Exp Neurobiol 2016; 25(3): 130-138
Published online June 30, 2016
© The Korean Society for Brain and Neural Sciences
Jeongeun Sim1#, Areum Jo1#, Bok-Man Kang2#, Sohee Lee1#, Oh Young Bang3,4,Chaejeong Heo1, Gil-Ja Jhon5, Youngmi Lee5 and Minah Suh1,2,4*
1Center for Neuroscience Imaging Research (CNIR), Institute for Basic Science (IBS), Suwon 16419, 2Department of Biomedical Engineering, Sungkyunkwan University, Suwon 16419, 3Department of Neurology, Neuroscience Center, Samsung Medical Center, Sungkyunkwan University School of Medicine,4Department of Health Sciences and Technology, SAIHST, Sungkyunkwan University, Seoul 06351, 5Department of Chemistry and Nano Science, Ewha Womans University, Seoul 03760, Koera
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-31-299-4496, FAX: 82-31-299-4506
#These authors contributed equally to this work
Ischemia can cause decreased cerebral neurovascular coupling, leading to a failure in the autoregulation of cerebral blood flow. This study aims to investigate the effect of varying degrees of ischemia on cerebral hemodynamic reactivity using
Keywords: Stroke, MCAO model, Optical intrinsic signal imaging, Hemodynamics, Arterial reactivity
Normal neurovascular coupling between neuronal activity and cerebral blood flow is critical to ensure the health of the brain, and this coupling is vulnerable to ischemic brain diseases. A reduction in cerebral blood flow (CBF) around occluded cerebral vessels leads to a series of biochemical, biomolecular, and functional damage that triggers neuronal cell death pathways [1,2]. These cascade actions eventually result in disrupted hemodynamic autoregulation and neurovascular coupling malfunctions , which are accompanied by ischemia-induced behavioral deficits . Moreover, the severity of ischemia is closely related to sensory stimulation-induced hemodynamic responses and the functional recovery of sensorimotor functions [5,6]. In addition, human stroke patients exhibit disruptive neurovascular coupling, which manifests as a reduction in cerebral blood flow [7,8]. Clinically, it is important to predict infarct regions following ischemia, and there has been an attempt to use reduced hemodynamics, such as CBF, as an early indicator of ischemic stroke.
The middle cerebral artery occlusion (MCAO) model is a common animal m odel o f ischemic s troke. MCAO induces cerebral ischemia and disrupts cerebral autoregulation. However, a dynamic evaluation of the failure in cerebral autoregulation has not yet been undertaken. To compensate for dysfunctional autoregulation, post-ischemic angiogenesis occurs in MCAO . As early as 12 h after ischemia, endothelial cells within the neurovascular coupling unit proliferate from preexisting vessels and make junctional structures in the surrounding infarcted brain tissue [10,11]. Because hemodynamics in the infarcted brain is affected by this disruption in autoregulation and postischemic angiogenesis, it is important to measure hemodynamics in an animal stroke model to evaluate the degree of ischemia. In addition, the degree of ischemia affects functional recovery surrounding the infarction following reperfusion from the ischemic state . The penumbra zone, a functionally impaired but not yet irreversibly injured tissue, is very dynamic and a crucial therapeutic target to improve clinical outcomes. We induced both moderate and severe infarct in MCAO models and measured whether the level of hemodynamic reactivity is dependent on the degree of ischemia.
Cortical electrical stimulation enhances cortical excitability and brings about robust cerebral blood volume (CBV) change, which mimics CBV changes during an epileptic event, which is a most hyper-excitable and hyper-synchronous brain activity . Optical recording of intrinsic signal (ORIS) is one of many functional perfusion-imaging technologies that can be utilized in conjunction with electrical stimulation that does not require injecting special chemicals [14,15]. ORIS with multi-wavelengths can be used to map neuronal activities derived from the oxygenation states of hemoglobin and changes in CBV . In addition, ORIS with a high-speed, charge-coupled device (CCD) is capable of creating continuous two-dimensional brain surface images, including pial vasculature. Therefore, in the MCAO ischemic models, we utilize ORIS with direct external stimulation to evaluate cortical hemodynamics and vessel reactivity in relation to the degree of ischemia. Our particular interest is whether the MCAO models with different degrees of ischemic severity exhibit differential hemodynamic responses in cerebral arteries and cortical tissue immediately after the application of an external electrical stimulus.
All animal preparations and procedures were performed in accordance with the Institutional Animal Care and Use Committee (IACUC) of Sungkyunkwan University. Male, 9-week-old, Sprague-Dawley rats (280~320g) were used (OrientBio, Seongnam, South Korea). All rats were housed in humidity- and temperature-controlled facilities with 50~60% humidity at 23℃ under a 12-h light/dark cycle.
For the MCAO procedure, the rats were anesthetized and maintained with 2% isoflurane and oxygen gas (VetEquip, CA, USA). A heating pad was used to maintain the animal's body temperature throughout the procedure (FHC, ME, USA). We carefully made an incision in the neck skin and exposed the right common carotid artery (CCA). A 4-0 monofilament nylon suture coated with silicon was introduced from the CCA into the middle cerebral artery (MCA). The insertion length of the suture (18~20 mm) and thickness of the silicon head (330~380 µm) varied according to the body weight of the animal . After MCA occlusion, the incision was temporarily closed and the animal was returned to home cage for either 60 min (
According to prior studies, the infarct volume of the MCAO model reached a maximum after 24 h of reperfusion . Therefore, we conducted the mNSS behavioral testing after 24 to 72 h of reperfusion. We focused on assessing primary sensory cortical functions to compare the hemodynamic changes in the primary sensory cortex and behavioral consequences in the MCAO model. The mNSS is composed of three different behavioral tests: motor, sensory, and beam balance tests (Fig. 1A). Detailed mNSS procedures were described by Chen et al. (2001) .
We performed the ORIS imaging right after completion of the behavioral test. We carefully removed the skull of both hemispheres under 2% isoflurane anesthesia. The animal's health was carefully monitored throughout the experiment. Two tungsten electrodes (Plastics One, Roanoke, VA, USA) were placed onto the intact dura mater of each hemisphere (Fig. 1B), which were connected to a pulse generator (Master-8, A.M.P.I., Jerusalem, Israel). Functional hemodynamic imaging was performed using an optical imaging system (Imager 3001, Optical Imaging, Rehovot, Israel) with an external light source (Leica Microsystems, Manheim, Germany). Reflected light was collected through two 50 mm tandem lenses (Nikon, Tokyo, Japan) and filtered using a 570±10 nm band-pass filter. The 570 nm is the isosbestic wavelength of total hemoglobin, which is equivalent to CBV changes. The images were acquired for 300 s at 3.33 Hz. The images recorded during the initial 30 s before applying 10 s stimulation (1 mA, 3.33 Hz, 10 ms pulse width) to the cortex were averaged and designated as the baseline image. Both the ipsilateral hemisphere (I.H), which had the infarction, and the opposite contralateral hemisphere (C.H) were alternatively imaged. In order to ensure hemodynamic recovery to baseline levels, the inter-trial interval was set at 900 s (Fig. 1C).
After ORIS imaging, the entire brain of the rat was extracted and sectioned into six coronal slices, 2 mm thick, using a rat brain matrix (World Precision Instruments, Sarasota, FL, USA). All coronal slices were stained with 2% 2,3,5-triphenyltetrazolium chloride (TTC) solution (Sigma-Aldrich, St. Louis, MO, USA) for 20 min and fixed with 4% paraformaldehyde at 4℃. After 24 h of initial staining, the six slices were photographed with a graduated ruler using a digital camera (Canon, Tokyo, Japan). The infarct volume of each slice was quantified using Fiji software (NI.H, MD, USA). We classified the MCAO models into two groups according to their infarct volume; the mild MCAO group had less than 90 mm3 of infarct area that was primarily located in the striatum, while the severe MCAO group had greater than 90 mm3 of infarct area that included areas in the cortex as well as the striatum.
The activation maps shown in Fig. 3A, C, and E were calculated by dividing every frame by the mean of the baseline frame. We measured the time course of CBV change in the region of interest (ROI, 7×7 pixels) of the cortical tissue, quantifying both the maximum value of CBV change and the time to reach that maximum value. The ROI that was selected included the tissue area that showed the strongest CBV changes but excluded the large vessels. The ORIS data were analyzed using custom-written MATLAB software (MathWorks, Natick, MA, USA).
To compare hemodynamic differences in the arteries between the MCAO and normal groups, we defined the reactive artery from images and quantified them as shown in Fig. 4A. First, the raw images were cropped (3.7×5.9 mm2) and segmented into cerebral blood vessels and surrounding tissues using the trainable Waikato Environment for Knowledge Analysis (WEKA) segmentation plugin, which combines a collection of machine learning algorithms in Fiji . Based on the segmentation results, the probability of each pixel in the raw image belonging to the segmented vessel was verified by the WEKA tool. The verified pixel probabilities, from 0% to 100%, were mapped in an intensity form. For accurate extraction of the blood vessel without intervention, such as a cerebral hemorrhage and unintended light reflection from the dura mater, we set the threshold value at 60% of the probability for vessel classification (Fig. 4A). This threshold value was a balance between the detection ratio and a false-positive ratio and was determined through an empirical analysis. In a sequence, the reactive region was defined by mapping the standard deviations (SD) of each pixel's value over time. All maps were filtered by a 3×3 Gaussian kernel to prevent the signals from interfering with the outliers. To exclude a low level of variation, which was defined as no response over time, a total of 1,500 pixels were gathered from the highest value in the calculated SD map and were binary coded. A binary coded image was multiplied to all frames of the vessel probability maps in order to extract the reactive vessels, which were defined as arteries, from the vessel probability map (Fig. 4A). The final step of quantification was to measure the time course of CBV change in the extracted artery in each frame. The measured intensity of artery was calculated to extract hemodynamic characteristics, such as maximum ΔCBV and time to peak to compare the differences between the groups using statistical analyses with Kruskal-Wallis nonparametric tests. SD mapping, binary coded images, and statistical analyses were all performed using the custom-written MATLAB software.
The MCAO rats were separated into two groups, a mild MCAO group (
In the normal group, CBV around the stimulating electrode increased immediately after the electrical stimulation and peaked in both hemispheres at about 105 s, as shown in two-dimensional images of the whole hemisphere (Fig. 3A, B). In the mild MCAO group, the CBV increase following electrical stimulation was also observed in both hemispheres (Fig. 3C, D). In the severe MCAO group, the CBV of the I.H decreased slightly in the region around the stimulating electrode, while the CBV of the C.H increased after electrical stimulation (Fig. 3E, F).
The maximum CBV change in both hemispheres increased by 30.13±1.16% at 104±4.16 s after stimulation in the normal group. As expected, CBV change in the I.H of the severe MCAO group (-8.98±0.72%) was significantly lower than CBV change in the corresponding C.H (30.83±0.94%) (average±SEM; p value<0.001, paired
To examine arterial responsiveness as the increase in CBV spreads throughout the infarcted brain, we segmented arteries using the WEKA methods (Fig. 4A). Representative images of the reactive artery (red color) are shown in Fig. 4B. However, segmented arteries could not be obtained from the I.H of the severe MCAO model because electrical stimulation to the I.H of the severe MCAO group did not cause a significant change in CBV. The averaged maximum change in CBV in arteries increased up to 39.91±1.57% at 93.85±4.87 s in the normal group (Fig. 4C). The CBV change in arteries of the mild MCAO group was faster (C.H, 78.45±7.19 s, p value=0.090; I.H, 53.70±14.10 s, p value=0.005) and smaller (C.H, 31.99±2.60%, p value=0.275; I.H, 26.65±7.38%, p value=0.379) than normal group in both hemispheres during electrical stimulation (Fig. 4D), which was distinct from the hemodynamic reactivity in tissue (Fig. 3G-H). In the C.H of the severe MCAO group, the reaction of the arteries was the slowest (109.45±3.58 s) and the smallest (27.75±2.34%) compared with the C.H of the other groups (Fig. 4E). The differences of the time to reach maximum CBV change of the artery were statistically significant in the C.H of the severe MCAO group compared with the normal (p value=0.023) and C.H in mild MCAO (p value=0.014) groups. However, the maximum CBV change of the tissue area in the C.H of the severe MCAO group was not significantly different among the three groups (Fig. 3G). These results suggest that in the severe MCAO group, the autoregulatory function of arteries in the contralateral hemisphere was disrupted.
We next investigated whether hemodynamic reactivity was correlated with the severity of ischemia in Fig. 5. We correlated pathophysical indexes (mNSS, weight loss, and infarct size) with the hemodynamic indexes (maximum change in pixel intensity and time to maximum pixel intensity) of segmented artery and tissue ROI. The time to peak ΔCBV in artery has the highest correlation score with infarct size among all correlations (r=0.738, p value=0.004) (Fig. 5A). Fig. 5B summarizes correlation coefficient values from correlation analysis of quantified hemodynamic values of the three groups along with pathophysical information.
The main purpose of our study was to explore the effect of cerebral ischemia on cerebral hemodynamics and the reactivity of the pial artery by employing
MCAO is an effective method to induce ischemic damage in rodent brains. With this technique, reperfusion can be easily achieved, and the duration of occlusion can be varied; thus, the degree of ischemia is effectively controlled in the MCAO model [17,21]. The gold standard to assess the effect of MCAO in rodent models is to measure neuro-behavioral scores as well as the infarct size using histopathological procedures after death . For more direct monitoring, numerous neuroimaging studies have been conducted that measured hemodynamic responses following MCAO including methodologies such as magnetic resonance imaging , positron emission tomography , and laser-Doppler flowmetry .
Our study differs from previous investigations because we assessed the severity of the MCAO and performed comparative vascular reactivity and functional hemodynamic studies based on
A dramatic hemodynamic feature of the severe MCAO animal is a decrease of CBV in the I.H following direct cortical electrical stimulation, which can induce strong neuronal activation. In the severe MCAO group, direct electrical stimulation induced neither increases in CBV nor dilations of the arteries on the I.H of the cortex. The degree of deduction greatly depends upon the level of ischemic s everity in t he MCAO group. Usually, direct cortical electrical stimulation causes an immediate CBV change in the stimulated brain hemispheres, as shown in Fig. 3. However, within the severe MCAO group, the hemisphere with the cortical infarct responded very little to direct cortical electric stimulation; if there was a response, the hemisphere exhibited a decrease in CBV. Perhaps the lack of response results from tissue failure, due to cellular losses such as the astrocytes and pericytes needed for neurovascular coupling, and from dysfunctional angiogenesis. Also, the decreased artery reactivity in the C.H of the severe MCAO group might be due to a broad infarction area in the cortex and, thus, reflects the severity of ischemia.
Overall, the hemodynamic measurements of pial artery responses in the MCAO groups are best correlated with infarct size and neurological severity scores. Therefore,