EFFECTS OF DIABETES MELLITUS ON THE BIOMECHANICAL PROPERTIES AND PHARMACOLOGICAL FUNCTION OF THE FEMALE RAT URETHRA EX-VIVO

EFFECTS OF DIABETES MELLITUS ON THE BIOMECHANICAL PROPERTIES AND PHARMACOLOGICAL FUNCTION OF THE FEMALE RAT URETHRA EX-VIVORachelle Lynn Prantil, MSUniversity of Pittsburgh, 2004Diabetic cystopathy results in a grossly distended, hypomotile bladder due to inefficient voiding. While the bladder has been extensively studied, little effort has been made towards the understanding of the urethra and the effects of this devastating disease. The current study is aimed to evaluate the effects of diabetes mellitus (DM) on the biomechanical properties and the pharmacological function of the female rat urethra ex vivo. DM was induced in female rats by injection of streptozotocin. At 3, 5,and 10 weeks, the urethras were excised and mounted into an ex-vivo system at in vivo length. For mechanical testing, urethras were subjected to stepwise increases of static, intraurethral pressure from 0 to 20 mmHg in both a baseline and passive state. Continuous outer diameter measurements were made using a laser micrometer at proximal, middle, and distal portions of the urethra. Compliance and beta stiffness were calculated from measured data. Pharmacological experiments involved assessments of mid urethral outer diameter response to Nč Nitro-L-arginine, phenylephrine, and EDTA. Age matched normal urethras served as controls. Statistical comparisons were made via ANOVA. Tissue was then processed for immuno- and histochemical quantification of smooth muscle, collagen, and elastin. For baseline healthy tissue, results showed a proximal to distal compliance gradient (proximal most compliant and distal least compliant), and the passive state enhanced the observation. Baseline beta stiffness values showed an increased stiffness in proximal and middle urethral portions by 5 and 10 weeks, and baseline compliance values showed at low pressures showed an increase in proximal compliance at 3 weeks and a decrease in proximal compliance at 5 weeks at high pressures. Passive beta stiffness and compliance values indicated proximal urethral stiffening by 10 weeks DM. Pharmacological studies revealed that DM abolishes endogenous nitric oxide release and increases the time to reach maximal relaxation. In cases of severe DM, alpha 1 adrenergic contraction was minimized. Little or no differences were found in the amount collagen, smooth muscle, and elastin. From these findings, it can be concluded that DM causes urethral stiffening and impaired contractile and relaxation urethral mechanism. Damaged urethral properties and function have serious implications for outlet resistance; thus, contributing to diabetic cystopathy

BIOMECHANICS AND FUNCTION OF THE FEMALE RAT URETHRA IN STRESS URINARY INCONTINENCE INDUCED BY BIRTH TRAUMA

Stress urinary incontinence (SUI) is common in women after vaginal delivery (VD) in childbirth or pelvic trauma, and may be associated with altered biomechanical or functional properties of the urethra. The goal of this dissertation was to identify biomechanical and functional changes in the urethra in a rat model of VD, as well as to understand the role of longitudinal smooth muscle in the healthy urethra. Female rat urethras were isolated in a rat model of SUI induced by VD. Controls were urethras isolated from normal rats. Our established ex vivo urethral testing system was utilized for biomechanical and pharmacological assessments. In this system, outer diameter was measured via a laser micrometer, and recorded along with applied intraluminal pressure to a computer. Urethral thickness was assessed histologically.Biomechancial properties of the urethra were markedly altered by VD for the baseline, passive (via calcium chelation), and active (stimulation via adrenergic and muscarinic receptors) states, most notably in the proximal urethra. Additionally, contractile responses to phenylephrine and bethanechol increased in the proximal urethra in VD rats compared to controls. There were also changes in the VD mid urethral segment. Functional and biomechanical parameters indicated that basal activity was increased for VD compared to controls in the middle segment, as well as adrenergic active biomechanical properties at low strains. VD impaired the basal tone distally compared to controls, but this was the only difference observed. VD urethras had evidence of altered collagen and elastin. Additionally, there was a lack of PGP 9.5, tyrosine hydroxylase, and vesicular acetylcholine transferase in the urethras of the VD group. This suggests that VD has mechanically damaging effects on urethral innervation.Finally, the role of the longitudinal smooth muscle in the urethra was further clarified via a modified urethral testing system. Circumferential and longitudinal testing of baseline, active, and passive urethral properties and function supported the idea that the role of longitudinally-oriented components of the urethra was to lengthen or shorten to enable the circumferential muscle to fully contract and shorten as required. In summary, this dissertation has provided evidence of damaged muscular, neural, and matrix components of the urethra associated with VD. The combination of these changes may contribute to SUI induced by VD

THE STRUCTURE OF THE TRANSVERSE CARPAL LIGAMENT: ITS COLLAGEN FIBER ORIENTATION AND THE EFFICACY OF COLLAGENASE IN DECREASING ITS STIFFNESS

Carpal tunnel syndrome (CTS) currently affects more than three million Americans each year. Hand surgeons treat CTS by targeting the transverse carpal ligament (TCL) which acts as the palmar roof of the carpal tunnel. Based on general observation, the TCL appears to be an inextensible collagenous matrix with fibers roughly oriented along the transverse direction. Several studies on the TCL’s configuration argue for different fiber orientations consisting of either oblique or transverse orientations; whereas, most findings were based on an observational methodology. Very few studies have determined such fiber orientations; whereas, even fewer studies have researched the ligament’s mechanical properties. Furthermore, the potential of altering the TCL’s microstructure may provide a potential, alternative to the currently accepted, invasive standards. Previous studies attempting to lengthen the TCL found that such procedures can effectively diminish CTS symptoms and also decrease the progression of several post-operative complications. However, these procedures consist of transecting the transverse carpal ligament in an attempt to increase its length. Furthermore, mechanical stimuli cannot alter the ligament because it is too stiff. In addition, such procedures also require invasive surgery and can cause complications that arise from carpal tunnel release. Therefore, a solution might lie in the application of collagenase where antecedent works have shown its capacity to reduce the v mechanical properties of a tissue. The following studies have emphasized the transverse carpal ligament’s collagen orientation and its mechanical response to subsequent collagenase treatment. The preferential collagen direction was quantified through the use of small angle light scattering (SALS). Results showed that transverse orientation was the most prevalent with minimal changes found within its orientation along its thickness. As for the TCL’s response to collagenase, standard concentrations of collagenase were applied to the TCL for each specimen through successive mechanical loading protocols along with successive observations to analyze the progressive changes within the ligament by slowly eliminating its collagen network. Collagenase effectively decreased the transverse carpal ligament’s stiffness without significantly changing its mechanical properties. Furthermore, these studies could contribute to a more sophisticated model of the TCL and lead to the development of a minimally invasive therapy contrary to current, invasive standards

Uropathic Observations in Mice Expressing a Constitutively Active Point Mutation in the 5-HT_(3A) Receptor Subunit

Mutant mice with a hypersensitive serotonin (5-HT)_(3A) receptor were generated through targeted exon replacement. A valine to serine mutation (V13′S) in the channel-lining M2 domain of the 5-HT_(3A) receptor subunit rendered the 5-HT₃ receptor ∼70-fold more sensitive to serotonin and produced constitutive activity when combined with the 5-HT_(3B) subunit. Mice homozygous for the mutant allele (5-HT_(3A)^(vs/vs)) had decreased levels of 5-HT_(3A) mRNA. Measurements on sympathetic ganglion cells in these mice showed that whole-cell serotonin responses were reduced, and that the remaining 5-HT₃ receptors were hypersensitive. Male 5-HT_(3A)^(vs/vs) mice died at 2-3 months of age, and heterozygous (5-HT_(3A)^(vs/+)) males and homozygous mutant females died at 4-6 months of age from an obstructive uropathy. Both male and female 5-HT_(3A) mutant mice had urinary bladder mucosal and smooth muscle hyperplasia and hypertrophy, whereas male mutant mice had additional prostatic smooth muscle and urethral hyperplasia. 5-HT_(3A) mutant mice had marked voiding dysfunction characterized by a loss of micturition contractions with overflow incontinence. Detrusor strips from 5-HT_(3A)^(vs/vs) mice failed to contract to neurogenic stimulation, despite overall normal responses to a cholinergic agonist, suggestive of altered neuronal signaling in mutant mouse bladders. Consistent with this hypothesis, decreased nerve fiber immunoreactivity was observed in the urinary bladders of 5-HT_(3A)^(vs/vs) compared with 5-HT_(3A) wild-type (5-HT_(3A)^(+/+)) mice. These data suggest that persistent activation of the hypersensitive and constitutively active 5-HT_(3A) receptor in vivo may lead to excitotoxic neuronal cell death and functional changes in the urinary bladder, resulting in bladder hyperdistension, urinary retention, and overflow incontinence

A microphysiological system model of therapy for liver micrometastases

Metastasis accounts for almost 90% of cancer-associated mortality. The effectiveness of cancer therapeutics is limited by the protective microenvironment of the metastatic niche and consequently these disseminated tumors remain incurable. Metastatic disease progression continues to be poorly understood due to the lack of appropriate model systems. To address this gap in understanding, we propose an all-human microphysiological system that facilitates the investigation of cancer behavior in the liver metastatic niche. This existing LiverChip is a 3D-system modeling the hepatic niche; it incorporates a full complement of human parenchymal and non-parenchymal cells and effectively recapitulates micrometastases. Moreover, this system allows real-time monitoring of micrometastasis and assessment of human-specific signaling. It is being utilized to further our understanding of the efficacy of chemotherapeutics by examining the activity of established and novel agents on micrometastases under conditions replicating diurnal variations in hormones, nutrients and mild inflammatory states using programmable microdispensers. These inputs affect the cues that govern tumor cell responses. Three critical signaling groups are targeted: the glucose/insulin responses, the stress hormone cortisol and the gut microbiome in relation to inflammatory cues. Currently, the system sustains functioning hepatocytes for a minimum of 15 days; confirmed by monitoring hepatic function (urea, α-1-antitrypsin, fibrinogen, and cytochrome P450) and injury (AST and ALT). Breast cancer cell lines effectively integrate into the hepatic niche without detectable disruption to tissue, and preliminary evidence suggests growth attenuation amongst a subpopulation of breast cancer cells. xMAP technology combined with systems biology modeling are also employed to evaluate cellular crosstalk and illustrate communication networks in the early microenvironment of micrometastases. This model is anticipated to identify new therapeutic strategies for metastasis by elucidating the paracrine effects between the hepatic and metastatic cells, while concurrently evaluating agent efficacy for metastasis, metabolism and tolerability.National Institutes of Health (U.S.) (Grant 1UH2TR000496-01)United States. Defense Advanced Research Projects Agency. Microphysiological Systems Program (W911NF-12-2-0039

Status of the LBNL normal-conducting CW VHF electron photo-gun

The fabrication and installation at the Lawrence Berkeley National Laboratory of a high-brightness high-repetition rate photo-gun, based on a normal conducting 187 MHz (VHF) RF cavity operating in CW mode, is in an advanced phase. The cavity will generate an electric field at the cathode plane of ∼ 20 MV/m to accelerate the electron bunches up to ∼ 750 keV, with peak current, energy spread and transverse emittance suitable for FEL and ERL applications. The gun vacuum system has been designed for achieving pressures compatible with the use of "delicate" high quantum efficiency semiconductor cathodes to generate up to a nC bunches at MHz repetition rate with present laser technology. Several photo-cathode/laser systems are under consideration, and in particular photo-cathodes based on K CsSb are being developed for the gun and have already achieved a QE of 8% at 532 nm wavelength, or close to 20% including the Schottky barrier lowering. The cathode will be operated by a μJ fiber laser in conjunction with refractive transverse beam shaping to create a flat top transverse profile, as well as a birefringent pulse stacker to create a flat top temporal profile. The present status and the plan for future activities are presented.

Modeling radiation injury-induced cell death and countermeasure drug responses in a human Gut-on-a-Chip

Studies on human intestinal injury induced by acute exposure to γ-radiation commonly rely on use of animal models because culture systems do not faithfully mimic human intestinal physiology. Here we used a human Gut-on-a-Chip (Gut Chip) microfluidic device lined by human intestinal epithelial cells and vascular endothelial cells to model radiation injury and assess the efficacy of radiation countermeasure drugs in vitro. Exposure of the Gut Chip to γ-radiation resulted in increased generation of reactive oxygen species, cytotoxicity, apoptosis, and DNA fragmentation, as well as villus blunting, disruption of tight junctions, and compromise of intestinal barrier integrity. In contrast, pre-treatment with a potential prophylactic radiation countermeasure drug, dimethyloxaloylglycine (DMOG), significantly suppressed all of these injury responses. Thus, the human Gut Chip may serve as an in vitro platform for studying radiation-induced cell death and associate gastrointestinal acute syndrome, in addition to screening of novel radio-protective medical countermeasure drugs

All-human microphysical model of metastasis therapy

The vast majority of cancer mortalities result from distant metastases. The metastatic microenvironment provides unique protection to ectopic tumors as the primary tumors often respond to specific agents. Although significant interventional progress has been made on primary tumors, the lack of relevant accessible model in vitro systems in which to study metastases has plagued metastatic therapeutic development - particularly among micrometastases. A real-time, all-human model of metastatic seeding and cancer cells that recapitulate metastatic growth and can be probed in real time by a variety of measures and challenges would provide a critical window into the pathophysiology of metastasis and pharmacology of metastatic tumor resistance. To achieve this we are advancing our microscale bioreactor that incorporates human hepatocytes, human nonparenchymal liver cells, and human breast cancer cells to mimic the hepatic niche in three dimensions with functional tissue. This bioreactor is instrumented with oxygen sensors, micropumps capable of generating diurnally varying profiles of nutrients and hormones, while enabling real-time sampling. Since the liver is a major metastatic site for a wide variety of carcinomas and other tumors, this bioreactor uniquely allows us to more accurately recreate the human metastatic microenvironment and probe the paracrine effects between the liver parenchyma and metastatic cells. Further, as the liver is the principal site of xenobiotic metabolism, this reactor will help us investigate the chemotherapeutic response within a metabolically challenged liver microenvironment. This model is anticipated to yield markers of metastatic behavior and pharmacologic metabolism that will enable better clinical monitoring, and will guide the design of clinical studies to understand drug efficacy and safety in cancer therapeutics. This highly instrumented bioreactor format, hosting a growing tumor within a microenvironment and monitoring its responses, is readily transferable to other organs, giving this work impact beyond the liver. © 2013 BioMed Central Ltd