Inflammation and Brain Repair

Question 1

Describe the difference between peripheral and CNS inflammation.

Answer 1

Periphery:
• In the periphery, when you have an acute injury, the aim of the inflammation is resolution – tissue repair and reestablishment of homeostasis
• There are some conditions that can become chronic if inflammation is uncontrolled – for some reason the response cant balance itself towards resolution.

CNS:
• If you have an acute injury, you have an acute inflammatory response but you have incomplete resolution – this is the reason you only get partial repair int eh cNS
• In chronic conditions, you have attempted repair (as seen in MS) – but you also have prolonged uncontrolled inflammation.

Emerging from this concept is that there is limited repair in the CNS after injury:

1. Most of the cells in the adult body are mature and terminally differentiated, but can dived to re-establish tissue homeostasis
2. Some organs like liver and skin quickly replenish dead cells
3. The CNS replenishes dead cells very inefficiently, and more neurological disorders are incurable.

Question 2

Describe the nervous system, immunity and regeneration during evolution.

Answer 2

• A simple organism, such as a sponge or jellyfish, has high regenerative capacity in the CNS – but they have low nervous and immune system complexity
− Just a few simple nerve rings that act as a CND
− Immunity is just of the innate arm – mostly phagocytic cells
• As you move through evolution, the nervous system and immune system become more complex
− Get the appearance of the neocortex and myelin, and presence of adaptive immunity
− However, these organisms have low capacity for CNS repair – eg, regeneration during the larval but not adult stage, or in the case of mammals, no CNS regeneration – just limited neurogenesis.

Question 3

What are the 4 mechanisms of brain repair?

Answer 3

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

Question 4

Describe the history of the discovery that neurogenesis occurs in the adult brain.

Answer 4

• 1913 ‘Ramon y Cajal’ → In adult centres, the nerve parhs are fixed, ended, immutable. Everything may die, nothing may be regenerated. It is for the science of the future to change, if possible, this harsh decree. It must work to moderate the gradual decay of neurons, to overcome the rigidity of their connections and to re-establish normal nerve paths.
• 1962 → Are new neurons formed in the brain of adult mammals? Science.
• 1992 ‘Reynolds and Weiss’ → Generation of neurons and astrocytes from isolated cells of the adult mammalian CNS. Science
− Plated out cells disaggregated from brains and cultured them in medium with EGP.
− Found that the cells expressed Nestin (associated with stem cells) and devolved into astrocytes and neurons
• 2000 → Neurogenesis in the adult brain: Death of a dogma. Nature Reviews Neuroscience

Question 5

How can we visualise neurogenesis?

Answer 5

• Use of radiolabelled bases, eg) 3H-thymidine or BrdU can be used to visualize cell replication
• This was the key finding in the 1962 paper – showed incorporation of radiolabelled bases in the granule cell layer of the dentate gyrus.

Question 6

Where does neurogenesis take place in the adult mouse brain?

Answer 6

1. Subgranular zone of the dentate gyrus
2. Posterior periventricular layer
3. Subventricular zone

Question 7

Describe neurogenesis in the sub granular zone of the dentate gyrus.

Answer 7

• Have proliferation of neuronal stem cells within the outer layer
• Newly formed differentiated cells migrate from the outer subgranular zon to the inner granule cell layer in. the dentate gyrus
• Here, neuronal fibres will integrate into a tissue

Question 8

Describe neurogenesis in the sub ventricular zone.

Answer 8

• In the SVZ, ependymal cells form a monolayer along the lateral ventricle with astrocytes, neuroblasts and transitory amplifying progenitors comprising the SVZ.
• The migration of the newly generated neuroblasts begins at the lateral ventricle, continues along the RMS and terminates in the olfactory bulb, where mature interneuron populations are generated.
• Astrocytes enseath the migrating neuroblasts along the RMS – restricting and containing them to their specific pathway
• Migrating neuroblasts enter the OB, migrate radially and give rise to granule or periglomerular cells → this process is thought to contribute to increased olfactory processes within the brain.

Question 9

Describe neurogenesisi following brain injury and repair.

Answer 9

• Following injury, neural progenitors from the SVZ leave the RMG and migrate laterally towards the damaged area.

How do they know where to go?

1. The injured brain secretes growth factors, cytokines, chemokines and mitogen. There are also endogenous signals from injured neurons (DAMPs)
2. These factors diffuse to the SVZ and induce progenitor migration
3. Newly formed cells migrate laterally from the SVZ to the damaged area
4. The increased proliferation and migration of neural progenitors continues for months after injury to the brain.

→ Isnt fast, takes months. Can correlate this with functional recovery of the patient.

Question 10

How can neurospheres be used to study neurogenesis, and what was found from this?

Answer 10

• Diaggregate cells from an embryonic mouse at day 16
• Can generate a neurosphere
• Neurospheres can be passaged – if NSCs present, they should be able to serially passage the neurospheres and replate at lower density.
− If you keep dissociating and replating, gives an exponential increase in the neurosphere population
− This suggests that there was a continually dividing cell present that could re-constitute the neurosphere
• Only neurospheres >2mm can be serially passaged –suggesting stem cells are the minority

What cells are present in the neurospheres?
• Neurons, glia, neuronal stem cells

Uses:
• Instead of using mice to generate the cell cultures, can grow these neurospheres as stem cells and passage indefinitely.
• Can introduce specific factors to the culture to induce a certain cell line
• Can also generate transgenic neurospheres, eg) IL-1B knockout.
• Really good for the 3Rs

IL-1B contribution to CNS renewal:
• Can see that with administration of IL-1B – there is much more BrdU incorporation – so appears to show that IL-1B drives neurogenesis in vitro
• But we have previously learnt that IL-1 is neurotoxic?
• Maybe there is a balance between its neurotoxic and neuroprotective effects (see later).

Question 11

What is angiogenesis/what are its beneficial effects?

Answer 11

• Biological process involving the growth of new blood vessels from pre-existing vessels
• Generally involved in wound-healing to restore blood supply to the injured tissue, or to sustain tumour development.
• Many factors are involved – chemokines, cytokines, integrins, ECM, growth factors
• Could be interpreted as a natural defense mechanism to help restore oxygen and nutrient supply to the affected brain tissue.
• Additonally, angiogenic vessels provide neurotrophic support to newly generated neurons.

Question 12

What are the mediators of angiogenesis?

Answer 12

• VEGF binding to VEGR2 is the most important for angiogenesis in repair
• Angiopoetin binding to Tie-2 is also important in regulating angiogenesis during inflammation
• VEGF binding to VEGFR1, Neuropilinns and ECM integrins are probably mostly important during embryonic vascular development.

Question 13

What changes occur in the growth factor mediators of angiogenesis after injury?

Answer 13

• Can see increased VEGF expression in the blood vessel in the ischaemic area compared to the non-ischamic area.
• Can see VEGFR2 expression following MCAO
• Endothelial cells are shown to be proliferating (using Ki67 marker of proliferation) in the infarct border 24 hours after MCAO

Extra reading:
• VEGF:
− the most important mitogen in the process of angiogenesis.
− The lack of a single VEGF allele shows already abnormal blood vessel development and leads to embryonic lethality
− Although there are multiple variants of both, the VEGF ligand and its receptor (see below), the angiogenic effects are mediated through VEGF with VEGF2
− VEGF is ubiquitously expressed in the normal brain, mainly by the choroid plexus, but also by astrocytes and neurons.
− Several groups described an upregulation of VEGF in the penumbra following experimental cerebral ischemia.
− VEGF protein induction could be demonstrated in astrocytes, neurons and microglia and also within microvessels in the peri-ischaemic region.
• VEGFR2:
− mediates the majority of the downstream angiogenic effects of VEGF
− Several groups described an upregulation of VEGFR-2 following experimental cerebral ischemia
− Lennmyr et al. showed an induction of VEGFR-2 in glial cells as well as endothelial cells in a rat model of cerebral ischemia.
− Induction of VEGFR-2 expression started 48 h after the insult in vessels at the border of the infarction. Later on also vessels invading the infarcted area became VEGFR-2 positive. Strong expression levels persisted up to 7 days
• Neuropilins:
− a rat model of focal cerebral ischemia, NP-1mRNA and protein were upregulated in endothelial cells of cerebral blood vessels at the border and in the core of the ischemic lesion 7 days after the ischemic insult.
− NP-1 gene expression persisted for at least 28 days after ischemia..
• Tie:
− Following cerebral ischemia microvessels in the ischemic lesion expressed Tie-1 as early as 2 h after the insult.
− Lin et al. [101] described a biphasic expression pattern of Tie-1 and Tie-2 following an ischemia-reperfusion model. A few hours after the insult they observed an upregulation of both receptors in capillaries inside the ischemic cortex. The second peak started 3 days after the insult and lasted up to 1 week post-MCAO.
• Angiopoetins:
− Ang-2 has been identified as the natural antagonist of Ang-1. It binds to Tie-2 without inducing signal transduction in Tie-2-expressing endothelial cells.
− In rat models of cerebral ischemia, Ang-2 levels increased during the first 24 h of ischemia in single cells localized in the infarct area and in the peri-infarct zone
− The majority of Ang-2 expressing cells could be identified as endothelial cells.
− Ang-2 expression was described to be increased up to 14 days [103] or even 28 days [188] following the ischemic insult.
− Angiopoietin-1 showed quite a different time course of induction - increased shortly after ischemia and found a massive increase 7–14 days after the insult, persisted up to 28 days.
• PDGF:
− originally believed to play its role following cerebral ischemia in neuroprotection.
− However, PDGF-B and its receptor PDGFR-beta are essential factors for the recruitment of pericytes to brain capillaries during embryonic development.
− Therefore, Renner et al. [127] suggested that the system may also support angiogenesis and vascular remodeling after cerebral ischemia by mediating interactions of endothelial cells with pericytes.
− Analysis of the expression pattern following experimental cerebral ischemia demonstrated that PDGFR-beta mRNA was specifically upregulated in vascular structures, mainly on pericytes, in the infarcted area 48 h after MCAO.
• Erythropoietin:
− Epo was originally thought to be exclusively produced in fetal liver and adult kidney.
− However, subsequent studies demonstrated that Epo mRNA is expressed in neurons in both rodent and primate brain, and its expression increases by hypoxia.
− Cerebral ischemia-induced Epo in endothelial cells located in the ischemic core 1 day after occlusion, the signal increased from 3 to 7 days.

Question 14

How does the ECM mediate the processes of angiogenesis?

Answer 14

• Afer injury, you get BBB leakage and ECM degradation. IL-1, VEGF and Ang1 production is triggered, and this leads to endothelial activation and proliferation
• This will cause some endothelial cells to grow out of the vessels
• Even though we have ECM degradation and disposition, we also have overexpression of the matrix
• The endothelial cells take advantage of this re-scaffolding of the matrix and use this as a scaffold to form new blood vessels
• This is a slow process – takes weeks/months to do
• When you get stabilisaton of the new blood vessels, you get re-attachment of the pericytes and astrocyte end-feet, giving a fully functioning blood vessel.

Question 15

How are neurogenesis and angiogenesis linked?

Answer 15

• In sites where you have neurogenesis, you need blood supply to drive this
• Angiognesis provides neurotrophic support for neurogenesis
• More neurogenesis therefore drives more angiogenesis, which in turn allows for more neurogenesis.

Question 16

Describe a role for endothelial progenitor cells in post-ischaemic angiogenesis.

Answer 16

• Neovascularisation was thought to exclusively result from angiogenesis (outgrowth of existing vessles)
• Later on, it became clear that EPCs contribute
• Circulating EPCs home ot sites of neovascularization and differentiate into endothelial cells in situ
• Bone-marrow derived EPCs have been shown to participate in cerebal neovascularization after FCI
• Several authors hypothesise that circulating EPCs might have prognostic value in stroke
• EPC levels have been found to be lower in patients with severe neurological impairment than in patients with less severe impairment

Question 17

What is the issue with therapeutic angiogenesis?

Answer 17

• Ischamie-induced angiogenesis can be boosted by a variety of agents
• However, almost all treatment strategies are not angiogenesis specific, but influence other post-ischaemic events too
• These side effects can either be detrimental (inflammation, vascular permeability) or beneficial (neurogenesis)
• The major problem is to accelerate cerebral angiogenesis without exacerbating brain edema in stroke patients
• Could use all the growth factors mentioned above.

Question 18

Describe the neuroprotective role of microglia in brain repair

Answer 18

Microglia are:
• The main resident immune cells of the brain
• Responseive to PAMPs and DAMPs (infection and injury) to trigger the inflammatory response
• Generally associated with neurotoxicity → if you remove them acutely, you limit brain damage

→ But this is in the acute phase. Research suggests that microglia can participate in brain repair.

Microglia contribute to CNS renewal:
• M1 and M2 phenotypes are well established in macrophages, less so in microglia, so called M1-like and M2-like.
• Following CNS injury, the microglia that are resting become activated by LPS, IFNy, TNFa etc… → primary activation
− In this acute phase, they secrete ROS, TNFa, IL-1, IL-6 and chemokines → this is neurotoxic
• However, microglia also have delayed activation by anti-inflammatory cytokines such as IL4, IL-13, IL-10, TGF-B
− In this recovery phase, the microglia produce anti-inflammatory cytokines and have a phagocytic phenotype
− They also produce neurotropic factors such as VEGF, PDGF and EGF → promotes angiogenesis and neurogenesis
− The end result is immune suppression and tissue regeneration and repair

→ The latest research therefore suggests that if you deplete the microglia later on, you get a worse outcome in patients.
→ You need to deplete them early on, and then restore function later on

Question 19

Describe the role of the glial scar in brain repair

Answer 19

• When you have necrotic cavitation, there is formation of a glial scar around the area
• The glial scar aims to form a barrier between the necrotic tissue and the healthy tissue

Is it beneficial or not?
• Some people say not, because it induces astrogliosis
• Astrogliosis (reactive astrocytosis) is an abnormal increase in the number of astrocytes due to the destruction of nearby neurons from CNS trauma, infection, ischamie, stroke, autoimmune responses and neurodegenerative disease.
• In healthy neural tissue, astrocytes play roles in energy provision, regulation of blood flow, homestasis of extracellular fluid and regulation of synapse function
• Astrogliosis changes the molecules expression and morphology of astrocytes, causing scar formation and in severe cases, inhibition of axon regeneration.

IL-1 induces astrogliosis:
• This then causes the production of MMP=9 and increased GFAP expression
• This is neurotoxic

Rolls et al, 2009: The bright side of glial scar in CNS repair:
• Every year more than 10,000 people in the United States alone become victims of spinal cord injury. Owing to the low regenerative capacity of the CNS, most such patients are left permanently paralyzed. For decades there was little hope for treatment. However, in the early 1990s neurologists began treating spinal cord injuries with steroids and other anti-inflammatory drugs. These drugs provided some hope, but they have a narrow therapeutic window and only a modest effect in the best of cases
• At approximately the same time, other research groups provided new grounds for optimism when they identified growth-inhibitory components in the injured CNS. Myelin-derived proteins such as nogo A (also known as RTN4), myelinassociated glycoprotein (MAG) and oligodendrocyte myelin glycoprotein (OMG) all inhibit axonal growth2–4. Subsequently, additional molecules were identified as growth inhibitors. These molecules have been associated with the glial scar that is actively formed following spinal cord injury.
• The glial scar consists predominately of reactive astrocytes, microglia/macrophages and extracellular matrix molecules, especially chondroitin sulfate proteoglycans (CSPGs). CSPGs are known mainly for their growth-inhibitory effects, and are secreted by almost all cell types at the injury site (especially astrocytes)7–9. The growth inhibitory effect of the scar tissue is considered to be a major obstacle for regeneration, and to partially explain the lack of effective CNS recovery.
• Various therapeutic approaches have attempted to eliminate and reorganize the chemical components of the glial scar or to regulate its negative effects. These include using degrading enzymes to eliminate scar components, blocking the activity of the growth inhibitors using specific antibodies14, blocking the receptors that recognize the growthinhibitory factors, inhibiting astrocyte proliferation to attenuate scar formation.
• Obviously, all of these approaches have been based on the perception that the glial scar is an obstacle to recovery that should be modified, eliminated, suppressed or circumvented. However, accumulating evidence indicates that scar tissue and its components might have an important role in the immediate response to CNS injury.

General features of the glial scar:
• Effects on axonal growth → Scar tissue, and especially CSPGs, is known for its inhibitory effect on axonal growth
• Sealing the site of injury and remodelling the tissue →
− In injured CNS tissue, neurons that were spared in the primary insult are exposed to a microenvironment that contains toxic factors, such as an imbalance in the levels of excitatory amino acids and ions, reactive oxygen species, free radicals and overwhelming inflammation.
− This results in further neuronal loss, a process known as secondary degeneration. In this degenerative environment, preventing the spread of toxicity requires the lesion site to be sealed and protective responses to be induced.
− We suggest that examining some of the features of the glial scar may reveal its potential to carry out part of this ‘SOS’ activity. After injury, astrocytes form a dense scar tissue that has been suggested to demarcate the lesion area and separate the injured tissue from its surroundings.
− An additional aspect of the glial scar that may be crucial for neuronal survival following injury relates to its activity in filling the gaps in the lesion area, creating a scaffold for the vascularization network.
− Astrocytes and matrix components stimulate and recruit endothelial cells and fibroblasts in the lesioned area and induce the formation of new capillaries at the site.
− Studies revealed that removal of astrocytes from the site of damage leads to larger lesions, local tissue disruption, severe demyelination and neuron and oligodendrocyte death.
• Temporal and spatial control of the local immune response →
− Numerous studies suggest that immune activity can accelerate tissue damage, and that in some cases the immune response itself is the cause of the initial injury.
− New lines of evidence indicate that the activity of the immune system in the CNS is more complicated than originally thought, and that neural tissue can benefit from the immune response if it is well regulated
− With the increasing understanding of immune activity in general, and specifically in the CNS, it has become clear that immune cells acquire diverse phenotypes following different types of stimulation. The phenotype acquired by immune cells and heir regulation (regardless of their phenotype) are crucial determinants of the functional outcomes of their activity. Thus, even immune cells that apparently have a beneficial and neuroprotective phenotype must be temporally and spatially restricted. Here, we suggest that the scar tissue can control the functional, temporal and spatial immune activity at sites of axonal injury.
• Controlling neurogenesis →
− Recently it became evident that CNS injuries trigger the proliferation of neural progenitors and stem cells. Increasing evidence indicates that the glial scar and its components are important players in this process.
− Both astrocytes and CSPGs have been associated with the regulation of neural progenitor cell proliferation and differentiation. Moreover, some data suggest that neural stem cells are actually a specialized type of astrocyte.
− Astrocytes have key roles in controlling multiple steps of adult neurogenesis, from proliferation and fate specification of neural progenitors to migration and integration of the neural progeny into pre-existing neuronal circuits in the adult brain

Reconciliation: timing and balance
• In our opinion the timing of scar generation and degradation are crucial in determining its effects. We suggest that the scar tissue is required in the acute phase after injury for sealing and cleaning the injury and restoring homeostasis. All of these processes are made possible by the unique features of the scar tissue.
• Consistent with this time-dependent view of the scar, application of xyloside on day 0, 2 and 7 after spinal cord injury resulted in destructive, beneficial or no effects, respectively67.
• Astrocytes in the acute phase after injury are crucial for recovery, whereas the presence of these cells in the chronic phase is inhibitory.
• None of these results contradict the fact that scar tissue inhibits growth and regeneration. What we suggest is that the growth inhibition itself might be beneficial, in a time-dependent manner. It is likely that growth inhibition in the early stages after the injury, rather than being an obstacle to recovery, is actually essential for the preservation of neurons that are capable of regrowth.
• Enabling axonal regrowth in the chaotic and metabolically unbalanced environment that is the injured CNS in its acute phase of recovery might cause more damage than benefit. Conversely, once homeostasis has been restored and balance achieved, recovery necessitates axonal regrowth and reconnection; this regrowth is inhibited by the scar tissue

Implications:
• Recognition of the beneficial aspects of the glial scar has major clinical implications. Most treatments that are currently being developed to eliminate scar formation in an attempt to support CNS recovery are designed for administration immediately after the injury.
• In our opinion, delaying the application of these treatments might allow the natural reparative properties of the scar to be manifested in the acute phase.
• We suggest that, at least during the first 24–48 hours following injury, the glial scar is crucial for recovery. Thus, treatments