The purpose of this article is to review relevant literature on the effect of mild traumatic brain injury (mTBI) and blast injury on the vestibular system. Dizziness and imbalance are common sequelae associated with mTBI, and in some individuals, these symptoms may last for six months or longer. In war-related injuries, mTBI is often associated with blast exposure. The causes of dizziness or imbalance following mTBI and blast injuries have been linked to white matter abnormalities, diffuse axonal injury in the brain, and central and peripheral vestibular system damage. There is some evidence that the otolith organs may be more vulnerable to damage from blast exposure or mTBI than the horizontal semicircular canals. In addition, benign paroxysmal positional vertigo (BPPV) is a common vestibular disorder following head injury that is treated effectively with canalith repositioning therapy. Treatment for (non-BPPV) mTBI-related vestibular dysfunction has focused on the use of vestibular rehabilitation (VR) augmented with additional rehabilitation methods and medication. New treatment approaches may be necessary for effective otolith organ pathway recovery in addition to traditional VR for horizontal semicircular canal (vestibulo-ocular reflex) recovery.

Objective: Cervical vestibular evoked myogenic potentials (cVEMPs) are short-latency electromyogram (EMG) evoked by high-level acoustic stimuli recorded from the activated sternocleidomastoid muscle and used to evaluate otolith organ function. The purpose of this study was to investigate the effects of aging on the cVEMP and on the sternocleidomastoid muscle EMG level. Design: A cross-sectional observational study was used to investigate differences in cVEMP and sternocleidomastoid muscle EMG level in a group of 24 younger and 24 older individuals. cVEMPs were recorded during activation of the sternocleidomastoid muscle at target EMG levels ranging from 0 to 90 μV and during maximum voluntary contraction of the sternocleidomastoid muscle. Results: The sternocleidomastoid muscle EMG amplitude increased as a function of target EMG level for both age groups; however, the mean EMG amplitude was greater for the younger group than the older group, and the variability of EMG amplitude was greater for the older group. The EMG amplitude at maximum voluntary contraction ranged from 88 to 279 μV for the younger subjects and from 32 to 230 μV for the older subjects, and the mean EMG amplitude at maximum voluntary contraction was significantly greater for the younger group than the older group. The cVEMP amplitude increased as a function of EMG target level for each age group. Although cVEMP amplitude increased as a function of target EMG level for both groups, the older group exhibited smaller cVEMP amplitudes, overall, compared with the younger group. To separate the influence of EMG level from aging on cVEMP amplitude, only the responses obtained at the 30 μV target EMG level were considered for the statistical analysis because there was no significant difference in EMG level between groups at the 30 μV target level. The mean cVEMP amplitudes at the 30 μV target level were 101 and 51 μV for the younger and older groups, respectively, and a statistical analysis indicated that cVEMP amplitude for the younger group was significantly greater than the older group. Conclusions: The findings suggest that the decrement in cVEMP amplitude is related to both age-related changes in the vestibular system and age-related changes in the sternocleidomastoid muscle.

Instrumental to forensic investigations is the ability to identify unknown human remains providing key evidence to criminal cases, resolution to missing persons, and assistance in mass or natural disasters. Identification of remains in an effort to determine age is an area of forensics that has received considerable attention. Traditional methods in age determination such as morphology are often biased, antiquated, and frequently result in a large margin of error. Conversely, the emergence of new forensic techniques provide promise to reduce the margin of error in determining age. One such technique has focused on relating the extent of amino acid racemization in teeth to age. Past research has focused primarily on the analysis of aspartic acid due to its high racemization rate. Our research indicates that glutamic acid also shows promise as related to age determination. Results will be presented illustrating optimization of gas chromatography using a chiral column for separation of amino acids found in dentin and their enantiomeric ratio quantification. Age correlation data will be presented on collected teeth ranging from mid-teens to early seventies.

Pain signaling involves transmission of nociceptive stimuli in the spinal cord where a critical balance between excitatory and inhibitory inputs determines the response to noxious stimuli. The neuropeptide, substance P (SP), mediates transmission of pain in part by binding to the tachykinin receptor (NK-1R) in the dorsal horn (DH) of the spinal cord. One of SP’s downstream effects is to modulate N-type Ca2+(N-) channels. While phospholipid breakdown is a part of the inflammatory process that accompanies tissue damage, the role of this metabolic pathway has not been completely described with respect to N-channel modulation during pain signaling. Despite the incomplete understanding of this modulation, pharmacological antagonists of both NK-1R and N-channels have been used to treat pain. In Chapter II, using whole-cell patch clamp recording techniques, the SP signaling cascade that mediates inhibition of recombinant N-channel activity was characterized. By adopting a pharmacological approach, I show that this pathway resembles the slow pathway that was earlier described for modulation of N-current by the M1 muscarinic receptor (M1R). M1R couples to Gq to stimulate phospholipid breakdown. Together with previous observations, the data presented in this chapter provide evidence for involvement of the extracellular receptor kinase (ERK1/2), phospholipase A2 and release of phospholipid metabolites in the modulation of N-current by SP. Overall, this chapter shows that phospholipid metabolism involved in modulation of N-currents is not specific to M1Rs but that other Gq-coupled receptors may also modulate N-currents via the same signal transduction pathway. In Chapter III, enhancement of N-current by SP was studied as part of a collaborative project to understand current enhancement that occurs when a palmitoylated accessory CaVβ2a subunit is co-expressed with the pore-forming subunit CaV2.2 and the accessory subunit α2δ-1. When CaVβ3 is present, SP inhibits N-current as described in Chapter II. However, when palmitoylated CaVβ2a is co-expressed with CaV2.2 (and α2δ-1), current enhancement is observed at negative test potentials, demonstrating that both M1Rs and NK-1Rs exhibit the same profile of N-current modulation. This change in modulation by muscarinic agonists is not observed in the presence of a depalmitoylated CaVβ2a. However a chimeric CaVβ2aβ1b subunit that contains the palmitoylated N-terminus from CaVβ2a confers enhancement. Normally expression of the β1b subunit resulted in current inhibition. These findings indicated that the palmitoylated CaVβ2a participates in enhancement of current. Our data support a model where inhibition dominates over enhancement; when inhibition is blocked, enhancement may be observed. Lastly, we show that N-current inhibition by SP is minimized when exogenous palmitic acid is applied to cells co-expressing CaVβ3 subunits with N-channels. These results indicate that the presence of palmitic acid can prevent N-current inhibition when SP is applied most likely by interacting with CaV2.2. We propose a model where palmitic acid occupies the inhibitory site and serves to antagonize inhibition by a lipid metabolite, which is most likely arachidonic acid. The CaVβ2a protein seems to have a role in positioning the palmitoyl groups near CaV2.2. This chapter provides a new role for protein palmitoylation where the palmitoyl groups of CaVβ2a are both necessary and sufficient to block inhibition of another protein: CaV2.2. In Chapter IV, I probe the role of the relative orientation of CaVβ2a and the pore-forming subunit of the N-channel in N-current modulation. Evidence is presented that shows that not just the presence of a palmitoylated CaVβ2a is necessary, but the relative orientation of CaVβ2a to CaV2.2 is critical for blocking inhibition. Using N-channel mutants that cause a change in the orientation of CaVβ2a relative to CaV2.2, I show that the block of inhibition is disrupted; inhibition by the slow pathway is rescued. These findings further support my model that the palmitoyl groups of CaVβ2a normally reside in a specific location that overlaps with the slow pathway inhibitory site on CaV2.2. Lastly I present data showing that the enhancement of N-current, observed when palmitoylated CaVβ2a is present, occurs via the slow pathway. In Chapter V the effect of CaVβ’s orientation on N-channel modulation by the dopamine D2 receptor is tested. In this form of modulation, inhibition is rapid and voltage-dependent. The signaling pathway is membrane-delimited since Gβγ, released after receptor stimulation, directly interacts with the N-channel at a site that overlaps with a high affinity binding site for CaVβs. While N-currents are modulated by this pathway, the deletion mutants show aberrant membrane-delimited modulation. The findings in this chapter further underscore the importance of proper positioning of CaVβ to CaV2.2 for eliciting proper N-current modulation after GPCR stimulation. Overall, the data presented in this dissertation provides a mechanistic approach into examining modulation of N-current by different GPCRs via two different signaling pathways as well as the role CaVβ subunits serve in each modulatory pathway.

Adenosine is a nucleoside that plays an important role in the regulation of contractility in the heart. Adenosine receptors are G-protein coupled and those implicated in regulation of contractility are presumed to act via modulating the activity of adenylyl cyclase and cAMP content of cardiomyocytes. Adenosine A1 receptors (A1R) reduce the contractile response of the myocardium to β-adrenergic stimulation. This is known as anti adrenergic action. The A2A adenosine receptor (A2AR) has the opposite effect of increasing contractile responsiveness of the myocardium. The A2AR also appears to attenuate the effects of A1R. The effects of these receptors have been primarily studied in the rat heart and with the utilization of cardiomyocyte preparations. With the increasing use of receptor knockout murine models and murine models of various pathological states, it is of importance to comprehensively study the effects of adenosine receptors on regulation of contractility in the murine heart. The following studies examine the adenosinergic regulation of myocardial contractility in isolated murine hearts. In addition, adenosinergic control of contractility is examined in hearts isolated from A2AR knockout animals. Responses to adenosinergic stimulation in murine isolated hearts are found to be comparable to those observed in the rat, with A1R exhibiting an anti adrenergic action and A2AR conversely enhancing contractility. A significant part of the A2AR effect was found to occur via inhibition of the A1R antiadrenergic action. A part of the anti adrenergic action of A1R has previously been shown to be the result of protein phosphatase 2A activation and localization to membranes. Additional experiments in the present study examine the effect of adenosinergic signaling on PP2A in myocardial extracts from wild type and A2AR knockout hearts. A2AR activation was found to decrease the activity of PP2A and enhance localization of the active enzyme to the cytosol; away from its presumed sites of action. In the A2AR knockout the response to A1R activation was enhanced compared with the wild type and basal PP2A activity was reduced. It is concluded that A2AR modulation of PP2A activity may account for the attenuation of the A1R effect by A2AR observed in the contractile studies.

Patients admitted to intensive care unit (ICU) are known to develop significant impairments in physical function. Patients with critical illness suffer up to 30% reductions in muscle size within the first ten days of admission to the ICU. Muscle strength testing, Medical Research Council-sum score, is current gold-standard to diagnosis ICU-acquired weakness and predicts risk of mortality and long-term physical function. Muscle power different from muscle strength in that it accounts for velocity of movement, is potentially a better independent predictor of function that has not been studied in this population. In addition, we hypothesize that muscle size and quality measured through ultrasound imaging has better applicability and prediction that strength testing. Therefore, we prospectively collected data surrounding these muscle parameters in patients admitted to the medicine ICU at University of Kentucky. Primary outcomes included physical function, muscle power with a novel assessment tool for the critically ill population, muscle strength, and muscle size and quality assess through ultrasound imaging. 36 patients admitted to ICU and 18 aged-matched controlled were enrolled. Patients had significantly lower scores on muscle power assessment at ICU discharge (33.6 ±19.0 W; t= 4.01, p < 0.001) and at hospital discharge (40.9 ±16.5 W; t= 4.81, p < 0.001) in comparison to controls (59.3± 14.7 W). Patients with better scores on muscle power assessment had significantly better scores on physical function measures (Six-minute walk test; rs = 0.548, p = 0.0001). Muscle size (cross-sectional area of rectus femoris muscle) and muscle power were strongly correlated (rs = 0.66, p < 0.0001). These data suggest that patients with critical illness have significantly reduced muscle power which directly related to deficits in physical function.

High temporal resolution of vision relies on the rapid kinetics of the photoresponse in the light-sensing photoreceptor neurons. It is well known that the rapid recovery of photoreceptor membrane potential at the end of light stimulation depends on timely deactivation of the visual transduction cascade within photoreceptors. Whether any extrinsic factor contributes to the termination speed of the photoresponse is unknown. In this thesis, using Drosophilaas a model system, I show that a feedback circuit mediated by both neurons and glia in the visual neuropile lamina is required for rapid repolarization of the photoreceptor at the end of the light response. In the first part of my thesis work, I provide evidence that lamina epithelial glia, the major glia in the visual neuropile, is involved in a retrograde regulation that is critical for rapid repolarization of the photoreceptor at the end of light stimulation. I identified the gene affected in a slrp (slow receptor potential) mutant that is defective in photoreceptor response termination, and found it needs to be expressed in both neurons and epithelial glia to rescue the mutant phenotype. The gene product SLRP, an ADAM (a disintegrin and metalloprotease) protein, is localized in a special structure of epithelial glia, gnarl, and is required for gnarl formation. This glial function of SLRP is independent of the metalloprotease activity. In the second part of my thesis work, I demonstrate that glutamatergic transmission from lamina intrinsic interneurons, the amacrine cells, to the epithelial glia is required for the rapid repolarization of photoreceptors at the end of the light response. From an RNAi-based screen, I identified a vesicular glutamate transporter (vGluT) in amacrine cells as an indispensable factor for the rapid repolarization of the photoreceptor, suggesting a critical role of glutamatergic transmission from amacrine cells in this retrograde regulation. Further, I found that loss of a glutamate-gated chloride channel GluCl phenocopies vGluT downregulation. Cell specific knockdown indicates that GluCl functions in both neurons and glia. In the lamina, a FLAG-tagged GluCl colocalized with the SLRP protein in the gnarl-like structures, and this localization pattern of GluCl depends on SLRP, suggesting that lamina epithelial glia receive glutamatergic input from amacrine cells through GluCl at the site of gnarl. Since the amacrine cell itself is innervated by photoreceptors, these observations suggest that a photoreceptor — amacrine cell — epithelial glia — photoreceptor feedback loop facilitates rapid repolarization of photoreceptors at the end of the light response. In summary, my thesis research has revealed a feedback regulation mechanism that helps to achieve rapid kinetics of photoreceptor response. This visual regulation contributes to the temporal resolution of the visual system, and may be important for vision during movement and for motion detection. In addition, this work may also advance our understanding of glial function, and change our concept about the effect of glutamatergic transmission.

Osteoclast-mediated focal articular bone erosion is a hallmark of rheumatoid arthritis, a disease of inflammation-induced bone loss. Inflammation in the bone microenvironment enhances osteoclast differentiation leading to bone erosion. Simultaneously, inflammation also inhibits osteoblast-mediated bone formation, further contributing to the net loss of bone. Previous studies have shown a paucity of mature osteoblasts at eroded bone surfaces correlating with suppression of bone formation and upregulation of antagonists of the Wnt pathway, a signaling cascade essential for osteoblast lineage commitment. Despite these observations, the exact pathogenesis of impaired bone formation in the setting of inflammation is not clearly understood. This dissertation aims to delineate the mechanisms by which inflammation suppresses osteoblast differentiation and activity in inflammatory arthritis. Specifically, this research elucidates how inflammation-induced alterations in the Wnt and bone morphogenetic protein (BMP) osteogenic signaling pathways contribute to bone loss and formation at distinct inflammatory microenvironments within the bone. Secondly, the means by which cellular mediators, including lymphocytes and macrophages, facilitate bone erosion and formation was addressed. Taken together, the research in this dissertation underscores the relationship between inflammation-induced bone loss and alterations in osteogenic signaling. Using an innovative murine inflammatory arthritis model, this study definitively demonstrates that resolving inflammation promotes osteoblast-mediated bone formation. Repair of erosions correlates with upregulation of synovial expression of Wnt10b, a Wnt agonist, and downregulation of sFRP1 and sFRP2, Wnt antagonists. This work also directly evaluates the contribution of sFRP1 to inflammation-induced bone destruction. Furthermore, this research demonstrates that expression of BMP3, a negative regulator of BMP signaling, is upregulated in osteoblasts by IL-17, a pro-inflammatory cytokine. BMP3-expressing osteoblasts are also observed at erosion sites in murine arthritis. Lastly, evaluation of the mediators of inflammation-induced periosteal bone formation implicates BMP2 as a means by which inflammation may positively regulate osteoblast function. This dissertation further elucidates the role of T cells and macrophages in the erosion and formation processes, respectively. In the absence of lymphocytes, bone erosion occurred normally, demonstrating that RANKL-expressing lymphocytes are not absolutely required for the bone erosion. Preliminary studies also suggest that M2 macrophages are potential mediators of bone formation via the expression of BMP2. In conclusion, this dissertation explores the ability of inflammation to act as a rheostat, which controls the fate of bone by modulating not only osteoclast differentiation, but also osteogenic signaling pathways and cellular mediators in the bone microenvironment. The soluble mediators and cell types identified in this research highlight novel mechanisms by which inflammation may regulate osteoblast activity within the bone microenvironment. Collectively, these data imply that strict control of inflammation may be necessary in order to create an anabolic environment that preserves bone architecture in diseases of inflammation-induced bone loss.

The purpose of this study was to document the effects that a regular yoga therapy practice has on the quality of life for a paraplegic individual. Due to the unique nature of spinal cord injury (SCI), this was a case study with one participant. For 5 weeks the subject practiced a standardized yoga routine three times a week with a yoga teacher, receiving private instruction. Interview questions were asked before and after the intervention to document the participant's quality of life. Each week, the subject completed a modified SF-36 questionnaire as well as Cohen's Perceived Stress Survey. Perceived pain and perceived stress were the two primary variables monitored in the study. The results of this study demonstrated a slight reduction in perceived stress, a reduction in perceived pain, and an improvement in overall quality of life. Additionally, the subject increased her strength significantly from the beginning to the end of the study. Based on the findings in this study, it appears that a regular adapted yoga routine is beneficial for paraplegic individuals.

Functional vasodilation in arterioles is impaired with chronic ischemia. We sought to examine the impact of chronic ischemia and age on skeletal muscle resistance artery function. To examine the impact of chronic ischemia, the femoral artery was resected from young (2-3mo) and adult (6-7mo) mice and the profunda femoris artery diameter was measured at rest and following gracilis muscle contraction 14 days later using intravital microscopy. Functional vasodilation was significantly impaired in ischemic mice (14.4±4.6% vs. 137.8±14.3%, p<0.0001 n=8) and non-ischemic adult mice (103.0±9.4% vs. 137.8±14.3%, p=0.05 n=10). In order to analyze the cellular mechanisms of the impairment, a protocol was developed to apply pharmacological agents to the experimental preparation while maintaining tissue homeostasis. Endothelial and smooth muscle dependent vasodilation were impaired with ischemia, 39.6 ± 13.6% vs. 80.5 ± 11.4% and 43.0 ± 11.7% vs. 85.1 ± 10.5%, respectively. From this data, it can be supported that smooth muscle dysfunction is the reason for the observed impairment in arterial vasodilation.