Abstract
Spreading depolarizations may contribute to delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage, but the effect of spreading depolarizations on brain lesion progression after subarachnoid hemorrhage has not yet been assessed directly. Therefore, we tested the hypothesis that artificially induced spreading depolarizations increase brain tissue damage in a rat model of subarachnoid hemorrhage. Subarachnoid hemorrhage was induced by endovascular puncture of the right internal carotid bifurcation. After one day, brain tissue damage was measured with T2-weighted MRI, followed by application of 1 M KCl (SD group, N = 16) or saline (no-SD group, N = 16) to the right cortex. Cortical laser-Doppler flowmetry was performed to record spreading depolarizations. MRI was repeated on day 3, after which brains were extracted for assessment of subarachnoid hemorrhage severity and histological damage. 5.0 ± 2.7 spreading depolarizations were recorded in the SD group. Subarachnoid hemorrhage severity and mortality were similar between the SD and no-SD groups. Subarachnoid hemorrhage-induced brain lesions expanded between days 1 and 3. This lesion growth was larger in the SD group (241 ± 233 mm3) than in the no-SD group (29 ± 54 mm3) (p = 0.001). We conclude that induction of spreading depolarizations significantly advances lesion growth after experimental subarachnoid hemorrhage. Our study underscores the pathophysiological consequence of spreading depolarizations in the development of delayed cerebral tissue injury after subarachnoid hemorrhage.
Keywords: Subarachnoid hemorrhage, spreading depression, magnetic resonance, animal models, brain imaging
Introduction
Aneurysmal subarachnoid hemorrhage (SAH) has a poor prognosis.1 A feared complication is the development of delayed cerebral ischemia (DCI), which occurs in approximately one-third of patients. The cause of DCI has been an ongoing matter of debate and suggested mechanisms include vasospasm, (micro-)thrombosis, and cortical spreading depolarization (SD).2,3 It has been shown in rats that accumulation of the hemolysis products hemoglobin and K+ in the subarachnoid space can induce spreading ischemia—an inverse hemodynamic response (i.e. transient hypoperfusion) to SD in tissue at risk—contributing to expanding cortical infarction.4 Furthermore, electrocorticography measurements in cortical tissue of SAH patients have revealed SDs in association with development of delayed ischemic damage.4
SDs are slow waves of neural cell depolarization, self-propagating through the cortex at a speed of 2–6 mm/min.5 Under normal conditions, a SD is a reversible phenomenon accompanied by an increase in perfusion to support restoration of the electrolyte balance.6 However, SDs may lead to irreversible damage in metabolically compromised brain tissue, such as after SAH or ischemic stroke.7 In rats, occasional SD-like phenomena have been detected acutely after experimental SAH.8,9 In a small series of 13 patients, electrocorticographic activity and perfusion were measured from a strip of optoelectrodes on the cortex after surgery for aneurysm clipping.10 Clusters of prolonged SDs, accompanied by transient hypoperfusion, were measured in close proximity to ischemic brain damage in five patients.10
Despite these observations, a direct link between the occurrence of SDs and (delayed) progression of cerebral tissue injury after SAH has not yet been demonstrated. Therefore, we tested the hypothesis that SDs, artificially induced in rats after SAH, increase delayed brain injury, measured with MRI and histology.
Materials and methods
Study design
This study was performed in accordance with guidelines of the European Communities Council Directive and approved by the Animal Experiments Committee of the University Medical Center Utrecht and Utrecht University. Data reporting is in compliance with the ARRIVE guidelines (www.nc3rs.org.uk/arrive-guidelines).
A sample size of 16 animals (male Wistar rats (200–250 g); Charles River, Sulzfeld, Germany) per group was a priori calculated based on a Chi-square test with a hypothesized SD-induced lesion growth from 200 ± 75 to 300 ± 75 mm3, and 35% mortality before day 3, based on a previous study from our group.11 Rats were housed under standard conditions and received daily intraperitoneal saline injections. Rats were excluded if no SAH was identified on postmortem investigation.
An additional six healthy control rats were used in a pilot study to measure the consequences of SD induction in healthy brain.
SAH model
Rats were anesthetized, endotracheally intubated and mechanically ventilated with 2% isoflurane in air/O2 (80%/20%). Intracranial endovascular perforation at the bifurcation of the right anterior cerebral artery and middle cerebral artery was induced by transiently advancing a sharpened prolene 3-0 suture through the right internal carotid artery, as described previously.9 After this, anesthesia was ended and rats were extubated.
Induction and recording of SDs
One day after SAH, rats were endotracheally intubated and mechanically ventilated with 2% isoflurane in air/O2 (80%/20%) for MRI (see below). Directly after MRI, rats remained anesthetized, and a 2 mm burr hole was drilled in the skull at 2 mm anterior of lambda and 2 mm right of the sagittal suture, and the underlying dura was opened. Laser-Doppler flowmetry (LDF) probes (Moor Instruments, Devon, UK) were positioned at 1 and 2 mm anterior of the burr hole (2 mm right of the sagittal suture) after skull thinning at these positions. A saline-soaked cotton ball was placed in the burr hole. After 10 min of baseline recording, the cotton ball was replaced by a cotton ball soaked in 1.0 M KCl (pilot study (N = 6) and SD group (N = 16)) or saline (no-SD group (N = 16)). LDF recording was continued for 50 min. Distinct transient increases in LDF were scored as SDs by an observer blinded to group assignment.
Sensorimotor function test
Functional status was assessed daily before any procedures with an inclination test (SD group: N = 11; no-SD group: N = 12).12 To this end, rats were placed on a triplex plane, of which the horizontal angle was increased in steps until the rat slid down.
MRI of brain lesions
On days 1 and 3 post-SAH, rats were endotracheally intubated and mechanically ventilated with 2% isoflurane in air/O2 (80%/20%) for MRI on a 4.7 T/40 cm MR system (Varian Inc., Palo Alto, CA, USA). A 90 mm Helmholtz volume coil and an inductively coupled surface coil (2.5 cm diameter) were used for excitation and detection of radio frequency signals, respectively. The MRI protocol included T2-weighted multiecho MRI (repetition time 3000 ms, echo times 12–144 ms in twelve 12 ms steps, field of view 32 × 32 mm2, data matrix 256 × 128, 19 slices of 1 mm, number of acquisitions 2).
T2 maps were calculated from a nonlinear least squares fitting routine. Images were registered to a reference T2-weighted image using FLIRT.13 Lesion regions, characterized by clear T2 hyperintensity, were drawn using FSL software (3.1.8, University of Oxford, Oxford, UK) by two independent observers who were blinded to group assignment, from which the intersection was taken. A cortical tissue volume of 2 × 2 × 1 mm3 below the burr hole was excluded from lesion volume calculation to prevent inclusion of tissue that was directly affected by KCl. Lesion growth was calculated as the difference between lesion volumes on day 3 and day 1.
SAH severity scoring
After spontaneous death or after sacrificing of the rat on day 3, brains were perfusion fixed with 4% paraformaldehyde and removed from the skull. Pictures of the ventral side of the brain were scored according to Sugawara’s SAH severity score,14 ranging from 0 (no subarachnoid blood) to 18 (large SAH).
Histology of tissue damage
Extracted brains were stored in phosphate-buffered saline with 0.5 g/l sodium azide (Sigma-Aldrich, St. Louis, MO, USA). We selected brain samples from rats (SD group: N = 4, no-SD group: N = 5) with different patterns of lesion development after SAH: MRI-detectable lesions on days 1 and 3, MRI-detectable lesions only on day 3, and no MRI-detectable lesions on days 1 and 3. The brains were cryoprotected by subsequent immersion in 15% sucrose (for 48 h) and 30% sucrose solutions (for 48 h). Coronal sections (30 µm) were cut on a freezing microtome, followed by Nissl staining according to standard protocols.15
Images of complete coronal sections corresponding with MRI slices were acquired using digital microscopy (Nanozoomer 2.0HT; Hamamatsu, Hamamatsu-shi, Shizuoka-ken, Japan). Further analysis was done on 20X images of four selected regions, characterized by: (i) T2 hyperintensity on post-SAH days 1 and 3 (“early lesion”), (ii) T2 hyperintensity only on post-SAH day 3 (“delayed injury”), and (iii) two contralesional counterparts (regions 1, 2, 3, and 4, respectively, in Figure 4(a)). Presence of neuronal injury/death, identified by pyknotic cell staining patterns, was scored for each quadrant of the respective regions by an observer blinded to group assignment, resulting in neuronal injury scores ranging from negative to ++++ for each region.
Figure 4.
Histology. (a) T2 maps of a brain slice of a rat from the SD group at post-SAH days 1 (top) and 3 (bottom). A subcortical lesion was present at post-SAH day 1, before SD induction, and a cortical lesion became apparent at post-SAH day 3, i.e. two days after SD induction. Regions of interest in early injured tissue (region 2), delayed injured tissue (region 4), and unaffected contralesional counterparts (regions 1 and 3, respectively) are displayed in the post-SAH day 3 image. (b) Nissl staining of four regions of interest (20X magnification) as displayed in Figure 3(a). Contralesional regions (1 and 3) show unaffected healthy tissue. Tissue with early injury after SAH (i.e. before SD induction) shows clear signs of blood extravasation into the parenchyma (reddish brown areas), neuronal damage and/or death, as shown by the majority of shrunken, pyknotic nuclei (region 4). The extent of tissue injury is milder in the region with delayed lesion manifestation (i.e. after SD induction) (region 2), with only a few dark, shrunken nuclei interspersed in the parenchyma. Scale bars: 300 µm.
Statistics
A repeated measures ANOVA with post hoc paired t-testing was used to analyze scores on the inclination test. An independent samples t-test was used for comparing T2 values between (sub)groups, and a paired samples t-test for comparing T2 values within individuals. Lesion volumes on MRI and SAH severity scores were compared with a Mann–Whitney U test. Lesion incidence and mortality were analyzed with a Chi-square test. Spearman’s Rho was calculated to measure correlation between SAH severity and mortality, and between number of SDs and lesion volume. Values are shown as mean ± SD. A p-value < 0.05 was considered statistically significant.
Results
One rat was excluded based on the absence of a SAH on postmortem investigation, resulting in final sample sizes of 16 (for the SD group) and 15 (for the no-SD group).
In the six healthy control rats, KCl application led to 6.7 ± 1.8 SDs, depicted by transient increases in blood flow as measured during the 50 min LDF recording. MRI of the underlying cortical tissue one day after KCl application showed no signs of tissue damage (T2 values: 58 ± 1 ms ipsilateral versus 59 ± 1 ms contralateral. In the SD group, 5.0 ± 2.7 SDs were measured in nine out of 12 surviving rats. Most of these SDs were characterized by transient hyperperfusion (Figure 1, top), similar to the observation in healthy control rats. However, in two rats, we recorded spreading hypoperfusion (Figure 1, bottom). In these two animals, cortical T2 values below the KCl application site were slightly elevated before SD induction (60 ± 3 ms) as compared to the animals with hyperemic responses (58 ± 1 ms), but this difference was not statistically significant (p = 0.32). LDF recordings were unsuccessful in three rats. No SDs were recorded in the no-SD group.
Figure 1.
T2 maps of lesions, and LDF recordings of SDs. T2 maps of a posterior brain slice at day 1 post-SAH, before SD induction (left panel), and at day 3 post-SAH, after SD induction (right panel), in two rats from the SD group. Arrows indicate the KCl application site (right panel). Middle panel: LDF recordings from the same two rats, showing KCl-induced SDs with associated transient flow increases (top row) or reductions (bottom row (recordings from both LDF probes)). Lesion growth between days 1 and 3 was larger in the animal with SD-associated transient hypoperfusions.
Sensorimotor function, as scored from the horizontal angle on the inclination test, was lower at day 1 (SD group: 35 ± 7; p = 0.002; no-SD group: 39 ± 6; p = 0.02) and day 3 post-SAH (SD group: 38 ± 7; p = 0.01; no-SD group: 40 ± 6; p = 0.03) as compared with pre-SAH (SD group: 49 ± 3; no-SD group: 46 ± 3). However, there were no statistically significant differences between the groups.
Mortality, lesion characteristics, and SAH severity are shown in Table 1. There were no statistically significant differences in mortality and SAH severity between the SD and no-SD groups.
Table 1.
Mortality, lesion characteristics, and SAH severity.
| Day 1 (pre-SD) | Day 3 (post-SD) | |||||
|---|---|---|---|---|---|---|
| Group | SD | No-SD | p | SD | No-SD | p |
| Mortality | 25% (N = 16) | 7% (N = 15) | 0.17 | 25% (N = 12) | 7% (N = 14) | 0.21 |
| Lesion occurrence | 50% (N = 12) | 29% (N = 14) | 0.26 | 100% (N = 9) | 31% (N = 13) | 0.001 |
| Lesion size (mm3) | 152 ± 295 (N = 12) | 49 ± 95 (N = 14) | 0.35 | 273 ± 275 (N = 9) | 60 ± 112 (N = 13) | 0.008 |
| Lesion growth (mm3) | N/A | N/A | N/A | 241 ± 233 (N = 9) | 29 ± 54 (N = 13) | 0.001 |
| SAH severity score | N/A | N/A | N/A | 11.9 ± 3.4 (N = 9) | 11.9 ± 2.7 (N = 13) | 0.84 |
Day 1 mortality was measured between pre-SAH and post-SAH day 1, i.e. before KCl or NaCl application. Day 3 mortality was measured in the interval between post-SAH days 1 (after KCl or NaCl application) and 3. Lesions were identified as clearly hyperintense brain tissue areas on T2 maps. Lesion occurrence and size were recorded on days 1 and 3. Lesion growth was defined as brain tissue area with a lesion on day 3 where no lesion was identified on day 1 (which could only be measured in animals that survived until day 3). SAH severity was measured from Sugawara’s scoring test.14
Lesions, identified as hyperintense tissue on T2-weighted MR images in ipsi- and contralateral cortical and subcortical areas, had significantly prolonged T2 values as compared with T2 values in unaffected tissue (SD group: 52 ± 1 ms; no-SD group: 52 ± 1 ms), at post-SAH day 1 (SD group: 75 ± 7 ms; p < 0.001; no-SD group: 68 ± 4 ms; p < 0.001) and day 3 (SD group: 62 ± 5 ms; p < 0.001; no-SD group: 61 ± 3 ms; p < 0.001). Figure 2 shows the lesion incidence maps of the groups at both time points, as well as the lesion growth. Figure 3 shows the lesion volumes of the individual animals at days 1 and 3 post-SAH in the SD and no-SD groups. Lesion occurrence and size were not statistically significantly different between groups at day 1 (before SD induction (Table 1)); admittedly this may also be due to the large variations in lesion size. At day 3, the lesion area had expanded, particularly in the SD group, which mostly (but not exclusively) involved ipsilateral cortical regions in both groups (Figures 2 and 3). Lesion occurrence at day 3 was higher in the SD group (100%) as compared to the no-SD group (31%) (p = 0.001). Moreover, lesion growth at day 3 was considerably larger in the SD group (241 ± 233 mm3) as compared to the no-SD group (29 ± 54 mm3) (p = 0.008). We found no statistically significant correlation between SAH severity and three-day lesion volume (Rho = 0.36; p = 0.10) or lesion growth (Rho = 0.33; p = 0.14). There was a high correlation between number of recorded SDs and lesion growth in the SD group (Rho = 0.69, p < 0.001).
Figure 2.
Maps of lesion incidence and growth. Multislice T2 maps of rat brain (top row), with the excluded region below the KCl application site in red, and voxel-based representations of fraction of rats with lesioned tissue identified on T2 maps at days 1 and 3, and the difference between these time points (“lesion growth”), in SD and no-SD groups. SD: spreading depolarization.
Figure 3.
Lesion size. Lesion volumes of individual animals at days 1 and 3 post-SAH in the SD (a) and no-SD groups (b). Each animal is represented by a different color.
Because of the large variation in lesion size, we performed a subgroup analysis based on the presence of a small lesion (size of ≤ 20 mm3) or a large lesion (size of > 20 mm3) on day 1. The lesion growth in animals with a small lesion at day 1 was significantly larger in the SD group (142 ± 119 mm3 (N = 6)) than in the no-SD group (4 ± 12 mm3 (N = 10)) (p = 0.001). In rats with a large lesion at day 1, lesion growth was not significantly different between the SD group (367 ± 298 mm3 (N = 6)) and the no-SD group (114 ± 55 mm3 (N = 4)) (p = 0.29); again this may be due to large variation in growth in the SD group.
Lesioned tissue, as identified with MRI on day 1, revealed pyknotic staining patterns with shrunken or absent nuclei on histological sections at day 3 (median injury score:+++) and presence of hemorrhages (Figure 4(b), region 4). In lesion growth regions, where tissue lesions were identified with MRI only at day 3, the extent of injury was smaller (median injury score: ++) (Figure 4(b), region 2). Neuronal injury was absent in regions identified as nonlesioned with MRI at day 3 (Figure 4(b), regions 1 and 3).
Discussion
Our study demonstrates that artificially induced SDs after experimental SAH in rats augment delayed brain injury. Our findings are in line with the hypothesis that SDs contribute to the development of DCI in SAH patients,10 which may be triggered by early brain injury leading to further progression of post-SAH tissue damage.
Cortical SDs have been previously recorded in humans up to two weeks post-SAH,10 and in laboratory animals within the first hours after SAH.8,16 The mechanism leading to SDs after SAH is unknown, but several pathologic conditions such as hypoxia, transient ischemia, high extracellular potassium levels, and free hemoglobin may trigger SDs.3,7,17 Under normal, physiological conditions experimentally induced SDs are followed by hyperemia to compensate for the increased energy metabolism. In accordance, we detected clear hyperemic responses with LDF after cortical application of 1 M KCl for less than an hour in healthy control rats, and no signs of tissue injury were detected with MRI one day thereafter. However, since cortical KCl application at higher dosage and for longer duration has been shown to cause direct local tissue damage,18 we excluded the underlying cortical volume from SAH lesion volume calculations. Nevertheless, possible further effects of KCl on tissue injury development in SAH-affected brain could not be ruled out.
SD-induced hyperemic responses, similar to those observed in healthy control rats, were found after KCl application to the perilesional cortex of rats at one day after SAH. However, in two out of nine animals, we detected waves of transient hypoperfusion, which suggests that the underlying tissue was already compromised. This confirms the hypothesis that under pathological conditions, such as after stroke or SAH, paradoxical hypoperfusion can occur after SDs, ultimately leading to ischemic tissue damage.7,19,20 Importantly, SD-induced hypoperfusion may also have occurred outside of our LDF recording region, i.e. in compromised areas with impaired neurovascular coupling, thereby contributing to post-SAH lesion expansion.
Despite large variation in lesion size after SAH, which is a feature of the endovascular puncture model in rats,21 the degree of SAH severity was similar between groups, and we measured statistically significant differences in lesion growth between the SD and no-SD groups. Our study is the first to relate lesion expansion to SDs beyond the first day after SAH in an experimental model. Lesion volume as measured by MRI expanded eight times more after induction of SDs, as compared to conditions without experimentally induced SDs. In a subgroup analysis of rats with small or large lesions on day 1 post-SAH, before cortical KCl or NaCl application, we found that the contribution of SDs to lesion growth was most significant in animals with small initial lesions. This suggests that under pathological conditions SD occurrence in relatively unaffected tissue can have critical impact on remote areas with neuronal or neurovascular impairment.
We did not detect any spontaneous SDs during the relatively short recording time frame of 1 h. Nevertheless, this does not exclude the occurrence of SAH-induced spontaneous SDs outside of the recording time or outside the observed region. Theoretically, the possible occurrence of spontaneous SDs—conceivably triggered by early brain injury—may have contributed to the 122% delayed lesion growth that was found in the no-SD group, but this could also be explained by other pathological factors that have been previously measured in this rat SAH model, such as vasospasm14,22,23 and luxury perfusion.11
Histological analysis revealed clear neuronal pyknosis in all areas identified as lesion areas on T2 maps at post-SAH day 3, which reflects ischemia-induced apoptosis and/or necrosis. No pyknosis was observed in nonlesioned areas, confirming that absence of MRI-detectable lesions corresponded with intact tissue. The extent of neuronal damage was related to the time of lesion occurrence on MRI, reflecting the different progression of early and delayed injury. However, more detailed histological assessments are required to accurately characterize the status of tissue damage in relation to the different aspects of SAH pathophysiology.
Despite the increased lesion expansion, we did not detect statistically significant effects on sensorimotor function between the SD and no-SD groups. This may be related to the relatively crude outcome measure of the inclined plane test, which suggests that more extensive and subtle behavioral tests should be included in future studies.
In conclusion, our study in rats demonstrates that KCl-induced SDs in perilesional cortical tissue aggravate (early) brain tissue damage after SAH leading to augmentation of delayed brain injury. Our animal model of modulation of brain injury by SD induction after SAH may be useful for future studies on the pathogenesis and treatment of evolving brain injury, which may include preclinical testing of therapeutic effects of SD inhibitors.
Funding
The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Dr Wermer was supported by personal grants from the Netherlands Organization for Scientific Research (ZonMW Veni grant), the Netherlands Heart Foundation (2011T055) and the Dutch Brain Foundation (project 2011(1)-102). This work was partly supported by the Utrecht University High Potential Program (R.M.D.) and the EU Marie Curie IAPP Program “BRAINPATH” (nr 612360) (A.M.J.M.v.d.M.) and the American Heart Association (A.M.S.).
Acknowledgements
The authors would like to thank Wouter Mol for his biotechnical support, and Lisha Ma, MD, for her contribution to the histological analyses.
Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Authors’ contributions
MJHW, RMD, WMB, AMJMM, MDF, SUR, AT, and AMS contributed substantially to the conception and design of the study.
AT supervised MR imaging, created lesion incidence maps, and wrote the methods section on MR imaging.
SUR advised on MR imaging acquisition, processing, and statistical analyses.
CL and AMS performed histology.
HJAO coscored SAH severity and brain lesions.
MJHW and RMD contributed substantially to interpretation of data and writing of the manuscript.
AMH performed the in vivo experiments and did the principal data analysis and writing of the manuscript.
All authors contributed to revising the article, and all authors approved the final version as submitted.
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