Acute CNS Disorders Flashcards
1
Q
What happens to the periphery when the CNS undergoes inflammation?
A
- Local inflammatory response
- Peripheral response to the CNS
- Acute phase response because production of chemokines from the injured brain to the peripheral compartment through the blood
- Liver amounts an acute phase response
- These signal to the bone marrow
- In CNS disease there is neuroadaptive immune cells that can get into the brain and contribute to immune inflammation (neutrophils, B cells, T cells)
2
Q
What is the neurovascular unit?
A
- Is a conceptual model where different elements contribute to brain homeostasis and function
- There are close interactions of brain cells (astrocytes, microglia and neurons) with the brain endothelium and the exctracellular matrix
3
Q
What are the components of the neurovascular unit?
A
- Astrocytes, microglia, neurons
- Endothelial cells and pericytes
- Extracellular matrix
4
Q
What are the two forms of the extracellular matrix?
A
- In the Parenchyma
- In the basement membrane on the apical part of the blood vessels (in the brain compartment not vascular)
5
Q
What do pericyte processes do?
A
Contribute to the stabalisation of blood vessels
6
Q
What do astrocyte end-feet do?
A
Send processes that are thick and make the whole structure compact and selective of nutrients
7
Q
What do the endothelilal cells do?
A
Make the vascular wall
8
Q
What are the tight junctions?
A
Adhesion molecules between endothelial cells causing tight sticking together
9
Q
How does the adhesion between two endothelia cells occur?
A
- First, on the luminal blood compartment side, tight junctions are present
- Then in the middle of the compartments are JAMs
- Then on the abluminal brain compartment side, is the adherens junction
10
Q
Outline how tight junctions work?
A
- Occludin and Claudin (two isoforms) that interact with each other
- They are expressed by two endothelial cells
- Within the cells, they are interacting with ZO-1 (Zona Occludens) and through beta actin, interact with the intracellular cytoskeleton
- This continues to the next tight junction/endothelial cell
- There is a continuous structure sticking all the endothelial cells together
11
Q
How do JAMs work?
A
They are an adhesion molecule that interacts with ZO-1 to interact with the cytoskeleton
12
Q
What is the adherins junction?
A
- Works due to familial molecule cadherins which bind together and interact intracellularly with adapter molecules catenins
- The catenins interact with the cytoskeleton
13
Q
How was the tight junction of the BBB visualised?
A
- Cultured endothelial cells in vitro
- Two compartments seperated by pores
- Can see ZO-1 staining between the two endothelial cells
- Using EM can identify the pore and the tight junction grabbing and sticking together the cells
- Can also see the position of the matrix, they make their own basement membrane in the culture as need to attach to something in order to extend their cellular processes
14
Q
What is the ECM comprised of?
A
- A complex network of extracellular
- Glycoproteins
- Proteoglycans
- That surround cells
15
Q
What is the structural role of the ECM?
A
Extracellular glue that holds cells together in the shape a tissue/organ
16
Q
What is the functional role of the ECM?
A
Important modulator of cell survival, differentiation and activation
If culture without a matrix, they are unlikely to survive
17
Q
What are glycoproteins and some examples?
A
- Long glycosylated polypeptides
- Polypeptide is large and have glycoprotein chains that attach to it
- Such as Laminin, fibronectin and collagen
18
Q
What are proteoglycans and some examples?
A
- The protein section is a shirt core with long chains of glycosaminoglycans (GAGs)
- The nature of the strucutre of these is very heterogenous depending on the length, structure and composition, may have different glycan matrices
- Such as heparan sulfate proteoglycan (HSPG), chondroitin sulfate proteoglycan (CSPG)
19
Q
What is the main component of the basement membrane and the parenchyma?
A
The glycoprotein Laminin
20
Q
Outline the strucutre of laminin
A
- Made of three isoforms of different subunits
- Alpha, beta and gamma
- Have an EGF-like repeat, globular domain and a helical domain
- The molecule is very sticky, he binding domains are used by cells or the matrix to bind onto
21
Q
Where is fibronectin found?
A
In the basement membrane and the brain paranchyma
22
Q
What is the strucutre of fibronectin?
A
- Two large proteins, heterodimer, linked by two sulphur bonds
- Has many binding domains
- RGD peptide is 3 amino acids which are binding domains for the EC matrix receptors
- LDV and REDV are adhesion moldeule binding domains
23
Q
What is the main component of the basement membrane?
A
Collagene 4 (collagen)
24
Q
What is the structure of collagen?
A
- Three-stranded coiled coil
- Always have a glycine in the third amino acids
- Depending on the other two (X, Y), there are two different isoforms
25
What are the ECM intracellular spaces mainly comprised of?
Proteiglycans but small amounts of the glycoproteins
26
What is the ECM basement membrane mainly comprised of?
Mostly glycoproteins as it is elastic
27
How do endothelia and astrocytes bind to the ECM?
- Bind to the ECM network dynamically
- Via ECM receptors
28
What are integrins?
- ECM receptors
- Have an alpha integrin subunit and a beta with different domains
29
What are the different integrin isoforms in mammals?
- 18 different integrin alpha subunits
- 8 different integrin beta subunits
- Making more that 24 different a/b dimers
30
What are two examplesof specific integrin dimers that are ECM specific receptors?
- Fibronectin- alpha 5 beta 1
- Laminin- alpha 1 beta 1
31
What occurs to the integrins when there is no matrix and if there is a matrix?
- When no matrix, there is a closed confirmation state, uniliganded so is inactive with low affinity
- But if grow cells onto an ECM, they bind and and are active
32
What is the binding site on te ECM that is recognised by the integrin to bind?
Arginine peptide
33
How dointegrin signalling mechainsms form?
- Have stabalisation of the cytoskeleton and recruitment of complex classes of molecules
- These make up the initial signalling transduction state such as focal adhesion kinase
- Have activation of other pathways such as PI3K
- Recruitment forms the initial state of integrin activations (focal adhesion)
- Causes a close state and have stabalisation of beta actin cytoskeleton stress fibres
34
What happens when astrocytes adhere to fibronectin?
- There is cellular spreading
- And formation of stress fibres/focal adhesions
- If there is no matrix, they do not expand their processes
35
Outline integrins outside in signal
If a cell binds onto the ECM there is intracellular signalling and cell survivial
36
Outline integrins inside-out signal
Can have cellular activation of focal adhesion which partially activates the integrins to prime and recognise the ECM
Can therefore regulate the ability to bind onto the matrix
37
What type of signalling does integrin perform?
Bi-directional
38
How do integrins maintain the integrity of the neurovascular unit?
- Need integrins expressed by endothelial cells to bind to the basement membrane
- This allows astrocyte end-feet to bind to the basement membrane too
39
What is the first response of the cerebrovascular unit in response to injury AND how does IL-1 influence this?
- The cells express adhesion molecules such as ICAM1 and VCAM1
- IL-1 is a key driver of vascular inflammation in the brain
- If repeat in an IL-1a and b KO in mice
- There is an increased response of these molecules
- Does not respond well if not present
40
What happens to the ECM after a stroke?
- There is degredation of the basement membrane
- The components all integrate
- There is degraded fibronectin and a loss of contact between endothelial cells and the ECM
41
What does IL-1 trigger in terms of BBB migration after injury?
- Infiltration of neutrophils from the blood compartment into the brain
- This triggers neuronal injury
- Increased expression, degredation of the ECM, opening of the BBB and toxicity due to neutrophil entry
42
How do the neutrophils surpass the endothelia into the brain compartment?
- Activation of the endothelial cells causes them to become sticky as they express adhesion molecules
- The neutrophil will stick to the cells and because there is continuous blood flow will start to roll
- This is partial adhesion rolling
- Eventually, adhesion arrests and the neutrophil will transmigrate throught the brain endothelium
- Degranulation and extravasation occurs
43
Why does partial adhesion/rolling occur?
- Due to the expression of adhesion selectins on the endothelial cells
- These interact with carbohydrates expressed on the surface of neutrophils
- Is not a strong interaction but due to the blood flow, the cell rolls
44
Why does arresting and adhesion of the neutrophil occur?
- Interaction of VCAM and ICAM with more specialised adhesion molecules on the surface of neutrophils (class of integrins)
- Such as alpha 4 beta 1 integrin interacts with VCAM and alpha L beta 2 integrin interacts with ICAM
- There is a strong interaction here, therefore the rolling will stop and transmigration will occur
45
How does degranulation and extravasation occur to neutrophils?
Involves MNPs (membrane protrusive nanotubules) which are key molecules in degrading the matrix
46
What is the response of the neurovascular unit **after** acute injury?
- Production of various inflammatory mediators
- Activation of endothelial cells and astrocytes
- Down-regulation of integrins, up-regulation of adhesion molecules
- Degredation of the ECM
- Looseining of physical interactions between the endothelia, astrocyte end feet and ECM
47
What does the loosening of physical interactions between the endothelia, astrocye end feet and the ECM cause?
- Leaky BBB
- Infiltration of circulating molecules
- Infiltration of neutrophils into the brain tissue
48
How does the neurovascular unit aid recivery after an acute injury?
- ECM are increased in astrocyte end-feet
- Integrin expression increases
- Partial structural and function recovery of the NVU
49
What is pentraxin-3?
Is an acute phase protein important in vascular inflammation/remodelling
It is expressed in the brain after an acute injury
50
What drives pentraxin 3 expression and how was it found that this happens?
IL-1
In KO IL-1 animals there is no penetraxin 3 upregulation after stroke
51
How was it found that PTX3 promotes brain repair?
- Is a long-term vasculature response, regulating long term repair mechanisms
- Looked at neurogenesis
- It is impaired if an animal has a stroke and is a PTX3 KO
- It compromises the neurogenic response and the formation of the glial scar
- Also compromises angiogenesis
52
How does IL-1a promote brain repair?
- Is neuroprotective, driving long term neurogenesis and angiogenesis
- If induce a stroke, and 3 days after inject IL-1a at a low dose, it promotes angiogenesis
- If delete IL-1a in microglial cells in the brain, it will imapir the glial scar formation
53
How many people people have a stroke in their lifetime?
1 in 4 over 25
54
Why is the incidence of stroke increasing?
Because ageing is the largest risk factor
55
How many strokes are there every year?
13.7 million
56
What are two current treatments for stroke?
- Thrombolysis
- Endovascular thrombectomy
57
What is a thrombolysis?
- Give TPA (tissue plasminogen activator)
- Activates plasmin which in turn activates fibrin which causes reperfusion in the tissue to break down the clot
58
What is an endovascular thrombectomy?
- If the clot is in a big vessel
- A stent retriever can pull the clot out of the artery and cause reperfusion in the tissue
59
What percentage of current patients receive treatment for ischaemic stroke?
<20% for reasons such as being on blood thinners etc
60
What is the pathophysiology of ischaemic stroke?
- Energy failure as no glucose and O2 in which membrane potential is reliant on
- Cell will depolarise (anoxic), excitotoxicity will occur as glutamate is released
- This causes oxidative stress and necrosis
- This then causes a peri-infarct depolarisaion, caclium overload and mitochondrial damage
- Leading to sterile inflammation in the tissue and then programmed cell death
61
How are stroke models made in animals?
- Middle cerebral artery occlusion
- FeCL3 model, damage to the endothelium causing a clot and damage
62
What is a major limitation of stroke symptomology study in animals?
Cannot study language
63
What is a benefit of using animal models to study stroke?
- In humans can only use blood samples to infer what is occuring in brain tissue
- Can examine brain tissue in animals
64
What are the three stages of a patient journey in stroke?
- Risk
- Ischaemic event
- post-stroke complications
65
How was it found that preceesing infection increases the risk of stroke?
- Looked at GP records in the UK
- If had a UTI or respiratory infection before
- Were three times more likely to have a stroke
66
How did COVID enhance stroke study?
- Had a very large database
- Risk of an ischaemic event is massive after infection of COVID
- Proposed mechanism is that SARS-COV2 binds to the endothelia cells causing damage and clots
67
What happens to stroke mice when injected with LPS or IL-1 before?
- Cause an infection using LPS or IL-1 making them inflammatory
- Then cause a stroke
- Doubled the brain damage with LPS
- When give an antagonist (IL-1RA) it abolished these effects, showing an IL-1 mechanism
68
Are IL-1 effects in stroke neutrophil dependent?
- Yes
- Post mortem brain had many neutrophils in the tissue
- Depleted these using an antibody (anti-PMN)
- The damage then returned to the level prior inflammatory challange
- The neutrophils drive damage
69
What happens to claudin-5 after stroke and what is it?
- Claudin-5 is a tight junction protein importent in the integrity of the BBB
- Caused inflammation in mice using IL-1B
- The stroke broke down the BBB, as seen by a loss of claudin-5
- By 8 hours the animals that are not inflammatory have some recovery of the BBB
- But the BBB remains disrupted for up to one day when have inflammation
- Therefore inflammation worsens and prolongs damage
70
What happened to stroke damage when given mice pneumonia?
- Are co-morbid mice for vascular risk factors: obese, insulin resistant with metabolic syndrome
- Worsens the damage when given a stroke
-If have inflammation, the damage is worse and BBB disruption is also worse
71
How do platelets play a role on infection effects in stroke?
- Platelets activated in infected mice prior to stroke
- Platelet aggregates are increased in the brain after MCAo
- The platelets adhere to the vasculature forming microthrombi and increasing damage
- If block GP1beta platelets, itreduces the damage
- So if someone has an underlying inflammatory status, their immune system is activated and have a worse outcome
72
What is the post-stroke inflammatory response?
- Neurons quickly become damaged
- Regions with little blood flow will die in minutes
- They release DAMPs activating innate receptors (TLR) cells and the endothelium
- There is a damaging inflammatory burst early on
- There is resolution but it is poorly characterised and understood
- The resolution may not be as good as in peripheral infection, the brain cannot resolbe as well as the peipheral tissue
- But there is some resolution and repair as people can recover some function
73
What are the immune mediators post stroke?
- Different molecules are involved
- Have early damage response driving the microglia and macrophages developing into different phenotypes
- The lymphocytes also produce responses.
74
Outline Anti-ICAM1 as a treatment for stroke
- One of the first immune interventions
- It is an adhesion molecule on the vasculature allowing immune cell entry
- In ICAM KO mice, they are protected against the stroke as it prevents the adhesion of leucocytes
- In people however, it was a negative trial, the people with the drug died more often that the placebo
- They gave a mice antobody to humans promoting an immune reaction
- Still could be a good target modifying ICAM but may never be tested again
75
What were the results of Natalizumab for strokes?
- Is an antobody for integrin, prevents the binding of the neutrophil to the VCAM
- Is a treatment for MS
- Found that it did not work, was a neutral trial, no reduced infarct growth
76
When is IL-1 expressed in stroke?
Is expressed a short time after the injury
77
What happens to the infarct of a stroke when peipheral IL-1Ra is given?
- If block IL-1 with IL1-Ra, it recovers bith therapeutic concentrations of CSF and blood
- It also reduces the damage by about 50%
78
When the mouse has a co-morbidity, is IL1Ra still effective?
- Yes
- Used old unhealthy animals
- Caused damage by blocking the artery
- Gave IL-1Ra and is still neuroprotective
- Suggests that IL-1 has prominent involvement
79
What is IL-1Ra's effect when given to stroke patients but delayed?
- Damage is often measured within 24 hours but need to measure after this as a drug could simply be slowing the process and ultimately lead to the same amount of amage
- Animals still sowed neuroprotection at 7 days
- Was given 3 hours after the stroke
80
What was the cross-lab study result of IL-1Ra
- Improves the power
- In all treatment sites, the drug waas protective
- Works against damage in the brain
81
How was it proved that IL-1 mediates the effect of stroke bia the cerebrovasculature?
- Genetically manipulated the receptors on the cells
- Deleted the signalling receptor for IL1
- Created mice where the receptor was removed from only brain endothelial cells
- Used tamoxifen to switch on the deletion
- The damage is less and in the BBB as well as improved behaviour
82
What was the result of phase 3 clinical trials of IL-1Ra in haemorrhagic stroke?
- Neutral
- There was also a sex effect, men showed improved outcome
83
What was the result of a phase 2 clinical trial on TLR4?
- LPS binds to TLR4
- Use Aptamer which is a DNA molecule that binds to TLR4
- There is reduced injury with the antagonist, can delay treatment and it still works
- People getting a thrombectomy also received the drug and improved immortality- positive effect
84
What occurs to the B cells post-stroke?
- Get post-stroke dementia
- May be because the immune system is involved
- Antibody against B cells
- In the context of post-stroke decline there are lots of B cells in the brain
- Can measure LTP in the animals (measure of learning)
- Causes an effect on LTP when have a stroke also other cognitive problems
85
What happens when B cells are blocked post-stroke?
- In mice who lack B cells, there is improved memory and cognition after stroke
- There are immune changes post stroke that change the oucome
86
What are the different X proteins in the coagulation cascade expressed as?
Zymogens- their inactive forms
They are cleaved to become activated
87
What are the two pathways of the coagulation cascade?
- Extrinsic pathway
- Intrinsic pathway
88
Outline what happens in the extrinsic coagulation cascade
- A cut to the finger
- Tissue factor is released locally into the blood stream to trigger this pathway
89
Outline the intrinsic pathway of the coagulation cascade
Inside of the vessel is damaged with no external damage such as in atherosclerosis
90
What happens when the internal and external coagulation cascade reach the common pathway?
- End up with activated 10a
- This generates thrombin which converts fibrinogen into fibrin
- This forms a clot/the meshwork of the strucutre
- Both pathways generate a small amount of thrombin and then this activates a whole host of other factors upstream (burst phase)
- Lots of thrombin is then generated and the clot foems
- Cross-linking then occurs by factor 13, cross-linking the clots together
91
What is an example of where the coagulation cascade can be inhibited?
- Many different points
- Such as the initial protein response
- Use TFP
92
How can an imbalance of the signalling cascade lead to stroke?
- If leans one way, there would be exessive clotting (ischaemic)
- But if slows down, this could cause haemorrhagic
93
What do current anti-coagulants try and target?
- Thrombin
- Are blunt tools and there is a massive risk of bleeding
94
What had to be accepted for thromboinflammation?
Blood clotting is more than stopping blood coming out of vessels but is part of the immune system
95
Outline how thromboinflammation is not just a clot
- If damage the endothelia at the same time as an infection there would be no barrier
- The formation of the clot itself stops this process
- It stops the infection from the blood to the tissue or vice versa
- Is a two way barrier
- Many things are incorporated into it such as immune cells and platelets playing an immune role
96
Why is Von Willebrand Factor key in thromboinflammation?
- Is key point of contact between the sub endothelial matrix and the endothelial layer exposed to damage
- Can try and target to bring the proteins under control during a stroke
97
What is immunothrombosis?
Is concerned with how the immune cells trigger thrombosis
98
What is thromboinflammation?
Formation of a clot influencing immune responses directly
99
How do immunothrombosis and thromboinflammation come together?
- PAMPs and DAMPs are strong activators of the immune system
- They contribute to triggering of the clot formation (immunothrombosis)
- Once the clot forms there are a number of factors that are released into the blood stream which have far reachiing influences on the immune system (thromboinflammation)
- If have a clot somewhere in the body, the bone marrow can change and become reprogrammed to produce different immune cells with different activities which are crucial to the immune system
100
What is a more focused example of coagulation effecting immune cell functions?
- Neutrophils can form nets- when they are hyperactivated, the kill themselves and release DNA
- The nets can be coated with a number of coagulation factors
- It can activate a kallikrein-kinin system which is part of the intrinsic pathway
- The formation of a net can trigger coagulation and trigger thrombin which has many different functions in the context of the immune system
- Thrombin generates the fibrin which is the mesh-work platform for immune cells to move into the tissues
- Coagulation promoted immune cell infultration into the tissues
101
Outline the role of platelets in thromboinflammation
- Are released from mega-karyocytes in the bone marrow and can be reprogrammed in the presence of a clot or infection
- The bone marrow changes and the phenotypes of the mega-karyocytes change
- Once they are in the blood they can directly interact with immune cells and release different signals (cytokines or chemokines)
- Platelet factor 4 is important as it changes the immune cells fundamentally like how T cells differentiate either dampening the immune response or proliferating to spill into the adaptive immune response
- The main role is within the innate immune response
- Can infleunce where these cells end up in the body through chemotaxis and adhesion
102
Where is VWF expressed?
- By endothelial cells mostly (also platelets and other places)
- Is expressed and stored in highly specialised organelles called viable pallardi bodies
103
How is VWF initially expressed and what can it become?
- Is a monomeric structure which has different domains
- Many of the domains trigger a dimerisation in these viable pallardi
- Then a higher order multimerization which can generate multimers of hundreds of VWF proteins
104
How does VWF leave the organelles to act?
- It is carefully fed out of the organelles and away from the endothelium into the blood stream
- The strings are choppped off and circulate in a highly regulated size
- This size is determined by blood flow, forces of blood flow cause unfolding and then cleavage by a protein called ADAMTS13
- The fragment then folds back up and circulates in its unreactive form
105
What are the consequences in KO ADAMST13 organisms?
- The size of VWF is too large
- It can then spontaneously unfold in the blood and cause clots to form
- This causes paradoxical platelet formation causing the body to haemorrhage
106
How does VWF influence platelets?
- It sticks out of the endothelium and catches circulating platelets when in an inactive form and bound to the sub-endothelial collagen
- This is transient and when the platelets are activated they upregulate adhesion molecules becomming more strongly tethered to the endothelium
- When there is damage to the endothelia, exposure to the sub-endothelia matrix, VWF unfolds and catches the platelets
- This causes large aggregation forming a physical barrier
107
Why is VWF and platelet functioning called primary haemostasis?
Its effects happen before coagulation
It is the first line of defence against the endothelial injury
108
How does VWF cause the priming of platelets?
- When VWF binds to a platelet through its normal process- GP1B receptor (complex of three integrins on the surface)
- It causes a specialised signalling sustem inside the platelet changing other cell surface receptors
- The alph2bBeta3 integrin becomes completely changed in its conirmation and allows it to bind directly to a neutrophil
- The receptor it binds to causes inside out signalling in the neutrophil which releases a net
- The net is coated in coagulation factors
109
How does VWF allow monocyte binding durectly to platelets?
- In the same way VWF primes the platelet for neutrophil binding
- Platelets have CD62P selecting protein on their surface
- Monocytes have PSGL1, these two bind
- When the platelet is primed due to VWF binding getting externalisation o fthe integrin, it can then bind ot MAC1 on the surface of the monocyte
- THis changes the processes inside and allows it to express tissue factor on the surface, feeding into coagulation
- If this is then near other coagulation factors, it will trigger fibrin
110
What is VWF's role in leukocyte recruitment?
- Different receptors on the suface of neutrophuls and macrophages that can be bound directly
- The immune cells roll along the endothelium and rely on the transient interaction such as the adhesion molecules to slow down and then bind to their receptors and extrapolate into the tissue
- The long strings of VWF provide a lot of this transient tethering to slow these down in circulation
111
How was VWF recruitment of leukocytes visualised?
- Looked at a culture of endothelial cells of the capillaries in the brain
- Are cultured and stimulated to provide an inflammatory environment
- They release VWF and an then take donor blood and pass this over the cell layer
- Can then see them slowing down when encountering a long string of VWF on the surface, allowing binding and sticking to their natural receptors on the endothelial layer
- Can then use a different set up to see them, once tethered, move through the endothelial layer
112
What conditions are VWF/ADAMST13 imbalances observed in?
- Peripheral infection
- Autoimmune disease
- Systemic inflammation
- Old age
- Hypertension
- Obesity and diabetes
**All major risk factors for stroke**
113
What was oberved in peripheral respiratory tract infection in terms of VWF content?
- Mice given subclinical infection where bacteria is carefully titrated to reach their lungs so they do not get sick (the infection does not travel anywhere else)
- In cross sections of the blood vesses, a green line is immunostaining for VWF, they light up when expressing the bacteria
- When take blood and measure VWF levels, it skyrockets
114
What was seen in comparisons with mice with a respiratory tract infection, and those without in terms of VWF and neutrophil content?
- Used a contrast agent specific for one antigen, can change the antibody depending on what we want to look at
- VWF causes a signal void on the MRI machine where the beads have stuck to the VWF in the vessels
- Can quantify this to see the massive accumulation of VWF in the brain (microvascular component)
- Downstream of this can see what it is doing once it is there
- Can see an antibody marker for neutrophils and their microvascular presence
- Can see upregulation in the brain of the infected animal
115
What is observed in stoke clots taken from mice and severity of the stroke when have increased VWF levels?
- Regulates stroke risk, higher risk of stroke
- When give a stroke experimentally, worse outcome and a larger size, are primed for more severe injury
- Can section the clots from a thrombectomy
- Was assumed that the majority of clots would be fibrin which can be targeted with TPA but actually is not that much (drug given clinically would therefore be useless)
- However, in the clot VWF makes up about 80% of it, so we should try and taret this protein to break the clot down and restore blood flow
116
What is the link between VWF/ADAMST13 and post-stroke cognitive decline?
- Longer reaching conseuences of a stroke
- Took a lot of immune cells and phenotyped them to find a signature for worse and longer-term outcomes
- The main outcome that was seen is post-stroke cognitive decline
- Think that the immune response to the stroke and the long term consequences of this cause this
- Looked at VWF and ADAMST13 in the plasma of patients
- The levels of these proteins in acute samples and at 6 months correlate well with cognition
- More ADAMST13 in the plasma 6 months post stroke, have less cognitive decline
- Want to boost the degredation of VWF at the point of injury to save from losing cognition down the line
117
What drug was developed using ADAMST13 and what were its results?
- Constitutively active variant of ADAMST13. Took the gene sequence for his and changed the amino acid sequence to make it more acive and active all the time (+caADAMST13)
- Mice are given a stroke, if give no drug, and monitor for an hour, the stroke does not change and the tissue dies
- if give the drug, an hour later and then measure, can see that there is a return of blood flow nearly completely to this area
- The injury is reduced by 40% after the drug
118
What is the inflammatory effect of +caADAMST13
- Has a profound anti-inflammatory effect
- By cleaving the VWF from the endothelial surface in these animals, it prevents the tethering of neutrophils
- Get much fewer infiltrating neutrophils and may contribute to the reduction in the size of the lesion
- If stain for neutrophils, there is much less in comparrison to those not treated
119
How could +caADAMST13 be used as a prophylactic?
- Looked at micce using a combination of the infection and stroke model
- looked longer term in the animals with the lung infection, even once resolved with antibiotics
- The VWF remains elevated fro 2 months after
- So if have an infeciton, 2 months later, stroke risk is still elevated
- In these animals, after the infection had peaked, gave them the drug or a control and waited 2 weeks to look at their brain
- Without the drug, still had lots of VWF so high risk of having a spontaneous stroke
- A single dose of the drug brings down the VWF to more manageable levels
120
What 2 things are emboliic infarction can be caused by?
- Atherosclerosis (mainly in the carotid and vertical arteries)
- Embolism of the heart
121
What are two aetiologies of ischaemic stroke?
- Embolic infarction
- Lacunar infarction
122
What is a lacunar infarction?
Small infarcts that occur in the white matter
123
Does infection have to be in the brain to increase the chance of a stroke?
No, it can also be in the periphery. The inflammation can either be activation of suppression of immunity too
124
What are the clinical applications of assessing inflammation?
- Understand risk factor profile for disease (a target for prevention)
- Understand pathophysiology of disease (target for acute treatment/modifying extent of injury
- An measurement tools to predict outcome and complications, to predict response to potential treatments and to evaluate biological effectiveness of potential treatments
125
What are some challanges of assessing neuroinflammation in patients?
- Access to CNS tissue/vasculature
- Complexity/expense of neuroimaging and emerging immunological techniques
- Validity of methods available
- Assessments in ill/confused patients
- Confounders
- Interpretation
126
What are the 4 methods available in patients to identify strokes?
- CNS/vascular functional imaging (MRI/PET/SPECT)
- Brain or vascular tissue (biopsy, surgical resection or post-mortem)
- Evaluation of CSF components and cells
- Evaluation of plasma/blood cells
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How can MRI be used to identify strokes?
- Can use cell specific contrast agents which define specific components of brain function
- Such as an injection of ultrasmall particles of iron oxide (USPIO)- which are preferentially phagocytosed by monocytes and macrophage lineages
- Gadolinium contrast for assessment of BBB leakage
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Outline how BBB leakage can be seen in acute ischaemia?
- A is a perfusion defect of an evolving stroke
- B is a map of the BBB leakage
- D shows more diffuse leakage
- E shows small vessel disease chronic changes
- Can see the BBB leakage is occuring in places of the white matter disease
- Shows there is more distant leakage, way off the anatomical locaiton of the stroke and this is associated with the small vessel disease
- Shows that the BBB may be associated with other areas of the brain too and not just the acute stroke
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How was macrophage infiltration in a left MCA territory infarction using an MRI seen?
- Used USPIO-enhanced MRI study
- Using a T1-W MRI image initially, showed no sign of the infarct
- After the injection (24hours) there are hints of a signal along the cortical border
- At 48 hours there is much more cortical enhancement mapping
- At day 5 T2 W image shows large cortical enhancement
- Shows that after a stroke, there is accumulation of cells from the monocyte macrophage compartment from within the area of infarct
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How is PET used to identify strokes?
- In-vivo quantification of cerebral blood flow, metabolism and receptor binding
- Injection of radiolabelled tracer
- Cyclotron accelerates proton beam: emission of positrons: photon production
- PET scanner detects photons generated
- This can be co-registered with a CT or MRI scan
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What are two common PET tracers used in strokes?
- 11C-PK11195 which is a specific peripheral benzodiazepine binding (PBBS) site (TSPO) ligand, binding to the translocator protein assocaited with mitochondria. This shows microglial activation
- 18F-2-deoxyglucose (18FDG) which shows glucose metabolism and is taken up by macrophage activation
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How has inflammation in a left carotid atherosclerotic stenosis been imaged using PET?
- Co-registered with a CT angiogram
- When register 18FDG PET and CT together, there is an area of high PET activity
- There is a carotid stenosis and active inflammation associated with this as evidenced by the macrophage activation
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How was microglial activation due to vascular risk imaged using PET?
- Flagged individuals with high risk and lower risk
- Those with higher risk had more CRP (commonly used inflammatory marker showing systemic inflammation)
- MRI co-registered with PK1195PET showed loads of diffuse microglial activation even though they had not have a stroke
- The low risk factor individual didn't have the widespread activation of microglial activity
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How has microglial activation been observed after stroke using PET in a left MCA territory infarction?
- Used PK11195
- Can see the evolution from 5 days to 13 days
- There is an increased signal and microglial activation in the area of infarct
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How was PET used to observe BBB breakdown after an intracerebral haemorrhagic stroke
- Used MRI, PET, gadolinium study
- There are areas of haematoma in the left hemisphere for one patient and in the basal ganglia for another
- There is also BBB leakage associated with this bleed
- There is little microglial activation associated wth the haemorrhage but there is microglial activation elsewhere (maybe connected regions of circuitry)
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How can single photon emission computed tomography (SPECT ) be used for strokes?
- Injection of radiolabelled tracer (gamma-emitting isotope)
- Gamma emission detected directly by a gamma camera
- Can also co-register with MRI and CT
- 111-Indium labelling of autologous leucocytes can happen where take out the blood of the patient, label and then reinject
- Technetium-99m Diethylenetriamine-pentacetic acid (99mTc-DTPA): BBB disruption
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What was a study to show changes in TSPO-binding in right MCA territory infarction using SPECT?
- Used 123I-CLINDE SPECT which is a tracer for SPECT used to image microglial activation
- There was microglial activation near the area of infarct
- As time goes on then the microglial activation drops below control levels, as this becomes scar tissue
- There is some activation in the oposite hemisphere
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How was BBB disruption in an intracerebral haemorrhagic stroke seen usign SPECT?
- Used DTPA-SPECT
- Co-registered with a CT scan
- Saw halos of BBB leakage, what we expect to see
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Why are brain post mortems limited in stroke research?
- Limited to post-morten or brain biopsy
- Has to be attained very quickly after sample taken
- Ethical and logistical issues
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Why are carotid examinations limited?
- Limited to endarterectomy specimens
- Can use correlation studies, imaging versus histopathological
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How was it seen that inflammatory cells are differentially associated with unstable carotid plaques?
- Unstable versus stable plaques
- They have different histological features
- Wanted to gather an understanding of the differential cell populations in these plaques using signle cells and microarrays
- Saw that macrophages, monocytes and T cells etc are particularly associated with unstable plaques
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What is the evolution of inflammation in the infarct/peri-infarct zone of a stroke?
- Looked at inflammatory markers
- IB1 is a stain for microglia, can see an increase in activation. At each time point in glia and in macrophages there is a higher proportional prevalnce of these cells
- Can see that IL-1 in macrophages, there is high expression relative to the normal tissue and TNFa in microglia too
- Tells us that in real brain tissue, there is evidence of inflammaiton and expression of inflammatory markers in certain cells
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Outline a study where CSF was measured in acute infarction
- Normally there is limited accessability
- Performed daily lumbar punctures and then another later on. Lastly a follow up
- IL-6 magnitude in the CSF is higher than IL-1
- There is a larger burden
- For both, there is a peak in concentration in the first few days which tails off
- This is consistent with the idea that there is an infarct driving the inflammation and this spills into the CSF
- This process then gets dampened down or switched off
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How can blood sampling be used in strokes?
- Plasma useful for cytokines/mediators released into systemic circulation
- There is an unknown value for mediators released at the site of inflammation
- It is an indirect measurement of neuroinflammation
- Whole blood or isolated immune cell populations for phenotype and function
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How was blood sampling used to genetically determine MCP-1 levels and ischaemic stroke?
- Wanted to know how genetically determined the concentration of cytokines in the blood are as risks for strokes
- Was a follow up study
- Found that MCP-1 has a key role in recruiting innate cells to sites of tissue injury
- This is consistently associated with the risk of stroke
- Embolic stroke was assocaited with levels of MCP-1 in the blood (not lacunar)
- Could then maybe measure MCP levels in those at high risk and make drugs to modify MCP?
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How was CRP found to be a risk for stroke using blood sampling?
- Used patient meta-analyses, a prospective study with no clinical vascular disease history at baseline
- Take people at relaive risk and then follow up
- Measure the CRP before and after, see if it independently increases the risk of stroke by controlling for many factors
- These are risk ratios
- The risk increase is modest, the more the we adjust for additional factors, the weaker the risk for CRP
- CRP shows dependency on these other factors
- Makes it hard to untangle association between these different factors
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What is the acute phase response of plasma CRP in acute stroke using blood sampling?
- In the classic acute inflammatory resopnse, CRP increases very quickly and is highly sensitive to changes
- But chronic remains elevated at a background level
- It is still elevated at 3 months
- May mean that higher CRP mens high microglia
- The baseline may reset for this higher level becoming the normal (continued activation)
- Cannot measure CRP before stroke
- This increase happens in both ischaemic and haemorrhagic strokes and is augmented after infection
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How does plasma CRP equate to survival/recurrance in acute ischaemic stroke and intracerebral haemorrhagic stroke?
- In ischaemic, can use to prognosticate
- If divide the concentrations of the CRP into three categories we can see if CRP in acute phase predicts recurrant stroke risk
- If have high CRP, there was increased risk by 50% so maybe inflammation is involved
- In ICH patients
- Looked at survival, for high medium and low CRP
- In the highest concentration, the likelihood of survival is only between 50-60%
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How was it seen that hyperacute ischaemic stroke altered peripheral monocyte and dendritic cell populations?
- Took blood within 6 hours of ischaemic stroke
- Even in the first 6 hours, expect more innate rather than adaptive immune activation (requires some innate to get going)
- Found that for different innate categories (moncytes and dendritic cells)
- Compared to controls, there is a diverse range of different changes in these compartments
- Some dentritic aren't changing and monocytes are going up
- Tells us there is a dynamic change in the innate cells in the periphery after a stroke
- Would help if could identify populations of cells within a few hours to treat
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Why do a lot of anti-inflammatory treatments fail in trials?
- Developed in experimental models of stroke but there is a translational gap in humans and animal patients
- How well do these experiemntal studies model and prepare for clinical trials
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What is an example of an anti-inflammatory drug that caused more harm than good and why?
- Enlimomab
- Murine antibody for ICAM-1
- If block inflammation, may be harmful
- If have a severe stroke, at a higher risk of getting pneumonia, if interfere with the immune system, may increase their risk of infection
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How does anakinra work?
Blocks IL-1 (IL1Ra)
There is no association with increased infection so is promising
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Outline how anakinra may be beneficial for acute ischaemic stroke?
- IL-1Ra competative inhibitor of IL-1R1
- Pre-clinical evaluation showed:
- Reduced infarct volume, reduced cytokine production, reduced BBB damage, reduced neutrophil infiltration, reduced microglial activation and improved functional outcomes
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What was the difference in phase 2 and SCIL-STROKE phase 2 anakinra trials?
- Phase 2 had no tPA which creates plasmin to break down proteins such as thrombolysis
- Phase 2 was IV but new is Sub-cut
- The abscence of thrombolysis (broken down by tPA) had loweR IL-16 and CRP but better outcome was not more likely, could be an interaction between tPA and anakinra
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How does anakinra act on inflammatory markers after an aute ischaemic stroke?
- It reduces inflammatory markers IL-16 and CRP
- Can see that for both of these, the treatment group compared to the placebo prevents the increase overtime
- Prevents the increase in pro-inflammatory markers in the blood, both are associated with worse outcome after stroke
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How could Natalizumab be used in acute ischaemic stroke?
- Monoclonal antibody to alpha integrin of VLA-4
- Pre-clinical evaluation showed:
- Reduced leucocyte infiltration to the brain (including through VCAM1) T cells
- Reduced cytokine production such as IFN-gamma
- Improved functional outcomes
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Was Natalizumab successful in phase 2 and 2b trials for acute ischaemic stroke?
- Giving the drug with a longer treatment window had no effect on infarct growth
- The odds of having a good outcome was less likely
- There is therefore no evidence that it is beneficial
- Estimates are on the side of no benefit
- When associated with thrombectomy, there is worse outcome even
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What is the difference between acute peripheral and CNS inflammation?
- Peripheral has an acute inflammatory response which aim is to restore homeostasis and repair
- CNS has incomplete resolution, there is initiation of inflammation and it never recovers to normal, there is neurodegeneration (low grade inflammatory response)
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What is the difference between chronic peripheral and CNS inflammation?
- Peripheral, there is initiation of inflammation and then an uncontrolled response where there is long lasting inflammation
- CNS has inflammation that is never resolved and sometimes worsens leading to neurodegeneration
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What is the relationship between an organisms ability to regenerate, the complexity of a nervous system and immune complexity?
As you increase the complexity of the nervous system and the immune response, there is reduced possibility for CNS regeneration
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What is the evidence of brain repair in MS?
- Relapsing-remitting
- The patient looks better during the remission phase, their motor and sensory abilities imprive
- There is likely a regenerative process here
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What are the 4 mechanisms of brain repair?
1. Restoration of the neuronal network (neurogenesis)
2. Restoration of the blood supply (angiogenesis)
3. Neuroprotective role of activated microglia
4. Protection of non-injured brain structures by glial scarring
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What is the history of the discovery of neurogenesis?
- In 1913 Cajal looked at post-mortem tissue and concluded that the brain cannot regenerate but postulated the posibility for science to reestablish normal nerve functions
- In 1962, new neurons were found to form in the brain of adult mammals
- In 2000 neurigenesis was confirmed
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How can neurogenesis be visualised?
- Take advantage of visualising DNA replication and strands opening, also the coupling of nucleotides into these DNA strands
- Can stain the nucleotide (3H-thymidine) with a marker
- Inject this into a rat ad can look under electron microsope and see the marker in cells
- Shows that they are about to divide into new neurons
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Where are the discrete subset of cell populations which can divide into neurons?
- Subgranular zone (SGZ) of the denate gyrus
- Posterior periventricular layer
- Subventricular zone (SVZ) of the third ventricle
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Where does neurogenesis start in the adult brain?
- Neural stem cells which can proliferate into more neural stem cells or newborn neural precursor cells
- These newborn neural precursor cells can differentiate into different cell types in the brain
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How does neurogenesis occur ni the subgranular zone?
- Have stem cells which start to differentiate and proliferate to become neuronal precursor cells
- They migrate from the subgranular zone to within the granule cell layer and make new neurons here
- They form a network with surrounding neurons and from other areas
- In particular the hippocampal denate gyrus which may be associated with new memory formation
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How does neurogenesis occur in an uninjured vs injured subventricular zone?
- In uninjured, neural proginators proliferate very slowly
- In injured, neural proginator cells proliferate and migrate laterally towards the damaged area
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How does neurogenesis affect brain injury and repair?
- In normal conditions, proliferating progenitors are in a mesh of astroglial processes in the SVZ
- When injured, the newly proliferated progenitor starts migating to the area of damage
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How are signals sent for neurogenesis in an injured brain and what does this cause?
- Injured brain area secretes many factors like growth factors, cytokines, chemokines and mitogens. Endogenous signals from injured neurons (DAMPS)
- These factors diffuse to the SVZ and induce proginator migration
- Newly formed cells migrate laterally from the subventricular zone to the damaged area
- The increased proliferation and migration of neural proginators continues for months after injury to the brain
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What is a quick summary of neurogenesis?
- Neurogenesis is a natural process that takes place in the normal adult brain
- Neurogenesis is increased in the injured brain and may participate to the repair process of the neuronal network
- Neurogenesis is induced by inflammatory mediators/growth factors
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What is agiogenesis?
- Is a biological process involved in the growth of new blood vessels from pre-existing vessels
- Generally involved in wound-healing to restore the blood supply to an injured tissue/tumour development
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What four factors are involved in angiogenesis after brain injury?
- Cytokines
- Chemokines
- Integrins: ECM (matrix is important for vascular remodelling)
- Growth factors
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What is the most important trigger for angiogenesis?
- Vascular endothelilal growth factor (VEGF)
- Important for endothelial proliferation and formation of new vessels
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What are 3 receptors for VEGF, where are they found and what are they involved in?
- VEGFR1 (some endothelia), involved in embryonic vascular development
- VEGFR2 (in all endothelia), recognised in injury conditions and tissue repair
- Neuropilins, NP1 and NP2, recognise VEGF during embryonic development
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What is angiopoetin-1 and 2 (Ang1) involved in?
Binds to Tie-2 receptor which is important in triggering the angiogenic response during inflammaiton
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What is ECM important for in angiogenesis?
- Binds to integrins
- Important in embryonic vascular development
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How was VEGF found to be important in angiogenesis?
- Detect the high level of expression of VEGF mRNA in blood vessels during ischaemia
- Also VEGFR2 immunohistochemistry showed increased levels which correlated to vascular proliferation and the growth of vessels in MCAO model
- Also stained for Ki67 which is a protein only expressed during cellular proliferation, so saw the growth (CD31, endothelial specific green marker) of the vessels due to VEGF 24 hours after MCAO
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Outline the pathway of ECM in mediating angiogenesis?
- When injury there is BBB, loss of interaction between the different components of the vascular unit
- Increased expression of IL-1, VEGF. Ang1 causing endothelial activation and proliferation
- ECM deposition, EC proliferation to new vessels and the involvement of integrins
- Vessel stabalisation, pericyte and astrocyte end-feet attachment
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How are angiogenesis and neurogenesis linked?
- Often occur together
- If forming new neural networks, they require blood supply
- So angiogenesis is required for neurogenesis to occur
- The initial starters are similar for both too
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Why is glial scar formation important?
- Is formed by astrocytes expressing GFAP
- Is important for containing the region and repairing the brain
- Is a balance between the timing of the acute and chronic inflammation
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How does IL-1B contribute to neurogenesis in the brain?
- Culture neuronal stem cells in a dish
- Can treat with IL-1 to see the incorporation of BrdU which is a cellular marker of proliferation
- Shows that IL-1 drives CNS renewal
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What are two ways in which IL-1 can drive neurogenesis?
- Through direct mechanisms
- OR indirectly through the expression of different mediators such as PTX3
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What 5 things is PTX3 important for, for neurogenesis?
- Activates the complement system
- Promotes extracellular matrix assembly
- Activates angiogenesis, blood vessel repair and remodelling
- Regulates inflammatory processes/leukocyte recruitment
- Microbe recognition and regulation of immunity
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What are 2 therapeutic strategies for improving neurogenesis after stroke?
- Targetting early, which has failed such as NXY Astra Zeneca compound which worked in animal models but no neuroprotective benefit in humans
- Promoting repair process, stem cell therapy, angiogenesis, ECM repair later on
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What does biphasic opening of the BBB mean during stroke?
- It opens quickly, within hours
- It then closes again
- After a few days it will reopen due to different mechanisms
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What two things happen within hours of a stroke?
- Initiation of immune response, due to damaged neurons, astrocytes and DAMPs such as HMGB1
- Innate immune response where danger is detected and respond by upregulating inflammatory cytokines (involved microglia, neutrophils and monocyte infiltration etc)
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What occurs within days of a stroke?
- The adaptive immue response with pro-inflammatory and anti-inflammatory lymphocytes
- T and B cells
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What occurs in weeks after stroke?
Recovery and resolution, may be some excess T and B cell actions
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What is the peri-infarct?
Area surrounding the core of the damage
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Which types of cells can promote neuroprotection in stroke?
IL-12 and IL-13 releasing cells (also neurotoxic)
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After a stroke, what actions do B cells have on cognitive decline?
Can worsen post-stroke cognitive decline
Mice KO for B cells do not suffer impairment
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During cerebral ischaemia, what can happen to the penumbra?
- Cells in the penumbra can die
- The artery supplying this area is blocked by the core of the stroke
- Blood flow has been reduced, can therefore be necrosis
- But the penumbra is also fed by a collateral flow so will have some small supply
- If blood flow is returned to this region quickly by removing the core, the cells may be saved
- But if the reduction stays, the cells will go onto die
- The core will always die but the penumbra may or may not
194
Which cells can be found in the brain in response to a stroke (monocyte surface receptors)?
- CD14
- CD16
- CCR2
- M2 microglia/macrophage
- M1 and 2 microglia/macrophage
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Outline classical proinflammatory monocytes
- M1 microglia
- High surface marker of CD14
- No CD16
- High CCR2
- In mice called Ly6C (high)
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Outline intermediate proinflammatory monocytes
- High surface markers of CD14
- Some CD16
- Some CCR2
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Outline nonclassical anti-inflammatory monocytes
- M2 microglia
- Some CD14 surface markers
- High CD16
- No CCR2
- In mice called Ly6C (low)
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Which type of monocyte is more likely to invade the brain after stroke?
- The classical monocytes
- Don't tend to get non-classical invasion
- They are found in the brain because the classical can chage the M2 cells or the microglia endogenous in the brain
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What is the concequence of acute peripheral immune changes after stroke?
- Get infiltration of immune cells in peripheral organs as well as the brain
- Causes immunosuppression
- Increases the risk of infection
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How can post-stroke immunodepression be characterised?
By reduced numbers of lymphocytes in the blood (lymphopenia)
In particular B and T cells
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Which occurs first, CNS chemokines upregulation or preipheral?
- Peripheral occurs quicker but is less prolonged, near immediate increase in chemokines
- CNS occurs within around 4 hours in the area of damage which can be sustained for 24 hours (can be neurotoxic or protective)
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What happens to CCR2 in experimental studies of stroke?
- Is a receptor which is mainly expressed on M1 microglia/macrophage/monocytes which responds to a chemokine CCL2
- Activation can lead to worse ischaemic damage
- Activation can lead to better outcome
- May be detrimental in acute phase but needed for recovery
- Is expressed on microglia/macrophages
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How do the two different types of neutrophils act?
- N1 is proinflammatory causing myeloperoxidase release (toxic to neurons) and MMPs which is an enzyme that breaks down the BBB through ECM
- Causes neurotoxicity
- N2 is anti-inflammatory causing debris clearance and neuroprotection
- But it also causes immunosuppression which increases risk of infection
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After stroke, how does IFN1 react?
Is released from Th1 cells and can exacerbate damage
