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Basic science
ORIGINAL RESEARCH
Focal middle cerebral artery ischemia in rats via a transfemoral approach using a custom designed microwire Afshin A Divani,1,2,3 Ricky Chow,3,4 Homayoun Sadeghi-Bazargani,5,6 Amanda J Murphy,1,7 Jessica A Nordberg,1 Julian V Tokarev,1,7 Mario Hevesi,1,7 Xiao Wang,8 Xiao-Hong Zhu,8 Tommy Acompanado,4 Peter A Edwards,4 Yi Zhang,8 Wei Chen8 1
Department of Neurology, University of Minnesota, Minneapolis, Minnesota, USA 2 Department of Neurological Surgery, University of Minnesota, Minneapolis, Minnesota, USA 3 Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota, USA 4 Lake Region Medical, Chaska, Minnesota, USA 5 Neurosciences Research Center, Tabriz University of Medical Sciences, Iran 6 Department of Public Health Sciences, Karolinska Institute, Stockholm, Sweden 7 Medical School, University of Minnesota, Minneapolis, Minnesota, USA 8 Department of Radiology, Center for Magnetic Imaging Research, University of Minnesota, Minneapolis, Minnesota, USA Correspondence to Dr AA Divani, University of Minnesota, Department of Neurology, MMC 295, 420 Delaware Street SE, Minneapolis, MN 55455, USA; [email protected] Received 22 December 2014 Revised 1 April 2015 Accepted 15 April 2015
ABSTRACT Objectives The aim of this study was to develop a reliable and repeatable method of inducing focal middle cerebral artery occlusion (MCAo) in rats without ligation of the external carotid artery (ECA), while reducing the risk of subarachnoid hemorrhage. Methods We prototyped microwires with different diameters (0.0120 inch, 0.0115 inch, 0.0110 inch), materials, and construction methods (coil-on-core, extruded polymer jacket-on-core). Under fluoroscopic guidance and using femoral artery access, the microwires were navigated into the internal carotid artery of male Wistar rats (n=50, weight 376±64 g) to induce MCAo for 1 or 2 h. We performed neurological assessments at baseline, and at 3, 24, 72, and 168 h after MCAo. MRI measurements were performed on a 9.4 T scanner at 1 and 7 days post-injury. Results The 0.0115 inch microwire with polymer jacket-on-core provided the most successful outcome. At 1 and 7 days post-injury, we observed similar infarction volumes for 1 and 2 h MCAo in the MRI study. Infarcted lesion volumes in both MCAo groups were significantly reduced at 7 days compared with 1 day post-injury. The trend in longitudinal changes for the scores of different neurological assessments was confirmed to be significant after the injury, but both groups showed a similar trend of neurological deficits over the course of the study. Conclusions We have developed a reliable and repeatable MCAo method in rats, allowing for precise occlusion of the MCA under direct fluoroscopic visualization without alteration of the cerebral hemodynamics associated with ECA ligation. The custom designed microwire can also be sized for targeted focal ischemia in larger animals.
INTRODUCTION
To cite: Divani AA, Chow R, SadeghiBazargani H, et al. J NeuroIntervent Surg Published Online First: [please include Day Month Year] doi:10.1136/ neurintsurg-2014-011607
Stroke is a major public health issue in the USA in terms of its impact on death rates and disability.1 Therefore, animal models of stroke, particularly ischemic stroke, are of great importance for elucidating the pathophysiology of disease progression and for evaluating new investigational treatments and therapies for minimizing such progression. Rodent models of middle cerebral artery occlusion (MCAo) are one of the most commonly studied models of stroke in the preclinical setting.2 Many different surgical techniques for temporary or permanent MCAo
have been proposed in the literature.3–9 With the recent advent of angiography procedures, endovascular approaches using commercially available microwires are suggested for temporary MCAo.10–13 In this study, we present the technical steps in prototyping custom made microwires with different diameters that allowed for precise occlusion of the MCA under direct fluoroscopic visualization. After determining the optimal microwire diameter/type, experiments were performed in Wistar rats to assess the performance of the designed microwire in inducing MCAo stroke.
METHODS Microwire design To determine the optimum microwire size and design, we evaluated a number of different microwire configurations. Nitinol cores (with a 0.0135 inch diameter at the proximal end) were ground down to approximately 0.0028 inch at the distal end over a 10 inch long taper. The distal end was then flattened to a 0.001 inch×0.007 inch rectangular shape to reduce tip stiffness and thus the likelihood of vessel perforation and dissection. Platinum coils with an outer diameter of 0.010 inch, 0.011 inch, and 0.012 inch where attached to the nitinol core wire, producing a coil-on-core configuration. A proprietary hydrophilic coating developed at Lake Region Medical (Chaska, Minnesota, USA) was then applied to ease microwire advancement and reduce potential mechanical damage to the endothelial lining that can lead to thrombus generation.14 15 Using the coil-on-core family of microwire configurations for MCAo induced subarachnoid hemorrhage (SAH) in over 30% of the preliminary experiments. Therefore, we discontinued the use of coil-on-core microwires and decided to apply a new design in constructing the microwire. For the second design, we used a polymer jacket-on-core construction. A 0.001 inch thick 80A polyurethane tube was adhered and partially reflowed onto the nitinol core with the previously described grind profile. The partially reflowed distal tip had a slight taper, exhibiting an outer diameter increasing from 0.010 inch at the very distal tip of the microwire to 0.0115 inch about 3 mm proximal. The distal tip of the microwire was J-shaped to allow for easier
Divani AA, et al. J NeuroIntervent Surg 2015;0:1–7. doi:10.1136/neurintsurg-2014-011607
1
Downloaded from http://jnis.bmj.com/ on May 12, 2015 - Published by group.bmj.com
Basic science navigation. A hydrophilic coating (Lake Region Medical) was also applied on the surface of the microwire to reduce endothelial damage. Figure 1 shows samples of the coil-on-core and polymer jacket-on-core microwires that were tested.
Animal experiments The study was approved and conducted according to the guidelines set by the Institutional Animal Care and Use Committee at the University of Minnesota. We produced and referenced resin replicas of rat cerebrovasculature for navigation planning of the designed microwires. Detailed descriptions of the vascular resin models have been published elsewhere.16 Figure 2 shows a sample of the rat cerebrovascular model in anterior–posterior and lateral views. The study was performed in two stages. First, the performance of the microwire was assessed by successfully navigating the microwire to induce MCA stroke without hemorrhage. Once a satisfactory outcome was obtained for the microwire design, we performed the second phase of the study by performing neurological examinations and MRI studies. For the coil-on-core microwire design, we observed a high incidence of SAH in 8 of 26 (30.8%) animals sacrificed within 24 h (data not presented). Therefore, we abandoned the use of the coil-on-core design and developed the second microwire model using a polymer jacket-on-core design. In the preliminary performance evaluation of the second microwire design, we did not observe any evidence of SAH among 17 animals used for testing (sacrificed within 24 h). We had four unrelated bleeding complications at the site of femoral ligation after withdraw of the microwire. Therefore, we decided to move forward with the use of the polymer jacket-on-core design for the second phase of the study. For this study, Wistar rats (n=50, weight 376±64 g) were used for inducing 1 (n=35) or 2 (n=15) h of MCAo. Each animal was anesthetized with 3% isoflurane in an induction chamber, which was maintained during the procedure with 2.0– 2.5% isoflurane (with 1 L/min flow of oxygen) using a custom designed nose cone. A rapid prototyping technique was
performed using a three-dimensional printer (see figure 1B). Heparin (100 U/kg) was administered subcutaneously as a bolus dose prior to the start of the procedure. Using an aseptic technique, the groin area was exposed in the right hind limb via skin incision. Using a surgical microscope (OPMI CS-NS; Carl Zeiss, Germany), the right femoral artery was isolated from the femoral vein and nerve. Drops of 2% lidocaine were used as a nerve block and to resolve vasospasm due to manipulation of the artery. An aneurysm clip was temporarily placed on the right common femoral artery to cease blood flow during insertion of the microwire into the right superficial femoral artery. In order to minimize injury, arterial access was established from the saphenous branch of the descending genicular artery, while leaving the popliteal, superficial epigastric, and common femoral arteries intact. The artery of access was distally ligated using a 3.0 silk ligature. Another ligature was loosely placed around the artery proximal to the site of access to permanently ligate the artery after withdrawing the microwire. Using dissecting micro-scissors, a 45° incision was made in the right saphenous artery to insert the microwire directly without using a femoral sheath. Once the tip of the microwire reached the aneurysm clip, the clip was removed to allow for advancement of the microwire into the aorta and subsequently into the right or left internal carotid artery. Navigation of the microwire was performed under fluoroscopic visualization. The position of the wire was confirmed using anterior– posterior and lateral views (see figure 3). We also documented changes in blood flow distribution before and during MCAo using digital subtraction angiography and by injecting 1 mL of contrast (Optiray 240) through the aorta in three animals (see figure 3). When the prescribed occlusion time was complete, the microwire was gently retracted under fluoroscopic visualization. Next, the superficial femoral artery was permanently ligated, and the skin was sutured. Ketoprofen (2.5 mg/kg), buprenorphine (0.05 mg/kg), enrofloxacin (10 mg/kg), and saline (5 mL) were administered subcutaneously intraoperatively. Throughout the procedure, body temperature was monitored and maintained at approximately 37±0.5°C using a rectal probe and a heating pad. After completion of the surgery, the animal was placed back into its cage for recovery, with free access to food and water. Postoperative analgesics and antibiotics were administered for 3 days. Animals were humanely euthanized after 7 days on completion of neurological and imaging studies.
Neurological assessments
Figure 1 (A) Sample images of the microwire design ( polymer jacket-on-core is shown on the right and the coil-on-core design is shown on the left). (B) Custom design nose cone using a rapid prototyping technique and a three-dimensional printer that was used for the experiment. 2
Neurological assessments were performed using three different methods (Garcia,17 Modo,18 and Longa19). Baseline assessments were conducted before injury followed by post-MCAo assessments at 3, 24, 72, and 168 h (7 days). We used five components from the original Garcia’s test17 that included spontaneous activity, symmetry in the movement of limbs, forepaw outstretching, body proprioception, and response to vibrissae touch. Three of the tests had a minimum score of 0 and two tests had a minimum score of 1. Thus the possible range of combined neurological scores was 2–15, as described elsewhere.20 21 We also used a modified version of the assessment suggested by Modo et al18 based on the presence or absence of six of the seven global neurological assessments with a total score ranging from 0 (signifying severe deficit) to 11 (signifying little to no deficit). The Modo’s assessments included spontaneous motility, righting reflex, grasping reflex, tilted cage top, placing reaction, and visual placing. For the third assessment, we used the method suggested by Long et al19 based on a Divani AA, et al. J NeuroIntervent Surg 2015;0:1–7. doi:10.1136/neurintsurg-2014-011607
Downloaded from http://jnis.bmj.com/ on May 12, 2015 - Published by group.bmj.com
Basic science
Figure 2 Resin replica of the rat cerebrovascular system. (A) Anterior–posterior view; (B) lateral view. 5 point scale, with 0 for no neurological deficit and 4 for no spontaneous walking and being depressed.
MRI studies At 1 and 7 days post-MCAo, MRI measurements were performed on a 9.4 T/31 cm animal scanner (Varian) with a 1H RF surface coil. For the MRI scans, animals were anesthetized with ∼2% isoflurane and mechanically ventilated to maintain normal physiological condition. T2 weighted images were acquired with a fast spin echo sequence (TE=10 ms; TR=4 s; FOV=3.2×3.2 cm; matrix=256×256; slice thickness=0.25 mm; 8 echo train length). Gradient echo diffusion images were acquired (TE=14 ms; TR=250 ms; FOV=3.2×3.2 cm; image matrix=128×128; 1 mm slice thickness) with two b factors (b1=10.8 and b2=1007 s/mm) for obtaining apparent diffusion coefficient maps.22 The continuous arterial spin labeling method23 was applied to obtain images for cerebral blood flow (CBF) with the following parameters: TE=20 ms, TR=3 s, FOV=3.2×3.2 cm, image matrix=64×64, and 1 mm slice thickness. Lesion volumes of the MCAo rats at 1 and 7 days post-MCAo were determined by one of the investigators ( JVT) who was blinded to the MCAo groups. Lesion area was calculated through tracing of hyperintense regions on each slice of the T2 weighted images using three-dimensional Doctor software (Able Software Corp, Lexington, Massachusetts, USA). Volumetric data were obtained by generating a complex surface rendering of the traced regions. Each series was also evaluated for midline shift, brain regions involved, and presence of cavitary changes at 7 days post-procedure; these items were recorded as either present or absent for each scanned animal.24 25
Statistical analysis Neurological scores (Garcia, Modo, and Longa) from the 1 and 2 h MCAo groups were compared using the Wilcoxon rank sum test. Infarction volume on MRI was compared between the two groups using the Mann–Whitney test. Tests comparing 1 day and 7 day post-procedure MRIs within each group were conducted using paired t-tests for continuous data whereas comparisons of proportions where made using a χ2 test with Yates’ continuity correction. Values are presented as mean±SD, unless otherwise noted. To consider potential lack of independence among multiple measurements of neurological assessments, we applied appropriate longitudinal analysis methods. Generalized linear models (GLM) and generalized estimating equations were applied which yielded similar results, and the results of generalized linear models were reported. A significant time×group interaction term was considered as a criterion for detecting a difference in response trends between the two groups over the experimentation time. Analyses were conducted in R V.3.0.2 (R Core Team. Vienna, Austria).
RESULTS The two populations of rats who underwent 1 and 2 h MCAo had similar weights and ratio of left/right MCAo (table 1), and both groups demonstrated visible infarctions on T2 weighted images at 1 and 7 days post-procedure (figures 4 and 5). Infarction volumes at 1 day after MCAo were calculated from the T2 weighted anatomic images and found to be 71.47±46.27 and 74.79±34.12 mm3 (p=0.91) for the 1 and 2 h MCAo groups, respectively. Similarly, at 7 days, there was no difference in infarction volumes between 1 and 2 h MCAo (28.55±21.61 mm3 and 30.59±19.19 mm3, respectively) ( p=0.64). Both groups
Divani AA, et al. J NeuroIntervent Surg 2015;0:1–7. doi:10.1136/neurintsurg-2014-011607
3
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Basic science
Figure 3 Fluoroscopy images of the microwire position in the right internal carotid artery (ICA). The tip of the microwire is extended into the anterior cerebral artery covering the opening of the middle cerebral artery (MCA). (A) Anterior–posterior view and (B) lateral view. Digital subtraction angiography images of cerebral blood flow, (C) before MCA occlusion (MCAo) and (D) during MCAo, while the microwire is positioned in the right ICA. demonstrated a statistically significant decrease in infarcted lesion volume between the 1 and 7 day assessments ( p=0.010 and
Basic science
ORIGINAL RESEARCH
Focal middle cerebral artery ischemia in rats via a transfemoral approach using a custom designed microwire Afshin A Divani,1,2,3 Ricky Chow,3,4 Homayoun Sadeghi-Bazargani,5,6 Amanda J Murphy,1,7 Jessica A Nordberg,1 Julian V Tokarev,1,7 Mario Hevesi,1,7 Xiao Wang,8 Xiao-Hong Zhu,8 Tommy Acompanado,4 Peter A Edwards,4 Yi Zhang,8 Wei Chen8 1
Department of Neurology, University of Minnesota, Minneapolis, Minnesota, USA 2 Department of Neurological Surgery, University of Minnesota, Minneapolis, Minnesota, USA 3 Department of Mechanical Engineering, University of Minnesota, Minneapolis, Minnesota, USA 4 Lake Region Medical, Chaska, Minnesota, USA 5 Neurosciences Research Center, Tabriz University of Medical Sciences, Iran 6 Department of Public Health Sciences, Karolinska Institute, Stockholm, Sweden 7 Medical School, University of Minnesota, Minneapolis, Minnesota, USA 8 Department of Radiology, Center for Magnetic Imaging Research, University of Minnesota, Minneapolis, Minnesota, USA Correspondence to Dr AA Divani, University of Minnesota, Department of Neurology, MMC 295, 420 Delaware Street SE, Minneapolis, MN 55455, USA; [email protected] Received 22 December 2014 Revised 1 April 2015 Accepted 15 April 2015
ABSTRACT Objectives The aim of this study was to develop a reliable and repeatable method of inducing focal middle cerebral artery occlusion (MCAo) in rats without ligation of the external carotid artery (ECA), while reducing the risk of subarachnoid hemorrhage. Methods We prototyped microwires with different diameters (0.0120 inch, 0.0115 inch, 0.0110 inch), materials, and construction methods (coil-on-core, extruded polymer jacket-on-core). Under fluoroscopic guidance and using femoral artery access, the microwires were navigated into the internal carotid artery of male Wistar rats (n=50, weight 376±64 g) to induce MCAo for 1 or 2 h. We performed neurological assessments at baseline, and at 3, 24, 72, and 168 h after MCAo. MRI measurements were performed on a 9.4 T scanner at 1 and 7 days post-injury. Results The 0.0115 inch microwire with polymer jacket-on-core provided the most successful outcome. At 1 and 7 days post-injury, we observed similar infarction volumes for 1 and 2 h MCAo in the MRI study. Infarcted lesion volumes in both MCAo groups were significantly reduced at 7 days compared with 1 day post-injury. The trend in longitudinal changes for the scores of different neurological assessments was confirmed to be significant after the injury, but both groups showed a similar trend of neurological deficits over the course of the study. Conclusions We have developed a reliable and repeatable MCAo method in rats, allowing for precise occlusion of the MCA under direct fluoroscopic visualization without alteration of the cerebral hemodynamics associated with ECA ligation. The custom designed microwire can also be sized for targeted focal ischemia in larger animals.
INTRODUCTION
To cite: Divani AA, Chow R, SadeghiBazargani H, et al. J NeuroIntervent Surg Published Online First: [please include Day Month Year] doi:10.1136/ neurintsurg-2014-011607
Stroke is a major public health issue in the USA in terms of its impact on death rates and disability.1 Therefore, animal models of stroke, particularly ischemic stroke, are of great importance for elucidating the pathophysiology of disease progression and for evaluating new investigational treatments and therapies for minimizing such progression. Rodent models of middle cerebral artery occlusion (MCAo) are one of the most commonly studied models of stroke in the preclinical setting.2 Many different surgical techniques for temporary or permanent MCAo
have been proposed in the literature.3–9 With the recent advent of angiography procedures, endovascular approaches using commercially available microwires are suggested for temporary MCAo.10–13 In this study, we present the technical steps in prototyping custom made microwires with different diameters that allowed for precise occlusion of the MCA under direct fluoroscopic visualization. After determining the optimal microwire diameter/type, experiments were performed in Wistar rats to assess the performance of the designed microwire in inducing MCAo stroke.
METHODS Microwire design To determine the optimum microwire size and design, we evaluated a number of different microwire configurations. Nitinol cores (with a 0.0135 inch diameter at the proximal end) were ground down to approximately 0.0028 inch at the distal end over a 10 inch long taper. The distal end was then flattened to a 0.001 inch×0.007 inch rectangular shape to reduce tip stiffness and thus the likelihood of vessel perforation and dissection. Platinum coils with an outer diameter of 0.010 inch, 0.011 inch, and 0.012 inch where attached to the nitinol core wire, producing a coil-on-core configuration. A proprietary hydrophilic coating developed at Lake Region Medical (Chaska, Minnesota, USA) was then applied to ease microwire advancement and reduce potential mechanical damage to the endothelial lining that can lead to thrombus generation.14 15 Using the coil-on-core family of microwire configurations for MCAo induced subarachnoid hemorrhage (SAH) in over 30% of the preliminary experiments. Therefore, we discontinued the use of coil-on-core microwires and decided to apply a new design in constructing the microwire. For the second design, we used a polymer jacket-on-core construction. A 0.001 inch thick 80A polyurethane tube was adhered and partially reflowed onto the nitinol core with the previously described grind profile. The partially reflowed distal tip had a slight taper, exhibiting an outer diameter increasing from 0.010 inch at the very distal tip of the microwire to 0.0115 inch about 3 mm proximal. The distal tip of the microwire was J-shaped to allow for easier
Divani AA, et al. J NeuroIntervent Surg 2015;0:1–7. doi:10.1136/neurintsurg-2014-011607
1
Downloaded from http://jnis.bmj.com/ on May 12, 2015 - Published by group.bmj.com
Basic science navigation. A hydrophilic coating (Lake Region Medical) was also applied on the surface of the microwire to reduce endothelial damage. Figure 1 shows samples of the coil-on-core and polymer jacket-on-core microwires that were tested.
Animal experiments The study was approved and conducted according to the guidelines set by the Institutional Animal Care and Use Committee at the University of Minnesota. We produced and referenced resin replicas of rat cerebrovasculature for navigation planning of the designed microwires. Detailed descriptions of the vascular resin models have been published elsewhere.16 Figure 2 shows a sample of the rat cerebrovascular model in anterior–posterior and lateral views. The study was performed in two stages. First, the performance of the microwire was assessed by successfully navigating the microwire to induce MCA stroke without hemorrhage. Once a satisfactory outcome was obtained for the microwire design, we performed the second phase of the study by performing neurological examinations and MRI studies. For the coil-on-core microwire design, we observed a high incidence of SAH in 8 of 26 (30.8%) animals sacrificed within 24 h (data not presented). Therefore, we abandoned the use of the coil-on-core design and developed the second microwire model using a polymer jacket-on-core design. In the preliminary performance evaluation of the second microwire design, we did not observe any evidence of SAH among 17 animals used for testing (sacrificed within 24 h). We had four unrelated bleeding complications at the site of femoral ligation after withdraw of the microwire. Therefore, we decided to move forward with the use of the polymer jacket-on-core design for the second phase of the study. For this study, Wistar rats (n=50, weight 376±64 g) were used for inducing 1 (n=35) or 2 (n=15) h of MCAo. Each animal was anesthetized with 3% isoflurane in an induction chamber, which was maintained during the procedure with 2.0– 2.5% isoflurane (with 1 L/min flow of oxygen) using a custom designed nose cone. A rapid prototyping technique was
performed using a three-dimensional printer (see figure 1B). Heparin (100 U/kg) was administered subcutaneously as a bolus dose prior to the start of the procedure. Using an aseptic technique, the groin area was exposed in the right hind limb via skin incision. Using a surgical microscope (OPMI CS-NS; Carl Zeiss, Germany), the right femoral artery was isolated from the femoral vein and nerve. Drops of 2% lidocaine were used as a nerve block and to resolve vasospasm due to manipulation of the artery. An aneurysm clip was temporarily placed on the right common femoral artery to cease blood flow during insertion of the microwire into the right superficial femoral artery. In order to minimize injury, arterial access was established from the saphenous branch of the descending genicular artery, while leaving the popliteal, superficial epigastric, and common femoral arteries intact. The artery of access was distally ligated using a 3.0 silk ligature. Another ligature was loosely placed around the artery proximal to the site of access to permanently ligate the artery after withdrawing the microwire. Using dissecting micro-scissors, a 45° incision was made in the right saphenous artery to insert the microwire directly without using a femoral sheath. Once the tip of the microwire reached the aneurysm clip, the clip was removed to allow for advancement of the microwire into the aorta and subsequently into the right or left internal carotid artery. Navigation of the microwire was performed under fluoroscopic visualization. The position of the wire was confirmed using anterior– posterior and lateral views (see figure 3). We also documented changes in blood flow distribution before and during MCAo using digital subtraction angiography and by injecting 1 mL of contrast (Optiray 240) through the aorta in three animals (see figure 3). When the prescribed occlusion time was complete, the microwire was gently retracted under fluoroscopic visualization. Next, the superficial femoral artery was permanently ligated, and the skin was sutured. Ketoprofen (2.5 mg/kg), buprenorphine (0.05 mg/kg), enrofloxacin (10 mg/kg), and saline (5 mL) were administered subcutaneously intraoperatively. Throughout the procedure, body temperature was monitored and maintained at approximately 37±0.5°C using a rectal probe and a heating pad. After completion of the surgery, the animal was placed back into its cage for recovery, with free access to food and water. Postoperative analgesics and antibiotics were administered for 3 days. Animals were humanely euthanized after 7 days on completion of neurological and imaging studies.
Neurological assessments
Figure 1 (A) Sample images of the microwire design ( polymer jacket-on-core is shown on the right and the coil-on-core design is shown on the left). (B) Custom design nose cone using a rapid prototyping technique and a three-dimensional printer that was used for the experiment. 2
Neurological assessments were performed using three different methods (Garcia,17 Modo,18 and Longa19). Baseline assessments were conducted before injury followed by post-MCAo assessments at 3, 24, 72, and 168 h (7 days). We used five components from the original Garcia’s test17 that included spontaneous activity, symmetry in the movement of limbs, forepaw outstretching, body proprioception, and response to vibrissae touch. Three of the tests had a minimum score of 0 and two tests had a minimum score of 1. Thus the possible range of combined neurological scores was 2–15, as described elsewhere.20 21 We also used a modified version of the assessment suggested by Modo et al18 based on the presence or absence of six of the seven global neurological assessments with a total score ranging from 0 (signifying severe deficit) to 11 (signifying little to no deficit). The Modo’s assessments included spontaneous motility, righting reflex, grasping reflex, tilted cage top, placing reaction, and visual placing. For the third assessment, we used the method suggested by Long et al19 based on a Divani AA, et al. J NeuroIntervent Surg 2015;0:1–7. doi:10.1136/neurintsurg-2014-011607
Downloaded from http://jnis.bmj.com/ on May 12, 2015 - Published by group.bmj.com
Basic science
Figure 2 Resin replica of the rat cerebrovascular system. (A) Anterior–posterior view; (B) lateral view. 5 point scale, with 0 for no neurological deficit and 4 for no spontaneous walking and being depressed.
MRI studies At 1 and 7 days post-MCAo, MRI measurements were performed on a 9.4 T/31 cm animal scanner (Varian) with a 1H RF surface coil. For the MRI scans, animals were anesthetized with ∼2% isoflurane and mechanically ventilated to maintain normal physiological condition. T2 weighted images were acquired with a fast spin echo sequence (TE=10 ms; TR=4 s; FOV=3.2×3.2 cm; matrix=256×256; slice thickness=0.25 mm; 8 echo train length). Gradient echo diffusion images were acquired (TE=14 ms; TR=250 ms; FOV=3.2×3.2 cm; image matrix=128×128; 1 mm slice thickness) with two b factors (b1=10.8 and b2=1007 s/mm) for obtaining apparent diffusion coefficient maps.22 The continuous arterial spin labeling method23 was applied to obtain images for cerebral blood flow (CBF) with the following parameters: TE=20 ms, TR=3 s, FOV=3.2×3.2 cm, image matrix=64×64, and 1 mm slice thickness. Lesion volumes of the MCAo rats at 1 and 7 days post-MCAo were determined by one of the investigators ( JVT) who was blinded to the MCAo groups. Lesion area was calculated through tracing of hyperintense regions on each slice of the T2 weighted images using three-dimensional Doctor software (Able Software Corp, Lexington, Massachusetts, USA). Volumetric data were obtained by generating a complex surface rendering of the traced regions. Each series was also evaluated for midline shift, brain regions involved, and presence of cavitary changes at 7 days post-procedure; these items were recorded as either present or absent for each scanned animal.24 25
Statistical analysis Neurological scores (Garcia, Modo, and Longa) from the 1 and 2 h MCAo groups were compared using the Wilcoxon rank sum test. Infarction volume on MRI was compared between the two groups using the Mann–Whitney test. Tests comparing 1 day and 7 day post-procedure MRIs within each group were conducted using paired t-tests for continuous data whereas comparisons of proportions where made using a χ2 test with Yates’ continuity correction. Values are presented as mean±SD, unless otherwise noted. To consider potential lack of independence among multiple measurements of neurological assessments, we applied appropriate longitudinal analysis methods. Generalized linear models (GLM) and generalized estimating equations were applied which yielded similar results, and the results of generalized linear models were reported. A significant time×group interaction term was considered as a criterion for detecting a difference in response trends between the two groups over the experimentation time. Analyses were conducted in R V.3.0.2 (R Core Team. Vienna, Austria).
RESULTS The two populations of rats who underwent 1 and 2 h MCAo had similar weights and ratio of left/right MCAo (table 1), and both groups demonstrated visible infarctions on T2 weighted images at 1 and 7 days post-procedure (figures 4 and 5). Infarction volumes at 1 day after MCAo were calculated from the T2 weighted anatomic images and found to be 71.47±46.27 and 74.79±34.12 mm3 (p=0.91) for the 1 and 2 h MCAo groups, respectively. Similarly, at 7 days, there was no difference in infarction volumes between 1 and 2 h MCAo (28.55±21.61 mm3 and 30.59±19.19 mm3, respectively) ( p=0.64). Both groups
Divani AA, et al. J NeuroIntervent Surg 2015;0:1–7. doi:10.1136/neurintsurg-2014-011607
3
Downloaded from http://jnis.bmj.com/ on May 12, 2015 - Published by group.bmj.com
Basic science
Figure 3 Fluoroscopy images of the microwire position in the right internal carotid artery (ICA). The tip of the microwire is extended into the anterior cerebral artery covering the opening of the middle cerebral artery (MCA). (A) Anterior–posterior view and (B) lateral view. Digital subtraction angiography images of cerebral blood flow, (C) before MCA occlusion (MCAo) and (D) during MCAo, while the microwire is positioned in the right ICA. demonstrated a statistically significant decrease in infarcted lesion volume between the 1 and 7 day assessments ( p=0.010 and
