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Tyrimo tikslas yra įvertinti lazerinės doplerografijos reikšmę nudegimo gylio diagnostikai ir įtaką gydymo metodo pasirinkimui. Darbo uždaviniai: 1. Lazerinės doplerografijos metu ištirti skirtingo laipsnio nudegusių audinių perfuzijos greitį bei spalvinę išraišką. 2. Ištirti lazerinės doplerografijos reikšmę nustatant 2A ir 2B laipsnio nudegimų žaizdų savaiminio sugijimo galimybes. 3. Nustatyti ir palyginti nudegimų gylio klinikinio vertinimo ir lazerinės doplerografijos tyrimo tikslumą, jautrumą ir specifiškumą. 4. Nustatyti ir palyginti nudegimų klinikinio vertinimo ir lazerinės doplerografijos tyrimo įtaką nudegusių pacientų stacionarizavimo trukmei bei gydymo išlaidoms. Darbo metodika: Perspektyvinis atsitiktinių imčių klinikinis tyrimas. Vertinta savaiminė nudegusių audinių epitelizacija ir operacinio gydymo poreikis. Prieš atsitiktinę atranką į grupes į klinikinį tyrimą įtrauktiems ligoniams buvo paimta biopsija iš nudegiminės žaizdos, siekiant nustatyti nudegimo gylį ir palyginti su klinikinio nudegimo vertinimu (KNV) bei lazerinės doplerografijos (LDG) tyrimu. Buvo vertintas KNV ir LDG tyrimų tikslumas, jautrumas ir specifiškumas. Po to ligoniai atsitiktinės atrankos būdu buvo suskirstyti į grupes pagal skirtingus nudegimo vertinimo metodus: KNV ir LDG. Suskirstymas į grupes buvo atliktas siekiant palyginti dviejų skirtingų nudegimo vertinimo metodų įtaką ligonių stacionarizavimo trukmei ir nudegimų gydymo kainai. Išvados: 1. LDG tyrimas gali tiksliai ir... [toliau žr. visą tekstą] / The aim of study was to evaluate the importance of laser doppler imaging of burn depth examination and selection of method of treatment. Objectives of the study: 1. To explore the perfusion velocity and color view of different de¬gree of burned wound during laser doppler imaging examination. 2. To explore the importance of laser doppler imaging to determine the sponteneous epithelization of 2A and 2B degree of burn wound. 3. To evaluate and compare the accuracy, sensitivity and specificity of clinical burn depth examination and laser doppler imaging. 4. To evaluate and compare the influence of clinical burn depth examination and laser doppler imaging to length of inpatient stay and the cost of treatment. Materials and Methods: Prospective randomized study. The spontaneous burn wound epithelization and requirement to surgery was estimate. The burn tissue biopsy was made for burned patients before their randomization into groups. The aim of biopsy was to deter¬mine the depth of burn and to define the correlation with clinical burn depth examination (CBDE) and laser doppler imaging (LDI). The accu¬racy, sensitivity and specificity was determined between different examination methods. After this the burned patients were randomized into two groups: CBDE and LDI. The aim of randomization was to compare the length of inpatient stay and cost of treatment of two different burn depth examination methods. Conclusions: 1. LDI scan allows to determine the perfusion velocity... [to full text]

Peripheral artery disease (PAD) affects more than 200 million people worldwide. PAD refers to illness due to a reduction or complete occlusion of blood flow in the artery, especially to the extremities in disease conditions, such as atherosclerosis or diabetes. Critical limb ischemia (CLI) is a severe form of PAD associated with high morbidity and mortality. Currently, no effective and permanent treatments are available for this disease. The current endovascular medications (e.g., angioplasty or stents) only relieve the clinical symptoms while the surgical therapies (e.g., bypass or endarterectomy) require grafting vessels from a healthy organ to the diseased limb of the patient. However, even with these therapeutic techniques, 30% of patients still undergo limb amputation within a year. Thus, understanding of disease mechanism and development of new therapeutic approaches are in urgent needs. Meis1 (myeloid ecotropic viral integration site 1) gene belongs to the three-amino-acid loop extension subclass of homeobox gene families, and it is a highly conserved transcription factor in all eukaryotes. Up to date, little is known about the role of Meis1 in regulating vascular remodeling under ischemic condition. In this study, we aim to investigate the role and underlying mechanism of Meis1 in the regulation of arteriogenesis and angiogenesis using hindlimb ischemia model of transgenic neonatal mice. The long-term goal is to develop a new treatment for patients with PAD. Three separate but related studies were planned to complete the proposed research aims. To better understand the role of Meis1, we reviewed, in the first chapter, all literature relevant to the recent advances of the Meis1 in normal hematopoiesis, vasculogenesis, and heart developments, which were mostly studied in zebrafish and mouse. Briefly, Meis1 is found to be highly expressed in the brain and retina in zebrafish and additional in the heart, nose, and limb in mouse during the very early developmental stage, and remains at a low level quickly after birth. Meis1 is necessary for both primitive and definitive hematopoiesis and required for posterior erythroid differentiation. The absence of Meis1 results in a severe reduction of the number of mature erythrocytes and weakens the heart beats in zebrafish. Meis1 deficiency mouse is dead as early as E11.5 due to the severe internal hemorrhage. In addition, Meis1 is essential in heart development. Knock-down of Meis1 can promote angiotensin II-induced cardiomyocytes (CMs) hypertrophy or CMs proliferation, which can be repressed by a transcription factor Tbx20. Meis1 appears to play a complicated role in the blood vessels. Although the major blood vessels are still normal when global deletion of Meis1, the intersegmental vessel cannot be formed in Meis1 morphants in the zebrafish, and the small vessels are either too narrow or form larger sinuses in Meis1 deficient mouse. The effects of Meis1 on the vascular network under normal and disease (ischemia) condition remain largely unknown, and the existing data in this field is limited. In the second chapter, we developed a method protocol to identify mice of all ages, especially neonates that we faced methodological difficulties to easily and permanently label prior to our major experiments. In this study, single- or 2-color tattooing (ear, tail, or toe or combinations) was performed to identify a defined or unlimited number of mice, respectively. Tail tattooing using both green and red pastes was suitable for identifying white-haired neonatal mice as early as postnatal day (PND) 1, whereas toe tattooing with green paste was an effective alternative approach for labeling black-haired mouse pups. In comparison, single-color (green) or 2-color (green and red) ear tattooing identified both white and black adult mice older than three weeks. Ear tattooing can be adapted to labeling an unlimited number of adult mice by adding the cage number. Thus, tattooing various combinations of the ears, tail, and toes provides an easy and permanent approach for identifying mice of all ages with minimal disturbance to the animals, which shows a new approach than any existing method to identify mouse at all ages, especially the neonatal pups used in the present study (Chapter 4). Various formation of hindlimb ischemia with ligations of femoral artery or vein or both have been reported in the literature. The ischemic severity varies dependent on mouse strains and ligation methods. Due to the tiny body size of our experimental neonatal mice (PND2), it is technically challenging to separate the femoral artery from femoral vein without potential bleeding. In the third chapter, we aimed to explore a suitable surgical approach that can apply to neonatal mice. To this end, we compared the effects of femoral artery/vein (FAV) excision vs. femoral artery (FA) excision on hindlimb model using adult CD-1 mice. We showed during the 4-week period of blood reperfusion, no statistically significant differences were found between FAV and FA excision-induced ischemia regarding the reduction of limb blood flow, paw size, number of necrotic toes, or skeletal muscle cell size. We conclude that FAV and FA excision in CD-1 mice generate a comparable severity of hindlimb ischemia. In other words, FAV ligation is no more severe than FA ligation. These findings provide valuable information for researchers when selecting ligation methods for their neonate hindlimb models. Based on these findings, we selected FAV ligation of hindlimb ischemia approach to study the function of Meis1 in vascular remodeling of neonatal mice. In the fourth chapter (the main part of my dissertation), we investigated the roles of Meis1 in regulating arteriogenesis and angiogenesis of neonatal mouse under the ischemic condition. To this end, endothelial cell-specific deletion of Meis1 was generated by cross-breeding Meis1flox/flox mice with Tie2-Cre mice. Wild-type (WT, Meis1f/f) and endothelial cell-specific knock-out (KO, Meis1ec-/-Tie2-Cre+) C57BL/6 mice at the age of PND2 were used. Under the anesthesia, the pups were subject to hindlimb ischemia by excising FAV. Laser Doppler Imager was used to measure the blood flow pre- and post-surgery up to 28 days. Toe necrosis, skeletal regeneration, and vascular distributions were examined at the end of experiments (PND28 post-ischemia). Surprisingly, during 4-week periods after ischemia, the blood flow ratios (ischemic vs. control limb) in KO mice significantly increased compared to WT on PND14 and PND28, suggesting the inhibitory effects of Meis1 on blood flow recovery under ischemic condition. Meanwhile, WT mice showed more severe necrotic limb (lower ratio of limb length and area, and higher necrotic scores at PND7) than those in the KO mice. Furthermore, significant increases in diameters of Dil-stained arterioles of the skin vessel and the vessels on the ligation site were observed in KO mice, indicating the enhanced arteriogenesis in KO mice. To investigate the underlying mechanism, RNA from the ischemia and control limb was extracted and q-PCR was used to study the potential genes involved in the mechanism. Casp3 and Casp8 were found downregulated showing less apoptosis in the KO mice. On the other hand, endothelial cells (ECs) were isolated from the lungs of 3-5 WT and KO neonates using CD31 Microbeads. CD31+ cells were plated and treated with 0, 0.5, and 1μM doxorubicin for 24 hours and analyzed with various assays. Meis1-KO ECs demonstrated higher cell viability and formed a higher number of vascular tubes than those in WT ECs following 0.5μM Dox treatment, presenting the potential ability of angiogenesis in KO-ECs. Furthermore, the increased viability in KO ECs may be due to the decreased expression or activities of Casp8 and Casp3. In conclusion, my present studies have developed a new methodology to easily and permanently identify all mice at any ages. The insignificant differences between FAV and FA ligations suggest that a relative-easy surgical approach could be used to generate hindlimb ischemic model, which potentially reduces the cost, decreases the surgical time and prevents damage of femoral nerve from surgical tools. More importantly, by using transgenic mice, we found that Meis1-KO dramatically increased blood flow and protected the ischemic hindlimb through vascular remodeling. Obviously, the molecular and cellular mechanisms underlying the above beneficial effects appear complicated and likely to involve multiple cellular remodeling processes and molecular signaling pathways to enhance arteriogenesis and angiogenesis and/or reduce cellular apoptosis through Meis1-mediated pathways. Our study demonstrated that under ischemic condition, knockout of Meis1 increases expression of Hif1a, which then activates Agt or VEGF, thus enhances arteriogenesis or angiogenesis; In addition, knockout of Meis1 activates Ccnd1, which subsequently promotes regeneration of skeletal muscle, and reduces expression of Casp8 and Casp3, thus preventing limb tissue from ischemia-induced apoptosis. Our innovative findings offer great potential to ultimately lead to new drug discovery or therapeutic approaches for prevention or treatment of PAD. / PHD / Peripheral artery disease (PAD), which affects more than 200 million people worldwide, commonly refers to the vascular diseases of legs or feet due to a reduction or even complete occlusion of blood flow to these areas. PAD is usually caused by blockage of main vessels in limbs under certain diseases, such as atherosclerosis. Unfortunately, no effective and permanent treatments are available for this disease. The current medications only relieve the clinical symptoms while the surgical therapy requires grafting vessels from a healthy organ to the diseased limb of the patient. In the present study, we aim to explore a new approach to facilitate the vessel formation in ischemic limb using Meis 1 transgenic mice. Meis 1 (myeloid ecotropic viral integration site 1) gene belongs to homeobox gene families, and it is a highly conserved transcription factor in all eukaryotes. My dissertation aims to understand how Meis 1 affects vascular remodeling in the mouse following induced hindlimb ischemia (to mimic PAD). To better understand the role of Meis 1, we first reviewed the literature studying the Meis 1 function on normal hematopoiesis, vasculogenesis, and heart development in zebrafish and mouse. The studies show that Meis 1 plays a complicated role in the blood vessels. When entirely deleting Meis 1 in the zebrafish, the intersegmental vessels cannot be formed. While in a mammal study, it is found that the major blood vessels are normal while the small vessels are either too narrow or form larger sinuses in Meis 1 deficient mouse. Thus, Mesi1 appears to play an important role in regulating vascular network, but the available information in this field is insufficient. The present projects aimed to study the roles of Meis 1 in regulating vascular remodeling following the hindlimb ischemia induced by ligation of main limb vessels (to mimic PAD). The transgenic mice with the deletion of Meis 1 (called knockout or KO mice) were generated by a Cre-loxP system (a gene manipulation method) to remove Meis 1 only in endothelial cells. The resulting KO mice were subject to the hindlimb ischemia and compared to those mice with the intact Meis 1 (called wild-type, or WT). To this end, the entire experiments contain three separate studies. In the first studies (Chapter 2), we developed an easy, but a permanent method to identify the mice at all age, especially the neonatal mice we used in the projects. Briefly, we used single- or 2-color tattooing to identify a defined or unlimited number of mice, respectively. We labeled our adult mice with ear tattooing combined with cage number and neonatal mice with toe tattooing if necessary to identify the individual animals. Next (Chapter 3), we determined the effects of femoral artery/vein (FAV) ligation vs. femoral artery (FA) ligation alone on hindlimb severities. The purpose of this study was to generate a suitable ligation model for the neonatal mice. Interestingly, no statistically significant differences were found between FAV and FA excision-induced ischemia, suggesting that FAV ligation could be applied to the neonatal hindlimb ischemia model in the rest of study. Upon the establishment of identification and ligation approaches for neonatal mice, we conducted systemic studies at both in vitro and in vivo settings to investigate the biological function of Meis 1 under ischemic condition. Briefly, two groups of Meis 1 mice at ages of postnatal day 2 were used in the study: WT (the normal mice), and endothelial specific knock-out (KO, Meis 1 gene was entirely deleted in endothelial cells). Under anesthesia, the postnatal day 2 pups were induced hindlimb ischemia, and blood flow was measured pre- and post-ischemia up to 4 weeks. Our data demonstrated that the blood flow was significantly higher in KO mice than WT mice, suggesting deletion of Meis 1 in endothelial cells can increase blood perfusion following ischemic injury. Moreover, the KO mice showed less toe damage compared with WT, thus showing protective benefit in rescuing the damaged limb. We also found that deletion of Meis 1 in endothelial cells increased cell viability and induced generation of more numbers of vessels under an induced apoptosis condition. These findings suggested that the deletion of Meis 1 in endothelial cells facilitates vessel formation and prevents the injured limbs from loss or undergoing apoptosis/necrosis, which may lead new drug discovery and development of effective therapy for treatments of PAD.

Cardiovascular disease is the leading cause of death in the United States, and coronary artery disease (CAD) kills over 370,000 people annually. There are available therapies that offer a temporary solution; however, only a heart transplant can fully resolve heart failure, and donor organ shortages severely limit this therapy. C-kit+ human cardiac stem cells (hCSCs) offers a viable alternative therapy to treat cardiovascular disease by replacing damaged cardiac tissue; however, low cell viability, low retention/engraftment, and uncontrollable in vivo differentiation after transplantation has limited the efficacy of stem cell therapy. Tissue engineering solutions offer potential tools to overcome current limitations of stem cell therapy. Materials derived from natural sources such as keratin from human hair offers innate cellular compatibility, bioactivity, and low immunogenicity. Keratin proteins extracted using oxidative chemistry known as keratose (KOS) have shown therapeutic potential in a wide range of applications including cardiac regeneration. My studies utilize KOS hydrogels to modulate c-kit+ hCSC differentiation, and explore the capability of differentiated cells to regenerate vascular tissue. In the first Chapter, we reviewed literature relevant to keratin-based biomaterials and their biomedical applications, the use of stem cells in cardiovascular research, and the differentiation of vascular smooth muscle cells (VSMCs). The section on biomedical applications of keratin biomaterials focuses on the oxidized form of keratin known as keratose (KOS), because this was the material used for our research. Since we planned to use this material in conjunction with c-kit+ hCSCs, we also briefly explored the use of stem cells in cardiovascular research. Additionally, we examined some key signaling pathways, developmental origins, and the cell phenotype of VSMCs for reasons that will become clear after observing results from chapters 2 and 3. Based upon our review of the literature, KOS biomaterials and c-kit+ hCSCs were determined to be promising as a combined therapeutic for the regeneration of cardiac tissue. Research in Chapter 2 focused on characterizing the effects of KOS hydrogel on c-kit+ hCSC cell viability, proliferation, morphology, and differentiation. Results demonstrated that KOS hydrogels could maintain hCSC viability without any observable toxic effects, but it modulated cell size, proliferation, and differentiation compared to standard tissue culture polystyrene cell culture (TCPS). KOS hydrogel produced gene and protein expression consistent with a VSMC phenotype. Further, we also observed novel "endothelial cell tube-like" microstructures formed by differentiated VSMCs only on KOS hydrogel, suggesting a potential capability of the hCSC-derived VSMCs for in vitro angiogenesis. Results from this study lead us to question what signaling pathways might be responsible for the apparent VSMC differentiation pattern we observed on KOS hydrogels. Research in Chapter 3 explored the time course of VSMC differentiation, cell contractility, inhibition of VSMC differentiation, and measured protein expression of transforming growth factor beta 1 (TGF-β1) and its associated peptides for hCSCs cultured on KOS hydrogels, tissue culture polystyrene, and collagen hydrogels. A review of VSMC differentiation signaling pathways informed our decision to investigate the role of TGF-β1 in VSMC differentiation. Results demonstrated that KOS hydrogel differentiated hCSCs significantly increased expression for all three vascular smooth muscle (VSM) markers compared to TCPS differentiated cells. Additionally, KOS differentiated hCSCs were significantly more contractile than cells differentiated on TCPS. Recombinant human (rh) TGF-β1 was able to induce VSM differentiation on TCPS. VSM differentiation was successfully inhibited using TGF-β NABs and A83-01. Enzyme-Linked Immunosorbent Assay (ELISA) analysis revealed that both TCPS and KOS hydrogel differentiated cells produced TGF-β1, with higher levels being measured at early time points on TCPS and later time points on KOS hydrogels. Results from supplementing rhTGF-β1 to TCPS and KOS hydrogels revealed that KOS seems to interact with TGF-β to a greater extent than TCPS. Western blot results revealed that latency TGFβ binding protein (LTBP-1) and latency associated peptide (LAP) had elevated levels early during differentiation. Further, the levels of LTBP-1 and LAP were higher on KOS differentiated hCSCs than TCPS hCSCs. This study reaffirms previous results of a VSM phenotype observed on KOS hydrogels, and provides convincing evidence for TGF-β1 inducing VSM differentiation on KOS hydrogels. Additionally, results from ELISA and western blot provide evidence that KOS plays a direct role in this pathway via interactions with TGF-β]1 and its associated proteins LTBP-1 and LAP. Results from chapter 2 and 3 offered significant evidence that our cells exhibited a VSMC phenotype, and that TGF-β1 signaling was a key contributor for the observed phenotype, but we still needed an animal model to explore the therapeutic potential of our putative VSMCs. Research in Chapter 4 investigated a disease model to test the ability of KOS hydrogel differentiated cells to regenerate vascular tissue. To measure vascular regenerative capability, we selected a murine model of critical limb ischemia (CLI). CLI was induced in 3 groups (n=15/group) of adult mixed gender NSG mice by excising the femoral artery and vein, and then treated the mice with either PBS (termed as PBS-treated), Cells differentiated on TCPS (termed as Cells from TCPS), or KOS hydrogel-derived VSMCs (termed as Cells from KOS). Blood perfusion of the hind limbs was measured immediately before and after surgery, then 14, and 28 days after surgery using Laser Doppler analysis. Tissue vascularization, cell engraftment, and skeletal muscle regeneration were measured using immunohistochemistry, 1,1'-Dioctadecyl3,3,3',3'-Tetramethylindocarbocyanine Perchlorate (DiL) vessel painting, and hematoxylin and eosin (HandE) pathohistological staining. During the 4-week period, both cell treatment groups showed significant increases in blood perfusion compared to the PBS-treated control, and at day 28 the Cells from KOS group had significantly better blood flow than the Cells from TCPS group. Additionally, the Cells from KOS group demonstrated a significant increase in the ratio of DiL positive vessels, capillary density, and a greater density of small diameter arterioles compared to the PBS-treated group. Further, both cell-treated groups had similar levels of engraftment into the host tissue. We conclude that Cells from KOS therapy increases blood perfusion in an NSG model of CLI, but does not lead to increased cell engraftment compared to other cell based therapies. Overall, the results from this dissertation demonstrated that KOS hydrogels produce VSMC differentiation from c-kit+ hCSCs mediated by TGF-β1 signaling, and that the differentiated cells are able to increase blood perfusion in a CLI model by increasing capillary density, suggesting enhanced angiogenesis. Future studies should explore potential protein-protein interactions between KOS, TGF-β1 and its associated proteins. Additionally, we should plan animal studies that examine the efficacy of our cells to regenerate cardiac tissue following an acute myocardial infarction (AMI). / PHD

<p>The present thesis proposes new methods for obtaining objective and accurate diagnoses in modern healthcare. Non-invasive techniques have been used to examine or diagnose three different medical conditions, namely neuropathy among diabetics, radiotherapy induced erythema (skin redness) among breast cancer patients and diagnoses of cutaneous malignant melanoma. The techniques used were Near-InfraRed spectroscopy (NIR), Multi Frequency Bio Impedance Analysis of whole body (MFBIA-body), Laser Doppler Imaging (LDI) and Digital Colour Photography (DCP).</p><p>The neuropathy for diabetics was studied in papers I and II. The first study was performed on diabetics and control subjects of both genders. A separation was seen between males and females and therefore the data had to be divided in order to obtain good models. NIR spectroscopy was shown to be a viable technique for measuring neuropathy once the division according to gender was made. The second study on diabetics, where MFBIA-body was added to the analysis, was performed on males exclusively. Principal component analysis showed that healthy reference subjects tend to separate from diabetics. Also, diabetics with severe neuropathy separate from persons less affected.</p><p>The preliminary study presented in paper III was performed on breast cancer patients in order to investigate if NIR, LDI and DCP were able to detect radiotherapy induced erythema. The promising results in the preliminary study motivated a new and larger study. This study, presented in papers IV and V, intended to investigate the measurement techniques further but also to examine the effect that two different skin lotions, Essex and Aloe vera have on the development of erythema. The Wilcoxon signed rank sum test showed that DCP and NIR could detect erythema, which is developed during one week of radiation treatment. LDI was able to detect erythema developed during two weeks of treatment. None of the techniques could detect any differences between the two lotions regarding the development of erythema.</p><p>The use of NIR to diagnose cutaneous malignant melanoma is presented as unpublished results in this thesis. This study gave promising but inconclusive results. NIR could be of interest for future development of instrumentation for diagnosis of skin cancer.</p>

The present thesis proposes new methods for obtaining objective and accurate diagnoses in modern healthcare. Non-invasive techniques have been used to examine or diagnose three different medical conditions, namely neuropathy among diabetics, radiotherapy induced erythema (skin redness) among breast cancer patients and diagnoses of cutaneous malignant melanoma. The techniques used were Near-InfraRed spectroscopy (NIR), Multi Frequency Bio Impedance Analysis of whole body (MFBIA-body), Laser Doppler Imaging (LDI) and Digital Colour Photography (DCP). The neuropathy for diabetics was studied in papers I and II. The first study was performed on diabetics and control subjects of both genders. A separation was seen between males and females and therefore the data had to be divided in order to obtain good models. NIR spectroscopy was shown to be a viable technique for measuring neuropathy once the division according to gender was made. The second study on diabetics, where MFBIA-body was added to the analysis, was performed on males exclusively. Principal component analysis showed that healthy reference subjects tend to separate from diabetics. Also, diabetics with severe neuropathy separate from persons less affected. The preliminary study presented in paper III was performed on breast cancer patients in order to investigate if NIR, LDI and DCP were able to detect radiotherapy induced erythema. The promising results in the preliminary study motivated a new and larger study. This study, presented in papers IV and V, intended to investigate the measurement techniques further but also to examine the effect that two different skin lotions, Essex and Aloe vera have on the development of erythema. The Wilcoxon signed rank sum test showed that DCP and NIR could detect erythema, which is developed during one week of radiation treatment. LDI was able to detect erythema developed during two weeks of treatment. None of the techniques could detect any differences between the two lotions regarding the development of erythema. The use of NIR to diagnose cutaneous malignant melanoma is presented as unpublished results in this thesis. This study gave promising but inconclusive results. NIR could be of interest for future development of instrumentation for diagnosis of skin cancer.