There is accumulating evidence, that ischemic preconditioning (PC) – a non-damaging ischemic challenge to the brain – confers a transient protection to a subsequent damaging ischemic insult. We established bilateral common carotid artery occlusion (BCCAO) as a preconditioning stimulus to induce early ischemic tolerance (IT) to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.
There is accumulating evidence, that ischemic preconditioning – a non-damaging ischemic challenge to the brain – confers a transient protection to a subsequent damaging ischemic insult. We have established bilateral common carotid artery occlusion as a preconditioning stimulus to induce early ischemic tolerance to transient focal cerebral ischemia in C57Bl6/J mice. In this video, we will demonstrate the methodology used for this study.
Ischemic stroke is a disease with a high mortality and an enormous socio-economic burden 1. Despite intensive experimental and clinical scientific efforts throughout recent decades, treatment options for acute ischemic stroke patients remain very limited 2. In contrast to that, increase in the percentage of elderly people in developed countries will dramatically increase incidences and prevalences of patients with ischemic stroke in the next decades 3. Therefore, there is a pressing need for new treatment strategies in patients with ischemic stroke.
One approach is to gain further understanding in mechanisms of endogenous adaptation of brain to cope with a damaging stimulus. This brain-derived neuroprotection is also known as ischemic preconditioning (PC) or ischemic tolerance (IT) and describes a phenomenon, in which a non-damaging noxious stimulus applied to the brain, induces a transient resistance against a subsequent damaging ischemic insult 4. IT occurs in two different time windows: early IT, which occurs within minutes to a few hours after PC and delayed IT, which needs a latency of a couple of hours to occur 4.
So far, research on IT in brain has focused on delayed IT. Much less is known on the mechanisms of early IT. The objective of this study is to establish bilateral common carotid artery occlusion (BCCAO) which is an established stimulus to induce delayed IT, as an adequate PC stimulus to induce early IT to transient focal cerebral ischemia (induced by middle cerebral artery occlusion, MCAO) in C57Bl6/J mice.
During both surgical procedures (BCCAO and MCAO), monitoring of cerebral blood flow (CBF) by laser Doppler flowmetry (LDF) was performed.
BCCAO was performed in anesthetized and spontaneously breathing mice. Both common carotid arteries (CCA) were exposed and occluded for 60 sec followed by 5 min of reperfusion. This BCCAO/reperfusion sequence was repeated twice. After surgery, mice recovered quickly and showed no signs of functional impairment. In order to rule out, that the BCCAO protocol described above did not result in delayed cell death in the brain, we performed a TdT-mediated dUTP-biotin nick end labelling (TUNEL) staining 72 hr after BCCAO (or sham surgery) in a separate group.
MCAO is a widely established model for induction of focal cerebral ischemia in rodents 5-7. The surgical procedures were carried out using standardized operating procedures (SOP) 8. After exposing left CCA, a silicon-covered monofilament was introduced into the distal CCA, advanced via the internal carotid artery (ICA) into the Circle of Willis until the anterior cerebral artery (ACA) and thereby, the origin of the middle cerebral artery (MCA) was occluded for a defined period of time. In this study, we used 45 min of MCAO, which typically results in an ischemic lesion in the MCA territory involving striatal and cortical brain areas.
In our study, we analyzed the temporal profile of early IT using BCCAO as the PC stimulus. Our data indicated, that the optimal time delay between BCCAO and MCAO to induce early IT is 30 min.
In this video, we will give a demonstration of both surgical procedures, i.e. BCCAO and MCAO.
All procedures described in this article were performed in accordance with the guidelines and regulations of the Landesamt für Gesundheit und Soziales, Berlin, Germany.
Pre-surgical procedures
1. Preparation of Monofilaments
Comment: Ideally, only sterilized or at least disinfected monofilaments should be used to ensure sterility at the surgical site. In practice, sterilization or disinfection of the hand-made monofilaments is very difficult because the quality of the monofilaments may worsen 9. Therefore, it might be preferable to use commercially available sterile monofilaments instead.
2. Preparation of Surgical Instruments and of the Animals
Surgical procedures
3. Positioning of the LDF Probe and LDF Monitoring
Comment: While turning the mouse around, the fiberoptic probe should be bent with care in order to avoid that it breaks off from the skull.
4. Bilateral Common Carotid Artery Occlusion
5. Middle Cerebral Artery Occlusion
Comment: There is another artery to be identified which turns to the right in caudal direction. This is the occipital artery, which variably originates either from the CCA bifurcation or from the proximal part of the ECA (Figure 4).
6. BCCAO Sham Surgery
For BCCAO sham procedures, mice were anaesthetized for the duration of a real BCCAO PC procedure, the salivation glands were mobilized and the CCA’s were exposed. It is crucial that direct manipulations to the carotid arteries or vagal nerves are avoided during sham procedure. Manipulations of the CCA can damage its endothelial layer, which can result in a release of vasoactive compounds from endothelium or induce local thrombosis within the arterial lumen. As a consequence, thromboembolic events might occur after sham surgery. This would be a major confounder of the procedure. On the other hand, manipulations of the vagal nerves might lead to transient or permanent dysfunction of the parasympathetic nerve system, which has the potential for the occurrence of significant cardiac arrhythmia or even irreversible cardiac arrest. Therefore, it is crucial to avoid any manipulations of the vagal nerves during BCCAO.
Post-surgical procedures
7. Infarct Volume Measurements
For assessment of infarct sizes, mice survived for 72 hr after MCAO. Afterwards animals were deeply anesthetized, brains were removed, snap-frozen in methylbutan, sectioned and stained with hematoxylin (using Papanicolau’s protocol) for infarct volume measurements. Brain sections were collected at 600 μm-intervals and infarct volumes were determined using an image analyzer (MCID core 7.0 Rev.2.0, GE Healthcare Niagara Inc) and corrected for edema according to the method of Lin et al. 7,11.
8. Inclusion Criteria
We included only animals which had a LDF-measured CBF reduction by more than 90% during each of the three BCCAO episodes. Ideally, LDF monitoring should be performed in both cerebral hemispheres during BCCAO. For practicability, we performed LDF monitoring only in the left hemispheres, which will be subjected to MCAO later on. This approach has been described before 6,12. Reperfusion after each BCCAO episode was required to be complete (i.e. return to baseline values). For reasons of reducing time duration mice were kept under anesthesia, we did not monitor CBF during MCAO surgery. In a pilot study with a separate group of animals however, we monitored CBF by LDF during MCAO surgery. In this group of mice, we consistently measured a reduction in CBF by 90% compared to baseline before CCA occlusion. Additionally, after removal of monofilaments CBF recovered to >90% of the respective baseline values after CCA occlusion. These LDF criteria are established in the literature and result in consistent infarct volumes 7. We only included animals which had a Bederson score of at least of 1 after recovery from anesthesia after MCAO. Furthermore, only animals which survived for 72 hr after MCAO and which showed no signs of intracranial hemorrhage were included in the study.
Our BCCAO protocol resulted in immediate and profound (>90%) CBF reductions when both CCA’s were occluded (Figure 1). Reopening of the CCA’s led to complete reperfusion as monitored by LDF (Figure 1). After PC, animals showed no signs of functional disability. Furthermore, there was no mortality. TUNEL staining performed 72 hr after BCCAO in a separate group of animals showed no signs of delayed cell death in the brain after BCCAO (data not shown).
When analyzing the temporal profile of the optimal time delay between BCCAO and MCAO for early IT (Figure 2), we found a significant reduction in the infarct volumes (reduction by 65%, n=10) compared to Sham group (n=10) when BCCAO was performed 30 min before MCAO. Furthermore, infarct volumes were reduced when BCCAO was performed 2 hr and 24 hr before MCAO (n=10). 24 hr represents the second (delayed) time window of IT 6.
Thus, it can be concluded that the optimal time window for early IT using BCCAO as a PC stimulus is 30 min before induction of transient focal cerebral ischemia (MCAO).
Figure 1. Course of mean CBF values measured by LDF during BCCAO and reperfusion (n=10).
Figure 2. Temporal profile of infarct volumes at different time delays of MCAO after BCCAO n=10/group, graphs display mean ± standard deviation, * p< 0,05 from sham, ** p< 0,01 from sham (One-way ANOVA with Bonferroni’s posthoc test).
Figure 3. Representative hematoxylin-stained brain sections derived from a BCCAO-preconditioned (A) and a sham-operated animal (B), which were subjected to 45 min MCAO 30 min after BCCAO/sham. Brains were removed and processed 72 hr after MCAO.
Figure 4. Scheme of the left carotid artery’s vascular anatomy including the procedures performed for BCCAO and MCAO.
In our study, we have shown that three sequences of BCCAO, each lasting for one minute and followed by reperfusion for five minutes, are an adequate ischemic PC stimulus to induce early IT.
Furthermore, we have demonstrated that the preconditioning protocol used is a safe and minor invasive procedure with no noteworthy postoperative morbidity, no signs of apoptosis in brain and no mortality. Several different preconditioning protocols have been described previously 6-7,12. Others have used 6 min of BCCAO as a preconditioning stimulus 13-14. However, due to the fact that one minute of BCCAO for three times resulted in a protective effect in the early time window of IT without any detectable ischemic brain injury, we decided to use this protocol.
For consistent results, duration of anesthesia and surgical procedures as well as other variables such as animal weights, brain temperatures during surgery, postsurgical fluid replenishment, postsurgical pain management as well as pre- and postsurgical cage enrichment should be standardized 15. Particular attention requires maintenance of normothermic conditions of the mice throughout the complete period of anesthesia because hypothermia itself has a preconditioning effect and can therefore interfere with the BCCAO effect 16. In awake and freely moving rodents, core temperatures are approximately between 0.5 and 1 degree Celsius higher than the corresponding brain temperatures 17. Resting brain temperatures in awake and freely moving rodents are approximately 36.5 degree Celsius 18. Since we did not measure brain temperatures in our experiments, we adjusted mice’ core temperatures to 37.0 ± 0.5 degree Celsius to achieve brain temperatures which resemble the above mentioned resting brain temperatures.
Theoretically, use of volatile anesthetics itself can be an additional confounder because it has been demonstrated that isoflurane itself can induce IT. However, isoflurane anesthesia becomes a relevant PC stimulus only when administered repetitively for several hours which was not the case in the procedures described in the current study 19-21. Furthermore, we also analyzed Sham-operated animals to control for the potential confounding effect of isoflurane anesthesia.
The size and shape of the monofilament used for induction of MCAO plays a critical role. Evenly silicone covered filaments improve the success rate of MCAO and decrease the rate of complications such as subarachnoid hemorrhages and should therefore be preferred compared to heat blunted filaments 22. Another crucial point is the filament length. Optimal filament lengths between 9-11 mm are reported 15. Due to the fact that our incisions were made proximal to the CCA bifurcation, we used a monofilament length of 13 mm and a thickness of approximately 200 μm.
Taken together, the type and quality of monofilament’s coating and its length are crucial to ensure a sufficient occlusion of the MCA and to avoid significant crossflow via anterior communicating artery. Substantial crossflow from the right side of the Circle of Willis results in an unwanted reduction of the infarct volume and thereby increases the variability of the results 23. Additional procedural standardizations with the intention to further reduce outcome variability include LDF-based CBF monitoring during MCAO and reperfusion and the use of commercially available industrial made, standardized monofilaments. By the use of commercially available monofilaments also their sterility can be ensured.
In addition, animals which do not have a >90% CBF reduction during BCCAO or MCAO, or which do not have a >90% reperfusion after re-opening of the arteries should be excluded from the study. This approach will help to further reduce variability in infarct volumes, which mainly occurs as a result of common variants in cerebral collateral circulation.
In summary, BCCAO is an established and easily applicable model to investigate effects of IT in early and delayed time windows. Studying the phenomenon of IT helps to get a better understanding of the mechanisms underlying endogenous protection in the brain. In perspective, this may help to develop alternative treatment opportunities for patients with acute ischemic stroke.
The authors have nothing to disclose.
Name of the instrument | Company | Catalogue number/model |
Binocular surgical microscope | Zeiss | Stemi 2000 C |
Light source for microscope | Zeiss | SteREO CL 1500 ECO |
Heating pad with rectal probe | FST | 21061-10 |
Stereotactic frame | David Kopf | Model 930 |
Scissors | FST | 91460-11 |
Dumont forceps #5 | FST | 11251-10 |
Dumont forceps #7 | FST | 11271-30 |
Mircovascular clamp | FST | 00398-02 |
Clamp applicator | FST | 00072-14 |
Springscissors | FST | 15372-62 |
Needle holder | FST | 12010-14 |
Needles | Feuerstein, Suprama | BER 562-20 |
5-0 silk suture | Feuerstein, Suprama | |
7-0 silk suture | Feuerstein,Suprama | |
8-0 silk suture | Feuerstein, Suprama |