Mammalian brain is challenging to study due to its heterogeneity and complexity. However, recent advances in molecular imaging, genomics and proteomics have contributed significantly to achieve insights into molecular basis of brain function and pathogenesis of neurological disorders. Efficient sample preparation is an integral part of a successful mass spectrometry (MS)-based proteomics. Apart from the identification, quantification of proteins is needed to investigate the alterations between proteome profiles from different sample sets. Therefore, this thesis investigates optimizing and application of the MS compatible sample preparation techniques for the identification and quantification of proteins from brain tissue. The central objective of this thesis was (i) to improve the extraction of proteins as well as membrane proteins (MPs) from the brain tissue and (ii) to apply the optimized method along with the stable isotope dimethyl labeling (DML) and label-free (LF) MS approaches for the relative quantification of the brain proteome profiles during neurological conditions such as Alzheimer’s disease (AD) and traumatic brain injury (TBI).  First study described in this thesis is focused on the qualitative aspects for the brain tissue sample preparation. The optimized extraction buffers from first study containing n-octyl-β-glucopyranside or triton X-114 were used in the further quantitative studies to extract the proteins from patient (AD or TBI) and control human brain samples. Triton X-114 has additional advantage of separating MPs into a micellar phase. Therefore we also investigated the possibility to apply this in combination with DML quantitation approach for enrichment of low abundant MPs from AD brains. AD and TBI causes severe socio-economic burden on the society and therefore there is a need to develop diagnostic markers to detect the early changes in the pathology of the disease. Analytical tools and techniques applied and discussed in this thesis for neuroproteomics applications proved to be powerful and reliable for analyzing complex biological samples to generate high-throughput screening and unbiased identification and quantitation of disease-specific proteins that are of great importance in understanding the disease pathology.

Inledning: Vid en förvärvad hjärnskada är delaktighet i rehabiliteringen en central del för patientens återanpassning till vardagen och eventuellt tillbakabildande av förlorade funktioner. Delaktighet i vårdförloppet leder till en ökad teoretisk förståelse och praktisk insikt hos patienten. Syfte: Syftet med denna studie var att beskriva hur vuxna personer med förvärvad hjärnskada upplever/har upplevt delaktighet under sin vårdtid på en rehabiliteringsavdelning. Metod: Detta är en kvalitativ studie med beskrivande design med semistrukturerade intervjuer som datainsamlingsmetod. Tio patienter som vårdats minst fem arbetsdagar på en rehabiliteringsavdelning intervjuades gällande frågor om delaktighet under vårdtiden på rehabiliteringsavdelningen. En kvalitativ innehållsanalys inspirerad av Graneheim och Lundman (2012) användes för att analysera insamlad data på latentnivå. Resultat: Utifrån temat ”delaktighet” uppstod tre kategorier: kompetent personal, informerad patient, samt självständighet. Temat ”icke-delaktighet” resulterade i två kategorier: bristfällig information och kontrollförlust. Det var flera av informanternas berättelser som liknade varandra i fråga om delaktighet på vårdavdelningen. Samtliga informanter beskrev att de upplevt sig vara delaktiga någon gång under vårdtiden på rehabiliteringsavdelningen. Det handlade bland annat om att de upplevt att de fått tillräcklig information från personalen angående sjukdom och vårdförlopp, att det fanns kompetent personal som anpassade sig efter patienternas individuella behov och önskemål samt möjlighet till självständighet under exempelvis träning. Ett flertal informanter beskrev även upplevelser av att inte vara delaktiga under vårdtiden. Bristfällig information från personalens sida samt en känsla av kontrollförlust var de huvudsakliga anledningarna till detta. Slutsats: Denna studie kan bidra till en ökad förståelse hos vårdpersonal angående delaktighet under rehabiliteringstiden för patienter med förvärvad hjärnskada. Då vårdtiderna kan vara långa på rehabiliteringsavdelningen är det särskilt viktigt med en god kontakt och bra kommunikation med patienterna för delaktighet och en patientcentrerad vård.

BACKGROUND:Diffuse axonal injury is a common consequence of traumatic brain injury (TBI) and often co-occurs with hypoxia, resulting in poor neurological outcome for which there is no current therapy. Here, we investigate the ability of the multifunctional compound erythropoietin (EPO) to provide neuroprotection when administered to rats after diffuse TBI alone or with post-traumatic hypoxia.METHODS:Sprague-Dawley rats were subjected to diffuse traumatic axonal injury (TAI) followed by 30minutes of hypoxic (Hx, 12% O2) or normoxic ventilation, and were administered recombinant human EPO-alpha (5000IU/kg) or saline at 1 and 24hours post-injury. The parameters examined included: 1) behavioural and cognitive deficit using the Rotarod, open field and novel object recognition tests / 2) axonal pathology (NF-200) / 3) callosal degradation (hematoxylin and eosin stain) / 3) dendritic loss (MAP2) / 4) expression and localisation of the EPO receptor (EpoR) / 5) activation/infiltration of microglia/macrophages (CD68) and production of IL-1beta.RESULTS:EPO significantly improved sensorimotor and cognitive recovery when administered to TAI rats with hypoxia (TAI+Hx). A single dose of EPO at 1hour reduced axonal damage in the white matter of TAI+Hx rats at 1day by 60% compared to vehicle. MAP2 was decreased in the lateral septal nucleus of TAI+Hx rats / however, EPO prevented this loss, and maintained MAP2 density over time. EPO administration elicited an early enhanced expression of EpoR 1day after TAI+Hx compared with a 7-day peak in vehicle controls. Furthermore, EPO reduced IL-1beta to sham levels 2hours after TAI+Hx, concomitant to a decrease in CD68 positive cells at 7 and 14days.CONCLUSIONS:When administered EPO, TAI+Hx rats had improved behavioural and cognitive performance, attenuated white matter damage, resolution of neuronal damage spanning from the axon to the dendrite, and suppressed neuroinflammation, alongside enhanced expression of EpoR. These data provide compelling evidence of EPO's neuroprotective capability. Few benefits were observed when EPO was administered to TAI rats without hypoxia, indicating that EPO's neuroprotective capacity is bolstered under hypoxic conditions, which may be an important consideration when EPO is employed for neuroprotection in the clinic.

The overall aim of this thesis was to describe occupational therapy practice for clients with cognitive impairment following acquired brain injury (CIABI) from the perspective of practicing occupational therapists (OTs). To fulfill this aim, qualitative and quantitative approaches were used including interviews (Study I) and questionnaires (Studies II -IV). Based on the qualitative descriptions generated in Study I, a questionnaire was developed and evaluated for content validity and test-rest validity (Study II). The questionnaire was then used in a survey (Study III). The reactive Delphi technique was used to empirically define the aspects that OTs found to be consistent with the concept of therapeutic use of self (Study IV). The results showed that a predominant practice pattern was the use of ADL activities for intervention regardless of whether limitations in occupational performance or cognitive function were assessed, or whether the approach to therapy was remedial or compensatory. General ADL-instruments were used more than instruments focused on impairment level. Therapies covering a wide range of cognitive impairments, and abilities important to organizing and executing occupational performance were commonly targeted. Therapies targeting clients’ activity limitations were prioritized before remediating impairment. Therapeutic use of self was regarded as being important and the results identified clientspecific aims not earlier described in relation to therapeutic use of self. Another prominent practice pattern was the collaborative approach toward clients, relatives, and other staff. Theories used to support practice were primarily general. Occupational therapy practice for clients with CIABI was found to be complex, and the practice patterns were affected by circumstances such as the ‘hidden’ nature of the cognitive impairments, perceived lack of knowledge, and organizational issues. The results of this thesis can be used as a foundation for further research on practice patterns or the specific therapies used. It can facilitate discussions on strengths and weaknesses witcurrent practice, the need for development, and research utilization.

Increased temperature is common after brain trauma and stroke, considered to be detrimental to outcome and usually treated with systemic cooling interventions. However, targeting cooling interventions at the head may be more logical. In addition to arterial blood, the human brain is cooled by heat loss through the skull and heat loss from the upper airways. It is these two mechanisms of heat loss which are the subject of this thesis. The initial research aim was to find out if restoring ‘normal’ airflow through the upper respiratory tracts of intubated, brain-injured patients could reduce brain temperature. Air at room temperature and humidity replicating normal resting minute volume was continuously administered nasally to 15 such patients. After a 30 minute baseline, they were randomised to receive airflow or no airflow for 6 hours and then crossed over for a further 6 hours. The airflow did not produce significant reductions in intracranial temperature (Mean -0.13 °C, SD 0.55 °C, 95% CI -0.43 to 0.17 °C). However, some evidence of heat loss through the skull was serendipitously observed. This was investigated formally in a randomised factorial trial, together with nasal airflow with enhancements (unhumidified air at twice minute volume with 20 ppm nitric oxide gas) intended to overcome some of the possible reasons for the neutral results with ‘normal’ airflow. After a 30 minute baseline, 12 intubated, brain-injured patients received enhanced nasal airflow, bilateral head fanning (8 m/s), both together and no intervention in randomised order. Each intervention was delivered for 30 minutes followed by 30 minutes washout. Mean brain temperature was reduced by 0.15 °C with nasal airflow (p=0.001, 95% CI 0.06 to 0.23 °C) and 0.26 °C with head fanning (p<0.001, 95% CI 0.17 to 0.34 °C). The estimate of the combined effect of airflow and fanning on brain temperature was 0.41 °C. Physiologically, this study demonstrated that heat loss through the upper airways and through the skull can reduce parenchymal brain temperature in brain-injured humans, that the effects are additive and the onset of temperature reduction is rapid. The most promising mechanism appeared to be heat loss through the skull and the final piece of research involved developing and initial (phase I) assessment of a convective head cooling device in healthy volunteers, with intracranial temperature measured non-invasively by magnetic resonance spectroscopy. After a 10 minute baseline, five healthy volunteers received 30 minutes head cooling followed by 30 minutes head and neck cooling via a hood and neck collar delivering 14.5 °C air at 42.5 L/s. The net brain temperature reduction with head cooling was 0.45 °C (SD 0.23 °C, p=0.01, 95% CI 0.17 to 0.74 °C) and with head and neck cooling 0.37 °C (SD 0.30 °C, p=0.049, 95% CI 0.00 to 0.74 °C). There was no significant reduction in cooling with progressive depth into the brain i.e. core brain was cooled. The main relevance of this research is physiological because it adds to knowledge and understanding of mechanisms of heat loss from the upper airways and through the skull in humans. Clinically, factors which enhance or inhibit these mechanisms may have an effect on brain temperature but the therapeutic relevance of head cooling by these methods requires further research.

The majority of the estimated three million traumatic brain injuries that occur each year are classified as “mild” and do not require surgical intervention. However, debilitating symptoms such as difficulties focusing on tasks, anxiety, depression, and visual deficits can persist chronically after a mild traumatic brain injury (TBI) even if an individual appears “fine”. These symptoms have been observed to worsen or be prolonged when an individual has suffered multiple mild TBIs. To test the hypothesis that increasing the amount of time between head injuries can reduce the histopathological and behavioral consequences of repeated mild TBI, a mouse model of closed head injury (CHI) was developed. A pneumatically controlled device with a silicone tip was used to deliver a diffuse, midline impact directly onto the mouse skull. A 2.0mm intended depth of injury caused a brief period of apnea and increased righting reflex response with minimal astrogliosis and axonal injury bilaterally in the entorhinal cortex, optic tract, and cerebellum. When five CHIs were repeated at 24h inter-injury intervals, astrogliosis was exacerbated acutely in the hippocampus and entorhinal cortex compared to a single mild TBI. Additionally, in the entorhinal cortex, hemorrhagic lesions developed along with increased neurodegeneration and microgliosis. Axonal injury was observed bilaterally in the white matter tracts of the cerebellum and brainstem. When the inter-injury interval was extended to 48h, the extent of inflammation and cell death was similar to that caused by a single CHI suggesting that, in our mouse model, extending the inter-injury interval from 24h to 48h reduced the acute effects of repeated head injuries. The behavioral consequences of repeated CHI at 24h or 48h inter-injury intervals were evaluated in a ten week longitudinal study followed by histological analyses. Five CHI repeated at 24h inter-injury intervals produced motor and cognitive deficits that persisted throughout the ten week study period. Based upon histological analyses, the acute inflammation, axonal injury, and cell death observed acutely in the entorhinal cortex had resolved by ten weeks after injury. However, axonal degeneration and gliosis were present in the optic tract, optic nerve, and corticospinal tract. Extending the inter-injury interval to 48h did not significantly reduce motor and cognitive deficits, nor did it protect against chronic microgliosis and neurodegeneration in the visual pathway. Together these data suggested that some white matter areas may be more susceptible to our model of repeated mild TBI causing persistent neuropathology and behavioral deficits which were not substantially reduced with a 48h inter-injury interval. In many forms of TBI, microgliosis persists chronically and is believed to contribute to the cascade of neurodegeneration. To test the hypothesis that post-traumatic microgliosis contributes to mild TBI-related neuropathology, mice deficient in the growth factor progranulin (Grn-/-) received repeated CHI and were compared to wildtype, C57BL/6 mice. Penetrating head injury was previously reported to amplify the acute microglial response in Grn-/- mice. In our studies, repeated CHI induced an increased microglial response in Grn-/- mice compared to C57BL/6 mice at 48h, 7d, and 7mo after injury. However, no differences were observed between Grn-/- and WT mice with respect to their behavioral responses or amount of axonal injury or ongoing neurodegeneration at 7 months despite the robust differences in microgliosis. Dietary administration of ibuprofen initiated after the first injury reduced microglial activation within the optic tract of WT mice 7d after repeated mild TBI. However, a two week ibuprofen treatment regimen failed to affect the extent of behavioral dysfunction over 7mo or decrease chronic neurodegeneration, axon loss, or microgliosis in brain-injured Grn-.- mice when compared to standard diet. Together these studies underscore that mild TBIs, when repeated, can result in long lasting behavioral deficits accompanied by neurodegeneration within vulnerable brain regions. Our studies on the time interval between repeated head injuries suggest that a 48h inter-injury interval is within the window of mouse brain vulnerability to chronic motor and cognitive dysfunction and white matter injury. Data from our microglia modulation studies suggest that a chronically heightened microglial response following repeated mild TBI in progranulin deficient mice does not worsen chronic behavioral dysfunction or neurodegeneration. In addition, a two week ibuprofen treatment is not effective in reducing the microglial response, chronic behavioral dysfunction, or chronic neurodegeneration in progranulin deficient mice. Our data suggests that microglia are not a favorable target for the treatment of TBI.

Glucocorticoid activation of two types of adrenocorticosteroid receptors (ACRs), themineralocorticoid receptor (MR) and the glucocorticoid receptor (GR), influences hippocampalneuron vulnerability to injury. Excessive activation of GR may compromise hippocampalneuron survival after several types of challenge including ischemic, metabolic, and excitotoxicinsults. In contrast, MR prevents adrenalectomy-induced loss of granule neurons in the dentategyrus. The present thesis addresses the respective roles of MR and GR in modulating neuronalsurvival following two forms of neuronal injury, excitotoxicity and traumatic brain injury. MaleSprague-Dawley rats were pretreated with MR antagonist spironolactone or GR antagonistmifepristone (RU486) and subsequently injected with kainic acid, an excitotoxic glutamateanalog, or injured with a controlled cortical impact. Twenty-four hours following injury,hippocampal neuron survival was measured to test the hypotheses that MR blockade wouldendanger and GR blockade would protect hippocampal neurons following injury. MessengerRNA levels of viability-related genes including bcl-2, bax, p53, BDNF, and NT-3 were alsomeasured to test the hypothesis that ACR regulation of these genes wouldcorrelate with neuronal survival. In addition, ACR mRNA levels were measured followingreceptor blockade and injury to test the hypothesis that glucocorticoid signaling is alteredfollowing neuronal injury via regulation of ACR expression.Mineralocorticoid receptor blockade with spironolactone increased neuronal vulnerability toexcitotoxic insult in hippocampal field CA3, and GR blockade with RU486 prevented neuronalloss after traumatic brain injury in field CA1. These results are consistent with the hypothesesthat MR protects and GR endangers hippocampal neurons. Adrenocorticosteroid receptorblockade decreased mRNA levels of the anti-apoptotic gene bcl-2 in select regions of uninjuredhippocampus, yet ACR regulation of bcl-2 did not consistently correspond with measures ofneuronal survival after injury. Kainic acid decreased MR mRNA levels in CA1 and CA3, whileboth kainic acid and controlled cortical impact dramatically decreased GR mRNA levels indentate gyrus. These data suggest that injury modulation of glucocorticoid signaling throughregulation of ACR expression may influence hippocampal neuron viability following injury.

Traumatic brain injury (TBI) is becoming a national epidemic, as it accounts for 1.5 million cases each year. This disorder affects primarily the young population and elderly. Currently, there is no treatment for TBI, which means that ~2% of the U.S. population is currently living with prolonged neurological damage and dysfunction. Recently, there have been many studies showing that TBI negatively impacts mitochondrial function. It has been proposed that in order to save the cell from destruction mitochondrial function must be preserved. The ketogenic diet, originally designed to mimic fasting physiology, is effective in treating epilepsy. Therefore, we have used fasting as a post injury treatment and attempted to elucidate its underlying mechanism. 24 hours of fasting after a moderate TBI increased tissue sparing, cognitive recovery, improved mitochondrial function, and decreased mitochondrial biomarkers of injury. Fasting results in hypoglycemia, the production of ketones, and the upregulation of free fatty acids (FFA). As such, we investigated the neuroprotective effect of hypoglycemia in the absence of fasting through insulin administration. Insulin administration was not neuroprotective and increased mortality in some treatment groups. However, ketone administration resulted in increased tissue sparing. Also, reduced reactive oxygen species (ROS) production, increased the efficiency of NADH utilization, and increased respiratory function. FFAs and uncoupling proteins (UCP) have been implicated in an endogenously regulated anti-ROS mechanism. FFAs of various chain lengths and saturation were screened for their ability to activate UCP mediated mitochondrial respiration and attenuate ROS production. We also measured FFA levels in serum, brain, and CSF after a 24 hour fast. We also used UCP2 transgenic overexpressing and knockout mice in our CCI injury model, which showed UCP2 overexpression increased tissue sparing, however UCP2 deficient mice did not show a decrease in tissue sparing, compared with their wild type littermates. Together our results indicate that post injury initiated fasting is neuroprotective and that this treatment is able to preserve mitochondrial function. Our work also indicates ketones and UCPs may be working together to preserve mitochondrial and cellular function in a concerted mechanism, and that this cooperative system is the underlying mechanism of fasting induced neuroprotection.

In the hours and days following acute CNS injury, a secondary wave of events is initiated that exacerbate spinal tissue damage and neuronal cell death. A potential mechanism driving these secondary events is opening of the mitochondrial permeability transition pore (mPTP) and subsequent release of several cell death proteins. Previous studies have shown that inhibition of cyclophilin D(CypD), the key regulating component in mPTP opening, was protective against insults that induce necrotic cell death. We therefore hypothesized that CypD-null mice would show improved functional and pathological outcomes following spinal cord injury (SCI) and traumatic brain injury (TBI). Moderate and severe spinal contusion was produced in wild-type (WT) and CypD-null mice at the T-10 level using the Infinite Horizon impactor. Changes in locomotor function were evaluated using the Basso Mouse Scale (BMS) at 3 days post-injury followed by weekly testing for 4 weeks. Histological assessment of tissue sparing and lesion volume was performed 4 weeks post SCI. Calpain activity, measured by calpain-mediated spectrin degradation, was assessed in moderate injury only by western blot 24 hours post SCI. Results showed that following moderate SCI, CypD-null mice had no significant improvement in locomotor recovery or tissue sparing compared to wild-type mice. Following severe SCI, CypD-null mice showed significantly lower locomotor recovery and decreased tissue sparing compared to WT mice. Calpain-mediated spectrin degradation was not significantly reduced in CypD-null mice compared to WT mice 24h post moderate SCI. The lack of protective effects in CypD-null mice suggests that more dominant mechanisms are involved in the pathology of SCI. In addition, CypD may have a pro survival role that is dependent on the severity of the spinal cord injury.

Traumatic brain injury (TBI) is a leading cause of death and long-term disability worldwide, and survivors are often left with cognitive deficits and significant problems with day to day tasks. To date, therapeutic pharmacological treatments of TBI remain elusive despite numerous clinical trials. An improved understanding of the molecular and cellular response to injury may help guide future treatment strategies. One promising marker for brain injury is the translocator protein (TSPO), which is normally expressed at a low level, but is highly expressed following brain damage and is associated with neuroinflammation. The isoquinoline carboxamide PK11195 binds selectively to the TSPO in many species, and has therefore become the most-studied TSPO ligand. To characterize the time-course of TSPO expression in the controlled cortical injury (CCI) model of TBI we subjected Sprague-Dawley rats to CCI and euthanatized them after 30 minutes, 12 hours, 1, 2, 4, or 6 days. Autoradiography with radiolabelled PK11195 was used to assess the time-course of TSPO binding following CCI. Autoradiographs were compared to adjacent tissue slices stained with the microglia/macrophage marker ED-1, with which a moderate positive correlation was discovered. PK11195 autoradiography was used as a tool with which to assess neuroinflammation following CCI and the administration of an α7 nAChR antagonist, methyllycaconitine (MLA). We hypothesized that blocking the calcium permeable α7 nAChR after brain injury would have a neuroprotective effect by attenuating excitotoxicity in the shortterm. Our study revealed clear dose-dependent tissue sparing in rats administered MLA after trauma and a modest improvement in functional outcome. The relatively modest recovery of function with MLA, which could be due to prolonged α7 nAChR blockade or downregulation lead us to explore the potential of α7 nAChR partial agonists in treating TBI. The α7 nAChR partial agonists tropisetron, ondansetron, and DMXB-A produced a moderate attenuation of cognitive deficits, but did not have a neuroprotective effect on tissue sparing. These studies show that following TBI, α7 nAChR modulation can have neuroprotective effects and attenuate cognitive deficits. Whether this modulation is best achieved through partial agonist treatment alone or a combination antagonist/agonist treatment remains to be determined.