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Normalization of T2 relaxation time and apparent diffusion coefficient in relation to the inflammatory changes in the substantia nigra of rats with focal cerebral ischemia Yan Mei Yang, Chan Chan Li, Le Kang Yin and XiaoYuan Feng Acta Radiol published online 26 September 2014 DOI: 10.1177/0284185114549496 The online version of this article can be found at: http://acr.sagepub.com/content/early/2014/09/26/0284185114549496
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Acta Radiol OnlineFirst, published on September 26, 2014 as doi:10.1177/0284185114549496
Original Article
Normalization of T2 relaxation time and apparent diffusion coefficient in relation to the inflammatory changes in the substantia nigra of rats with focal cerebral ischemia
Acta Radiologica 0(0) 1–7 ! The Foundation Acta Radiologica 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0284185114549496 acr.sagepub.com
Yan Mei Yang*, Chan Chan Li*, Le Kang Yin and XiaoYuan Feng
Abstract Background: Focal cerebral ischemia results in delayed neurodegeneration in remote brain regions, such as the substantia nigra. To date, a reasonable explanation is still lacking regarding the correlation of magnetic resonance (MR) signal pseudo-normalization following a transient abnormal change and subsequent progressive pathological damage. Purpose: To characterize the substantia nigra following middle cerebral artery occlusion and to evaluate the potential pathophysiological changes associated with the pseudo-normalization of MR signals in the substantia nigra at the subacute stage after stroke onset. Material and Methods: Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion. During the occlusion of single middle cerebral artery, computed tomography (CT) perfusion was acquired to observe the blood flow perfusion in the primary ischemic striatum and ipsilateral substantia nigra. Next, the MR T2 relaxation time and apparent diffusion coefficient changes within the substantia nigra were determined on days 1, 3, 7, and 14 after stroke onset, and compared with immunohistochemistry for microglia activation and astrogliosis. Results: Twenty-four rats with strong hypoperfusion in the primary ischemic territory and no alterations of the perfusion in the ipsilateral substantia nigra detected both visually and measurably during the middle cerebral artery occlusion were further studied. All animals showed MR pseudo-normalization with T2 relaxation time and apparent diffusion coefficient recovered in the ipsilateral substantia nigra at the subacute phase following focal cerebral ischemia. Normalization of the MR signals corresponded well with the spatio-temporal occurrence of microglia activation and astrogliosis. Conclusion: The pseudo-normalization of T2 relaxation time and apparent diffusion coefficient reflects the neuroinflammatory changes that accompany activation of microglia and astrocytes in the ipsilateral substantia nigra following middle cerebral artery occlusion.
Keywords Substantia nigra, cerebral ischemia, inflammation, magnetic resonance imaging (MRI), immunohistochemistry Date received: 4 March 2014; accepted: 13 July 2014
Introduction It is well known that focal cerebral ischemia causes neuronal damage in the territory of disturbed cerebral vessels. In addition to this neuronal injury, cerebral ischemia triggers changes in remote areas that are separated from the affected vessels. The substantia nigra (SN) is nourished by the perforating branches of the posterior cerebral artery (PCA), which originate from
Department of Radiology, Huashan Hospital, Fudan University, Shanghai, PR China *Equal contributors. Corresponding author: Yan Mei Yang, Department of Radiology, Huashan Hospital, Fudan University, 12 Wulumuqi Middle Road, Shanghai 200040, PR China. Email: [email protected]
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the initial portion of the superior cerebellar artery (SCA) in rats. In the SN, middle cerebral artery (MCA) occlusion in rats induces extensive delayed SN neurodegeneration, and stroke patients with striatal damage also display SN degeneration following the onset of ischemia (1,2). The SN is a key structure involved in motor control, and has bi-directional neuronal connections with the striatum, which becomes ischemic after MCA occlusion (3). Although the mechanisms underlying remote injury are largely unclear, it has been proposed that post-ischemic alterations of the striato-nigral gamma-aminobutyric acid (GABA) system are associated with neuronal loss in the SN and subsequent motor deficits. Imaging techniques, such as computed tomography (CT) and magnetic resonance imaging (MRI) play an important role in the diagnosis and therapeutic decision-making for ischemic stroke (4). Using T2-weighted (T2W) imaging and diffusion-weighted imaging (DWI), previous studies (1,5–8) have detected remote neurodegeneration of the ipsilateral SN following focal cerebral ischemia, which resulted in T2W imaging and DWI hyperintensity signals within 3 to 5 days in rats and at 1 to 4 weeks after striatal ischemia in patients. These results share a common characteristic that T2W imaging and DWI are associated with a delayed normointensity in the SN after a transient abnormal signal both in patients and animals. However, studies performed in rat have demonstrated that focal cerebral ischemia can cause severe and prolonged neuronal damage in the ipsilateral SN, and these pathological changes are not static but progressive (2). To date, a reasonable explanation is still lacking regarding the correlation of MR signal pseudo-normalization and the subsequent progressive pathological damage. Thus, we performed CT perfusion to confirm successful focal cerebral ischemia and to ensure stable blood flow perfusion in the SN. We investigated the MR signal in the remote SN over time after a unilateral MCA occlusion and compared these MR data with immunohistochemistry analyses.
Material and Methods Middle cerebral artery occlusion and protocol This study was performed according to the National Institutes of Health guidelines for the care and use of laboratory animals. All protocols were approved by the medical experiment animal administrative committee of Shanghai. All efforts were made to minimize animal suffering and to reduce the number of animals used. Healthy adult male Sprague Dawley rats (180–230 g) from the Shanghai Experimental Animal Centre were used in this study. Rats were anesthetized with an
intraperitoneal injection of chloral hydrate (350– 400 mg/kg). The rectal temperature was monitored and maintained at 37 0.5 C using a controlled water flow system throughout the procedure. Rats were subjected to focal cerebral ischemia induced by a transient left MCA occlusion as previously described (5,9). Briefly, the left external carotid artery (ECA) was initially ligated and transected to generate an ECA stump. Next, a nylon filament was introduced into the ECA stump and advanced into the internal carotid artery (ICA) to block blood flow to MCA. During MCA occlusion, computed tomography (CT) perfusion was performed to observe blood flow perfusion of the MCA territory and SN. Twenty-four rats were further studied, which showed impaired perfusion in the striatum and constant perfusion in the SN and were randomly assigned to each time point groups (n ¼ 6 animals each). Post-ischemic reperfusion was performed at 90 min after MCA occlusion by suture removal until its tip reached the ECA stump. In six rats from each time point, T2W imaging, T2 map, and DWI were performed on days 1, 3, 7, and 14 using a MR scanner. After MRI, the brains were removed and processed for histological examination.
CT perfusion imaging and analysis CT perfusion imaging was performed using a multislice CT scanner (Light-Speed 16, GE Healthcare, Milwaukee, WI, USA). The anesthetized rat was placed in a supine position in a custom-made board on the CT scanning table. The animal’s venous catheter was inserted into the femoral vein and connected to an automated injector. An un-enhanced CT scan was first obtained to localize the lesion. The parameters were: 120 kV; 100 mA; slice thickness, 2.5 mm; field of view (FOV), 9.6 9.6 cm; matrix, 512 512; scanning time, 1 s/scan. Next, perfusion scanning was performed using the cine scan technique, which covered the frontal lobe to the medulla. A non-ionic type CT contrast agent (Iohexol, 300 mg I/mL, GE Healthcare) was applied via the femoral vein using the automated injector at a dose of 2 mL with an injection flow rate of 1 mL/s. In addition, perfusion imaging was scanned and repeated 50 times. The parameters of cine scanning were the same as the un-enhanced scanning. The CT perfusion raw data were transferred to a dedicated workstation (Advantage Windows 4.1, GE Healthcare). For all datasets, perfusion analysis was performed using the CT Perfusion 3.0 software. The basilar artery and superior sagittal sinus were selected as the input artery and output vein (regions of interest range, 18–25 mm2), respectively. The images produced by the software were represented as three parametric maps: cerebral blood flow (CBF), cerebral blood
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volume (CBV), and mean transit time (MTT). Regions of interest (ROIs) were placed onto the striatum and substantia nigra of the ischemic and intact side on the perfusion parametric maps. Representative parametric values obtained from two radiologists with years of experience of CT perfusion imaging were averaged. The relative perfusion parameters (ipsilateral/contralateral region) were expressed as the mean standard deviation.
MRI and analysis We performed MRI scan using a GE Signa VH/i system equipped with a 3.0-T superconducting magnet (GE Healthcare) (9). Multi-slice T2W fast spin-echo imaging was performed and resulted in 12 contiguous slices in the coronal plane: TR, 4000 ms; TE, 120 ms; slice thickness, 2 mm; spacing, 0; FOV, 60 60 mm; matrix, 256 192; NEX, 4; scanning time, 3 min 24 s. Multi-echo T2W imaging was used to acquire the T2 map: TR, 3000 ms; TE, 159.9/146.6/133.2/119.9/106.6/ 93.3/79.9/66.6/53.3/40.0/26.6/13.3 ms; slice thickness, 2 mm; spacing, 0; FOV, 60 60 mm; matrix, 256 160; NEX, 1; scanning time, 5 min 20 s. DWI with diffusion-sensitive gradients in three dimensions (single-shot spin-echo echo planar images) was used: TR, 3500 ms; TE, 94.4 ms; slice thickness, 2 mm; spacing, 0; FOV, 40 40 mm; matrix, 64 96; NEX, 8; b, 0,1000 s/mm2; scanning time, 1 min 56 s. For images analysis, MR digital imaging and communications in medicine format (DICOM) images were transferred from the picture archive and communications system (PACS) to a separate personal computer (MacPro, Apple, Cupertino, CA, USA). OsiriX (version 4.1, OsiriX Foundation, Geneva, Switzerland), and free and open source postprocessing and fusion software tool (free down load from http://www.osirixviewer.com) were used. For the quantification analysis, the sync series analyze tool was used to synchronize the cursor on T2W imaging to the T2 map and ADC map onto the same slice image. The ROIs were drawn on the ischemic ipsilateral and contralateral SN. ROIs were visually matched between sequences to avoid the outermost pixels and to minimize partial volume effects. The ROI analyze tool was used to store the ROIs and to determine the average T2 relaxation time (ms), ADC (mm2/s), and standard deviation values from each region.
Tissue processing and staining On days 1, 3, 7, and 14, after all imaging experiments, six rats from each time point were anesthetized and perfused through the left ventricle with 50 mL of saline, followed by 100 mL of 4% paraformaldehyde.
The brains were rapidly removed, and immersion-fixed overnight in 4% paraformaldehyde in phosphate buffer and embedded in paraffin at 4 C until further use. Subsequently, 10-mm-thick coronal sections corresponding to each MR slice images were cut using a microtome, and sections in the SN were stained using Hematoxylin and Eosin (H&E). Microglia (CD68 immuno-positive cells) and astrocytes (GFAP immuno-positive cells) were detected using standard diaminobenzidine (DAB) immunohistochemisty with the CD68 monoclonal antibody (Biolegend, San Diego, CA, USA) and glial fibrillary acidic protein (GFAP) antibody (GeneTex, San Antonio, TX, USA), respectively. The number of CD68-positive cells and GFAP-positive cells were quantified at a magnification in a blind manner using a computerized image-analysis system consisting of a NIS camera installed onto an Olympus light microscope, which was attached to a personal computer. Data were expressed as the mean standard deviation numbers of cells (i.e. cell number/mm3).
Statistical analysis Statistical analyses were performed using SPSS software version 16 (SPSS Inc., Chicago, IL, USA). The relative CT perfusion parameters, T2 relaxation times, ADC, CD68- and GFAP-positive cell number were compared using one-way analysis of variance (ANOVA) for separated measures. Comparisons between ipsilateral and contralateral regions were then made using Student’s t-test. The level of statistical significance was P < 0.05 throughout this study.
Results Cerebral blood flow perfusion changes in the striatum and substantia nigra during MCA occlusion During MCA occlusion, CT perfusion was performed to monitor blood flow perfusion in the brain. Heterogeneous decreases of tissue perfusion in the MCA territory were detected in ischemic animals. At 30 min after MCA occlusion, the CBF and CBV in the ischemic striatum decreased compared with the contralateral striatum, and the MTT was prolonged (Fig. 1a–c), which confirmed a successful MCA occlusion. The relative perfusion values of the bilateral striatum at 30 min were rCBF 0.23 0.08, rCBV 0.75 0.09, rMTT 4.44 0.11. In contrast, the CT perfusion data showed constant blood flow perfusion in the SN (Fig. 1d–f) with relative values of rCBF of 1.03 0.06, rCBV 0.98 0.07, rMTT 1.01 0.05. Thus, the premise of studying remote changes was established. Compared between the perfusion
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Fig. 1. CT perfusion CBF map (a), CBV map (b), and MTT map (c) in the striatum (ST); the section was obtained from a representative rat acquired at 30 min during MCA occlusion and shows the cerebral perfusion deficit in the MCA territory. There is reduced CBF and CBV, and prolonged MTT compared with the contralateral striatum. CT perfusion CBF map (d), CBV map (e), and MTT map (f) from the same rat in the substantia nigra (SN); this section shows that the blood flow perfusion had no visible and measurable difference between the bilateral SN at each perfusion image.
parameters after MCA occlusion, there was statistical significance in the CBF, CBV, and MTT in the bilateral striatum (P < 0.05), but no significant differences between the ipsilateral and contralateral SN (P > 0.05).
Temporal profile of magnetic resonance T2 relaxation time and ADC in the substantia nigra after MCA occlusion The primary ischemic lesion was developed in the striatum in all grouped MCA occlusion rats. In the SN section, there were no visible and measurable differences between the bilateral SN in T2 relaxation time (80.2 8.1 ms vs. 77.7 9.8 ms) and ADC value (7.88 0.78 104 mm2/s vs. 8.09 1.02 104 mm2/s) on day 1 after stroke (Fig. 2a and f). On day 3, the ipsilateral SN to the MCA occlusion showed a T2 hyperintensity signal and ADC low signal compared to the contralateral SN (Fig. 2b, g, h) (T2 relaxation time 105.7 9.2 ms vs. 74.1 6.8 ms, ADC value 5.34 1.08 104 mm2/s vs. 7.74 0.89 104 mm2/s, respectively). At the subacute stage, days 7 and 14, abnormal changes could not be discriminated in both the qualitative and quantitative determination on the T2 map and ADC map (Fig. 2c, d, i) (T2 relaxation time 83.4 10.1 ms vs. 79.5 8.8 ms, ADC value 7.72 0.97 104 mm2/s vs. 7.61 1.11 104 mm2/s on day 7, T2 relaxation time 81.2 10.8 ms vs. 82.4 9.8 ms, ADC value 7.60 0.88 104 mm2/s vs. 7.59 0.89 104 mm2/s on day 14, respectively). The dynamic changes of T2 relaxation time and ADC in the SN after MCA occlusion are shown in Fig. 2e and j. Compared with the T2 relaxation time and ADC between the bilateral SN after MCA occlusion, there were statistical significances on day 3 (*P < 0.05), but
no significant differences on days 1, 7, and 14 (P > 0.05).
Morphological observation of H&E staining in the substantia nigra after MCA occlusion In the ipsilateral SN with respect to the MCA occluded side, most neurons in the pars reticulata lacked an abnormal appearance as assessed using H&E staining and a light microscope on day 1 after MCA occlusion (Fig. 3a). On day 3 post ischemia, the neuronal cells were swollen, the neuropil was slightly vacuolated, and the perivascular spaces were enlarged (Fig. 3b). The pars reticulata of the SN became smaller compared to the contralateral side after 7 and 14 days. The reactive glia gradually increased in number on days 7 and 14 (Fig. 3c, d).
Proliferation of CD68 and GFAP immuno-positive cell in the substantia nigra after MCA occlusion We next investigated whether immunohistochemical changes occurred in the SN after focal cerebral ischemia. Representative microphotographs of CD68 immunostaining in the SN after stroke are shown in Fig. 4a–d and GFAP immunostaining in Fig. 4e–h. The number of CD68 and GFAP positive cells were 25 7 /mm3 and 35 9 /mm3 on day 1, 30 5 /mm3 and 42 8 /mm3 on day 3, 42 5 /mm3 and 62 10 / mm3 on day 7, and 52 9 /mm3 and 71 9 /mm3 on day 14, respectively. In the contralateral SN, a small number of CD68 and GFAP immuno-positive cells were observed at each time point, in which the number of CD68 and GFAP positive cells were 20 5 /mm3 and 32 7 /mm3 on day 1, 25 5 /mm3 and
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Fig. 2. Alteration of T2 (a–d, g) and ADC (f, h, i) signals in the SN after MCA occlusion. On day 1 post ischemia, there is no abnormal signal on both sides of the SN. On day 3 after stroke, high T2 and low ADC signal can be visualized in ischemic ipsilateral SN (b, g, h). On days 7 and 14, the T2 elevation and ADC decline had recovered (c, d, i). The line diagrams illustrated the temporal profile of T2 relaxation time (e) and ADC (j) in the ischemic ipsilateral and intact SN on days 1, 3, 7, and 14. *P < 0.05, significantly different compared to the contralateral region.
Fig. 3. H&E staining on ischemic ipsilateral SN sections on day 1 (a, 400), day 3 (b, 400), day 7 (c, 400), and day 14 (d, 400) after MCA occlusion. Using light microscopy, there was no abnormal change in the SN on day 1. On day 3 after stroke, the neuronal cells were swollen, neuropils are vacuolated, and the perivascular spaces are enlarged with light microscope observation (b). Subsequently, vacuolation of neuropil became milder, and the reactive glia gradually increased in number (c, d).
Fig. 4. CD68 (a–c) and GFAP (e–g) immunohistochemical staining in the ischemic ipsilateral SN after stroke 1 day (a, 40; e, 40) and 14 days (b, 40; c, 400; f, 40; g, 400) are shown. The number of CD68 immunopositive cell increases in the ipsilateral SN from 7 days up to 14 days after focal cerebral ischemia (b, c) compared to days 1 (a) and 3. Similarly, the expression of GFAP immunoreaction increased in the ipsilateral SN on 7 and 14 days (f, g) compared to day 1 (e) and 3. The line diagrams illustrated the CD68 (d) and GFAP positive cell number (h) in the ipsilateral and contralateral SN on days 1, 3, 7, and 14. *P < 0.05, significantly different compared to contralateral intact region.
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31 6 /mm3 on day 3, 26 4 /mm3 and 34 6 /mm3 on day 7, and 28 6 /mm3 and 32 7 /mm3 on day 14. Compared with the SN on the ischemic and intact side, on days 1 and 3 post ischemia, there were no significant differences in both immunostaining results (P > 0.05). In contrast, there was a significantly increased number of CD68 positive microglia and GFAP positive astrocytes within the ipsilateral SN compared to the contralateral SN 7–14 days after focal cerebral ischemia (*P < 0.05).
Discussion This study was performed in a rat model of focal cerebral ischemia. Our data revealed MR signal pseudonormalization phenomenon with T2 relaxation time and apparent diffusion coefficient recovered in the ipsilateral SN at the subacute phase after stroke onset. Normalization of the T2 relaxation time and apparent diffusion coefficient corresponded well with the spatiotemporal occurrence of microglia activation and astrogliosis. With the present data, three aspects need to be addressed. The first issue involves the essential study of the precondition of remote degeneration following focal cerebral ischemia. To the best of our knowledge, this is the first study to employ CT perfusion to study remote degeneration after MCA occlusion. Impaired hemodynamic changes in the MCA territory were clearly demonstrated using the CT perfusion technique. However, in the SN, there was no visibly and measurably abnormal perfusion on all perfusion parametric maps. The different tissue perfusions between the striatum and SN at the early stage after focal cerebral ischemia demonstrated success of the animal model and enabled studies on pathogenesis in the remote site. The second issue relates to the immunohistochemistry feature in the SN following striatal ischemia. A significant increase in the number of microglia and astrocyte was observed in the pars reticulata of the ipsilateral SN 7–14 days after focal cerebral ischemia. Thus, specific evidence supports that inflammatory processes participate in the pathogenesis and execution of remote neurodegeneration. Activated microglia and astrocytes spread in the pars reticulata in a diffuse pattern, which might be due to the selective neuronal loss in the absence of necrosis in the ischemic remote region. The third issue involves changes in the T2 relaxation time and ADC in the SN following MCA occlusion. First, the present study effectively illustrated neurodegeneration of the ipsilateral SN. On day 3 after stroke, degeneration was demonstrated as a prolonged T2 relaxation time and decreased ADC. It is well known the reduced ADC is the most sensitive indication of swollen cells after focal ischemic brain damage, and
T2 elevation is a good method to image brain edema after infarction. These current results were consistent with results obtained in previous experimental studies (7,8,10–12). These signal changes were present later compared to the primary ischemic lesion and may be caused by swelling astrocytes and neurons. Thus, the MR signal can reflect the secondary neuronal swelling in the ipsilateral SN after MCA occlusion. Subsequently, the T2 relaxation time and ADC of the ipsilateral SN recovered to the same extent as the contralateral side, which corresponded well with the spatio-temporal distribution of astrogliosis and microglia activation. In fact, this correlation between MR signals and inflammatory cell proliferation has been observed in primary ischemic studies (13–16). In a primary ischemic lesion, glial activation and infiltration of hematogenous cells induced ADC normalization and reduced T2 elevation at the sub-acute phase following focal cerebral ischemia. The intense areas of inflammatory cell proliferation showed MR fogging presentation with normal tissue. Thus, changes in the T2 relaxation time and ADC could undergo characteristic changes and differentiate distinct stages of the inflammatory response and tissue remodeling after stroke both in primary and remote sites. In conclusion, pseudo-normalization of the T2 relaxation time and apparent diffusion coefficient reflects the neuroinflammatory changes in the activation of microglia and astrocytes in the ipsilateral substantia nigra after middle cerebral artery occlusion. Acknowledgments We gratefully acknowledge the contribution and endeavor of Mr. Tong Jin Sun.
Conflict of interest We declare that we have no conflict of interest.
Funding This study was supported by the grant of National Natural Science Foundation of China (No. 81371521) and Shanghai Natural Science Foundation of China (No. 09ZR1405100).
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Normalization of T2 relaxation time and apparent diffusion coefficient in relation to the inflammatory changes in the substantia nigra of rats with focal cerebral ischemia Yan Mei Yang, Chan Chan Li, Le Kang Yin and XiaoYuan Feng Acta Radiol published online 26 September 2014 DOI: 10.1177/0284185114549496 The online version of this article can be found at: http://acr.sagepub.com/content/early/2014/09/26/0284185114549496
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On behalf of: Nordic Society of Medical Radiology
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Acta Radiol OnlineFirst, published on September 26, 2014 as doi:10.1177/0284185114549496
Original Article
Normalization of T2 relaxation time and apparent diffusion coefficient in relation to the inflammatory changes in the substantia nigra of rats with focal cerebral ischemia
Acta Radiologica 0(0) 1–7 ! The Foundation Acta Radiologica 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/0284185114549496 acr.sagepub.com
Yan Mei Yang*, Chan Chan Li*, Le Kang Yin and XiaoYuan Feng
Abstract Background: Focal cerebral ischemia results in delayed neurodegeneration in remote brain regions, such as the substantia nigra. To date, a reasonable explanation is still lacking regarding the correlation of magnetic resonance (MR) signal pseudo-normalization following a transient abnormal change and subsequent progressive pathological damage. Purpose: To characterize the substantia nigra following middle cerebral artery occlusion and to evaluate the potential pathophysiological changes associated with the pseudo-normalization of MR signals in the substantia nigra at the subacute stage after stroke onset. Material and Methods: Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion. During the occlusion of single middle cerebral artery, computed tomography (CT) perfusion was acquired to observe the blood flow perfusion in the primary ischemic striatum and ipsilateral substantia nigra. Next, the MR T2 relaxation time and apparent diffusion coefficient changes within the substantia nigra were determined on days 1, 3, 7, and 14 after stroke onset, and compared with immunohistochemistry for microglia activation and astrogliosis. Results: Twenty-four rats with strong hypoperfusion in the primary ischemic territory and no alterations of the perfusion in the ipsilateral substantia nigra detected both visually and measurably during the middle cerebral artery occlusion were further studied. All animals showed MR pseudo-normalization with T2 relaxation time and apparent diffusion coefficient recovered in the ipsilateral substantia nigra at the subacute phase following focal cerebral ischemia. Normalization of the MR signals corresponded well with the spatio-temporal occurrence of microglia activation and astrogliosis. Conclusion: The pseudo-normalization of T2 relaxation time and apparent diffusion coefficient reflects the neuroinflammatory changes that accompany activation of microglia and astrocytes in the ipsilateral substantia nigra following middle cerebral artery occlusion.
Keywords Substantia nigra, cerebral ischemia, inflammation, magnetic resonance imaging (MRI), immunohistochemistry Date received: 4 March 2014; accepted: 13 July 2014
Introduction It is well known that focal cerebral ischemia causes neuronal damage in the territory of disturbed cerebral vessels. In addition to this neuronal injury, cerebral ischemia triggers changes in remote areas that are separated from the affected vessels. The substantia nigra (SN) is nourished by the perforating branches of the posterior cerebral artery (PCA), which originate from
Department of Radiology, Huashan Hospital, Fudan University, Shanghai, PR China *Equal contributors. Corresponding author: Yan Mei Yang, Department of Radiology, Huashan Hospital, Fudan University, 12 Wulumuqi Middle Road, Shanghai 200040, PR China. Email: [email protected]
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the initial portion of the superior cerebellar artery (SCA) in rats. In the SN, middle cerebral artery (MCA) occlusion in rats induces extensive delayed SN neurodegeneration, and stroke patients with striatal damage also display SN degeneration following the onset of ischemia (1,2). The SN is a key structure involved in motor control, and has bi-directional neuronal connections with the striatum, which becomes ischemic after MCA occlusion (3). Although the mechanisms underlying remote injury are largely unclear, it has been proposed that post-ischemic alterations of the striato-nigral gamma-aminobutyric acid (GABA) system are associated with neuronal loss in the SN and subsequent motor deficits. Imaging techniques, such as computed tomography (CT) and magnetic resonance imaging (MRI) play an important role in the diagnosis and therapeutic decision-making for ischemic stroke (4). Using T2-weighted (T2W) imaging and diffusion-weighted imaging (DWI), previous studies (1,5–8) have detected remote neurodegeneration of the ipsilateral SN following focal cerebral ischemia, which resulted in T2W imaging and DWI hyperintensity signals within 3 to 5 days in rats and at 1 to 4 weeks after striatal ischemia in patients. These results share a common characteristic that T2W imaging and DWI are associated with a delayed normointensity in the SN after a transient abnormal signal both in patients and animals. However, studies performed in rat have demonstrated that focal cerebral ischemia can cause severe and prolonged neuronal damage in the ipsilateral SN, and these pathological changes are not static but progressive (2). To date, a reasonable explanation is still lacking regarding the correlation of MR signal pseudo-normalization and the subsequent progressive pathological damage. Thus, we performed CT perfusion to confirm successful focal cerebral ischemia and to ensure stable blood flow perfusion in the SN. We investigated the MR signal in the remote SN over time after a unilateral MCA occlusion and compared these MR data with immunohistochemistry analyses.
Material and Methods Middle cerebral artery occlusion and protocol This study was performed according to the National Institutes of Health guidelines for the care and use of laboratory animals. All protocols were approved by the medical experiment animal administrative committee of Shanghai. All efforts were made to minimize animal suffering and to reduce the number of animals used. Healthy adult male Sprague Dawley rats (180–230 g) from the Shanghai Experimental Animal Centre were used in this study. Rats were anesthetized with an
intraperitoneal injection of chloral hydrate (350– 400 mg/kg). The rectal temperature was monitored and maintained at 37 0.5 C using a controlled water flow system throughout the procedure. Rats were subjected to focal cerebral ischemia induced by a transient left MCA occlusion as previously described (5,9). Briefly, the left external carotid artery (ECA) was initially ligated and transected to generate an ECA stump. Next, a nylon filament was introduced into the ECA stump and advanced into the internal carotid artery (ICA) to block blood flow to MCA. During MCA occlusion, computed tomography (CT) perfusion was performed to observe blood flow perfusion of the MCA territory and SN. Twenty-four rats were further studied, which showed impaired perfusion in the striatum and constant perfusion in the SN and were randomly assigned to each time point groups (n ¼ 6 animals each). Post-ischemic reperfusion was performed at 90 min after MCA occlusion by suture removal until its tip reached the ECA stump. In six rats from each time point, T2W imaging, T2 map, and DWI were performed on days 1, 3, 7, and 14 using a MR scanner. After MRI, the brains were removed and processed for histological examination.
CT perfusion imaging and analysis CT perfusion imaging was performed using a multislice CT scanner (Light-Speed 16, GE Healthcare, Milwaukee, WI, USA). The anesthetized rat was placed in a supine position in a custom-made board on the CT scanning table. The animal’s venous catheter was inserted into the femoral vein and connected to an automated injector. An un-enhanced CT scan was first obtained to localize the lesion. The parameters were: 120 kV; 100 mA; slice thickness, 2.5 mm; field of view (FOV), 9.6 9.6 cm; matrix, 512 512; scanning time, 1 s/scan. Next, perfusion scanning was performed using the cine scan technique, which covered the frontal lobe to the medulla. A non-ionic type CT contrast agent (Iohexol, 300 mg I/mL, GE Healthcare) was applied via the femoral vein using the automated injector at a dose of 2 mL with an injection flow rate of 1 mL/s. In addition, perfusion imaging was scanned and repeated 50 times. The parameters of cine scanning were the same as the un-enhanced scanning. The CT perfusion raw data were transferred to a dedicated workstation (Advantage Windows 4.1, GE Healthcare). For all datasets, perfusion analysis was performed using the CT Perfusion 3.0 software. The basilar artery and superior sagittal sinus were selected as the input artery and output vein (regions of interest range, 18–25 mm2), respectively. The images produced by the software were represented as three parametric maps: cerebral blood flow (CBF), cerebral blood
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volume (CBV), and mean transit time (MTT). Regions of interest (ROIs) were placed onto the striatum and substantia nigra of the ischemic and intact side on the perfusion parametric maps. Representative parametric values obtained from two radiologists with years of experience of CT perfusion imaging were averaged. The relative perfusion parameters (ipsilateral/contralateral region) were expressed as the mean standard deviation.
MRI and analysis We performed MRI scan using a GE Signa VH/i system equipped with a 3.0-T superconducting magnet (GE Healthcare) (9). Multi-slice T2W fast spin-echo imaging was performed and resulted in 12 contiguous slices in the coronal plane: TR, 4000 ms; TE, 120 ms; slice thickness, 2 mm; spacing, 0; FOV, 60 60 mm; matrix, 256 192; NEX, 4; scanning time, 3 min 24 s. Multi-echo T2W imaging was used to acquire the T2 map: TR, 3000 ms; TE, 159.9/146.6/133.2/119.9/106.6/ 93.3/79.9/66.6/53.3/40.0/26.6/13.3 ms; slice thickness, 2 mm; spacing, 0; FOV, 60 60 mm; matrix, 256 160; NEX, 1; scanning time, 5 min 20 s. DWI with diffusion-sensitive gradients in three dimensions (single-shot spin-echo echo planar images) was used: TR, 3500 ms; TE, 94.4 ms; slice thickness, 2 mm; spacing, 0; FOV, 40 40 mm; matrix, 64 96; NEX, 8; b, 0,1000 s/mm2; scanning time, 1 min 56 s. For images analysis, MR digital imaging and communications in medicine format (DICOM) images were transferred from the picture archive and communications system (PACS) to a separate personal computer (MacPro, Apple, Cupertino, CA, USA). OsiriX (version 4.1, OsiriX Foundation, Geneva, Switzerland), and free and open source postprocessing and fusion software tool (free down load from http://www.osirixviewer.com) were used. For the quantification analysis, the sync series analyze tool was used to synchronize the cursor on T2W imaging to the T2 map and ADC map onto the same slice image. The ROIs were drawn on the ischemic ipsilateral and contralateral SN. ROIs were visually matched between sequences to avoid the outermost pixels and to minimize partial volume effects. The ROI analyze tool was used to store the ROIs and to determine the average T2 relaxation time (ms), ADC (mm2/s), and standard deviation values from each region.
Tissue processing and staining On days 1, 3, 7, and 14, after all imaging experiments, six rats from each time point were anesthetized and perfused through the left ventricle with 50 mL of saline, followed by 100 mL of 4% paraformaldehyde.
The brains were rapidly removed, and immersion-fixed overnight in 4% paraformaldehyde in phosphate buffer and embedded in paraffin at 4 C until further use. Subsequently, 10-mm-thick coronal sections corresponding to each MR slice images were cut using a microtome, and sections in the SN were stained using Hematoxylin and Eosin (H&E). Microglia (CD68 immuno-positive cells) and astrocytes (GFAP immuno-positive cells) were detected using standard diaminobenzidine (DAB) immunohistochemisty with the CD68 monoclonal antibody (Biolegend, San Diego, CA, USA) and glial fibrillary acidic protein (GFAP) antibody (GeneTex, San Antonio, TX, USA), respectively. The number of CD68-positive cells and GFAP-positive cells were quantified at a magnification in a blind manner using a computerized image-analysis system consisting of a NIS camera installed onto an Olympus light microscope, which was attached to a personal computer. Data were expressed as the mean standard deviation numbers of cells (i.e. cell number/mm3).
Statistical analysis Statistical analyses were performed using SPSS software version 16 (SPSS Inc., Chicago, IL, USA). The relative CT perfusion parameters, T2 relaxation times, ADC, CD68- and GFAP-positive cell number were compared using one-way analysis of variance (ANOVA) for separated measures. Comparisons between ipsilateral and contralateral regions were then made using Student’s t-test. The level of statistical significance was P < 0.05 throughout this study.
Results Cerebral blood flow perfusion changes in the striatum and substantia nigra during MCA occlusion During MCA occlusion, CT perfusion was performed to monitor blood flow perfusion in the brain. Heterogeneous decreases of tissue perfusion in the MCA territory were detected in ischemic animals. At 30 min after MCA occlusion, the CBF and CBV in the ischemic striatum decreased compared with the contralateral striatum, and the MTT was prolonged (Fig. 1a–c), which confirmed a successful MCA occlusion. The relative perfusion values of the bilateral striatum at 30 min were rCBF 0.23 0.08, rCBV 0.75 0.09, rMTT 4.44 0.11. In contrast, the CT perfusion data showed constant blood flow perfusion in the SN (Fig. 1d–f) with relative values of rCBF of 1.03 0.06, rCBV 0.98 0.07, rMTT 1.01 0.05. Thus, the premise of studying remote changes was established. Compared between the perfusion
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Fig. 1. CT perfusion CBF map (a), CBV map (b), and MTT map (c) in the striatum (ST); the section was obtained from a representative rat acquired at 30 min during MCA occlusion and shows the cerebral perfusion deficit in the MCA territory. There is reduced CBF and CBV, and prolonged MTT compared with the contralateral striatum. CT perfusion CBF map (d), CBV map (e), and MTT map (f) from the same rat in the substantia nigra (SN); this section shows that the blood flow perfusion had no visible and measurable difference between the bilateral SN at each perfusion image.
parameters after MCA occlusion, there was statistical significance in the CBF, CBV, and MTT in the bilateral striatum (P < 0.05), but no significant differences between the ipsilateral and contralateral SN (P > 0.05).
Temporal profile of magnetic resonance T2 relaxation time and ADC in the substantia nigra after MCA occlusion The primary ischemic lesion was developed in the striatum in all grouped MCA occlusion rats. In the SN section, there were no visible and measurable differences between the bilateral SN in T2 relaxation time (80.2 8.1 ms vs. 77.7 9.8 ms) and ADC value (7.88 0.78 104 mm2/s vs. 8.09 1.02 104 mm2/s) on day 1 after stroke (Fig. 2a and f). On day 3, the ipsilateral SN to the MCA occlusion showed a T2 hyperintensity signal and ADC low signal compared to the contralateral SN (Fig. 2b, g, h) (T2 relaxation time 105.7 9.2 ms vs. 74.1 6.8 ms, ADC value 5.34 1.08 104 mm2/s vs. 7.74 0.89 104 mm2/s, respectively). At the subacute stage, days 7 and 14, abnormal changes could not be discriminated in both the qualitative and quantitative determination on the T2 map and ADC map (Fig. 2c, d, i) (T2 relaxation time 83.4 10.1 ms vs. 79.5 8.8 ms, ADC value 7.72 0.97 104 mm2/s vs. 7.61 1.11 104 mm2/s on day 7, T2 relaxation time 81.2 10.8 ms vs. 82.4 9.8 ms, ADC value 7.60 0.88 104 mm2/s vs. 7.59 0.89 104 mm2/s on day 14, respectively). The dynamic changes of T2 relaxation time and ADC in the SN after MCA occlusion are shown in Fig. 2e and j. Compared with the T2 relaxation time and ADC between the bilateral SN after MCA occlusion, there were statistical significances on day 3 (*P < 0.05), but
no significant differences on days 1, 7, and 14 (P > 0.05).
Morphological observation of H&E staining in the substantia nigra after MCA occlusion In the ipsilateral SN with respect to the MCA occluded side, most neurons in the pars reticulata lacked an abnormal appearance as assessed using H&E staining and a light microscope on day 1 after MCA occlusion (Fig. 3a). On day 3 post ischemia, the neuronal cells were swollen, the neuropil was slightly vacuolated, and the perivascular spaces were enlarged (Fig. 3b). The pars reticulata of the SN became smaller compared to the contralateral side after 7 and 14 days. The reactive glia gradually increased in number on days 7 and 14 (Fig. 3c, d).
Proliferation of CD68 and GFAP immuno-positive cell in the substantia nigra after MCA occlusion We next investigated whether immunohistochemical changes occurred in the SN after focal cerebral ischemia. Representative microphotographs of CD68 immunostaining in the SN after stroke are shown in Fig. 4a–d and GFAP immunostaining in Fig. 4e–h. The number of CD68 and GFAP positive cells were 25 7 /mm3 and 35 9 /mm3 on day 1, 30 5 /mm3 and 42 8 /mm3 on day 3, 42 5 /mm3 and 62 10 / mm3 on day 7, and 52 9 /mm3 and 71 9 /mm3 on day 14, respectively. In the contralateral SN, a small number of CD68 and GFAP immuno-positive cells were observed at each time point, in which the number of CD68 and GFAP positive cells were 20 5 /mm3 and 32 7 /mm3 on day 1, 25 5 /mm3 and
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Fig. 2. Alteration of T2 (a–d, g) and ADC (f, h, i) signals in the SN after MCA occlusion. On day 1 post ischemia, there is no abnormal signal on both sides of the SN. On day 3 after stroke, high T2 and low ADC signal can be visualized in ischemic ipsilateral SN (b, g, h). On days 7 and 14, the T2 elevation and ADC decline had recovered (c, d, i). The line diagrams illustrated the temporal profile of T2 relaxation time (e) and ADC (j) in the ischemic ipsilateral and intact SN on days 1, 3, 7, and 14. *P < 0.05, significantly different compared to the contralateral region.
Fig. 3. H&E staining on ischemic ipsilateral SN sections on day 1 (a, 400), day 3 (b, 400), day 7 (c, 400), and day 14 (d, 400) after MCA occlusion. Using light microscopy, there was no abnormal change in the SN on day 1. On day 3 after stroke, the neuronal cells were swollen, neuropils are vacuolated, and the perivascular spaces are enlarged with light microscope observation (b). Subsequently, vacuolation of neuropil became milder, and the reactive glia gradually increased in number (c, d).
Fig. 4. CD68 (a–c) and GFAP (e–g) immunohistochemical staining in the ischemic ipsilateral SN after stroke 1 day (a, 40; e, 40) and 14 days (b, 40; c, 400; f, 40; g, 400) are shown. The number of CD68 immunopositive cell increases in the ipsilateral SN from 7 days up to 14 days after focal cerebral ischemia (b, c) compared to days 1 (a) and 3. Similarly, the expression of GFAP immunoreaction increased in the ipsilateral SN on 7 and 14 days (f, g) compared to day 1 (e) and 3. The line diagrams illustrated the CD68 (d) and GFAP positive cell number (h) in the ipsilateral and contralateral SN on days 1, 3, 7, and 14. *P < 0.05, significantly different compared to contralateral intact region.
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31 6 /mm3 on day 3, 26 4 /mm3 and 34 6 /mm3 on day 7, and 28 6 /mm3 and 32 7 /mm3 on day 14. Compared with the SN on the ischemic and intact side, on days 1 and 3 post ischemia, there were no significant differences in both immunostaining results (P > 0.05). In contrast, there was a significantly increased number of CD68 positive microglia and GFAP positive astrocytes within the ipsilateral SN compared to the contralateral SN 7–14 days after focal cerebral ischemia (*P < 0.05).
Discussion This study was performed in a rat model of focal cerebral ischemia. Our data revealed MR signal pseudonormalization phenomenon with T2 relaxation time and apparent diffusion coefficient recovered in the ipsilateral SN at the subacute phase after stroke onset. Normalization of the T2 relaxation time and apparent diffusion coefficient corresponded well with the spatiotemporal occurrence of microglia activation and astrogliosis. With the present data, three aspects need to be addressed. The first issue involves the essential study of the precondition of remote degeneration following focal cerebral ischemia. To the best of our knowledge, this is the first study to employ CT perfusion to study remote degeneration after MCA occlusion. Impaired hemodynamic changes in the MCA territory were clearly demonstrated using the CT perfusion technique. However, in the SN, there was no visibly and measurably abnormal perfusion on all perfusion parametric maps. The different tissue perfusions between the striatum and SN at the early stage after focal cerebral ischemia demonstrated success of the animal model and enabled studies on pathogenesis in the remote site. The second issue relates to the immunohistochemistry feature in the SN following striatal ischemia. A significant increase in the number of microglia and astrocyte was observed in the pars reticulata of the ipsilateral SN 7–14 days after focal cerebral ischemia. Thus, specific evidence supports that inflammatory processes participate in the pathogenesis and execution of remote neurodegeneration. Activated microglia and astrocytes spread in the pars reticulata in a diffuse pattern, which might be due to the selective neuronal loss in the absence of necrosis in the ischemic remote region. The third issue involves changes in the T2 relaxation time and ADC in the SN following MCA occlusion. First, the present study effectively illustrated neurodegeneration of the ipsilateral SN. On day 3 after stroke, degeneration was demonstrated as a prolonged T2 relaxation time and decreased ADC. It is well known the reduced ADC is the most sensitive indication of swollen cells after focal ischemic brain damage, and
T2 elevation is a good method to image brain edema after infarction. These current results were consistent with results obtained in previous experimental studies (7,8,10–12). These signal changes were present later compared to the primary ischemic lesion and may be caused by swelling astrocytes and neurons. Thus, the MR signal can reflect the secondary neuronal swelling in the ipsilateral SN after MCA occlusion. Subsequently, the T2 relaxation time and ADC of the ipsilateral SN recovered to the same extent as the contralateral side, which corresponded well with the spatio-temporal distribution of astrogliosis and microglia activation. In fact, this correlation between MR signals and inflammatory cell proliferation has been observed in primary ischemic studies (13–16). In a primary ischemic lesion, glial activation and infiltration of hematogenous cells induced ADC normalization and reduced T2 elevation at the sub-acute phase following focal cerebral ischemia. The intense areas of inflammatory cell proliferation showed MR fogging presentation with normal tissue. Thus, changes in the T2 relaxation time and ADC could undergo characteristic changes and differentiate distinct stages of the inflammatory response and tissue remodeling after stroke both in primary and remote sites. In conclusion, pseudo-normalization of the T2 relaxation time and apparent diffusion coefficient reflects the neuroinflammatory changes in the activation of microglia and astrocytes in the ipsilateral substantia nigra after middle cerebral artery occlusion. Acknowledgments We gratefully acknowledge the contribution and endeavor of Mr. Tong Jin Sun.
Conflict of interest We declare that we have no conflict of interest.
Funding This study was supported by the grant of National Natural Science Foundation of China (No. 81371521) and Shanghai Natural Science Foundation of China (No. 09ZR1405100).
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