- Email: [email protected]
Bilateral common carotid artery occlusion induced brain lesions in rats: A longitudinal diffusion tensor imaging study
Magnetic Resonance Imaging 33 (2015) 551–558
Contents lists available at ScienceDirect
Magnetic Resonance Imaging journal homepage: www.mrijournal.com
Bilateral common carotid artery occlusion induced brain lesions in rats: A longitudinal diffusion tensor imaging study Xuxia Wang, Fuchun Lin, Yunling Gao, Hao Lei ⁎ State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, P. R. China
a r t i c l e
i n f o
Article history: Received 30 October 2014 Revised 14 February 2015 Accepted 15 February 2015 Keywords: Diffusion tensor imaging Axial diffusivity Radial diffusivity Demyelination Axon damage
a b s t r a c t Bilateral common carotid artery occlusion (BCCAO) has been widely used to reproduce the white matter (WM) and gray matter (GM) damage associated with chronic cerebral hypoperfusion (CCH). This study investigated whether diffusion tensor imaging (DTI) could be used at the early stages of disease to assess brain damage induced by BCCAO. To this end, DTI, together with histological methods, was used to evaluate the progression of WM lesions and GM neurodegeneration following BCCAO. The DTI was sufficiently sensitive to detect WM abnormalities in selected regions of the brain at 4 weeks after BCCAO. These abnormalities may indicate damage to the myelin and axons in the optic nerve (ON) and optic tract (OT). Our longitudinal results showed that DTI could be used to detect abnormalities of the WM and GM in select regions of the brain as early as 2 days after ligation. The DTI parameter patterns of change were region-specific throughout the detection time course. Lesions of the external capsule (EC) and periventricular hypothalamic nucleus (Pe) have not been thoroughly studied before. We found that the EC and Pe were both vulnerable to BCCAO and that the associated lesions could be detected using DTI. The current study demonstrated that in vivo DTI could potentially be used to measure WM damage evolution in a BCCAO rat model as well as early brain injury following CCH. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Chronic cerebral hypoperfusion (CCH) is an important factor in brain damage associated with aging and vascular dementia [1–5]. For example, white matter (WM) lesions often accompany CCH in the aging brain [6]. A rat model with permanent bilateral common carotid artery occlusion (BCCAO) has been proposed to reproduce the effects of CCH. In the rat brain, BCCAO usually leads to WM lesions and gray matter (GM) neurodegeneration. Previous studies [7,8] showed that the lesions included myelin rarefaction and vacuolization, axon damage, microglial activation, and astrogliosis in the optic nerve (ON) and optic tract (OT). These lesions were long-lasting. The GM hippocampus, neocortex, and superior colliculus suffered delayed neuronal loss [9], transient gliosis [10] and long-lasting gliosis [11], respectively, after BCCAO. Thus, there is a need for improved, noninvasive, and highly sensitive imaging methods to be developed for the early detection of brain damage as well as its evolution following BCCAO in vivo. These techniques would be ⁎ Corresponding author at: Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, West No.30 Xiao Hong Shan, Wuhan 430071, P. R. China. Tel.: +86 27 8719 8542; fax: +86 27 8719 9291. E-mail address: [email protected] (H. Lei). http://dx.doi.org/10.1016/j.mri.2015.02.010 0730-725X/© 2015 Elsevier Inc. All rights reserved.
especially useful if they could be combined with histopathological methods. They could also be used in pathological assessments of imaging indices in aging as well as vascular dementia studies. A recent study by Sorial et al. [12] showed that magnetic resonance imaging (MRI) tools such as diffusion tensor imaging (DTI) could be used to detect the diffusion of subtle WM and GM abnormalities at 7 weeks after BCCAO. This indicated that DTI could potentially be used to longitudinally track the development of CCH-induced brain damage. A dynamic DTI evaluation of a BCCAO rat model is important not only in understanding the course of CCH-induced brain lesions, but also in developing imaging biomarkers in the evaluation of new neuroprotective drugs. Up to now, few studies have used the DTI technique to longitudinally assess rat brain damage induced by BCCAO. In the present study, we measured longitudinal DTI index changes in a rat model of BCCAO. The measurements were performed at 2 days, 1 week, 2 weeks, and 4 weeks after BCCAO. Histological evaluations were performed at 4 weeks after BCCAO to verify DTI findings. The primary objective was to determine whether DTI measurements were sufficiently sensitive to detect abnormalities in the rat brain in the early stages following BCCAO and to determine whether DTI could provide useful information regarding the nature and time course of CCH-induced brain lesions.
552
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
2. Material and methods 2.1. Animal preparation and surgical procedures The animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All of the animal protocols were approved by the institutional animal care committee of the Wuhan Institute of Physics and Mathematics. A total of 16 male Wistar rats, approximately 8 weeks of age and weighing 200–230 g, were used. The rats were housed in a room with an ambient temperature of 23 ± 1 °C and a 12-h light–dark cycle. They had free access to food and water. Each rat was anesthetized with an intraperitoneal injection of 5% choral hydrate solution (6 ml/kg body weight). A median incision was made in their necks. Their bilateral common carotid arteries were separated and isolated. For the rats assigned to the BCCAO group (BCCAO, n = 9), the bilateral carotid arteries were double-ligated with 4-0 sutures. For the rats in the control group (CON, n = 7), the bilateral carotid arteries were manipulated as in the BCCAO group, but without being ligated. The wounds were then closed. The body temperature of the rats was maintained (with a heating lamp) at 37 ± 1 °C during surgery as well as during recovery from anesthesia. 2.2. Magnetic resonance imaging measurements MRI measurements were performed at 2 days, 1 week, 2 weeks, and 4 weeks after ligation on a 7 T/20 cm Bruker Biospec scanner. A volume coil was used for radiofrequency pulse transmission and a quadrature surface coil was used for signal detection. The rats were anesthetized by isoflurane in pure O2 (3% for induction, 1.5%–2% for maintenance) delivered via a nose cone during the imaging sessions with their respiration monitored continuously. DTI was performed with a spin-echo 4-shot echo-planar imaging (EPI) sequence, an encoding scheme of 30 gradient directions homogenously distributed on the unit sphere, and the following acquisition parameters: TR 5000 ms, TE 26 ms, FOV 3 cm × 3 cm, slice thickness of 0.8 mm, matrix size of 128 × 128, Δ 14 ms, δ 3 ms, two b values (0 and 800 s/mm 2) and 4 averages.
15 μm or 30 μm. Slices at Bregma 2.52 mm (including the ON) were sectioned continuously for Luxol fast blue (LFB) staining, immunocytochemical glial fibrillary acidic protein (GFAP) staining, and hyperphosphorylated neurofilament (SMI-31) staining respectively. Slices at Bregma 1.44 mm (including the external capsules (EC), striatum (CPu), and motor and sensory cortexs (CTX)) were sectioned continuously for LFB staining, Nissl staining, immunocytochemical GFAP staining, and SMI-31 staining respectively. Slices at Bregma −2.4 mm (including the OT and periventricular hypothalamic nucleus (Pe)) were sectioned continuously for LFB staining, Nissl staining, immunocytochemical GFAP staining, and SMI-31 staining respectively. For LFB staining, 15 μm-thick brain sections were incubated in a 0.1% LFB solution overnight at 60 °C. After reaching room temperature, these sections were first rinsed with 95% ethanol and then washed in distilled water. Differentiation was completed by briefly rinsing them in 0.05% lithium carbonate and 70% ethanol and then returning them to water until the contrast between the white and gray matter was maximized. The LFB-stained sections were then counterstained with toluidine blue. For Nissl staining, 15 μm-thick brain tissue sections were immersed in a 0.1% toluidine blue solution for approximately 1 min, washed with distilled water, and then rinsed in 80% ethanol, 95% ethanol, 100% ethanol,100% ethanol, and 100% xylene subsequently for 5 min followed by mounting. For immunohistochemistry, antigen retrieval was performed with 30 μm-thick brain sections. After cooling to room temperature, the endogenous peroxidases were inactivated via immersion in 3% H2O2 for 15 min, 3 washes in PBS, and incubation with 0.2% Triton X-100 for 30 min at 37 °C. After 3 rinses with PBS, the slides were blocked with 10% normal goat serum for 1 h to block nonspecific antibody interactions. Then, the diluted primary antibodies (GFAP, 1:100 PBS, and SMI-31, 1:500 PBS) were incubated for 2 h at 37 °C. After washing, the GFAP immunolabeling was detected with Histostain SP kit from Zymed with diaminobenzidine as the substrate (except for the ON); for the ON, the GFAP immunolabeling was detected with fluorescein isothiocyanateconjugated goat anti-rabbit; the SMI-31 immunolabeling was detected with a secondary antibody (Alexa Fluor 488 goat anti-mouse). 2.4. DTI data analysis
2.3. Histological evaluations After the MRI examination at 4 weeks, the anesthetized rats were perfused with 300 ml saline and subsequently 300 ml 4% paraformaldehyde dissolved in 100 mmol/l phosphate-buffered saline (PBS, pH = 7.4). Their brains were removed, post-fixed in the same fixative for at least 24 h, and stored in a refrigerator at 4 °C until examined. The brains were sectioned on a cryostat microtome (Leica, Germany). Coronal brain sections were obtained with a thickness of
Fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) maps were acquired using the toolbox provided by the Bruker PARAVISON 5.0 software. Using the PARAVASION 5.0 software, the Regions of Interest (ROIs), representing different anatomical structures, were traced manually on the FA (i.e., without interpolation) maps for each rat with guidance from the Paxinos digital atlas [13]. Then, the ROIs were shifted to the identical position on the MD, AD and RD maps, and the values of DTI
Fig. 1. The ROIs are manually drawn from the FA maps of the rat brain. ON: optic nerve, CTX: motor and sensory cortex, EC: external capsule, CPu: striatum, OT: optic tract, Pe: periventricular hypothalamic nucleus.
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
553
Fig. 2. A quantitative analysis of the DTI indices in the WM ROIs of the rat brain at 4 weeks after ligation. * p b 0.05, statistical inter-group significance at 4 weeks after BCCAO. ON: optic nerve, OT: optic tract, EC: external capsule.
indices were recorded. Student’s unpaired t-tests were performed to compare the DTI indices values in different brain regions at each time point between the BCCAO group and the control group.
3. Results 3.1. DTI parameter variation and histological evaluation of WM at 4 weeks after BCCAO Fig. 1 presents the positions of the ROIs. Fig. 2 shows the ROI analysis of the WM in the rat brain at 4 weeks after BCCAO. Compared
with the control, the FA values showed a marked reduction in the ON and OT, as well as an increase in the EC; the MD values were substantially decreased in the EC. The AD values had a significant reduction in the ON and OT; there was no apparent alteration in the EC. The RD values were elevated in the ON and OT and reduced in the EC. Fig. 3 shows the corresponding histological evaluations of the WM at 4 weeks after BCCAO. The LFB staining showed severe myelin rarefaction and vacuoles in the ON and OT and slightly reduced myelin rarefaction and vacuoles in the EC compared with the control. The immunoreactivity of axon marker for SMI-31 was significantly reduced in the ON and OT but not in the EC and obvious astrogliosis was observed in the ON, OT and EC.
Fig. 3. Histological evaluations of WM lesions at 4 weeks after BCCAO. LFB: Luxol fast blue staining, SMI-31: hyperphosphorylated neurofilament immunostaining, GFAP: glial fibrillary acidic protein immunostaining. ON: optic nerve, OT: optic tract, EC: external capsule. Magnification = ×400 in all of the images.
554
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
Fig. 4. The changes in the DTI indices of the WM ROIs over time. * p b 0.05 statistical significance between the BCCAO and control group at each time point. ON: optic nerve, OT: optic tract, EC: external capsule.
3.2. Evolution of DTI indices in WM after BCCAO
3.3. Evolution of DTI indices and histological evaluation in GM after BCCAO
To assess the evolution of WM damage after BCCAO, we assessed 4 time points (Fig. 4) following BCCAO. With the exception of the 2 day time point for the ON, the evolution of the FA values in the ON and OT showed a progressive reduction from 2 days to 4 weeks after BCCAO (compared with the control). However, in the EC the FA values significantly increased from 1 week to 4 weeks. The MD values were significantly reduced at 2 days and 4 weeks after BCCAO in the EC, and there were no significant changes in the ON or OT. The AD values in the ON and OT decreased from 1 week to 4 weeks after ligation; in the EC the AD value was markedly smaller only at 2 days after BCCAO. The RD values in the ON and OT significantly increased from 1 week to 4 weeks after ligation; however, the RD values in the EC were lower than those in the control at all time points (Fig. 4).
The evolution of the FA, MD, AD and RD was also investigated in three GM regions of the rat brains (Fig. 5). The FA values were markedly elevated in the CPu at 2 days after ligation and had no significant changes in Pe and CTX. The MD values were notably reduced at 2 days and elevated at 4 weeks in the Pe; the MD values were significantly reduced from 2 days to 4 weeks in the CPu (with the exception of the 2 week time point) and were markedly reduced at 2 days in the CTX. The AD values were significantly decreased at 2 days in the Pe and CTX as well as from 1 week to 2 weeks in the CPu. The RD values were significantly reduced at 2 days and notably elevated at 4 weeks following ligation in the Pe, substantially decreased from 2 days to 4 weeks following ligation in the CPu (with the exception of the 2 week time point), and reduced at 2 days following ligation in the CTX.
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
555
Fig. 5. The changes in the DTI indices of the GM ROIs over time. * p b 0.05 statistical significance between the BCCAO and control group at each time point. Pe: periventricular hypothalamic nucleus, CPu: striatum, CTX: motor and sensory cortex.
Fig. 6 shows the histological evaluations of the GM at 4 weeks after BCCAO. The Nissl staining showed that sparse cell arrangements occurred in the CPu and that neuronal shrinkage occurred in the Pe and CTX. The immunostaining for GFAP showed obvious gliosis in the Pe, CPu and CTX.
4. Discussion To the best of our knowledge, this is the first longitudinal study combining DTI with histological methods developed to detect the evolution of BCCAO brain injury in rat models. DTI is a sensitive and valid tool to investigate the abnormalities of rat brains [14,15] and may be used non-invasively to assess the spatiotemporal course of pathological changes of the BCCAO rat brain. At 4 weeks after BCCAO,
an increased RD value and reduced AD and FA values were shown in the ON and OT. From 2 days to 4 weeks after ligation, our results showed that DTI could potentially be used to detect an abnormality of rat brains as early as 2 days and the change patterns of the DTI parameters were region-specific among these selected brain regions. MRI findings in a BCCAO rat model using conventional T2-weighted imaging (T2WI) have been reported [16] and indicated few abnormalities in rat brains. This may have been because the T2WI was not sufficiently sensitive or because of a lower field strength. At 4 weeks after ligation, our results showed that DTI was sufficiently sensitive to detect an abnormality of the selected WM regions (Fig. 2). As a DTI index, the variation of FA indicated a change in WM microstructure integrity. A reduction of the FA has been found in numerous animal models, such as those involving retinal ischemia [17], neonatal rat hypoxia–ischemia [18], and a cuprizone mouse
556
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
Fig. 6. The histological evaluations of the GM lesions at 4 weeks after BCCAO. GFAP: glial fibrillary acidic protein immunostaining. Pe: periventricular hypothalamic nucleus, CPu: striatum, CTX: motor and sensory cortex. Magnification = ×400 in all images.
model [19]. Occasionally, FA increased [20–22]. In the current rat model, the FA values of the ON and OT were decreased at 4 weeks after ligation, which reflected a loss of directionality. In the EC, the FA value increased with a reduced RD at 4 weeks after ligation; this could be related to the glial scar formation that had previously been found to play a role in elevated FA [22]. The FA variations indicated that the selected WM regions were all vulnerable to BCCAO. The AD and RD have also been widely studied in recent years. Many reports [23,24] have recently found that impaired myelin could increase RD. Our study and previous reports [7,25] found that the ON and OT presented severe vacuoles and an increased crookedness of myelin fibers at 4 weeks after ligation. This suggests that the elevated RD values of the ON and OT maybe associated with myelin damage. Although the LFB staining showed abnormal results with small myelin fiber vacuoles in the EC at 4 weeks after the ligation, the RD value did not increase but was reduced compared with the control. This indicated that another mechanism may play a key role in reducing the RD value and supported the hypothesis [26] that the relationship between demyelination and RD is more complicated than previously thought. Multiple studies [17,27,28] support the hypothesis that increased RD was a specific marker of myelin injury. However, our EC data demonstrated a potentially reduced sensitivity of RD for detecting demyelination. Decreased AD is related to axon swelling [27] and neurofilament dysfunction [19]. In a BCCAO rat model, Wakita et al. [7] found increased immuno-
reactivity of the amyloid β/A4 precursor protein (APP) and chromogranin A (CgA), in the ON and OT. Additionally, many fibers showed a morphology typical of axon swelling. In our data, the AD values were significantly reduced in the presence of decreased SMI-31 immunoreactivity in the ON and OT. This suggested that axon damage may play a role in the varied AD values. In the EC, we found no significant change of the AD with relatively normal SMI-31 staining compared with the control. The differences in the AD and RD between the OT (or ON) and EC after BCCAO indicated regionspecific injury effects in the selected WM regions. In a vascular dementia animal model, the WM regions suffered a combination of demyelination [7], axon injury [7], astrogliosis [11] (Fig. 3), microglial activation [25], and low cerebral blood flow (CBF) [29]which could result in competing influences on the DTI indices [30,31]. If this is true, interpreting changes in DTI indices requires further study. The longitudinal changes in the DTI indices of the ON, OT and EC are shown in Fig. 4 and the evolution patterns of the DTI indices in the ON (or OT) and EC were not the same after BCCAO. DTI was sufficiently sensitive to detect the BCCAO-induced WM lesions as early as 2 days after ligation. This early detection showed that the DTI technique could potentially be used to determine the approximate time course of WM lesions after BCCAO. From the first to the fourth week, the significantly changed DTI indices of the ON and OT were consistent with an earlier pathological observation [7],
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
indicating WM rarefaction (via Klüver–Barrera staining in the ON and OT) and an increased expression of APP and encephalitogenic peptide (EP) 3 days after ligation (which persisted for up to at 30 days). These indicated that the DTI could potentially be used to detect WM lesions in the current rat model. In contrast with the ON and OT, the EC showed an increased FA and reduced RD. This indicated that the pathological factor that strongly influenced the DTI indices in the EC may differ from that in the ON or OT. The pathological changes in the EC after ligation were previously of little scientific concern and the relevant pathological processes were unclear and required further study. A temporal alteration of the DTI parameters was observed not only in the brain WM but also in the GM sites (Fig. 5) and there were region-specific DTI index evolution patterns in the selected GM regions. Our results demonstrated that DTI was also sensitive in the early detection of brain abnormalities in the selected GM regions. The changes in the values of the DTI indices demonstrated that the injury induced by BCCAO continued in the CPu from 1 week to 4 weeks following ligation. There were no significant CTX DTI changes. However, neuronal shrinkage may result in an increase in MD [32] as well as astrogliosis (Fig. 6), thereby producing a reduction in MD [33]. These factors exert competing influences on diffusion tensor indices. The DTI in the CTX was not sufficiently sensitive during this period. In the Pe, (and in contrast with the CTX), the MD and RD values were increased with obvious neuronal shrinkage and astrogliosis at 4 weeks. This indicated that the pathological factors that played an important role in altering the MD differed from those of the CTX. Compared with the reduced MD and RD values of the Pe at 2 days, the increased MD and RD values at 4 weeks implied that the primary factor affecting the MD and AD values may have changed between the 2 day and 4 week time points. The hypothalamus is a center of autonomic control and is involved in regulating appetite. However, the hypothalamus has received little attention in BCCAO rat models. The Pe is a part of the hypothalamus and is vulnerable to BCCAO (Figs. 5 and 6), and previous research [34] indicated that BCCAO animals regain weight slowly compared with those in control groups. The decreased or increased MD values that we observed in the GM regions were affected by numerous pathological processes, such as neuron loss or shrinkage (Fig. 6), gliosis (Fig. 6), microglial activation [10], low CBF [35] and others, all of which could differentially affect the movement of water molecules. Although it is difficult to determine (due to the abnormal MD value) which pathological process occurred, these abnormal MD values provide useful information that furthers our understanding of the pathological processes of the GM in the BCCAO rat brain. In the present study, we monitored rat brain injury at different time points after ligation and discovered that the change patterns of the DTI parametric values were region-specific among select brain regions. The current results indicate that DTI may be a sensitive and valid means for assessing BCCAO-induced WM lesions. The results also provide a basic understanding of brain injury evolution after BCCAO. DTI may be helpful in obtaining useful and accurate information regarding early-stage vascular dementia diagnosis. Acknowledgments The authors express their gratitude to Mrs. Erin Beatson for proof reading. This work was supported by the Natural Science Foundation of China (No. 81000598). References [1] Choi JY, Morris JC, Hsu CY. Aging and cerebrovascular disease. Neurol Clin 1998; 16:687–711.
557
[2] Claus JJ, Breteler MM, Hasan D, Krenning EP, Bots ML, Grobbee DE, et al. Regional cerebral blood flow and cerebrovascular risk factors in the elderly population. Neurobiol Aging 1998;19:57–64. [3] Farkas E, Luiten PG, Bari F. Permanent, bilateral common carotid artery occlusion in the rat: a model for chronic cerebral hypoperfusion-related neurodegenerative diseases. Brain Res Rev 2007;54:162–80. [4] Kim SK, Cho KO, Kim SY. White matter damage and hippocampal neurodegeneration induced by permanent bilateral occlusion of common carotid artery in the rat: comparison between Wistar and Sprague–Dawley strain. Korean J Physiol Pharmacol 2008;12:89–94. [5] Yoshizaki K, Adachi K, Kataoka S, Watanabe A, Tabira T, Takahashi K, et al. Chronic cerebral hypoperfusion induced by right unilateral common carotid artery occlusion causes delayed white matter lesions and cognitive impairment in adult mice. Exp Neurol 2008;210:585–91. [6] Fernando MS, Simpson JE, Matthews F, Brayne C, Lewis CE, Barber R, et al. White matter lesions in an unselected cohort of the elderly: molecular pathology suggests origin from chronic hypoperfusion injury. Stroke 2006;37:1391–8. [7] Wakita H, Tomimoto H, Akiguchi I, Matsuo A, Lin JX, Ihara M, et al. Axonal damage and demyelination in the white matter after chronic cerebral hypoperfusion in the rat. Brain Res 2002;924:63–70. [8] Cho KO, La HO, Cho YJ, Sung KW, Kim SY. Minocycline attenuates white matter damage in a rat model of chronic cerebral hypoperfusion. J Neurosci Res 2006; 83:285–91. [9] Ni J, Ohta H, Matsumoto K, Watanabe H. Progressive cognitive impairment following chronic cerebral hypoperfusion induced by permanent occlusion of bilateral carotid arteries in rats. Brain Res 1994;653:231–6. [10] Schmidt-Kastner R, Aguirre-Chen C, Saul I, Yick L, Hamasaki D, Busto R, et al. Astrocytes react to oligemia in the forebrain induced by chronic bilateral common carotid artery occlusion in rats. Brain Res 2005;1052:28–39. [11] Wakita H, Tomimoto H, Akiguchi I, Kimura J. Glial activation and white matter changes in the rat brain induced by chronic cerebral hypoperfusion: an immunohistochemical study. Acta Neuropathol 1994;87:484–92. [12] Soria G, Tudela R, Marquez-Martin A, Camon L, Batalle D, Munoz-Moreno E, et al. The ins and outs of the BCCAo model for chronic hypoperfusion: a multimodal and longitudinal MRI approach. PLoS One 2013;8:e74631. [13] George Paxinos CW. The rat brain in stereotaxic coordinates. 2nd ed. San Diego: Academic Press; 1996. [14] Dror V, Eliash S, Rehavi M, Assaf Y, Biton IE, Fattal-Valevski A. Neurodegeneration in thiamine deficient rats—a longitudinal MRI study. Brain Res 2010;1308: 176–84. [15] Wang S, Wu EX, Qiu D, Leung LH, Lau HF, Khong PL. Longitudinal diffusion tensor magnetic resonance imaging study of radiation-induced white matter damage in a rat model. Cancer Res 2009;69:1190–8. [16] Plaschke K, Bardenheuer HJ, Martin E, Sartor K, Heiland S. Evolution of apparent diffusion coefficient and transverse relaxation time (T2) in the subchronic stage of global cerebral oligemia in different rat models. Exp Brain Res 2006;169: 361–8. [17] Song SK, Sun SW, Ju WK, Lin SJ, Cross AH, Neufeld AH. Diffusion tensor imaging detects and differentiates axon and myelin degeneration in mouse optic nerve after retinal ischemia. Neuroimage 2003;20:1714–22. [18] Wang S, Wu EX, Cai K, Lau HF, Cheung PT, Khong PL. Mild hypoxic–ischemic injury in the neonatal rat brain: longitudinal evaluation of white matter using diffusion tensor MR imaging. AJNR Am J Neuroradiol 2009;30:1907–13. [19] Sun SW, Liang HF, Trinkaus K, Cross AH, Armstrong RC, Song SK. Noninvasive detection of cuprizone induced axonal damage and demyelination in the mouse corpus callosum. Magn Reson Med 2006;55:302–8. [20] Bockhorst KH, Narayana PA, Liu R, Ahobila-Vijjula P, Ramu J, Kamel M, et al. Early postnatal development of rat brain: in vivo diffusion tensor imaging. J Neurosci Res 2008;86:1520–8. [21] Van Camp N, Blockx I, Verhoye M, Casteels C, Coun F, Leemans A, et al. Diffusion tensor imaging in a rat model of Parkinson's disease after lesioning of the nigrostriatal tract. NMR Biomed 2009;22:697–706. [22] Budde MD, Janes L, Gold E, Turtzo LC, Frank JA. The contribution of gliosis to diffusion tensor anisotropy and tractography following traumatic brain injury: validation in the rat using Fourier analysis of stained tissue sections. Brain 2011; 134:2248–60. [23] Wang S, Wu EX, Tam CN, Lau HF, Cheung PT, Khong PL. Characterization of white matter injury in a hypoxic-ischemic neonatal rat model by diffusion tensor MRI. Stroke 2008;39:2348–53. [24] Xie M, Tobin JE, Budde MD, Chen CI, Trinkaus K, Cross AH, et al. Rostrocaudal analysis of corpus callosum demyelination and axon damage across disease stages refines diffusion tensor imaging correlations with pathological features. J Neuropathol Exp Neurol 2010;69:704–16. [25] Lee JH, Park SY, Shin YW, Hong KW, Kim CD, Sung SM, et al. Neuroprotection by cilostazol, a phosphodiesterase type 3 inhibitor, against apoptotic white matter changes in rat after chronic cerebral hypoperfusion. Brain Res 2006;1082: 182–91. [26] Van Camp N, Blockx I, Camon L, de Vera N, Verhoye M, Veraart J, et al. A complementary diffusion tensor imaging (DTI)-histological study in a model of Huntington's disease. Neurobiol Aging 2012;33:945–59. [27] Song SK, Yoshino J, Le TQ, Lin SJ, Sun SW, Cross AH, et al. Demyelination increases radial diffusivity in corpus callosum of mouse brain. Neuroimage 2005;26:132–40. [28] Song SK, Sun SW, Ramsbottom MJ, Chang C, Russell J, Cross AH. Dysmyelination revealed through MRI as increased radial (but unchanged axial) diffusion of water. Neuroimage 2002;17:1429–36.
558
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
[29] Tsuchiya M, Sako K, Yura S, Yonemasu Y. Cerebral blood flow and histopathological changes following permanent bilateral carotid artery ligation in Wistar rats. Exp Brain Res 1992;89:87–92. [30] Assaf Y, Pasternak O. Diffusion tensor imaging (DTI)-based white matter mapping in brain research: a review. J Mol Neurosci 2008;34:51–61. [31] Ding AY, Hui ES, Wu EX. The effects of hypercapnia on DTI quantification in anesthetized rat brain. Conf Proc IEEE Eng Med Biol Soc 2009;2009:2711–4. [32] Douaud G, Behrens TE, Poupon C, Cointepas Y, Jbabdi S, Gaura V, et al. In vivo evidence for the selective subcortical degeneration in Huntington's disease. Neuroimage 2009;46:958–66.
[33] Anderova M, Vorisek I, Pivonkova H, Benesova J, Vargova L, Cicanic M, et al. Cell death/proliferation and alterations in glial morphology contribute to changes in diffusivity in the rat hippocampus after hypoxia–ischemia. J Cereb Blood Flow Metab 2011;31:894–907. [34] Farkas E, de Wilde MC, Kiliaan AJ, Meijer J, Keijser JN, Luiten PG. Dietary long chain PUFAs differentially affect hippocampal muscarinic 1 and serotonergic 1A receptors in experimental cerebral hypoperfusion. Brain Res 2002;954:32–41. [35] Otori T, Katsumata T, Muramatsu H, Kashiwagi F, Katayama Y, Terashi A. Longterm measurement of cerebral blood flow and metabolism in a rat chronic hypoperfusion model. Clin Exp Pharmacol Physiol 2003;30:266–72.
Contents lists available at ScienceDirect
Magnetic Resonance Imaging journal homepage: www.mrijournal.com
Bilateral common carotid artery occlusion induced brain lesions in rats: A longitudinal diffusion tensor imaging study Xuxia Wang, Fuchun Lin, Yunling Gao, Hao Lei ⁎ State Key Laboratory of Magnetic Resonance and Atomic and Molecular Physics, Wuhan Center for Magnetic Resonance, Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, Wuhan 430071, P. R. China
a r t i c l e
i n f o
Article history: Received 30 October 2014 Revised 14 February 2015 Accepted 15 February 2015 Keywords: Diffusion tensor imaging Axial diffusivity Radial diffusivity Demyelination Axon damage
a b s t r a c t Bilateral common carotid artery occlusion (BCCAO) has been widely used to reproduce the white matter (WM) and gray matter (GM) damage associated with chronic cerebral hypoperfusion (CCH). This study investigated whether diffusion tensor imaging (DTI) could be used at the early stages of disease to assess brain damage induced by BCCAO. To this end, DTI, together with histological methods, was used to evaluate the progression of WM lesions and GM neurodegeneration following BCCAO. The DTI was sufficiently sensitive to detect WM abnormalities in selected regions of the brain at 4 weeks after BCCAO. These abnormalities may indicate damage to the myelin and axons in the optic nerve (ON) and optic tract (OT). Our longitudinal results showed that DTI could be used to detect abnormalities of the WM and GM in select regions of the brain as early as 2 days after ligation. The DTI parameter patterns of change were region-specific throughout the detection time course. Lesions of the external capsule (EC) and periventricular hypothalamic nucleus (Pe) have not been thoroughly studied before. We found that the EC and Pe were both vulnerable to BCCAO and that the associated lesions could be detected using DTI. The current study demonstrated that in vivo DTI could potentially be used to measure WM damage evolution in a BCCAO rat model as well as early brain injury following CCH. © 2015 Elsevier Inc. All rights reserved.
1. Introduction Chronic cerebral hypoperfusion (CCH) is an important factor in brain damage associated with aging and vascular dementia [1–5]. For example, white matter (WM) lesions often accompany CCH in the aging brain [6]. A rat model with permanent bilateral common carotid artery occlusion (BCCAO) has been proposed to reproduce the effects of CCH. In the rat brain, BCCAO usually leads to WM lesions and gray matter (GM) neurodegeneration. Previous studies [7,8] showed that the lesions included myelin rarefaction and vacuolization, axon damage, microglial activation, and astrogliosis in the optic nerve (ON) and optic tract (OT). These lesions were long-lasting. The GM hippocampus, neocortex, and superior colliculus suffered delayed neuronal loss [9], transient gliosis [10] and long-lasting gliosis [11], respectively, after BCCAO. Thus, there is a need for improved, noninvasive, and highly sensitive imaging methods to be developed for the early detection of brain damage as well as its evolution following BCCAO in vivo. These techniques would be ⁎ Corresponding author at: Wuhan Institute of Physics and Mathematics, Chinese Academy of Sciences, West No.30 Xiao Hong Shan, Wuhan 430071, P. R. China. Tel.: +86 27 8719 8542; fax: +86 27 8719 9291. E-mail address: [email protected] (H. Lei). http://dx.doi.org/10.1016/j.mri.2015.02.010 0730-725X/© 2015 Elsevier Inc. All rights reserved.
especially useful if they could be combined with histopathological methods. They could also be used in pathological assessments of imaging indices in aging as well as vascular dementia studies. A recent study by Sorial et al. [12] showed that magnetic resonance imaging (MRI) tools such as diffusion tensor imaging (DTI) could be used to detect the diffusion of subtle WM and GM abnormalities at 7 weeks after BCCAO. This indicated that DTI could potentially be used to longitudinally track the development of CCH-induced brain damage. A dynamic DTI evaluation of a BCCAO rat model is important not only in understanding the course of CCH-induced brain lesions, but also in developing imaging biomarkers in the evaluation of new neuroprotective drugs. Up to now, few studies have used the DTI technique to longitudinally assess rat brain damage induced by BCCAO. In the present study, we measured longitudinal DTI index changes in a rat model of BCCAO. The measurements were performed at 2 days, 1 week, 2 weeks, and 4 weeks after BCCAO. Histological evaluations were performed at 4 weeks after BCCAO to verify DTI findings. The primary objective was to determine whether DTI measurements were sufficiently sensitive to detect abnormalities in the rat brain in the early stages following BCCAO and to determine whether DTI could provide useful information regarding the nature and time course of CCH-induced brain lesions.
552
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
2. Material and methods 2.1. Animal preparation and surgical procedures The animal experiments were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. All of the animal protocols were approved by the institutional animal care committee of the Wuhan Institute of Physics and Mathematics. A total of 16 male Wistar rats, approximately 8 weeks of age and weighing 200–230 g, were used. The rats were housed in a room with an ambient temperature of 23 ± 1 °C and a 12-h light–dark cycle. They had free access to food and water. Each rat was anesthetized with an intraperitoneal injection of 5% choral hydrate solution (6 ml/kg body weight). A median incision was made in their necks. Their bilateral common carotid arteries were separated and isolated. For the rats assigned to the BCCAO group (BCCAO, n = 9), the bilateral carotid arteries were double-ligated with 4-0 sutures. For the rats in the control group (CON, n = 7), the bilateral carotid arteries were manipulated as in the BCCAO group, but without being ligated. The wounds were then closed. The body temperature of the rats was maintained (with a heating lamp) at 37 ± 1 °C during surgery as well as during recovery from anesthesia. 2.2. Magnetic resonance imaging measurements MRI measurements were performed at 2 days, 1 week, 2 weeks, and 4 weeks after ligation on a 7 T/20 cm Bruker Biospec scanner. A volume coil was used for radiofrequency pulse transmission and a quadrature surface coil was used for signal detection. The rats were anesthetized by isoflurane in pure O2 (3% for induction, 1.5%–2% for maintenance) delivered via a nose cone during the imaging sessions with their respiration monitored continuously. DTI was performed with a spin-echo 4-shot echo-planar imaging (EPI) sequence, an encoding scheme of 30 gradient directions homogenously distributed on the unit sphere, and the following acquisition parameters: TR 5000 ms, TE 26 ms, FOV 3 cm × 3 cm, slice thickness of 0.8 mm, matrix size of 128 × 128, Δ 14 ms, δ 3 ms, two b values (0 and 800 s/mm 2) and 4 averages.
15 μm or 30 μm. Slices at Bregma 2.52 mm (including the ON) were sectioned continuously for Luxol fast blue (LFB) staining, immunocytochemical glial fibrillary acidic protein (GFAP) staining, and hyperphosphorylated neurofilament (SMI-31) staining respectively. Slices at Bregma 1.44 mm (including the external capsules (EC), striatum (CPu), and motor and sensory cortexs (CTX)) were sectioned continuously for LFB staining, Nissl staining, immunocytochemical GFAP staining, and SMI-31 staining respectively. Slices at Bregma −2.4 mm (including the OT and periventricular hypothalamic nucleus (Pe)) were sectioned continuously for LFB staining, Nissl staining, immunocytochemical GFAP staining, and SMI-31 staining respectively. For LFB staining, 15 μm-thick brain sections were incubated in a 0.1% LFB solution overnight at 60 °C. After reaching room temperature, these sections were first rinsed with 95% ethanol and then washed in distilled water. Differentiation was completed by briefly rinsing them in 0.05% lithium carbonate and 70% ethanol and then returning them to water until the contrast between the white and gray matter was maximized. The LFB-stained sections were then counterstained with toluidine blue. For Nissl staining, 15 μm-thick brain tissue sections were immersed in a 0.1% toluidine blue solution for approximately 1 min, washed with distilled water, and then rinsed in 80% ethanol, 95% ethanol, 100% ethanol,100% ethanol, and 100% xylene subsequently for 5 min followed by mounting. For immunohistochemistry, antigen retrieval was performed with 30 μm-thick brain sections. After cooling to room temperature, the endogenous peroxidases were inactivated via immersion in 3% H2O2 for 15 min, 3 washes in PBS, and incubation with 0.2% Triton X-100 for 30 min at 37 °C. After 3 rinses with PBS, the slides were blocked with 10% normal goat serum for 1 h to block nonspecific antibody interactions. Then, the diluted primary antibodies (GFAP, 1:100 PBS, and SMI-31, 1:500 PBS) were incubated for 2 h at 37 °C. After washing, the GFAP immunolabeling was detected with Histostain SP kit from Zymed with diaminobenzidine as the substrate (except for the ON); for the ON, the GFAP immunolabeling was detected with fluorescein isothiocyanateconjugated goat anti-rabbit; the SMI-31 immunolabeling was detected with a secondary antibody (Alexa Fluor 488 goat anti-mouse). 2.4. DTI data analysis
2.3. Histological evaluations After the MRI examination at 4 weeks, the anesthetized rats were perfused with 300 ml saline and subsequently 300 ml 4% paraformaldehyde dissolved in 100 mmol/l phosphate-buffered saline (PBS, pH = 7.4). Their brains were removed, post-fixed in the same fixative for at least 24 h, and stored in a refrigerator at 4 °C until examined. The brains were sectioned on a cryostat microtome (Leica, Germany). Coronal brain sections were obtained with a thickness of
Fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity (RD) maps were acquired using the toolbox provided by the Bruker PARAVISON 5.0 software. Using the PARAVASION 5.0 software, the Regions of Interest (ROIs), representing different anatomical structures, were traced manually on the FA (i.e., without interpolation) maps for each rat with guidance from the Paxinos digital atlas [13]. Then, the ROIs were shifted to the identical position on the MD, AD and RD maps, and the values of DTI
Fig. 1. The ROIs are manually drawn from the FA maps of the rat brain. ON: optic nerve, CTX: motor and sensory cortex, EC: external capsule, CPu: striatum, OT: optic tract, Pe: periventricular hypothalamic nucleus.
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
553
Fig. 2. A quantitative analysis of the DTI indices in the WM ROIs of the rat brain at 4 weeks after ligation. * p b 0.05, statistical inter-group significance at 4 weeks after BCCAO. ON: optic nerve, OT: optic tract, EC: external capsule.
indices were recorded. Student’s unpaired t-tests were performed to compare the DTI indices values in different brain regions at each time point between the BCCAO group and the control group.
3. Results 3.1. DTI parameter variation and histological evaluation of WM at 4 weeks after BCCAO Fig. 1 presents the positions of the ROIs. Fig. 2 shows the ROI analysis of the WM in the rat brain at 4 weeks after BCCAO. Compared
with the control, the FA values showed a marked reduction in the ON and OT, as well as an increase in the EC; the MD values were substantially decreased in the EC. The AD values had a significant reduction in the ON and OT; there was no apparent alteration in the EC. The RD values were elevated in the ON and OT and reduced in the EC. Fig. 3 shows the corresponding histological evaluations of the WM at 4 weeks after BCCAO. The LFB staining showed severe myelin rarefaction and vacuoles in the ON and OT and slightly reduced myelin rarefaction and vacuoles in the EC compared with the control. The immunoreactivity of axon marker for SMI-31 was significantly reduced in the ON and OT but not in the EC and obvious astrogliosis was observed in the ON, OT and EC.
Fig. 3. Histological evaluations of WM lesions at 4 weeks after BCCAO. LFB: Luxol fast blue staining, SMI-31: hyperphosphorylated neurofilament immunostaining, GFAP: glial fibrillary acidic protein immunostaining. ON: optic nerve, OT: optic tract, EC: external capsule. Magnification = ×400 in all of the images.
554
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
Fig. 4. The changes in the DTI indices of the WM ROIs over time. * p b 0.05 statistical significance between the BCCAO and control group at each time point. ON: optic nerve, OT: optic tract, EC: external capsule.
3.2. Evolution of DTI indices in WM after BCCAO
3.3. Evolution of DTI indices and histological evaluation in GM after BCCAO
To assess the evolution of WM damage after BCCAO, we assessed 4 time points (Fig. 4) following BCCAO. With the exception of the 2 day time point for the ON, the evolution of the FA values in the ON and OT showed a progressive reduction from 2 days to 4 weeks after BCCAO (compared with the control). However, in the EC the FA values significantly increased from 1 week to 4 weeks. The MD values were significantly reduced at 2 days and 4 weeks after BCCAO in the EC, and there were no significant changes in the ON or OT. The AD values in the ON and OT decreased from 1 week to 4 weeks after ligation; in the EC the AD value was markedly smaller only at 2 days after BCCAO. The RD values in the ON and OT significantly increased from 1 week to 4 weeks after ligation; however, the RD values in the EC were lower than those in the control at all time points (Fig. 4).
The evolution of the FA, MD, AD and RD was also investigated in three GM regions of the rat brains (Fig. 5). The FA values were markedly elevated in the CPu at 2 days after ligation and had no significant changes in Pe and CTX. The MD values were notably reduced at 2 days and elevated at 4 weeks in the Pe; the MD values were significantly reduced from 2 days to 4 weeks in the CPu (with the exception of the 2 week time point) and were markedly reduced at 2 days in the CTX. The AD values were significantly decreased at 2 days in the Pe and CTX as well as from 1 week to 2 weeks in the CPu. The RD values were significantly reduced at 2 days and notably elevated at 4 weeks following ligation in the Pe, substantially decreased from 2 days to 4 weeks following ligation in the CPu (with the exception of the 2 week time point), and reduced at 2 days following ligation in the CTX.
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
555
Fig. 5. The changes in the DTI indices of the GM ROIs over time. * p b 0.05 statistical significance between the BCCAO and control group at each time point. Pe: periventricular hypothalamic nucleus, CPu: striatum, CTX: motor and sensory cortex.
Fig. 6 shows the histological evaluations of the GM at 4 weeks after BCCAO. The Nissl staining showed that sparse cell arrangements occurred in the CPu and that neuronal shrinkage occurred in the Pe and CTX. The immunostaining for GFAP showed obvious gliosis in the Pe, CPu and CTX.
4. Discussion To the best of our knowledge, this is the first longitudinal study combining DTI with histological methods developed to detect the evolution of BCCAO brain injury in rat models. DTI is a sensitive and valid tool to investigate the abnormalities of rat brains [14,15] and may be used non-invasively to assess the spatiotemporal course of pathological changes of the BCCAO rat brain. At 4 weeks after BCCAO,
an increased RD value and reduced AD and FA values were shown in the ON and OT. From 2 days to 4 weeks after ligation, our results showed that DTI could potentially be used to detect an abnormality of rat brains as early as 2 days and the change patterns of the DTI parameters were region-specific among these selected brain regions. MRI findings in a BCCAO rat model using conventional T2-weighted imaging (T2WI) have been reported [16] and indicated few abnormalities in rat brains. This may have been because the T2WI was not sufficiently sensitive or because of a lower field strength. At 4 weeks after ligation, our results showed that DTI was sufficiently sensitive to detect an abnormality of the selected WM regions (Fig. 2). As a DTI index, the variation of FA indicated a change in WM microstructure integrity. A reduction of the FA has been found in numerous animal models, such as those involving retinal ischemia [17], neonatal rat hypoxia–ischemia [18], and a cuprizone mouse
556
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
Fig. 6. The histological evaluations of the GM lesions at 4 weeks after BCCAO. GFAP: glial fibrillary acidic protein immunostaining. Pe: periventricular hypothalamic nucleus, CPu: striatum, CTX: motor and sensory cortex. Magnification = ×400 in all images.
model [19]. Occasionally, FA increased [20–22]. In the current rat model, the FA values of the ON and OT were decreased at 4 weeks after ligation, which reflected a loss of directionality. In the EC, the FA value increased with a reduced RD at 4 weeks after ligation; this could be related to the glial scar formation that had previously been found to play a role in elevated FA [22]. The FA variations indicated that the selected WM regions were all vulnerable to BCCAO. The AD and RD have also been widely studied in recent years. Many reports [23,24] have recently found that impaired myelin could increase RD. Our study and previous reports [7,25] found that the ON and OT presented severe vacuoles and an increased crookedness of myelin fibers at 4 weeks after ligation. This suggests that the elevated RD values of the ON and OT maybe associated with myelin damage. Although the LFB staining showed abnormal results with small myelin fiber vacuoles in the EC at 4 weeks after the ligation, the RD value did not increase but was reduced compared with the control. This indicated that another mechanism may play a key role in reducing the RD value and supported the hypothesis [26] that the relationship between demyelination and RD is more complicated than previously thought. Multiple studies [17,27,28] support the hypothesis that increased RD was a specific marker of myelin injury. However, our EC data demonstrated a potentially reduced sensitivity of RD for detecting demyelination. Decreased AD is related to axon swelling [27] and neurofilament dysfunction [19]. In a BCCAO rat model, Wakita et al. [7] found increased immuno-
reactivity of the amyloid β/A4 precursor protein (APP) and chromogranin A (CgA), in the ON and OT. Additionally, many fibers showed a morphology typical of axon swelling. In our data, the AD values were significantly reduced in the presence of decreased SMI-31 immunoreactivity in the ON and OT. This suggested that axon damage may play a role in the varied AD values. In the EC, we found no significant change of the AD with relatively normal SMI-31 staining compared with the control. The differences in the AD and RD between the OT (or ON) and EC after BCCAO indicated regionspecific injury effects in the selected WM regions. In a vascular dementia animal model, the WM regions suffered a combination of demyelination [7], axon injury [7], astrogliosis [11] (Fig. 3), microglial activation [25], and low cerebral blood flow (CBF) [29]which could result in competing influences on the DTI indices [30,31]. If this is true, interpreting changes in DTI indices requires further study. The longitudinal changes in the DTI indices of the ON, OT and EC are shown in Fig. 4 and the evolution patterns of the DTI indices in the ON (or OT) and EC were not the same after BCCAO. DTI was sufficiently sensitive to detect the BCCAO-induced WM lesions as early as 2 days after ligation. This early detection showed that the DTI technique could potentially be used to determine the approximate time course of WM lesions after BCCAO. From the first to the fourth week, the significantly changed DTI indices of the ON and OT were consistent with an earlier pathological observation [7],
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
indicating WM rarefaction (via Klüver–Barrera staining in the ON and OT) and an increased expression of APP and encephalitogenic peptide (EP) 3 days after ligation (which persisted for up to at 30 days). These indicated that the DTI could potentially be used to detect WM lesions in the current rat model. In contrast with the ON and OT, the EC showed an increased FA and reduced RD. This indicated that the pathological factor that strongly influenced the DTI indices in the EC may differ from that in the ON or OT. The pathological changes in the EC after ligation were previously of little scientific concern and the relevant pathological processes were unclear and required further study. A temporal alteration of the DTI parameters was observed not only in the brain WM but also in the GM sites (Fig. 5) and there were region-specific DTI index evolution patterns in the selected GM regions. Our results demonstrated that DTI was also sensitive in the early detection of brain abnormalities in the selected GM regions. The changes in the values of the DTI indices demonstrated that the injury induced by BCCAO continued in the CPu from 1 week to 4 weeks following ligation. There were no significant CTX DTI changes. However, neuronal shrinkage may result in an increase in MD [32] as well as astrogliosis (Fig. 6), thereby producing a reduction in MD [33]. These factors exert competing influences on diffusion tensor indices. The DTI in the CTX was not sufficiently sensitive during this period. In the Pe, (and in contrast with the CTX), the MD and RD values were increased with obvious neuronal shrinkage and astrogliosis at 4 weeks. This indicated that the pathological factors that played an important role in altering the MD differed from those of the CTX. Compared with the reduced MD and RD values of the Pe at 2 days, the increased MD and RD values at 4 weeks implied that the primary factor affecting the MD and AD values may have changed between the 2 day and 4 week time points. The hypothalamus is a center of autonomic control and is involved in regulating appetite. However, the hypothalamus has received little attention in BCCAO rat models. The Pe is a part of the hypothalamus and is vulnerable to BCCAO (Figs. 5 and 6), and previous research [34] indicated that BCCAO animals regain weight slowly compared with those in control groups. The decreased or increased MD values that we observed in the GM regions were affected by numerous pathological processes, such as neuron loss or shrinkage (Fig. 6), gliosis (Fig. 6), microglial activation [10], low CBF [35] and others, all of which could differentially affect the movement of water molecules. Although it is difficult to determine (due to the abnormal MD value) which pathological process occurred, these abnormal MD values provide useful information that furthers our understanding of the pathological processes of the GM in the BCCAO rat brain. In the present study, we monitored rat brain injury at different time points after ligation and discovered that the change patterns of the DTI parametric values were region-specific among select brain regions. The current results indicate that DTI may be a sensitive and valid means for assessing BCCAO-induced WM lesions. The results also provide a basic understanding of brain injury evolution after BCCAO. DTI may be helpful in obtaining useful and accurate information regarding early-stage vascular dementia diagnosis. Acknowledgments The authors express their gratitude to Mrs. Erin Beatson for proof reading. This work was supported by the Natural Science Foundation of China (No. 81000598). References [1] Choi JY, Morris JC, Hsu CY. Aging and cerebrovascular disease. Neurol Clin 1998; 16:687–711.
557
[2] Claus JJ, Breteler MM, Hasan D, Krenning EP, Bots ML, Grobbee DE, et al. Regional cerebral blood flow and cerebrovascular risk factors in the elderly population. Neurobiol Aging 1998;19:57–64. [3] Farkas E, Luiten PG, Bari F. Permanent, bilateral common carotid artery occlusion in the rat: a model for chronic cerebral hypoperfusion-related neurodegenerative diseases. Brain Res Rev 2007;54:162–80. [4] Kim SK, Cho KO, Kim SY. White matter damage and hippocampal neurodegeneration induced by permanent bilateral occlusion of common carotid artery in the rat: comparison between Wistar and Sprague–Dawley strain. Korean J Physiol Pharmacol 2008;12:89–94. [5] Yoshizaki K, Adachi K, Kataoka S, Watanabe A, Tabira T, Takahashi K, et al. Chronic cerebral hypoperfusion induced by right unilateral common carotid artery occlusion causes delayed white matter lesions and cognitive impairment in adult mice. Exp Neurol 2008;210:585–91. [6] Fernando MS, Simpson JE, Matthews F, Brayne C, Lewis CE, Barber R, et al. White matter lesions in an unselected cohort of the elderly: molecular pathology suggests origin from chronic hypoperfusion injury. Stroke 2006;37:1391–8. [7] Wakita H, Tomimoto H, Akiguchi I, Matsuo A, Lin JX, Ihara M, et al. Axonal damage and demyelination in the white matter after chronic cerebral hypoperfusion in the rat. Brain Res 2002;924:63–70. [8] Cho KO, La HO, Cho YJ, Sung KW, Kim SY. Minocycline attenuates white matter damage in a rat model of chronic cerebral hypoperfusion. J Neurosci Res 2006; 83:285–91. [9] Ni J, Ohta H, Matsumoto K, Watanabe H. Progressive cognitive impairment following chronic cerebral hypoperfusion induced by permanent occlusion of bilateral carotid arteries in rats. Brain Res 1994;653:231–6. [10] Schmidt-Kastner R, Aguirre-Chen C, Saul I, Yick L, Hamasaki D, Busto R, et al. Astrocytes react to oligemia in the forebrain induced by chronic bilateral common carotid artery occlusion in rats. Brain Res 2005;1052:28–39. [11] Wakita H, Tomimoto H, Akiguchi I, Kimura J. Glial activation and white matter changes in the rat brain induced by chronic cerebral hypoperfusion: an immunohistochemical study. Acta Neuropathol 1994;87:484–92. [12] Soria G, Tudela R, Marquez-Martin A, Camon L, Batalle D, Munoz-Moreno E, et al. The ins and outs of the BCCAo model for chronic hypoperfusion: a multimodal and longitudinal MRI approach. PLoS One 2013;8:e74631. [13] George Paxinos CW. The rat brain in stereotaxic coordinates. 2nd ed. San Diego: Academic Press; 1996. [14] Dror V, Eliash S, Rehavi M, Assaf Y, Biton IE, Fattal-Valevski A. Neurodegeneration in thiamine deficient rats—a longitudinal MRI study. Brain Res 2010;1308: 176–84. [15] Wang S, Wu EX, Qiu D, Leung LH, Lau HF, Khong PL. Longitudinal diffusion tensor magnetic resonance imaging study of radiation-induced white matter damage in a rat model. Cancer Res 2009;69:1190–8. [16] Plaschke K, Bardenheuer HJ, Martin E, Sartor K, Heiland S. Evolution of apparent diffusion coefficient and transverse relaxation time (T2) in the subchronic stage of global cerebral oligemia in different rat models. Exp Brain Res 2006;169: 361–8. [17] Song SK, Sun SW, Ju WK, Lin SJ, Cross AH, Neufeld AH. Diffusion tensor imaging detects and differentiates axon and myelin degeneration in mouse optic nerve after retinal ischemia. Neuroimage 2003;20:1714–22. [18] Wang S, Wu EX, Cai K, Lau HF, Cheung PT, Khong PL. Mild hypoxic–ischemic injury in the neonatal rat brain: longitudinal evaluation of white matter using diffusion tensor MR imaging. AJNR Am J Neuroradiol 2009;30:1907–13. [19] Sun SW, Liang HF, Trinkaus K, Cross AH, Armstrong RC, Song SK. Noninvasive detection of cuprizone induced axonal damage and demyelination in the mouse corpus callosum. Magn Reson Med 2006;55:302–8. [20] Bockhorst KH, Narayana PA, Liu R, Ahobila-Vijjula P, Ramu J, Kamel M, et al. Early postnatal development of rat brain: in vivo diffusion tensor imaging. J Neurosci Res 2008;86:1520–8. [21] Van Camp N, Blockx I, Verhoye M, Casteels C, Coun F, Leemans A, et al. Diffusion tensor imaging in a rat model of Parkinson's disease after lesioning of the nigrostriatal tract. NMR Biomed 2009;22:697–706. [22] Budde MD, Janes L, Gold E, Turtzo LC, Frank JA. The contribution of gliosis to diffusion tensor anisotropy and tractography following traumatic brain injury: validation in the rat using Fourier analysis of stained tissue sections. Brain 2011; 134:2248–60. [23] Wang S, Wu EX, Tam CN, Lau HF, Cheung PT, Khong PL. Characterization of white matter injury in a hypoxic-ischemic neonatal rat model by diffusion tensor MRI. Stroke 2008;39:2348–53. [24] Xie M, Tobin JE, Budde MD, Chen CI, Trinkaus K, Cross AH, et al. Rostrocaudal analysis of corpus callosum demyelination and axon damage across disease stages refines diffusion tensor imaging correlations with pathological features. J Neuropathol Exp Neurol 2010;69:704–16. [25] Lee JH, Park SY, Shin YW, Hong KW, Kim CD, Sung SM, et al. Neuroprotection by cilostazol, a phosphodiesterase type 3 inhibitor, against apoptotic white matter changes in rat after chronic cerebral hypoperfusion. Brain Res 2006;1082: 182–91. [26] Van Camp N, Blockx I, Camon L, de Vera N, Verhoye M, Veraart J, et al. A complementary diffusion tensor imaging (DTI)-histological study in a model of Huntington's disease. Neurobiol Aging 2012;33:945–59. [27] Song SK, Yoshino J, Le TQ, Lin SJ, Sun SW, Cross AH, et al. Demyelination increases radial diffusivity in corpus callosum of mouse brain. Neuroimage 2005;26:132–40. [28] Song SK, Sun SW, Ramsbottom MJ, Chang C, Russell J, Cross AH. Dysmyelination revealed through MRI as increased radial (but unchanged axial) diffusion of water. Neuroimage 2002;17:1429–36.
558
X. Wang et al. / Magnetic Resonance Imaging 33 (2015) 551–558
[29] Tsuchiya M, Sako K, Yura S, Yonemasu Y. Cerebral blood flow and histopathological changes following permanent bilateral carotid artery ligation in Wistar rats. Exp Brain Res 1992;89:87–92. [30] Assaf Y, Pasternak O. Diffusion tensor imaging (DTI)-based white matter mapping in brain research: a review. J Mol Neurosci 2008;34:51–61. [31] Ding AY, Hui ES, Wu EX. The effects of hypercapnia on DTI quantification in anesthetized rat brain. Conf Proc IEEE Eng Med Biol Soc 2009;2009:2711–4. [32] Douaud G, Behrens TE, Poupon C, Cointepas Y, Jbabdi S, Gaura V, et al. In vivo evidence for the selective subcortical degeneration in Huntington's disease. Neuroimage 2009;46:958–66.
[33] Anderova M, Vorisek I, Pivonkova H, Benesova J, Vargova L, Cicanic M, et al. Cell death/proliferation and alterations in glial morphology contribute to changes in diffusivity in the rat hippocampus after hypoxia–ischemia. J Cereb Blood Flow Metab 2011;31:894–907. [34] Farkas E, de Wilde MC, Kiliaan AJ, Meijer J, Keijser JN, Luiten PG. Dietary long chain PUFAs differentially affect hippocampal muscarinic 1 and serotonergic 1A receptors in experimental cerebral hypoperfusion. Brain Res 2002;954:32–41. [35] Otori T, Katsumata T, Muramatsu H, Kashiwagi F, Katayama Y, Terashi A. Longterm measurement of cerebral blood flow and metabolism in a rat chronic hypoperfusion model. Clin Exp Pharmacol Physiol 2003;30:266–72.