Clinical Assessment of Neuroinflammation Flashcards
What are CT scans
• Used to confirm stroke
• Good for giving structural and density information
− Low density → fluid (black)
− High density → bone (white)
• Has low sensitivity, often don’t detect features of infarction early on
What are the reasons to clinically assess neuroinflammation in patients?
• Understand risk factor profile for disease → target for prevention
• Understand pathophysiology of disease → target for acute treatment/modifying extent of oinjury
• Measurement tools:
− To predict outcome (including complications)
− To predict response to potential treatments
− To evaluate biological effectiveness of treatments
What are the challenges of assessing neuroinflammation in patients?
• Access to CNS tissue/vasculature
− Can either do it post mortem (not ideal methodologically and in terms of interpretation)
− Can do it by biopsy material (possible when stroke patients have surgery, but isn’t common)
• Complexity and expense of neuroimaging techniques
− Technology limited to certain sites, eg) in Manchester we only have PET scanner for neurological disease at the Christie. Not good for patients further away
• Validity of methods available
• Assessment in ill/confused patients – may not want to transfer them around hospitals
• Confounders
• Interpretation
What are the methods available to assess neuroinflammation?
- CNS/vascular functional imaging (MRI/PET/SPECT)
- Brain tissue biopsy
- Evaluation of CSF components
− Don’t always routinely have access to CSF
− Patients with SAH will have a drain going into the CSF, but doing lumbar puncture or spinal tap in patients just to get CSD isn’t really viable, particularly for repeated measurements - Evaluation of plasma/blood cells → emerging as a good surrogate for CSF
− Very easy to get hold of
− Gives us an indirect measure of what is happening in the brain – there is communication between the brain and peripheral component
− There may be measures in the blood that we can back-translate from our knowledge of physiology, allowing us to say for example, that molecule X is representing ____ occurring in the brain
Describe MRI as a functional imaging assessment of neuroinflammation.
• Generally available in most hostpitals
• Attractive as it does not use radiation
• However, accessing MRI in the acute phase of stroke is difficult in the clinic, and even more so for research
• If we inject contrast agents, the information we can get from MRI is even greater:
− Injection of USPIO → preferentially phagocytosed by monocytes
− Can visualize the uptake of this contrast using MRI
Macrophage infiltration in L-MCA territory infarction – USPIO enhanced MRI study:
• Use systemically administered USPIO to track macrophage infiltration into the area
• In T1 MRI, fluid is black
− In plain T1 MRI, cant really see the infarct, not very useful
− Inject USPIO – can see areas of white, these are macrophages starting to infiltrate
− At 48hrs, can see much greater intensity – represents ongoing infiltration of macrophages and resident microglia
• in T2 MRI, fluid is white
− Can easily see infarct area with this
− Can correlate with the macrophage infiltration seen on the T1 USPIO – coregisters well
→ This approach could be compromised by passive diffusion of free USPIO through a defective BBB.
Describe PET as a functional imaging method of assessing neuroinflammation.
• In vivo quantification of cerebral blood flow, metabolism and receptor binding
• Involves injection of radiolabelled tracer (positron emitting isotope)
• Cyclotron accelerates proton beam – emission of positrons – photon production
• PET scanner detects photons
• 3D-images of tracer concentration are then constructed by computer analysis, often aided by a CT scan
• Much more complex than MRI:
− Only one in Manchester, in the Christie, and highly weighted towards cancer
− Need a high level of technical and interpretive expertise
Common PET tracers:
• [11C]-PK11195 (specific TSPO ligand) → microglial activation
− Upregulation of TSPO is a marker of activated microglia
• 18FDG → macrophage activation
Evolution of microglial activation in L-MCA territory infarction – PK11195 PET:
• 5 days after stroke, begin to see some changes in sginal
• However, 13 days after there is much more signal within the L-MCA infarct territory
• Much more delayed reponse than what was seen with USPIO MRI – could suggest different temporal activation pattern between circulating monocytes and resident microglia, or could just reflect the differences between the two models of assessment.
• In experimental models of stroke, activated microglia revealed by PK11195 are located mainly in the core
Inflammatory R-carotid atherosclerotic stenosis – 18FDG PET:
• More tenous application, and is looking at carotid atheroma
• In this model, showed high signal in the area of the right carotid artery, showing macrophage activation
• Co-registered well with T1 MRI image showing white signal around right carotid artery
→ These images are qualitative over quantitative – not really able to assess severity of the damage
→ Also usually performed in a select cohort, and they usually publish the ‘best’ images
Extra reading:
• Using PET, it was shown that in a rat model of transient ischaemia, expression of TSPO in microglia and macrophages increased until 7 to 11 days after stroke, and decreased later
• Migration of astrocytes towards the lesion reflecting formation of a scar was correlated with TSPO expression
• Interestingly, in a model of migraine in rats, PK11195 PET showed TSP activation after induction, indicating that microglia cells are activated also in response to nociceptive stimulus
• PK11195 PET has been performed in small groups of patients, and between 3 and 150 days after stroke, microglial activation can be observed at the primary lesion site, in peri-infarct regions as well as in remote locations, and even in the contralateral hemisphere.
• This suggests therapeutic targeting of neuroinflammation can be extended to late time windows
Describe SPECT as a functional imaging method of assessing neuroinflammation.
- Injection of a radiolabelled tracer
- Gamma emission detected directly by a gamma camera
Common tracers:
• 111-Indium → autologous leukocytes
• 99mTc-DTPA → BBB disruption
→ Our access to this within Europe is limited to a small number of centres –there are places in Germany that have PET and SPECT scanners in their stroke units, this is a clear advantage.
Cerebral neutrophil recruitment: 111-Indium labeled neutrophil SPECT study:
• Signal of neutrophil presences that co-registers with two large areas of acute infarction on a CT scan
BBB-disruption in DTPA SPECT study:
• Shows halos of abnormal signal, represents massive BBB disruption almost perfectly co-registered with haemorrhage.
Describe examination of biopsy tissue as a method of clinical assessment of neuroinflammation.
Brain:
• Limited to post-mortem or brain biopsy
• Ethical/logistical issues:
− Difficult to get access immediately after death
− The way different cultures proceed after death determines how easy it is to get access to brain samples
Carotids:
• Limited to edarterectomy speciments (surgical removal of the inner lining of an artery, together with any obstructive deposits)
→ Biopsies allow us to perform correlation studies – functional imaging vs. histopathological
Brain CRP expression pattern in human stroke:
• The histology ICH stainings show massive CRP staining in the brain in response to haemorrhage. when compared with ischemia and control
• So in terms of CRP, there is a difference between haemorrhagic and ischaemic stroke
• Could just be because there is a lot of the blood in the brain in haemorrhagic, and CRP is in the blood – but there is also staining within the blood vessels and neuronal cells not seen in ischaemia
Describe evaluation of CSF as a method of clinical assessment of neuroinflammation.
• Limited access, as unethical to do lumbar puncture
• One study has been performed by Tarkowski et al, 1995
− Results appear to illustrate that the magnitude of cytokine production in the CSF is greater than that in the serum (periphery)
− Induction of IL-6 is greater than IL-1
− There is little peripheral IL-1 (classic tissue cytokine) compared to IL-6 (classic systemic cytokine)
− When you look at the time course, there is still an elevation at day 90 compared with controls
→ Tells us that during stroke, there is a massive cytokine response in the CSF – indicating cytokines are being synthesized within the brain.
Describe plasma sampling as a method of clinical assessment of neuroinflammation.
- Useful for cytokines/mediators released into the systemic circulation (eg, IL-6, CRP)
- Little value for mediators released at the site of inflammation (eg, TNF, IL1)
- Indirect measurement of neuroinflammation (eg, plasma IL-6 upregulated by IL-1)
Plasma CRP in acute stroke:
• In ischemic stroke, CRP levels increase steadily for up to 5 days after the stroke , and the level was still elevated above the control 1 year after
• It wasn’t measured above 72 hours in haemorrhagic, but shows a trend for ongoing CRP release.
• This could be important in terms of recovery from stroke, or risk of future stroke.
→ So CRP is a potentially useful biomarker for predicting outcome.
Plasma CRP in acute stroke survival/recurrence:
Split CRP concentration into tertiles:
• Ischaemic stroke → lowest tertile has lowest mortality, highest has highest
• Haemorrhagic → lowest tertile has highester survival, highest has highest.
Effect of IV IL-1RA in acute stroke plasma inflammatory markers:
• As we have seen, those with high CRP tend to have the worst outcomes – so would be useful to have a drug that could lower CRP
• Those administered IL-1RA had reduced neutrophil counts and CRP concentrations
CRP and risk of stroke – beyond conventional risk factors?
The Emerging Risk Factors Collaboration, 2010:
• 160,000 patient meta-analysis
• 54 prospective studies
• No clinical vascular disease history at baseline
• Range of vascular risk
• Baseline CRP
• → Was found that as you get old, risk of stroke increases, and also risk of CRP increases
→ So, potential link between CRP and stroke could be confounded by age
→ Has actually been found that the more you adjust for other potential risk factors (sex, BP, smoking, diabetes, BMI, cholesterol) – the lower the association seems to be.
Describe the evidence for stroke-associated immune suppression.
Measuring pro-inflammatory mediators:
• Plasma proinflammatory mediators (IL6, CRP)
• Plasma stress mediators (eg, cortisol and catechols)
• Immune cell counts (neutrophils)
Measuring immune suppression:
• Immune cell counts (monocytes, lymphocytes)
• Immune cell function (ability of induction of IL-1, TNF, IL-6 and IFNy)
• Plasma anti-inflammatory mediators (eg, IL-10)
Stroke induces peripheral lymphopenia:
• In both experimental and acute stroke, B and T cells are decreased
• This does improve gradually, but even by day 7-14 in patients, not quite back to normal when compared with controls
• These experimetns indicate adaptive immune suppression and shows mirror image findings between experimental and clinical studies
Stroke suppresses perioheral cytokine production:
• Take whole blood samples, culture with LPS and perform an ELISA
• For LPS-stimulated IL-1 and TNFa production, there is profound suppression of cytokine production after stroke
• If this is reflecting what is happening in vivo, then the circulating monocytes are suppressed, and they aren’t producing cytokines when stimulated
Stress response and immune suppression are associated with stroke-associated infection:
• Plasma cortisol levels increase in response to infection
• This is associated with decreased TNF and IL-1 induction
Describe imaging in MS.
- Involves immune cells passing through the BBB and attacking the myelin sheath → imaging cell migration across the BBB will be useful.
- Various MRI-based techniques can assess disease activity and effect of therapy in patients with MS.
- Conventional T1-weighted Gd-enhanced MRI addresses acute disease activity and T2-weighted MRI quantifies overall tissue alterations depicted as hyperintense lesions (NI and neuroaxonal damage).
- In addition to conventional MR sequences, molecular NI-targeted imaging technologies have used USPIO particles to image macrophage and Tcell infiltration in EAE models of MS → has been used to follow T1-weighted signal increase in areas of active lesions with myeloperoxidase expression. More and smaller lesions are detected by this approach than with conventional T1- and T2-weighted MRI
- Experiments based on genetically engineered T cells expressing luciferase have been used
- Direct visualization of the local interaction between immune and neuronal cells was shown by two-photon microscopy in living mice subjected to EAE.
- By combining intravital microscopy with confocal and electron microscopy it could be excellently shown that Th17 cells induce severe, localized, and partially reversible fluctuations in neuronal intracellular Ca2 + concentration as an early sign of neuronal damage, pointing at the key role of the Th17-cell effector phenotype for neuronal dysfunction in chronic inflammation
- Microglial activation in patients with MS has been studied with 11C-PK11195 and PET but so far only in a limited number of patients. Radiotracer binding was increased in areas of acute and relapse-associated inflammation.
Describe imaging in neurodegenerative disaeses.
• while sustained inflammatory responses involving microglia and astrocytes are likely to contribute to disease progression, microglia also have a neuroprotective role by mediating the clearance of Ab. Therefore, a still unresolved question is how to control and modulate microglial activity, in a safe and efficient manner, to slow down or reverse the course of ND.
• Positron-emission tomography and MRI have contributed a broad spectrum of imaging findings in ND:
− altered cerebral glucose consumption (AD)
− altered neuronal transmission within the cholinergic, dopaminergic, serotonergic, and other neurotransmitter systems (AD and PD)
− accumulation of Ab and tau proteins (AD and PD)
− degeneration of central motor neurons (ALS)
• Imaging studies of NI in ND have mostly focused on microglial activation.
• 11C-PK11195 showed quantitatively the in-vivo microglial activation
• In double tracer studies of patients with AD using [ 11C]PIB together with 11C-PK11195, the distribution of the amyloid load and the microglial activation were correlated with the cognitive status
Describe imaging in epilepsy.
So far, imaging of NI in the context of epilepsy has only been performed in single patients.
• [11C]-(R)-PK11195 uptake was observed in regions of epileptic foci and focal cortical dysplasia as determined by highresolution MRI, electroencephalography recording and intraictal and interictal [18F]FDG-PET
