The protocol presented here shows a technique to create a rodent model of brain injury. The method described here uses laser irradiation and targets motor cortex.
A common technique for inducing stroke in experimental rodent models involves the transient (often denoted as MCAO-t) or permanent (designated as MCAO-p) occlusion of the middle cerebral artery (MCA) using a catheter. This generally accepted technique, however, has some limitations, thereby limiting its extensive use. Stroke induction by this method is often characterized by high variability in the localization and size of the ischemic area, periodical occurrences of hemorrhage, and high death rates. Also, the successful completion of any of the transient or permanent procedures requires expertise and often lasts for about 30 minutes. In this protocol, a laser irradiation technique is presented that can serve as an alternative method for inducing and studying brain injury in rodent models.
When compared to rats in the control and MCAO groups, the brain injury by laser induction showed reduced variability in body temperature, infarct volume, brain edema, intracranial hemorrhage, and mortality. Furthermore, the use of a laser-induced injury caused damage to the brain tissues only in the motor cortex unlike in the MCAO experiments where destruction of both the motor cortex and striatal tissues is observed.
Findings from this investigation suggest that laser irradiation could serve as an alternative and effective technique for inducing brain injury in the motor cortex. The method also shortens the time for completing the procedure and does not require expert handlers.
Globally, stroke is the second leading cause of death and the third leading cause of disability1. Stroke also leads to severe disability, often requiring extra care from medical staff and relatives. There is, therefore, a need to understand the complications associated with the disorder and improve the potential for more positive outcomes.
The use of animal models is the initial step to understanding diseases. To ensure the best research outcomes, a typical model would include a simple technique, affordability, high reproducibility, and minimal variability. The determinants in ischemic stroke models include brain edema volume, infarct size, the extent of the blood-brain barrier (BBB) breakdown, and functional impairment generally evaluated via neurological severity score2.
The most widely used stroke induction technique in rodent models occludes the middle cerebral artery (MCA) transiently or permanently3. This technique produces a stroke model similar to the ones in humans: it has a penumbra surrounding the stroked area, is highly reproducible, and regulates ischemia duration and reperfusion4. Nevertheless, the MCAO method has some complications. The technique is prone to intracranial hemorrhage and injury to the ipsilateral retina with a dysfunction of the visual cortex and common hyperthermia that often lead to additional outcomes5,6,7. Other limitations include high variations in induced stroke (arising from the probable extension of the ischemia to unintended regions, like the external carotid artery region), insufficient occlusion of the MCA, and premature reperfusion. Also, rats of different strains and sizes exhibit various infarct volumes8. In addition to all the disadvantages mentioned, MCAO model cannot induce small isolated strokes in deep brain areas, because it is limited technically in terms of its requirement of minimum vessel size for catheterization. This makes the need for an alternative model all the more critical. Another method, photothrombosis, provides a possible alternative to MCAO procedures but does not improve on the efficiency9. This technique targets stroke with light and offers some improvements on the previous models. However, photothrombosis requires an invasive craniotomy that is associated with secondary compications9.
In the light of outlined shortcomings, the protocol presented here provides a capable alternative laser technique for inducing brain injury in rodents. The mechanism of action of the laser technique is based on the laser’s photothermal effects imparted on living tissues, which leads to the absorption of light beams by body tissues and their conversion into heat. The advantages of using a laser technique are its safety and ease of manipulation. A laser’s ability to produce heat to stop bleeding makes it very important in medicine, while its ability to amplify different beams at a given meet point ensures that lasers avoid destroying healthy tissues that stands in the way of the target point10. The laser beam used in this protocol can pass through a low liquid medium, such as bone, without emitting its energy and/or causing any destruction. Once it reaches a high liquid medium, such as brain tissues, it uses up its energy to destroy the target tissues. The technique, therefore, can induce brain injury only in the appropriate area of the brain.
The technique presented here showed a tremendous amount of ability to regulate its levels of irradiation, producing the chosen variations of brain injury intended from the start. Unlike the original MCAO that impacts both the cortex and striatum, the laser technique was able to regulate the impact of brain injury, inducing injury only on the intended motor cortex. Herein, the laser-induced brain injury protocol and a summary of representative results for the procedure performed on the cerebral cortex of rats are provided.
The following procedure was conducted according to the Guidelines of the Use of Experimental Animals of the European Community. The experiments were also approved by the Animal Care Committee at the Ben-Gurion University of the Negev.
1. Animal selection and preparation
2. MCAO procedure
3. Laser-induced brain injury experimental procedure
4. Neurological severity score (NSS)
5. Post-injury manipulations
6. Evaluation of the brain injury
No deaths or SAH were registered in either the control or experimental groups (Table 1). The MCAO group had a 20% rate of both mortality and SAH.
The relative body temperature changes in the rats of both groups were also similar, despite a difference in the variability of both groups (Table 1).
There was a significantly worse NSS in both the laser (16 ± 1.1) and MCAO (20 ± 1.5) models, compared to the sham-operated control group (1 ± 0.3; Table 1; p<0.01).
The laser-induced brain injury also caused a significant increase in infarct volume at the target hemisphere, compared to the sham-operated control group (2.4% ± 0.3 vs 0.5% ± 0.1; Table 2 and Figure 1A; p<0.01), per the Mann-Whitney U test. However, the infarct volume of the laser model was smaller in comparison to the MCAO technique (2.4% ± 0.3 vs 9.9% ± 2.9).
Brain edema determined 24 h after brain injury are shown in Figure 1B and Table 2. There was no difference in brain edema between the laser-induced brain injury model and the sham-operated control group (3.4% ± 0.6 vs 0.7% ± 1.2). There was a significant difference in brain edema between the laser model and the MCAO technique (3.4 ± 0.6 vs 7 ± 2.6†). Data are presented as mean ± SEM.
Compared to the sham-operated control group, the laser-induced brain injury and MCAO technique both caused a significant increase in BBB breakage at the non-injured hemisphere (563 ng/g ± 66 and 1176 ng/g ± 168, respectively, vs 141 ng/g ± 14; Figure 2A and Table 2; p<0.01) and target hemisphere (2204 ng/g ± 280 and 2764 ng/g ± 256, respectively, vs 134 ng/g ± 11; Figure 2B and Table 2; p<0.01).
Histological examination of rats’ brains are shown in Figure 3.
NSS | Temperature, °C | SAH, % | Mortality, % | |||
Groups | mean ± SEM | variability, % | mean ± SEM | variability, % | ||
Sham-operated control | 1 ± 0.3 | 97 | 37.2 ± 0.1 | 59 | 0 | 0 |
Laser 50J x10 | 16 ± 1.1* | 30 | 37.4 ± 0.1 | 84 | 0 | 0 |
p-MCAO | 20 ± 1.5* | 37 | 38.3 ± 0.1* | 129 | 20* | 20* |
Table 1: Assessment of NSS, body temperature, subarachnoid hemorrhage, and mortality. * = p < 0.01
BBB | Infarcted Volume | Brain Edema | ||||
Groups | mean ± SEM | variability, % | mean ± SEM | variability, % | mean ± SEM | variability, % |
Sham-operated control | 134 ± 11 | 25 | 0.5 ± 0.1 | 77 | 0.7 ± 1.2 | 573 |
Laser 50J x10 | 2204 ± 280* | 40 | 2.4 ± 0.3* | 34 | 3.4 ± 0.6 | 58 |
p-MCAO | 2764 ± 256* | 29 | 9.9 ± 2.9* | 92 | 7 ± 2.6* | 115 |
Table 2: Assessment of BBB breakdown, infarct zone, and brain edema. * = p < 0.01
Figure 1: Assessment of brain injury in the laser model 24 h after the injury compared to the MCAO model and sham-operated control. (A) Assessment of infarct volume. There was an increase in infarct volume in the laser model compared to the sham-operated control (*p<0.01). However, the infarct volume in the laser model was smaller compared to the MCAO model (*p<0.01). (B) Assessment of total brain edema. There was an increase in brain edema in the MCAO model compared to either the laser model or sham-operated control. There was no difference in brain edema between the laser model and sham-operated control. The data are measured as % to the contralateral hemisphere and expressed as mean ± SEM. Please click here to view a larger version of this figure.
Figure 2: The extent of BBB breakdown compared to sham controls. (A) Contralateral (non-injured) hemisphere. Both the laser and MCAO models, led to a significant increase in BBB breakage at the non-injured hemisphere compared to the sham-operated control group (*p<0.01). (B) Ipsilateral (injured) hemisphere. There was a difference in ipsilateral BBB breakdown in the laser and MCAO models compared to the sham-operated control (*p<0.01). Please click here to view a larger version of this figure.
Figure 3: Histological examination of rats’ brains from sham, laser and MCAO groups. Please click here to view a larger version of this figure.
It is fair to assume that the laser technique is minimally invasive, given that no deaths or SAH occurred in the laser group. The primary cause of death and SAH is the damage to blood vessels that leads to an elevation of intracranial pressure (ICP), as shown in the original MCAO techniques10. The absence of death and SAH in the laser group is likely due to the specific effects of lasers: they do not have direct impact on blood vessels and can induce coagulation in case of leakage. Low infarct volume and brain edema also help minimize the risk of death. The use of lasers should be considered as a suitable technique for inducing brain injury with minimal adverse outcomes, given that the original MCAO techniques for triggering stroke (both transient and permanent) have been shown to produce deaths and SAH6.
Low body temperature findings in the laser group show that the laser technique does not occlude the hypothalamic artery that regulates body temperature, as the original MCAO does typically7, supporting the theory that the laser technique is more targeted. Low variability across the board of parameters investigated indicated consistency in the use of lasers to induce brain injury, but such fine results depend very much on the choice of power. Sufficient power provides desired outcomes, while little or surplus calibrations can cause under- or over-performance, which in either case is detrimental. Nevertheless, the ability to aim for the target still makes the technique less risky. Hence, correct handling makes it easier to obtain results with precision using the laser technique, as well as to regulate the method for desirable effects.
The precision and efficacy of the laser technique were evident in its ability to strike only the motor cortex without causing damage to the striatum, suggesting that the laser technique can produce localized injury that is almost impossible to achieve with MCAO10. This achievable outcome with the laser technique is due to the ability to regulate the laser beam and its power and makes the laser method a model technique for inducing smaller, peripheral, and deep and defined brain injury that cannot be obtained with MCAO. The simplicity of manipulating a laser machine makes it very desirable. Unlike MCAO techniques that demand arduous training and experts, using lasers is more simple, requiring no experts or expensive training. The use of the laser technique could boost research and help to uncover better outcomes more quickly than the MCAO method alone.
In terms of limitations of the laser technique, the use of laser beams does not produce brain injuries that are perfectly similar to acute vascular occlusive strokes. Specifically, lasers produce immediate tissue scars at the target site that are comparable to a vascular occlusive stroke that is several days old. The technique might, therefore, not be suitable for evaluating drugs that aim to prevent the spread of stroke but should be ideal in assessing isolated motor cortex stroke on prolonged motor, cognitive, and behavioral impairments. The use of a small number of rats for this research was also a limitation, with only half the number of rats (n = 10) in each group used for brain harvesting and examination of the size of the stroke, the extent of brain edema, BBB breakage, and SAH presence.
The lack of comparisons between our technique and other laser methods may also be deemed a limitation. We deliberated on performing comparative methods but decided not to do so because assessing the damage caused by these other laser methods is difficult. For example, the photothrombosis technique6 causes weak damage that makes it challenging to evaluate brain swelling and other conditions that may occur. Also, the use of craniotomy in the laser technique for ischemia is problematic because craniotomy is very invasive and can increase BBB’s permeability, causing additional brain injury that is not associated with stroke. Assessing such damage for comparison with our method is nearly impossible. The laser model induces stroke with radiation through the skull without craniotomy.
Like many models, the laser model has its benefits and limitations, with the most glaring drawback is its inability to mimic perfectly human stroke as precisely as other models. Nevertheless, the low variability in primary outcomes of most parameters, its precision, affordability, ability to induce smaller brain injuries, and its straightforward application makes it a suitable alternative technique for brain injury in rodents.
The authors have nothing to disclose.
We would like to thank the Department of Anesthesiology of Soroka University Medical Center and the laboratory staff of Ben-Gurion University of the Negev for their help in the performance of this experiment.
2,3,5-Triphenyltetrazolium chloride | SIGMA – ALDRICH | 298-96-4 | |
50% trichloroacetic acid | SIGMA – ALDRICH | 76-03-9 | |
Brain & Tissue Matrices | SIGMA – ALDRICH | 15013 | |
Cannula Venflon 22 G | KD-FIX | 1.83604E+11 | |
Centrifuge Sigma 2-16P | SIGMA – ALDRICH | Sigma 2-16P | |
Compact Analytical Balances | SIGMA – ALDRICH | HR-AZ/HR-A | |
Digital Weighing Scale | SIGMA – ALDRICH | Rs 4,000 | |
Dissecting scissors | SIGMA – ALDRICH | Z265969 | |
Eppendorf pipette | SIGMA – ALDRICH | Z683884 | |
Eppendorf Tube | SIGMA – ALDRICH | EP0030119460 | |
Ethanol 96 % | ROMICAL | Flammable Liquid | |
Evans Blue 2% | SIGMA – ALDRICH | 314-13-6 | |
Fluorescence detector | Tecan, Männedorf Switzerland | model Infinite 200 PRO multimode reader | |
Heater with thermometer | Heatingpad-1 | Model: HEATINGPAD-1/2 | |
Infusion Cuff | ABN | IC-500 | |
Isofluran, USP 100% | Piramamal Critical Care, Inc | NDC 66794-017 | |
Multiset | TEVA MEDICAL | 998702 | |
Olympus BX 40 microscope | Olympus | ||
Optical scanner | Canon | Cano Scan 4200F | |
Petri dishes | SIGMA – ALDRICH | P5606 | |
Scalpel blades 11 | SIGMA – ALDRICH | S2771 | |
Sharplan 3000 Nd:YAG (neodymium-doped yttrium aluminum garnet) laser machine | Laser Industries Ltd | ||
Stereotaxic head holder | KOPF | 900LS | |
Sterile Syringe 2 ml | Braun | 4606027V | |
Syringe-needle 27 G | Braun | 305620 |