Traumatic Brain Injury (TBI) is an increasingly diagnosed constellation of injuries derived from acute mechanical trauma to the brain. With the rise of advanced neuroimaging techniques recent focus has oriented primarily towards the mild-moderate range of TBI which previously was missed diagnostically. Characteristically, these advances have shown increasing areas of micro-hemorrhage in susceptible areas of the brain and to date there are no treatment modalities targeting micro-hemorrhages or their sequelae. This dissertation explores the effects of the resulting heme processing response in the days following injury with a particular focus on inducing early heme clearance from the parenchyma using a rat central fluid percussion injury model in the mild-moderate injury range. Since heme is released ~24-48 hours post-injury and is known to be cytotoxic we observed there may be a critical window for treatment to clear heme before it is spontaneously released and to increase the buffering capacity of the tissue. We targeted heme clearance by using drugs known to increased expression of Nrf2, an upstream transcriptional regulator of the canonical heme processing protein heme oxygenase 1 (HO-1), and tracking expression of HO-1, the iron sequestration/storage proteins Lipocalin 2 (LCN2) and Ferritin (FTL), as well as the activity of matrix metalloproteinases 2 and 9 (MMP2, MMP9). We examined both tissue known to be frankly hemorrhagic (the neocortex) as well as tissue lacking any identifiable bleed (the hippocampus). We demonstrated that using the HO-1 inducers Hemin and Sulforaphane in a single dose paradigm given 1 hour post-injury heme clearance was accelerated in the neocortex with the majority of heme pigment processed by 24 hours post-injury. Further there was significant attenuation of protein expression in HO-1 and ferritin as well as the enzyme activity of MMP2 and MMP9 in both the neocortex and the hippocampus. Behavioral attenuation was also seen in both rotarod and Morris water maze tests. While we intended to target hemorrhagic processing after injury, and indeed demonstrated improved clearance of heme from post-injury hemorrhagic regions of the brain, in both tissues studied we observed remarkably similar responses to the drugs utilized in protein expression, enzyme activity, and behavioral improvement which may suggest a globally improved pathologic state or that there are unidentified pathologic micro-hemorrhages or leaky vessels which extend further into the brain parenchyma than currently identified.

A growing body of evidence implicates the importance of the endogenous cannabinoid 2-arachidonyl glycerol (2-AG) in memory regulation. The biosynthesis of 2-AG occurs primarily through the diacylglycerol lipases (DAGL-α and -β), with 2-AG serving as a bioactive lipid to both activate cannabinoid receptors and as a rate limiting precursor for the production of arachidonic acid and subsequent pro-inflammatory mediators. Gene deletion of DAGL-α shows decrements in synaptic plasticity and hippocampal neurogenesis suggesting this biosynthetic enzyme may be important for processes of normal spatial memory. Additionally, 2-AG is elevated in response to pathogenic events such as traumatic brain injury (TBI), suggesting its regulatory role may extend to conditions of neuropathology. As such, this dissertation investigates the in vivo role of DAGL-α and -β to regulate spatial learning and memory in the healthy brain and following neuropathology (TBI). The first part of this dissertation developed a mouse model of learning and memory impairment following TBI, using hippocampal-dependent tasks of the Morris water maze (MWM). We found modest, but distinct differences in MWM performance between left and right unilateral TBI despite similar motor deficits, histological damage, and glial reactivity. These findings suggest that laterality in mouse MWM deficit might be an important consideration when modeling TBI-induced functional consequences. The second part of this dissertation work evaluated DAGL-β as a target to protect against TBI-induced learning and memory deficit given its selective expression on microglia and the role of 2-AG as a precursor for eicosanoid production. The gene deletion of DAGL-β did not protect against TBI-induced MWM or motor deficits, but unexpectedly produced a survival protective phenotype. These findings suggest that while DAGL-β does not contribute to injury-induced memory deficit, it may contribute to TBI-induced mortality. The third and final set of experiments investigated the role of DAGL-α in mouse spatial learning and memory under physiological conditions (given the predominantly neuronal expression of DAGL-α). Complementary pharmacological and genetic manipulations produced task specific impaired MWM performance, as well as impaired long-term potentiation and alterations to endocannabinoid lipid levels. These results suggest that DAGL-α may play a selective role in the integration of new spatial information in the normal mouse brain. Overall, these data point to DAGL-α, but not DAGL-β, as an important contributor to hippocampal-dependent learning and memory. In contrast, DAGL-β may contribute to TBI-induced mortality.

According to the Center for Disease Control’s (CDC) report to the Congress, there are 2.2 million emergency department visits; 80,000 hospitalizations; and 50,000 deaths each year due to traumatic brain injury. Adults 65 years and older account substantially for the majority of the hospitalization and deaths. Over 70% of the traumatic brain injuries of the older adults are classified as mild to moderate; however, even with these milder injuries, older adults present with a significantly higher morbidity and mortality compared to all other age groups (LeBlanc et al., 2006). With that in mind, it seems essential to develop a deeper understanding of the causes behind higher mortality and morbidity of traumatic brain injury in the elder population. It is well documented that increased age is accompanied by increased CNS inflammation. Recently, our laboratory showed that inflammation drives brain pathology. Specifically, we reported that the axon initial segment of cortical neurons was structurally and functionally compromised in an inflamed CNS environment. With this in mind, we proposed that age-related inflammation predisposes that brain to exacerbated pathologic consequence. To test this hypothesis, we administered a mild to moderate central fluid percussion brain injury in aged and young adult mice. Using immunocytochemical labeling against the axon initial segment protein ankyrinG combined with laser scanning confocal microscopy, we quantitatively compared axon initial segment number and length between age groups and within age groups with and without injury. Additionally, we also quantified global axonal pathology by immunolabeling for amyloid precursor protein (APP) positive swelling as an indicator of compromised axonal transport. We proposed that ankyrinG labeling will be both reduced in the aged injured mice compared against aged uninjured, young adult injured and young adult non-injured. We observed a significant increase in APP accumulations due to injury independent of aging, and due to aging independent of injury. No significant changes in the effect of injury between young and aged injured mice were observed. Although AIS length was not altered between age groups following injury, our results demonstrate that the elderly population presents with significantly shorter initial segments. The consequence of this shortening is not clear but may reflect compensatory changes in the brain to maintain homeostasis.

2.5 million people sustain a traumatic brain injury (TBI) annually in the United States. Although there is potential for functional recovery following TBI, there is no definitive treatment to improve recovery after TBI. Our lab has shown that TBI enhances an endogenous neurogenic response in the subventricular zone and hippocampus. TBI-induced neural stem cells (NSCs) can integrate into regions such as the hippocampus and olfactory bulb. Although the mechanism behind TBI-enhanced neurogenesis remains unknown, the Notch signaling pathway has been implicated as a regulator in the maintenance and survival of NSCs. This thesis explores the effects of Notch pathway manipulation on functional recovery following TBI. We hypothesize that Notch signaling plays a critical role in recovery after TBI. Activation of this pathway via a Notch agonist (Notch1) will facilitate post-injury recovery while inhibition of this pathway via a Notch antagonist (recombinant Jagged-1 Fc) will deter post-injury recovery. Functional recovery was assessed within 30 days or 60 days post-injury in two different cohorts of animals. The behavior assays conducted in this study included motor, cognitive, and olfactory assessment. In the 30-day phase, Notch pathway manipulation following TBI did not affect functional performance. In the 60-day study, significant group differences were found. While the FPI+Vehicle animals exhibited a functional recovery in Morris water maze, injured animals with Notch inhibition failed to show this cognitive recovery, indicating the involvement of the Notch pathway in cognitive recovery at the chronic stage following TBI. Motor and olfaction were not significantly affected by Notch pathway manipulation.

Severe traumatic brain injury (TBI) is a pervasive and devastating condition, often resulting in permanent alterations in cognition, behavior, and personality. As such, survivors of severe TBI usually rely on the assistance of caregivers to navigate situations of daily living throughout their life span. Spouses of individuals with TBI have been shown to experience greater levels of burden than parental caregivers of TBI survivors (Kreutzer et al., 1994; Mauss-Clum & Ryan, 1981) and subsequently rates of divorce and separation have been documented within the research literature to be high (Thomsen, 1984; Wood et al., 2005). Although marital breakdown has been frequently studied, research on marital satisfaction and coping within long-term marriages where one spouse has survived a severe TBI remains insufficient. The purpose of this study was to provide a descriptive analysis of long-term spousal caregivers of individuals with severe TBI. First, demographic variables of caregiving spouses of individuals with severe TBI, who remained married at least 10 years post injury, were described and comparisons to other spousal caregiver samples were addressed. Second, the relationship between marital adjustment and demographic variables, coping, and quality of life was examined. Finally, an analysis of primary stressors was conducted to better understand the caregiving spouses' continued experience of stressors. Twenty-one spousal caregivers who remained married for at least 10 years post injury to their spouse who sustained a severe TBI participated in the study. Analyses revealed that spouses had remained married an average of 19 years post-injury and in general were mildly dissatisfied within their marriages. Furthermore, marital adjustment (Dyadic Adjustment Scale total score) of long-term spousal caregivers of severe TBI was not significantly different than spousal caregivers of individuals with chronic illness. Higher marital satisfaction scores were associated with higher endorsement of emotion-focused coping strategies and greater mental health quality of life. In addition, marital satisfaction was positively associated with the longevity of the marital relationship, the age of the spouse at the time of the injury, and the length of the marriage at the time when the injury was sustained. As expected, higher scores of marital satisfaction were associated with higher levels of mental health quality of life. There were no significant relationships between physical health quality of life and marital satisfaction. An analysis of primary stressors show that long-term caregiving spouses continue to report similar types of stressors as indicated in the research literature from caregiving spouses during the first 5 to 8 years post injury. Findings were discussed in relation to implications for practice and additional research.

<p>Traumatic brain injury (TBI) renders the brain more vulnerable to secondary insults. The increased vulnerability can probably be explained by a combination of disturbances in hemodynamics, metabolism and craniospinal dynamics. Reduced ability to compensate for added intracranial volume, i.e. reduced intracranial compliance (IC), is one possible mechanism. The <i>aim</i> of this thesis was to study the role of IC on the effect of secondary insults after TBI. </p><p>A rat TBI model was developed where IC could be altered without causing pathological increases in intracranial pressure (ICP). Reduction of IC was made by placing rubber film between the dura mater and bilateral bone flaps. A reduction of IC in terms of reduced Pressure Volume Index was confirmed. Microdialysis (MD) of extracellular fluid was used to monitor neurochemical changes. Reduced IC after TBI proved to increase the vulnerability of the brain to secondary intracranial volume insults according to neurochemical microdialysis markers. Reduced IC or intracranial volume insults alone did not cause any metabolic changes as compared to controls. Moderate posttraumatic hypotension (50mmHg for 30 min) induced 2 hrs after TBI, did not aggravate posttraumatic extracellular neurochemical changes significantly, irrespective of the level of IC. Although controversial, a mild to moderate hypotensive insult after initial posttraumatic stabilization may not be as detrimental as earlier believed.</p><p>The Spiegelberg Compliance Monitor and MD were simultaneously used in 10 TBI patients to get an impression of the clinical value of IC monitoring and the relationship between IC, temperature and MD Lactate/Pyruvate ratio. IC and MD could be monitored simultaneously in TBI patients. Higher L/P ratios were seen when IC was low. Patients with induced coma treatment had significantly higher average L/P ratios, possibly due to their poorer neurological condition. An indication was also found that in TBI patients with high temperatures, L/P ratio rose as IC decreased, but in patients with low temperature there was no effect of IC on L/P ratio. These data suggest the importance of avoiding hyperthermia in TBI patients, especially in patients with low or decreased IC (monitored or anticipated).</p>

Traumatic brain injury (TBI) renders the brain more vulnerable to secondary insults. The increased vulnerability can probably be explained by a combination of disturbances in hemodynamics, metabolism and craniospinal dynamics. Reduced ability to compensate for added intracranial volume, i.e. reduced intracranial compliance (IC), is one possible mechanism. The aim of this thesis was to study the role of IC on the effect of secondary insults after TBI. A rat TBI model was developed where IC could be altered without causing pathological increases in intracranial pressure (ICP). Reduction of IC was made by placing rubber film between the dura mater and bilateral bone flaps. A reduction of IC in terms of reduced Pressure Volume Index was confirmed. Microdialysis (MD) of extracellular fluid was used to monitor neurochemical changes. Reduced IC after TBI proved to increase the vulnerability of the brain to secondary intracranial volume insults according to neurochemical microdialysis markers. Reduced IC or intracranial volume insults alone did not cause any metabolic changes as compared to controls. Moderate posttraumatic hypotension (50mmHg for 30 min) induced 2 hrs after TBI, did not aggravate posttraumatic extracellular neurochemical changes significantly, irrespective of the level of IC. Although controversial, a mild to moderate hypotensive insult after initial posttraumatic stabilization may not be as detrimental as earlier believed. The Spiegelberg Compliance Monitor and MD were simultaneously used in 10 TBI patients to get an impression of the clinical value of IC monitoring and the relationship between IC, temperature and MD Lactate/Pyruvate ratio. IC and MD could be monitored simultaneously in TBI patients. Higher L/P ratios were seen when IC was low. Patients with induced coma treatment had significantly higher average L/P ratios, possibly due to their poorer neurological condition. An indication was also found that in TBI patients with high temperatures, L/P ratio rose as IC decreased, but in patients with low temperature there was no effect of IC on L/P ratio. These data suggest the importance of avoiding hyperthermia in TBI patients, especially in patients with low or decreased IC (monitored or anticipated).

Traumatic brain injury (TBI) is the most common cause of mortality and disability in the younger (&lt;50 years) Swedish population with an incidence rate of 20,000 cases per year. This thesis aims to increase the understanding of brain injury mechanisms, especially in a molecular and cellular context. Bone morphogenetic protein (BMP) signalling was examined in three genetically modified mice (two “loss-of-function”, one “gain-of-function”) exposed to TBI (controlled cortical impact, CCI) with CaMKII used as promoter for Cre-driven recombination in postnatal forebrain neurons. The mice survived, developed normally and did not show any obvious phenotypes except for an upregulation in Mtap2 mRNA in mice with impaired BMP signalling. Reactive Gfap and Timp1 mRNA expression measured using quantitative RT-PCR (qRT-PCR) was reduced in the mice overexpressing BMP signals. The BMP signalling pathway was further studied in cultured PC12 cells with BMP4 and NGF added. Egr3 expression was substantially increased by these growth factors. Blocking Egr or Junb functions reduced neurite outgrowth. TBI-induced mRNA expression changes in 100 selected genes in C57BL/6J mouse neocortex and hippocampus were measured using qRT-PCR at different time points post-injury. Several distinct gene clusters with similar expression patterns were identified. GeneChip analysis (Affymetrix) of the injured mouse neocortex at three days revealed 146 transcripts significantly upregulated, confirming and extending the qRT-PCR results. The findings demonstrate marked increases after injury among chemokine transcripts and activation of many genes involved in inflammation. In conclusion, the present study has revealed transcriptional changes in specific signalling pathways after brain injury. The results may help to identify novel targets for neuroprotective interventions after traumatic brain injury.

Neural stem and progenitor cells (NSPC) can differentiate to neurons and glial cells. NSPC are easily propagated in vitro and are therefore an attractive tool for tissue regeneration. Traumatic brain injury (TBI) is a common cause for death and disabilities. A fundamental problem following TBI is tissue loss. Animal studies aiming at cell replacement have encountered difficulties in achieving sufficient graft survival and differentiation. To improve outcome of grafted cells after experimental TBI (controlled cortical impact, CCI) in mice, we compared two transplantation settings. NSPC were transplanted either directly upon CCI to the injured parenchyma, or one week after injury to the contralateral ventricle. Enhanced survival of transplanted cells and differentiation were seen when cells were deposited in the ventricle. To further enhance cell survival, efforts were made to reduce the inflammatory response to TBI by administration of ibuprofen to mice that had been subjected to CCI. Inflammation was reduced, as monitored by a decrease in inflammatory markers. Cell survival as well as differentiation to early neuroblasts seemed to be improved. To device a 3D system for future transplantation studies, NSPC from different ages were cultured in a hydrogel consisting of hyaluronan and collagen. Cells survived and proliferated in this culturing condition and the greatest neuronal differentiating ability was seen in cells from the newborn mouse brain. NSPC were also used in a model of peripheral nervous system injury, and xeno-transplanted to rats where the dorsal root ganglion had been removed. Cells survived and differentiated to neurons and glia, furthermore demonstrating their usefulness as a tool for tissue regeneration.

The overall aim of the research presented here was to expand the knowledge on metabolic course and nutritional outcome in patients with severe traumatic brain injury and to analyze the use and accuracy of different methods of assessment. Study I, a systematic review of 30 articles demonstrated consistent data on increased metabolic rate, of catabolism and of upper gastrointestinal intolerance in the majority of the patients during early post injury period. Data also indicated a tendency of less morbidity and mortality in early fed patients. Study II, a retrospective survey, based on medical records of 64 patients from three regions in Sweden, showed that the majority of patients regained their independence in eating within six months post injury. However, energy intake was set at a low level and 68 % of the patients developed malnutrition with 10 to 29 % loss of initial body mass during the first and second month post injury. Study III, a questionnaire based study addressed to 74 care units caring for patients with severe traumatic brain injury showed that resources in terms of qualified staff members were reportedly good, but nutritional guidelines were adopted in less than half of the units, screening for malnutrition at admission was rarely performed and surveillance of energy intake declined when oral intake began. Moreover, assessment of energy requirements relied on calculations and the profession in charge to estimate energy requirement varied depending on nutritional route and unit speciality. At transferral between units nutritional information was lost. Study IV and V, a prospective descriptive study on metabolic course, energy balance and methods of assessment in six patients showed that patients were in negative energy balance from 3rd week post injury and lost 8-19 % of their initial body weight. Concurrent nutritional problems were difficulties in retaining enteral and/or parenteral nutrition delivery routes until oral feeding was considered satisfactory. The majority of methods for predicting energy expenditure agreed poorly with measured energy expenditure.  The Penn-State equation from 1998 was the only valid predictive method during mechanical ventilation. This thesis concludes that patients with moderate or severe traumatic brain injury exhibit a wide range of increased metabolic rate, catabolism and upper gastrointestinal intolerance during the early post-injury period. Most patients regain independence in eating, but develop malnutrition. Suggested explanations, other than the systemic disturbances early post injury, could be the use of inaccurate predictions of energy expenditure, deficient nutritional routines and difficulties in securing alternative nutritional routes until oral feeding is satisfactory. The impact of timing, content and ways of administration of nutritional support on neurological outcome after a severe traumatic brain injury remains to be demonstrated.