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- All Subjects: Microglia
- All Subjects: RASopathies
- Creators: Newbern, Jason
- Creators: Hilbert, Alexander Robert
Neuroinflammation Following Experimental Diffuse Brain Injury in Pre-pubertal and Peri-pubertal Rats
Description
Traumatic brain injury is the leading cause of mortality and morbidity in children and adolescents. Adolescence is a critical time in development where the body and brain undergoes puberty, which not only includes reproductive maturation, but also adult social and cognitive development. Brain-injury-induced disruptions can cause secondary inflammation processes and as a result, pediatric TBI can lead to significant life-long and debilitating morbidities that continue long after initial injury. In this study, neuroinflammation following diffuse brain injury was explored in prepubertal and peripubertal rats using an adapted method of midline fluid percussion injury (mFPI) for juvenile rats to further understand the relationship between pediatric TBI and puberty disruption due to endocrine dysfunction. We expect the adapted mFPI model to be effective in producing diffuse, moderate brain injury in juvenile rats and hypothesize that pre-pubertal rats (PND35) will have increased neuroinflammation compared to peri-pubertal rats (PND17) and shams because of the potential neuroprotective nature of sex steroids. Male Sprague-Dawley rats (n=90) were subjected to either a diffuse midline fluid percussion injury (mFPI) or sham injury at post-natal day (PND) 17 (pre-puberty) or PND35 (peri-puberty). Animals were sacrificed at different time points defined as days post injury (DPI) including 1DPI, 7DPI and 25DPI to represent both acute and chronic time points, allowing for comparisons within groups (injury vs. sham) and across groups (PND17 vs PND35). Body weight of the rats was measured postoperatively at various time points throughout the study to follow recovery. Tissue was collected and subjected to Heamatoxylin and Eosin (H&E) stain to visualize histology and evaluate the application of diffuse mFPI to juvenile rats. In addition, tissue underwent immunohistochemical analysis using 3,3'-diaminobenzidine (DAB) to stain for ionized calcium binding proteins (Iba1) in order to assess injury-related neuroinflammation in the form of microglia activation. Diffuse brain injury using the mFPI model did not affect rat body weight or cause overt cell death, suggesting adaption of the adult mFPI model for juvenile rats is representative of moderate diffuse brain injury. In addition, diffuse TBI lead to morphological changes in microglia suggesting there is an increased inflammatory response following initial insult, which may directly contribute to improper activation of pubertal timing and progression in adolescent children affected. Since there is little literature on the full effects of puberty dysfunction following TBI in the pediatric population, there is a significant need to further assess this area in order to develop improved interventions and potential therapies for this affected population.
ContributorsNewbold, Kelsey Bevier (Author) / Newbern, Jason (Thesis director) / Rowe, Rachel (Committee member) / Ortiz, J. Bryce (Committee member) / School of Mathematical and Natural Sciences (Contributor) / Department of Psychology (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Rasopathies are a family of developmental syndromes that exhibit craniofacial abnormalities, cognitive disabilities, developmental delay and increased risk of cancer. However, little is known about the pathogenesis of developmental defects in the nervous system. Frequently, gain-of-function mutations in the Ras/Raf/MEK/ERK cascade (aka ERK/MAPK) are associated with the observed pathogenesis. My research focuses on defining the relationship between increased ERK/MAPK signaling and its effects on the nervous system, specifically in the context of motor learning. Motor function depends on several neuroanatomically distinct regions, especially the spinal cord, cerebellum, striatum, and cerebral cortex. We tested whether hyperactivation of ERK/MAPK specifically in the cortex was sufficient to drive changes in motor function. We used a series of genetically modified mouse models and cre-lox technology to hyperactivate ERK/MAPK in the cerebral cortex. Nex:Cre/NeuroD6:Cre was employed to express a constitutively active MEK mutation throughout all layers of the cerebral cortex from an early stage of development. RBP4:Cre, caMEK only exhibited hyper activation in cortical glutamatergic neurons responsible for cortical output (neurons in layer V of the cerebral cortex). First, the two mouse strains were tested in an open field paradigm to assess global locomotor abilities and overall fitness for fine motor tasks. Next, a skilled motor reaching task was used to evaluate motor learning capabilities. The results show that Nex:Cre/NeuroD6:Cre, caMEK mutants do not learn the motor reaching task, although they performed normally on the open field task. Preliminary results suggest RBP4:Cre, caMEK mutants exhibit normal locomotor capabilities and a partial lack of learning. The difference in motor learning capabilities might be explained by the extent of altered connectivity in different regions of the corticospinal tract. Once we have identified the neuropathological effects of various layers in the cortex we will be able to determine whether therapeutic interventions are sufficient to reverse these learning defects.
ContributorsRoose, Cassandra Ann (Author) / Newbern, Jason M. (Thesis director) / Olive, Foster (Committee member) / Bjorklund, Reed (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Neuroinflammation is an important secondary injury response occurring after traumatic brain injury (TBI). Anxiety-like disorders are commonly exacerbated after TBI and are mediated through the amygdala; however, the amygdala remains understudied despite its important contribution in processing emotional and stressful stimuli. Therefore, we wanted to study neuroinflammation after experimental TBI using midline fluid percussion in rodent models. We assessed microglia morphology over time post-injury in two circuit related nuclei of the amygdala, the basolateral amygdala (BLA) and central amygdala of the nucleus (CeA), using skeletal analysis. We also looked at silver staining and glial fibrillary acidic protein (GFAP) to evaluate the role of neuropathology and astrocytosis to evaluate for neuroinflammation in the amygdala. We hypothesized that experimental diffuse TBI leads to microglial activation in the BLA-CeA circuitry over time post-injury due to changes in microglial morphology and increased astrocytosis in the absence of neuropathology. Microglial cell count was found to decrease in the BLA at 1 DPI before returning to sham levels by 28 DPI. No change was found in the CeA. Microglial ramification (process length/cell and endpoints/cell) was found to decrease at 1DPI compared to sham in the CeA, but not in the BLA. Silver staining and GFAP immunoreactivity did not find any evidence of neurodegeneration or activated astrocytes in the respectively. Together, these data indicate that diffuse TBI does not necessarily lead to the same microglial response in the amygdala nuclei, although an alternative mechanism for a neuroinflammatory response in the CeA likely contributes to the widespread neuronal and circuit dysfunction that occurs after TBI.
ContributorsHur, Yerin (Author) / Newbern, Jason (Thesis director) / Thomas, Theresa Currier (Committee member) / Beitchman, Joshua (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
The RAS/MAPK (RAS/Mitogen Activated Protein Kinase) pathway is a highly conserved, canonical signaling cascade that is highly involved in cellular growth and proliferation as well as cell migration. As such, it plays an important role in development, specifically in development of the nervous system. Activation of ERK is indispensable for the differentiation of Embryonic Stem Cells (ESC) into neuronal precursors (Li z et al, 2006). ERK signaling has also shown to mediate Schwann cell myelination of the peripheral nervous system (PNS) as well as oligodendrocyte proliferation (Newbern et al, 2011). The class of developmental disorders that result in the dysregulation of RAS signaling are known as RASopathies. The molecular and cell-specific consequences of these various pathway mutations remain to be elucidated. While there is evidence for altered DNA transcription in RASopathies, there is little work examining the effects of the RASopathy-linked mutations on protein translation and post-translational modifications in vivo. RASopathies have phenotypic and molecular similarities to other disorders such as Fragile X Syndrome (FXS) and Tuberous Sclerosis (TSC) that show evidence of aberrant protein synthesis and affect related pathways. There are also well-defined downstream RAS pathway elements involved in translation. Additionally, aberrant corticospinal axon outgrowth has been observed in disease models of RASopathies (Xing et al, 2016). For these reasons, this present study examines a subset of proteins involved in translation and translational regulation in the context of RASopathy disease states. Results indicate that in both of the tested RASopathy model systems, there is altered mTOR expression. Additionally the loss of function model showed a decrease in rps6 activation. This data supports a role for the selective dysregulation of translational control elements in RASopathy models. This data also indicates that the primary candidate mechanism for control of altered translation in these modes is through the altered expression of mTOR.
ContributorsHilbert, Alexander Robert (Author) / Newbern, Jason (Thesis director) / Olive, M. Foster (Committee member) / Bjorklund, Reed (Committee member) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
The ERK1/2 cell signaling pathway is highly conserved and a prominent regulator of processes like cell proliferation, differentiation, and survival. During nervous system development, the ERK1/2 cascade is activated by the binding of growth factors to receptor tyrosine kinases, leading to the sequential phosphorylation of intracellular protein kinases in the pathway and eventually ERK1 and ERK2, the effectors of the pathway. Well-defined germline mutations resulting in hyperactive ERK1/2 signaling have been implicated in a group of neurodevelopmental disorders called RASopathies. RASopathic individuals often display features such as developmental delay, intellectual disability, cardio-facial abnormalities, and motor deficits. In addition, loss-of-function in ERK1/2 can lead to neurodevelopmental disorders such as autism spectrum disorder (ASD) and intellectual disability. To better understand the pathology of these neurodevelopmental disorders, the role of ERK1/2 must be examined during the development of specific neuronal and glial subtypes. In this study, we bred transgenic mice with conditional deletion of ERK1/2 in cholinergic neuronal populations to investigate whether ERK1/2 mediates the survival or activity of basal forebrain and striatal cholinergic neurons during postnatal development. By postnatal day 10, we found that ERK1/2 did not seem to mediate cholinergic neuron number within the basal forebrain or striatum. In addition, we showed that expression of FosB, a neuronal activity-dependent transcription factor and target of ERK1/2, was not yet observed in cholinergic neurons within either of these anatomical regions by P10. Finally, our preliminary data suggested that FosB expression within layer IV of the somatosensory cortex, a target domain for basal forebrain cholinergic projections, also did not appear to be mediated by ERK1/2 signaling. However, since cholinergic neuron development is not yet complete by P10, future work should explore whether ERK1/2 plays any role in the long-term survival and function of basal forebrain and striatal cholinergic neurons in adulthood. This will hopefully provide more insight into the pathology of neurodevelopmental disorders and inform future therapeutic strategies.
ContributorsBalasubramanian, Kavya (Author) / Newbern, Jason (Thesis director) / Velazquez, Ramon (Committee member) / Rees, Katherina (Committee member) / Barrett, The Honors College (Contributor) / Department of Psychology (Contributor)
Created2023-05
Description
Of the 2.87 million traumatic brain injuries (TBI) sustained yearly in the United States, 75% are diffuse injuries. A single TBI can have acute and chronic influences on the neuroendocrine system leading to hypothalamic-pituitary-adrenal axis (HPA) dysregulation and increased affective disorders. Preliminary data indicate TBI causes neuroinflammation in the hippocampus, likely due to axonal damage, and in the paraventricular nucleus of the hypothalamus (PVN), where no axonal damage is apparent. Mechanisms regulating neuroinflammation in the PVN are unknown. Furthermore, chronic stress causes HPA dysregulation and glucocorticoid receptor (GR)-mediated neuroinflammation in the PVN. The goal of this project was to evaluate neuroinflammation in the HPA axis and determine if GR levels change at 7 days post-injury (DPI).
Adult male and female Sprague Dawley rats were subjected to midline fluid percussion injury. At 7 DPI, half of each brain was post-fixed for immunohistochemistry (IBA-1) and half biopsied for gene/protein analysis. IBA-1 staining was analyzed for microglia activation via skeleton analysis in the hypothalamus and hippocampus. Extracted RNA and protein were used to quantify mRNA expression and protein levels for GRs. Data indicate increased microglia cell number and decreased endpoints/cell and process length in the PVN of males, but not females. In the dentate gyrus, both males and females have an increased microglia cell number after TBI, but there is also an interaction between sex and injury in microglia presentation, where males exhibit a more robust effect than females. Both sexes have significant decreases of endpoints/cell and process length. In both regions, GR protein levels decreased for injured males, but in the hippocampus, GR levels increased for injured females. Data indicate that diffuse TBI causes alterations in microglia morphology and GR levels in the hypothalamus and hippocampus at 7 DPI, providing a potential mechanism for HPA axis dysregulation at a sub-acute time point.
Adult male and female Sprague Dawley rats were subjected to midline fluid percussion injury. At 7 DPI, half of each brain was post-fixed for immunohistochemistry (IBA-1) and half biopsied for gene/protein analysis. IBA-1 staining was analyzed for microglia activation via skeleton analysis in the hypothalamus and hippocampus. Extracted RNA and protein were used to quantify mRNA expression and protein levels for GRs. Data indicate increased microglia cell number and decreased endpoints/cell and process length in the PVN of males, but not females. In the dentate gyrus, both males and females have an increased microglia cell number after TBI, but there is also an interaction between sex and injury in microglia presentation, where males exhibit a more robust effect than females. Both sexes have significant decreases of endpoints/cell and process length. In both regions, GR protein levels decreased for injured males, but in the hippocampus, GR levels increased for injured females. Data indicate that diffuse TBI causes alterations in microglia morphology and GR levels in the hypothalamus and hippocampus at 7 DPI, providing a potential mechanism for HPA axis dysregulation at a sub-acute time point.
ContributorsRidgway, Samantha (Author) / Thomas, Theresa C (Thesis advisor) / Newbern, Jason (Thesis advisor) / Bimonte-Nelson, Heather A. (Committee member) / Arizona State University (Publisher)
Created2019