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Browsing by Author "Theus, Michelle H."

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    Aberrant hippocampal neurogenesis contributes to learning and memory deficits in a mouse model of repetitive mild traumatic brain injury
    Greer, Kisha (Virginia Tech, 2019-10-02)
    Adult hippocampal neurogenesis, or the process of creating new neurons in the dentate gyrus (DG) of the hippocampus, underlies learning and memory capacity. This cognitive ability is essential for humans to operate in their everyday lives, but cognitive disruption can occur in response to traumatic insult such as brain injury. Previous findings in rodent models have characterized the effect of moderate traumatic brain injury (TBI) on neurogenesis and found learning and memory shortfalls correlated with limited neurogenic capacity. While there are no substantial changes after one mild TBI, research has yet to determine if neurogenesis contributes to the worsened cognitive outcomes of repetitive mild TBI. Here, we examined the effect of neurogenesis on cognitive decline following repetitive mild TBI by utilizing AraC to limit the neurogenic capacity of the DG. Utilizing a BrdU fate-labeling strategy, we found a significant increase in the number of immature neurons that correlate learning and memory impairment. These changes were attenuated in AraC-treated animals. We further identified endothelial cell (EC)-specific EphA4 receptor as a key mediator of aberrant neurogenesis. Taken together, we conclude that increased aberrant neurogenesis contributes to learning and memory deficits after repetitive mild TBI.
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    Abrogation of atypical neurogenesis and vascular-derived EphA4 prevents repeated mild TBI-induced learning and memory impairments
    Greer, Kisha; Gudenschwager-Basso, Erwin K.; Kelly, Colin; Cash, Alison; Kowalski, Elizabeth A.; Cerna, Steven; Ocampo, Collin Tanchanco; Wang, Xia; Theus, Michelle H. (2020-09-21)
    Brain injury resulting from repeated mild traumatic insult is associated with cognitive dysfunction and other chronic co-morbidities. The current study tested the effects of aberrant neurogenesis in a mouse model of repeated mild traumatic brain injury (rmTBI). Using Barnes Maze analysis, we found a significant reduction in spatial learning and memory at 24 days post-rmTBI compared to repeated sham (rSham) injury. Cell fate analysis showed a greater number of BrdU-labeled cells which co-expressed Prox-1 in the DG of rmTBI-injured mice which coincided with enhanced cFos expression for neuronal activity. We then selectively ablated dividing neural progenitor cells using a 7-day continuous infusion of Ara-C prior to rSham or rmTBI. This resulted in attenuation of cFos and BrdU-labeled cell changes and prevented associated learning and memory deficits. We further showed this phenotype was ameliorated in EphA4f.(/f)/Tie2-Cre knockout compared to EphA4f.(/f) wild type mice, which coincided with altered mRNA transcript levels of MCP-1, Cx43 and TGF beta. These findings demonstrate that cognitive decline is associated with an increased presence of immature neurons and gene expression changes in the DG following rmTBI. Our data also suggests that vascular EphA4-mediated neurogenic remodeling adversely affects learning and memory behavior in response to repeated insult.
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    Atypical Neurogenesis, Astrogliosis, and Excessive Hilar Interneuron Loss Are Associated with the Development of Post-Traumatic Epilepsy
    Gudenschwager-Basso, Erwin Kristobal; Shandra, Oleksii; Volanth, Troy; Patel, Dipan C.; Kelly, Colin; Browning, Jack L.; Wei, Xiaoran; Harris, Elizabeth A.; Mahmutovic, Dzenis; Kaloss, Alexandra M.; Correa, Fernanda Guilhaume; Decker, Jeremy; Maharathi, Biswajit; Robel, Stefanie; Sontheimer, Harald; VandeVord, Pamela J.; Olsen, Michelle L.; Theus, Michelle H. (MDPI, 2023-04-25)
    Background: Traumatic brain injury (TBI) remains a significant risk factor for post-traumatic epilepsy (PTE). The pathophysiological mechanisms underlying the injury-induced epileptogenesis are under investigation. The dentate gyrus—a structure that is highly susceptible to injury—has been implicated in the evolution of seizure development. Methods: Utilizing the murine unilateral focal control cortical impact (CCI) injury, we evaluated seizure onset using 24/7 EEG video analysis at 2–4 months post-injury. Cellular changes in the dentate gyrus and hilus of the hippocampus were quantified by unbiased stereology and Imaris image analysis to evaluate Prox1-positive cell migration, astrocyte branching, and morphology, as well as neuronal loss at four months post-injury. Isolation of region-specific astrocytes and RNA-Seq were performed to determine differential gene expression in animals that developed post-traumatic epilepsy (PTE+) vs. those animals that did not (PTE−), which may be associated with epileptogenesis. Results: CCI injury resulted in 37% PTE incidence, which increased with injury severity and hippocampal damage. Histological assessments uncovered a significant loss of hilar interneurons that coincided with aberrant migration of Prox1-positive granule cells and reduced astroglial branching in PTE+ compared to PTE− mice. We uniquely identified Cst3 as a PTE+-specific gene signature in astrocytes across all brain regions, which showed increased astroglial expression in the PTE+ hilus. Conclusions: These findings suggest that epileptogenesis may emerge following TBI due to distinct aberrant cellular remodeling events and key molecular changes in the dentate gyrus of the hippocampus.
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    Cross-Talk and Subset Control of Microglia and Associated Myeloid Cells in Neurological Disorders
    Mills, Jatia; Ladner, Liliana; Soliman, Eman; Leonard, John; Morton, Paul D.; Theus, Michelle H. (MDPI, 2022-10-25)
    Neurological disorders are highly prevalent and often lead to chronic debilitating disease. Neuroinflammation is a major driver across the spectrum of disorders, and microglia are key mediators of this response, gaining wide acceptance as a druggable cell target. Moreover, clinical providers have limited ability to objectively quantify patient-specific changes in microglia status, which can be a predictor of illness and recovery. This necessitates the development of diagnostic biomarkers and imaging techniques to monitor microglia-mediated neuroinflammation in coordination with neurological outcomes. New insights into the polarization status of microglia have shed light on the regulation of disease progression and helped identify a modifiable target for therapeutics. Thus, the detection and monitoring of microglia activation through the inclusion of diagnostic biomarkers and imaging techniques will provide clinical tools to aid our understanding of the neurologic sequelae and improve long-term clinical care for patients. Recent achievements demonstrated by pre-clinical studies, using novel depletion and cell-targeted approaches as well as single-cell RNAseq, underscore the mechanistic players that coordinate microglial activation status and offer a future avenue for therapeutic intervention.
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    Divergent age-dependent peripheral immune transcriptomic profile following traumatic brain injury
    Hazy, Amanda; Bochicchio, Lauren; Oliver, Andrea; Xie, Eric; Geng, Shuo; Brickler, Thomas; Xie, Hehuang David; Li, Liwu; Allen, Irving C.; Theus, Michelle H. (Springer Nature, 2019-06-12)
    The peripheral immune system is a major regulator of the pathophysiology associated with traumatic brain injury (TBI). While age-at-injury influences recovery from TBI, the differential effects on the peripheral immune response remain unknown. Here, we investigated the effects of TBI on gene expression changes in murine whole blood using RNAseq analysis, gene ontology and network topology-based key driver analysis. Genome-wide comparison of CCI-injured peripheral whole blood showed a significant increase in genes involved in proteolysis and oxidative-reduction processes in juvenile compared to adult. Conversely, a greater number of genes, involved in migration, cytokine-mediated signaling and adhesion, were found reduced in CCI-injured juvenile compared to CCI-injured adult immune cells. Key driver analysis also identified G-protein coupled and novel pattern recognition receptor (PRR), P2RY10, as a central regulator of these genes. Lastly, we found Dectin-1, a c-type lectin PRR to be reduced at the protein level in both naive neutrophils and on infiltrating immune cells in the CCI-injured juvenile cortex. These findings demonstrate a distinct peripheral inflammatory profile in juvenile mice, which may impact the injury and repair response to brain trauma.
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    Efferocytosis is restricted by axon guidance molecule EphA4 via ERK/Stat6/MERTK signaling following brain injury
    Soliman, Eman; Leonard, John; Basso, Erwin K. G.; Gershenson, Ilana; Ju, Jing; Mills, Jatia; de Jager, Caroline; Kaloss, Alexandra M.; Elhassanny, Mohamed; Pereira, Daniela; Chen, Michael; Wang, Xia; Theus, Michelle H. (2023-11-09)
    Background Efferocytosis is a process that removes apoptotic cells and cellular debris. Clearance of these cells alleviates neuroinflammation, prevents the release of inflammatory molecules, and promotes the production of anti-inflammatory cytokines to help maintain tissue homeostasis. The underlying mechanisms by which this occurs in the brain after injury remain ill-defined. Methods We used GFP bone marrow chimeric knockout (KO) mice to demonstrate that the axon guidance molecule EphA4 receptor tyrosine kinase is involved in suppressing MERTK in the brain to restrict efferocytosis of resident microglia and peripheral-derived monocyte/macrophages. Results Single-cell RNAseq identified MERTK expression, the primary receptor involved in efferocytosis, on monocytes, microglia, and a subset of astrocytes in the damaged cortex following brain injury. Loss of EphA4 on infiltrating GFP-expressing immune cells improved functional outcome concomitant with enhanced efferocytosis and overall protein expression of p-MERTK, p-ERK, and p-Stat6. The percentage of GFP+ monocyte/macrophages and resident microglia engulfing NeuN+ or TUNEL+ cells was significantly higher in KO chimeric mice. Importantly, mRNA expression of Mertk and its cognate ligand Gas6 was significantly elevated in these mice compared to the wild-type. Analysis of cell-specific expression showed that p-ERK and p-Stat6 co-localized with MERTK-expressing GFP + cells in the peri-lesional area of the cortex following brain injury. Using an in vitro efferocytosis assay, co-culturing pHrodo-labeled apoptotic Jurkat cells and bone marrow (BM)-derived macrophages, we demonstrate that efferocytosis efficiency and mRNA expression of Mertk and Gas6 was enhanced in the absence of EphA4. Selective inhibitors of ERK and Stat6 attenuated this effect, confirming that EphA4 suppresses monocyte/macrophage efferocytosis via inhibition of the ERK/Stat6 pathway. Conclusions Our findings implicate the ERK/Stat6/MERTK axis as a novel regulator of apoptotic debris clearance in brain injury that is restricted by peripheral myeloid-derived EphA4 to prevent the resolution of inflammation.
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    EGR1 recruits TET1 to shape the brain methylome during development and upon neuronal activity
    Sun, Zhixiong; Xu, Xiguang; He, Jianlin; Murray, Alexander; Sun, Ming-an; Wei, Xiaoran; Wang, Xia; McCoig, Emmarose; Xie, Evan; Jiang, Xi; Li, Liwu; Zhu, Jinsong; Chen, Jianjun; Morozov, Alexei; Pickrell, Alicia M.; Theus, Michelle H.; Xie, Hehuang David (2019-08-29)
    Life experience can leave lasting marks, such as epigenetic changes, in the brain. How life experience is translated into storable epigenetic information remains largely unknown. With unbiased data-driven approaches, we predicted that Egr1, a transcription factor important for memory formation, plays an essential role in brain epigenetic programming. We performed EGR1 ChIP-seq and validated thousands of EGR1 binding sites with methylation patterns established during postnatal brain development. More specifically, these EGR1 binding sites become hypomethylated in mature neurons but remain heavily methylated in glia. We further demonstrated that EGR1 recruits a DNA demethylase TET1 to remove the methylation marks and activate downstream genes. The frontal cortices from the knockout mice lacking Egr1 or Tet1 share strikingly similar profiles in both gene expression and DNA methylation. In summary, our study reveals EGR1 programs the brain methylome together with TET1 providing new insight into how life experience may shape the brain methylome.
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    Emerging Developments in Microbiome and Microglia Research: Implications for Neurodevelopmental Disorders
    Lebovitz, Yeonwoo; Ringel-Scaia, Veronica M.; Allen, Irving C.; Theus, Michelle H. (Frontiers, 2018-09-03)
    From immunology to neuroscience, interactions between the microbiome and host are increasingly appreciated as potent drivers of health and disease. Epidemiological studies previously identified compelling correlations between perinatalmicrobiome insults and neurobehavioral outcomes, the mechanistic details of which are just beginning to take shape thanks to germ-free and antibiotics-based animal models. This review summarizes parallel developments from clinical and preclinical research that suggest neuroactive roles for gut bacteria and their metabolites. We also examine the nascent field of microbiome-microglia crosstalk research, which includes pharmacological and genetic strategies to inform functional capabilities of microglia in response to microbial programming. Finally, we address an emerging hypothesis behind neurodevelopmental disorders, which implicates microbiome dysbiosis in the atypical programming of neuroimmune cells, namely microglia.
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    Endothelial Cell-Specific Knockout of Meis1 Protects Ischemic Hindlimb Through Vascular Remodeling
    Chen, Miao (Virginia Tech, 2018-06-28)
    Peripheral artery disease (PAD) affects more than 200 million people worldwide. PAD refers to illness due to a reduction or complete occlusion of blood flow in the artery, especially to the extremities in disease conditions, such as atherosclerosis or diabetes. Critical limb ischemia (CLI) is a severe form of PAD associated with high morbidity and mortality. Currently, no effective and permanent treatments are available for this disease. The current endovascular medications (e.g., angioplasty or stents) only relieve the clinical symptoms while the surgical therapies (e.g., bypass or endarterectomy) require grafting vessels from a healthy organ to the diseased limb of the patient. However, even with these therapeutic techniques, 30% of patients still undergo limb amputation within a year. Thus, understanding of disease mechanism and development of new therapeutic approaches are in urgent needs. Meis1 (myeloid ecotropic viral integration site 1) gene belongs to the three-amino-acid loop extension subclass of homeobox gene families, and it is a highly conserved transcription factor in all eukaryotes. Up to date, little is known about the role of Meis1 in regulating vascular remodeling under ischemic condition. In this study, we aim to investigate the role and underlying mechanism of Meis1 in the regulation of arteriogenesis and angiogenesis using hindlimb ischemia model of transgenic neonatal mice. The long-term goal is to develop a new treatment for patients with PAD. Three separate but related studies were planned to complete the proposed research aims. To better understand the role of Meis1, we reviewed, in the first chapter, all literature relevant to the recent advances of the Meis1 in normal hematopoiesis, vasculogenesis, and heart developments, which were mostly studied in zebrafish and mouse. Briefly, Meis1 is found to be highly expressed in the brain and retina in zebrafish and additional in the heart, nose, and limb in mouse during the very early developmental stage, and remains at a low level quickly after birth. Meis1 is necessary for both primitive and definitive hematopoiesis and required for posterior erythroid differentiation. The absence of Meis1 results in a severe reduction of the number of mature erythrocytes and weakens the heart beats in zebrafish. Meis1 deficiency mouse is dead as early as E11.5 due to the severe internal hemorrhage. In addition, Meis1 is essential in heart development. Knock-down of Meis1 can promote angiotensin II-induced cardiomyocytes (CMs) hypertrophy or CMs proliferation, which can be repressed by a transcription factor Tbx20. Meis1 appears to play a complicated role in the blood vessels. Although the major blood vessels are still normal when global deletion of Meis1, the intersegmental vessel cannot be formed in Meis1 morphants in the zebrafish, and the small vessels are either too narrow or form larger sinuses in Meis1 deficient mouse. The effects of Meis1 on the vascular network under normal and disease (ischemia) condition remain largely unknown, and the existing data in this field is limited. In the second chapter, we developed a method protocol to identify mice of all ages, especially neonates that we faced methodological difficulties to easily and permanently label prior to our major experiments. In this study, single- or 2-color tattooing (ear, tail, or toe or combinations) was performed to identify a defined or unlimited number of mice, respectively. Tail tattooing using both green and red pastes was suitable for identifying white-haired neonatal mice as early as postnatal day (PND) 1, whereas toe tattooing with green paste was an effective alternative approach for labeling black-haired mouse pups. In comparison, single-color (green) or 2-color (green and red) ear tattooing identified both white and black adult mice older than three weeks. Ear tattooing can be adapted to labeling an unlimited number of adult mice by adding the cage number. Thus, tattooing various combinations of the ears, tail, and toes provides an easy and permanent approach for identifying mice of all ages with minimal disturbance to the animals, which shows a new approach than any existing method to identify mouse at all ages, especially the neonatal pups used in the present study (Chapter 4). Various formation of hindlimb ischemia with ligations of femoral artery or vein or both have been reported in the literature. The ischemic severity varies dependent on mouse strains and ligation methods. Due to the tiny body size of our experimental neonatal mice (PND2), it is technically challenging to separate the femoral artery from femoral vein without potential bleeding. In the third chapter, we aimed to explore a suitable surgical approach that can apply to neonatal mice. To this end, we compared the effects of femoral artery/vein (FAV) excision vs. femoral artery (FA) excision on hindlimb model using adult CD-1 mice. We showed during the 4-week period of blood reperfusion, no statistically significant differences were found between FAV and FA excision-induced ischemia regarding the reduction of limb blood flow, paw size, number of necrotic toes, or skeletal muscle cell size. We conclude that FAV and FA excision in CD-1 mice generate a comparable severity of hindlimb ischemia. In other words, FAV ligation is no more severe than FA ligation. These findings provide valuable information for researchers when selecting ligation methods for their neonate hindlimb models. Based on these findings, we selected FAV ligation of hindlimb ischemia approach to study the function of Meis1 in vascular remodeling of neonatal mice. In the fourth chapter (the main part of my dissertation), we investigated the roles of Meis1 in regulating arteriogenesis and angiogenesis of neonatal mouse under the ischemic condition. To this end, endothelial cell-specific deletion of Meis1 was generated by cross-breeding Meis1flox/flox mice with Tie2-Cre mice. Wild-type (WT, Meis1f/f) and endothelial cell-specific knock-out (KO, Meis1ec-/-Tie2-Cre+) C57BL/6 mice at the age of PND2 were used. Under the anesthesia, the pups were subject to hindlimb ischemia by excising FAV. Laser Doppler Imager was used to measure the blood flow pre- and post-surgery up to 28 days. Toe necrosis, skeletal regeneration, and vascular distributions were examined at the end of experiments (PND28 post-ischemia). Surprisingly, during 4-week periods after ischemia, the blood flow ratios (ischemic vs. control limb) in KO mice significantly increased compared to WT on PND14 and PND28, suggesting the inhibitory effects of Meis1 on blood flow recovery under ischemic condition. Meanwhile, WT mice showed more severe necrotic limb (lower ratio of limb length and area, and higher necrotic scores at PND7) than those in the KO mice. Furthermore, significant increases in diameters of Dil-stained arterioles of the skin vessel and the vessels on the ligation site were observed in KO mice, indicating the enhanced arteriogenesis in KO mice. To investigate the underlying mechanism, RNA from the ischemia and control limb was extracted and q-PCR was used to study the potential genes involved in the mechanism. Casp3 and Casp8 were found downregulated showing less apoptosis in the KO mice. On the other hand, endothelial cells (ECs) were isolated from the lungs of 3-5 WT and KO neonates using CD31 Microbeads. CD31+ cells were plated and treated with 0, 0.5, and 1μM doxorubicin for 24 hours and analyzed with various assays. Meis1-KO ECs demonstrated higher cell viability and formed a higher number of vascular tubes than those in WT ECs following 0.5μM Dox treatment, presenting the potential ability of angiogenesis in KO-ECs. Furthermore, the increased viability in KO ECs may be due to the decreased expression or activities of Casp8 and Casp3. In conclusion, my present studies have developed a new methodology to easily and permanently identify all mice at any ages. The insignificant differences between FAV and FA ligations suggest that a relative-easy surgical approach could be used to generate hindlimb ischemic model, which potentially reduces the cost, decreases the surgical time and prevents damage of femoral nerve from surgical tools. More importantly, by using transgenic mice, we found that Meis1-KO dramatically increased blood flow and protected the ischemic hindlimb through vascular remodeling. Obviously, the molecular and cellular mechanisms underlying the above beneficial effects appear complicated and likely to involve multiple cellular remodeling processes and molecular signaling pathways to enhance arteriogenesis and angiogenesis and/or reduce cellular apoptosis through Meis1-mediated pathways. Our study demonstrated that under ischemic condition, knockout of Meis1 increases expression of Hif1a, which then activates Agt or VEGF, thus enhances arteriogenesis or angiogenesis; In addition, knockout of Meis1 activates Ccnd1, which subsequently promotes regeneration of skeletal muscle, and reduces expression of Casp8 and Casp3, thus preventing limb tissue from ischemia-induced apoptosis. Our innovative findings offer great potential to ultimately lead to new drug discovery or therapeutic approaches for prevention or treatment of PAD.
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    Endothelial-Specific EphA4 Negatively Regulates Native Pial Collateral Formation and Re-Perfusion following Hindlimb Ischemia
    Okyere, Benjamin; Giridhar, Kaavya; Hazy, Amanda; Chen, Miao; Keimig, David; Bielitz, Robert C.; Xie, Hehuang; He, Jia-Qiang; Huckle, William R.; Theus, Michelle H. (PLOS, 2016-07-28)
    Leptomeningeal anastomoses play a critical role in regulating vascular re-perfusion following obstruction, however, the mechanisms regulating their development remains under investingation. Our current findings indicate that EphA4 receptor is a novel negative regulator of collaterogenesis. We demonstrate that EphA4 is highly expressed on pial arteriole collaterals at post-natal day (P) 1 and 7, then significantly reduced by P21. Endothelial cell (EC)-specific loss of EphA4, EphA4f/f/Tie2::Cre (KO), resulted in an increase in the density but not diameter of pial collaterals compared to WT mice. ECs isolated from KO mice displayed a 3-fold increase in proliferation, enhanced migration, tube formation and elevated levels of phospho(p)-Akt compared to WT ECs. Attenuating p-Akt, using LY294002, reduced the proliferative and migration effects in the KO ECs. RNAseq analysis also revealed altered expression patterns for genes that regulate cell proliferation, vascular development, extracellular matrix and immune-mediate responses, namely MCP-1, MMP2 and angiopoietin-1. Lastly, we show that induction of hindlimb ischemia resulted in accelerated re-perfusion, collateral remodeling and reduced tissue necrosis in the absence of ECspecific EphA4 compared to WT mice. These findings demonstrate a novel role for EphA4 in the early development of the pial collateral network and suggests a role in regulating vascular remodeling after obstruction.
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    Eph-mediated restriction of cerebrovascular arteriogenesis
    Okyere, Benjamin (Virginia Tech, 2019-04-26)
    Stroke is a leading cause of morbidity and long-term neurological disability in the U.S. Ischemic stroke, which accounts for approximately 90% of all strokes, is the result of an occlusion in the arteriole cerebrovascular network. No effective treatment options exist to provide neuroprotection from occlusion, and limited success has been seen clinically when attempting to restore blood flow to vulnerable neural tissue regions. Enhancement of pial collateral remodeling (Arteriogenesis) has recently been shown to improve blood flow and mitigate neural tissue damage following stroke (1-3). Arteriogenesis is the remodeling of pre-existing arteriole vessel which are able to re-route blood to blood-deprived regions of tissue. Arteriogenesis requires endothelial cell (EC) and smooth muscle cell proliferation, extracellular matrix degradation and recruitment of circulating bone marrow-derived cells (4-6). Unlike spouting angiogenesis, which requires weeks following occlusion to develop, arteriogenesis begins as early as 24-48hrs post-stroke (7, 8) and can expeditiously enhance blood flow to ischemic regions, making it an attractive target for therapeutic intervention. Our preliminary studies, in an EphA4 global knockout mouse model, indicated that EphA4 receptor tyrosine kinase severely limits pial arteriole collateral formation. The preliminary work also showed that activation of EC EphA4 receptor in vitro inhibited vascular formation. Additionally, ECs lining the collateral vessel have been shown to play a role in collateral remodeling (9). Taken together, the objective of this dissertation was to elucidate the cell autonomous role of the EphA4 receptor and given the central role of the EC in collateral remodeling, we postulated that EphA4 receptor on ECs the limits pial collateral formations. Using a cell-specific loss-of-function approach, we tested the hypothesis that EC-specific EphA4 plays an important role in pial collateral development and remodeling after induced stroke. The results from this dissertation show that (1) EphA4 expression on ECs suppress the formation of pial collaterals during development and limits EC growth via suppression of p-Akt in vitro (2) EC-specific EphA4 ablation leads to increased collateral remodeling, enhanced blood flow recovery, tissue protection and improved neurological behavioral outcomes after stroke and (3) Mechanistically, EphA4 limits pial collateral remodeling via attenuation of the Tie2/Angiopoietin-2 signaling pathway. The work presented in this dissertation demonstrate that EphA4 can be targeted therapeutically to increase pial collateral remodeling to alleviate neurological deficits after ischemic stroke.
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    EphA4 Influences Blood Brain Barrier Disruption and Endothelial Cell Response following Traumatic Brain Injury in a Mouse Model
    Cash, Alison M. (Virginia Tech, 2022)
    An astonishing number of deaths and related disabilities are attributed to traumatic brain injury (TBI) in the United States per year. Due to the unforeseeable nature of TBI and its association with the sequelae of other neurological comorbidities, research is centered around the secondary responses of brain mechanisms proceeding the initial mechanical injury. Blood brain barrier disruption is a well described driver of this secondary injury response and predictive marker of prognosis following TBI. Although BBB disruption plays a role in subsequent edema, inflammation, and the overall TBI outcome, the molecular mechanisms responsible for its regulation remain to be investigated. A large family of receptor tyrosine kinases, known as Eph receptors, that are important for axon growth and guidance embryonically and early-postnatally have been implicated in brain insults. Previous findings have shown that Eph expression is upregulated at the mRNA and protein level immediately following TBI. Moreover, ablation of Eph receptors on endothelial cells (ECs) revealed improved blood flow to the lesioned cortex in knockout (KO) mice compared to wild type (WT). Based on these results, we hypothesize that Eph receptors negatively regulate BBB permeability leading to neural dysfunction and motor deficits following TBI. To investigate this hypothesis, we characterized the temporal profile of the BBB, evaluated the EC-specific effects of Eph receptors, and used RNA sequencing to assess the cell-specific contributions following TBI in WT compared to KO mice. Our results show that EC-specific loss of Eph expression ameliorated BBB permeability at 6hr, 1-, 4-, and 7-days post injury (dpi) correlating with improved motor function at 7- and 14-dpi. Furthermore, mechanistic studies revealed increased mRNA expression of Tie2, Ang1, and the tight junction proteins Zona Occludens and Occludin in KO mice compared to WT. As well as, connection with neuronal processes. Based off of these findings, we utilized a soluble Tie2 inhibitor to elucidate the influence of Eph receptors on the Tie2/Ang pathway, and their role in mediating the effects seen. Tie2 inhibition of the KO mice revealed similar BBB disruption and lesion volume as WT 1dpi, attenuating the previous protection KO mice demonstrated. Future studies are necessary to understand other pathways that may be implicated in Eph receptor influence on endothelial cells such as inflammatory mediators and neurovascular crosstalk. This data provides evidence that Eph receptors negatively mediate EC response through downstream signaling of the Tie2/Ang pathway and may be a means of therapeutic target in the future.
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    Extracellular Matrix Contributions to Early Vascular Development and Pericyte Precursor Dynamics
    Hoque, Maruf M. (Virginia Tech, 2023-07-24)
    The vasculature is a highly intricate system of "highways" that shuttles blood from the heart to every tissue and organ in the human body. These vessels are responsible for carrying oxygen, trafficking hormones, delivering nutrients, and removing waste products from the body. The formation of a functioning vascular system depends on the close coordination of many cell types and, on the capillary level, specifically endothelial cells and pericytes as well as the surrounding protein microenvironment, known as the extracellular matrix (ECM). Impaired coordination amongst the cellular and protein constituents results in the improper functioning of the vascular network and can eventually contribute to the failure of organ systems. This dissertation research focuses on how improper ECM deposition affects vascular assembly. We utilized several approaches to affect ECM composition, specifically: 1) hypoxia exposure and 2) reducing ECM pharmacologically and utilizing lentiviral-mediated silencing of Type IV Collagen (Col-IV, gene Col4a1) expression. In these experimental settings, we observed downstream changes in the coordination between endothelial cells and pericytes while forming vascular networks. In short, this dissertation work suggests that excess ECM deposition, and particularly that of Col-IV, has unique deleterious effects on the developing vasculature as compared to reduced ECM deposition. The findings from this work suggest mechanisms underlying how the vasculature may be destabilized in hypoxia-associated pathologies, such as preeclampsia.
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    Immunoregulatory and neutrophil-like monocyte subsets with distinct single-cell transcriptomic signatures emerge following brain injury
    Gudenschwager Basso, Erwin K.; Ju, Jing; Soliman, Eman; de Jager, Caroline; Wei, Xiaoran; Pridham, Kevin J.; Olsen, Michelle L.; Theus, Michelle H. (2024-02-03)
    Monocytes represent key cellular elements that contribute to the neurological sequela following brain injury. The current study reveals that trauma induces the augmented release of a transcriptionally distinct CD115+/Ly6Chi monocyte population into the circulation of mice pre-exposed to clodronate depletion conditions. This phenomenon correlates with tissue protection, blood–brain barrier stability, and cerebral blood flow improvement. Uniquely, this shifted the innate immune cell profile in the cortical milieu and reduced the expression of pro-inflammatory Il6, IL1r1, MCP-1, Cxcl1, and Ccl3 cytokines. Monocytes that emerged under these conditions displayed a morphological and gene profile consistent with a subset commonly seen during emergency monopoiesis. Single-cell RNA sequencing delineated distinct clusters of monocytes and revealed a key transcriptional signature of Ly6Chi monocytes enriched for Apoe and chitinase-like protein 3 (Chil3/Ym1), commonly expressed in pro-resolving immunoregulatory monocytes, as well as granule genes Elane, Prtn3, MPO, and Ctsg unique to neutrophil-like monocytes. The predominate shift in cell clusters included subsets with low expression of transcription factors involved in monocyte conversion, Pou2f2, Na4a1, and a robust enrichment of genes in the oxidative phosphorylation pathway which favors an anti-inflammatory phenotype. Transfer of this monocyte assemblage into brain-injured recipient mice demonstrated their direct role in neuroprotection. These findings reveal a multifaceted innate immune response to brain injury and suggest targeting surrogate monocyte subsets may foster tissue protection in the brain.
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    The Influence of Biomechanics on Acute Spatial and Temporal Pathophysiology Following Blast-Induced Traumatic Brain Injury
    Norris, Caroline Nicole (Virginia Tech, 2023-06-21)
    Blast-induced traumatic brain injury (bTBI) remains a significant problem among military populations. When an explosion occurs, a high magnitude positive pressure rapidly propagates away from the detonation source. Upon contact, biological tissues throughout the body undergo deformation at high strain rates and then return to equilibrium following a brief negative pressure phase. This mechanical disruption of the tissue is known to cause oxidative stress and neuroinflammation in the brain, which can lead to neurodegeneration and consequently poor cognitive and behavioral outcomes. Further, these clinical outcomes, which can include chronic headaches, problems with balance, light and noise sensitivity, anxiety, and depression, may be sustained years following blast exposure and there are currently no effective treatments. Thus, there is a need to investigate the acute molecular responses following bTBI in order to motivate the development of effective therapeutic strategies and ultimately improve or prevent long-term patient outcomes. It is important to not only understand the acute molecular response, but how the brain tissue mechanics drive these metabolic changes. The objective of this work was to identify the interplay between the tissue-level biomechanics and the acute bTBI pathophysiology. In a rodent bTBI model, using adult rats, intracranial pressure was mapped throughout the brain during blast exposure where frequency contributions from skull flexure and wave dynamics were significantly altered between brain regions and were largely dependent on blast magnitude. These findings informed the subsequent spatial and temporal changes in neurometabolism. Amino acid molecular precursor concentrations decreased at four hours post-blast in the cortex and hippocampus regions. This motivates further investigation of amino acids as therapeutic targets aimed to reduce oxidative stress and prevent prolonged injury cascades. However, neurochemical changes were not consistent across blast magnitudes, which may be explained by the disparities in biomechanics at lower blast pressures. Lastly, we investigated the acute changes in metabolic regulators influencing excitotoxicity where it was found that astrocytes maintained normal clearance of excitatory and inhibitory neurotransmitters prior to astrocyte reactivity. Outcomes of this work provide improved understanding of blast mechanics and associated acute pathophysiology and inform future therapeutic and diagnostic approaches following bTBI.
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    Integrative Analyses of Environmental Factors Impacting Animal and Human Health Through Perturbations of Microbial Communities
    Caswell, Clayton C.; Ahmed, S. Ansar; Sriranganathan, Nammalwar; Allen, Irving C.; Luo, Xin; Meng, Xiang-Jin; Theus, Michelle H.; Yuan, Lijuan; Hungerford, Laura L.; Pierson, Bill; Rist, Cassidy (Virginia Tech, 2017-05-15)
    The term ‘microbiome’ defines the vast microscopic communities collectively composed of bacteria, viruses, fungi, and eukaryotic protozoans that inhabit myriad niches, including environmental locales, as well as the surfaces and organ systems of animals and humans. Recent empirical evidence clearly demonstrates the substantial role that microbiomes play in facilitating the homeostasis of complex biological systems, and as such, perturbation of these microbial communities can lead to dysregulation of environmental ecosystems, significant declines in animal and human health, and the emergence of detrimental conditions, such as infectious diseases, inflammatory disorders, and neurodegenerative aliments. A variety of factors are involved in shifting the composition and complexity (i.e., the functionality) of microbiomes, including the contamination of soil, water, and food sources with toxicants, pharmaceuticals, and antimicrobial compounds...
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    Investigating Novel Targets to Inhibit Cancer Cell Survival
    Pridham, Kevin J. (Virginia Tech, 2018-04-18)
    Cancer remains the second leading cause of death in the United States and the world, despite years of research and the development of different treatments. One reason for this is cancer cells are able to survive through adaptation to their environment and aberrantly activated growth signaling. As such, developing new therapies that overcome these hurdles are necessary to combat cancer. Previous work in our laboratory using RNA interference screening identified genes that regulate the survival of glioblastoma (GBM) or autophagy in chronic myelogenous leukemia (CML) cancer cells. One screen identified Phosphatidylinositol-4,5-bisophosphate 3-kinase catalytic subunit beta (PIK3CB) in the family of Phosphatidylinositol 3-kinases (PI3K) as a survival kinase gene in GBM. Work contained in this dissertation set out to study PIK3CB mediated GBM cell survival. We report that only PIK3CB, in its family of other PI3K genes, is a biomarker for GBM recurrence and is selectively important for GBM cell survival. Another screen identified the long non-coding RNA, Linc00467, as a gene that regulates autophagy in CML. Autophagy is a dynamic survival process used by all cells, benign and cancerous, where cellular components are broken down and re-assimilated to sustain survival. Work contained in this dissertation set out to characterize the role that Linc00467 serves in regulating autophagy in a myriad of cancers. Collectively our data have showed Linc00467 to actively repress levels of autophagy in cancer cells. Further, our data revealed an important role for Linc00467 in regulating the stability of the autophagy regulating protein serine-threonine kinase 11 (STK11). Because of the unique role that Linc00467 serves in regulating autophagy we renamed it as, autophagy regulating long intergenic noncoding RNA or ARLINC. Taken together the work in this dissertation unveils the inner-workings of two important cancer cell survival pathways and shows their potential for development into therapeutic targets to treat cancer.
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    Leptomeningeal anastomoses: Mechanisms of pial collateral remodeling in ischemic stroke
    Kaloss, Alexandra M.; Theus, Michelle H. (Wiley, 2022-02-03)
    Arterial collateralization, as determined by leptomeningeal anastomoses or pial collateral vessels, is a well-established vital player in cerebral blood flow restoration and neurological recovery from ischemic stroke. A secondary network of cerebral collateral circulation apart from the Circle of Willis, exist as remnants of arteriole development that connect the distal arteries in the pia mater. Recent interest lies in understanding the cellular and molecular adaptations that control the growth and remodeling, or arteriogenesis, of these pre-existing collateral vessels. New findings from both animal models and human studies of ischemic stroke suggest a multi-factorial and complex, temporospatial interplay of endothelium, immune and vessel-associated cell interactions may work in concert to facilitate or thwart arteriogenesis. These valuable reports may provide critical insight into potential predictors of the pial collateral response in patients with large vessel occlusion and may aid in therapeutics to enhance collateral function and improve recovery from stroke. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology
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    Mechanisms of Blood–Brain Barrier Dysfunction in Traumatic Brain Injury
    Cash, Alison; Theus, Michelle H. (MDPI, 2020-05-08)
    Traumatic brain injuries (TBIs) account for the majority of injury-related deaths in the United States with roughly two million TBIs occurring annually. Due to the spectrum of severity and heterogeneity in TBIs, investigation into the secondary injury is necessary in order to formulate an effective treatment. A mechanical consequence of trauma involves dysregulation of the blood–brain barrier (BBB) which contributes to secondary injury and exposure of peripheral components to the brain parenchyma. Recent studies have shed light on the mechanisms of BBB breakdown in TBI including novel intracellular signaling and cell–cell interactions within the BBB niche. The current review provides an overview of the BBB, novel detection methods for disruption, and the cellular and molecular mechanisms implicated in regulating its stability following TBI.
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    Modeling Neural Stem Cell Dynamics in Congenital Heart Disease
    Porter, Demisha Donei Lasha (Virginia Tech, 2023-06-28)
    Neural stem/progenitor cells (NSPCs) play a crucial part in the evolutionary development of the human neocortex. During early postnatal development, NSPCs give rise to immature neurons called neuroblasts within the subventricular zone (SVZ) that utilize unique migratory streams to integrate widely in the cerebral cortex. However, the cellular mechanisms enabling these unique migratory routes through the compacted cellular landscape remain unknown. Special emphasis has been placed on understanding the susceptibility of these brain regions to severe conditions such as congenital heart disease (CHD), resulting in poor neurological outcomes. Owing to its reminiscent complexity to humans, the neonatal piglet (Sus scrofa domesticus), which possesses a highly evolved gyrencephalic neocortex and an expansive outer SVZ, provides a powerful translational model system for the study of how heart dysfunction impacts cortical development from both a modern and evolutionary perspective. The present study provides a detailed characterization of neuroblast migration along their associate substrates in the piglet cortex under normal physiological conditions and how reduced oxygenation (i.e., hypoxia) can impact their vulnerability and/or resistance to injury during a critical period of postnatal development. In this thesis, I investigated the spatiotemporal distribution and developmental origin of SVZ-derived neuroblasts. Following BrdU tracing, multiplex labeling, and confocal microscopy, I show that the porcine brain contains populations of newly generated (BrdU+/DCX+) neurons in the prefrontal cortex that are produced postnatally. Regional analyses using immunohistochemical staining for doublecortin (DCX), a marker expressed by immature neurons, revealed that DCX+ clusters co-express markers of neuronal cell migration (PSA-NCAM), GABAergic interneuron marker (GABA+), and specific transcription factors (SCGN+SP8+) associated with the caudal- and lateral ganglionic eminence progenitor domains in the ventral forebrain. Moreover, I found that DCX+ neuroblasts are encased by astrocytic processes and tightly associated with blood vessels in the SVZ. Additionally, this thesis describes the use of chronic hypoxia as a model to profile neuroblast migration along associated substrates in pathological conditions related to CHD. Together, this work serves as a framework for the functional utilization of the neonatal piglet to understand the impact of substrate-dependent neuronal migration on brain maturation and neurodevelopmental diseases.