Kimberly
R. Byrnes
Neuroscience Program
Doctor
of Philosophy
2003
Major Advisor: Juanita J. Anders, Ph.D., Associate Professor of Anatomy, Physiology, and Genetics, and Neuroscience
Dissertation Title: 810 NM Light Treatment of Acute Spinal Cord Injury Alters the Immune Response and Improves Axonal Regeneration and Functional Recovery
ABSTRACT
Spinal cord injury (SCI) results in substantial and often permanent impairment of function due to the lack of regeneration of damaged axons. Despite vigorous research, no cure for SCI has been found. Light therapy (LT), through the absorption of light by target tissue, improves healing in a number of injury models. However, no study to date has assessed the ability of LT to facilitate the regeneration of specific spinal cord tracts. Our hypothesis was that transcutaneous application of 810 nm light promotes axonal regeneration and functional reinnervation following transection of the corticospinal tract (CST) by changing the extracellular milieu of the spinal cord. Three studies were implemented to investigate this hypothesis. First, anterograde and retrograde tract tracing techniques were used to investigate axonal regrowth after SCI and LT. LT (810 nm) was applied at the site of acute injury to the CST of adult rats. Anterograde tract tracing demonstrated that LT improved axonal regrowth after injury, with significant increases in axon number (199 +/- 12) and distance of regrowth (8.7 +/- 0.8 mm) as compared to controls (p<0.01). Double label retrograde tract tracing revealed that transected axons regrew and reinnervated motor neurons in the lumbar spinal cord in the light treated group only (p<0.05). Functional analyses revealed that this regeneration was coupled with significant improvement in 2 tests of CST performance, angle of rotation and ladder beam cross time (p<0.05). Second, to explore the effect of LT on the spinal cord cellular environment, we investigated the inflammatory response after SCI, using quantitative immunohistochemistry techniques. This study revealed that LT suppressed the invasion/activation of macrophages, microglia and T lymphocytes after SCI (p<0.001) and delayed the activation of astrocytes. The third study explored gene expression after SCI and LT. A number of cytokines and chemokines were assessed using reverse transcriptase-polymerase chain reaction (RT-PCR). Expression of interleukin 6, monocyte chemoattractant protein 1 (MCP-1) and inducible nitric oxide synthase (iNOS) was suppressed at 6 hours post-injury by LT (p<0.01). These results demonstrate that LT has an anti-inflammatory effect on the spinal cord after injury and significantly improves axonal regeneration and functional recovery.
Doctor of
Philosophy
2003
Major Advisor: Joseph T. McCabe, Ph.D., Vice-Chair of Department of Anatomy, Physiology & Genetics; and Professor of Molecular and Cellular Biology, and Neuroscience
Dissertation Title: Cullin 5 Expression in the Rat: Cellular and Tissue Distribution, and Changes in Response to Water Deprivation and Hemorrhagic Shock
ABSTRACT
Protein degradation by ubiquitination and the 26S proteasome is used to modulate
the steady-state levels of proteins and to regulate cellular processes. Proteins
become targets of the proteasome by covalent attachment of polyubiquitin chains,
which requires three main enzymes (E1, E2, and E3). It is the E3 ubiquitin
ligases that control the selection and specificity of substrate ubiquitination.
Cullin-5 (Cul-5), a member of the cullin family of E3 ubiquitin ligases, remains
obscure. The goals of this research project were to characterize Cul-5, and
investigate its response to cellular stresses of water deprivation and hemorrhagic
shock in the rat.
Northern blotting of poly(A)+ RNA from various rat tissues demonstrated the
cul-5 transcript is approximately 6.3 kb. Reverse transcription-polymerase
chain reaction (RT-PCR) indicated cul-5 mRNA is present in twelve tissues
examined: brainstem, cerebral cortex, cerebellum, hypothalamus, aorta, gastrointestinal
tract, heart, kidney medulla, liver, lung, skeletal muscle, and spleen. Quantitative
realtime PCR confirmed RT-PCR results that Cul-5 mRNA is ubiquitously expressed
and that levels are similar in all tissues. Cellular specificity examination
showed cul-5 mRNA expression in rodent neuronal, glial, and vascular endothelial
cells in the central nervous system (CNS) via RT-PCR. We corroborated these
data by immunocytochemical techniques demonstrating Cul-5 protein presence
in neurons, astrocytes, blood vessels, and choroid plexus in rat.
Functional assays measured cul-5 mRNA expression responses to water deprivation and hemorrhagic shock. Quantitative realtime PCR showed significant cul-5 mRNA elevations in the rat cerebral cortex (3 fold, p<0.001), hypothalamus (2 fold, p<0.007), and kidney (1.5 fold, p<0.04) following 48 hours of water deprivation. Water deprivation for 24 hours or rehydration (24 hours access to water following 48 hours of water deprivation) also increased kidney cul-5 mRNA levels (1.5 fold, p<0.04 and 3 fold, p<0.001 respectively). Hemorrhagic shock was used as a second in vivo cellular stress model. Rats were subjected to volume controlled (27 ml/kg) hemorrhage over 10 minutes and kept in shock for 60 minutes. Levels of cul-5 mRNA were significantly increased in the brainstem and cerebellum (1.6 fold, p<0.01 and 1.5 fold, p<0.05 respectively), and decreased in the hypothalamus (0.5 fold, p<0.05) compared to sham-treated rats.
We determined that Cul-5 is synthesized in all tissues and organs we examined, and in neurons, glia, and endothelial cells in the CNS. Using two paradigms of cellular stress, we found cul-5 mRNA levels in the CNS are altered by water deprivation and by hemorrhagic shock. However, much remains to be revealed concerning what precise physiological role(s) Cullin-5 plays in various cellular processes.
A. Tamara Crowder
Neuroscience Program
Doctor of Philosophy
2003
Major Advisor: Thomas E. Cote, Ph.D., Department of Pharmacology
Dissertation Title: Co-expression of Regulator of G Protein Signalling
4 (RGS4)
and the mu opioid receptor in regions of rat brain:
Evidence that RGS4 attenuates mu opioid receptor signalling
ABSTRACT
Regulators of G protein Signalling (RGS) proteins influence G protein-coupled receptor signal transduction by enhancing the intrinsic GTPase activity of G proteins. The RGS-enhanced GTPase activity of G proteins may be responsible for the desensitization of certain G protein-coupled receptors, including the mu opioid receptor. The goal of this research was to evaluate the ability of recombinant RGS4 to affect mu opioid receptor-mediated cellular signalling and to identify regions of the rat brain in which both RGS4 and the mu opioid receptor are co-expressed.
We evaluated the ability of recombinant RGS4 to affect [D-Ala2, N-Me-Phe4, gly-ol] enkephalin (DAMGO)-mediated inhibition of adenylyl cyclase activity in membranes of SH-SY5Y cells, a cell line that express endogenous mu receptors. Recombinant RGS4 caused a concentration-dependent attenuation of DAMGO-mediated inhibition of adenylyl cyclase activity.
RGS4 diminished the efficacy, but not the potency, of DAMGO in inhibiting adenylyl cyclase activity. In contrast, RGS4 had no effect on the ability of GTP S, a nonhydrolyzable analogue of GTP, to inhibit adenylyl cyclase activity. RGS4 also had no effect on DAMGO stimulated [35S]GTP S binding in SH-SY5Y membranes. Additionally, RGS4 was tested for its ability to affect [3H]DAMGO binding to the mu receptor. RGS4 failed to affect either the KD of the Bmax of [3H]DAMGO in saturation binding experiments.
Antibodies generated against rat RGS4 and the rat mu opioid receptor were used in immunohistochemical staining to identify specific regions of rat brain where the two proteins are co-expressed. Both RGS4 and mu opioid receptor proteins were present in many of the same regions of the brain. Further, we demonstrated that RGS4 is primarily localized to the nucleus, but that administration of fentanyl, a potent mu opioid agonist, induces translocation out of the nucleus, to the cytoplasm in the hippocampal CA3 pyramidal neurons.
Together, these findings are consistent with the proposal that RGS4 can desensitize mu opioid receptor by increasing the intrinsic GTPase of Gi-type G proteins associated with the mu opioid receptor and that, in vivo, RGS4 and the mu opioid receptor are co-expressed in many of the same regions of the rat brain.
Holly
H. Nash
Neuroscience Program
Doctor of
Philosophy
2002
Major Advisor: Juanita J. Anders, Ph.D., Department of Anatomy, Physiology, and Genetics
Dissertation Title: Regeneration of the Adult Rat Spinal Cord in Response to Ensheathing Cells and Methylprednisolone
ABSTRACT
Axons fail to regenerate after spinal cord injury (SCI) in adult mammals, leading to permanent loss of function. Following SCI, ensheathing cells promote recovery in animal models, whereas methylprednisolone study, a new method of purifying ensheathing cells was developed, resulting in a final population of ensheathing cells that were 93% pure. In the second study, the ability of a modified directed forepaw reaching (DFR) apparatus to accurately assess function of the corticospinal tract (CST) was examined. The data demonstrated that the modified apparatus prevented extinguishing of DFR behavior after SCI. In addition, the modified apparatus allowed for the collection of quantitative data to accurately assess CST function after bilateral, cervical spinal cord lesions. In the third study, the effectiveness of combining ensheathing cells and methylprednisolone after SCI was investigated. After lesioning the CST in adult rats, a purified population of ensheathing cells was transplanted into the lesion, and methylprednisolone was administered for 24 hours. At six weeks post injury, functional recovery was assessed by measuring successful DFR performance. Axonal regeneration was analyzed by counting the number of anterogradely labeled CST axons caudal to the lesion. Lesioned control rats, receiving either no treatment or vehicle, had abortive axonal regrowth (1 mm) and poor DFR success (38% and 42%, respectively). Compared to controls, rats treated with methylprednisolone for 24 hours had significantly more axons at 7 mm caudal to the lesion, and DFR performance was significantly improved (57%). Rats that received ensheathing cells with methylprednisolone had significantly more regrowing axons than all other lesioned rats up to 13 mm caudal to the lesion. Successful DFR performance was significantly higher in rats with ensheathing cell transplants, both without (72%) and with (78%) methylprednisolone, compared to other lesioned rats. These data confirm previous reports that ensheathing cells promote axonal regeneration and functional recovery after spinal cord lesions in a rat model. In addition, this research provides new evidence that, when used in combination, methylprednisolone and ensheathing cells improve axonal regrowth up to 13 mm caudal to the lesion.promotes neurological recovery in humans. The aim of this research was to explore the effectiveness of ensheathing cells and methylprednisolone after acute SCI in the adult rat.
Major Bruce
A. Schoneboom
Neuroscience Program
Doctor of
Philosophy
2002
Major Advisor: Franziska B. Grieder, DVM, Ph.D., Microbiology and Immunology
Dissertation Title: Neuro-Immune Mechanisms in Response to Venezuelan Equine Encephalitis Virus Infection
ABSTRACT
Venezuelan equine encephalitis virus (VEE) is an emerging pathogen with epizootics
and epidemics occurring in the Western Hemisphere. Recent outbreaks in South
America have caused significant morbidity and mortality among domesticated
livestock and surrounding human communities. VEE pathogenesis is characterized
by infection of the central nervous system (CNS) where the virus targets neurons,
resulting in significant neurodegeneration. VEE encephalitis can result in
profound neurological deficits or even death. Because of the devastating nature
of this disease and the lack of interventional therapies, it is important
to understand the intricate details of VEE neuropathogenesis in order to identify
targets for treatment to effect a cure.
Inflammation has recently been implicated as a component of neurodegeneration. Inflammation in the CNS in response to acute infections is a protective mechanism that attempts to contain and clear neuro-invasive pathogens, however this upregulation of pro- inflammatory genes may be deleterious to surrounding neurons. The combined effects of direct infection and inflammation may be additive or synergistic in the amount of injury sustained in the CNS.
Glial cells are of particular importance in the CNS immune response. These resident cells of the CNS have intimate associations with neurons and regulate the CNS milieu. One type of glial cell is the astrocyte. Astrocytes are found in vast numbers in the CNS and have essential functional roles in maintaining a healthy environment for neurons. Further, astrocytes playa role in the pro-inflammatory innate immune response.
To identify the role of astrocytes in VEE infection, I characterized astrocyte susceptibility to VEE infection using an in vitro culture system and have further described their pro-inflammatory responses following VEE infection. Specifically, inducible nitric oxide synthase, tumor necrosis factor-alpha, and interleukin-6 are upregulated in response to VEE infection in primary astrocyte cultures as shown by reverse transcriptase-polymerase chain reaction and analyses of protein synthesis. I have also demonstrated that there were quantitative differences in the upregulation of these responses between virulent and attenuated strains of VEE.
To characterize the pro-inflammatory response in vivo, I measured cytokine gene expression in the CNS using a murine model of VEE infection. The cytokine responses to virulent VEE resulted in the upregulation of multiple genes important in inflammation and apoptosis. In contrast, cytokine responses in the CNS were delayed or absent following infection with attenuated VEE, depending on the specific mutant VEE strain.
Finally, CNS tissue from mice infected with VEE was double-labeled for astrocytosis and apoptosis, and stained for VEE antigen in adjacent tissue sections. Apoptosis occurs not only in areas of the brain where VEE antigen could be detected, but also in areas of acute astrogliosis, where no VEE antigen could be demonstrated. This association of apoptosis and astrogliosis suggests that inflammation may be contributing to neuronal degeneration in response to VEE infection.
William D.
Watson.
Neuroscience Program
Doctor of
Philosophy
2000
Major Advisor: Ajay Verma, M.D./Ph.D., Department of Neurology
Dissertation Title: A Thapsigargin-insensitive Intracellular Calcium Sequestering Compartment in Rat Brain
ABSTRACT
Calcium
plays a central regulatory role in the normal function of all cells. Electrical,
secretory, and metabolic activities of cells in the brain require fine control
over ionized cytoplasmic calcium levels. Intracellular calcium levels are
controlled by a diverse set of cytoplasmic and membrane-associated mechanisms
including calcium binding proteins, channels, pumps, and exchangers. The endoplasmic
reticulum (ER) calcium stores have a major impact on neuronal intracellular
signaling. Most of the ER in neurons and glia appears to accumulate calcium
by energy driven ion pumps known as sarco/endoplasmic reticulum calcium ATPases
(SERCAs), which are potently and selectively inhibited by thapsigargin. However,
the ER represents a heterogeneous network of cisternae in which calcium-accumulating
subcompartments may be spatially and functionally distinct. We describe here
the characterization of a novel calcium accumulating subcompartment of rat
brain ER, which is insensitive to thapsigargin. This compartment accumulates
calcium in a magnesium and ATP-dependent manner and is distinguished from
thapsigargin-sensitive calcium pools with respect to anion permeability I
inhibitor sensitivity, sensitivity to calcium mobilizers, and brain anatomical
distribution.