Background and Purpose: The lack of reliable rodent model for repeated ischemia-reperfusion (I-R) hampers experimental research on stroke. Therefore, the objective of the present study was to develop a mouse model for repeated I-R cycles in a single animal on different days.
Methods and Results: The right common carotid artery (CCA) was ligated and both vertebral arteries were coagulated. A customized vascular occluder, with its actuating tubing glued to a microport, was cuffed around the isolated left CCA and secured. Inflating of the occluder diaphragm via the microport restricted the blood flow via the left CCA and reduced the cerebral blood flow (CBF) by 75% in both hemispheres, while deflating allowed for the CBF restoration. Two minutes of forebrain ischemia followed by a 24 h reperfusion period was tolerated by animals for 5 cycles. Importantly, repeated 2 min I-R cycles attenuated the infarct volume induced by occlusion of the middle cerebral artery.
Conclusions: The described model is a reliable method to induce transient I-R events in the forebrain. The model mimics transient ischemic attacks and allows for controlling the ischemic durations, intervals, and numbers of I-R cycles.
Multiple animal models have been developed to investigate possible therapies for stroke (Yamashita et al., 2009; Wang et al., 2010; Hoyte et al., 2006; Hossmann, 1998; Zhang et al., 2008; Zhao, 2009; Hossmann, 2008; Taguchi et al., 2010). Nevertheless, only limited stroke models have been established that allow repeated I-R cycles in a single animal on different days (Hossmann, 2008). Among different animal models of human diseases, rodent models are especially valuable due to their relatively low cost and wide spectrum of transgenic strains (Hossmann, 1998; Hossmann, 2008; Zhang et al., 2008; Zhao, 2009; Del Zoppo et al., 1986; Pulsinelli et al., 1988; Stenzel-Poore et al., 2007; Durukan et al., 2008). In the present study, we describe a novel experimental model of I-R using a customized vascular occluder. The main advantages of this model are the ability to repeat the I-R cycles multiple times and to fully control the ischemic duration and frequency.
In principle, our method is based on a four vessel occlusion model. However, in our model only three vessels, namely both vertebral arteries and the contralateral CCA, are coagulated or ligated. CBF is maintained through the ipsilateral CCA, which is under reliable control of the installed occluder. Indeed, inflating and deflating the occluder produces a dependable model for repeated transient forebrain I-R cycles.
To our knowledge, the procedure described in the present study is the first rodent model that allows repeated I-R cycles over an extensive period of time. Importantly, the model requires only a single surgery; then, I-R cycles are induced by inflating and deflating the occluder through an easily accessible microport. While we developed this model in mice, it is also applicable for rats.
For humane and experimental quality control reasons, we established the following stringent criteria for a successful surgery:
- Ligation of the contralateral CCA should result in a less than 30% decrease in CBF, indicating an intact Willis circle.
- Installation of the occluder around the CCA should not reduce CBF. On the other hand, inflation of the occluder diaphragm should decrease the blood flow over 75%, producing forebrain ischemia.
- CBF should restore to at least 80% of the initial values 20 min after diaphragm deflation.
- No stroke symptoms or other complications should occur during the I-R cycles. Thus, any animals exhibiting eyelid ptosis, hemiplegia, rotating, circling, abnormal postures, and/or loss of body weight over 30% post surgery should be excluded from the study.
The described model can be employed in a variety of stroke and brain ischemia-related experiments, providing opportunities to explore novel mechanisms involved in stroke development and/or therapeutic interventions. Examples of such studies include evaluation of the effects of preconditioning, postconditioning, or transient ischemic attack on stroke development. It has been widely accepted that preconditioning and postconditioning treatments can attenuate damage from more severe ischemic insults (Corbett & Crooks, 1997; Hoyte et al., 2006; Lee et al., 2008; Li et al., 2005; Ravati et al., 2000; Dirnagl et al., 2009; Zhang et al., 2008; Zhao, 2009; Stenzel-Poore et al., 2007; Pignataro et al., 2009; Degracia, 2010). Nevertheless, the mechanisms underlying these effects are still elusive. Some of the important but unanswered problems include a) the appropriate ischemic duration, b) the number and c) the frequency of I-R cycles that allow the brain to achieve optimal protection while minimizing their adverse effects (Li et al., 2005; Tanay et al., 2006; Zhan et al., 2008; Wegener et al., 2004; Stenzel-Poore et al., 2007; Stenzel-Poore et al., 2003). These problems can be easily addressed using our novel experimental model. Despite losing ~18% of mice due to complications of transient forebrain ischemia, we determined that five 2 min I-R cycles provided excellent protection against permanent ischemia. On the other hand, 10 min I-R intervals resulted in a high mortality of mice, which reached 50% after 5 cycles.
The observed mouse responses to the repeated I-R cycles in many ways resembled those in patients with transient ischemic attacks. The similarities include transient brain ischemic incidents and recovery without or only with mild neuronal and behavioral deficit (Lloyd-Jones et al., 2010; Wegener et al., 2004; Ratan et al., 2004). Approximately 10–25% patients with transient ischemic attacks develop stroke in 90 days after their first transient ischemic occurrence (Wegener et al., 2004), which also is mimicked in our model. Thus, our novel animal model may be a valuable tool to study the brain responses and pathological processes involved in repeated I-R.
In summary: we report a novel experimental model for repeated I-R cycles resulting in forebrain ischemia. The model is based on coagulation of vertebral arteries and ligation of the contralateral CCA, while controlling the flow through ipsilateral CCA via a customized occluder and microport set. The main advantage of this model is the ability to control ischemic duration and frequency of intervals. The model can be used in studies on pre- or postconditioning and in research on transient ischemic attacks.