Published online before print March 6, 2008, doi: 10.1161/STROKEAHA.107.503565
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(Stroke. 2008;39:1541.)
© 2008 American Heart Association, Inc.
Original Contributions |
MRI Detection of Secondary Damage After Stroke
Chronic Iron Accumulation in the Thalamus of the Rat Brain
From the In-vivo-NMR-Laboratory (C.J., P.R.-C., M.H.), Max Planck Institute for Neurological Research, Cologne, Germany; Brain Ischemia and Neurodegeneration (C.J.), IIBB-CSIC, IDIBAPS, Barcelona, Spain; and Clinical Neurosciences Research Laboratory (P.R.-C.), Hospital Clínico Universitario, Santiago de Compostela, Spain.
Correspondence to Prof Dr Mathias Hoehn, In-vivo-NMR-Laboratory, Max-Planck-Institute for Neurological Research, Gleuelerstrasse 50, D-50931 Cologne, Germany. E-mail mathias{at}nf.mpg.de
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Abstract |
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Background and Purpose— Iron plays a central role in many metabolic processes. Under certain pathological situations it accumulates, producing negative effects such as increasing damage by oxidative stress. The present study examined long-term iron accumulation in a stroke model with secondary degeneration, using MRI and histological techniques.
Methods— Male Wistar rats (n=22) were subjected to 60 minutes MCA occlusion. MR images (T2- and T2*-weighted) were obtained weekly between weeks 1 and 7 after reperfusion, and at weeks 10, 14, 20, and 24. Histological iron detection and immunohistochemical examination for different markers (NeuN, GFAP, OX-42, HO-1, and APP) were performed at the 3 survival time points (3, 7, and 24 weeks).
Results— Infarcts affecting MCA territory were evident on T2-weighted imaging, and all animals showed deficits on behavioral tests. In the thalamus, T2 hyperintensity was detected 3 weeks after stroke, and disappeared around week 7 when T2*-weighted images showed a marked hypointensity in that area. Histology revealed neuronal loss in the thalamus, accompanied by strong microglial reactivity and microglial HO-1 expression. APP deposits were detected in the thalamus from week 3 on and persisted until week 24. Iron storage was detected in microglia at week 3, in the parenchyma at week 7, and around APP deposits at week 24.
Conclusions— T2*-weighted MRI allows the detection of secondary damage in the thalamus after MCAO. Iron accumulation in the thalamus is mediated by HO-1 expression in reactive microglia.
Key Words: transient cerebral ischemia • thalamus • magnetic resonance imaging • iron • heme-oxigenase-1
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Introduction |
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Reduction of regional cerebral blood flow may result in brain infarction. The most common experimental brain infarction model is the occlusion of the middle cerebral artery (MCAO), directly affecting ipsilateral cerebral cortex and caudate putamen. Focal brain ischemia also leads to changes in regions remote from the primary affected areas.1,2 These changes include vasogenic edema3,4 and excitotoxicity,5–7 which can contribute to secondary damage early after the insult. Cerebral infarct is one of the most common causes of Wallerian anterograde degeneration. After proximal axonal or somal injury, a degeneration of axons and myelin sheaths is observed. This axonal degeneration and myelin clearance give rise to accumulation of proteins and ions in the distal affected regions,8 contributing to the secondary lesion. Secondary damage also generates chronic microglial responses in the thalamus.9,10
Iron is essential for cell function and is involved in many important processes such as oxygen transport, storage, and activation. Iron storage is mediated by ferritin. High plasma ferritin levels have been related to poorer prognosis after stroke11 because ferritin is a source of free iron. Accumulation of iron, after hypoxia/ischemia in newborns, can be detected with MRI techniques.12 This iron accumulation causes pronounced susceptibility effects, thus reducing local T2 relaxation time, which results in hypointensity on T2*-weighted (T2*-W) images. High iron content of blood cells allows the detection of different events such as hemorrhages in the brain parenchyma and hemorrhagic transformation after brain infarct. Other pathologies, such as Alzheimer disease, can lead to iron accumulation in the brain. Different metal ions (iron, copper, zinc) have been found to accumulate around senile plaques.13
Heme-containing proteins are a source of iron during brain injury. Inducible heme oxygenase (HO-1) is a heat shock protein that responds to stress, catabolizing free heme into iron, carbon monoxide, and biliverdin (an antioxidant). Thus, HO-1 has been suggested to be protective against oxidative stress.14
The aim of this work was to study, by MRI techniques, the different events taking place during and after thalamic secondary degeneration. Our hypothesis was that HO-1, transiently expressed by reactive microglia in the thalamus, contributes to chronic iron accumulation in the thalamus and is detectable by MRI.
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Materials and Methods |
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Animals
Male Wistar rats (n=22; 280 to 320 g body weight at the beginning of the study; Harlan; Borschen, Germany) were used for the longitudinal study of secondary degeneration after stroke. One animal with abnormal T2* MRI before stroke induction was excluded from the study. Rats were euthanized at 3 different survival time points: 3 weeks (n=5), 7 weeks (n=8), and 24 weeks (n=8). All experiments were performed in accordance with the National Institute of Health animal protection guidelines and approved by the local governmental authorities.
Induction of Ischemia
Focal cerebral ischemia was produced by 1-hour intraluminal occlusion of MCA followed by reperfusion. Rats were kept under 1 to 1.5% halothane in nitrous oxide/oxygen (70:30) during all surgical procedures. The right common carotid artery was exposed, and branches of the external carotid artery (ECA) were electrocoagulated and sectioned. The pterygopalatine artery was tied close to the internal carotid artery. The common carotid artery was permanently occluded with a silk thread (3/0). A filament (4/0 Ethicon sutures), coated with silicon, was introduced through the ECA to the level of the MCA branching point. During MCAO rats showed a decrease in cortical blood flow measured by LDF with normal blood flow recovery after reperfusion. All animals showed infarction in the right parietal cortex and caudate-putamen (MCA territory) on T2-weighted MRI at 48 hours after stroke induction. Other brain regions, irrigated by anterior and posterior cerebral arteries, were not affected.
Adhesive Removal Test
Somatosensory deficits were assessed with the adhesive removal test. Experiments were done during the dark part of the day/night cycle, when animals show higher activity. The adhesive removal test was performed as described by Schallert and colleagues15 and applied in our laboratory.16 Briefly, 2 adhesive paper dots (12 mm diameter) were accurately attached to the distal-radial region on the wrist of each forelimb. Both the time to contact each paw and the time to remove paper dots were recorded 3 times, with a minimal interval of 5 minutes between consecutive trials. Before the first (preischemic) MRI session animals were trained for 3 consecutive days. Tests were always carried out the day before each MRI experiment.
MRI
MRI experiments were conducted on 4.7 T and 7 T horizontal bore magnets (Bruker BioSpin, Germany) with 20-cm wide actively shielded gradient coils (100 and 200 mT/m, respectively). Radiofrequency transmission was achieved with home-built Helmholtz coils (12 cm diameter); signal was detected using 2.2 cm surface coils, positioned over the head of the animal, which was fixed with tooth bar, ear plugs, and adhesive tape. Transmission and reception rf coils were actively decoupled from each other. Gradient-echo pilot scans were performed at the beginning of each imaging session for accurate positioning of the animal inside the magnet bore. T2-weighted (T2-W) images were acquired using a multi-slice multi-echo spin-echo sequence with the following acquisition parameters: field-of-view=3.2x3.2 cm2, image matrix=128x128 (isotropic in-plane resolution of 250 µm), 16 consecutive slices of 1 mm thickness, repetition time TR=3 seconds, 16 echoes with echo time TE of 7.5 ms (7T) or 12 ms (4.7T). T2*-weighted 3D data sets were acquired only at 4.7 T (to take advantage of the shorter T1 values at lower field) using gradient-echo sequence with the following acquisition parameters: field-of-view=2.0x2.0x1.0 cm3, image matrix=160x160x40 (zero-filled to 256x256x64, spatial resolution: 78x78x156 µm), TR/TE=200/20 ms, excitation pulse angle=30°. All images were processed using ImageJ (Rasband, W.S., ImageJ, NIH, http://rsb.info.nih.gov/ij).
Immunohistochemistry
Brains of all animals were processed for histological procedures. Expression of HO-1 and APP (Amyloid Precursor Protein) and identification of neurons, astrocytes, and microglia was performed by immunohistochemistry. Rats were perfused with 0.1 mol/L sodium phosphate buffer (PBS) pH 7.2, followed by 4% paraformaldehyde in the same buffer. Brains were postfixed overnight with the same fixative at 4°C and cryoprotected in 30% sucrose solution for 3 days. 40-µm-thick coronal sections were cut with a freezing microtome (Leica, Nussloch, Germany) and stored in a cryoprotectant solution. Free-floating sections were used for immunohistochemical detection for neurons, glial cells, inducible heme-oxygenase, and amyloid precursor protein.
Endogenous peroxidases were blocked with methanol-H2O2 and unspecific-binding sites with 3% normal horse serum for 2 hours at room temperature. Sections were incubated at 4°C with a mouse monoclonal antibody against either NeuN (neuronal marker; Chemicon Int, diluted 1:400), GFAP (astrocyte marker; Advanced Immunochemicals Inc, diluted 1:400), OX42 (microglial marker; Stressgen, diluted 1:500), HO-1 (Chemicon Int, diluted 1:500), and APP (Chemicon Int, diluted 1:400). This was followed by biotinylated goat antimouse antibody (Vectastain, Vector) diluted 1:200 for 1 hour at room temperature and the avidin-biotin complex (ABC kit, Vector Laboratories; 1:100) for 1 hour. The reaction was developed with 0.05% diaminobenzidine and 0.03% H2O2.
Double immunohistochemistry was carried out after the first immunoreaction with OX42 or APP. The second primary antibodies were: a mouse monoclonal antibody against HO-1 (diluted 1:500) and a mouse monoclonal antibody against OX42 (diluted 1:500). Sections previously reacted with the first primary antibody were incubated with the second primary antibody, followed by a corresponding biotinylated secondary antibody. All sections were then incubated with the avidin-biotin complex, washed with 0.01 mol/L sodium phosphate buffer (pH 6), and preincubated for 10 minutes with 0.01% benzidine dihydrochloride and 0.025% sodium nitroferricyanide in 0.01 mol/L sodium phosphate buffer (pH 6). The reaction was developed with this solution containing 0.005% H2O2. All sections were mounted, dehydrated, and coverslipped with mounting medium (Enthelan, Fluka).
Images were acquired using Leica MZ FL III and Leica DM RB light microscopes, both equipped with a CCD camera.
Pearl Prussian Blue Staining
Coronal sections (40 µm) from all animals were processed for iron detection. Sections were mounted on glass slides. Hydrochloric acid (2%) and potassium hexacyanoferrat (2%) were mixed just before use (working solution). Sections were rinsed in working solution for 20 minutes. After washing in distilled water sections were either counterstained with nuclear fast red or processed for OX42 immunostaining.
Image Analysis
Infarct volume was determined from quantitative T2 maps recorded at 3 weeks after stroke. Lesion volume was defined as an increase of T2 by more than 2 standard deviations above the homotopic contralateral values.
Statistical Analysis
The statistical comparison of lesion volumes at 3 weeks after stroke and of response times of the behavioral test was performed for all studied animals at weeks 3, 7, and 24 using Student t test. A probability value of <0.05 was considered significant.
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Figure 1A shows the experimental design. Behavioral tests indicated significant differences between healthy and impaired hemispheres. Time-to-contact and time-to-remove (Figure 1B) the sticky tape increased dramatically on the left forepaw (contralateral to the lesion), whereas the right forepaw did not express significant changes.
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Hyperintensities of T2-W images showed similar infarct extents as well as locations affected. For calculation of infarct volumes quantitative T2 maps were constructed from the multi-echo train for every time point studied from pre-MCAO to 6 months after occlusion. Statistical analysis of infarct volumes, based on T2 maps, was performed at 3 weeks when vasogenic edema as a confounding factor was already dissolved again. Infarct volumes did not show significant differences between groups (155±61, 134±46, 178±53 mm3 for 3-, 7-, and 24-weeks group, respectively). T2 signal of infarcted areas was homogeneously high in caudate-putamen and parietal cortex from the third week after infarct to the last observation time point, indicating cystic transformation of the infarct core. Thalamic T2-W signal changes started the third week after stroke, showing a hyperintensity in thalamic nuclei ipsilateral to the lesion (Figure 2A). Contralateral thalamic nuclei did not show changes on T2-W signal intensity. Affected thalamic regions were identified as ventroposteromedial (VPM) and ventroposterolateral (VPL) nuclei by comparison with the Paxinos and Watson atlas.17 Thalamic hyperintensity was not detected from 7 to 24 weeks after MCAO (Figure 2B and 2C). At the same experimental sessions 3D T2*-weighted sequences were acquired. Evolution of primary infarct showed irregular hypointensities in the infarct core according to T2*-W imaging, indicating microbleedings, reactive microglia, or infiltrating macrophages.
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The same MR modalities showed patterns of evolution in the thalamus different from those in primarily affected cortical and striatal regions. Thalamic T2*-W images were apparently normal 3 weeks after the infarct (Figure 2D), whereas an intense hypointensity became evident 7 weeks after stroke (Figure 2E), persisting up to 24 weeks (Figure 2F). These regions with signal intensity decrease coincided with regions showing T2-W hyperintensity at 3 weeks after the ischemic insult (VPL and VPM).
Three weeks after ischemia neuronal loss was evident in VPL and VPM thalamic nuclei and remained until the end of the experiment (24 weeks; Figure 3A, 3E, and 3I). Glial reactivity was detectable from the third week on (Figure 3B and 3C). Astrocytic reaction was localized mainly around the VPL and VPM at 7 and 24 weeks after the infarct, forming an astrocytic scar (Figure 3F and 3J). Microglial reaction dramatically increased at 7 weeks (Figure 3G) compared to the expression at 3 weeks (Figure 3C). Microglial OX42 expression was still very important 24 weeks after stroke (Figure 3K). HO-1 expression in the ipsilateral thalamus was increased 3 weeks after MCAO compared to later time points, when only a small number of cells were detectable and these only barely (Figure 3D, 3H, and 3L). Cells expressing HO-1 at 3 weeks were identified by coexpression of microglial markers (Figure 4C).
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Reactive microglia in the thalamus showed iron content at this time point (Figure 4B). Iron accumulation in the thalamus was evident from the third week after the onset of ischemia (Figure 4A). Iron was first clearly localized intracellularly (Figure 5A), whereas 7 weeks after infarct the iron showed a parenchymal distribution (Figure 5B). After 6 months iron was localized around thalamic structures similar to Alzheimer plaques (Figure 5C).
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APP expression was localized as small dots in the VPL and VPM 3 weeks after ischemia. An increase in APP deposition was visually evident from week 7 after ischemia and was present until the end of the experiment (Figure 6A, 6B, and 6C). Double staining showed high density of microglial cells around the APP deposits (Figure 6D, 6E, and 6F).
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Discussion |
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We have shown that high-resolution MRI is ideally suited to study long-term changes in brain regions remote from the primary infarct. We demonstrate here for the first time that T2*W-MRI permits to detect dynamics of iron accumulation in the thalamus, long after the onset of the ischemic episode. We have provided possible mechanisms that explain changes detected by MRI in the thalamus, including inflammation, oxidative stress, and microglial reactivity, among others.
One-hour occlusion of the MCA causes infarct formation in its supplying territory affecting ipsilateral basal ganglia and parietal cortex. These regions were affected in all animals in this study. But brain infarct is a dynamic process that includes anterograde and retrograde fiber degeneration of the axon, finally affecting parts remote from the primary infarct.1 Neuronal secondary damage is a phenomenon that follows a long time course. MRI changes in remote areas have been reported in models of permanent MCA occlusion, indicating early alterations in thalamic nuclei and substantia nigra.18,19 Using a model of transient MCA occlusion we were able to detect dynamic MRI changes in different brain regions, such as VPL and VPM thalamic nuclei, internal capsule, substantia nigra, and medial geniculate complex, among others. Thalamic neuronal death is detectable from 4 to 7 days after cortical lesion in mice and rats5,20 and is delayed, appearing from 1 to 7 months after cortical lesion, in rabbits.21 In a transient MCAO model in rats neuronal death in the thalamus was not detectable before 7 days, whereas a primary astrocytic and microglial reaction appeared already 3 days after the insult.22 In our model we detected extensive neuronal death and glial reactivity, both events causing localized edema in the VPL and VPM nuclei 3 weeks after the ischemic insult. Increase in extracellular water content is detectable by increase in T2 values. Later permanent decrease in T2* values indicates a chronic accumulation of iron in the same thalamic nuclei.
Fetal cortical transplants reduce thalamic atrophy attributable to retrograde degeneration in thalamic nuclei ipsilateral to cortical lesions. This suggests that some trophic support is needed for the survival of thalamic neurons.23 Altered levels of trophic factors causes degeneration of neurons and oligodendrocytes, both highly sensitive to such changes, inducing myelin degeneration.24 This neuronal and oligodendrocytic degeneration leads to changes of connected regions, remote from the primary infarct, such as the thalamus and induces a delayed microglial reactivity and heme oxygenase expression.25 Inducible heme-oxygenase (HO-1 or HSP-32) is a chaperone, hardly expressed under normal conditions but highly induced under oxidative stress,26 degrading heme groups and generating biliverdin (a powerful antioxidant), CO, and iron.14 We found transient expression of HO-1 at 3 weeks, at the same time that inflammation, delayed neuronal death, and microglial reaction took place. HO-1 expression has been used as an index of oxidative stress in neurodegenerative diseases such as Alzheimer disease27 or amyotrophic lateral sclerosis,28 suggesting that HO-1 could be an indicator of chronic oxidative stress. Different mechanisms try to neutralize damage caused by oxidative stress. The brain is particularly vulnerable to oxidative stress because of its high metabolic rate, using 20% of basal oxygen consumption. Inflammatory processes generate oxygen free radicals. The increase in HO-1 might contribute to an increased susceptibility to oxidative stress in elderly people.29,30 Long-term expression of HO-1 is associated with Wallerian degeneration. HO-1 induction could also provide an index of microglial and macrophage stress response to injury.26
Mononuclear phagocyte lineage cells express ferritin. Light-chain ferritin, localized mainly in microglia in rats, is implicated in long-term iron storage.26,31,32 HO-1 is found in both glial cells and neurons, whereas ferritin is only expressed by glia. Microglial cells have the capacity to sequester free iron, limiting iron mediated cell injury. Iron is potentially toxic, catalyzing hydroxyl radical formation via the Fenton reaction.33
Brain iron promotes protein oligomerization resulting in highly prevalent age-related proteinopathies such as Alzheimer disease (AD), Parkinson disease (PD), and Dementia with Leavy Bodies (DLB).34,35 Protein accumulation is a widely observed phenomenon in neurodegenerative diseases. APP is an integral membrane protein expressed in many tissues and concentrated in the synapses of neurons. Amyloid beta (Aβ) is generated by APP proteolysis and is one of the primary components of amyloid plaques. APP production is present under normal conditions and at all ages but is increased under stress situation. APP is a marker of axonal degeneration, accumulates because of the disruption of axonal transport,36 and is highly expressed by astrocytes in the ischemic border.37 APP and Aβ chronic deposition have been reported in the thalamus after MCAO in rats38 and humans.39 There exists a balance between Aβ production and elimination acting through different mechanisms (microglial cells, astrocytes, insulin-degrading enzyme, among others). Thalamic APP accumulation could be the consequence of an increased production, or a decrease in elimination, or a combination of both mechanisms. We have found chronic APP expression, mainly by microglial cells from the third week after stroke on, in the thalamus ipsilateral to the infarct, in agreement with other reports.38 APP accumulation shows a time-dependent varying pattern. At the earliest time point studied here APP was localized mainly as small dots, probably attributable to axonal degeneration. After 7 weeks APP adopted a plaque-like morphology with iron accumulation around the plaques. Alzheimer disease is linked to inflammation, with microglial and astrocytic increased reactivity, and metal biology, with presence of ions such as copper, zinc, and iron.13,30 These metals accelerate Aβ peptide aggregation and enhance metal-catalyzed oxidative stress associated with amyloid plaque formation. The presence of an iron-responsive element (IRE) in the 5'UTR of the APP (APP 5'UTR) provided the first molecular biological support for the present model that APP of AD is a metalloprotein.40 Neural toxicity is significantly attenuated after pretreatment of Aβ with iron-chelators, suggesting that the toxicity of Aβ is mediated via redox-active iron that enhances cellular oxidative stress.41 Thus, iron can contribute to APP and Aβ toxicity and accumulation associated to stroke.
Summary
In the present investigation, we describe, for the first time, long-term changes detected by MRI in thalamic nuclei after MCAO in rats. These changes include neuronal loss and glial reactivity accompanied by edema, and followed by iron deposition, providing a mechanism that helps explain the observed temporal profile of T2*-weighted contrast on MRI.
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Acknowledgments |
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Help with the immunohistochemical staining from Christiane Sprenger is gratefully acknowledged.
Sources of Funding
The investigation was supported by grants from the EU-FP6 program: DiMI (LSHB-CT-2005-512146), EMIL (LSHC-CT-2004-503569) and StemStroke (LSHB-CT-2006-037526).
Disclosures
None.
Received August 31, 2007; revision received October 1, 2007; accepted October 3, 2007.
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