The infarct-limiting effect of cerebral ischaemic postconditioning in rats depends on the middle cerebral artery branching pattern

Abstract

Cerebral ischaemic postconditioning (PostCon) is a recently discovered endogenous neuroprotective phenomenon that occurs after several brief bouts of reperfusion/ischaemia instituted immediately after prolonged cerebral ischaemia. Data on the extent of PostCon-mediated infarct size limitation in models of focal cerebral ischaemia–reperfusion are controversial. In this study, we investigated the infarct-limiting effect of PostCon in the rat model of focal cerebral ischaemia–reperfusion. The relationship between anatomic pattern of the middle cerebral artery (MCA) and infarct size was also studied. The protocol of PostCon consisting of five episodes each of 10-s ischaemia and 10-s reperfusion was protective in terms of infarct size limitation only in animals with the typical bifurcating MCA branching pattern. The anatomic pattern of the MCA should be considered as one of the important factors influencing the outcome of neuroprotection studies.

Stroke continues to be a leading cause of mortality and disability in industrialized countries. In the United States, stroke accounts for 16.7% of deaths due to cardiovascular disease (Roger et al. 2011). Therefore, prevention of cerebral ischaemic injury remains among the most important goals of medical care. With the advent of reperfusion strategies such as thrombolysis, both survival rates and prognosis have improved in stroke patients. Along with the obvious favourable effects, reperfusion therapy has brought to light the problem of irreversible reperfusion injury, including neuronal necrosis and apoptosis (for a review, see Reza Noorian et al. 2011 and references therein). Although the underlying mechanisms have been extensively studied, reliable clinical treatment strategies are still lacking. On the other hand, a promising approach may be derived from animal models, as is suggested by the abundant data on cerebral preconditioning and postconditioning (PreCon and PostCon, respectively), procedures aimed at injury reduction. Cerebral PreCon describes a phenomenon of increased brain tolerance to prolonged ischaemia after several brief episodes of ischaemia–reperfusion (Kitagawa et al. 1990). Recently, another mode of brain protection against ischaemia–reperfusion injury, termed ‘PostCon’, has been developed (Zhao et al. 2006). PostCon is a repetitive mechanical interruption of blood flow that is applied immediately after the onset of cerebral reperfusion. PostCon has a major advantage over PreCon with regard to potential clinical applications because PostCon, either in the form of direct mechanical manoeuvre or as a pharmacological intervention applied in lieu of stuttering reperfusion, might be used for cerebral protection in patients undergoing cerebral reperfusion via thrombolysis.

In rat models of focal cerebral ischaemia, the protective effect of PostCon has been shown to depend on both the PostCon protocol and the duration of test ischaemia (Zhao et al. 2006). One should be aware, however, that the effectiveness of PostCon in rat models of middle cerebral artery (MCA) ligation through craniectomy might also be influenced by endogenous factors such as gender, age, presence of other disease states and interindividual variability in the anatomic characteristics of the arteries that supply blood to the brain. Previous studies in rats clearly indicate that the MCA anatomic pattern can vary significantly between individual animals, with up to 6 distinct types of branching patterns identified (Rubino & Young 1988; Fox et al. 1993). Also, the distinct MCA patterns can differently affect both perfusion deficit area and infarct size in models of surgical MCA ligation (Rubino & Young 1988). In the present study, the effect of ischaemic PostCon on cerebral infarct size was analysed in Wistar rats having different MCA branching patterns.

Materials and methods

Animals

All experiments were performed on male Wistar rats weighting 250–260 g. The animals were maintained on a 12-h light/dark cycle and were provided food and water ad libitum. The animals were anesthetized with chloral hydrate administered intraperitoneally at a dose of 450 mg/kg. Core body temperature was maintained at 37.0 ± 0.5 °C by a feedback-controlled heating pad (TCAT-2LV controller; Physitemp Instruments Inc., Clifton, NJ, USA).

Ethical Approval

The procedures were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institute of Health (USA), and were approved by the ethics committees at V.A. Almazov Federal Heart, Blood and Endocrinology Center and I.P. Pavlov Federal Medical University, St. Petersburg, Russian Federation.

Surgical procedures

Focal cerebral ischaemia was induced by permanent left MCA (LMCA) occlusion combined with 30-min left common carotid artery (LCCA) occlusion (Fau et al. 2007). To secure access to the left MCA (LMCA), an inverse V-shaped scalp incision was made between the left ear and left eye, with the base of the skin flap located at the level of the left upper eyelid. After partial excision of the temporalis muscle, three burr holes of 1.5 mm in diameter were drilled through the squamosal bone at the upper caudal angle of the surgical field. Craniectomy was performed between the holes, followed by incision of the dura mater for visual inspection of the LMCA. A somewhat simplified classification of MCA branching was applied that included only three primary and most apparent patterns of MCA anatomy: bifurcating (bMCA, two relatively thick branches), multiple-branching (mbMCA, MCA immediately giving rise to multiple thin branches) and combined (cMCA, two thick branches that further divide into several thin ones) (Figure 1). Under microscopic control, all visible branches of the distal MCA appearing in the cranial window were carefully inspected and electrocoagulated (Fotec E-80, Russian Federation) above the rhinal fissure. The LCCA was accessed through a ventral midline cervical incision with blunt dissection of the sternohyoid and sternomastoid muscles. The LCCA was then occluded for 30 min with a microvascular clip. The wounds were closed, and the animals were allowed to recover from anaesthesia.

Figure 1.

Schematic depiction of the major middle cerebral artery branching patterns. (a) bifurcating MCA pattern; (b) multiple-branching MCA pattern; (c) combined MCA pattern. (1) Middle cerebral artery; (2) site(s) of MCA electrocoagulation; (3) borders of the craniectomy window.

Experimental protocol

Animals with three distinct types of MCA branching were allocated into the following groups:

• Controls-bMCA (n = 10): Electrocoagulation of LMCA plus 30-min LCCA occlusion in animals with a bifurcating MCA pattern;

• PostCon-bMCA (n = 8): See previous group; immediately after 30-min LCCA occlusion, five cycles of LCCA reperfusion/re-occlusion (10/10 s each) were performed;

• Controls-mbMCA (n = 7): Focal cerebral ischaemia was induced in animals with a multiple-branching MCA pattern in the same manner as in the Controls-bMCA group;

• PostCon-mbMCA (n = 6): See previous group; PostCon protocol was similar to that in the PostCon-bMCA group;

• Controls-cMCA (n = 7): Focal cerebral ischaemia was induced in animals with a combined MCA pattern in the same manner as in the Controls-bMCA group;

• PostCon-cMCA (n = 6): See previous group; PostCon protocol was similar to that in the PostCon-bMCA group.

The animals were sacrificed 48 h later for histochemical determination of infarct size.

Infarct size measurement

The brain was cut into five frontal slices, 3 mm thick. Infarct size was determined after incubation of the brain slices in 2.0% 2,3,5-triphenyltetrazolium chloride (MP Biomedicals, Solon, OH, USA) at 37 °C (pH 7.4) for 15 min followed by fixation in 10% buffered formaldehyde solution for 24 h (Chen et al. 1986). The slices were photographed and digitized using ImageJ software. The following volumes were calculated for each animal: volume of the non-infarcted cortex of ipsilateral hemisphere (V_N) and total volume of the cortex of contralateral hemisphere (V_C). Infarct size (%V_I) was determined following correction for oedema according to the method of Swanson et al. (1990), using the following formula:%V_I = 100 × (V_C – V_N)/V_C. The infarct size was determined by an investigator blinded to the group assignments.

Statistical analysis

All data are presented as mean standard deviation. Statistical differences in infarct size were evaluated with statistica 6.0 software (exact Fisher's exact test and nonparametric Mann–Whitney U test). The differences were considered significant at P < 0.05.

Results

Mortality

At the end of the study, results from 38 of 44 rats (86.4%) could be obtained. The mortality rates were not significantly different between groups at 1/10, 1/8, 1/7, 1/6, 1/7 and 1/6 in groups 1–6 respectively.

Infarct size

All the control groups expectedly indicated the development of neocortical infarction. However, infarct size differed among the animals according to the anatomic pattern of the distal MCA. Infarct size averaged 18.9 ± 1.49%, 9.8 ± 5.26% and 12.3 ± 2.33% in the Controls-bMCA, Controls-mbMCA and Controls-cMCA groups respectively (Figure 2). Of note, infarct size was significantly smaller in the Controls-mbMCA group as compared to Controls-bMCA (P < 0.05). In the Controls-cMCA group, infarct size also tended to be smaller than in Controls-bMCA; however, the difference was not statistically significant. Moreover, the infarct size values in the Controls-mbMCA group were the most variable.

Figure 2.

Cerebral infarct size in rats with different patterns of middle cerebral artery branching subjected to ischaemic postconditioning. The data are presented as dot plots with median values. Significant infarct size reduction after postconditioning is observed only in animals with the typical bifurcating pattern of the middle cerebral artery.

PostCon significantly reduced infarct size in the PostCon-bMCA group (8.4 ± 2.64%, P < 0.05 vs. PostCon-bMCA). In contrast, application of PostCon in the animals with a multiple-branching MCA pattern did not cause significant infarct size limitation (10.6 ± 5.04%, P > 0.05 vs. PostCon-mbMCA). Infarct size tended to be lower in the PostCon-cMCA group as compared to Controls-cMCA, although the difference between these two groups was not statistically significant (10.2 ± 2.11%, P > 0.05).

Discussion

In the present study, it has been shown for the first time that the infarct-limiting effect of ischaemic PostCon can be influenced by the anatomic pattern of the arteries involved in the blood supply to the brain. We also demonstrated that cerebral infarct size can differ significantly in animals with different MCA branching patterns. In particular, infarct size was significantly smaller in the animals with the multiple-branching MCA pattern in comparison with those having the typical bifurcating pattern. This observation could be explained by the different efficiency of collateral blood supply to the ischaemic territory of the brain in animals with different anatomic variants of MCA. It is conceivable that the numerous minor cortical MCA branches observed in the Controls-mbMCA group have better connections with cortical anastomoses that provide collateral blood flow from the ipsilateral anterior and posterior cerebral arteries. Furthermore, we cannot exclude the possibility that certain procedure-related factors might influence the resulting infarct size. For instance, the preserved function of additional small branches of MCA located outside the cranial window might contribute to a smaller area at risk in the mbMCA group.

The proportion of animals with different MCA branching within the single experimental group should be considered when interpreting the results of neuroprotection studies utilizing the model of surgical MCA ligation, because the occasional predominance of animals with the multiple-branching MCA pattern could potentially lead to false-positive results. Moreover, exclusion of animals with the multiple-branching distal MCA pattern from the final analysis might be recommended, because of poor reproducibility of infarct size data in this particular cohort of animals.

The present study demonstrated the apparent infarct-limiting effect of an ischaemic PostCon protocol consisting of five episodes of 10-s LCCA occlusion, each separated by 10-s reperfusion, performed immediately after 30-min LCCA occlusion in animals with a bifurcating MCA pattern. These findings are consistent with the data of Zhao et al. (2006), who used a slightly different PostCon protocol in which three cycles of 30-s bilateral CCA occlusion and 10-s reperfusion performed after permanent MCA ligation in Sprague-Dawley rats, combined with bilateral CCA occlusion for 15, 30 and 60 min, resulted in infarct size reduction by 80%, 51% and 17% respectively. As with our study, focal cerebral ischaemia was elicited in this work by permanent ligation of the distal MCA, while PostCon episodes were induced by transient ischaemia–reperfusion in the CCA vascular bed. Thus, in both studies, the neuroprotective effect of PostCon was limited to the penumbral area, that is, the potentially reparable tissue area around the ischaemic core.

Previous studies demonstrated that the effectiveness of ischaemic PostCon strongly depends on the specific protocol employed. For example, Kim et al. (2010) did not note any neuroprotective effect of PostCon elicited by three episodes of 10-s MCA occlusion and 30-s reperfusion after 60-min intraluminal MCA occlusion in rats. In contrast, a robust neuroprotective effect was found after application of six cycles of 30-s ischaemia/30-s reperfusion in the same model (Xing et al. 2008). Application of a PostCon protocol consisting of three 30-s cycles of MCA occlusion and 30-s reperfusion after 90-min MCA occlusion also resulted in infarct size limitation and reduction in neurological deficits (Liu et al. 2011). The above-mentioned controversies might be explained by the differences in PostCon protocols, by the duration of the reperfusion period prior to assessment of endpoints and also by the natural variability in the anatomic characteristics of MCA. Interestingly, the application of the same PostCon protocol in animals with different MCA anatomy resulted in distinct neuroprotective responses. Several hypothetical explanations might be suggested to account for the lack of PostCon effect in the animals with multiple-branching and combined MCA patterns. First, the infarct-sparing effect of PostCon is probably not additive to that provided by better collateralization of the ischaemic area in these two groups. It is generally accepted that the extent of collateral support through the circle of Willis is one of the crucial determinants of final infarct size in rat models of MCA occlusion through craniectomy. The impact of ancillary perfusion through both primary and secondary collateral pathways on cerebral infarct size was convincingly demonstrated in adult (Longa et al. 1989) and neonatal (Bonnin et al. 2011) rats, as well as in humans (for review, see Liebeskind 2003). We presume that better collateral blood supply to the affected MCA territory in the mbMCA and cMCA groups may result in a smaller volume of ischaemic core and greater penumbral volume vs. the bifurcating MCA group, although the net anatomic area at risk is comparable among all groups. Second, it might be hypothesized that the lack of neuroprotective effect of PostCon in animals with specific anatomic MCA patterns is explained not only by differences in collateral blood supply but also by the genetic association of certain anatomic patterns with metabolic profiles crucial for neuroprotection. After rigorous experimental testing, these data should be considered for planning and interpretation of preclinical and clinical trials.

In theory, both primary and secondary collateral pathways can be involved in compensating for reduced cerebral blood flow in the ischaemic area after simultaneous occlusion of the MCA and ipsilateral CCA. In 7-day-old rats, occlusion of the MCA plus the CCA was shown to result in a rapid increase in blood flow velocity in both the basilar trunk and the contralateral internal carotid artery (Bonnin et al. 2011). The diversion of blood flow in the circle of Willis towards the affected area, along with maximal dilation of the small vessels within the ischaemic zone, contributes to the opening of cortical (leptomeningeal) anastomoses. Cortical anastomoses might provide retrograde filling of the smallest distal branches of occluded MCA from several vascular territories including those of the ipsilateral posterior cerebral artery, the anterior cerebral artery and branches of the MCA proximal to the occlusion (Brozici et al. 2003). However, the density and diameter of cortical anastomoses may vary significantly between species, strains and individual animals (Oliff et al. 1997). Also, it was reported that cortical anastomoses do not open immediately but ≥40 min after a major arterial occlusion (Brozici et al. 2003). One obvious limitation of the present study is that collateral blood flow using laser or ultrasound Doppler flowmetry was not measured during the experiments. Information on the extent of collateral flow after MCA occlusion in animals with differing MCA anatomy would add much to the understanding of the underlying mechanisms of both infarct size variability and PostCon effectiveness/ineffectiveness. Future studies will address this important issue.

In conclusion, the results of the present study indicate that ischaemic PostCon exerts a significant infarct-limiting effect in the model of focal cerebral ischaemia in Wistar rats with a typical bifurcating MCA branching pattern. A similar PostCon protocol failed to protect the brain in animals with multiple-branching and combined MCA patterns. The anatomic pattern of the MCA should be considered one of the important factors influencing the outcome of neuroprotection studies in this particular experimental model.

Funding source

This work was supported by the grant of the President of the Russian Federation for support of leading scientific groups 2359.2012.7.

Conflict of interest

The authors declare that they have no conflict of interest.