In vivo analysis of neuroinflammation in the late chronic phase after experimental stroke

doi:10.1016/j.neuroscience.2015.02.024

Neuroscience

Volume 292, 30 April 2015, Pages 71-80

https://doi.org/10.1016/j.neuroscience.2015.02.024 Get rights and content

Highlights

•
Induction of pMCAO in rats by the macrosphere model.
•
In vivo analysis of neuroinflammation until day 56.
•
Imaging by USPIO-MRI and [¹¹C]PK11195-PET.
•
Quantification of phagocytic activity and other inflammatory processes.
•
Prediction of tissue fate.

Abstract

Background and purpose: In vivo imaging of inflammatory processes is a valuable tool in stroke research. We here investigated the combination of two imaging modalities in the chronic phase after cerebral ischemia: magnetic resonance imaging (MRI) using intravenously applied ultra small supraparamagnetic iron oxide particles (USPIO), and positron emission tomography (PET) with the tracer [¹¹C]PK11195. Methods: Rats were subjected to permanent middle cerebral artery occlusion (pMCAO) by the macrosphere model and monitored by MRI and PET for 28 or 56 days, followed by immunohistochemical endpoint analysis. To our knowledge, this is the first study providing USPIO-MRI data in the chronic phase up to 8 weeks after stroke. Results: Phagocytes with internalized USPIOs induced MRI-T2^∗ signal alterations in the brain. Combined analysis with [¹¹C]PK11195-PET allowed quantification of phagocytic activity and other neuroinflammatory processes. From 4 weeks after induction of ischemia, inflammation was dominated by phagocytes. Immunohistochemistry revealed colocalization of Iba1+ microglia with [¹¹C]PK11195 and ED1/CD68 with USPIOs. USPIO-related iron was distinguished from alternatively deposited iron by assessing MRI before and after USPIO application. Tissue affected by non-phagocytic inflammation during the first week mostly remained in a viably vital but remodeled state after 4 or 8 weeks, while phagocytic activity was associated with severe injury and necrosis accordingly. Conclusions: We conclude that the combined approach of USPIO-MRI and [¹¹C]PK11195-PET allows to observe post-stroke inflammatory processes in the living animal in an intraindividual and longitudinal fashion, predicting long-term tissue fate. The non-invasive imaging methods do not affect the immune system and have been applied to human subjects before. Translation into clinical applications is therefore feasible.

Graphical abstract

USPIO-MRI revealed macrophages after stroke (red). Combined analysis with [¹¹C]PK11195-PET presented dominance of non-phagocytic neuroinflammation in the acute phase and increasing phagocytic activity in the chronic phase after stroke. Tissue affected by phagocytic activity, was associated with severe injury and necrosis.

Introduction

Cerebral ischemia is accompanied by various cellular and molecular processes, which contribute to restoration of brain function after stroke (Wieloch and Nikolich, 2006, Murphy and Corbett, 2009). Especially inflammation impacts on neuroplasticity and long-term recovery (del Zoppo, 2010, Morrison and Filosa, 2013, Ruscher et al., 2013). In vivo visualization of inflammatory reactions may facilitate translation of novel therapies into clinical studies (Fumagalli et al., 2013, Heiss, 2014, Quattromani et al., 2014). Among others, magnetic resonance imaging (MRI) using ultra small supraparamagnetic iron oxide particles (USPIO) (Deddens et al., 2012, Marinescu et al., 2013) and positron emission tomography (PET) with the radiotracer [¹¹C]PK11195 (Schroeter et al., 2009, Jacobs et al., 2012) represent attractive approaches.

The lipophilic tracer [¹¹C]PK11195 binds to the 18-kDa translocator protein (TSPO), a cholesterol-transporter found on the membrane of mitochondria (Papadopoulos et al., 2006). After brain injury, its expression on microglia, astrocytes and macrophages increases, representing a target for imaging (Stephenson et al., 1995, Chen and Guilarte, 2008). Unfortunately the method is limited due to short half-life of ¹¹C and low signal-to-noise ratio (Jucaite et al., 2012, Dickens et al., 2014). Moreover radiosynthesis of [¹¹C]PK11195 is very time consuming and expensive.

In MRI, intravenously applied USPIOs lead to hypointense T2^∗-signal changes in the lesioned central nervous system (CNS) by invasion of USPIO-loaded macrophages (Nighoghossian et al., 2007, Desestret et al., 2013). This was shown to occur independently of a potentially associated blood–brain barrier breakdown (Stoll and Bendszus, 2010, Yang et al., 2013). Translational approaches demonstrated USPIO+ phagocytes also in the human CNS after stroke (Saleh et al., 2004, Saleh et al., 2007). However, these findings have been questioned by studies using transient stroke models in high-field MRI (Desestret et al., 2009, Farr et al., 2011, Harms et al., 2013). In addition dysregulation of natural neuroinflammatory responses by USPIOs has been discussed (Siglienti et al., 2006, Hsiao et al., 2008). Recent results furthermore suggest that iron deposition naturally occurs after stroke (Danielisova et al., 2004, Li et al., 2009, Hagemeier et al., 2012) and other diseases associated with persistent microglia activation (Zivadinov et al., 2011).

The present study was conducted to evaluate the validity of combined imaging by USPIO-MRI and [¹¹C]PK11195-PET as a method to longitudinally and intraindividually analyze stroke-induced inflammatory processes in the living organism.

Section snippets

Experimental design

Thirteen male Wistar rats underwent permanent middle cerebral artery occlusion (pMCAO) by the macrosphere model, which closely resembles the dynamic patterns of neuroinflammatory procedures in human stroke (Gerriets et al., 2003, Walberer et al., 2010). At day 6 (d6), d27, and d55, animals were subjected to both MRI (T2, T2^∗) and PET, using the tracer [¹¹C]PK11195 to investigate neuroinflammatory processes. Directly afterward, USPIOs (300 μmol Fe/kg) were injected intravenously (iv) followed by

Dynamics of post-stroke inflammation

During the observation period of 56 days, the inflammatory signal by [¹¹C]PK11195, decreased over time and moved from regions directly adjacent to the infarct toward the thalamus (d28), and further toward midbrain and pons (d56) (Fig. 2A, B). In line with previous results, post-stroke inflammation peaked at d7.

Immunohistochemistry

Focal signals for iron (Fe) were present in all USPIO+ areas corresponding to the infarct-margin and periinfarct region (Fig. 2C). Iba1-stained tissue confirmed the presence of microglia

Discussion

To date, the detailed pathomechanisms underlying stroke-induced inflammatory processes still remain elusive and especially the distinction of various cellular subtypes contributing to postischemic reactions is matter of vivid debates (Prinz et al., 2011). Cellular and genetic labeling as well as functional studies have shown that in particular the microglia/macrophage population is highly heterogeneous in respect to origin, activity and marker expression (Morrison and Filosa, 2013, Perego et

Conclusion

The combination of USPIO-MRI and [¹¹C]PK11195-PET allows longitudinal and intra-individual analysis of inflammatory processes in the chronic post-stroke phase, facilitating valid predictions about regional tissue fate. Hereby valuable information about mechanisms of repair and recovery after stroke is provided. As both methods have successfully been applied in humans, translation of this multi-modal imaging protocol into clinical routine is feasible and may help to monitor new therapeutic

Sources of funding

This study was supported by the Köln-Fortune-Programme and the European Community’s Seventh Framework Programme, project number 2780006, “NeuroFGL”.

Disclosures

The authors declare no competing financial interests.

Acknowledgment

None.

References (58)

M.K. Chen et al.
Translocator protein 18 kDa (TSPO): molecular sensor of brain injury and repair
Pharmacol Ther
(2008)
J. Cizek et al.
Fast and robust registration of PET and MR images of human brain
Neuroimage
(2004)
T.A. Evans et al.
High-resolution intravital imaging reveals that blood-derived macrophages but not resident microglia facilitate secondary axonal dieback in traumatic spinal cord injury
Exp Neurol
(2014)
G. Fleige et al.
Magnetic labeling of activated microglia in experimental gliomas
Neoplasia
(2001)
T. Gerriets et al.
The macrosphere model: evaluation of a new stroke model for permanent middle cerebral artery occlusion in rats
J Neurosci Methods
(2003)
H. Kettenmann et al.
Microglia: new roles for the synaptic stripper
Neuron
(2013)
A.A. Lammertsma et al.
Simplified reference tissue model for PET receptor studies
Neuroimage
(1996)
L. Li et al.
Quantitative analysis of iron concentration and expression of ferroportin 1 in the cortex and hippocampus of rats induced by cerebral ischemia
J Clin Neurosci
(2009)
V. Papadopoulos et al.
Translocator protein (18 kDa): new nomenclature for the peripheral-type benzodiazepine receptor based on its structure and molecular function
Trends Pharmacol Sci
(2006)
M.J. Quattromani et al.
Enriched housing down-regulates the Toll-like receptor 2 response in the mouse brain after experimental stroke
Neurobiol Dis
(2014)

M. Schilling et al.

Predominant phagocytic activity of resident microglia over hematogenous macrophages following transient focal cerebral ischemia: an investigation using green fluorescent protein transgenic bone marrow chimeric mice

Exp Neurol

(2005)

M. Schilling et al.

Effects of monocyte chemoattractant protein 1 on blood-borne cell recruitment after transient focal cerebral ischemia in mice

Neuroscience

(2009)

F. Shah et al.

Synthesis of the enantiomers of [N-methyl-¹¹C]PK 11195 and comparison of their behaviours as radioligands for PK binding sites in rats

Nucl Med Biol

(1994)

I. Siglienti et al.

Cytokine profile of iron-laden macrophages: implications for cellular magnetic resonance imaging

J Neuroimmunol

(2006)

R. Tanaka et al.

Migration of enhanced green fluorescent protein expressing bone marrow-derived microglia/macrophage into the mouse brain following permanent focal ischemia

Neuroscience

(2003)

T. Wieloch et al.

Mechanisms of neural plasticity following brain injury

Curr Opin Neurobiol

(2006)

V. Danielisova et al.

Iron deposition in the brain following the ischemia in a rat model of ischemic tolerance

Acta Medica (Hradec Kralove)

(2004)

L.H. Deddens et al.

Imaging neuroinflammation after stroke: current status of cellular and molecular MRI strategies

Cerebrovasc Dis

(2012)

G.J. del Zoppo

Acute anti-inflammatory approaches to ischemic stroke

Ann N Y Acad Sci

(2010)

V. Desestret et al.

Early-stage investigations of ultrasmall superparamagnetic iron oxide-induced signal change after permanent middle cerebral artery occlusion in mice

Stroke

(2009)

V. Desestret et al.

In vitro and in vivo models of cerebral ischemia show discrepancy in therapeutic effects of M2 macrophages

PLoS One

(2013)

A.M. Dickens et al.

Detection of microglial activation in an acute model of neuroinflammation using PET and radiotracers 11C-(R)-PK11195 and 18F-GE-180

J Nucl Med

(2014)

T.D. Farr et al.

Challenges towards MR imaging of the peripheral inflammatory response in the subacute and chronic stages of transient focal ischemia

NMR Biomed

(2011)

S. Fumagalli et al.

CX3CR1 deficiency induces an early protective inflammatory environment in ischemic mice

Glia

(2013)

M. Gauberti et al.

Molecular magnetic resonance imaging of brain–immune interactions

Front Cell Neurosci

(2014)

J. Hagemeier et al.

Brain iron accumulation in aging and neurodegenerative disorders

Expert Rev Neurother

(2012)

K. Hamacher et al.

Efficient stereospecific synthesis of no-carrier-added 2-[¹⁸F]-fluoro-2-deoxy-d-glucose using aminopolyether supported nucleophilic substitution

J Nucl Med

(1986)

C. Harms et al.

Certain types of iron oxide nanoparticles are not suited to passively target inflammatory cells that infiltrate the brain in response to stroke

J Cereb Blood Flow Metab

(2013)

W.D. Heiss

PET imaging in ischemic cerebrovascular disease: current status and future directions

Neurosci Bull

(2014)

Cited by (42)

Acute minocycline treatment inhibits microglia activation, reduces infarct volume, and has domain-specific effects on post-ischemic stroke cognition in rats
2023, Behavioural Brain Research
Ischemic stroke affects millions of individuals worldwide and a high prevalence of survivors experience cognitive deficits. At present, the underlying mechanisms that drive post-stroke cognitive decline are not well understood. Microglia play a critical role in the post-stroke inflammatory response, but experimental studies show that an accumulation of chronically activated microglia can be harmful and associates with cognitive impairment. This study assessed the effect of acute post-stroke minocycline treatment on chronic microglia and astrocyte expression within the infarct and remote white matter regions, as well as its effect on various domains of cognitive function post-stroke. Nine-month-old male rats received an injection of endothelin-1 into the right dorsal striatum to induce transient focal ischemia, and then were treated with minocycline or saline for 4 days post-stroke. Rats were tested using a series of lever-pressing tasks and the Morris water maze to assess striatal-based learning, cognitive flexibility, and spatial learning and reference memory. We found that minocycline-treated rats had smaller stroke-induced infarcts and less microglia activation in the infarct area and remote white matter regions compared to saline-treated rats at 28 days post-stroke. The behavioural testing results differed according to the cognitive domain; whereas minocycline-treated rats trended towards improved striatal-based learning in a lever-pressing task, but cognitive flexibility was unaffected during the subsequent set-shifting task. Furthermore, minocycline treatment unexpectedly impaired spatial learning, yet it did not alter reference memory. Collectively, we show that post-stroke minocycline treatment can reduce chronic microglia activation even in remote brain regions, with domain-specific effects on cognitive function.
Imaging of microglia in post-stroke inflammation
2023, Nuclear Medicine and Biology
Microglia constantly survey the central nervous system microenvironment and maintain brain homeostasis. Microglia activation, polarization and inflammatory response are of great importance in the pathophysiology of ischemic stroke. For exploring biochemical processes in vivo, positron emission tomography (PET) is a superior imaging tool. Translocator protein 18 kDa (TSPO), is a validated neuroinflammatory biomarker which is widely used to evaluate various central nervous system (CNS) pathologies in both preclinical and clinical studies. TSPO level can be elevated due to peripheral inflammatory cells infiltration and glial cells activation. Therefore, a clear understanding of the dynamic changes between microglia and TSPO is critical for interpreting PET studies and understanding the pathophysiology after ischemic stroke. Our review discusses alternative biological targets that have attracted considerable interest for the imaging of microglia activation in recent years, and the potential value of imaging of microglia in the assessment of stroke therapies.
A pilot [<sup>11</sup>C]PBR28 PET/MRI study of neuroinflammation and neurodegeneration in chronic stroke patients
2021, Brain, Behavior, and Immunity - Health
Citation Excerpt :
Our findings provide evidence of extensive glial activation in chronic stroke patients, leading to the possibility that chronic neuroinflammation may play a role in secondary poststroke pathologies such as depression and fatigue (Barritt and Smithard, 2011; Shi et al., 2019; Spalletta et al., 2006). Previous studies in patients and animal models with subacute MCA stroke have observed glial activation in the ipsilesional thalamus and ipsilesional internal capsule, regions with known neuroanatomical connections to the damaged tissue (Arlicot et al., 2010; Gerhard et al., 2005; Justicia et al., 2008; Pappata et al., 2000; Radlinska et al., 2009; Thiel et al., 2010; Walberer et al., 2014; Walter et al., 2015). Case studies of patients who incurred an MCA stroke several months prior have also observed glial activation in more distal connections of the damaged tissue, including in the midbrain and pons (Gerhard et al., 2005; Walter et al., 2015).
Neuroinflammation occurs in response to acute ischemic stroke, and has been speculated to underlie secondary poststroke pathologies, such as depression, that often develop over time poststroke. However, no study has examined whether neuroinflammation is present in chronic stroke patients (e.g., ≥ 1 year poststroke). This study tested whether neuroinflammation is present in chronic stroke patients, and is associated with neurodegeneration, using [¹¹C]PBR28 PET and diffusion MRI.
Eight patients with middle cerebral artery (MCA) ischemic stroke incurred 1–3 years prior and 16 healthy controls underwent [¹¹C]PBR28 PET to measure glial activation and diffusion MRI to measure microstructural integrity by mean diffusivity (MD) and fractional anisotropy (FA) using an integrated PET/MRI scanner. Group differences in [¹¹C]PBR28 binding, MD and FA were analyzed voxelwise across the whole brain excluding the infarct zone defined as voxels containing the infarct in any patient.
Compared to controls, patients showed elevations in [¹¹C]PBR28 binding in several brain regions outside the infarct zone, including regions with presumed direct neuroanatomical connections to the infarct (e.g., ipsilesional internal capsule and thalamus) and those without known direct connections (e.g., contralesional thalamus and cingulate gyrus). Patients also showed widespread elevations in MD, with a subset of these regions having reduced FA. In patients, MD was more elevated in regions with co-localized elevations in [¹¹C]PBR28 binding than in contralateral regions without elevations in [¹¹C]PBR28 binding.
This pilot study supports the presence of extensive glial activation along with widespread loss in microstructural integrity in non-infarcted tissue in a cohort of patients with chronic MCA stroke. The loss in microstructural integrity was greater in regions with co-localized glial activation. It is possible that stroke risk factors (e.g., hypertension) contributed to these tissue changes in patients.
Global brain inflammation in stroke
2019, The Lancet Neurology
Citation Excerpt :
The aforementioned spatial and temporal evolution profile of microglia in patients was mirrored in rodent models of brain ischaemia induced by middle cerebral artery occlusion. Although focal microglial activation peaked at about 1 week after ischaemia and subsequently ablated over 1 month,44–47 the distribution of activated microglia progressively developed in the midbrain, pons, and contralateral regions in subsequent months.48,49 In an endothelin-1-induced prefrontal infarct rat model, activated microglia were detected in remote cortex and white matter at 28 days after ischaemia; this finding was associated with neuronal loss in these areas.50
Stroke, including acute ischaemic stroke and intracerebral haemorrhage, results in neuronal cell death and the release of factors such as damage-associated molecular patterns (DAMPs) that elicit localised inflammation in the injured brain region. Such focal brain inflammation aggravates secondary brain injury by exacerbating blood–brain barrier damage, microvascular failure, brain oedema, oxidative stress, and by directly inducing neuronal cell death. In addition to inflammation localised to the injured brain region, a growing body of evidence suggests that inflammatory responses after a stroke occur and persist throughout the entire brain. Global brain inflammation might continuously shape the evolving pathology after a stroke and affect the patients' long-term neurological outcome. Future efforts towards understanding the mechanisms governing the emergence of so-called global brain inflammation would facilitate modulation of this inflammation as a potential therapeutic strategy for stroke.
Inflammation-induced brain endothelial activation leads to uptake of electrostatically stabilized iron oxide nanoparticles via sulfated glycosaminoglycans
2017, Nanomedicine: Nanotechnology, Biology, and Medicine
Citation Excerpt :
Differing from polymer-coated particles, citrate-coated and thereby electrostatically stabilized VSOP were shown to interact with cell surface bound GAG.25 Thus, contrary to polymer-coated particles that target primary phagocytes,8,40–42 the VSOP iron oxide cores lose their citrate coating in the GAG environment and the cationic core surface is bound by the strongly complexing moieties of the GAG, such as the carboxyl and sulfate groups.25 GAG sulfation pattern and chain length are characteristic for particular cell types, and determine the binding and protection from proteolysis of numerous biologically relevant proteins such as growth factors, chemokines, or cytokines.43
Based on our previous data on the presence of very small superparamagnetic iron oxide nanoparticles (VSOP) on brain endothelial structures during experimental autoimmune encephalomyelitis (EAE), we investigated the mechanisms of VSOP binding on inflamed brain endothelial cells in vivo and in vitro. After intravenous application, VSOP were detected in brain endothelial cells of EAE animals at peak disease and prior to clinical onset. In vitro, inflammatory stimuli increased VSOP uptake by brain endothelial bEnd.3 cells, which we confirmed in primary endothelial cells and in bEnd.3 cells cultured under shear stress. Transmission electron microscopy and blocking experiments revealed that during inflammation VSOP were endocytosed by bEnd.3. Modified sulfated glycosaminoglycans (GAG) on inflamed brain endothelial cells were the primary binding site for VSOP, as GAG degradation and inhibition of GAG sulfation reduced VSOP uptake. Thus, VSOP-based MRI is sensitive to visualize early neuroinflammatory processes such as GAG modifications on brain endothelial cells.
Drivers of Chronic Pathology Following Ischemic Stroke: A Descriptive Review
2024, Cellular and Molecular Neurobiology

View all citing articles on Scopus

^†: Both authors contributed equally to this study.

View full text

In vivo analysis of neuroinflammation in the late chronic phase after experimental stroke

Highlights

Abstract

Graphical abstract

Introduction

Section snippets

Experimental design

Dynamics of post-stroke inflammation

Immunohistochemistry

Discussion

Conclusion

Sources of funding

Disclosures

Acknowledgment

Pharmacol Ther

Neuroimage

Exp Neurol

Neoplasia

J Neurosci Methods

Neuron

Neuroimage

J Clin Neurosci

Trends Pharmacol Sci

Neurobiol Dis

Exp Neurol

Neuroscience

Nucl Med Biol

J Neuroimmunol

Neuroscience

Curr Opin Neurobiol

Iron deposition in the brain following the ischemia in a rat model of ischemic tolerance

Acta Medica (Hradec Kralove)

Imaging neuroinflammation after stroke: current status of cellular and molecular MRI strategies

Cerebrovasc Dis

Acute anti-inflammatory approaches to ischemic stroke

Ann N Y Acad Sci

Early-stage investigations of ultrasmall superparamagnetic iron oxide-induced signal change after permanent middle cerebral artery occlusion in mice

Stroke

In vitro and in vivo models of cerebral ischemia show discrepancy in therapeutic effects of M2 macrophages

PLoS One

Detection of microglial activation in an acute model of neuroinflammation using PET and radiotracers 11C-(R)-PK11195 and 18F-GE-180

J Nucl Med

Challenges towards MR imaging of the peripheral inflammatory response in the subacute and chronic stages of transient focal ischemia

NMR Biomed

CX3CR1 deficiency induces an early protective inflammatory environment in ischemic mice

Glia

Molecular magnetic resonance imaging of brain–immune interactions

Front Cell Neurosci

Brain iron accumulation in aging and neurodegenerative disorders

Expert Rev Neurother

Efficient stereospecific synthesis of no-carrier-added 2-[18F]-fluoro-2-deoxy-d-glucose using aminopolyether supported nucleophilic substitution

J Nucl Med

Certain types of iron oxide nanoparticles are not suited to passively target inflammatory cells that infiltrate the brain in response to stroke

J Cereb Blood Flow Metab

PET imaging in ischemic cerebrovascular disease: current status and future directions

Neurosci Bull

Efficient stereospecific synthesis of no-carrier-added 2-[¹⁸F]-fluoro-2-deoxy-d-glucose using aminopolyether supported nucleophilic substitution