Inflammation in Acute Brain Injury

Question 1

What is stroke and what are the types of stroke?

Answer 1

• Stroke (aka cerebrovascular accident) is where poor blood flow to the brain results in cell death
• The brain is metabolically demanding – uses 25% of cardiac output.
• Neurons cannot function without oxygen and glucose – they lack the capacity to function and die
• Ischaemic → The vessel becomes blocked
• Haemorrhagic → The vessel bursts

Question 2

What is the burden of stroke on society?

Answer 2

• 2nd leading cause of death worldwide for people >60
• 5th leading cause for those aged 15-59 (incidence in creasing in young people, likely because of an increase in the prevalence of stroke drivers, eg, diabetes)
• Leading cause of long-term disability
• 15 million have a stroke each year
• World Stroke Organisation say 1 in 6 will have a stroke
• Every 6 seconds someone will die from stroke
• Global problem – incidience is very high in Asia and Russa
• Around £7billion a year in the UK
• In the US, costs expected to rise rom $80-$180billion

Question 3

What are the risk factors for stroke?

Answer 3

• High blood pressure
• Lack of physical activity
• Obesity
• Diabetes
• High cholesterol
• Smoking
• Stress and depression
• High alcohol intake
• Cardiac disorders

→ All risk factors for CVD. Stroke is essentially a CVD.

Question 4

What are the current stroke treatments?

Answer 4

• If stroke suspected, brain scan to see if haemorrhagic or ischaemic
• If ischaemic – tissue plasminogen activator (thrombolytic agent) → currently the only acute treatment
− If given in haemorrhagic, would cause death
• Currently only a small proportion of patients treated
− Needs to be given within 4.5 hours, after this the risk of haemorrhage and mortality increases
− Stroke services need to be optimal for this to happen – have to be able to get to hospital quick enough and get a scan. Cant do this in remote locations or poor countries
• Reperfusion injury can be detrimental – results in inflammation and oxidative stress
• Increased risk of haemorrhage
• New treatments still required

Endovascular therapy new option?
• Inserts a fine wire into the vessel to pull out the clot
• Limited application – needs highly trained interventional neuroradiologists
• Only a small % would have access
• Could break up the clot and send smaller clots up other arteries

Question 5

Describe a temporal profile of inflammation in stroke.

Answer 5

Preceeding events:
• Conventional risk factors such as athersosclerosis
• Acute and chronic infection are a risk factor, leads to…
• APPs, blood becoming more pro-coagula endothelial activation and atherosclerotic instability

Post stroke:
• HPA axis activation
• Neurological impairment and dysphagia
• Possible brain-induced immunosuppression

Temporal profile:
• In the years preceeeding stroke, you can have risk factors such as LDL accumulation or a chronic inflammatory diseases
• Much closer to the time of stroke you could have an infection or surgery
• At the point of stroke, you have the thrombus formation and within minutes you have large amounts of tissue damage due to hypoxia activating inflammation.
• Cellular damage triggers innate immunity within hours, with leukocytes responding to hypoxia and tissue damage
• Within days adaptive immunity is engaged
• In the weeks/months following stroke you have resolution of inflammation, clearing of dead cells and promotion of cellular regrowth
• However, in the years following stroke it is possible you may now have chronic CNS inflammation and immune activation, with persistent autoimmune responses to brain antigens as a result of antibody generation
• There is also evidence for chronic immune suppression following stroke

→ Note that a confounding factor is that the risk factors are associated with raised inflammatory profiles – so the inflammatory status after stroke will be influenced by their pre-exisitng inflammatory profile.

Question 6

What is the experimental evidence for a role of central IL-1 production in stroke?

Answer 6

• IL-1 increases after experimental stroke
− Luheshi et al, 2011
− 60 minutes after MCAO
− Co-localised with the microglia – so the microglia are expressing IL-1
• Inhibiting IL-1 reduces ischaemic injury
− IL-1RA (delayed, after 3 hrs)
− Anti-IL1 antibody
− Knockout of IL-1 genes
− Caspase 1 inhibitor

Clinical trial of Anakinra:
• Phase II – 34 patients within 6 hrs of stroke, 17 placebo and 17 anakinra
• Double blind, placebo controlled, randomized
• Primary outcome is safety and feasibility in this patient population
• Outcome:
− IL-1RA reduced the neutrophil increase
− IL-1RA reduces IL-6 increase
− IL-1RA caused an increase from 14 to 30% of people having full recovery, and a decrease from 28 to 20% mortalities

Question 7

Anakinra didn’t progress to phase III for stroke. Bangle et al concluded that you need to demonstrate efficacy at later time points, in animals with comorbidities and in animals other than rodents. What were the experiments that showed this?

Answer 7

Modelling co-morbidity:
• Ob/Ob mice, corpulent mice, aged mice, atherosclerotic mice or spontaneously hypertensive rats
• Have a phenotype closer to that what is seen in man
Peripheral IL-1RA is also protective – Greenhalgh et al, 2010:
• IL-1RA gets into the plasma within minutes, and into the CSF within minutes (100-fold lower concentrations, but still protective)
• After just a single administration, remains in the brain for around 8 hours before levels begin to decrease
• Reduces infarct volume around 50%
• This IL-1RA was given at the time of stroke, so not idea as in the clinic it would be given a bit after
• However, many studies in human still fail to get it into the brain

IL-1RA is effective with co-morbidity – Pradillo et al, 2012:
• 25mg/kg IL-1RA subcutaneously in corpulent rats was protective
• First demonstration of a stroke drug working in a co-morbid animal

Delayed IL-1RA is protective:
• Drug administered 3 and 6 hours post stroke
• 24 hours after administration, there was still protection
• Could be argued that the drug is merely delaying the damage – but 7 days after administration there was still protection.

Long-term functional recovery – Girard et al, 2014:
• A criticism of most of the experimental studies is that although they show a protective effect in terms of damage, they did not assess to see whether this translates to functional recovery
• This is what is important – not the damage, but whether it can restore function
• You can train a rat to reach a food pellet through a Perspex screen – requires fine motor skill
• If you give the animal stroke, they can no longer do the task
• If you give IL-1RA at the time of reperfusion, it recovers function
• If you wait one day after reperfusion, there is no functional recovery

Cross-lab study – Mayasami et al, 2015:
• A big criticism of stroke research is its replication
• The same treatment was tested in labs around the world, and in every study (apart from one in Finland) – it worked

Question 8

Describe a role for microglia in stroke

Answer 8

• Microglia are the first responders in the brain
• They become recruited to the infarct
• The rapid loss of neuronal integrity in the ischaemic core may control the activation state of microglia
• Classical activation – expression of IL-1B, TNFa and iNOS was achieved in response to mildly hypoxic neurons
• Severely hypoxic neurons induced the expression of neuroprotective factors (BDNF, GDNF)
• Difficulty in understanding microglia function in stroke is caused by several confounding factors:
− Microglia quickly respond to changes in the brain, so the degree of activation upon ischaemia can be a contributor to or reflection of the severity of injury
− due to BBB damage, centrally expressed factors can leak into the circulation and peripherally secreted mediators can gain access to the parenchyma
• Several studies have found correlation between microglial activation and central cytokine expression with stroke:
− Tetracyclines inhibit microglia and protect hippocampal neurons against global ischemia
− Over-expression of the anti-inflammatory IL-10 on microglia cells reduces infact size and downregulates IL-1, TNFa
− Microglial phagocytosis of invading neutrophils after ischaemia has been shown to be protective, and microglial TNFa has also shown to be neuroprotective in some studies
➢ So microglia can be both beneficial and detrimental

Question 9

Describe a role for astrocytes in stroke

Answer 9

• Produce IL-1, ROS, chemokines, MMPs and vasoactive mediators

Question 10

Describe what happens to the brain endothelium in stroke

Answer 10

• Activation may be induced by hypoxia, resident glia and circulating cells
• Upregulate E-selectin, ICAM-1 and VCAM-1, reduce expression of occludins and claudins → allows recruitment of peripheral cells

Question 11

Describe a role of TLRs in stroke

Answer 11

• TLR receptors on neurons are increased in response to energy deprivation
• Neurons from mice lacking TLR2/4 are protected against energy-deprivation induced injury
• Microglial TLR critical for expression of IL-1

Question 12

Describe the role of ROS in stroke

Answer 12

• Generated during mitochondrial dysfuncton in neurons and contribute to neurotoxicity by mediating pro-apoptotic signaling cascades
• iNOS expressed by glia are sources of ROS
• Hypoxia can induce microglial iNOS expression mediated by HIF signaling
• Agents that scavange free radicals rescue neurons from ischaemic insults

Question 13

Describe the clinical evidence (other than Anakinra) for central inflammation in stroke.

Answer 13

• Using PET and [11C]PK11195, found no increase in microglia signal 3 days after stroke, but an increase thereafter up to 30 days
• Provides evidence that there are chronic processes occurring – but that microglia are maybe more reparative in stroke?

Question 14

What did Emsley and Chapman find about systemic inflammation post stroke?

Answer 14

Emsley et al, 2003:
• APPs increase
• WBCs increase
• Erythrocyte sedimental rate increase (blood more pro-coagulant)
• IL-6 increases in the plasma after 4 hours

→ So there is a peripheral inflammatory response occurring

Chapman et al, 2009:
• CXCL1 (neutrophil attractant chemokine) increased in the plasma after 4 hours
• Goes away after 24 hours
• Expressed in the brain cortex and striatum after 4 hours, and still sustained after 24 hours

The question is – does the peripheral inflammation drive the CNS inflammation?
• The hypothesis is that the peripheral inflammation drives a chemokine gradient where the peripheral CXCL1 decreases, and then increase in the brain
• Creates a gradiant so that neutrophils come out of the bone marrow and migrate into the brain

• Blocking IL-6 prevents the increase in CXCL1

Question 15

What is the role of peripheral leukocytes in stroke?

Answer 15

• One of the earliest inflammatory responses to stroke in patients is increased peripheral leukocytes
• Observed within 24 hours, some studies report sustained elevation for a week (Emsley)
• Their levels correlate with infarct volume
• Labelling of neutrophils shows they are recruited within 24hrs of symptoms
• Monocytes also increase, but delayed

Question 16

What is the link between peripheral adhesion molecules and stroke?

Answer 16

• Already seen how adhesion molecules increase on the brain endothelial
• Also been found that circulating levels of soluble adhesion molecules increase

Question 17

What is the role of APPs in stroke?

Answer 17

• Increased CRP – attracted attention as a potential biomarker predictive of outcome
• As with leukocytes, sustained CRP observed a week after stroke
• JUPITER trial indicated patients with elevated high-sensitivie CRP benefit from statin therapy regardless of LDL level

Question 18

Summarise the role of peripheral cytokines in stroke

Answer 18

• In an experimental setting, a large number of cytokines has been investigated, but in the clinical situation the focus has been mostly on IL-6
• Several clinical studies report early (24h) and sustained (7d) release of iL-6 correlating with outcome
• Other studies assessing intrathecal and serum IL-6 show that only CSF levels correlate with outcome
• In the same study, increased IL-1B was found in the CSF and not serum
• Other studies report no increase in serum TFNa, whereas some report both serum and CSF TNFa increases
• Reasons for differences could be due to pre-existing levels of cytokine

Question 19

Summarise the role of peripheral derived cells in stroke

Answer 19

Innate:
• So, they key things that we think are happening in terms of the acute phase of stroke, is we believe there is a migration of neutrophils into the brain, activation of microglial cells which release IL-1, and this contributes to the death of neurons. If you inhibit the IL-1, it is protective.
• Neutrophils and neutrophil-derived factors have been shown to mediate BBB breakdown and can be involved in the formation of ischaemic damage.
• Post-ischaemic administration of anti-Mac-1 antibody results in significant reductions in ischaemic cell damage.
• Depletion of neutrophils showed that these cells are a major source of oxygen radicals during reperfusion after focal CI

It is important to determine whether leukocyte infiltration into the ischaemic brain area is essential for augmented pathology.
• it is not clear whether neutrophils accumulate in the brain early enough to locally modify the maturation of the primary ischaemic damage in all experimental stroke models.
• Current data indicate that blood-derived myeloid leukocytes and their products can be potentially harmful in stroke, but these cells do not necessarily infiltrate into the brain during the formation of primary ischaemic damage and do not necessarily have a detrimental role in all experimental models of acute CI.

Adaptive:
• Several studies showed that absence of adaptive immune cells can reduce the ischaemic damage. Rag1/, CD4+ T-cell/, CD8+ T-cell/ and IFN-c/ mice exhibit significant reductions in platelet, leukocyte adhesion and infarct volume
• Similarly, T- and B-cell-deficient (SCID) mice have reduced lesion size and central inflammation after experimental stroke
• Regulatory T cells can reduce brain inflammation via IL-10 after experimental stroke
• Systemic depletion of regulatory T cells (Tregs), which are capable of dampening the pro-inflammatory response, exacerbates delayed brain damage. Mice lacking Tregs displayed increased post-ischaemic activation of resident and invading inflammatory cells, elevated central expression of IL-1b and TNFa and elevated serum TNFa and IFNc
• Targeting the adaptive immune system, however, is not without potential risk for the organism, as stroke-induced immunosuppression can predispose to post-stroke infections, which increase morbidity and mortality after the ischaemic event.

Question 20

What is the link between inflammation and coagulation in stroke?

Answer 20

• Cross-talk between these two pathways is likele to be of pivotal importance in cerebral ischaemia
• Pro-inflammatory cytokines downregulate endogenous inhibitors of coagulation, including tissue factor pathway inhibitor, antithrombin and the protein C csystem
• The coagulation cascade may also directly activate cellular inflammatory processes
• Platelets express pro-inflammatory molecules including IL-1
• Platelet P-selectin also supports leukocyte infiltration
• Platelets are the main target of current stroke therapy ⇒ TPA works to reduce platelet clot formation

Question 21

What is the link between chronic inflammatory disease and stroke?

Answer 21

• Systemic inflammation is associated with all known co-morbidities
• Adipose tissue is now recognized as an abundant source of multiple inflammatory mediators, and obesity is now considered a state of chronic inflammation with significantly elevated levels of cytokines including TNFa and IL-6
• Evidence that brain cell cultures from animals with pre-existing inflammatory disease have exacerbated cell loss when given ischaemic challenges

Question 22

What is the link between infection and stroke?

Answer 22

Smeeth et al, 2014:
• Looked back at GP databases in the UK
• In the group of people idenfitied as having an infection, they looked at incidence of stroke
• Found that in systemic respiratory tract infection and UTI, patients were 3 fold more likely to have a stroke 1-3 days after infection
• Other studies have shown that if you have an infection, the outcome in stroke is worse

What is the mechanism?

McColl et al, 2007:
• When you administer LPS, it makes the infarct volume worse along with the neurological deficit
• If you co-administer IL-1RA, it makes it better
• This shows that the damage from infection is IL-1 mediated
• Administering iL-1 increases damage
• Co-administering anti-PMN neutrophil depleting antibody reduces the damage and neurological deficit
• IL-1 effects are MMP-9 dependent:
− Using an MMP-9 inhibitor shows you get reduced breakdown of the BBB (by looking at Claudin-5 expression)
− Also showed you get reduced brain damage

→ LPS induces IL-1 production via TLRs.
→IL-1 acts on astrocytes to release MMP-9 – neurotoxic, and induces BBB breakdown
→ Allows neutrophils in

Systemic inflammation alters kinetics of BBB breakdown:
• BBB disrupted for longer – without inflammation, disruption resolves around 8 hours after reperfusion.

• A criticism of this work is whether giving LPS 30 minutes before stroke mimics the real infection (probably not)
• So, work was done where animals were given pneumonia
− Data mimics what is seen with LPS
− IN both lean and corpulent rats, infection causes an increase in brain infarct and BBB damage
− Just as with LPS, effects of pneumonia dependent on IL-1

Question 23

What is the link between ageing and stroke?

Answer 23

• Relative risk of stroke increases by age.
• Cerebral blood vessels undergo profound changes with aging including reduced capillary density and the formation of thickened and fibrotic basement membranes.
• In the cerebral cortex microhaemorrhages become frequent
• Aged microvessels exhibit increased oxidative stress including the producuction superoxide anions and enhanced NADPH oxidase and inducible NO synthase expression
• Similar vascular changes are seen in aged rodents, including reduced basal blood flow in arteriole-arteriole anastomoses and structural changes in basal lamina and endothelial cells
• Experimental stroke studies reported increased mortality rate, increased BBB disruption, reduced hypoxia-induced microvascular growth and impaired neurogenesis in aged rodents.

Question 24

What is meant by inflammatory pre-conditioning and tolerance in stroke?

Answer 24

• Pre-conditioning uses a noxious stimulus near to but below threshold of damage, which grants resistance to the same or a similar subsequent stimulus beyond threshold of damage.
• Ischaemic pre-conditioning → (sub-threshold ischaemic challenge prior to experimental stroke) can be neuroprotective, reducing ischaemic damage, neuronal apoptosis and improving recovery.
• Inflamamtory pre-conditioning → Similarly to ischaemic pre-conditioning, toll-like receptor (TLR) ligands (potent stimulators of innate immune cells) administered systemically induce a state of tolerance to subsequent ischaemic injury. This action is most likely mediated through the hyporesponsiveness or re-programming of TLR-mediated signalling, suppressing pro-inflammatory and stimulating anti-inflammatory pathways.
− Needs to be administered early enough to avoid direct affect of the LPS on the infarct.
• Induction of immunological tolerance has a similarly protective effect. For example, T cells primed to a CNS antigen myelin basic protein prevent CNS autoimmunity and mediate improved outcome after experimental stroke (Gee et al., 2008). Mucosal tolerance to a vascular antigen, E-selectin, protects against white matter damage in a common carotid artery ligation model of hypertensive stroke-prone rats.

Question 25

How can we target the inflammatory response in cerebral ischaemia?

Answer 25

• As we have already seen, in rodent models of acute CI, administration of exogenous (intracerebroventricular or intraparenchymal) IL-1b exacerbates ischaemic injury, whilst inhibition of endogenous IL-1 with IL-1 receptor antagonist (IL-1RA) protects against ischaemic injury.
• Similarly, inhibition of IL-1b converting enzyme (or deletion of IL-1b and IL-1a results in markedly reduced ischaemic damage and neuronal death.
• Deficiency of inflammatory chemokines such as MCP- or fractalkine (CX3CL1) causes mice to be less susceptible to ischaemic injury. Deletion of fractalkine receptor (CX3CR1) not only reduces brain damage and neuronal apoptosis, but also results in blunted central IL-1b and TNFa expression after focal CI (Denes et al., 2008).
• Systemic administration of an anti-inflammatory tetracycline derivative, minocycline, is protective in experimental focal or global CI.

Question 26

What are the problems with anti-inflammatory intervention in stroke?

Answer 26

• The complexity of the systemic response to stroke requires us to consider various parameters, including timing, interactions of different pathways and long term effects of early interventions.
• It is not well understood why a rapid pro-inflammatory response is initiated in acute CI, given the very limited replacement of neurones and their high vulnerability to inflammatory injury in the brain.
• One aspect of the systematic view suggests that execution of a pro-inflammatory response is a prerequisite for vascular remodelling and wound healing. Following external carotid artery ligation in mice, macrophage depletion prevents flow-mediated inward vascular remodelling.
• Thus, similar processes appear to occur in peripheral and central inflammation, but their sustained effects on cell survival and recovery may be different in the brain and the periphery. This makes interpreting experimental data or finding selective targets for intervention difficult.
• The systemic pro-inflammatory response is also crucial in defence against pathogens after stroke. This is best emphasised by the profound immunosuppression induced in response to CI, both in patients and in experimental animals. These changes include splenic atrophy, reduced B- and T-cell counts and proliferation, and reduced pro-inflammatory protein expression by blood cells. The underlying mechanisms may be similar to those leading to systemic immunodepression after brain trauma.
• The extensive apoptotic loss of lymphocytes, and a shift from T helper cell (Th)1 to Th2 cytokine production result in the development of spontaneous septicaemia and pneumonia in mice after acute CI. Indeed, preventative antibacterial treatment improved the general medical and neurological outcome in a mouse model of stroke. Development of pneumonia is one of the most frequent complications after stroke and an important independent contributor to poor outcome in patients (Therefore dampening the general pro-inflammatory response as a treatment for stroke is not without risk.