Rho Kinase (Rho-K) has been implicated in the pathophysiology of many deleterious conditions and its inhibition was shown to ameliorate these compromising effects. It is unclear; however, whether inhibition of Rho-K would decrease infarct size in hearts after ischemia/reperfusion. Adult ICR mice were randomized to 1 of 4 treatments: saline, fasudil (Rho-K inhibitor (10 mg/kg i.p.), Fasudil+L-NAME (Nitric Oxide synthase inhibitor, 15 mg/kg), and L-NAME. Hearts were isolated, perfused in Langendorff mode and subjected to 30 min stabilization before 30 min ischemia and 60 min reperfusion. Left ventricular (LV) function was monitored. Hearts were stained and infarct size measured. Fasudil reduced infarct size as compared with control hearts; however, this protective effect was abolished by L-NAME. LV function mirrored these trends. The loss of cardioprotection after L-NAME administration indicates that cardioprotection by Rho-K inhibition is mediated through nitric oxide-dependent pathway. Furthermore, Fasudil administration at and throughout reperfusion showed similar cardioprotection.

Many neurological disorders are a result of widespread changes in the excitability of brain tissue. The specific changes in neuronal excitability produces or worsens many of the symptoms associated with these disorders. Pharmacological methods are effective, but their associated side-effects are substantial, and often approximate the severity of the symptoms of the original disorder. The ketogenic diet, a high-fat, low-carbohydrate diet, has been shown to improve many neurological diseases by reducing hyperexcitability without the aforementioned side effects. The current study tested the hypothesis that a ketogenic diet would be associated with alterations in cerebral excitability. Animals were fed either a control or ketogenic diet for at least 21 days prior to experimentation. Power spectral analysis was conducted using EEG data across frequency bands, and compared between diet groups. Current source density analysis was also performed to visualize potential alterations in cerebral excitability.. In the second part of the experiment, a non-invasive ischemic stroke was delivered, and the excitability of the contralateral cortex was monitored. No significant differences were observed between ketogenic and control experiments in regards to overall excitability, although ketogenic diet experiments showed a significantly higher number of acute EEG depressions. No cortical spreading depression events were observed in contralateral recordings. Our findings are in contrast with data showing that ketogenic diets change overall basal excitability, but are in concert with other studies that show that ketogenic diets may not be associated with changes in excitability, but in changes in neuroplasticity.

Indiana University-Purdue University Indianapolis (IUPUI) / Recent clinical outcome studies demonstrate that Sodium glucose cotransporter 2 inhibitors (SGLT2i) significantly reduce major adverse cardiovascular events and heart failure outcomes in subjects with type 2 diabetes mellitus. At present, several hypotheses have been proposed to explain the observed cardiovascular benefit of SGLT2i, however, the mechanisms responsible remain to be elucidated. This investigation tested the hypothesis that SGLT2i improves cardiac function and efficiency during acute, regional ischemia/reperfusion injury via preferential shifts in myocardial substrate selection and/or inhibition of cardiac sodium/hydrogen exchanger-1 (NHE-1). Our initial investigation evaluated the effects of 24 hour pretreatment of the SGLT2i canagliflozin on cardiac contractile function, substrate utilization, and efficiency before and during regional myocardial ischemia/reperfusion injury in healthy swine. At the onset of ischemia, canagliflozin increased left ventricular end diastolic and systolic volumes which returned to baseline with reperfusion. This increased end diastolic volume was directly associated with increased stroke volume and stroke work relative to controls during ischemia. Canagliflozin also increased cardiac work efficiency during ischemia relative to control swine. No differences in myocardial substrate uptake of glucose, lactate, fatty acids or ketones were detected between groups. In separate experiments using a longer 60 min coronary occlusion, canagliflozin significantly diminished myocardial infarct size. Subsequent studies investigated the effect of an acute administration (15-30 min pre-treatment) of canagliflozin and the NHE-1i cariporide on cardiac contractile function efficiency in response to myocardial ischemia/reperfusion injury. Similar to our initial studies, canagliflozin increased diastolic filling, stroke work and improved cardiac work efficiency relative to untreated control hearts during the ischemic period. In contrast, cariporide did not alter ventricular filling volume, cardiac output or work efficiency at any time point. Additional examination of AP-1 cells transfected with wild-type NHE-1 showed dose-dependent inhibition of NHE-1 activity by cariporide, while canagliflozin had minimal effect on overall activity. This investigation demonstrates that SGLT2i improves cardiac function and efficiency during acute, regional ischemia in healthy swine. However, the present data fail to support the hypothesis that these SGLT2i-mediated improvements involve either preferential alterations in myocardial substrate utilization or the inhibition of NHE-1 activity.

PURPOSE: Previous work suggests that Dihydroorotate dehydrogenase (DHODH) inhibition via teriflunomide (TERI) may provide protection in multiple disease models. To date, little is known about the effect of TERI on the heart. This study was performed to assess the potential effects of TERI on cardiac ischemia reperfusion injury. METHODS: Male and female rat hearts were subjected to global ischemia (25 min) and reperfusion (120 min) on a Langendorff apparatus. Hearts were given either DMSO (VEH) or teriflunomide (TERI) for 5 min prior to induction of ischemia and during the reperfusion period. Left ventricular pressure, ECG, coronary flow, and infarct size were determined using established methods. Mitochondrial respiration was assessed via respirometry. RESULTS: Perfusion of hearts with TERI led to no acute effects in any values measured across 500 pM-50 nM doses. However, following ischemia-reperfusion injury, we found that 50 nM TERI-treated hearts had an increase in myocardial infarction (p < 0.001). In 50 nM TERI-treated hearts, we also observed a marked increase in the severity of contracture (p < 0.001) at an earlier time-point (p = 0.004), as well as reductions in coronary flow (p = 0.037), left ventricular pressure development (p = 0.025), and the rate-pressure product (p = 0.008). No differences in mitochondrial respiration were observed with 50 nM TERI treatment (p = 0.24-0.87). CONCLUSION: This study suggests that treatment with TERI leads to more negative outcomes following cardiac ischemia reperfusion, and administration of TERI to at-risk populations should receive special considerations.

A Thesis submitted to The University of Arizona College of Medicine - Phoenix in partial fulfillment of the requirements for the Degree of Doctor of Medicine. / Current stroke imaging protocol at Barrow Neurological Institute calls for a noncontrast computed tomography (NCCT), a computed tomography angiography (CTA), and a computed tomography perfusion (CTP) at the time of presentation to the emergency department (ED), and follow up imaging includes magnetic resonance diffusion weighted imaging (MR‐DWI). This information is used to determine the appropriateness and safety of tissue plasminogen activator (tPA) administration. Previous studies have shown the risk for post‐tPA hemorrhagic conversion rises significantly as the size of the infarct core increases. Thus, it is of great importance to have an accurate method of measuring core infarct size in patients presenting with acute ischemic stroke. The purpose of our study is to determine if CTP correctly identifies the infarct core and if post‐tPA hemorrhagic conversion is related to the size of the infarct core and/or the accuracy of CTP in identifying the infarct core. The ultimate goal is to improve patient outcomes by decreasing the morbidity and mortality associated with tPA administration. This study is a retrospective chart review of all patients who presented to the ED during a one year period with signs and symptoms of acute ischemic stroke who then subsequently received tPA. Imaging was also reviewed, including the NCCT, CTA, CTP, and MRDWI for each patient. In this study, MR‐DWI is used as the gold standard for determining the presence or absence of an infarct core. CTP and MR‐DWI are in agreement of the presence of an infarct core in 7 patients, or 10 percent of the time. Similarly, CTP and MR‐DWI are in agreement of the absence of an infarct core in 31 patients, or 44 percent of the time. In the other 32 patients, CTP and MR‐DWI are in disagreement. The percent correlation between CTP and MR‐DWI was found to be 24 percent with a p‐value < 0.05. As for post‐tPA hemorrhagic conversion, 12 percent of patients had hemorrhagic conversion, and when the hemorrhage rate was compared to the size of the infarct core, the odds of post‐tPA hemorrhagic conversion were 56 times higher in the group of patients with infarct cores larger than one‐third of a vascular territory than in patients with smaller infarct cores with a p‐value < 0.001. Although no significant correlation was found between the accuracy of CTP data and the rate of post‐tPA hemorrhagic conversion, patients with concordant CTP and MR data had a 46% lower likelihood of post‐tPA hemorrhagic conversion than did patients with contradictory CTP and MR‐DWI data. Conclusion: Because patients with infarct cores larger than one‐third of a vascular territory are 56 times more likely to hemorrhage than patients with smaller infarct cores and CTP is less accurate than MR‐DWI in identifying the infarct core in patients presenting with acute ischemic stroke, CTP studies should not be part of the acute stroke imaging protocol. Another imaging modality, such as MR‐DWI, may be preferential in the setting of acute ischemic stroke to identify the infarct core.

Recent studies have shown that the potent phosphodiesterase-5 (PDE-5) inhibitor, sildenafil citrate, induces a powerful cardioprotective effect against ischemia-reperfusion (I/R) injury in rabbit and mouse hearts. However, the effect of this drug in inducing long-term protection against I/R injury remains unknown. The goal of this study was to identify the duration of the protective window of sildenafil citrate as well as vardenafil, a more potent PDE-5 inhibitor. Rabbits were treated with sildenafil (0.7 mg/kg, iv), vardenafil (0.143 mg/kg), or an equivalent volume of saline. After 24 hrs, 48 hrs, 96 hrs, or 7 days of sildenafil treatment, the hearts were subjected to I/R. In the vardenafil groups, the hearts were subjected to I/R at 24 hrs and 7 days after administration of the drug. To evaluate the role of nitric oxide (NO) in cardioprotection, a non-selective blocker of nitric oxide synthase, L-NAME (15 mg/kg, iv) was administered 10 minutes prior to I/R. The results show significant reductions in infarct size in hearts treated with sildenafil and vardenafil as compared to the corresponding saline controls at all time points. The protective effects of sildenafil and vardenafil were abrogated in animals treated with L-NAME. L-NAME had no effect on infarct size in saline treated control rabbits. These data suggest that both sildenafil and vardenafil induce a long-term protective effect against myocardial infarction which is mediated via a NO-dependent pathway. These studies are important in exploiting the clinical potential of PDE-5 inhibitors in terms of protection against ischemia/reperfusion injury in patients with coronary artery disease.

There is a growing epidemic of chronic heart failure (CHF) in the developed world. The costs associated with providing care is profound and despite our best efforts, new, more effective treatments for CHF are needed; 50% of patients diagnosed with CHF are dead within 5 years. Current paradigms rely heavily on pharmacologic interventions, which merely help manage the disease. Surgical interventions may also be considered for late stage CHF patients such as heart transplant or left ventricular assist device (LVAD) but require burdensome and invasive surgical procedures. In addition they are costly, and require the need for life long immunosuppressive and anticoagulant therapies respectively. Despite our best intentions, the long-term prognosis for CHF patients remains poor. With over a decade of clinical investigation taken place, data from cell-based therapy trials remains inconsistent. While demonstrating safety, limited efficacy has been reported and to date, no stem cell therapy has been approved by the FDA. Despite these shortcomings important lessons have been learned that can be applied to future developments. Retrospective analysis of early cell-based clinical trial data has suggested that variations in isolated cell number, viability, and potency from donor to donor in autologous preparations yielded wide discrepancies in functional outcomes. In addition, sub culturing adult stem cells, even for short periods of time in 2D polystyrene environments void of complementary cell populations and extra cellular matrix protein interactions, may alter the therapeutic potential of a given cell. As a solution, allogeneic approaches where donor cell quality and potency can be assessed and optimized may help achieve functional benefits. Furthermore, co-dosing with multiple cell populations or developing 3D sub-culture environments that more closely mimic the in vivo milieu may ultimately yield more potent therapeutic cell populations. While these alterations may improve cell-based therapy outcomes, other solutions have been proposed such as tissue engineering. While the concept of tissue engineering is not new, advancements in biomaterials, bioreactor design and cell sources have greatly enhanced the reality of these preparations. Previously, one of the greatest limitations to tissue engineering is overcoming the cell requirements for developing and testing where millions if not billions of cells are required. Cell sourcing limitations appear to have been solved with the discovery and development of induced pluripotent stem cell (iPSC) derived cell populations. First reported in 2007, they have the ability to generate embryonic like pluripotent stem cells without the ethical concerns of embryonic stem cells. These iPSCs hold tremendous potential for drug toxicology / screening, personalized medicine and cell therapies. The body of work described in this dissertation looks at developing and testing a tissue engineered cardiac patch to treat heart failure. For which, an emphasis has been to provide 1) structural support for engrafted cells and 2) a rapidly inducible vascular supply once implanted in vivo. Biomaterials were sourced that facilitate infill by multiple cell populations in 3D culture and the establishment of extra cellular matrix deposits. Together, these patches enhanced cellular development in vitro and result in long term functional improvements in small animal models for CHF. Additional feasibility work was performed in large animal models to permit upscaling and development of surgical implantation techniques to demonstrate clinical applicability

"Recent evidence suggests that delivering human mesenchymal stem cells (hMSCs) to the infarcted heart reduces infarct size and improves ventricular performance. However, cell delivery systems have critical limitations such as inefficient cell retention and poor survival, and lack targeted localization. Our laboratories have recently developed a method to produce discrete fibrin microthreads that can be attached to a needle and delivered to a precise location within the heart wall. We hypothesize that fibrin microthreads will support hMSC proliferation, survival and retention of multipotency, and may therefore facilitate targeted hMSC delivery to injured tissues such as infarcted myocardium. To test this hypothesis, we bundled 100 μm diameter microthreads to provide grooves to encourage initial cell attachment. We seeded hMSCs onto the microthread bundles by applying 50,000 cells in 100 μL of media. The number of cells adhered to the microthreads was determined up to 5 days in culture. Cell density on the fibrin microthreads increased over time in culture, achieving an average density of 730 ± 101 cells/mm2. A LIVE/DEAD assay confirmed that the cells were viable and Ki-67 staining verified the increase in cell number over time was due to proliferation. Additionally, functional differentiation assays proved that the hMSCs cultured on microthreads retained their ability to differentiate into adipocytes and osteocytes. The results of this study demonstrate that delivering 1 to 4 cell seeded microthread bundles to the infarcted rat myocardium has the potential to produce a positive improvement in mechanical function and these microthreads support hMSC proliferation and survival. Additionally these findings suggest that cell-seeded microthreads may serve a platform technology to improve localized delivery of viable cells to infarcted myocardium to promote functional tissue regeneration. "

Made available in DSpace on 2016-04-28T20:18:29Z (GMT). No. of bitstreams: 1 2008 - Daniel de Castro Trindade.pdf: 735793 bytes, checksum: 3d6718dbe1ec23e27fbeb49005d34856 (MD5) Previous issue date: 2009-02-28 / The myocardial infarction was induced in rats by the permanent occlusion of left coronary artery in two different parts. In the first, infarcted rats were treated (CAPoral, 2g/L water) or not (INF) with oral captopril immediately after infarct and the whole experiment (21 days). In the second part, the infarcted rats were treated with captopril (CAPicv, 2 ?L 25 mg/mL/ 12-12 hours) or saline (SAL) intracerebroventricular (icv) during five consecutive days. The functional assessments were performed by electro (ECG) and echocardiogram before and after the experiment. The behavior study of water or hypertonic saline ingestion was performed in individual metabolic cages during the whole period of icv injections. The post-mortem assessment was performed in the end of each part. The ECG recorded from INF, CAPoral, SAL and CAPicv showed similar and indicative values of large myocardial infarction: decrease of QRS index amplitude, presence of Q wave in D1 and rightward deviation of the QRS axis. The main differences in the end of the treatment between INF and CAPoral groups were the prevention of P wave increase and attenuation in rightward deviation of the QRS axis in CAPoral. In the second part, there were no significant differences in ECG exam between infarcted groups. The ECO performed in the first part showed attenuation of the left atrial and ventricular dilatation, ejection fraction improvement and normalization of left ventricular filling only in CAPoral group. In the second part, ECO also showed that captopril treatment induced significative attenuation of left ventricular dilatation and improvement of ventricular filling similarly as captopril treatment by oral route. The study of fluids ingestion showed that CAPicv group exhibited less water ingestion if compared to SAL group. The hypertonic saline ingestion was not different between SAL and CAPicv groups. CAPoral group exhibited smaller scar tissue if compared to INF group. On the other hand, CAPicv group showed similar infarcted area to SAL group in histological study. / O infarto do mioc?rdio foi induzido em ratos pela oclus?o permanente da art?ria coron?ria esquerda em duas diferentes etapas. Na primeira, os ratos infartados foram tratados (CAPoral, 2g/L ?gua) ou n?o (INF) com captopril por via oral imediatamente ap?s o infarto e durante todo per?odo do experimento (21 dias). Na segunda etapa, os ratos infartados foram tratados com captopril (CAPicv, 2 ?L 25mg/mL/ 12-12 horas) ou salina (SAL) intracerebroventricular (icv) durante cinco dias consecutivos. As avalia??es funcionais foram realizadas por eletrocardiograma (ECG) e ecocardiografia (ECO) antes e ao final do experimento. O estudo comportamental de ingest?o de ?gua ou salina hipert?nica foi realizado em gaiolas metab?licas individuais durante todo per?odo de inje??es icv. A avalia??o post-mortem foi realizada no final de cada etapa. Os ECGs dos grupos INF, CAPoral, SAL e CAPicv apresentaram valores similares e indicativos de presen?a de infarto extenso do mioc?rdio como: diminui??o da amplitude do ?ndice QRS, presen?a de onda Q em D1 e desvio do vetor QRS para direita. As principais diferen?as ao final do tratamento entre os grupos INF e CAPoral foram a preven??o do aumento da onda P no grupo CAPoral e a atenua??o do desvio do vetor QRS para direita. Em rela??o aos animais da segunda etapa, n?o houve diferen?as significativas entre os grupos. No ECO realizado na primeira etapa, o grupo CAPoral mostrou atenua??o das dilata??es do ?trio e ventr?culo esquerdos, melhora na fra??o de eje??o e normaliza??o do padr?o de enchimento ventricular analisados pela t?cnica de Doppler. Na segunda etapa, o ECO mostrou que o tratamento com captopril promoveu redu??o significativa da dilata??o do ventr?culo esquerdo e melhora do enchimento ventricular. O estudo da ingest?o de fluidos mostrou que o grupo CAPicv apresentou menor ingest?o de ?gua quando comparado ao grupo SAL. O consumo de salina hipert?nica n?o foi significativamente diferente entre os grupos SAL e CAPicv. O grupo CAPoral apresentou menor tamanho de infarto quando comparado ao grupo INF, o que n?o foi observado no grupo CAPicv, que apresentou tamanho de infarto similar ao grupo SAL no estudo histol?gico.

<p>This dissertation investigates the feasibility of a real-time transthoracic Acoustic Radiation Force Impulse (ARFI) imaging system to measure myocardial function non-invasively in clinical setting. Heart failure is an important cardiovascular disease and contributes to the leading cause of death for developed countries. Patients exhibiting heart failure with a low left ventricular ejection fraction (LVEF) can often be identified by clinicians, but patients with preserved LVEF might be undetected if they do not exhibit other signs and symptoms of heart failure. These cases motivate development of transthoracic ARFI imaging to aid the early diagnosis of the structural and functional heart abnormalities leading to heart failure.</p><p>M-Mode ARFI imaging utilizes ultrasonic radiation force to displace tissue several micrometers in the direction of wave propagation. Conventional ultrasound tracks the response of the tissue to the force. This measurement is repeated rapidly at a location through the cardiac cycle, measuring timing and relative changes in myocardial stiffness. ARFI imaging was previously shown capable of measuring myocardial properties and function via invasive open-chest and intracardiac approaches.</p><p>The prototype imaging system described in this dissertation is capable of rapid acquisition, processing, and display of ARFI images and shear wave elasticity imaging (SWEI) movies. Also presented is a rigorous safety analysis, including finite element method (FEM) simulations of tissue heating, hydrophone intensity and mechanical index (MI) measurements, and thermocouple transducer face heating measurements. For the pulse sequences used in later animal and clinical studies, results from the safety analysis indicates that transthoracic ARFI imaging can be safely applied at rates and levels realizable on the prototype ARFI imaging system. </p><p>Preliminary data are presented from <italic>in vivo</italic> trials studying changes in myocardial stiffness occurring under normal and abnormal heart function. Presented is the first use of transthoracic ARFI imaging in a serial study of heart failure in a <italic>porcine</italic> model. Results demonstrate the ability of transthoracic ARFI to image cyclically-varying stiffness changes in healthy and infarcted myocardium under good B-mode imaging conditions at depths in the range of 3-5 cm. Challenging imaging scenarios such as deep regions of interest, vigorous lateral motion and stable, reverberant clutter are analyzed and discussed.</p><p>Results are then presented from the first study of clinical feasibility of transthoracic cardiac ARFI imaging. At the Duke University Medical Center, healthy volunteers and patients having magnetic resonance imaging-confirmed apical infarcts were enrolled for the study. The number of patients who met the inclusion criteria in this preliminary clinical trial was low, but results showed that the limitations seen in animal studies were not overcome by allowing transmit power levels to exceed the FDA mechanical index (MI) limit. The results suggested the primary source of image degradation was clutter rather than lack of radiation force. Additionally, the transthoracic method applied in its present form was not shown capable of tracking propagating ARFI-induced shear waves in the myocardium.</p><p>Under current instrumentation and processing methods, results of these studies support feasibility for transthoracic ARFI in high-quality B-Mode imaging conditions. Transthoracic ARFI was not shown sensitive to infarct or to tracking heart failure in the presence of clutter and signal decorrelation. This work does provide evidence that transthoracic ARFI imaging is a safe non-invasive tool, but clinical efficacy as a diagnostic tool will need to be addressed by further development to overcome current challenges and increase robustness to sources of image degradation.</p> / Dissertation