The purpose of this study is to establish and validate an animal model for research in the recovery and sequela stages of brain ischemia by testing brain infarction and sensorimotor function after middle cerebral artery occlusion/reperfusion (MCAO/R) after 1-90 days in rats.
The purpose of this study was to establish and validate an animal brain ischemia model in the recovery and sequela stages. A middle cerebral artery occlusion/reperfusion (MCAO/R) model in male Sprague-Dawley rats was chosen. By changing the rat's weight (260−330 g), the thread bolt type (2636/2838/3040/3043) and the brain infarct time (2-3 h), a higher Longa's score, a larger infarct volume and a greater model success ratio were screened using the Longa's score and TTC staining. The optimum model condition (300 g, 3040 thread bolt, 3 h brain infarct time) was acquired and used in a 1-90 day observation period after reperfusion via assessment of sensorimotor functions and infarct volume. At these conditions, the bilateral asymmetry test had a significant difference from 1 to 90 days, and the grid-walking test had a significant difference from 1 to 60 days; both differences could be a suitable sensorimotor functional test. Thus, the most appropriate condition of a novel rat model in the recovery and sequela stages of brain ischemia was found: 300 g rats that underwent MCAO with a 3040 thread bolt for a 3 h brain infarct and then reperfused. The appropriate sensorimotor functional tests were a bilateral asymmetry test and a grid-walking test.
Brain ischemia is divided into three stages with different post-stroke indicators: the acute stage (within 1 week), the recovery stage (1 week to 6 months), and the sequelae stage (more than 6 months). Presently, most studies focus on the acute stage of brain ischemia because of its significant effect and multi-relative research models1,2,3. However, the recovery and sequelae stages of brain ischemia cannot be ignored due to their long-term complication of disabilities. Therefore, the purpose of this study is to explore a stable, reliable and relatively simple animal model to research the recovery and sequela stages of brain ischemia.
Among the many experimental brain ischemia models, we use middle cerebral artery occlusion (MCAO) via thread bolt insertion into the right middle cerebral artery (MCA). This model is similar to human stroke, which can produce larger infarct volumes, result in many behavioral disorders related to stroke, and can allow blood reperfusion (R) by removing the thread bolt4,5,6. MCAO/R is also considered the gold standard animal model of brain ischemia7. Furthermore, the severity of the brain injury depends on the diameter and the insertion length of the thread bolt, the duration of brain ischemia, and the animal weight (larger rats have bigger brains and thicker cerebral vessels)8. Therefore, by changing the thread bolt type, the infarct time, and the rat weight, a suitable model can be found for the recovery and sequela stages of brain ischemia in MCAO/R rats. To validate the rat model, we performed a 1-day, 35-day, 60-day, and 90-day study of the MCAO/R model using TTC staining and sensorimotor function experiments (a bilateral asymmetry test, a grid-walking test, a rotarod test and a lifting rope test).
The procedure and use of animal subjects have been approved by the National Institute of Health for the care and use of laboratory animals. This protocol is specifically adjusted for the tests of middle cerebral artery occlusion/reperfusion (MCAO/R) and sensorimotor function.
1. Experimental design and grouping
2. Establishment of a unilateral MCAO/R model in rats9
NOTE: During the operation, use the microforceps gently to prevent breakage of the blood vessel. Avoid damage to the nerves and other blood vessels in the neck of the rat when the vessel is isolated. Care must be taken to present appropriate aseptic technique for all survival surgical procedures. The technique illustrated later in the video should be practiced through the entire procedure.
3. TTC staining
NOTE: The rat brain slice mold and blade must be pre-cooled in a -20 °C refrigerator before use to prevent adhesion caused by a large temperature difference. During staining, prevent adhesion between the brain slices and the culture plate, which can result in insufficient staining.
4. Assessment of sensorimotor function
NOTE: Rats (300 g, 3040 thread bolt, 3 h brain infarct time) with a Longa's score of 2−3 were selected to perform the sensorimotor function experiments from 1-90 days. Keep quiet and do not disturb the animals during this period of study. The data were analyzed by two-way ANOVA. Values shown represent mean ± S.D. P < 0.05 indicate difference.
Using the abovementioned procedure for a MCAO/R model with a Longa's score and TTC staining, different treatments of average weight (275/300/320 g), bolt types (2636/2838/3040/3043; Table 1) and ischemic times (2-3 h) and 1 day reperfusion were used to screen for the optimal brain ischemia model in rats. Model parameters of 300 g weight, 3040 thread bolt, and 3 h brain infarct time were the most suitable for the largest cerebral infarction, highest Longa's score and greatest model success ratio. This was significantly improved on the conventional treatment of a 275 g weight, 2636 thread bolt, and 2 h brain infarct time (Figure 1).
Furthermore, rats with 300 g weight, 3040 thread bolt, 3 h brain infarct time and a 2−3 Longa's score underwent sensorimotor function tests (a bilateral asymmetry test, a grid-walking test, a rotarod test, and a lifting rope test) and TTC staining to study the recovery status of brain ischemia from 1-90 days. The infarct and shrink volume were 23.4%, 19.6%, 16.1% (P < 0.01, compared with the first day) and 15.7% (P < 0.01, compared with the first day) after 1, 35, 60, and 90 days post MCAO/R, respectively (Figure 2). On the first day after MCAO/R, infarct volume was biggest. In time, the infarct volume became smaller and the shrink volume became larger. The infarct and shrink volume no longer changed after 60 days of MCAO/R.
The sensorimotor bias in the bilateral asymmetry test, the grid-walking error times in the grid-walking test and the lifting rope score in the lifting rope test all significantly increased, while the rotarod time in the rotarod test decreased significantly after 1 day of MCAO/R (Figure 3), which indicated that all four tests were meaningful in the stage of acute brain ischemia. However, only sensorimotor bias maintained large functional disorders with a time-dependent manner after 35, 60 and 90 days of MCAO/R. There were significant differences of grid-walking error times in the grid-walking test after 35 and 60 days of MCAO/R. These results indicated that the bilateral asymmetry test and the grid-walking test could be suitable sensorimotor function tests for the stage of recovery and sequela in rats.
Figure 1: 300 g weight, 3040 thread bolt, 3 h brain infarct time may be the optimum condition of the brain ischemic injury induced by MCAO/R. (A,B) Pictures and cartogram of infarct volume of brain tissue (n = 9−12). (C) Longa's score (n = 9−12). (D) The statistics of model success ratio of rats (n = 10−15). Model success ratio = (total number of rats – death rats after MCAO/R – failure rats after MCAO/R)/total number of rats. Failure rats are the model rats that do not have a suitable Longa's score. Error bars represent S.D., *P < 0.05, **P < 0.01. This figure has been modified from Liu et al.15. Please click here to view a larger version of this figure.
Figure 2: The infarct and shrink volume gradually decreased from 1 to 90 days after MCAO/R. (A) The TTC staining of rat brain tissue. (B) The cartogram of infarct and shrink volume (n = 16−19). Error bars represent S.D., **P < 0.01 vs. the first day after MCAO/R. This figure has been modified from Liu et al.15. Please click here to view a larger version of this figure.
Figure 3: Bilateral asymmetry test and grid-walking test were the suitable sensorimotor function tests in the recovery and sequela stage of brain ischemia. (A) The right limb tearing favorability in debonding experiment. (B) The grid-walking error times in grid-walking test. (C) The length of time in rotarod test. (D) The score in lifting rope test. Error bars represent S.D., n = 15−19, *P < 0.05, ***P < 0.001. This figure has been modified from Liu et al.15. Please click here to view a larger version of this figure.
Type | The diameter of thread bolt | The diameter of thread bolt head | Recommended weight of rat | Level |
2636 | 0.26 mm | 0.36 mm | 250-280 g | A4 |
2838 | 0.28 mm | 0.38 mm | 280-350 g | A4 |
3040 | 0.30 mm | 0.40 mm | 360-400 g | A4 |
3043 | 0.30 mm | 0.43 mm | >400 g | A4 |
Note: A4 level thread bolt is the standard that the head end is hemispherical, the front end is covered with poly-lysine, marked, sterilized, and buy-on-use without any treatment (This Table has been modified from Liu et al., 2018). |
Table 1: Thread blot information. This table has been modified from Liu et al.15.
Many models establishing methods and behavioral indicators that are well used in acute cerebral ischemia may not have significant changes in the recovery and sequela stages of brain ischemia16,17. However, the number of patients with brain ischemic in the recovery and sequela stages is the greatest. It is essential to select a suitable animal model for the recovery and sequela stages of ischemia stroke.
We use the MCAO/R model in rats to screen the suitable weight of rats (260−330 g), the type of thread bolt (2636/2838/3040/3043), and the time of brain infarct (2-3 h) for the most severe infarct injury, a high model success ratio, and visible behavioral indicators, which will be suitable for the recovery and sequelae stages of brain ischemia.
Rats that weigh 300 g with a 3040 thread bolt and 3 h brain infarct time have larger infarct volumes, more severe behavioral defects, and a greater model success ratio (Figure 1). Furthermore, we provided validation methods of this rat model by TTC staining and sensorimotor function tests (bilateral asymmetry test, grid-walking test, rotarod test and lifting rope test) 1-90 days after reperfusion. We found that the bilateral asymmetry test and grid-walking test could be used to research the recovery and sequela stages of ischemia because the significant differences of these indicators last 90 days and 60 days, respectively. The larger the infarct and shrink volume is, the more severe the sensorimotor deficits, which can be seen in Figure 2 and Figure 3.
This method is mainly suitable for brain ischemia caused by MCAO. However, the model has differences in brain anatomies between humans and rats, such as the grade of collateral circulation. Another limitation is that white matter recovery cannot be seen by TTC staining. Further studies of collateral circulation and white matter recovery with MR imaging or other methods can confirm the predictive value of this model.
The most critical matter is that the skill of creating a MCAO/R model in rats is not easy and requires practice. Before the experiment, confirm an acceptable and parallel model success ratio. More instruments and methods are needed to test the sensorimotor function in the recovery and sequela stages of stroke. If a more difficult task, such as increasing the speed from 10 to 30 rpm was used, a longer period of deficit may appear in the rotarod test. Other behavioral tests may be also suitable for this model, such as gait detection. More precise detection methods should be used for patients in the recovery and sequela stages of brain ischemia, which can identify the effect of drug or other therapeutic tools.
As a new animal model to study brain ischemia in the recovery and sequela stages, the method presented here is meaningful and deserves popularization.
The authors have nothing to disclose.
This work was supported by the National Natural Science Foundation of China (81603315, 81603316), Key R & D plan of Jiangxi province in China (20171ACH80001), Industrial and academic cooperation projects in colleges and universities of Fujian province in China (2018Y41010011).
Anatomical Microscope | Leica (Germany) | S8 | Microscopic operating instrument |
Blade | Gellette | / | Cutting brain sections |
Constant Temperature Shaking Bed | Taicang Experimental Equipment Factory | THZ-C | To keep the brain sections stained evenly and at a constant temperature |
Digital Camera | Canon | 700D | For taking pictures of TTC staining |
Electric Shaver | Shanghai Yuyan Scientific Instruments Co., Ltd. | 3000# | Removal of hair from the neck of rats |
Forceps Hamostatic | Shanghai Medical device Co., Ltd. | 14 cm | Using for brain removing |
Image Pro Plus Software | Media Cybernetics Inc. | 6.0 | Analyze the infarct volume |
Isoflurane | RWD Life Science | 217170702 | Anesthetic gas |
Microforceps | Shanghai Jinzhong Medical Devices Co., Ltd. | 10 cm | Vascular micromanipulation |
Microshear | Shanghai Jinzhong Medical Devices Co., Ltd. | 10 cm | Vascular micromanipulation |
Ophthalmic Forceps | Shanghai Jinzhong Medical Devices Co., Ltd. | 10 cm | Auxiliary skin and muscle anatomy |
Pphthalmic Scissors | Shanghai Jinzhong Medical Devices Co., Ltd. | 10 cm | Using for cutting the skin of neck |
Rat Brain Slice Mold | Shanghai Yuyan Scientific Instruments Co., Ltd. | 400 g | For standard, uniform cutting of brain tissue |
Rat Rotating Bar Fatigue Apparatus | Anhui Zhenghua Biological Instrument and Equipment Co., Ltd. | ZH-300B | To test the sensorimotor function |
Small Animal Anaesthesia Machine | Shanghai Yuyan Scientific Instruments Co., Ltd. | ABM3000 | A gas anesthetic machine |
Small Animal Thermostat | Beijing Damida Technology Co., Ltd. | DM.7-YLS-20A | To maintain animal body temperature constant during operation |
Surgical Scissors | Shanghai Medical device Co., Ltd. | 16 cm | Using for decapitate and brain removing |
Suture | Shanghai Jinhuan Medical Devices Co., Ltd. | 4-0 / 5-0 | Using for skin and muscle sutures / Using for vascular ligations |
Thread Bolt | Beijing Cinontech Co. Ltd. | 2636/2838/3040/3043-A4 | Blockage of the middle cerebral artery in rats |
5-triphenyl-2H-tetrazolium chloride (TTC) | Sigma | LOT#BCBP3272V | Brain section staining reagent |