Magnetic Resonance Imaging xxx (2015) xxx–xxx
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Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling Chien-Hsiang Huang a, b, Yen-Yu Ian Shih c, Tiing-Yee Siow d, Yi-Hua Hsu b, Chiao-Chi V. Chen b, Teng-Nan Lin b, Fu-Shan Jaw a,⁎, Chen Chang b,⁎⁎ a
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan Institute of Biomedical Sciences, Academic Sinica, Taipei, Taiwan Experimental Neuroimaging Laboratory, Department of Neurology and Biomedical Research Imaging Center, University of North Carolina, Chapel Hill, NC, USA d Department of Medical Imaging and Intervention, Chang-Gung Memorial Hospital, Chang-Gung University College of Medicine, Taoyuan, Taiwan b c
a r t i c l e
i n f o
Article history: Received 13 October 2014 Revised 13 March 2015 Accepted 26 April 2015 Available online xxxx Keywords: Cerebral ischemia MRI Proangiogenesis Vascular functionality Vascular reactivity
a b s t r a c t Postischemic angiogenesis is an important recovery mechanism. Both arteries and veins are upregulated during angiogenesis, but eventually there are more angiogenic veins than arteries in terms of number and length. It is critical to understand how the veins are modulated after ischemia and then transitioned into angiogenic vessels during the proangiogenic stage to finally serve as a restorative strength to the injured area. Using a rat model of transient focal cerebral ischemia, the hypercapnic blood oxygen level-dependent (BOLD) response was used to evaluate vascular reactivity, while the hyperoxic BOLD and tissue oxygen level-dependent (TOLD) responses were used to evaluate the vascular functionality at 1, 3, and 7 days after ischemia. Vessel-like venous signals appeared on R2* maps on days 3 and 7, but not on day 1. The large hypercapnic BOLD responses on days 3 and 7 indicated that these areas have high vascular reactivity. The temporal correlation between vascular reactivity and the immunoreactivity to desmin and VEGF further indicates that the integrity of vascular reactivity is associated with the pericyte coverage as regulated by the VEGF level. Vascular functionality remained low on days 1, 3, and 7, as reflected by the small hyperoxic BOLD and large hyperoxic TOLD responses, indicating the low oxygen consumption of the ischemic tissues. These functional changes in proangiogenic veins may be critical for angiogenesis. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Postischemic angiogenesis is an important recovery mechanism that helps restore the affected tissue [1,2]. Both arteries and veins are increased during angiogenesis, but there is a differential regulation by angiogenic molecules on these two categories of blood vessels. This eventually results in more angiogenic veins than arteries in terms of number and length [3]. Blood oxygen level-dependent (BOLD) MRI is a useful technique to monitor venous vessels, and their ability to capture post-ischemic angiogenesis has been demonstrated previously [4,5]. These earlier studies show that enhanced venous angiogenesis is associated with increased cerebral blood flow (CBF), axonal remodeling, and better prognosis.
⁎ Correspondence to: F. S. Jaw, Institute of Biomedical Engineering, National Taiwan University, Taipei, 10051, Taiwan. Fax: +886 2 33665268. ⁎⁎ Correspondence to: C. Chang, Institute of Biomedical Sciences, Academia Sinica, Taipei, 11529, Taiwan. Fax: + 886 2 27887641. E-mail addresses: [email protected] (F-S. Jaw), [email protected] (C. Chang).
The proangiogenic stage is a critical phase during which the vascular function of the original vessels is modified and the angiogenic vessels are induced [6]. This period occurs before the completion of angiogenesis, which is reportedly to take place at 2–28 days [4,7–9] after a stroke event, with the delay varying with the stroke model and method of investigation. Investigations into the proangiogenic stage provide critical information for understanding how the veins are modulated after ischemia and then transitioned into angiogenic vessels to serve as a restorative strength to the injured area. Previous studies have found enlarged vessel size at the proangiogenic stage [8,10,11], resulting in an elevated cerebral blood volume and CBF [5,12]. Interestingly, increased vascular permeability [8,13] and pericyte loss [14,15] have also been reported in these vessels. However, whether the hyperperfusion during the proangiogenic period indeed cause the supplied tissue to become highly oxygenated is unknown since the vasoregulation is highly compromised at the time. The vascular reactivity and functionality, which determine the vascular capacity to deliver oxygen to tissues, have yet to be established in the proangiogenic stage. Vascular reactivity is defined as the ability of the vasculature to dilate in response to an elevated partial pressure of carbon dioxide
http://dx.doi.org/10.1016/j.mri.2015.04.009 0730-725X/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
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(pCO2) level [16]. BOLD MRI measures vascular reactivity by observing changes in vascular oxygen saturation (sO2) in response to a hypercapnic challenge [17]. The hypercapnic challenge induces vasodilation in intact vessels and thus increases CBF, sO2, and the BOLD signal [17]. Previous studies have demonstrated reduced vascular reactivity after stroke in the ischemic region and its recovery within 24 hours after a mild ischemic insult [18–20]. However, the physiological significance of the improved vascular reactivity and its relation to proangiogenic vascular remodeling remain to be determined. Vascular functionality is defined as the ability of the vasculature to increase sO2 in response to an elevated partial pressure of oxygen (pO2). Oxyhemoglobin enters the brain and delivers oxygen to a region with normal or high consumption, resulting in the accumulation of deoxyhemoglobin in the vessels. After a hyperoxic challenge, the elevated pO2 level converts the deoxyhemoglobin into oxyhemoglobin, and once the sO2 level is saturated the additional oxygen dissolves in the plasma to form molecular oxygen according to the oxygen dissociation curve [21]. The basal sO2 level is high in regions with low oxygen consumption, and a larger amount of molecular oxygen will be produced during the hyperoxic challenge [21]. BOLD MRI is sensitive to the changes in sO2 level, while tissue oxygen level-dependent (TOLD) MRI is sensitive to alterations in the molecular-oxygen level during the hyperoxic challenge [21,22]. Shen et al. reported an exaggerated hyperoxic BOLD response in the penumbra at 30 minutes after ischemia, and a reduced hyperoxic BOLD response and increased TOLD response in the core at 24 hours after reperfusion. The vascular functionality has been explored for acute stroke [23–26], but it remains poorly identified during the proangiogenic stage. The aim of the present study was to elucidate the venous reactivity and functionality during the proangiogenic stage after ischemia. To achieve this goal, the hypercapnic BOLD response and the hyperoxic BOLD and TOLD responses were measured from the subacute to the proangiogenic stage in a 60-minute three-vessel-occlusion rat model. Furthermore, evaluations of vessel density, pericyte coverage, and vascular remodeling by immunohistochemistry were performed to correlate these features with the MRI results.
2. Materials and methods 2.1. Stroke model All experimental procedures were approved by the Institute of Animal Care and Utilization Committee at Academia Sinica, Taipei, Taiwan. Six male Sprague–Dawley rats were used for the MRI experiments. The rats were subjected to focal cerebral ischemia at 8 weeks old, as described previously [27]. In brief, the right middle cerebral artery of each rat was transiently ligated under stereomicroscopy, and then the common carotid arteries on both sides were also occluded using nontraumatic aneurysm clips. After 60 minutes, reperfusion was accomplished by releasing all of the arterial occlusions. The rectal temperature of the anesthetized rats was maintained at 37.0 ± 0.5 °C using a homeothermic blanket (Harvard, Holliston, MA, USA). To assure successful hypercapnic and hyperoxic challenges while minimizing the disturbance of the MRI measurements, a second batch of nine rats (n = 3 for each time point) were prepared identically to those used in the imaging studies for measurement of physiological parameters. The femoral artery was cannulated with PE-50 tubing to allow blood pressure monitoring, and arterial blood samples were drawn from the same femoral artery for blood gas analysis of pO2, pCO2, and sO2, which was achieved using a blood gas analyzer (ABL5, Radiometer America, Westlake, OH, USA), in each gas-inhaling condition. The physiological data are summarized in Table 1. To correlate MRI parameters and immunohistological data, a third batch of nine rats were included in the experiment. Immediately
after the MRI acquisition, three rats were sacrificed at each time point for immunohistology. 2.2. MRI experiments All experiments were performed on a 7 T PharmaScan 70/16 MR scanner (Bruker, Germany) with an active shielding gradient (30 G/cm in 80 μs). Images were acquired using a 72-mm birdcage transmitter coil for excitation and a separate quadratic surface coil for signal detection. Each rat was initially anesthetized with 5% isoflurane at an air flow of 2 L/minute. When fully anesthetized, the animals were placed in a prone position and fitted with a custom-designed head holder inside the magnet. Anesthesia was maintained by the administration of 2% isoflurane via a nose piece throughout the experiments. The temperature was maintained at 37.0 ± 0.5 °C, and the breathing rate was kept at 55 ± 5 breaths/min. Images were acquired at 1, 3, and 7 days after reperfusion, the period at proangiogenic stage according to our previous study [8]. T2-weighted images (T2WIs) were used to identify the lesioned region using a RARE sequence with the following parameters: TR = 6000 ms, TEeff = 80 ms, echo-train length = 8 ms, FOV = 2.56 × 2.56 cm 2, slice thickness = 0.5 mm, acquisition matrix = 256 × 256, and 2 averages. Diffusion-weighted images (DWIs) were acquired using a EPIDWI sequence with the following parameters: TR = 10000 ms, TE = 24 ms, segments = 8, δ = 4 ms, Δ = 12 ms, b values = 0 and 1100 s/mm2 along each of the 3 orthogonal diffusion gradient axes (x, y, and z), FOV = 2.56 × 2.56 cm2, slice thickness = 0.5 mm, acquisition matrix = 128 × 128 (zero-filling to 256 × 256), and 1 average. To assess hypercapnic and hyperoxic BOLD responses, an experimental pipeline introduced by Abramovitch et al. [28] was used. Hypercapnia and hyperoxia were imposed by having the animals sequentially inhale air, 5% CO2 in air (5% CO2), and 5% CO2 in oxygen (carbogen). A multigradient-echo (MGE) sequence was applied to determine the transverse relaxation rate (R2*) in each inhalation condition. Acquisition was delayed by 15 minutes after gas transition to allow complete gas exchange. MGE images were acquired using the following parameters: TR = 200 ms, initial TE = 5 ms, echo spacing = 5 ms, total number of echoes = 12, flip angle = 25°, FOV = 2.56 × 2.56 cm 2, slice thickness = 0.5 mm, acquisition matrix = 256 × 256, and 12 averages. The hyperoxic TOLD response was evaluated using ΔR1, which was determined by acquiring T1-weighted images (T1WIs) in 5%-CO2- and carbogen-inhaling conditions. T1WIs were acquired using a FLASH sequence with the following parameters: TR = 250 ms, TE = 3 ms, flip angle = 60°, FOV = 2.56 × 2.56 cm 2, slice thickness = 1 mm, acquisition matrix = 128 × 128, and 16 averages. Fig. 1 shows a schematic diagram of the experimental procedure. 2.3. Data analysis Apparent diffusion coefficient (ADC) and R2* maps were calculated from DWIs and MGE on a pixel-by-pixel basis using linear leastsquares regression with MRVision software (MRVision, Winchester, MA, USA). Vascular reactivity and vascular functionality were calculated according to the following equations:
Vascular reactivity ¼ hypercapnic ΔR2 ¼ R2 ðairÞ −R2 ð5%CO2 Þ
Vascular functionality ¼ hyperoxic ΔR2 ¼ R2 ð5%CO2 Þ −R2 ðcarbogenÞ
ð1Þ
ð2Þ
where ΔR2* is the change in R2*, and R2*(air), R2*(5%CO2), and R2*(carbogen) are the R2* maps in air-, 5%-CO2-, and carbogen-inhaling
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
C-H. Huang et al. / Magnetic Resonance Imaging xxx (2015) xxx–xxx
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Table 1 Physiological parameters. Day 1
Day 3
Air BP (mm Hg) pCO2 (mm Hg) pO2 (mm Hg) sO2 (%)
94.5 43.8 96.8 93.4
5% CO2 ± ± ± ±
11.4 6.5 17.3 2.2
92.1 62.5 103.0 98.0
± ± ± ±
10.5 5.6⁎⁎ 11.7 0.4⁎⁎
Carbogen
Air
99.5 68.6 385.4 99.6
92.1 44.1 96.1 93.6
± ± ± ±
10.6 12.5 10.3†† 0.5††
Day 7 5% CO2
± ± ± ±
16.4 9.0 24.1 2.8
90.5 60.3 100.8 98.3
± ± ± ±
13.5 7.2⁎⁎ 15.9 0.8⁎⁎
Carbogen
Air
91.8 58.6 381.6 99.6
101.0 42.0 101.3 94.0
± ± ± ±
11.2 6.1 21.3†† 0.5††
5% CO2 ± ± ± ±
7.4 7.8 10.5 3.6
91.2 62.6 110.0 98.6
± ± ± ±
Carbogen 9.1 5.5⁎⁎ 14.9 0.5⁎⁎
89.5 64.0 383.5 99.6
± ± ± ±
11.4 6.9 15.4†† 0.5††
BP, blood pressure. Data are mean and SD values. ⁎⁎ P b 0.01 for comparison between air- and 5%-CO2-inhaling conditions. †† P b 0.01 for comparison between 5%-CO2- and carbogen-inhaling conditions.
conditions, respectively. The molecular-oxygen level was calculated using MRVision according to the following equation: ! Scarbogen 1 ln S5%CO2 TR
Molecular oxygen level ¼ hyperoxic ΔR1 ¼
ð3Þ
where Scarbogen and S5%CO2 are the signal intensities on T1WIs in the 5%-CO2- and carbogen-inhaling conditions, respectively. The remodeled venous vessels were identified in the lesioned cortex based on the evolutions of hyperintense signals on R2* maps, as described by Ding et al. [4]. Regions of interest (ROIs) in the lesioned area were defined manually according to the hyperintense signals on T2WI, as shown in Fig. 2a. The ROIs were used to measure R2* and derive the ratio of the pixels indicative of hyperintense signals on R2* maps at all imaging time points. The hyperintense signals on R2* maps were extracted using MATLAB (MathWorks, Natick, MA, USA) by applying an adaptive threshold filter with a window size of 7 × 7 to suppress the background signal enhancement due to edema [29]. Pixels with R2* values higher than the median within the 7 × 7 matrix were colored red and defined as hyperintense signals. The ratio of the number of pixels indicating hyperintense signals was then calculated by dividing the number of extracted pixels by the total number of pixels in each ROI. The hypercapnic and hyperoxic ΔR2* values of the extracted pixels were quantified in both cortices. The pixels for vessels in a corresponding area in the contralateral cortex were extracted using the same procedure as described above. The ΔR1 was measured throughout the ROIs in both cortices. 2.4. Immunohistological analysis The rats after measurement of physiological parameters were sacrificed by transcardial perfusion with normal saline under chloral
hydrate (360 mg/kg body weight) anesthesia followed by 4% paraformaldehyde. The brains were removed and kept in the 4% paraformaldehyde for post-fixation by 6 hours. After the post-fixation, brains were paraffin-embedded and sectioned at 5 μm. To evaluate the immunoreactivity to endothelial cells, pericytes, and vascular remodeling, anti-RECA-1 (1:500, Abcam, MA, USA), anti-desmin (1:1000; Dako, CA, USA), and anti-VEGF (1:400, Thermo Scientific, CA, USA) were stained in the brain sections respectively. The stained sections were examined by a light microscopy (BX51; Olympus, Tokyo, Japan). The vessel density derived from the immunoreactivity to RECA-1 and percentage of area covered by immunoreactive signals of desmin and VEGF were analyzed by ImageJ (NIH, Bethesda, Maryland, USA). 2.5. Statistical analysis Data are presented as mean ± standard deviation (SD) values. The vessel density, area fraction of hyperintensities on R2* maps, and area fraction of immunoreactivity to desmin and VEGF in the lesioned cortex at different time points were compared using Student's t-tests. Hypercapnic ΔR2*, hyperoxic ΔR2*, and hyperoxic ΔR1 were compared between ipsilateral and contralateral cortices at different time points using two-way ANOVA followed by post hoc Fisher's protected t-tests. The level of statistical significance was set at P b 0.05. The correlations were analyzed using the linear regression model. The statistical significance was determined by Pearson's test of correlation. 3. Results The evolutions of the lesioned area on T2WIs and DWIs are shown in Fig. 2A and B respectively as reported previously [12]. Fig. 2C shows R2* maps obtained in air-inhaling conditions over 7 days. In the lesioned cortex, few hyperintense signals were observed on day 1, with an R2* value of 19.74 ± 2.11 s −1 (Fig. 2C, left). This reflects
Fig. 1. Schematic diagram of the experimental procedure. The MGE sequence was used to acquire data in air-, 5%-CO2-, and carbogen-inhaling conditions to derive hypercapnic and hyperoxic BOLD responses. T1-weighted FLASH sequences were used to acquire data in 5%-CO2- and carbogen-inhaling conditions to derive hyperoxic TOLD responses.
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
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Fig. 2. Temporal changes on T2WIs, DWIs, and R2* maps. (A) Temporal evolution of T2WIs. (B) Temporal evolution of DWIs. (C) Temporal evolution of R2* maps. The representative ROIs in contralateral and ipsilateral cortices are indicated by dotted lines on T2WI image on day 1. (D) Hyperintense-R2* voxels extracted according to the adaptive threshold in the lesioned cortex on days 1, 3, and 7. (E) Quantitative analysis of the venous area over 7 days. Data are mean and SD values; **P b 0.01.
the edema and low concentration of deoxyhemoglobin in this area on day 1. However, increased hyperintense signals in the hypointense area were evident on day 3, with an R2* value of 26.13 ± 4.33 s −1 (Fig. 2C, middle). The hyperintense signals were reduced on day 7, with an R2* value of 35.26 ± 3.15 s −1 (Fig. 2C, right). The evolutions of R2* signals over time suggest the occurrence of vessels with proangiogenic remodeling on days 3 and 7 along with the changes in edema. Fig. 2D shows the hyperintense-R2* voxels on R2* maps. The ischemic area occupied by the hyperintense-R2* voxels in the ipsilateral cortex
was 8.87 ± 9.61% on day 1, 37.84 ± 6.49% on day 3, and 39.16 ± 7.90% on day 7 (Fig. 2E). The percentage was low in the ipsilateral cortex and significantly increased on days 3 and 7 (P b 0.01 from day 1 to day 3 and from day 1 to day 7; Fig. 2E). These data also indicate the proangiogenic vascular remodeling on days 3 and 7. The temporal evolutions of vascular reactivity revealed by hypercapnic BOLD response are shown in Fig. 3. Two-way ANOVAs indicate the significant interaction between time and cortical sides (F(2,30) = 50.218, P b 0.0001). Fisher's post hoc tests indicate that
Fig. 3. Temporal changes in hypercapnic BOLD response. (A) Hypercapnic BOLD response represented by the ΔR2* maps derived from R2* maps in air- and 5%-CO2-inhaling conditions on days 1, 3, and 7. (B) Quantitative analysis of hypercapnic BOLD response over 7 days. Data are mean and SD values; *P b 0.05; **P b 0.01.
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
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the hypercapnic BOLD response was lower in ipsilateral than the contralateral vessels on day 1 (P b 0.01), implying that the vascular reactivity was impaired. The hypercapnic BOLD response in ipsilateral vessels was significantly increased on day 3 and then decreased on day 7 (P b 0.01 from day 1 to day 3 and from day 1 to day 7; P b 0.05 from day 3 to day 7). The hypercapnic BOLD response in ipsilateral vessels surpassed that in the contralateral cortex on days 3 and 7 (P b 0.05 on day 3; P b 0.01 on day 7), suggesting the exaggerated vascular reactivity. Changes in hypercapnic BOLD response in contralateral vessels from day 1 to day 7 were not statistically significant (P N 0.05). The pCO2 significantly increased from air- to 5%-CO2-inhaling condition at each time point, indicating successful hypercapnic challenge (P b 0.01, Table 1). No significant change in blood pressure, pO2, or sO2 was observed between air- and 5%-CO2-inhaling conditions (P N 0.05). The temporal evolutions of vascular functionality revealed by hyperoxic BOLD response are shown in Fig. 4. The hyperoxic BOLD response remained small from day 1 to day 7 in the ipsilateral compared to the contralateral vessels (F(1,30) = 206.955, P b 0.0001; P b 0.01), indicating low vascular functionality. Although the vascular functionality was increased from day 3 to day 7 (F(2,30) = 3.771, P b 0.05; P b 0.05 from day 3 to day 7), the increaments are much smaller compared to the contralateral vessels. This suggests that the sO2 level was saturated before the hyperoxic challenge in the ipsilateral vessels. No significant difference in hyperoxic BOLD response was observed over time in the contralateral vessels (P N 0.05). The pO2 and sO2 significantly increased from 5%-CO2- to carbogen-inhaling condition at each time point, indicating successful hyperoxic challenge (P b 0.01, Table 1). No significant change in blood pressure or pCO2 was observed between 5%-CO2 and carobgen-inhaling conditions (P N 0.05). The hyperoxic TOLD response was determined by calculating ΔR1 between the 5%-CO2- to carbogen-inhaling conditions (Fig. 5). A threefold increase in ΔR1 was observed on days 1 and 3 in the ipsilateral cortex (F(1,30) = 1070.757, P b 0.0001; P b 0.01) compared with contralateral cortex. Consistent with the result of hyperoxic BOLD response, this also implies that the sO2 level was saturated in the lesioned cortex, and excess oxygen dissolved in the plasma after the hyperoxic challenge. On day 7, the ΔR1 observed in the ipsilateral cortex was significantly decreased relative to that observed on days 1 and 3 (F(2,30) = 20.123, P b 0.0001; P b 0.01 from day 1 to day 7 and from day 3 to day 7), but was still much higher than that on the contralateral side (P b 0.01). This indicates that the amount of excess oxygen was reduced on day 7. No significant difference in ΔR1 was observed over time in the contralateral cortex (P N 0.05). The vessel density was evaluated by immunoreactivity to RECA-1 (Fig. 6B), which was observed in a moderate quantity on days 1 and 3 and significantly increased on day 7 (P b 0.01 from day 1 to day 7 and from day 3 to day 7) as reported previously [8]. The pericyte
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coverage was evaluated by immunoreactivity to desmin (Fig. 6C), which was rarely observed in the lesioned cortex on day 1, significantly increased on day 3, and decreased by two fold on day 7 (P b 0.01 from day 1 to days 3 and 7 and from day 3 to day 7). VEGF was scarcely distributed in the neural cells in the lesioned cortex on day 1, significantly increased in the shrunken cells and perivascular regions on day 3, and then became less observed on day 7 (Fig. 6D). The evolutions in immunoreactivity to VEGF have a similar pattern as that of desmin (P b 0.01 from day 1 to days 3 and 7 and from day 3 to day 7; Fig. 6D). Fig. 7 shows the correlations between the MRI parameters and immunohistological data. Strong positive correlations were observed between hypercapnic ΔR2* and desmin area fraction (R2 = 0.8751, P b 0.0001, Fig. 7A) and between hyperoxic ΔR2* and vessel density (R2 = 0.8241, P b 0.0001, Fig. 7B). However, weak correlations were observed between hypercapnic ΔR2* and vessel density (R2 = 0.0496, P N 0.05, data not shown) as well as between hyperoxic ΔR2* and desmin area fraction (R2 = 0.0053, P N 0.05, data not shown). Additionally, a significant correlation was found between ADC and hypercapnic ΔR2* (R2 = 0.4366, P b 0.001, correlation plot not shown). 4. Discussion This study used BOLD and TOLD MRI with hypercapnic and hyperoxic challenges to investigate the venous reactivity and functionality during the proangiogenic stage after ischemia. The vessel-like venous signals on R2* maps appeared on days 3 and 7, but not on day 1. The large hypercapnic BOLD response on days 3 and 7 indicated that these areas have high vascular reactivity. The temporal correlation between vascular reactivity and the immunoreactivity to desmin further indicates that the integrity of vascular reactivity is associated with the pericyte coverage. Vascular functionality remained low on days 1, 3, and 7, as reflected by the small hyperoxic BOLD and large hyperoxic TOLD responses, indicating the low oxygen consumption of the ischemic tissues. The impaired vascular reactivity after ischemia and its recovery have been reported previously [18–20], but the underlying mechanism and its relation to proangiogenic venous remodeling remain obscure. Recently, Wegner et al. [30] attributed the improved vascular reactivity to the presence of newly formed vessels at 14 days after reperfusion in an intraluminal thread stroke model. Here we suggest that the venous reactivity could be associated with the pericyte coverage after ischemia, which is the key component in the regulation of vasodilation and vasoconstriction [31]. The impaired vascular reactivity was observed in the present study on day 1, and the reduced pericyte coverage was also demonstrated by scarce immunoreactivity to desmin with a moderate vessel density. As suggested by recent studies, the decreased pericyte coverage may be due to the early loss of pericytes after stroke [14,15]. The venous reactivity had improved
Fig. 4. Temporal changes in hyperoxic BOLD response. (A) Hyperoxic BOLD response represented by the ΔR2* maps derived from R2* maps in 5%-CO2- and carbogen-inhaling conditions on days 1, 3, and 7. (B) Quantitative analysis of hyperoxic BOLD response over 7 days. Data are mean and SD values; *P b 0.05; **P b 0.01.
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
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Fig. 5. Temporal changes in hyperoxic TOLD response. (A) Hyperoxic TOLD response represented by the ΔR1 maps derived from T1WIs in 5%-CO2- and carbogen-inhaling conditions on days 1, 3, and 7. (B) Quantitative analysis of hyperoxic TOLD response over 7 days. Data are mean and SD values; **P b 0.01.
significantly by day 3, in association with the increased pericyte coverage and VEGF level. The increased VEGF level may play a critical role in pericyte recruitment, as reported by Zechariah et al. [32]. Parallel increased VEGF and pericyte coverage after ischemia has also been discussed by ElAli et al. [33]. Furthermore, the increased pericyte coverage may be a prerequisite for the subsequent angiogenesis [34,35]. The vessel density was increased on day 7, while both vascular reactivity and pericyte coverage were decreased, suggesting that the angiogenic vessels lack pericytes, possibly due to the reduction in VEGF. These findings are supported by the strong correlations between hypercapnic ΔR2* and desmin area fraction (R2 = 0.8751, P b 0.0001,
Fig. 7A), and between desmin and VEGF area fraction (R2 = 0.8345, P b 0.0001, correlation plot not shown). In addition, VEGF production was known to be induced by hypoxia [36], and thus the reduction of VEGF from day 3 to day 7 may be due to the lower hypoxic stress relieved by angiogenesis. Inflammation, a common process after stroke, may also affect the vascular reactivity through the endothelial dysfunction [37] and the activation of astrocyte endfeet [38]. The endothelial dysfunction after ischemia has been reported to be associated with decreased nitride oxide availability, where both the basal CBF and the vascular reactivity would be decreased [37]. The activated astrocyte endfeet
Fig. 6. Immunohistochemistry of the vessel density, pericyte coverage, and vascular remodeling. (A) Representative image of the selected two regions in each brain slice for quantification as indicated by the rectangular. Temporal changes in immunoreactivity to RECA-1 (B), desmin (C), and VEGF (D) and quantitative analysis over 7 days. Images were magnified from the regions 2, 4, and 6, respectively. Data are mean and SD values; **P b 0.01.
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
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Fig. 7. Correlations among the MRI parameters and immunohistological data. Two ROIs as shown in Fig. 6 were measured in each rat. (A) The correlation between hypercapnic ΔR2* and desmin area fraction. (B) The correlation between hyperoxic ΔR2* and vessel density as indicated by RECA-1.
after ischemia has recently been reported to cause vasodilation and increased basal CBF [38], which may result in an influenced vascular reactivity as well. These inflammatory markers were not investigated in the present study, but should be taken into consideration when interpreting the results of vascular reactivity. The detectability of DWIs in ischemic tissue at acute stage has been shown in preclinical [39] and clinical [40] studies, whereas its usage for detecting the remodeled vessels at the proangiogenic stage has not been demonstrated. In the present study, the significant correlation between ADC and hypercapnic ΔR2* at the proangiogenic stage was observed (R 2 = 0.4366, P b 0.001, correlation plot not shown). Previous studies indicated that the vascular reactivity was impaired while the ADC was already recovered within 24 hours after reperfusion [19,41]. In addition, Shen et al. reported that the impaired vascular reactivity preceded the ADC decrease at 30 minutes after ischemia [42]. These studies reported the changes between ADC and vascular reactivity at the acute stage. Our results further reveal the correlation between ADC and vascular reactivity at the proangiogenic stage, but its underlying mechanism remains to be studied. Reduced venous functionality may be caused by a high basal sO2 level in the regions with low oxygen consumption. For example, as shown in the previous studies, the oxygen consumption is low in the ischemic core and the hyperoxic BOLD response is markedly reduced in the acute phase of stroke [23–26]. Similarly, the small hyperoxic BOLD response in the present study from day 1 to day 7 may reflect a low oxygen consumption in the lesioned cortex. The large hyperoxic TOLD response from day 1 to day 7 represents further evidence of the high basal sO2 level, since the molecular-oxygen level is more directly related to tissue pO2 and ΔR1 is a more sensitive parameter for detecting fully oxygenated tissues [43–45]. Similarly, Shen et al. [26] reported a small hyperoxic BOLD response with a large ΔR1 in the ischemic core at 24 hours after reperfusion. The findings of the present study demonstrate that this phenomenon is actually maintained until day 7. In previous studies, the hypercapnic and hyperoxic BOLD responses have together been used to identify the immature tumor vessels [28,46,47]. These studies described that immature vessels lack pericyte coverage with a high oxygen extraction fraction, resulting in low vascular reactivity and enhanced vascular functionality. In the present study, the proangiogenic venous vessels with extensive pericyte coverage on day 3 showed high vascular reactivity and low vascular functionality, both indicating maturity of these vessels. From day 3 to day 7, the vascular reactivity was decreased, and the vascular functionality was increased, which may be attributed to the formation of new immature vessels. This is supported by the strong correlation between hyperoxic ΔR2* and vessel density by RECA-1 (R2 = 0.8241, P b 0.0001, Fig. 7B). Furthermore, to our knowledge the present study is the first to investigate both hypercapnic and hyperoxic BOLD responses in the remodeled vessels after ischemia. Many factors may have influenced the measurements in this study. First, the hyperintense signals on R2* maps were based on
BOLD contrast, and vessels of arterial origins might not be detected using this method due to the high sO2 level [17]. The vessel-like hyperintense signals on R2* maps were absent at 1 day but appeared at 3 and 7 days after ischemia (Fig. 2). However, RECA-1 shows a moderate vessel density on days 1 and 3 and significantly increased vessel density on day 7 (Fig. 6). The discrepancy may be due to the vascular signals on R2* maps mainly originating from veins, while RECA-1 reveals both arterial and venous vessels. Second, T1-shortening effects (e.g., the inflow and enhancement of molecular oxygen) during hypercapnic and hyperoxic challenges can increase the signal intensities on T2*WIs and cause erroneous ΔR2* values. Therefore, curve-fitted R2* maps were used to derive ΔR2* values in the present study in order to reduce the influence of such T1-shortening effects [48,49]. Third, isoflurane used in the present study is a vasodilator, and the increase in basal CBF by isoflurane may cause overestimation of the T2 and T2* values [50]. In addition, the repeated isoflurane exposure of the rats may affect the cerebral autoregulation [51]. However, the optimized anesthesia protocol with minimal physiological disturbance remains a challenge. In conclusion, the present study found that vessel-like venous signals on R2* maps appeared at 3 and 7 days after ischemia, but not after 1 day, and that these areas have high vascular reactivity and reduced vascular functionality. The integrity of the venous reactivity may be associated with the pericyte coverage as regulated by the VEGF level. The reduced venous functionality may be due to the low oxygen consumption of the ischemic tissue. Information about these vascular variables in proangiogenic vessels could be critically useful when developing novel angiogenic therapies for stroke. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments Y.Y.I.S. was supported by AHA 15SDG23260025, NIH R01NS091236, NARSAD Young Investigator Award, NCTraCS 550KR81420 and 550KR91413 (parent award: 1UL1TR001111), UNC S14 University Research Council Award and UNC Junior Faculty Development Award. Y.Y.I.S. is also an Ellen Schapiro and Gerald Axelbaum Investigator supported by the Brain & Behavior Research Foundation. References [1] Seevinck PR, Deddens LH, Dijkhuizen RM. Magnetic resonance imaging of brain angiogenesis after stroke. Angiogenesis 2010;13:101–11. [2] Jiang Q, Zhang ZG, Chopp M. MRI of stroke recovery. Stroke 2010;41:410–4. [3] Blebea J, Vu JH, Assadnia S, McLaughlin PJ, Atnip RG, Zagon IS. Differential effects of vascular growth factors on arterial and venous angiogenesis. J Vasc Surg 2002; 35:532–8.
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
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Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
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Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling Chien-Hsiang Huang a, b, Yen-Yu Ian Shih c, Tiing-Yee Siow d, Yi-Hua Hsu b, Chiao-Chi V. Chen b, Teng-Nan Lin b, Fu-Shan Jaw a,⁎, Chen Chang b,⁎⁎ a
Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan Institute of Biomedical Sciences, Academic Sinica, Taipei, Taiwan Experimental Neuroimaging Laboratory, Department of Neurology and Biomedical Research Imaging Center, University of North Carolina, Chapel Hill, NC, USA d Department of Medical Imaging and Intervention, Chang-Gung Memorial Hospital, Chang-Gung University College of Medicine, Taoyuan, Taiwan b c
a r t i c l e
i n f o
Article history: Received 13 October 2014 Revised 13 March 2015 Accepted 26 April 2015 Available online xxxx Keywords: Cerebral ischemia MRI Proangiogenesis Vascular functionality Vascular reactivity
a b s t r a c t Postischemic angiogenesis is an important recovery mechanism. Both arteries and veins are upregulated during angiogenesis, but eventually there are more angiogenic veins than arteries in terms of number and length. It is critical to understand how the veins are modulated after ischemia and then transitioned into angiogenic vessels during the proangiogenic stage to finally serve as a restorative strength to the injured area. Using a rat model of transient focal cerebral ischemia, the hypercapnic blood oxygen level-dependent (BOLD) response was used to evaluate vascular reactivity, while the hyperoxic BOLD and tissue oxygen level-dependent (TOLD) responses were used to evaluate the vascular functionality at 1, 3, and 7 days after ischemia. Vessel-like venous signals appeared on R2* maps on days 3 and 7, but not on day 1. The large hypercapnic BOLD responses on days 3 and 7 indicated that these areas have high vascular reactivity. The temporal correlation between vascular reactivity and the immunoreactivity to desmin and VEGF further indicates that the integrity of vascular reactivity is associated with the pericyte coverage as regulated by the VEGF level. Vascular functionality remained low on days 1, 3, and 7, as reflected by the small hyperoxic BOLD and large hyperoxic TOLD responses, indicating the low oxygen consumption of the ischemic tissues. These functional changes in proangiogenic veins may be critical for angiogenesis. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Postischemic angiogenesis is an important recovery mechanism that helps restore the affected tissue [1,2]. Both arteries and veins are increased during angiogenesis, but there is a differential regulation by angiogenic molecules on these two categories of blood vessels. This eventually results in more angiogenic veins than arteries in terms of number and length [3]. Blood oxygen level-dependent (BOLD) MRI is a useful technique to monitor venous vessels, and their ability to capture post-ischemic angiogenesis has been demonstrated previously [4,5]. These earlier studies show that enhanced venous angiogenesis is associated with increased cerebral blood flow (CBF), axonal remodeling, and better prognosis.
⁎ Correspondence to: F. S. Jaw, Institute of Biomedical Engineering, National Taiwan University, Taipei, 10051, Taiwan. Fax: +886 2 33665268. ⁎⁎ Correspondence to: C. Chang, Institute of Biomedical Sciences, Academia Sinica, Taipei, 11529, Taiwan. Fax: + 886 2 27887641. E-mail addresses: [email protected] (F-S. Jaw), [email protected] (C. Chang).
The proangiogenic stage is a critical phase during which the vascular function of the original vessels is modified and the angiogenic vessels are induced [6]. This period occurs before the completion of angiogenesis, which is reportedly to take place at 2–28 days [4,7–9] after a stroke event, with the delay varying with the stroke model and method of investigation. Investigations into the proangiogenic stage provide critical information for understanding how the veins are modulated after ischemia and then transitioned into angiogenic vessels to serve as a restorative strength to the injured area. Previous studies have found enlarged vessel size at the proangiogenic stage [8,10,11], resulting in an elevated cerebral blood volume and CBF [5,12]. Interestingly, increased vascular permeability [8,13] and pericyte loss [14,15] have also been reported in these vessels. However, whether the hyperperfusion during the proangiogenic period indeed cause the supplied tissue to become highly oxygenated is unknown since the vasoregulation is highly compromised at the time. The vascular reactivity and functionality, which determine the vascular capacity to deliver oxygen to tissues, have yet to be established in the proangiogenic stage. Vascular reactivity is defined as the ability of the vasculature to dilate in response to an elevated partial pressure of carbon dioxide
http://dx.doi.org/10.1016/j.mri.2015.04.009 0730-725X/© 2015 Elsevier Inc. All rights reserved.
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
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(pCO2) level [16]. BOLD MRI measures vascular reactivity by observing changes in vascular oxygen saturation (sO2) in response to a hypercapnic challenge [17]. The hypercapnic challenge induces vasodilation in intact vessels and thus increases CBF, sO2, and the BOLD signal [17]. Previous studies have demonstrated reduced vascular reactivity after stroke in the ischemic region and its recovery within 24 hours after a mild ischemic insult [18–20]. However, the physiological significance of the improved vascular reactivity and its relation to proangiogenic vascular remodeling remain to be determined. Vascular functionality is defined as the ability of the vasculature to increase sO2 in response to an elevated partial pressure of oxygen (pO2). Oxyhemoglobin enters the brain and delivers oxygen to a region with normal or high consumption, resulting in the accumulation of deoxyhemoglobin in the vessels. After a hyperoxic challenge, the elevated pO2 level converts the deoxyhemoglobin into oxyhemoglobin, and once the sO2 level is saturated the additional oxygen dissolves in the plasma to form molecular oxygen according to the oxygen dissociation curve [21]. The basal sO2 level is high in regions with low oxygen consumption, and a larger amount of molecular oxygen will be produced during the hyperoxic challenge [21]. BOLD MRI is sensitive to the changes in sO2 level, while tissue oxygen level-dependent (TOLD) MRI is sensitive to alterations in the molecular-oxygen level during the hyperoxic challenge [21,22]. Shen et al. reported an exaggerated hyperoxic BOLD response in the penumbra at 30 minutes after ischemia, and a reduced hyperoxic BOLD response and increased TOLD response in the core at 24 hours after reperfusion. The vascular functionality has been explored for acute stroke [23–26], but it remains poorly identified during the proangiogenic stage. The aim of the present study was to elucidate the venous reactivity and functionality during the proangiogenic stage after ischemia. To achieve this goal, the hypercapnic BOLD response and the hyperoxic BOLD and TOLD responses were measured from the subacute to the proangiogenic stage in a 60-minute three-vessel-occlusion rat model. Furthermore, evaluations of vessel density, pericyte coverage, and vascular remodeling by immunohistochemistry were performed to correlate these features with the MRI results.
2. Materials and methods 2.1. Stroke model All experimental procedures were approved by the Institute of Animal Care and Utilization Committee at Academia Sinica, Taipei, Taiwan. Six male Sprague–Dawley rats were used for the MRI experiments. The rats were subjected to focal cerebral ischemia at 8 weeks old, as described previously [27]. In brief, the right middle cerebral artery of each rat was transiently ligated under stereomicroscopy, and then the common carotid arteries on both sides were also occluded using nontraumatic aneurysm clips. After 60 minutes, reperfusion was accomplished by releasing all of the arterial occlusions. The rectal temperature of the anesthetized rats was maintained at 37.0 ± 0.5 °C using a homeothermic blanket (Harvard, Holliston, MA, USA). To assure successful hypercapnic and hyperoxic challenges while minimizing the disturbance of the MRI measurements, a second batch of nine rats (n = 3 for each time point) were prepared identically to those used in the imaging studies for measurement of physiological parameters. The femoral artery was cannulated with PE-50 tubing to allow blood pressure monitoring, and arterial blood samples were drawn from the same femoral artery for blood gas analysis of pO2, pCO2, and sO2, which was achieved using a blood gas analyzer (ABL5, Radiometer America, Westlake, OH, USA), in each gas-inhaling condition. The physiological data are summarized in Table 1. To correlate MRI parameters and immunohistological data, a third batch of nine rats were included in the experiment. Immediately
after the MRI acquisition, three rats were sacrificed at each time point for immunohistology. 2.2. MRI experiments All experiments were performed on a 7 T PharmaScan 70/16 MR scanner (Bruker, Germany) with an active shielding gradient (30 G/cm in 80 μs). Images were acquired using a 72-mm birdcage transmitter coil for excitation and a separate quadratic surface coil for signal detection. Each rat was initially anesthetized with 5% isoflurane at an air flow of 2 L/minute. When fully anesthetized, the animals were placed in a prone position and fitted with a custom-designed head holder inside the magnet. Anesthesia was maintained by the administration of 2% isoflurane via a nose piece throughout the experiments. The temperature was maintained at 37.0 ± 0.5 °C, and the breathing rate was kept at 55 ± 5 breaths/min. Images were acquired at 1, 3, and 7 days after reperfusion, the period at proangiogenic stage according to our previous study [8]. T2-weighted images (T2WIs) were used to identify the lesioned region using a RARE sequence with the following parameters: TR = 6000 ms, TEeff = 80 ms, echo-train length = 8 ms, FOV = 2.56 × 2.56 cm 2, slice thickness = 0.5 mm, acquisition matrix = 256 × 256, and 2 averages. Diffusion-weighted images (DWIs) were acquired using a EPIDWI sequence with the following parameters: TR = 10000 ms, TE = 24 ms, segments = 8, δ = 4 ms, Δ = 12 ms, b values = 0 and 1100 s/mm2 along each of the 3 orthogonal diffusion gradient axes (x, y, and z), FOV = 2.56 × 2.56 cm2, slice thickness = 0.5 mm, acquisition matrix = 128 × 128 (zero-filling to 256 × 256), and 1 average. To assess hypercapnic and hyperoxic BOLD responses, an experimental pipeline introduced by Abramovitch et al. [28] was used. Hypercapnia and hyperoxia were imposed by having the animals sequentially inhale air, 5% CO2 in air (5% CO2), and 5% CO2 in oxygen (carbogen). A multigradient-echo (MGE) sequence was applied to determine the transverse relaxation rate (R2*) in each inhalation condition. Acquisition was delayed by 15 minutes after gas transition to allow complete gas exchange. MGE images were acquired using the following parameters: TR = 200 ms, initial TE = 5 ms, echo spacing = 5 ms, total number of echoes = 12, flip angle = 25°, FOV = 2.56 × 2.56 cm 2, slice thickness = 0.5 mm, acquisition matrix = 256 × 256, and 12 averages. The hyperoxic TOLD response was evaluated using ΔR1, which was determined by acquiring T1-weighted images (T1WIs) in 5%-CO2- and carbogen-inhaling conditions. T1WIs were acquired using a FLASH sequence with the following parameters: TR = 250 ms, TE = 3 ms, flip angle = 60°, FOV = 2.56 × 2.56 cm 2, slice thickness = 1 mm, acquisition matrix = 128 × 128, and 16 averages. Fig. 1 shows a schematic diagram of the experimental procedure. 2.3. Data analysis Apparent diffusion coefficient (ADC) and R2* maps were calculated from DWIs and MGE on a pixel-by-pixel basis using linear leastsquares regression with MRVision software (MRVision, Winchester, MA, USA). Vascular reactivity and vascular functionality were calculated according to the following equations:
Vascular reactivity ¼ hypercapnic ΔR2 ¼ R2 ðairÞ −R2 ð5%CO2 Þ
Vascular functionality ¼ hyperoxic ΔR2 ¼ R2 ð5%CO2 Þ −R2 ðcarbogenÞ
ð1Þ
ð2Þ
where ΔR2* is the change in R2*, and R2*(air), R2*(5%CO2), and R2*(carbogen) are the R2* maps in air-, 5%-CO2-, and carbogen-inhaling
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
C-H. Huang et al. / Magnetic Resonance Imaging xxx (2015) xxx–xxx
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Table 1 Physiological parameters. Day 1
Day 3
Air BP (mm Hg) pCO2 (mm Hg) pO2 (mm Hg) sO2 (%)
94.5 43.8 96.8 93.4
5% CO2 ± ± ± ±
11.4 6.5 17.3 2.2
92.1 62.5 103.0 98.0
± ± ± ±
10.5 5.6⁎⁎ 11.7 0.4⁎⁎
Carbogen
Air
99.5 68.6 385.4 99.6
92.1 44.1 96.1 93.6
± ± ± ±
10.6 12.5 10.3†† 0.5††
Day 7 5% CO2
± ± ± ±
16.4 9.0 24.1 2.8
90.5 60.3 100.8 98.3
± ± ± ±
13.5 7.2⁎⁎ 15.9 0.8⁎⁎
Carbogen
Air
91.8 58.6 381.6 99.6
101.0 42.0 101.3 94.0
± ± ± ±
11.2 6.1 21.3†† 0.5††
5% CO2 ± ± ± ±
7.4 7.8 10.5 3.6
91.2 62.6 110.0 98.6
± ± ± ±
Carbogen 9.1 5.5⁎⁎ 14.9 0.5⁎⁎
89.5 64.0 383.5 99.6
± ± ± ±
11.4 6.9 15.4†† 0.5††
BP, blood pressure. Data are mean and SD values. ⁎⁎ P b 0.01 for comparison between air- and 5%-CO2-inhaling conditions. †† P b 0.01 for comparison between 5%-CO2- and carbogen-inhaling conditions.
conditions, respectively. The molecular-oxygen level was calculated using MRVision according to the following equation: ! Scarbogen 1 ln S5%CO2 TR
Molecular oxygen level ¼ hyperoxic ΔR1 ¼
ð3Þ
where Scarbogen and S5%CO2 are the signal intensities on T1WIs in the 5%-CO2- and carbogen-inhaling conditions, respectively. The remodeled venous vessels were identified in the lesioned cortex based on the evolutions of hyperintense signals on R2* maps, as described by Ding et al. [4]. Regions of interest (ROIs) in the lesioned area were defined manually according to the hyperintense signals on T2WI, as shown in Fig. 2a. The ROIs were used to measure R2* and derive the ratio of the pixels indicative of hyperintense signals on R2* maps at all imaging time points. The hyperintense signals on R2* maps were extracted using MATLAB (MathWorks, Natick, MA, USA) by applying an adaptive threshold filter with a window size of 7 × 7 to suppress the background signal enhancement due to edema [29]. Pixels with R2* values higher than the median within the 7 × 7 matrix were colored red and defined as hyperintense signals. The ratio of the number of pixels indicating hyperintense signals was then calculated by dividing the number of extracted pixels by the total number of pixels in each ROI. The hypercapnic and hyperoxic ΔR2* values of the extracted pixels were quantified in both cortices. The pixels for vessels in a corresponding area in the contralateral cortex were extracted using the same procedure as described above. The ΔR1 was measured throughout the ROIs in both cortices. 2.4. Immunohistological analysis The rats after measurement of physiological parameters were sacrificed by transcardial perfusion with normal saline under chloral
hydrate (360 mg/kg body weight) anesthesia followed by 4% paraformaldehyde. The brains were removed and kept in the 4% paraformaldehyde for post-fixation by 6 hours. After the post-fixation, brains were paraffin-embedded and sectioned at 5 μm. To evaluate the immunoreactivity to endothelial cells, pericytes, and vascular remodeling, anti-RECA-1 (1:500, Abcam, MA, USA), anti-desmin (1:1000; Dako, CA, USA), and anti-VEGF (1:400, Thermo Scientific, CA, USA) were stained in the brain sections respectively. The stained sections were examined by a light microscopy (BX51; Olympus, Tokyo, Japan). The vessel density derived from the immunoreactivity to RECA-1 and percentage of area covered by immunoreactive signals of desmin and VEGF were analyzed by ImageJ (NIH, Bethesda, Maryland, USA). 2.5. Statistical analysis Data are presented as mean ± standard deviation (SD) values. The vessel density, area fraction of hyperintensities on R2* maps, and area fraction of immunoreactivity to desmin and VEGF in the lesioned cortex at different time points were compared using Student's t-tests. Hypercapnic ΔR2*, hyperoxic ΔR2*, and hyperoxic ΔR1 were compared between ipsilateral and contralateral cortices at different time points using two-way ANOVA followed by post hoc Fisher's protected t-tests. The level of statistical significance was set at P b 0.05. The correlations were analyzed using the linear regression model. The statistical significance was determined by Pearson's test of correlation. 3. Results The evolutions of the lesioned area on T2WIs and DWIs are shown in Fig. 2A and B respectively as reported previously [12]. Fig. 2C shows R2* maps obtained in air-inhaling conditions over 7 days. In the lesioned cortex, few hyperintense signals were observed on day 1, with an R2* value of 19.74 ± 2.11 s −1 (Fig. 2C, left). This reflects
Fig. 1. Schematic diagram of the experimental procedure. The MGE sequence was used to acquire data in air-, 5%-CO2-, and carbogen-inhaling conditions to derive hypercapnic and hyperoxic BOLD responses. T1-weighted FLASH sequences were used to acquire data in 5%-CO2- and carbogen-inhaling conditions to derive hyperoxic TOLD responses.
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
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Fig. 2. Temporal changes on T2WIs, DWIs, and R2* maps. (A) Temporal evolution of T2WIs. (B) Temporal evolution of DWIs. (C) Temporal evolution of R2* maps. The representative ROIs in contralateral and ipsilateral cortices are indicated by dotted lines on T2WI image on day 1. (D) Hyperintense-R2* voxels extracted according to the adaptive threshold in the lesioned cortex on days 1, 3, and 7. (E) Quantitative analysis of the venous area over 7 days. Data are mean and SD values; **P b 0.01.
the edema and low concentration of deoxyhemoglobin in this area on day 1. However, increased hyperintense signals in the hypointense area were evident on day 3, with an R2* value of 26.13 ± 4.33 s −1 (Fig. 2C, middle). The hyperintense signals were reduced on day 7, with an R2* value of 35.26 ± 3.15 s −1 (Fig. 2C, right). The evolutions of R2* signals over time suggest the occurrence of vessels with proangiogenic remodeling on days 3 and 7 along with the changes in edema. Fig. 2D shows the hyperintense-R2* voxels on R2* maps. The ischemic area occupied by the hyperintense-R2* voxels in the ipsilateral cortex
was 8.87 ± 9.61% on day 1, 37.84 ± 6.49% on day 3, and 39.16 ± 7.90% on day 7 (Fig. 2E). The percentage was low in the ipsilateral cortex and significantly increased on days 3 and 7 (P b 0.01 from day 1 to day 3 and from day 1 to day 7; Fig. 2E). These data also indicate the proangiogenic vascular remodeling on days 3 and 7. The temporal evolutions of vascular reactivity revealed by hypercapnic BOLD response are shown in Fig. 3. Two-way ANOVAs indicate the significant interaction between time and cortical sides (F(2,30) = 50.218, P b 0.0001). Fisher's post hoc tests indicate that
Fig. 3. Temporal changes in hypercapnic BOLD response. (A) Hypercapnic BOLD response represented by the ΔR2* maps derived from R2* maps in air- and 5%-CO2-inhaling conditions on days 1, 3, and 7. (B) Quantitative analysis of hypercapnic BOLD response over 7 days. Data are mean and SD values; *P b 0.05; **P b 0.01.
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
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the hypercapnic BOLD response was lower in ipsilateral than the contralateral vessels on day 1 (P b 0.01), implying that the vascular reactivity was impaired. The hypercapnic BOLD response in ipsilateral vessels was significantly increased on day 3 and then decreased on day 7 (P b 0.01 from day 1 to day 3 and from day 1 to day 7; P b 0.05 from day 3 to day 7). The hypercapnic BOLD response in ipsilateral vessels surpassed that in the contralateral cortex on days 3 and 7 (P b 0.05 on day 3; P b 0.01 on day 7), suggesting the exaggerated vascular reactivity. Changes in hypercapnic BOLD response in contralateral vessels from day 1 to day 7 were not statistically significant (P N 0.05). The pCO2 significantly increased from air- to 5%-CO2-inhaling condition at each time point, indicating successful hypercapnic challenge (P b 0.01, Table 1). No significant change in blood pressure, pO2, or sO2 was observed between air- and 5%-CO2-inhaling conditions (P N 0.05). The temporal evolutions of vascular functionality revealed by hyperoxic BOLD response are shown in Fig. 4. The hyperoxic BOLD response remained small from day 1 to day 7 in the ipsilateral compared to the contralateral vessels (F(1,30) = 206.955, P b 0.0001; P b 0.01), indicating low vascular functionality. Although the vascular functionality was increased from day 3 to day 7 (F(2,30) = 3.771, P b 0.05; P b 0.05 from day 3 to day 7), the increaments are much smaller compared to the contralateral vessels. This suggests that the sO2 level was saturated before the hyperoxic challenge in the ipsilateral vessels. No significant difference in hyperoxic BOLD response was observed over time in the contralateral vessels (P N 0.05). The pO2 and sO2 significantly increased from 5%-CO2- to carbogen-inhaling condition at each time point, indicating successful hyperoxic challenge (P b 0.01, Table 1). No significant change in blood pressure or pCO2 was observed between 5%-CO2 and carobgen-inhaling conditions (P N 0.05). The hyperoxic TOLD response was determined by calculating ΔR1 between the 5%-CO2- to carbogen-inhaling conditions (Fig. 5). A threefold increase in ΔR1 was observed on days 1 and 3 in the ipsilateral cortex (F(1,30) = 1070.757, P b 0.0001; P b 0.01) compared with contralateral cortex. Consistent with the result of hyperoxic BOLD response, this also implies that the sO2 level was saturated in the lesioned cortex, and excess oxygen dissolved in the plasma after the hyperoxic challenge. On day 7, the ΔR1 observed in the ipsilateral cortex was significantly decreased relative to that observed on days 1 and 3 (F(2,30) = 20.123, P b 0.0001; P b 0.01 from day 1 to day 7 and from day 3 to day 7), but was still much higher than that on the contralateral side (P b 0.01). This indicates that the amount of excess oxygen was reduced on day 7. No significant difference in ΔR1 was observed over time in the contralateral cortex (P N 0.05). The vessel density was evaluated by immunoreactivity to RECA-1 (Fig. 6B), which was observed in a moderate quantity on days 1 and 3 and significantly increased on day 7 (P b 0.01 from day 1 to day 7 and from day 3 to day 7) as reported previously [8]. The pericyte
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coverage was evaluated by immunoreactivity to desmin (Fig. 6C), which was rarely observed in the lesioned cortex on day 1, significantly increased on day 3, and decreased by two fold on day 7 (P b 0.01 from day 1 to days 3 and 7 and from day 3 to day 7). VEGF was scarcely distributed in the neural cells in the lesioned cortex on day 1, significantly increased in the shrunken cells and perivascular regions on day 3, and then became less observed on day 7 (Fig. 6D). The evolutions in immunoreactivity to VEGF have a similar pattern as that of desmin (P b 0.01 from day 1 to days 3 and 7 and from day 3 to day 7; Fig. 6D). Fig. 7 shows the correlations between the MRI parameters and immunohistological data. Strong positive correlations were observed between hypercapnic ΔR2* and desmin area fraction (R2 = 0.8751, P b 0.0001, Fig. 7A) and between hyperoxic ΔR2* and vessel density (R2 = 0.8241, P b 0.0001, Fig. 7B). However, weak correlations were observed between hypercapnic ΔR2* and vessel density (R2 = 0.0496, P N 0.05, data not shown) as well as between hyperoxic ΔR2* and desmin area fraction (R2 = 0.0053, P N 0.05, data not shown). Additionally, a significant correlation was found between ADC and hypercapnic ΔR2* (R2 = 0.4366, P b 0.001, correlation plot not shown). 4. Discussion This study used BOLD and TOLD MRI with hypercapnic and hyperoxic challenges to investigate the venous reactivity and functionality during the proangiogenic stage after ischemia. The vessel-like venous signals on R2* maps appeared on days 3 and 7, but not on day 1. The large hypercapnic BOLD response on days 3 and 7 indicated that these areas have high vascular reactivity. The temporal correlation between vascular reactivity and the immunoreactivity to desmin further indicates that the integrity of vascular reactivity is associated with the pericyte coverage. Vascular functionality remained low on days 1, 3, and 7, as reflected by the small hyperoxic BOLD and large hyperoxic TOLD responses, indicating the low oxygen consumption of the ischemic tissues. The impaired vascular reactivity after ischemia and its recovery have been reported previously [18–20], but the underlying mechanism and its relation to proangiogenic venous remodeling remain obscure. Recently, Wegner et al. [30] attributed the improved vascular reactivity to the presence of newly formed vessels at 14 days after reperfusion in an intraluminal thread stroke model. Here we suggest that the venous reactivity could be associated with the pericyte coverage after ischemia, which is the key component in the regulation of vasodilation and vasoconstriction [31]. The impaired vascular reactivity was observed in the present study on day 1, and the reduced pericyte coverage was also demonstrated by scarce immunoreactivity to desmin with a moderate vessel density. As suggested by recent studies, the decreased pericyte coverage may be due to the early loss of pericytes after stroke [14,15]. The venous reactivity had improved
Fig. 4. Temporal changes in hyperoxic BOLD response. (A) Hyperoxic BOLD response represented by the ΔR2* maps derived from R2* maps in 5%-CO2- and carbogen-inhaling conditions on days 1, 3, and 7. (B) Quantitative analysis of hyperoxic BOLD response over 7 days. Data are mean and SD values; *P b 0.05; **P b 0.01.
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
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Fig. 5. Temporal changes in hyperoxic TOLD response. (A) Hyperoxic TOLD response represented by the ΔR1 maps derived from T1WIs in 5%-CO2- and carbogen-inhaling conditions on days 1, 3, and 7. (B) Quantitative analysis of hyperoxic TOLD response over 7 days. Data are mean and SD values; **P b 0.01.
significantly by day 3, in association with the increased pericyte coverage and VEGF level. The increased VEGF level may play a critical role in pericyte recruitment, as reported by Zechariah et al. [32]. Parallel increased VEGF and pericyte coverage after ischemia has also been discussed by ElAli et al. [33]. Furthermore, the increased pericyte coverage may be a prerequisite for the subsequent angiogenesis [34,35]. The vessel density was increased on day 7, while both vascular reactivity and pericyte coverage were decreased, suggesting that the angiogenic vessels lack pericytes, possibly due to the reduction in VEGF. These findings are supported by the strong correlations between hypercapnic ΔR2* and desmin area fraction (R2 = 0.8751, P b 0.0001,
Fig. 7A), and between desmin and VEGF area fraction (R2 = 0.8345, P b 0.0001, correlation plot not shown). In addition, VEGF production was known to be induced by hypoxia [36], and thus the reduction of VEGF from day 3 to day 7 may be due to the lower hypoxic stress relieved by angiogenesis. Inflammation, a common process after stroke, may also affect the vascular reactivity through the endothelial dysfunction [37] and the activation of astrocyte endfeet [38]. The endothelial dysfunction after ischemia has been reported to be associated with decreased nitride oxide availability, where both the basal CBF and the vascular reactivity would be decreased [37]. The activated astrocyte endfeet
Fig. 6. Immunohistochemistry of the vessel density, pericyte coverage, and vascular remodeling. (A) Representative image of the selected two regions in each brain slice for quantification as indicated by the rectangular. Temporal changes in immunoreactivity to RECA-1 (B), desmin (C), and VEGF (D) and quantitative analysis over 7 days. Images were magnified from the regions 2, 4, and 6, respectively. Data are mean and SD values; **P b 0.01.
Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
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Fig. 7. Correlations among the MRI parameters and immunohistological data. Two ROIs as shown in Fig. 6 were measured in each rat. (A) The correlation between hypercapnic ΔR2* and desmin area fraction. (B) The correlation between hyperoxic ΔR2* and vessel density as indicated by RECA-1.
after ischemia has recently been reported to cause vasodilation and increased basal CBF [38], which may result in an influenced vascular reactivity as well. These inflammatory markers were not investigated in the present study, but should be taken into consideration when interpreting the results of vascular reactivity. The detectability of DWIs in ischemic tissue at acute stage has been shown in preclinical [39] and clinical [40] studies, whereas its usage for detecting the remodeled vessels at the proangiogenic stage has not been demonstrated. In the present study, the significant correlation between ADC and hypercapnic ΔR2* at the proangiogenic stage was observed (R 2 = 0.4366, P b 0.001, correlation plot not shown). Previous studies indicated that the vascular reactivity was impaired while the ADC was already recovered within 24 hours after reperfusion [19,41]. In addition, Shen et al. reported that the impaired vascular reactivity preceded the ADC decrease at 30 minutes after ischemia [42]. These studies reported the changes between ADC and vascular reactivity at the acute stage. Our results further reveal the correlation between ADC and vascular reactivity at the proangiogenic stage, but its underlying mechanism remains to be studied. Reduced venous functionality may be caused by a high basal sO2 level in the regions with low oxygen consumption. For example, as shown in the previous studies, the oxygen consumption is low in the ischemic core and the hyperoxic BOLD response is markedly reduced in the acute phase of stroke [23–26]. Similarly, the small hyperoxic BOLD response in the present study from day 1 to day 7 may reflect a low oxygen consumption in the lesioned cortex. The large hyperoxic TOLD response from day 1 to day 7 represents further evidence of the high basal sO2 level, since the molecular-oxygen level is more directly related to tissue pO2 and ΔR1 is a more sensitive parameter for detecting fully oxygenated tissues [43–45]. Similarly, Shen et al. [26] reported a small hyperoxic BOLD response with a large ΔR1 in the ischemic core at 24 hours after reperfusion. The findings of the present study demonstrate that this phenomenon is actually maintained until day 7. In previous studies, the hypercapnic and hyperoxic BOLD responses have together been used to identify the immature tumor vessels [28,46,47]. These studies described that immature vessels lack pericyte coverage with a high oxygen extraction fraction, resulting in low vascular reactivity and enhanced vascular functionality. In the present study, the proangiogenic venous vessels with extensive pericyte coverage on day 3 showed high vascular reactivity and low vascular functionality, both indicating maturity of these vessels. From day 3 to day 7, the vascular reactivity was decreased, and the vascular functionality was increased, which may be attributed to the formation of new immature vessels. This is supported by the strong correlation between hyperoxic ΔR2* and vessel density by RECA-1 (R2 = 0.8241, P b 0.0001, Fig. 7B). Furthermore, to our knowledge the present study is the first to investigate both hypercapnic and hyperoxic BOLD responses in the remodeled vessels after ischemia. Many factors may have influenced the measurements in this study. First, the hyperintense signals on R2* maps were based on
BOLD contrast, and vessels of arterial origins might not be detected using this method due to the high sO2 level [17]. The vessel-like hyperintense signals on R2* maps were absent at 1 day but appeared at 3 and 7 days after ischemia (Fig. 2). However, RECA-1 shows a moderate vessel density on days 1 and 3 and significantly increased vessel density on day 7 (Fig. 6). The discrepancy may be due to the vascular signals on R2* maps mainly originating from veins, while RECA-1 reveals both arterial and venous vessels. Second, T1-shortening effects (e.g., the inflow and enhancement of molecular oxygen) during hypercapnic and hyperoxic challenges can increase the signal intensities on T2*WIs and cause erroneous ΔR2* values. Therefore, curve-fitted R2* maps were used to derive ΔR2* values in the present study in order to reduce the influence of such T1-shortening effects [48,49]. Third, isoflurane used in the present study is a vasodilator, and the increase in basal CBF by isoflurane may cause overestimation of the T2 and T2* values [50]. In addition, the repeated isoflurane exposure of the rats may affect the cerebral autoregulation [51]. However, the optimized anesthesia protocol with minimal physiological disturbance remains a challenge. In conclusion, the present study found that vessel-like venous signals on R2* maps appeared at 3 and 7 days after ischemia, but not after 1 day, and that these areas have high vascular reactivity and reduced vascular functionality. The integrity of the venous reactivity may be associated with the pericyte coverage as regulated by the VEGF level. The reduced venous functionality may be due to the low oxygen consumption of the ischemic tissue. Information about these vascular variables in proangiogenic vessels could be critically useful when developing novel angiogenic therapies for stroke. Conflict of interest The authors declare that there are no conflicts of interest. Acknowledgments Y.Y.I.S. was supported by AHA 15SDG23260025, NIH R01NS091236, NARSAD Young Investigator Award, NCTraCS 550KR81420 and 550KR91413 (parent award: 1UL1TR001111), UNC S14 University Research Council Award and UNC Junior Faculty Development Award. Y.Y.I.S. is also an Ellen Schapiro and Gerald Axelbaum Investigator supported by the Brain & Behavior Research Foundation. References [1] Seevinck PR, Deddens LH, Dijkhuizen RM. Magnetic resonance imaging of brain angiogenesis after stroke. Angiogenesis 2010;13:101–11. [2] Jiang Q, Zhang ZG, Chopp M. MRI of stroke recovery. Stroke 2010;41:410–4. [3] Blebea J, Vu JH, Assadnia S, McLaughlin PJ, Atnip RG, Zagon IS. Differential effects of vascular growth factors on arterial and venous angiogenesis. J Vasc Surg 2002; 35:532–8.
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Please cite this article as: Huang C-H, et al, Temporal assessment of vascular reactivity and functionality using MRI during postischemic proangiogenenic vascular remodeling, Magn Reson Imaging (2015), http://dx.doi.org/10.1016/j.mri.2015.04.009
