Leonard:Stop it! You cannot blow up my head with your mind.Sheldon:Then I’ll settle for an aneurysm!The Big Bang Theory – S01E09

List of Papers

This thesis is based on the following papers, which are referred to in the text by their Roman numerals.

I Hånell A, Clausen F, Björk M, Jansson K, Philipson O, Nilsson

L, Hillered L, Weinreb P, Lee D, McIntosh T, Gimbel D, Strittmatter S and Marklund N. (2010) Genetic Deletion and Pharmacological Inhibition of Nogo-66 Receptor Impairs Cog-nitive Outcome after Traumatic Brain Injury in Mice. Journal of Neurotrauma, 27:1297–1309 (July).

II Hånell A, Clausen F, Nylund A, Vallstedt A, Israelsson C, Lar-hammar M, Björk M, Kullander K and Marklund N. Functional and Histological Outcome following Focal Traumatic Brain In-jury in Conditional EphA4-knockout mice. Submitted manu-script.

III Clausen F, Hånell A, Björk M, Hillered L, Mir A, Gram H and Marklund N. (2009) Neutralization of interleukin-1 modifies the inflammatory response and improves histological and cogni-tive outcome following traumatic brain injury in mice. Euro-pean Journal of Neuroscience, 30:385–396.

IV Clausen F, Hånell A, Israelsson C, Hedin J, Ebendal T, Mir A, Gram H and Marklund N. Neutralization of Interleukin-1 Re-duces Cerebral Edema and Tissue loss and Improves Late Cog-nitive Outcome Following Traumatic Brain Injury in Mice. Submitted manuscript.

Reprints were made with permission from the respective publishers.

Contents

Introduction .................................................................................................. 11Traumatic Brain Injury ........................................................................... 11Plasticity ................................................................................................. 13Inflammation ........................................................................................... 17Inflammatory Effects on Plasticity ......................................................... 20

Aims ............................................................................................................. 22Materials and Methods ................................................................................. 23

Animals ................................................................................................... 23Experimental Traumatic Brain Injury ..................................................... 23Histological Evaluation .......................................................................... 23Functional Evaluation ............................................................................. 25

Results .......................................................................................................... 28Paper I ..................................................................................................... 28Paper II .................................................................................................... 28Paper III .................................................................................................. 28Paper IV .................................................................................................. 29

Discussion .................................................................................................... 30Enhancing plasticity to treat TBI ............................................................ 30Modifying inflammation to treat TBI ..................................................... 31Behavioral testing ................................................................................... 32Why the unsuccessful clinical trials? ...................................................... 33

Conclusions .................................................................................................. 35Sammanfattning på svenska ......................................................................... 36Acknowledgements ...................................................................................... 37References .................................................................................................... 39

Abbreviations

APP Amyloid Precursor ProteinBBB Blood Brain Barrier cAMP Cyclic Adenosine MonophosphateCAPS Cryopyrin Associated Periodic SyndromeCCI Controlled Cortical ImpactCNS Central Nervous SystemCSF Cerebrospinal FluidCT Computerized TomographyDAMP Danger Associated Molecular PatternDRG Dorsal Root GanglioneGOS Extended Glasgow Outcome ScaleGAG GlucoseaminoglycansGCS Glasgow Coma ScaleGFAP Glial Fibrillary Acidic ProteinGOS Glasgow Outcome ScaleICP Intracranial PressureIFN InterferonIL InterleukinIL-1R Interleukin 1 ReceptorIL-1ra Interleukin 1 Receptor AntagonistIL-1RAcP Interleukin 1 Receptor Accessory ProteinL-FPI Lateral Fluid Percussion InjuryLGI1 Leucine Rich Glioma Inactivated 1LINGO LRR and Ig Domain Containing, Nogo- Receptor-Interacting ProteinMAG Myelin-associated GlycoproteinMAI Myelin-associated InhibitorsMHC Major Histocompatibility ComplexMRI Magnetic Resonance ImagingMWM Morris Water MazeNgR Nogo ReceptorNTR Neurotrophin ReceptorOMgp Oligodendrocyte Myelin GlycoproteinPAMP Pathogen Associated Molecular PatternPirB Paired-immunoglobulin like receptor-BPOSH Plenty of SH3:s

PRR Pattern Recognition ReceptorRLS Reaction Level ScaleSCI Spinal Cord InjuryTAJ Toxicity and Jnk InducerTBI Traumatic Brain InjuryTLR Toll-like Receptor

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Introduction

Traumatic Brain Injury

EpidemiologyThe annual incidence of traumatic brain injury (TBI) is estimated to between 100 and 250 per 100 000 in the western world1, 2. The major risk factors for TBI are age, gender and low socioeconomic status with young persons be-ing overrepresented in traffic accidents and the elderly in fall accidents. TBI often occurs in combination with ethanol intoxication3, and males are more likely to sustain a TBI, presumably because of a higher degree of risk taking behavior4. Other common causes for TBI include sports accidents, assault, abuse, and gunshot wounds. In recent years blast injuries, typically caused by roadside bombs, combat activities or terrorist attacks, have been recognized as a new form of TBI. Here the injury is caused by a pressure wave, rather than mechanical impact or head movement, and thus represents a new injury mechanism5.

Secondary Brain InjuryThe primary brain injury that occurs at the moment of impact can be pre-vented, for example by improved traffic safety, but not treated with medical care. The initial damage however initiates a sequence of events, some last-ing for minutes and other for days or years, which markedly exacerbates the initial injury. Since this occurs after the primary injury it may be attenuated with medical intervention. These effects were first noticed in patients who at admission had minor neurological deficits but later deteriorated and died, known as “talk and die cases”6. Several secondary brain injury mechanisms have since been identified and include edema formation, release of excitatory amino acids, formation of radical oxygen species, and inflammation7. The sec-ondary damage evolves after the initial injury, making medical intervention possible, and much TBI research has been focused on inhibition of secondary injury mechanisms. Secondary insults, such as episodes of hypotension, hy-poxia, fever and seizures, also exacerbate the brain injury and modern clinical

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care tries to limit their effects.

DiagnosisAssessment of the level of consciousness and neurological status, brain imag-ing and intracranial pressure (ICP) measurements are the most important meth-ods used to determine the type and severity of TBI. The level of consciousness and neurological status are usually expressed using the Glasgow Coma Scale (GCS)8, while some Swedish hospitals use the Reaction Level Scale (RLS)9. A neurological examination is informative but the use of computerized tomog-raphy (CT) and magnetic resonance imaging (MRI) has allowed a much more detailed evaluation of the location and type of brain injuries. ICP is a critical parameter for clinical decision making and can be continuously monitored us-ing surgically implanted pressure sensors in patients with severe TBI. Current research efforts try to find biomarkers in cerebrospinal fluid, plasma and the extracellular compartment of the brain which can give additional information about the injured brain10.

TreatmentWhen life-threatening extracranial injuries resulting in hypotension and hy-poxia have been stabilized one main focus of acute TBI treatment is to main-tain an adequate supply of oxygen and nutrients to the brain. This is done by securing a sufficient cerebral blood flow, usually achieved by controlling ICP, and/or by reducing brain metabolism. Strategies to control ICP include head elevation, hyperventilation, and sedation using propofol, midazolam or barbiturates11, 12. In the emergency situation, mannitol may rapidly lower ICP by reducing edema. In the most severely injured patients with uncontrollable ICP, surgical evacuation of focal contusions or a hemicraniectomy can be per-formed. Reducing the extent of secondary insults is important and fever is treated with antipyretics13 and seizures with anti-epileptic drugs. Despite a large research effort and several clinical trials there is, unfortunately, no drug available for clinical use which specifically targets secondary brain injury mechanisms and improves outcome.

OutcomeOutcome is usually assessed using the Glasgow Outcome Scale (GOS)14 or the extended version (eGOS) and is mainly determined by the severity of the brain injury while high age is recognized as an important, negative, prognostic factor15. Women have been suggested to fare better following TBI, supported by the improved outcome following progesterone treatment in animal mod-els, but this remains controversial. The introduction of standardized treatment guidelines and specialized neurointensive care units has markedly improved

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the outcome following TBI16, 17, and a limited recovery of function is usually seen in the first months post-injury. Still most survivors suffer from lifelong detrimental effects of the injury such as depression, memory problems, per-sonality changes, epilepsy and fatigue.

PlasticityIn this thesis plasticity refers to the brains ability to adapt to new circum-stances and injury18 but sometimes the term has a more narrow use19 and the origin of the word has been discussed20, 21. The brain has a remarkable capacity to adapt to changes in circumstances, for example can persons who lose one sense augment others and blind persons reading Braille rely on the occipital cortex which is normally used for vision22, 23.

On the cellular and molecular level plastic changes can be caused by several mechanisms. Synapses can be strengthened or reduced and new ones can be formed. Formation of new axons or dendrites on short distances is usually referred to as sprouting while long distance growth is termed axonal regenera-tion. These changes results in altered connections between neurons and can result in a relocation of function between some areas of the brain.

According to the Kennard principle immature nervous systems recover better following injury compared to mature ones24, 25 and phylogenetically less de-veloped organisms lacking myelin recover better from central nervous system (CNS) injury. It is not known if adult humans benefit from this decreased plasticity but it probably has some function. It might improve our nervous system in a way we are not aware of or it could be a trade off for other advan-tages, such as more efficient action potential transmission following myelina-tion. Accordingly, increasing plasticity could be beneficial following injury but might also be detrimental, and lack of Nogo receptor 1 (NgR1) signaling, which may increase plasticity, has been implicated to cause schizophrenia-like symptoms26, 27.

In the injured CNS plasticity is reduced by the formation of a glial scar, my-elin-associated inhibitors (MAI), inhibitory axon guidance cues, lack of neu-rotrophic factors and a reduced intrinsic growth capacity in neurons28, 29.

The Glial ScarFollowing CNS injury a glial scar made up of reactive astrocytes, activated microglia and extracellular matrix components is formed. This is believed to be beneficial by sealing off damaged areas of the brain but might limit re-covery by inhibiting axonal growth29. Most of the extracellular matrix com-

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ponents are chondroitin sulphate proteoglycans which can be broken down using chondroitinase ABC. This treatment improves outcome following ex-perimental SCI30 and cause a limited increase in functional recovery in TBI models31. The cause may be increased plasticity or the growth promoting ef-fects of soluble glucoseaminoglycan (GAG) chains32, 33, which are cleaved off from chondroitin sulphate proteoglycans. Preventing the glial scar formation by ablating reactive astrocytes impairs outcome following experimental TBI, indicating that the glial scar is overall beneficial34. The boundary created by the glial scar limits the movement of excitatory amino acids, free radicals and immune cells which is valuable early in the disease process while the inhibition of plasticity likely is a negative factor after the acute phase. When attempting to reduce glial scarring timing is therefore important and treatment should probably begin after the acute phase29.

Myelin-associated InhibitorsIt has been known for a long time that axonal regeneration in the injured pe-ripheral nervous system is efficient in contrast to the CNS35. The reasons for this discrepancy began to be unraveled by the finding that CNS axons can regenerate through a bridge of peripheral myelin36, 37. It was later found that CNS myelin contains inhibitory factors, which cause axonal growth cone col-lapse, and that blocking them can promote axonal regeneration38-40. Several of these factors, the MAI:s, have now been identified and some evaluated as potential drug targets41 (Fig 1).

Nogo exists in three different splice forms named Nogo-A, Nogo-B and No-go-C and is also known as reticulon 4. Nogo contain two sequences which cause growth cone collapse, Nogo-66 by binding to NgR142 and amino-Nogo via binding to integrins43. Administration of anti-Nogo antibodies improves outcome in experimental models of spinal cord injury (SCI)40 and stroke44 and these are now being evaluated in a clinical trial for SCI (www.clinical-trials.gov; NCT00406016). When administration of an anti-Nogo antibody was initiated 24 hours following experimental TBI cognitive outcome was improved45, 46 but on the other hand aged Nogo A/B knockout mice have im-paired outcome following TBI in mice47. A detrimental effect of Nogo inhibi-tion early following TBI might explain the discrepancy48.

Myelin-associated glycoprotein (MAG) exists in two splice variants, small MAG (S-MAG) and large MAG (L-MAG), and is typically attached to the in-nermost myelin sheath49. MAG binds NgR1, NgR2 and gangliosides and the interaction with NgR1 causes growth cone collapse50, 51 in the adult CNS but not during development. MAG directed antibodies improve function follow-ing experimental TBI52 and stroke53.

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Oligodendrocyte-myelin glycoprotein (OMgp) is expressed in myelin and when it interacts with NgR1 it causes growth cone collapse54. OMgp was pre-viously thought to be expressed at the Nodes of Ranvier and inhibit nodal sprouting, but this result has been questioned55, 56. The expression of OMgp is increased following experimental SCI57 and OMgp null mice on mixed 129/BL6 background have improved outcome following SCI, even though this is not the case for mice on a BL/6 background58. Inhibition of OMgp has not yet been evaluated in models of brain trauma.

Leucine Rich Glioma Inactivated 1 (LGI1) is a secreted endogenous inhibitor of NgR1 and promotes axonal growth on myelin substrates59. Mutations in LGI1 which stop the secretion of the protein can cause epilepsia60 and so far the use of LGI1 as a drug target for CNS trauma has not been evaluated.

Nogo receptor (NgR) exists in three iso-forms, NgR1, NgR2 and NgR3, coded by three different genes, where NgR1 is often referred to as the Nogo-66 re-ceptor. NgR1 binds Nogo, MAG and OMgp while NgR2 only binds MAG and NgR3 none of the three42. The fact that NgR1 binds three structurally very different ligands, all inhibiting axonal regeneration, makes it a logical target for CNS injury therapies. Inhibiting NgR1 have improved outcome following both SCI61 and stroke62 but lack of NgR1 can also influence normal memory function, synapse structure and cause schizophrenia-like behavior63. NgR1 also binds to amyloid precursor protein (APP) and treatment with soluble NgR reduces Aβ levels and improves outcome in a model of Alzheimers disease64,

65. The effect of interfering with NgR1 signaling was evaluated in Paper I of this thesis.

LRR and Ig Domain Containing, Nogo Receptor-Interacting Protein (Lingo-1) is a co-receptor for NgR166 and is important in the myelination process67. In-

NgR1 NgR2 NgR3PirB

Nogo-66Amino-Nogo MAG OMgp

LGI1

LingoTroyp75NTR

Integrins SialicAcid

Figure 1. Signaling relevant to myelin inhibition. Myelin-associated glycoprotein (MAG), Oligodendrocyte Myelin glycoprotein (OMgp), Paired-immunoglobulin like receptor-B (PirB), Nogo receptor (NgR).

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hibiting Lingo-1 function resulted in improved outcome in models of multiple sclerosis68 and SCI69 but has so far not been evaluated in experimental TBI.

p75NTR and Troy, also known as Toxicity and Jnk Inducer (TAJ), are both co-receptors for NgR1 but have not been evaluated as a drug target following CNS disease or injury. Inhibition of p75NTR may also reduce axonal growth since it functions as a neurotrophin receptor.

Paired-immunoglobulin like receptor-B (PirB) is a receptor for Nogo, MAG and OMgp which is structurally unrelated to NgR170. Similar to NgR1 it regu-lates ocular dominance plasticity and axon growth on myelin substrates71 and PirB knockout mice have been tested in a TBI model but the recovery of mo-tor function was not improved72.

Lynx1 resembles snake venom toxins and binds to acetyl choline receptors73. It was recently discovered that Lynx1 regulates ocular dominance plasticity in a way similar to NgR and PirB74 which makes it potentially interesting as a drug target for TBI.

Intracellular signaling downstream of NgR is beginning to be unraveled. It involves cAMP, kalirin9, POSH, Rock and Rho and the end result is modi-fication of the cytoskeleton, growth cone collapse and termination of axon growth75-77.

Axon Guidance CuesA number of receptor/ligand pairs exist which guide the growing axons of the developing nervous system to their correct targets by attracting or repuls-ing the growth cone. These include the secreted slit proteins and their Robo receptors, the ephrins and their eph-receptors, the netrins which bind to UNC and DCC receptors and the semaphorins which interacts with plexins78. These proteins are mainly expressed during development but some maintain their expression in the adult brain and inhibitory axon guidance cues might limit plasticity following trauma79. For example EphB3 is expressed in myelin in the adult brain and activation results in growth cone collapse80 and interfering with EphA4 function is beneficial following SCI81. The effect of conditional ephA4 knockout in mice subjected to experimental TBI was evaluated in Pa-per II.

Intrinsic Regenerative CapacityCNS axons can regenerate in through myelin from the peripheral nervous sys-tem, demonstrating that they have an intrinsic regenerative capacity82. Dor-sal root ganglion (DRG) cells send axons both to the peripheral and central

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nervous systems and if both axons of a DRG cell are cut simultaneously the peripheral axon will regenerate but not the one in the CNS. However, if the peripheral axon is cut first, and then the central, both will regenerate83. The reason for this might be the increase in intrinsic regenerative capacity caused by the rise of intracellular cAMP following the initial lesion84. Accordingly a similar increase in regeneration is observed following cAMP elevation using rolipram85.

InflammationIt was previously believed that inflammation in the CNS was not possible because of the lack of resident immune cells and the impenetrable blood-brain barrier (BBB)86, 87. It is now known that microglia function as immune cells and that several types of immune cells can cross the BBB. The involvement of the immune system in CNS diseases is well established for multiple sclerosis88 and has been implicated in narcolepsy89, Alzheimer’s disease and Parkinson’s disease87. There are two ways in which the immune system can cause dam-age to the host. Surrounding tissue can be damaged when the immune system performs a functional task, for example the release of toxic substances to at-tack bacteria or parasites. The host can also sustain damage if the immune system mistakes endogenous or harmless proteins for pathogens and initiates an immune response against them, which occurs in autoimmune diseases and allergy90.

Initiation of Inflammation after TBIAn inflammatory response occurs even after a closed head injury when no pathogens are present. This is due to events early following TBI that can trig-ger an immune response even in this sterile environment. Damage to the en-dothelium of blood vessels is a known pathway for initiation of inflammation and the tearing of cell walls and the leakage of intracellular proteins to the ex-tracellular environment is also likely to be immunogenic. The innate immune system relies on pattern recognition receptors (PRR) to identify pathogens. In the CNS these are primarily expressed on microglia but also on astrocytes. Binding of endogenous proteins to PRR and the ensuing activation of microg-lia has been suggested as one of the first steps in the inflammatory cascade. PRR recognize pathogen associated molecular patterns (PAMPs) during in-fections but are believed to recognize danger associated molecular patterns (DAMPs)87 during brain trauma.

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ComplementThe complement system is a group of plasma proteins that can enhance phago-cytosis and kill bacterial cells by creating pores in their plasma membrane. Components of the complement system are normally not present in the CNS but can pass the damaged BBB following TBI and potentially cause damage91,

92. Interfering with the regulation of complement activation worsen outcome after experimental TBI93 while inhibiting activation improves outcome94-96.

NeutrophilsNeutrophils are phagocytic cells that exist in large numbers in normal plasma that respond rapidly to tissue injury and infection. When activated they can release reactive oxygen species, proinflammatory cytokines and the cytotox-ic compounds stored in their granules97, 98. This is functional when targeting rapidly dividing bacteria but potentially detrimental at a site of sterile tissue injury. During inflammation endothelial cells express receptors which interact with neutrophils and make them roll along the endothelium, adhere to it, and finally transmigrate into the tissue97. Following TBI infiltration of neutrophils was associated with BBB breakdown99 but this has been disputed100. Follow-ing TBI neutrophils responds rapidly and reach their maximum number in brain tissue after one or two days99, 101. Mice lacking some mediators of neu-trophil infiltration have reduced brain edema but neutrophil infiltration and functional outcome was not altered102.

MicrogliaMicroglia in the healthy brain have extended processes and are called ramified microglia but following infection or brain trauma they are activated, adopt a more globular shape, and are referred to as ameboid microglia103. Microglia are thought to function primarily as immune cells but their involvement in ex-perimental obsessive compulsive disorder suggests that they might have other functions as well104. The processses of microglia in the healthy brain continu-ously monitor the local environment105 and they can be activated via Toll-like receptor 2 (TLR2) and TLR4106 which requires the presence of interleukin-1β (IL-1β)107.

MacrophagesWhen monocytes from the bloodstream enter peripheral tissue they transform into macrophages which are the primary phagocytes of the immune system. Some tissues have their own macrophage like cells, like the Kupfer cells of the liver, and activated microglia can be considered to be tissue macrophages of the CNS108. Macrophages can be divided into interferon-γ (IFN-γ) activat-ed pro-inflammatory macrophages (M1) and pro-regenerative macrophages

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(M2) activated in the presence of IL-4 and and IL-13109, but the amount and effect of each subtype following TBI is not known. Since activated microglia resemble monocyte-derived macrophages it is not known whether the increase in microglia/macrophages result from proliferation and activation of resident microglia or from infiltration of cells from the blood stream110.

Adaptive ImmunityThe cytotoxic T-cells (TC) and B-cells of the adaptive immunity directs their actions against a single specific antigen while the T-helper (TH) and regulatory T-cells (TReg) controls the degree and type of the inflammatory response111-113. The number of T-cells peak between one114 and five115 days following TBI. B-cells secreting auto reactive antibodies are potentially harmful even when outside the brain116 and mice unable to produce antibodies have improved out-come following SCI117. Autoreactive antibodies directed against β-tubulin118, neurons and basal lamina119 have been detected following experimental TBI.

IL-1R1

IL-1β

IL-1α

IL-1RAcP IL-1R2

Canakinumaband rilonacept

IL-1ra(anakinra)

Cellular response No effectFigure 2. Members of the Interleukin 1 family. Interleukin (IL), interleukin-1recep-tor (IL-1R), IL-1 receptor antagonist (IL-1ra), IL-1 receptor accessory protein (IL-1RAcP).

The IL-1 familyIL-1 is recognized as an important regulator of the immune system and two subtypes exists, IL-1α and IL-1β, where IL-1α is usually associated to the cell membrane while IL-1β is secreted. IL-1 is expressed as a pro-form and is activated by protease cleaving at an activated inflammasome120. The acti-vated form bind to the IL-1 receptor type 1 (IL-1R1) which exert the biologi-cal effect after it combines with IL-1 receptor accessory protein (IL-1RAcP). The effect of IL-1 is limited by the decoy receptor IL-1R2 which binds IL-1

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without causing a cellular response. The effects of IL-1β can also be limited by IL-1 receptor antagonist (IL-1ra), an endogenous antagonist to IL-1R1121 (Fig 2).

The levels of IL-1β increase following TBI in both rodents122 and humans123,

124 and IL-1β is considered to be detrimental since it induces fever and upregu-lates the pro-inflammatory cytokine IL-6 as well as the nitric oxide-producing enzyme iNOS125. Administration of IL-1β impairs outcome126 while increased levels of IL-1ra improves outcome127-129 following experimental TBI, support-ing the negative effects of IL-1β. The effect of an IL-1β directed antibody following CCI in the mouse was evaluated in Papers III and IV.

Given the central role of IL-1β in inflammation, drugs which inhibit it are interesting for to the treatment of several diseases, and so far three drugs have been approved for clinical use. Canakinumab (Ilaris) is a humanized monoclonal antibody directed against IL-1β which is approved for treatment of Cryopyrin Associated Periodic Syndrome (CAPS)130. Anakinra (Kineret) consist of recombinantely produced IL-1ra and is approved for treatment of rheumatoid arthritis in patients not responding to standard treatment. Rilona-cept (Arcalyst) is a fusion protein consisting of IL-1RI, IL-1RAcP and the Fc part of IgG, and is approved for the treatment of CAPS131. Canakinumab has a half-life of 26 days, considerably longer than the 4-6 hours of anakinra130 and approximately one week of rilonacept131.

Inflammatory Effects on PlasticityThe immune system is responsible for eradicating pathogens and clearing cel-lular debris but is also important for scar formation and healing of the dam-aged tissue. Several proteins normally associated with the immune system are also important for plasticity and growth of axons and some immune cells secrete factors that promote plasticity. The immune response is considered to be both detrimental and beneficial following TBI with some of the benefit probably due to increased tissue regeneration and plasticity.

PirB was first recognized as a part of the immune system132 and only recently identified as a mediator of ocular dominance plasticity71 and growth cone col-lapse70. The possible effects of PirB having a role in both the immune system and axonal growth have not been investigated in models of CNS inflammation or injury. MHC class 1 is another well known member of the immune system, important for the adaptive immune response against viruses and intracellular bacteria. Surprisingly it is also involved in plasticity and the regulation of ocular dominance plasticity133.

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M2 macrophages can promote tissue repair109 and shifting macrophage, and perhaps microglial, differentiation towards the M2 phenotype might be ben-eficial following CNS injury. Inflammation is also known to stimulate regen-eration of the optic nerve, in part mediated by macrophage derived protein oncomodulin134, 135.

NgR1 is well established as a mediator of growth cone collapse but is also ex-pressed on macrophages136. This might ensure that macrophages are repulsed from healthy, myelinated, axons137 but might impede debris clearance in the CNS, where Wallerian degeneration notably is very slow138, 139.

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Aims

The aim of this thesis is to improve the understanding of plasticity and inflam-mation following TBI and how to modify them with the long term goal of finding drugs that limit secondary damage or promote recovery. No such drug exists today and if one is found it would be a huge benefit to TBI patients.

The aim of Paper I was to determine the effect of genetic absence and phar-macological inhibition of NgR1 following Controlled Cortical Impact (CCI) in mice and evaluate outcome using Morris Water Maze (MWM), the cylinder test, rotarod, tissue loss, Timm stain and Aβ immunostaining. Based on the positive results in experimental stroke and SCI the hypothesis was that inter-fering with NgR signaling would be beneficial.

The aim of Paper II was to determine the effect of a conditional knock out of ephA4 in mice subjected to CCI and evaluate outcome using MWM, the cyl-inder test, rotarod, tissue loss, Timm stain and Glial Fibrillary Acidic Protein (GFAP) immunostaining. Since EphA4 is a repulsive guidance cue expressed in the adult CNS and knockout of ephA4 is beneficial following SCI an im-proved outcome was expected.

The aim of Paper III was to determine the effect of intracerebroventricular ad-ministration of an IL-1β directed antibody following CCI in mice and evaluate outcome using MWM, the cylinder test, rotarod, tissue loss and immunostain-ings for microglia, T-cells and neutrophils. Treatment with IL-1ra is beneficial following brain injury and a similar result using antibody mediated inhibition of IL-1β was expected.

The aim of Paper IV was to evaluate the clinically more relevant intraperito-neal administration route for the antibody used in Paper III utilizing a similar study design with the addition of edema formation evaluation. The hypothesis was that the drug would reach the injured brain, reduce edema formation and improve cognitive outcome.

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Materials and Methods

AnimalsAdult C57/BL6 mice were used in the thesis, in Paper I with a NgR knockout and in Paper II with a conditional ephA4 knockout. Male mice were used in all studies except for the genetically modified mice which were both male and female.

Experimental Traumatic Brain InjuryCCI is a mainly focal model of TBI widely used in both rats140 and mice141. A craniectomy is performed on the anaesthetized animal and a pneumatically driven metal piston strikes the exposed dura mater. The bone flap was reat-tached using tissue adhesive in Papers II-IV as the procedure otherwise would mimic a decompressive craniectomy142-144. The impact speed, penetration depth and diameter of the metal piston can be varied to control the severity of the impact. The injury causes a short apnea and results in the formation of a cerebrospinal fluid (CSF) filled cavity in the impacted cortex and cell loss in the underlying hippocampus145 and impairs motor and cognitive function.

Histological EvaluationImmunostaining relies on antibody binding to a specific protein and subse-quent visualization using fluorescence or an enzymatic reaction. Background signals from endogenous enzymes and fluorescent molecules as well as incor-rect antibody binding can cause erroneous results. This has to be controlled for by relevant control experiments such as omission of the primary antibody, identical results from two or more antibodies, staining sections from knockout animals, staining sections where the target protein is known to be present and verify that the sub cellular location is correct if it is previously known146-150. Performing adequate controls is very important and failure to do so can lead to erroneous results55, 56. Immunostaining for GFAP was performed in Paper II and evaluated using densitometry. In Paper III the number of activated mi-

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croglia, neutrophils and macrophages, and in Paper IV the number of acti-vated microglia, were evaluated using immunostainings and cell counting.

Lesion volume determination can be used both as a measure of the injury level and as an indicator of a neuroprotective drug effect and was used in Papers I-IV. Several sections, usually five to ten, obtained at a regular interval throughout damaged area are stained with hematoxylin and eosin (Fig 3). The areas of healthy and lost tissue are then measured and the corresponding vol-umes calculated. To correct for methodological errors the contralateral hemi-sphere is usually used as a control151.

The Timm stain detects the zink-ions in the terminal area of the dentate gran-ule cells152 and was used in Papers I and II (Fig 4). This staning is widely used to detect mossy fibers sprouting which is believed to be important in epi-lepsia153. Following both CCI154 and lateral fluid percussion injury (L-FPI)155 sprouting of mossy fibers has been detected but not to the same extent as in models of epilepsia.

Edema can be measured using the dry weight/wet weight method where a tissue sample is weighed, dried and weighed again. The water content is cal-culated as (wet weight-dry weight)/wet weight.

Figure 3. Hematoxylin and eosin stained coronal sections from different bregma levels used to determine the amount of tissue loss following the controlled cortical impact injury model.

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Functional EvaluationMWM is a test to assess learning and memory in rodents, developed by Rich-ard Morris in 1984156 and was used in Papers I-IV. The test is performed by placing the animal in a pool of water containing a platform hidden just below the surface. Animals that fail to locate the platform are guided to it and by repeating the process the animal will eventually learn the location of the plat-form157. After the animals have learned the location of the platform a probe trial is usually performed where the platform has been removed. The main function of the probe trial is to distinguish between animals that are using a search strategy and the ones that rely on the provided spatial cues. If there’s a time lag between the acquisition trials and the probe trial it is also possible to distinguish between learning/short term memory and long term memory. To verify that the animals’ visual capacity is intact and that they are trying to find the platform a trial with the platform made visible is performed. The MWM gives reliable information and is widely used following experimental TBI us-ing both rats158 and mice159.

The rotarod test is used to assess motor function in both rats160 and mice161 and was used in Papers I-IV (Fig 5). It is performed by placing the animal on a rotating rod and the animal is forced to run on the rod to avoid falling down. The rotation of the rod can be set to different constant speeds or to accelerate. Motivation to run is provided by the fear of falling which can vary consider-able between individuals and results are often presented as percentage of pre-

Sham

Veh

icle

Trau

ma

Vehi

cle

Figure 4. The Timm stain reveals a band of black granules formed after experimen-tal TBI indicating mossy fiber sprouting. This can be seen in the higher magnifica-tion images. From Paper I.

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injury performance. The rotarod is widely used in TBI research and has been suggested to more sensitive than the beam balance and beam walk tests162, 163.

Neuroscore is a test of neurological function used for both rats164 and mice165,

166 which consists of several different parts. Forelimb and hindlimb flexion is evaluated when the animal is suspended by the tail. Lateral pulsion is per-formed by having one hand on each side of the animal and pushing it from side to side, healthy animals will try to resist the push. Circling behavior and ability to stand on an inclined plane can also be included164. The performance in each test is evaluated on a scale and the sum of the subscores is the main result.

Figure 5. The rotarod test, the mice have to run to remain on the rotating rod. Latency to fall is the primary endpoint of the test.

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The cylinder test measures asymmetries in forelimb use and is used in several models of CNS disease167 (Fig 6). It is performed by placing the animal in a glass cylinder where it spontaneously will rear and use the forepaws as support against the cylinder wall. Ideally, two mirrors are placed at an angle behind the cylinder to allow viewing from three sides. The experiment is filmed to allow evaluation in slow motion and give the possibility to re-examine rears. Evaluation is done by scoring a rear or a single frame of the film as normal, left forelimb impairment or right forelimb impairment167, 168. The test can be used in any experimental model where there is a unilateral injury to brain regions used for motor control such as stroke, spinal cord injury, Parkinson’s disease and TBI.

Figure 6. A mouse in the cylinder test, viewed from three angles with the help of mirrors. The number in the top left corner allows the evaluator to be blinded with regard not only to injury status and treatment but also to time after injury.

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Results

Paper IThe promising results previously obtained when testing NgR1 inhibition in models of SCI and stroke was the rationale for testing NgR1 inhibition in TBI. Both NgR1 knockout mice and mice treated with intracerebroventricularly injected soluble NgR1, which blocks the interaction between NgR1 and its ligands, were subjected to the CCI TBI model and evaluated with histological and behavioral tests. Motor function was evaluated using the cylinder test, rotarod and neuroscore without revealing a treatment effect. Cognitive func-tion was measured in the MWM, and using both the genetic knockout and pharmacological inhibition learning and memory was unexpectedly impaired. Brain trauma caused sprouting of mossy fibers and tissue loss but this was not affected by the treatment. A link between NgR1 and Aβ formation has been reported64, 65 but no Aβ was detected in this TBI model.

Paper IITargeting EphA4 have resulted in improved outcome following SCI and re-duced glial scarring81. Conventional knockouts of EphA4 have a hopping gait that makes evaluation of motor function difficult and therefore a conditional knockout model was used where EphA4 is absent from embryonic day 16. Mice were subjected to CCI and their motor behavior and cognitive function were evaluated but no differences were found between brain-injured wildtype controls and cKO mice. The extent of the glial scar was measured with im-munostaining for GFAP but no effect of genotype was detected. EphA4 is a repulsive axon guidance cue and the effect on mossy fiber sprouting was ex-amined using Timm staining without revealing any effect of EphA4 absence.

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Paper IIIIL-1β is one of the central regulators of the immune response and previous studies have shown that it is involved in the immune response following TBI. An antibody to IL-1β was infused into the contralateral ventricle using os-motic minipumps implanted following the CCI injury. Cognitive function was measured using the MWM two weeks post-injury and found to be improved by this treatment. Motor function was evaluated in the cylinder test and ro-tarod but not affected by treatment. The loss of tissue and the number of ac-tivated microglia, neutrophils and T-cells was reduced in anti-IL-1β treated animals providing a possible mechanism for the improved cognition.

Paper IVDrug administration via the intracerebroventricular route is not clinically fea-sible and this study was done to examine if the anti-IL1β antibody would have an effect when administered outside the BBB. The drug was administrered intraperitoneally 30 minutes after CCI or sham injury and verified to reach the CNS. Cognitive function was evaluated at both an early, 48 hours, and a late, 19 days, following injury and was improved at the late timepoint but not the early. Similar to Paper III, no effect on motor function, evaluated in the cylin-der test and rotarod, was detected but both tissue loss and the number of acti-vated microglia were reduced. Edema, one of the main reasons for increased ICP, was examined using the dry weight/wet weight method and found to be reduced following anti-IL1β antibody treatment.

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Discussion

Enhancing plasticity to treat TBIResearch on increased plasticity as a way to treat TBI is still in its infancy and it is still uncertain if it will result in improved clinical practice. Should it work it will on the other hand be a way to restore lost function while other methods focus on reducing loss of function via neuroprotection. Initiation of treatment in the acute phase may not be required, and might even be detrimental for this type of drugs. This facilitates treatment initiation within the therapeutic window, something which can be a problem for neuroprotective treatments.

In Paper I both genetic deletion and pharmacological inhibition of NgR1 was evaluated using CCI in mice. Given the positive results in SCI61 and stroke62 models the impaired cognitive outcome was unexpected. Cognitive outcome has unfortunately not been evaluated following NgR1 inhibition in stroke models which makes comparison difficult. No effect on recovery of motor function was found in Paper I, in contrast to the results in stroke models. It is possible that recovery was improved with our treatments but not detected with the motor tests used. Even though stroke and TBI have similarities they differ on several points169 which may explain the discrepancy. Inhibition of both MAG52 and Nogo45 is beneficial following experimental TBI and it is unclear why NgR1 inhibition was detrimental. Timing might be important and NgR1 inhibition following TBI could be beneficial if there is a delay between injury and treatment initiation. Even though mossy fiber sprouting was not increased, it is possible that the inhibition of NgR1, which also results in in-hibition of Nogo, MAG and OMgp, results in excessive or aberrant plasticity in other areas of the brain.

In Paper II the effect of conditional EphA4 knockout in mice subjected to the CCI injury was evaluated. The role of EphA4 has previously not been evalu-ated in TBI but improves outcome and reduces the glial scar in SCI models81. In Paper II no effect was however found on cognitive outcome, motor function or the extent of the glial scar. The brain and spinal cord are both part of the CNS but differ in several aspects which might explain the disparate results. Since conventional EphA4 knockout mice adopt a hopping gate a conditional

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knockout model was used in Paper II. The lack of effect in this model could be due to residual EphA4 expression or compensatory upregulation of other members of the ephrin family and pharmacological inhibition of EphA4 might give a different result.

Several potential targets for drug development have not been evaluated in experimental TBI models, including Troy and OMgp, and for PirB only mo-tor function has been evaluated72. For most other possible targets only limited investigations have been performed, often just a single study. Accordingly, a substantial amount of additional research is required before a reliable conclu-sion about the usefulness of plasticity-enhancing treatments for TBI can be made.

Interfering with the brain’s natural restriction of plasticity can potentially cause serious side effects. Some experimental results support this view, for example, NgR knockout has been implicated to cause schizophrenia-like symptoms27, loss of LGI1 function can cause epilepsy170, 171 and blocking EphA4 upregula-tion might cause peripheral pain following SCI172. By including behavioral tests to detect these problems already in the preclinical phase they may be avoided in clinical trials.

Modifying inflammation to treat TBIThe immune system contains several cell types regulated by a multitude of interleukins, chemokines and other signaling molecules. In the body this com-plexity is only superseded by the brain and the details of immune signaling is only beginning to be understood. The immune response following TBI is incompletely understood but mainly consists of activation of microglia and infiltration of neutrophils and T-cells. The effort to identify beneficial and det-rimental processes and establishing when in the disease process they should be targeted has to be continued.

The effect of an IL-1β directed antibody was evaluated in Paper III and Pa-per IV. Previous work interfering with IL-1β signaling improved outcome173 and administration of IL-1β worsen outcome in experimental TBI126. These results were confirmed in Paper III and the possibility to use a clinically more relevant administration route was evaluated in Paper IV. The tissue sparing effects of IL-1β inhibition are well established while the improvement of late functional outcome has to be verified in other species and models. The thera-peutic window and the optimal duration of IL-1β inhibition also remain to be established. In this context, the long half-life of canakinumab, 26 days, can be practical for long treatment times but may be a major limitation if IL-1β inhibition is not desired late in the injury process.

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The state of the immune response is reflected in the cytokine environment and the application of methods that simultaneously measures a large amount of different peptides would be useful to follow the dynamics of neuroinflam-mation. Comparing the temporal profile of a cytokine in different compart-ments such as brain tissue, CSF and serum, could also reveal whether it is produced locally or systemically. Cytokines that are upregulated in plasma shortly after trauma could also be used as biomarkers for an initial assess-ment of injury severity.

Behavioral testingAssessment of the behavioral outcome is used to verify that treatments lead to functional improvements and not only improved morphology. A large number of tests have been devised to evaluate cognition and motor function in rodents following TBI162, 174-177.

It’s a cornerstone of scientific work that the same result is obtained regardless of where or by whom the experiments are performed. This is however not the case when comparing the behavior of different mouse strains178, 179, and this might be a problem in TBI research as well. Differences between labs such as living conditions at the breeder, transportation to the lab, food, drinking wa-ter, cage size, lightning condition, background sounds and schedule for cage cleaning could all alter the outcome of experiments. It is currently debated if this problem can be solved by standardizing the lab environment or system-atically change environmental variables180-184. Either way, performing experi-ments in several species, strains, experimental models, gender and labs will probably provide more robust results.

Rodents not used to human contact consider them dangerous and animal-ex-perimenter interaction most likely influences the outcome of experiments185. This effect can be reduced by handling the animals and thereby accustom them to the experimenter186. Another option is to use test with less contact between animal and experimenter, such as the IntelliCage187, or to use tests where animal-experimenter interaction is less likely to affect the outcome, such as the cylinder test167.

Results from behavioral testing often have a high variability which requires large groups of animals to detect treatment effects. In TBI research variabil-ity can come from three sources, differences between animals before the ex-periment, unintended differences in the injury severity and differences aris-ing during the behavioral testing. Pre-injury variability is probably largely affected by social status, which can influence for example hypothalamic IL-1β levels188. Many of the commonly used rat stocks are also outbred, and there-

33

fore genetically diverse, and fetal position in the uterus affects hormonal ex-posure189. Variability is also caused by unintended differences in injury sever-ity, which can be substantial even with a skilled operator. Variability during behavioral testing might be caused by the order in which the animals are tested and subtle differences in the way the animals are handeled and placed in the testing apparatus. Reducing variability is no easy task, but if possible it would be of great benefit to experimental TBI research. Performing behavioral test and exclude outliers before the injury might be a way forward.

Why the unsuccessful clinical trials?A number of major clinical trials7, 190, based on promising preclinical data, for TBI have been performed but so far none has resulted in a drug approved for clinical use in TBI patients. The reasons for the discrepancy between preclini-cal and clinical results is currently discussed145, 191 and it is unlikely that any single factor is the sole cause.

When targeting early secondary effects of TBI, such as glutamate release and free radical formation192, the time between injury and drug administration is crucial. After the patient is brought to the hospital, diagnosed and informed consent obtained the opportunity to limit secondary damage might be lost or diminished. How long after injury a drug can be administered and still have an effect can be examined preclinically, as has been done for Cyclosporin A193, which can be useful when trying to find the best candidates for clinical evaluation.

The heterogeneity of TBI makes the design of clinical trials difficult and the injury can be classified based on severity, type and injury mechanism194. By analyzing the subtypes of TBI separately drugs which improve some but not all types of TBI might be found. The final outcome is usually measured using the GOS or eGOS, which is sometimes dichotomized in the final analysis. Trials using a more detailed description of the outcome, including separate values for memory, motor function, personality and other important aspects of recovery, might stand a better chance to detect a beneficial effect.

Randomizing subjects to a treatment avoids bias and is the standard in clinical trials but seldom done in preclinical work195. Randomizing individual animals to a treatment may result in a cage where all animals belong to a single group. This may result in systematic errors and can be avoided by instead random-izing manually created balanced groups. Using a good way to assign animals to treatment groups rather than picking them at random avoids this potential risk of bias196.

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In addition to elucidation of disease mechanisms, preclinical experiments are used to find the best candidates for clinical evaluation. To do this, the inves-tigator has to consider not only the statistical significance of an improved outcome, but also if the improvement is likely to translate into a meaningful clinical benefit. By relying on functional testing performed throughout the disease process, rather than histological findings and functional tests in the acute phase, the chances of a successful clinical trial will probably increase.

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Conclusions

In Papers I and II genetic and pharmacological methods were used to increase plasticity following experimental TBI to improve recovery, but no beneficial effects on behavior was observed. The impaired outcome found in Paper I when interfering with NgR1 signaling was unexpected but might be explained by excessive or aberrant plasticity and a different result might be obtained with a delayed initiation of therapy. The lack of improved outcome in Paper II might be caused by compensatory upregulation of other members of the eph-rin family and pharmacological inhibition of EphA4 might be a better option.

The improved outcome observed in Papers III and IV following administra-tion of an IL-1β directed antibody is encouraging. If the therapeutic window is found to be long enough, and the results replicable in other species, antibody mediated IL-1β inhibition is a promising candidate for a clinical trial.

All clinical trials in TBI so far have failed to detect an improved outcome. Future efforts would probably benefit from improved methods for functional outcome evaluation and determination of the therapeutic window in the pre-clinical phase as well as an improved classification of TBI and a more detailed description of the functional outcome in clinical trials.

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Sammanfattning på svenska

Traumatisk hjärnskada drabbar främst unga personer i trafikolyckor och äldre i fallolyckor. Modern neurointensivvård har förbättrat prognosen men många drabbade lider fortfarande av depression, minnesproblem, personlighetsför-ändringar, epilepsi och trötthet. Den ursprungliga skadan förvärras under framför allt de första dagarna och trots flera kliniska prövningar finns inget läkemedel som förhindrar detta. De flesta patienter återfår delvis förlorad för-måga men det finns inget läkemedel som stimulerar återhämtningen.

En del av förbättringen kan bero på plasticitet, hjärnans förmåga att anpassa sig till nya förutsättningar. Att öka plasticiteten genom att underlätta tillväx-ten av nya axon kan eventuellt förbättra återhämtningen efter en hjärnskada. Arbete I och II undersöker två sätt att öka plasticeten, men tyvärr ledde detta endast till en försämring i minne och inlärning i Arbete I. Det är mycket som inte är känt när det gäller återhämtningen efter en hjärnskdada och trots de nedslående resultaten i Arbete I och II förtjärnar området fortsatta undersök-ningar.

En hjärnskada leder till en inflammation och vissa inslag i den tros kunna or-saka ytterligare skador. I Arbete III och IV undersöktes effekten av att minska inflammationen vilket resulterade i minskad vävnadsförlust och förbättrad inlärningsförmåga. Resultaten i Arbete III och IV är hoppingivande och det finns goda möjligheter att hitta en behandling baserad på minskad inflamma-tion.

37

Acknowledgements

To my supervisor Niklas Marklund, who always brings inspiration and works tirelessly to take TBI research forward.

Lars Hillered, my co-supervisor and boss, who’s leadership creates a friendly and relaxed atmosphere.

Fredrik Clausen, my almost-supervisor, for lunch company and lots of scien-tific advice.

Sara Ekmark-Lewen, Anna Erlandsson, Johann Hedin, Lotta Israelsson, Anders Lewen, Camilla Lööv, Anders Nylund, Karin Skoglund, Inger Ståhl-Myllyaho and Ulrika Wallenquist in the neurosurgery group for making it a great place to work.

Maria Björk, for making an amazing work with the cylinder test evaluation.

To Veronica Fjellström, Kristine Jansson, Caroline Hagbohm, Olivia Ki-wanuka, Gudrun Andrea Fridgeirsdottir and Shabnam Salimi and all other members of the vast horde of students who have worked in the lab.

To Petra Millbert, Gun Schönning and Maude Karlsdotter for keeping things together.

Much appreciation to Klas Kullander and Anna Vallstedt for providing the ephA4 cKO mice, to Ola Philipson and Lars Nilsson for the Aβ-work, to Lotta Israelsson and Ted Ebendahl for the RT-PCR analysis and all other co-authors of the papers presented in the thesis.

To Per Enblad and the clinical neurosurgery group for providing insights about the clinical management of TBI patients.

To everyone working at CKMF, especially Annika, Jessica, Olle, Gry and Navia for organizing the pub evenings.

Thanks to Anna-Lena Ask for valuable comments on my thesis summary.

38

My former supervisors Lars-Inge, Martin Malmsten, Johan Sällström, Erik Persson, Henrik Gold, Karin Forsberg-Nilsson and Jimmy Larsson who in-troduced me to science.

To my family and friends for reminding me of the life outside work.

The Task Ahead

This is the most complex object in the known universe.

Fascinating! Are we going to find out

how it works?

No! We’re going to repair it.

?!

The Task Ahead. In remembrance of ”Flat Cat” a priceless artwork lost forever.

39

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