Brain–specific proteins (BSP) are each relatively specific for particular cell–types within the nervous system. The BSP studied were glial fibrillary acidic protein (GFAP) and S100B for the astrocyte, ferritin for microglia and neurofilaments (Nf) for the axon. BSP are released into the extracellular fluid (ECF) following cellular destruction and during phases of high cellular activity such as astrocytic or microglial activation. ECF BSP equilibrate with those in the cerebrospinal fluid (CSF). This allows us to quantify BSP from the CSF and estimate the overall average of axonal damage (CSF Nf), astrocytic and microglia activation (respectively CSF S100B, CSF ferritin) and astrogliosis (CSF GFAP). New enzyme linked immunoabsorbant assays (ELISA) have been developed for measuring Nf and GFAP in the CSF. Previously established ELISAs have been used to measure S100B and ferritin. It has been shown that spinal cord atrophy in a mouse model of autoimmune encephalomyelitis (EAE) was paralleled by a decrease of Nf indicating loss of axons, and an increase in GFAP indicating astrogliosis. These findings have been confirmed and extended in a human post–mortem study where BSP levels were quantified in multiple sclerosis (MS) lesions of different age and activity. S100B and Nf were associated with acute lesions, ferritin was elevated in all lesion types, while GFAP was increased in both acute and chronic lesions. CSF BSP levels were then quantified in a cross–sectional study of MS patients with the aim of distinguishing clinical subgroups, such as relapsing remitting (RR), primary progressive (PP) and secondary progressive (SP) disease. In addition an attempt was made to relate CSF BSP levels to grades of disability using clinical scales including Kurtzke’s EDSS, an ambulation index (AI) and the 9–hole PEG test (9HPT). It was shown that CSF S100B was higher in RR MS while CSF ferritin was elevated in PP MS patients. The S100B:ferritin ratio emphasised the distinction between the MS subtypes. CSF GFAP was higher in poorly ambulating (AI) and severely disabled (EDSS) patients. CSF GFAP correlated with the EDSS in SP MS patients. This suggests that gliosis is an important feature in SP MS. CSF Nf levels were quantified in a longitudinal study at baseline and at 3–year follow–up. It was shown that more SP/PP than RR MS patients experienced an increase in CSF Nf levels over this time, suggesting cumulative axonal damage in this subgroup. RR MS patients who had elevated CSF Nf levels at baseline had a worse clinical course, suggesting that initial high CSF Nf levels in RR MS patients are a poor prognostic sign. CSF Nf levels at follow–up correlated with the EDSS, AI and 9HPT suggesting that axonal pathology in MS is a dynamic process possibly balancing features of de- and regenerative activities.

Study 1. Using a cognitive-process approach, 25 schizophrenic patients were matched with 25 healthy volunteers and compared on tests of memory and executive function. The schizophrenia group was found to have a significant impairment in immediate memory with relatively spared long-delay and recognition memory. Memory deficits were irrespective of the encoding strategies used and were unrelated to chronicity. In addition, the schizophrenic patients performed worse than controls on tests of executive function which was supported by some significant correlations between aspects of memory and executive function. The pattern of performance resembled that found in patients with subcortical or frontal lesions. Study 2. To examine further executive aspects of memory, an attempt to demonstrate confabulation in schizophrenia was made. Twelve schizophrenic patients were matched with 12 volunteers, 8 of whom were normal healthy subjects, with the remained being depressed patients. The subjects were asked to recall a set of experimental narratives, with confabulation being defined as the recall of ideas not present in the narrative. Subjects were also examined on a number of neuropsychological tests and the patients were assessed on the Krawiecka scale. Variable amounts of confabulation were observed in all the schizophrenic patients while only one control subject confabulated. The form of confabulation differed from those observed in other patients in that the original ideas were spontaneously rearranged to produce new ones. Confabulation was found to be related to difficulties in suppressing inappropriate responses and formal thought disorder. Study 3. Three schizophrenic patients previously identified as confabulators, were intensively studied to establish the mechanisms of narrative confabulation in schizophrenia. Patients were administered experimental tasks as well as standard neuropsychological tests of memory and executive function. Assessment of current symptoms was made using the SANS and SAPS scales. The severity of cognitive impairment was found to reflect the severity of confabulation, but memory impairment was neither nor sufficient to account for confabulation. Within the spectrum of executive deficits, impairments in response suppression and response monitoring, but not planning or generation were consistently associated with confabulation. The findings from the experimental tasks suggest that faults occur at both input and output. At the input stage, narrative material is encoded in a disorganised manner while at the output stage, this disorganisation is compounded by faulty editing processes. Study 4. Four schizophrenic patients who were known confabulators with narrative material, were subjected to an experimental autobiographical questionnaire designed to establish whether schizophrenic patients confabulate in response to questions calling on the recollection of personal facts and events. In addition, a number of neuropsychological tests were administered and current symptoms was assessed with the SANS and SAPS scales. All patients were observed to confabulate to varying degrees, particularly in response to questions relating to personal episodes rather than facts. For two patients, personal delusional systems were found to play a role in confabulation by providing a framework on which to base certain confabulatory recollections. Memory impairment was not found to be a necessary component to autobiographical confabulation but deficits in response suppression and response monitoring were observed to be related to the verification process performed during this task. Study 5. In an attempt to establish which anatomical regions may be at fault in schizophrenia when patients are engaged in response suppression tasks, six normal subjects were studied using positron emission tomography (PET) to identify anatomical regions involved when performing the Hayling Test. Subjects were also required to perform a control condition in which they had to read out the last word of given sentences. Compared to the control task, response initiation was associated with left sided activation of the frontal operculum, inferior frontal gyrus, middle temporal gyrus and right anterior cingulate gyrus, whereas response suppression was associated with left frontal operculum, inferior frontal gyrus and right anterior cingulate gyrus activation only. The difference between the two parts of the Hayling Test was in the increased activation of the left middle temporal gyrus and the left inferior frontal region (Brodmann's area 44/6) during response initiation.

The two of major pathways that cause ischemic damage are oxidative stress and inflammation. To decreasing oxidative stress and inflammation, new anti-oxidant and anti-inflammatory agents are tested in ischemic models. In order to study ALRX828C anti-inflammatory properties, an in vivo six-day old air pouch model of inflammation was used to evaluate the anti-inflammatory potential of ALRX828C. Also, the dose response of ALRX828C for TNFα (IC50 = 30 μM) and IL-17 (IC50 = 1.3 μM) were determined by using human peripheral blood mononuclear cell cultures stimulated with ionomycin and PMA. To examine ALRX828C anti-inflammatory effect in neuroinflammation, a neurodegenerative model was used to evaluate its potential. I also showed that reducing oxidative stress with a potent antioxidant, Idebenone in nano-emulsion form, can effectively reduce tissue damage during ischemia in organotypic slice culture subjected to oxygen-glucose depravation (OGD). In conclusion, reducing oxidative stress and inflammation after stroke can reduce ischemic damage substantially.

The two of major pathways that cause ischemic damage are oxidative stress and inflammation. To decreasing oxidative stress and inflammation, new anti-oxidant and anti-inflammatory agents are tested in ischemic models. In order to study ALRX828C anti-inflammatory properties, an in vivo six-day old air pouch model of inflammation was used to evaluate the anti-inflammatory potential of ALRX828C. Also, the dose response of ALRX828C for TNFα (IC50 = 30 μM) and IL-17 (IC50 = 1.3 μM) were determined by using human peripheral blood mononuclear cell cultures stimulated with ionomycin and PMA. To examine ALRX828C anti-inflammatory effect in neuroinflammation, a neurodegenerative model was used to evaluate its potential. I also showed that reducing oxidative stress with a potent antioxidant, Idebenone in nano-emulsion form, can effectively reduce tissue damage during ischemia in organotypic slice culture subjected to oxygen-glucose depravation (OGD). In conclusion, reducing oxidative stress and inflammation after stroke can reduce ischemic damage substantially.

Chronic traumatic encephalopathy (CTE) is a neurodegenerative disorder caused by repeated concussive or subconcussive blows to the head. Clinically, this disease is characterized by cognitive dysfunction, short-term memory loss, and motor deficits. Pathologically, deposition of the abnormal protein tau, cerebral atrophy, and white matter degeneration is common. CTE has been categorized into Stages I-IV based on increased severity of protein deposition and cerebral atrophy. Acutely, mild traumatic brain injury (TBI) damages the long white matter tracks in the corpus callosum. In addition, it initiates a neuroinflammatory cascade aimed at protecting healthy tissue by clearing any toxic or damaging debris. This cascade results predominantly from the activation of the resident immune cells of the brain, microglia. Inflammation begins immediately and then subsides weeks or months after injury. However, pathological chronic activation of microglia can occur that can cause cell death and degeneration. Several studies have linked traumatic brain injury as well as chronic neuroinflammation to a variety of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, and chronic traumatic encephalopathy (CTE). The present study quantifies the level of inflammation found in the brains of those diagnosed with varying stages of CTE compared to normal, healthy controls. The thickness of the corpus callosum was measured to investigate the correlation between microglial density and white matter degeneration. Cases were selected from the donated brains of former athletes and military veterans who had a history of repetitive mild TBI. Eleven healthy control cases, ten early stage (Stage I/II), and nine late stage (Stage III/IV) CTE cases were selected for analysis. Tissue sections of the anterior and posterior cingulate of each case were stained for microglia, reactive astrocytes, and macrophages using IBA-1, GFAP, and CD68 markers. The percent area stained of each section was calculated to compare inflammatory cell density across progressive stages of the disease. Analysis showed a significant thinning of the corpus callosum of Stage III/IV CTE cases compared to normal controls. There was a significant decrease in microglia and reactive astrocytes of both the anterior and posterior portions of the corpus callosum in both early and advanced stage CTE cases compared to healthy controls. Corpus callosum thickness was significantly decreased in advanced stage (III-IV), but not early stage (I-II) disease. Overall, this suggests that neuroinflammation is decreased in the corpus callosum in CTE despite marked degeneration. Repetitive mild TBI might impair mechanisms of brain inflammation and repair.

Indiana University-Purdue University Indianapolis (IUPUI) / Toxoplasma gondii is an intracellular parasite that causes persistent, lifelong infection in one-third of humans worldwide. The parasite converts from a lytic, actively replicating form (tachyzoite) into a latent tissue cyst form (bradyzoite) that evades host immunity and is impervious to current drugs. While acute infection can be life threatening to immunosuppressed individuals, chronic infection has been linked to behavioral changes in rodents and neurological disease in humans. Notably, chronic infection in mice leads to hyperactivity in an open field. Whether these behavioral changes are due to parasite manipulation of the host or the host response to infection remains an outstanding question. We have previously shown that the anti-hypertensive drug guanabenz reduces Toxoplasma cyst burden in the brains of BALB/c mice, providing a means to examine whether brain cyst depletion reverses behavioral changes. We used two mouse strains (BALB/c and C57BL/6) differing in their susceptibility to infection. Following drug treatment of chronically infected mice, locomotor activity in an open field was assessed. In both mouse strains, the increased hyperactivity seen during chronic infection returned to normal levels following guanabenz treatment. Guanabenz reduced brain cyst burden ~70% in BALB/c mice as expected, but it increased cyst burden 49% in C57BL/6 mice. Examination of the brains showed that guanabenz decreased inflammation and perivascular cuffing in both infected mouse strains. Our study shows for the first time that it is possible to reverse a key behavioral change associated with chronic Toxoplasma infection. Surprisingly, the rescue from parasite-induced hyperactivity correlates with a decrease in neuroinflammation instead of cyst counts, suggesting that some behavioral changes arise from host responses to infection rather than a parasite-driven process. / 2022-03-10

Despite being a common problem, there are many gaps in the understanding of mild traumatic brain injury (mTBI). Its pathophysiology is unclear, diagnostic criteria are variable, and the associated symptomatology is non-specific. As a result, there are challenges associated with precise mTBI diagnosis and treatment. The dissertation seeks to identify distinctive features, both clinical and pathophysiological, exclusively associated with mTBI. In addition, the neuroinflammatory component of mTBI is explored in detail in the context of inflammatory cytokines’ potential use as prognostic biomarkers and development of a targeted treatment. Three studies were conducted to explore mTBI. We conducted a retrospective chart review to identify the clinical presentation exclusively associated with mTBI that sets it apart from other similar conditions. This was accomplished through symptomatology comparison between the patients with head injuries that meet the ACRM (1993) criteria for mTBI diagnosis vs. those who do not. The results of this study showed that 20.5% of patients with chronic post-concussive symptoms do not meet the ACRM (1993) criteria of mTBI despite sustaining a head injury. In addition, no symptom specific differences were found between the two populations. A detailed systematic review and meta-analysis were also conducted to identify the common inflammatory cytokines associated with mTBI and to explore their potential use as prognostic biomarkers. The results show significantly elevated blood IL-6, IL-1RA, IFN-γ (at <24 hrs.) and MCP-1/CCL2 (within a week) levels in patients with mTBI compared to healthy controls in majority of the included studies. A meta-analysis was further conducted that supported these findings by showing significantly elevated IL-6, MCP-1/CCL2, and IL-1β levels in patients with mTBI in the acute stages (<7 days). In addition, elevated IL-6, TNF-α, IL-1RA, IL-10, and MCP-1/CCL2 levels were associated with poor prognosis in patients with mTBI. In addition, a systematic review was conducted to identify the inflammatory cytokines associated with adverse psychological outcome in population with mTBI. The results show that IL-6, TNF-α, IL-10, and CRP are associated with PTSD and/or depression in the population with mTBI, particularly in the chronic stages. Collectively, these studies show that all symptomatic patients with head trauma, whether or not they meet the subjective criteria of mTBI, should be managed and offered early rehabilitation to avoid long tern adverse consequences. In addition, this thesis supports the neuro-inflammatory hypothesis of mTBI and identifies inflammatory cytokines that could be potentially utilized as prognostic biomarkers and for the development of mTBI treatment. / Thesis / Doctor of Philosophy (PhD)

Les processus de neuro-inflammation jouent un rôle majeur dans la maladie d'Alzheimer (MA). Des études génétiques récentes démontrent cette association entre neuro-inflammation et MA et impliquent notamment un gène, TREM2, qui code pour un récepteur exprimé à la surface de la microglie. La tauopathie est une lésion caractéristique de la MA. Elle se traduit par l’hyperphosphorylation et l’agrégation intraneuronale de la protéine Tau. Les travaux sur le rôle de TREM2 dans le développement de la pathologie Tau sont peu nombreux et donnent des résultats contradictoiresAinsi, l’objectif de ma thèse est d’étudier le rôle de la neuroinflammation et de TREM2 dans la progression de la tauopathie, dans deux modèles différents. Le premier est obtenu par injection stéréotaxique de vecteurs AAV dans la couche CA1 de l’hippocampe de souris déficientes ou non en TREM2. Ces vecteurs entrainent la surexpression de différentes formes de la protéine Tau humaine et permettent de récapituler les différents stades de la tauopathie.Nous avons en parallèle utilisé un modèle transgénique plus progressif de tauopathie, la souris THY-Tau22, afin d’étudier l’influence du stade de la pathologie dans l’effet provoqué par une déficience en TREM2 sur l’évolution de la pathologie. Notre étude a mis en évidence la toxicité des formes solubles de Tau dans le modèle AAV par rapport à ses formes agrégées. Le modèle transgénique THY-Tau22 nous a permis de mettre en évidence une augmentation des lésions tauopathiques dans les souris déficientes en TREM2 par rapport aux souris qui ne le sont pas, uniquement à un stade avancé. Cela suggère que, à l’instar des modèles amyloïdes, l’effet de la déficience en TREM2 sur le décours de la tauopathie est différent en fonction du stade considéré. / Neuroinflammation processes appear to play a major role in Alzheimer's disease (AD). Recent genetic studies support this correlation between neuroinflammation and AD and include a gene, TREM2, expressed on microglial surface. Tauopathy is a characteristic lesion of AD. It results in hyperphosphorylation and intraneuronal aggregation of Tau protein. In the literature, only few articles describe the role of TREM2 in the development of Tau pathology, and they report contradictory results. We therefore do not know for sure whether a deficiency in TREM2 has a deleterious effect or not on tauopathy. Thus, the goal of my thesis is to study the role of neuroinflammation and TREM2 in the progression of tauopathy, in two different models. The first is obtained by stereotaxic injection of AAV vectors into the CA1 layer of the hippocampus of TREM2-deficient or non-deficient mice. These vectors lead to the overexpression of different forms of the human tau protein, thus making it possible to recapitulate the different tauopathy stages.In parallel, we used a more progressive trangenic model of tauopathy, the THY-Tau22 mouse, to study the influence of TREM2 deficiency at different stage of the pathology. Our study demonstrated the toxicity of Tau soluble forms in the AAV model compared to its aggregated forms. The THY-Tau22 transgenic model allowed us to demonstrate an increase in tauopathic lesions in TREM2 deficient mice compared to wild type mice, at late stage only. This suggests that, similar to amyloid models, the effect of TREM2 deficiency on the course of tauopathy is influenced by the stage of the disease.

Several studies have shown that dietary omega-3 polyunsaturated fatty acids (n-3 PUFA) are beneficial in neurodegenerative diseases, although the mechanism of action is not agreed upon. Because most neurodegenerative diseases have an inflammatory component, it is possible that docosahexaenoic acid (DHA) is anti-inflammatory in the brain as it is known to be in several non-neural tissues. Specialized pro-resolving mediators (SPM) are metabolized from DHA and are leading candidates to explain the anti-inflammatory effects of DHA. The goal of this work was to investigate the role and potential mechanisms of action of DHA in neuroinflammation. In our first approach, fat-1 transgenic mice had higher phospholipid and unesterified DHA levels in their hippocampi, and attenuated lipopolysaccharide (LPS)-induced neuroinflammation, compared to wildtype littermates. Feeding wildtype littermates n-3 PUFA mimicked hippocampal DHA levels and LPS-induced neuroinflammatory responses of fat-1 mice, indicating DHA is anti-neuroinflammatory whether derived from the diet or the activity of the fat-1 protein. In an attempt to further augment hippocampal DHA levels, feeding n-3 PUFA adequate mice an n-3 PUFA diet increased phospholipid but not unesterified DHA levels, and did not attenuate LPS-induced neuroinflammation, highlighting the potential importance of unesterified DHA. Directly infusing unesterified DHA into a cerebral ventricle throughout LPS-induced neuroinflammation mimicked several aspects of the attenuated neuroinflammatory response seen with our chronic dietary and transgenic models, as did infusing its 17S-hydroperoxy-DHA (17S-HpDHA) derivative, a precursor to SPM. The metabolism of DHA to SPM in the brain was found to be distinct from non-neural tissues, characterized by the presence of protectin D1 and maresin 1, and the absence of resolvin D1 or D2. Further, infusing 17S-HpDHA increased protectin D1 concurrent to attenuating neuroinflammation, suggesting protectin D1 is responsible for some of the anti-neuroinflammatory effects of DHA. In conclusion, DHA is anti-neuroinflammatory in a mouse model of neuroinflammation, in part, via its metabolism to SPM.

Neuroinflammation and excitotoxicity are frequently regarded as the classical hallmarks of all major central nervous system (CNS) diseases such as stroke and neurodegenerative disorders. However, the limited number of current clinical options for the treatment of these diseases and the side effects associated with these treatment options indicate that there is an urgent and important need to develop drugs that delay neurological diseases. Although the molecular mechanisms underlying these neurological diseases remain poorly understood, it is widely accepted that alterations in microglia function is the key causative factor. It was recently reported that granulocyte colony-stimulating factor (G-CSF) and a natural marine compound, GB9, show great potential as anti-inflammatory agents. In the present study, we used a model of neuroinflammation to investigate the neuroprotective effects of G-CSF and GB9, and whether they exert an anti-neuroinflammatory effect on IFN-£^-stimulated microglia (BV2). Our results revealed that both G-CSF and GB9 attenuate the upregulation of proinflammatory mediators such as inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) in IFN-£^-stimulated microglia. Furthermore, Western blot and immunohistochemical analyses revealed that G-CSF or GB9 prevent downregulation of the glutamate transporter (Glu-Asp transporter, GLAST) and activation of the glutamate receptor in the IFN-£^-stimulated microglia. Additionally, our in vivo analyses revealed that centrally administered G-CSF could reverse the increase of OX-42 immunoactivity, which is the marker of IFN-£^-stimulated microglia. In summary, our findings support the hypotheses that G-CSF and the marine compound, GB9, possess anti-neuroinflammatory properties and could be pursued as potential therapeutic agents for CNS diseases.