Anatomy, Cell Biology & Physiology Department Theses and Dissertations

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IUPUI Department of Anatomy, Cell Biology & Physiology

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Inhibitory synpatic transmission in striatal neurons after transient cerebral ischemia
(2009-10) Li, Yan; Xu, Zao C.; Cummins, Theodore R.; Yang, Charles R.; Zhou, Feng C.
In the striatum, large aspiny (LA) interneurons survive transient cerebral ischemia while medium spiny (MS) neurons die. Excitotoxicity is believed to be the major cause for neuronal death after ischemia. Since inhibitory tone plays an important role in the control of neuronal excitability, the present study is aimed at examining if there are any changes in inhibitory synaptic transmission in striatal neurons after ischemia and the possible mechanisms. Transient forebrain ischemia was induced in male Wistar rats using the four-vessel occlusion method. Inhibitory postsynaptic currents (IPSCs) were evoked intrastriatally and whole-cell voltage-clamp recording was performed on striatal slices. The expression of glutamate decarboxylase65 (GAD65) was analyzed using immunohistochemical studies and Western blotting. Muscimol (a specific GABAA receptor agonist) was injected intraperitoneally to the rats (1 mg/kg) to observe ischemic damage, evaluated by counting the survived cells in the striatum after hematoxylin & eosin (HE) staining. The amplitudes of evoked IPSCs were significantly increased in LA neurons while depressed in MS neurons after ischemia. This enhancement was due to the increase of presynaptic release. Muscimol (1 μM) presynaptically facilitated inhibitory synaptic transmission in LA neurons at 24 h after ischemia. The optical density of GAD65-positive terminals and the number of GAD65-positive puncta was significantly increased in the striatum at both 1 day and 3 days after ischemia. Consistently, data from western blotting suggested an increased expression of GAD65 in the striatum after ischemia. For the rats treated with muscimol, the number of survived cells in the striatum was greatly increased compared to the non-treatment group. The present study demonstrates an enhancement of inhibitory synaptic transmission in LA neurons after ischemia, which is contributed by two mechanisms. One is the increased presynaptic release of GABA mediated by presynaptic GABAA receptors. The other is the increased expression of GAD. Facilitation of inhibitory synaptic transmission by muscimol protects striatal neurons against ischemia. Therefore, the enhancement of inhibitory synaptic transmission might reduce excitotoxicity and contribute to the selective survival of LA neurons after ischemia.
The Effects of Instrument-Assisted Cross Fiber Massage on Ligament Healing
(2010-05) Loghmani, Mary T.; Warden, Stuart J.; Burr, David B.; Robling, Alex G.; Seifert, Mark Frederick; Turner, Charles H.
Ligament injury is one of the most prevalent musculoskeletal disorders that may lead to disability or disease, such as osteoarthritis. Conservative interventions which accelerate or augment ligament healing are needed to enhance therapeutic outcomes. The purpose of this research agenda was to investigate the tissue level effects of a type of manual therapy, cross fiber massage (CFM), in particular instrument-assisted CFM (IACFM), on ligament healing. Bilateral knee medial collateral ligament (MCL) injuries were created using an established rodent model where one MCL received IACFM treatment and the other untreated MCL served as a within subjects control. The short and long term effects of IACFM on the biomechanical and histological properties of repairing ligaments were investigated. Tensile mechanical testing was performed to determine ligament mechanical properties. Ligament histology was examined under light microscopy and scanning electron microscopy. IACFM was found to accelerate early ligament healing (4 weeks post-injury), possibly via favorable effects on collagen formation and organization, but minimal improvement was demonstrated in later healing (12 weeks post-injury). Regional blood flow and angiogenesis were investigated as possible mechanisms underlying the accelerated healing found in IACFM-treated ligaments. Laser Doppler perfusion imaging was used to investigate vascular function. Micro-computed tomography was used to determine vascular structural parameters. Compared to untreated contralateral injured controls, IACFM-treated injured knees demonstrated a delayed increase in blood flow and altered microvascular structure, possibly suggesting angiogenesis. Mechanotransduction is discussed as a mechanism for the beneficial effects of CFM in that application of a mechanical force was found to enhance biomechanical and histological properties as well as vascular function and structure acutely in healing ligaments. Although this thesis focused on IACFM treatment of injured knee ligaments, it is plausible for concepts to apply to other manual modalities that offer conservative alternatives to invasive procedures or pharmaceuticals in the treatment of soft tissue injuries.
THE HAND1 LINEAGE REVEALS DISTINCT ROLES FOR HAND FACTORS DURING CARDIOVASCULAR DEVELOPMENT
(2010-10) Barnes, Ralston M.; Firulli, Anthony B.; Bidwell, Joseph P.; Conway, Simon J.; Field, Loren J.
The basic Helix-Loop-Helix (bHLH) transcription factors Hand1 and Hand2 play critical roles in the development of multiple organ systems during embryogenesis. The dynamic expression patterns of these two factors within developing tissues obfuscates their respective unique and redundant organogenic functions. To define cell lineages potentially dependent upon Hand gene expression, we generated a mutant allele in which the coding region of Hand1 is replaced by Cre recombinase. Subsequent Cre-mediated activation of β-galactosidase or eYFP reporter alleles enabled lineage trace analyses that clearly define the fate of Hand1-expressing cells. Comparisons between Hand1 expression and Hand1-lineage greatly refine our understanding of its dynamic spatio-temporal expression domains and raise the possibility of novel Hand1 functions in structures not thought to be Hand1-dependent. To genetically examine functional overlap between Hand1 and Hand2, we conditionally deleted Hand2 from Hand1-expressing cells. Hand2 conditional knockout mice die midgestation and exhibit cardiovascular and limb defects. Moreover, Hand2 lineage-restricted deletion from the proepicardial organ results in defective epicardialization and failure to form coronary arteries. Together, these novel data demonstrate a hierarchal relationship whereby transient Hand1 expression within the septum transversum defines epicardial precursors that depend upon subsequent Hand2 function.
Contribution of rankl regulation to bone resorption induced by PTH receptor activation in osteocytes
(2012-10-19) Ben-awadh, Abdullah Nasser; Bellido, Teresita M.; Plotkin, Lilian I.; Allen, Matthew R.
PTH increases osteoclasts by upregulating RANKL in cells of the osteoblastic lineage, but the precise differentiation stage of the PTH target cell remains undefined. Recent findings demonstrate that PTH regulates gene expression in osteocytes and that these cells are an important source of RANKL. We therefore investigated whether direct regulation of the RANKL gene by PTH in osteocytes is required to stimulate osteoclastic bone resorption. To address this question, we examined bone resorption and RANKL expression in transgenic mice in which PTH receptor signaling is activated only in osteocytes (DMP1-caPTHR1) crossed with mice lacking the distal control region regulated by PTH in the RANKL gene (DCR -/-). Longitudinal analysis of circulating C-terminal telopeptide (CTX) in male mice showed elevated resorption in growing mice that progressively decreased to plateau at 3-5 month of age. Resorption was significantly higher (~100%) in DMP1-caPTHR1 mice and non-significantly lower (15-30%) in DCR -/-mice, versus wild type littermates (WT) across all ages. CTX in compound DMP1-caPTHR1; DCR -/-mice was similar to DMP1-caPTHR1 mice at 1 and 2 months of age, but by 3 months of age, was significantly lower compared to DMP1-caPTHR1 mice (50% higher than WT), and by 5 months, it was undistinguishable from WT mice. Micro-CT analysis revealed lower tissue material density in the distal femur of DMP1-caPTHR1 mice, indicative of high remodeling, and this effect was partially corrected in compound vi mice. The increased resorption exhibited by DMP1-caPTHR1 mice was accompanied by elevated RANKL mRNA in bone at 1 and 5 months of age. RANKL expression levels displayed similar patterns to CTX levels in DMP1-caPTHR1; DCR -/-compound mice at 1 and 5 month of age. The same pattern of expression was observed for M-CSF. We conclude that resorption induced by PTH receptor signaling requires direct regulation of the RANKL gene in osteocytes, but this dependence is age specific. Whereas DCR-independent mechanisms involving gp130 cytokines or vitamin D 3 might operate in the growing skeleton, DCR-dependent, cAMP/PKA/CREB-activated mechanisms mediate resorption induced by PTH receptor signaling in the adult skeleton.
Dissecting the cellular and molecular mechanisms mediating neurofibromatosis type 1 related bone defects
(2013-06) Rhodes, Steven David; Yang, Feng-Chun; Clapp, D. Wade; Robling, Alexander G.; Bidwell, Joseph P.
Skeletal manifestations including short stature, osteoporosis, kyphoscoliosis, and tibial dysplasia cumulatively affect approximately 70% of patients with neurofibromatosis type 1 (NF1). Tibial pseudarthrosis, the chronic non-union of a spontaneous fracture, is a debilitating skeletal malady affecting young children with NF1. These non-healing fractures respond poorly to treatment and often require amputation of the affected limb due to limited understanding of the causative mechanisms. To better understand the cellular and molecular pathogenesis of these osseous defects, we have established a new mouse model which recapitulates a spectrum of skeletal pathologies frequently observed in patients with NF1. Nf1flox/-;Col2.3Cre mice, harboring Nf1 nullizygous osteoblasts on a Nf1+/- background, exhibit multiple osseous defects which are closely reminiscent of those found in NF1 patients, including runting (short stature), bone mass deficits, spinal deformities, and tibial fracture non-union. Through adoptive bone marrow transfer studies, we have demonstrated that the Nf1 haploinsufficient hematopoietic system pivotally mediates the pathogenesis of bone loss and fracture non-union in Nf1flox/-;Col2.3Cre mice. By genetic ablation of a single Nf1 allele in early myeloid development, under the control of LysMCre, we have further delineated that Nf1 haploinsufficient myeloid progenitors and osteoclasts are the culprit lineages mediating accelerated bone loss. Interestingly, conditional Nf1 haploinsufficiency in mature osteoclasts, induced by CtskCre, was insufficient to trigger enhanced lytic activity. These data provide direct genetic evidence for Nf1’s temporal significance as a gatekeeper of the osteoclast progenitor pool in primitive myelopoiesis. On the molecular level, we found that transforming growth factor-beta1 (TGF-β1), a primary mediator in the spatiotemporal coupling of bone remodeling, is pathologically overexpressed by five- to six- fold in both NF1 patients and in mice. Nf1 deficient osteoblasts, the principal source of TGF-β1 in the bone matrix, overexpress TGF-β1 in a gene dosage dependent fashion. Moreover, p21Ras dependent hyperactivation of the Smad pathway accentuates responses to pathological TGF-β1 signals in Nf1 deficient bone cells. As a proof of concept, we demonstrate that pharmacologic TβRI kinase inhibition can rescue bone mass defects and prevent tibial fracture non-union in Nf1flox/-;Col2.3Cre mice, suggesting that targeting TGF-β1 signaling in myeloid lineages may provide therapeutic benefit for treating NF1 skeletal defects.
Remediation Trends in an Undergraduate Anatomy Course and Assessment of an Anatomy Supplemental Study Skills Course
(2014-01-15) Schutte, Audra Faye; O'Loughlin, Valerie Dean; Brokaw, James J.; Flinders, David J., 1955-; Mescher, Anthony L.
Anatomy A215: Basic Human Anatomy (Anat A215) is an undergraduate human anatomy course at Indiana University Bloomington (IUB) that serves as a requirement for many degree programs at IUB. The difficulty of the course, coupled with pressure to achieve grades for admittance into specific programs, has resulted in high remediation rates. In an attempt to help students to improve their study habits and metacognitive skills Medical Sciences M100: Improving Learning Skills in Anatomy (MSCI M100) was developed. MSCI M100 is an undergraduate course at IUB which is taught concurrently with Anat A215, with the hopes of promoting academic success in Anat A215. This multifaceted study was designed to analyze the factors associated with students who remediate Anat A215, to predict at-risk students in future semesters, and assess the effectiveness of MSCI M100. The first facet involved analysis of Anat A215 students’ demographic information and class performance data from the spring semester of 2004 through the spring semester of 2010. Results of data analysis can be used by IUB instructors and academic advisors to identify students at risk for remediating, as well as provide other undergraduate anatomy instructors across the U.S. with potential risk factors associated with remediation. The second facet of this research involved analyzing MSCI M100 course assignments to determine if there are improvements in student study habits and metacognitive skills. This investigation involved quantitative analysis of study logs and a learning attitudes survey, as well as a thorough inductive analysis of students’ weekly journal entries. Lastly, Anat A215 exam scores and final course grades for students who completed MSCI M100 and students who did not complete MSCI M100 were compared. Results from these analyses show promising improvements in students’ metacognition and study habits, but further research will better demonstrate the efficacy of MSCI M100.
A Psychometric Evaluation of Script Concordance Tests for Measuring Clinical Reasoning
(2013-06) Wilson, Adam Benjamin; Pike, Gary R. (Gary Robert), 1952-; Humbert, Aloysius J.; Brokaw, James J.; Seifert, Mark F.
Purpose: Script concordance tests (SCTs) are assessments purported to measure clinical data interpretation. The aims of this research were to (1) test the psychometric properties of SCT items, (2) directly examine the construct validity of SCTs, and (3) explore the concurrent validity of six SCT scoring methods while also considering validity at the item difficulty and item type levels. Methods: SCT scores from a problem solving SCT (SCT-PS; n=522) and emergency medicine SCT (SCT-EM; n=1040) were used to investigate the aims of this research. An item analysis was conducted to optimize the SCT datasets, to categorize items into levels of difficulty and type, and to test for gender biases. A confirmatory factor analysis tested whether SCT scores conformed to a theorized unidimensional factor structure. Exploratory factor analyses examined the effects of six SCT scoring methods on construct validity. The concurrent validity of each scoring method was also tested via a one-way multivariate analysis of variance (MANOVA) and Pearson’s product moment correlations. Repeated measures analysis of variance (ANOVA) and one-way ANOVA tested the discriminatory power of the SCTs according to item difficulty and type. Results: Item analysis identified no gender biases. A combination of moderate model-fit indices and poor factor loadings from the confirmatory factor analysis suggested that the SCTs under investigation did not conform to a unidimensional factor structure. Exploratory factor analyses of six different scoring methods repeatedly revealed weak factor loadings, and extracted factors consistently explained only a small portion of the total variance. Results of the concurrent validity study showed that all six scoring methods discriminated between medical training levels in spite of lower reliability coefficients on 3-point scoring methods. In addition, examinees as MS4s significantly (p<0.001) outperformed their MS2 SCT scores in all difficulty categories. Cross-sectional analysis of SCT-EM data reported significant differences (p<0.001) between experienced EM physicians, EM residents, and MS4s at each level of difficulty. When considering item type, diagnostic and therapeutic items differentiated between all three training levels, while investigational items could not readily distinguish between MS4s and EM residents. Conclusions: The results of this research contest the assertion that SCTs measure a single common construct. These findings raise questions about the latent constructs measured by SCTs and challenge the overall utility of SCT scores. The outcomes of the concurrent validity study provide evidence that multiple scoring methods reasonably differentiate between medical training levels. Concurrent validity was also observed when considering item difficulty and item type.
Understanding interprofessional education : a multiple-case study of students, faculty, and administrators
(2013-07) Henkin, Katherine; Helfenbein, Robert J.; Ebright, Patricia R.; Shew, Ronald L.; Torbeck, Laura J.; Wilbur, Lee G.
Although interprofessional education (IPE) opportunities can help prepare students for future practice and patient-centered care, many health professions students in the country are not educated in an environment with opportunities to learn with, from, or about students from other health professions. With upcoming curricular changes at the Indiana University School of Medicine (IUSM) and the Indiana University School of Nursing (IUSN), IPE remains at the forefront of these changes in both schools. To date, few studies have explored student, faculty, and administrators’ conceptualizations of IPE prior to formal implementation. Additionally, previous studies have not compared IPE conceptualizations across these groups. This multiple-case study explores and compares how groups of stakeholders from the IUSM (Indianapolis) and the IUSN (Indianapolis) conceptualize IPE. Data collection included the examination of discipline-specific public documents and one-on-one interviews (N=25) with pre-licensure students, clinical faculty, and administrators from each school. Coding and extraction of themes transpired through within-case and cross-case analysis and data supported the following findings: the ‘business of medicine’ may prevent IPE from becoming a priority in education; stakeholders’ conceptualizations of IPE are shaped through powerful experiences in education and practice; students desire more IPE opportunities at the institution; stakeholders at the IUSN have a long-standing investment in IPE; and the institution requires a ‘culture shift’ in order to sustain IPE efforts. The findings suggest that IPE belongs in all education sectors and IPE efforts deserve reward and reimbursement. The findings also insinuate that leadership, roles, and team training education belong in IPE and IPE culture requires all individuals’ (e.g., student, faculty, administrators, patients) commitment. Importantly, the institution must continue IPE development, research, and dissemination. These findings can help shape curricula as time progresses, increase the likelihood of developing a successful new curriculum, and prompt ongoing reflection about IPE. This information can influence how institutions approach IPE and may lead to a more successful and informed IPE curriculum in the first years of implementation. And, hopefully what is learned through IPE will be translated into healthcare practice environments.
Targeting acute phosphatase PTEN inhibition and investigation of a novel combination treatment with Schwann cell transplantation to promote spinal cord injury repair in rats
(2013-07) Walker, Chandler L.; Xu, Xiao-Ming; Zhou, Feng C.; Jin, Xiao-Ming; Cummins, Theodore R.
Human traumatic spinal cord injuries (SCI) are primarily incomplete contusion or compression injuries at the cervical spinal level, causing immediate local tissue damage and a range of potential functional deficits. Secondary damage exacerbates initial mechanical trauma and contributes to function loss through delayed cell death mechanisms such as apoptosis and autophagy. As such, understanding the dynamics of cervical SCI and related intracellular signaling and death mechanisms is essential. Through behavior, Western blot, and histological analyses, alterations in phosphatase and tensin homolog (PTEN)/phosphatidylinositol-3-kinase (PI3K) signaling and the neuroprotective, functional, and mechanistic effects of administering the protein tyrosine phosphatase (PTP) inhibitor, potassium bisperoxo (picolinato) vanadium ([bpV[pic]) were analyzed following cervical spinal cord injury in rats. Furthermore, these studies investigated the combination of subacute Schwann cell transplantation with acute bpV(pic) treatment to identify any potential additive or synergistic benefits. Although spinal SC transplantation is well-studied, its use in combination with other therapies is necessary to complement its known protective and growth promoting characteristics. v The results showed 400 μg/kg/day bpV(pic) promoted significant tissue sparing, lesion reduction, and recovery of forelimb function post-SCI. To further clarify the mechanism of action of bpV(pic) on spinal neurons, we treated injured spinal neurons in vitro with 100 nM bpV(pic) and confirmed its neurprotection and action through inhibition of PTEN and promotion of PI3K/Akt/mammalian target of rapamycin (mTOR) signaling. Following bpV(pic) treatment and green fluorescent protein (GFP)-SC transplantation, similar results in neuroprotective benefits were observed. GFP-SCs alone exhibited less robust effects in this regard, but promoted significant ingrowth of axons, as well as vasculature, over 10 weeks post-transplantation. All treatments showed similar effects in forelimb function recovery, although the bpV and combination treatments were the only to show statistical significance over non-treated injury. In the following chapters, the research presented contributes further understanding of cellular responses following cervical hemi-contusion SCI, and the beneficial effects of bpV(pic) and SC transplantation therapies alone and in combination. In conclusion, this work provides a thorough overview of pathology and cell- and signal-specific mechanisms of survival and repair in a clinically relevant rodent SCI model.
The individual and combined effects of exercise and collagenase on the rodent Achilles tendon
(2013-10) Dirks, Rachel Candace; Warden, Stuart J.; Allen, Matthew R.; Fuchs, Robyn K.; Robling, Alexander G.
Tendinopathy is a common degenerative pathology that is characterized by activity related pain, focal tendon tenderness, intratendinous imaging changes, and typically results in changes in the histological, mechanical, and molecular properties of the tendon. Tendinopathy is difficult to study in humans, which has contributed to limited knowledge of the pathology, and thus a lack of appropriate treatment options. However, most believe that the pathology is degenerative as a result of a combination of both extrinsic and intrinsic factors. In order to gain understanding of this pathology, animal models are required. Because each tendon is naturally exposed to different conditions, a universal model is not feasible; therefore, an appropriate animal model must be established for each tendon susceptible to degenerative changes. While acceptable models have been developed for several tendons, a reliable model for the Achilles tendon remains elusive. The purpose of this dissertation was to develop an animal model of Achilles tendinopathy by investigating the individual and combined effects of an intrinsic and extrinsic factor on the rodent Achilles tendon. Rats selectively bred for high capacity running and Sprague Dawley rats underwent uphill treadmill running (an extrinsic factor) to mechanically overload the Achilles tendon or served as cage controls. Collagenase (intrinsic factor) was injected into one Achilles tendon in each animal to intrinsically break down the tendon. There were no interactions between uphill running and collagenase injection, indicating that the influence of the two factors was independent. Uphill treadmill running alone failed to produce any pathological changes in the histological or mechanical characteristics of the Achilles tendon, but did modify molecular activity. Intratendinous collagenase injection had negative effects on the histological, mechanical, and molecular properties of the tendon. The results of this dissertation demonstrated that the combined introduction of uphill treadmill running and collagenase injection did not lead to degenerative changes consistent with human Achilles tendinopathy. Intratendiouns collagenase injection negatively influenced the tendon; however, these changes were generally transient and not influenced by mechanical overload. Future studies should consider combinations of other intrinsic and extrinsic factors in an effort to develop an animal model that replicates human Achilles tendinopathy.

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