edema: a new experimental model of cerebral embolism in rats in which recirculation can be introduced in the ischemic area, Japanese Journal of Stroke 8 (1986) 1–8.
[9] E. Kilic, M. Bähr, D.M. Hermann, Effects of recombinant tissue plasminogen activator after intraluminal thread occlusion in mice: role of hemodynamic alterations, Stroke 32 (2001) 2641–2647. [10] E.Z. Longa, P.R. Weinstein, S. Carlson, R. Cummins, Reversible middle cerebral artery occlusion without craniectomy in rats, Stroke 20 (1989) 84–91. [11] N. Shimamura, G. Matchett, T. Tsubokawa, H. Ohkuma, J. Zhang, Comparison of silicon-coated nylon suture to plain nylon suture in the rat middle cerebral artery occlusion model, Journal of Neuroscience and Methods 156 (2006) 161–165. [12] R. Schmid-Elsaesser, S. Zausinger, E. Hungerhuber, A. Baethmann, H.J. Reulen, A critical reevaluation of the intraluminal thread model of focal cerebral ischemia: evidence of inadvertent premature reperfusion and subarachnoid hemorrhage in rats by laser-Doppler flowmetry, Stroke 29 (1998) 2162–2170. [13] T. Suzuki, Y. Zhang, Y.-F. Zhang, F. Schlachetzki, W.M. Pardridge, Imaging gene expression in regional brain ischemia in vivo with a targeted [111in]-antisense radiopharmaceutical, Molecular Imaging 3 (2004) 356–363. [14] Z.A. Shah, K. Namiranian, J. Klaus, K. Kibler, S. Dore, Use of an optimized transient occlusion of the middle cerebral artery protocol for the mouse stroke model, Journal of Stroke and Cerebrovascular Disease 15 (2006) 133–138. [15] T.A. Woolsey, C.M. Rovainen, S.B. Cox, M.H. Henegar, G.E. Liang, D.Q. Liu, Y.E. Moskalenko, J. Sui, L. Wei, Neuronal units linked to microvascular modules in cerebral cortex: response elements for imaging the brain, Cerebral Cortex 6 (1996) 647–660. [16] Y. Zhang, W.M. Pardridge, Conjugation of brain-derived neurotrophic factor to a blood–brain barrier drug targeting system enables neuroprotection in regional brain ischemia following intravenous injection of the neurotrophin, Brain Research 889 (2001) 49–56.
Contents lists available at SciVerse ScienceDirect
Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet
Nylon filament coated with paraffin for intraluminal permanent middle cerebral artery occlusion in rats Xia-lin Zuo 1 , Ping Wu 1 , Ai-min Ji ∗ Center for Drug Research and Development, Zhujiang Hospital, Southern Medical University, Guangzhou 510282, PR China
h i g h l i g h t s
In this study we tested the effects of paraffin-coated nylon filament for rat MCAO model. Rats models of MCAO with paraffin-coated nylon filament (cMCAO) have larger infarct areas. Rats models in cMCAO group have lower mortality. The success rate in cMCAO group is 100% (n = 20). The paraffin-coated nylon filament was applicable to making MCAO model.
a r t i c l e
i n f o
Article history: Received 5 January 2012 Received in revised form 2 May 2012 Accepted 3 May 2012 Keywords: Nylon filament Middle cerebral artery occlusion Rats
a b s t r a c t A variety of intraluminal nylon filament has been used in rat middle cerebral artery occlusion (MCAO) models. However the lesion extent and its reproducibility vary among laboratories. The properties of nylon filament play a part of reasons for these variations. In the present study, we used paraffin-coated nylon filament for rat MCAO model, tested the effects and advanced improvement for making the rat MCAO. Forty male Sprague-Dawley (SD) rats were randomized into two groups, MCAO with traditional uncoated nylon filament (uMCAO) and MCAO with paraffin-coated nylon filament (cMCAO), three rats as normal group and sham group respectively. Assessment included mortality rates, model success rates, neurological deficit evaluation, and infarct volume. The study showed two rats died in uMCAO group, no rat died in cMCAO group within the 12 h. The model success rate of uMCAO was 100%, while the uMCAO group was 55% (n = 20, two died within 12 h, seven rats were excluded as the brain slices showed no TTC staining due to subarachanoid hemorrhage). Neurological evaluation demonstrated group cMCAO had more worse neurological outcomes than group uMCAO, and the difference was statistically signification (p < 0.05). TTC staining cMCAO group had significantly larger infarct volumes than uMCAO group, and also showed statistically significant difference (p < 0.05). The result demonstrated that the paraffincoated nylon filament intraluminal occlusion provide better occlusion of middle cerebral artery than the uncoated nylon filament, improve the consistent of model, and raise the success rate to reduce the number of experimental animals. These positive results are much encouraging and interesting. © 2012 Elsevier Ireland Ltd. All rights reserved.
Stroke as one of the leading causes of death and adult disability in the worldwide, it has produced significant family and society burdens [1]. However, treatment options to date are very limited. To meet the need for validating the novel therapeutic approaches and understanding the physiopathology of the ischemic brain injury, experimental strokes were must used research tools. There are many kinds of experimental strokes, however, the most frequently used is cerebral ischemia model due to its a closer approximation
∗ Corresponding author. Tel.: +86 02061643500; fax: +86 02084300639. E-mail address: [email protected] (A.-m. Ji). 1 These authors contributed equally to this work and should be considered co-first authors. 0304-3940/$ – see front matter © 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.05.017
to clinical ischemic stroke. The animal species for experimental stroke is usually limited to laboratory species, rat and mice are most commonly used as they are relatively inexpensive and their cranial circulatory anatomy is similar to humans [15]. In the methods of cerebral ischemic stroke, intraluminal occluding the middle cerebral artery (MCA) is the most frequently used; the vessel can be occluded either permanently or transiently, and damage results from both ischemia and reperfusion [5,6,8,12,14]. However, the properties of filaments, including diameter, flexibility, surface finish and tip end smoothness affect the results of model, especially the smoothness of tip end is critical for the model making due to insert blood vessel and leading the filament pass through the blood vessel, a little bit of tip end unevenness increase the risk of damage the blood wall causing hemorrhage. Researchers had
X.-l. Zuo et al. / Neuroscience Letters 519 (2012) 42–46
awarded this issue long time ago, various methods had been taken to improve it, such as heating the tip of nylon filament to make it blunted, silicone or glue coated the filament [2,4,7]. Coated filament showed improved results as the experimental animals with larger infarct areas/low mortality rate than uncoated nylon filament [2,11]. Coated nylon filament have been used in MCAO related studies, however, the coated materials vary in different laboratories and no detail description the preparation methods were introduced. For this reason, a detailed study of the coated nylon filament for MCAO is much needed. Paraffin is high carbon mineral grease, with the advantage of strong inertia, good stability and biocompatibility, covered on the nylon filament surface easily and adhere well; simple preparation is practical that can be applied to in other laboratories. In this article, we investigated the paraffin-coated nylon filament for permanent MCAO model in rats, compared the results with the traditional uncoated nylon filament.
1. Materials and methods 1.1. Animals Adult male Sprague-Dawley rats, 10–12 W age, weight 250–280 g, were purchased from animal center of Southern Medical University, Guangzhou, China (No. 0080980). Rats were house in cages maintained in a regulated environment (12 h light/dark cycle), supplied with food and water without restriction.
1.2. Preparation of paraffin-coated nylon filament Nylon filament (diameter, 0.26 mm) was cut into 70 mm long under the microscope. The nylon filament was inserted into polyethylene tubing (inner diameter = 0.28 mm) at the length of 8 mm, the tubing was filled with melting paraffin for 10 s, then the tubing was removed after the paraffin coagulated. Put the paraffincoated filament in the bechtop irradiation with UV light for 30 min before using. To maintain consistency, it is recommended that one person prepare all of the paraffin-coated nylon filaments.
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1.3. Permanent middle cerebral artery occlusion (pMCAO) Middle cerebral artery occlusion (MCAO) was induced as described in previous studies. In brief, rats were anesthetized with chloral hydrate (300 mg/kg), maintained the body temperature at 37 ◦ C during surgery procedures until the rats awoke from surgery by a heating lamp. Disinfected the skin and surrounding fur with 70% ethyl alcohol, after a midline neck incision, pulled apart the soft tissues and exposed the left common carotid artery (CCA), gently separated it from the vagal nerves, the left external carotid artery (ECA) and the left internal carotid artery (ICA) were carefully dissected free from the surrounding nerves, then ligated the CCA and ECA with 3-0 silk thread. A small hole was cut in the CCA before it bifurcates to the ECA and the ICA [13,16]. The paraffin-coated nylon filament or uncoated nylon filament (Fig. 2B) was then introduced into the ICA to ∼17 mm until resistance was felt, lodged the filament in the ACA and blocked blood flow to the MCA (Fig. 1B), then incision was sutured. The animal was kept warm at 37 ◦ C with a heating lamp until came awake. In sham operations group, the filament is introduced into the ICA about 10 mm (Fig. 1C), the subsequent operation is identical to the experiment group. After 6 h, evaluating the neurological status as described by Longa et al. [10], then the rats were killed at 12 h after permanent MCAO and taken the brain tissue as quickly as possible. 1.4. Evaluate the neurological deficit scores Neurological deficits evaluations were carried out at 6 h after MAC occlusion by an observer masked to the identity of experimental groups using the following criteria as described by Longa et al. [10]: 0, no neurologic deficit or normal function; 1, left Horner s syndrome, 2, failure to extend right forepaw fully, 3, turning to right and 4, circling to right or absence of spontaneous motor activity. Hence, the higher the score the poor the neurological function is. 1.5. Triphenyltetrazolium chloride staining Animals were reanesthetized with chloral hydrate (300 mg/kg) and decapitated after 12 h of modeling. The rat brains were
Fig. 1. (A) Schematic illustration of the cerebral artery in the rats. Anterior cerebral artery (ACA), middle cerebral artery (MCA), posterior cerebral artery (PCA), internal carotid artery (ICA), external carotid artery (ECA), common carotid artery (CCA). (B) Schematic illustration of MCAO by nylon filament (black) is shown. (C) Schematic illustration of the sham operations.
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Fig. 2. Microscope observation the tip of nylon filament (40×). Traditional uncoated nylon filament (A); paraffin-coated nylon filament (B).
quickly removed and was frozen at −30 ◦ C for 10 min, dissected into 5 coronal slices and immediately incubated in 2% 2,3,5triphenyltetrazolium chloride (TTC) at 37 ◦ C for 10 min as described as previous reported [9]. The TTC stained viable brain tissue was dark red, whereas infracted tissue was unstained. After TTC staining, the slices were fixed in 4% paraformaldehyde. The border between infarcted and noninfarcted tissue was outlined and analysis with the Image J software, and the area of infarction was measured by subtracting the area of the nonischemic ipsilateral hemisphere from that of the contralateral side. Infarct area were calculated and expressed as a percentage of infarct area to total hemispheric area for each slice. The unsuccessful rat models had to be excluded in this study. 1.6. Statistical analysis The data were expressed as mean ± standard deviation (SD). Statistical analysis of two groups of MCAO (uncoated filament or coated filament method) was performed with variance analysis, and p < 0.05 was considered statistically significant. 2. Results 2.1. Preparation of paraffin-coated nylon filament Nylon filament was processed by coating paraffin at the tip of nylon filament. Under the microscope, the tip of uncoated nylon filaments were sharp (Fig. 2A) that can easy damage artery wall, however, the tip of paraffin-coated nylon filament (Fig. 2B) were blunted and with smoother surface.
Fig. 3. Neurological deficit scores of each studied group. The group of cMCAO had worse neurological outcomes than the group of uMCAO. There was significant difference in MCAO with paraffin-coated nylon filament group (cMCAO) and MCAO with traditional uncoated nylon filament (uMCAO) (p < 0.05). Data are mean ± SD (n = 20). *p < 0.05.
2.2. Mortality of pMCAO In uMCAO group, there were two rats died 10 h later of modeling, none of the rats died within the 12 h in cMCAO group. Autopsy found the reason for the died rats were intracranial hemorrhage and edema. The overall mortality was 10% in uMCAO group.
score of cMCAO group was 3.5 ± 0.7 (n = 20) versus uMCAO group was 2.63 ± 0.67 (n = 20). cMCAO group had worse neurological deficits than group uMCAO, there was a statistically significant difference between the two groups (p = 0.01) (Fig. 3).
2.3. Success rate of model In uMCAO group, there were two rats died of intracranial hemorrhage and edema in 10 h after modeling, and seven rats were excluded from the success rate account as these rats were found intracranial hemorrhage when sacrificed at 12 h, even though these rats showed neurological deficit. The success rate of uMCAO group in this study is 55.0% (n = 20). In cMCAO group, there was no rat died after modeling and no rat suffered intracranial hemorrhage, the success rate of model is 100% (n = 20). 2.4. Neurological deficits Neurologic examinations were performed at 6 h after MCA occlusion. Neurological evaluation demonstrated the neurological
2.5. TTC staining Rats were sacrificed 12 h after permanent MCAO and assessed the infarct areas with TTC staining. The result showed, in the uMCAO group, 11 rats brain had infarcts areas (two death, and the brain of seven rats suffered intracranial hemorrhage without showing TTC staining), whereas in the cMCAO group, all rats had infarcts (n = 20). cMCAO group had larger infarct areas than uMCAO group (24.28 ± 11.31% vs. 11.57 ± 5.56%, respectively) (Fig. 4). The mean of infarct areas was 209.97% larger compared with uMCAO group, and there was a statistically significant difference between the two groups (p = 0.015).
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Fig. 4. Representative TTC staining brain samples 12 h after doing different treatments. MCAO with paraffin-coated nylon filament (cMCAO), MCAO with traditional uncoated nylon filament (uMCAO), sham operation (sham) and normal group are show. (B) Infraction area (%) was shown for rats MCAO model with traditional uncoated nylon filament and paraffin-coated nylon filament. Data are mean ± SD. *p < 0.05.
3. Discussion In previous experiments, we also found the success rate of using uncoated nylon filament for MCAO model was rather low, accordance with the previous research that the success rate of middle cerebral artery model with uncoated filament in rats was less than 60% [3]. Thus, we tried to do some improvements, blunted the tip or paraffin-coated nylon for filament respectively, the results showed the paraffin-coated nylon has much more promising improvements than tip blunted method, which was in line with the previous research by Longa et al. [10], their study showed tip blunted method without making significant improvement, the MCAO model was still relatively low reproducibility and success rate was only 56%. A previous study demonstrated the silicone coated-nylon filament showed better results [11]. Hence, doing a systematic research to standardize the process of paraffin-coated nylon filament for MCAO model is much interesting. In our present study, we chose paraffin as coating material. Paraffin belongs to high carbon mineral grease, a mixture of alkanes that falls within the 20 ≤ n ≤ 40 range; enter the liquid phase past approximately 37 ◦ C and solid at the room temperate quickly. In addition, paraffin with good lubricity, chemical stability, biocompatibility, and covered on the nylon filament surface easily and stick well. Simple preparing method is practical for popularity. We systematically studied the paraffin-coated filament in the permanent MCAO model. The reason we chose permanent MCAO model is because the inducing damage only by the occlusion effects, exclude the reperfusion induced damage by temporary occlusion method. TTC-defined core expands to involve most of the cortical tissue supplied by the MCAO, this occurs quite rapidly and is complete (by TTC) within 6–12 h after stroke onset. We chose 6 h after the modeling to evaluate the acute phase damage effects by assessing the neurological defects. Certainly by 12 h, all of the territories supplied by the MCA is irreversibly damaged, TTC staining can show the infract areas to reflect the finally occlusion effects. Our study results demonstrated that the mortality of cMCAO group within 12 h was 0% (0/20), while in the uMCAO group was 10% (2/20), the reason for the died rats were intracranial hemorrhage and edema, this suggested the paraffin coated-nylon filament with good biocompatibility for blood vessel. The model success rate of uMCAO
was 100%, while the uMCAO group was 55% (n = 20, two died within 12 h, seven rats were excluded as the brain slices showed no TTC staining that means they were not true sense of ischemia stroke model). Neurological defects evaluation after 6 h of modeling, the cMCAO group have more severe defects than uMCAO group and showed statistically significant between two groups (p = 0.01). The most encouraging in our study was we found in cMCAO group, TTC staining showed lager infract area, the mean infarct areas was 209.97% than group uMCAO. In uMCAO group, seven rats suffered intracranial hemorrhage when decapitated at 12 h, and TTC staining showed no infarct areas of these rat brains, although these rats showed neurological defects, so these rats should be exclude from cerebral ischemia study to avoid false positive results. Also, we added an additional experiment in this study. The results showed the mortality of this model (cMCAO) within 12–24 h was 12.5% (2/16); 50% within 24–48 h (8/16); 37.5% within 48–84 h((6/16)). 12.5% animals model were survival less than 24 h and more than 87.5% animals model were survival at 24 h or more longer, such survival time and mortality would be enough to test novel therapies. In summary, we conclude that the paraffin-coated nylon filament for intraluminal permanent middle cerebral artery occlusion provide better occlusion than the traditional uncoated nylon filament in the aspects of model success rate, unify infarct area and neurological defects, by virtue of thickness, blunt tip and smooth surface. Presently, there is limited published data to support the use of one type of nylon filament to the exclusion of the other. In this study, we demonstrated that paraffin-coated filament for intraluminal permanent middle cerebral artery occlusion is worth popularity. Acknowledgements Authors acknowledge the support given by the National Science and Technology Major Project (Project No. 2009ZX09103605); Joint Project of the Chinese Academy of Sciences and the Guangdong Province Guangdong Science and Technology plan Project (Project No. 2009B091300129); and the Science and Technology Development project of Guangzhou (Project No. 2010UI-E00531-7).
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