or placebo o relative to sham instrumented control hearts at top high lfts due to methotrexate left panel, top right and min bottom left panel mean lvdp of hearts from to min of reperfusion are shown in the bottom right panel tetrazolium chloride stained heart slices showed that hearts treated with csil at , and min had similar injury as that of the sham operated hearts � , � and � , and � respectively p � ns the infarct size of hearts treated with csil even at min of global ischemia was � of the ventricles this was significantly smaller than its corresponding control p , whereas hearts treated with control iggl at , and min of global ischemia were � , � and � respectively fig , left panel the corresponding nitroblue tetrazolium chloride stained heart slices are shown in fig , right panel another parameter of myocardial injury high lfts due to methotrexate that was determined was mitochondrial size although mitochondrial swelling is a hallmark of high lfts due to methotrexate ischemic injury, irreversible injury cannot be directly extrapolated from just observation of mitochondrial high lfts due to methotrexate size nevertheless, in view of the myocardial functional and his tochemical evidences, mitochondrial high lfts due to methotrexate size assessment from transmission electron micrographs add additional support for myocardial preservation in csil treated hearts, relative to iggl or placebo treated hearts figure shows the comparison of fig mean infarct sizes of rat hearts treated with csil or iggl or placebo at and min of global ischemia left panel the corresponding nitroblue tetrazolium chloride stained mid slices of rat hearts treated with high lfts due to methotrexate csil or iggl at , and min of global ischemia minimal injury was seen in and dangers of quitting taking cymbalta min csil treated hearts, but injury was evident in the min csil treated heart slice injury is evident in all heart slices treated with iggl right panel, bottom two rows �i i normal cut csil csil csejo igsl tg& iflsi placebo v fig mean mitochondrial size of normal, csil, high lfts due to methotrexate iggl or placebo treated hearts treatment was as indicated in the text mitochondrial size of normal hearts, csil treatment at and min of global ischemia, as well as iggl treated hearts at , and min of global ischemia and with placebo no difference in mitochondrial size was observed between normal myocardium � mean number of pixels � sem and myocardium treated at and min high lfts due to methotrexate of global ischemia � , � , � and � respectively p = ns however, high lfts due to methotrexate mitochondria of hearts treated with iggl at , and min of global ischemia or placebo � , � , � and � respectively were larger than mitochondria of csil high lfts due to methotrexate treated hearts p these studies showed that myocardial viability preservation is not restricted to embryonic cardiocytes in cultures adult hearts are also amenable to structural high lfts due to methotrexate and functional preservation, following cell membrane lesion sealing in a timedependent manner during ischemia this method of cell membrane lesion sealing has also been reported high lfts due to methotrexate to preserve the integrity of vascular endothelium with antiactinimmunoliposomes a question that remains concerning the utility of csil is whether immunoliposomes can retain the protective functions in the presence of plasma proteins in vivo, since experiments have demonstrated that cells in culture and adult hearts perfused with nonprotein oxygenated buffer were prevented from undergoing myocardial necrosis, following cell membrane lesion sealing intervention with cytoskeletal high lfts due to methotrexate antigen specific immunoliposomes to demonstrate that cell membrane lesion sealing also occur in high lfts due to methotrexate vivo, rabbits with experimental myocardial infarction were used in this study, rabbits were injected with antimyosin csil, plain liposomes or saline at the time of left circumflex coronary artery occlusion by intracoronary infusion the occlusion was kept for high lfts due to methotrexate min followed by hrs of reperfusion the hearts were excised, sliced into � slices parallel to the short axis and stained with nitroblue tetrazolium chloride the infarct was approximately to of the infarcts of the control plain liposome high lfts due to methotrexate or saline treated rabbit hearts subsequently, comparison to iggliposome treated hearts with acute myocardial infarction demonstrated that the csil treatment resulted in significantly smaller infarct size, high lfts due to methotrexate as was observed in comparison to plain liposome or saline treated hearts high lfts due to methotrexate thus, cytoskeletalantigen specific immunoliposomes, consisting of antimyosin or antiactinimmunoliposomes, were demonstrated to be able to preserve cell viability and integrity its potential utility in the cardiovascular system would be enhanced once its efficacy following intravenous delivery has been high lfts due to methotrexate demonstrated however, the study of asahi et al showed that intravenous delivery of high lfts due to methotrexate the antiactin immunoliposomes enabled preservation of the integrity of the endothelial cells of high lfts due to methotrexate the cerebral vessels csil as targeted gene or drug delivery due to the proposed mechanism of cell membrane lesion sealing, we also proposed that if drugs or gene constructs were to be included in the immunoliposomes such as high lfts due to methotrexate csils, then these drugs or gene constructs should be delivered directly into the cytoplasm fig this route should bypass the endocytic route of drugs or gene construct delivery, thereby reducing destruction of the delivered cargo by the lysosomal enzymes, after formation of endolysosomes using silver grains as model fig transmission electron micrographs of embryonic cardiocyte treated with silver grains impregnated csil left and plain high lfts due to methotrexate liposome impregnated with silver grains right � = jim drugs, we demonstrated that these drugs can be delivered directly into the cytoplasm of hypoxic cardiocytes treated with silver grains loaded csils figure left shows a transmission electron micrograph of a cardiocyte treated with silver grains impregnated csils silver grains in groups of concentration at about nm were observed however, in cells treated with high lfts due to methotrexate silver grains impregnated plain liposomes, very few cells were viable of one such cell detected by transmission electron microscopy, the silver grains were observed in the extracellular space [fig right] fig diagrammatic representation of delivery of intraliposomally entrapped genetic construct or drugs directly into the cytoplasm of target cell nmuin ftivoih wiiit mi ��� when the silver grains were replaced with genetic constructs, pgl and psv gal vectors, hypoxic cardiocytes treated with csil impregnated with either vectors showed luciferase activity or bacterial jgalactosidase activity the successful transfection of the high lfts due to methotrexate hypoxic cardiocytes with pgl, a vector for firefly luciferase enzyme � � fig relative light units of luciferase activity of cardiocytes treated with various preparations and controls in csils is shown in fig as relative light units rlus rlus were determined by the use of a luminometer as can be seen, only hypoxic cardiocytes treated with pglcsils showed increased rlus significantly above normal cells high lfts due to methotrexate with treatment with no vectors similarly, normoxic cardiocytes treated with pglcsil, hypoxic cardiocytes and normoxic cardiocytes treated with plain liposomes, or with only vectors, showed high lfts due to methotrexate no significant gene transcription and expression when hypoxic cardiocytes were treated with csil high lfts due to methotrexate with entrapped psvgal vectors, almost all cells in the field of view under light microscopy exhibited bacterial �galactosidase enzyme activity, following reaction with xgal bromo chloroindolylbetadgalactopyranoside, nm mgcl,mm iqfetcn h, mm kfecn in phosphate buffered saline ph high lfts due to methotrexate [fig a] when this mode of gene expression was compared with transfection of high lfts due to methotrexate psvjgal vector with cationic liposomes, cationic liposome transfection according to the manufacturers protocol resulted in transfection of only a few cells per field of view [fig b], in this micrograph, two cells with intense ��galactosidase activity were observed high lfts due to methotrexate quantitation of the number of cells in the field of view that was high lfts due to methotrexate successfully trans fected with psvjgal vector in csil, cationic liposome, iggliposomes, plain high lfts due to methotrexate liposome and vector alone are shown in fig c only csil and cationic high lfts due to methotrexate transfection showed gene expression csiltransfection or csilfection was more than times more efficient high lfts due to methotrexate in transfecting cells than cationic liposomes although the intensity of gene expression was low with csilfection, using the initial vector concentration of xg of vectors in mg lipids in ml, when the vector concentration was increased to � zg, also in mg lipids in ml, the intensity of gene transfection was high lfts due to methotrexate increased [fig d], this study showed that approximately x xg ��� � m t� !
or placebo o relative to sham instrumented control hearts at top high lfts due to methotrexate left panel, top right and min bottom left panel mean lvdp of hearts from to min of reperfusion are shown in the bottom right panel tetrazolium chloride stained heart slices showed that hearts treated with csil at , and min had similar injury as that of the sham operated hearts � , � and � , and � respectively p � ns the infarct size of hearts treated with csil even at min of global ischemia was � of the ventricles this was significantly smaller than its corresponding control p , whereas hearts treated with control iggl at , and min of global ischemia were � , � and � respectively fig , left panel the corresponding nitroblue tetrazolium chloride stained heart slices are shown in fig , right panel another parameter of myocardial injury high lfts due to methotrexate that was determined was mitochondrial size although mitochondrial swelling is a hallmark of high lfts due to methotrexate ischemic injury, irreversible injury cannot be directly extrapolated from just observation of mitochondrial high lfts due to methotrexate size nevertheless, in view of the myocardial functional and his tochemical evidences, mitochondrial high lfts due to methotrexate size assessment from transmission electron micrographs add additional support for myocardial preservation in csil treated hearts, relative to iggl or placebo treated hearts figure shows the comparison of fig mean infarct sizes of rat hearts treated with csil or iggl or placebo at and min of global ischemia left panel the corresponding nitroblue tetrazolium chloride stained mid slices of rat hearts treated with high lfts due to methotrexate csil or iggl at , and min of global ischemia minimal injury was seen in and dangers of quitting taking cymbalta min csil treated hearts, but injury was evident in the min csil treated heart slice injury is evident in all heart slices treated with iggl right panel, bottom two rows �i i normal cut csil csil csejo igsl tg& iflsi placebo v fig mean mitochondrial size of normal, csil, high lfts due to methotrexate iggl or placebo treated hearts treatment was as indicated in the text mitochondrial size of normal hearts, csil treatment at and min of global ischemia, as well as iggl treated hearts at , and min of global ischemia and with placebo no difference in mitochondrial size was observed between normal myocardium � mean number of pixels � sem and myocardium treated at and min high lfts due to methotrexate of global ischemia � , � , � and � respectively p = ns however, high lfts due to methotrexate mitochondria of hearts treated with iggl at , and min of global ischemia or placebo � , � , � and � respectively were larger than mitochondria of csil high lfts due to methotrexate treated hearts p these studies showed that myocardial viability preservation is not restricted to embryonic cardiocytes in cultures adult hearts are also amenable to structural high lfts due to methotrexate and functional preservation, following cell membrane lesion sealing in a timedependent manner during ischemia this method of cell membrane lesion sealing has also been reported high lfts due to methotrexate to preserve the integrity of vascular endothelium with antiactinimmunoliposomes a question that remains concerning the utility of csil is whether immunoliposomes can retain the protective functions in the presence of plasma proteins in vivo, since experiments have demonstrated that cells in culture and adult hearts perfused with nonprotein oxygenated buffer were prevented from undergoing myocardial necrosis, following cell membrane lesion sealing intervention with cytoskeletal high lfts due to methotrexate antigen specific immunoliposomes to demonstrate that cell membrane lesion sealing also occur in high lfts due to methotrexate vivo, rabbits with experimental myocardial infarction were used in this study, rabbits were injected with antimyosin csil, plain liposomes or saline at the time of left circumflex coronary artery occlusion by intracoronary infusion the occlusion was kept for high lfts due to methotrexate min followed by hrs of reperfusion the hearts were excised, sliced into � slices parallel to the short axis and stained with nitroblue tetrazolium chloride the infarct was approximately to of the infarcts of the control plain liposome high lfts due to methotrexate or saline treated rabbit hearts subsequently, comparison to iggliposome treated hearts with acute myocardial infarction demonstrated that the csil treatment resulted in significantly smaller infarct size, high lfts due to methotrexate as was observed in comparison to plain liposome or saline treated hearts high lfts due to methotrexate thus, cytoskeletalantigen specific immunoliposomes, consisting of antimyosin or antiactinimmunoliposomes, were demonstrated to be able to preserve cell viability and integrity its potential utility in the cardiovascular system would be enhanced once its efficacy following intravenous delivery has been high lfts due to methotrexate demonstrated however, the study of asahi et al showed that intravenous delivery of high lfts due to methotrexate the antiactin immunoliposomes enabled preservation of the integrity of the endothelial cells of high lfts due to methotrexate the cerebral vessels csil as targeted gene or drug delivery due to the proposed mechanism of cell membrane lesion sealing, we also proposed that if drugs or gene constructs were to be included in the immunoliposomes such as high lfts due to methotrexate csils, then these drugs or gene constructs should be delivered directly into the cytoplasm fig this route should bypass the endocytic route of drugs or gene construct delivery, thereby reducing destruction of the delivered cargo by the lysosomal enzymes, after formation of endolysosomes using silver grains as model fig transmission electron micrographs of embryonic cardiocyte treated with silver grains impregnated csil left and plain high lfts due to methotrexate liposome impregnated with silver grains right � = jim drugs, we demonstrated that these drugs can be delivered directly into the cytoplasm of hypoxic cardiocytes treated with silver grains loaded csils figure left shows a transmission electron micrograph of a cardiocyte treated with silver grains impregnated csils silver grains in groups of concentration at about nm were observed however, in cells treated with high lfts due to methotrexate silver grains impregnated plain liposomes, very few cells were viable of one such cell detected by transmission electron microscopy, the silver grains were observed in the extracellular space [fig right] fig diagrammatic representation of delivery of intraliposomally entrapped genetic construct or drugs directly into the cytoplasm of target cell nmuin ftivoih wiiit mi ��� when the silver grains were replaced with genetic constructs, pgl and psv gal vectors, hypoxic cardiocytes treated with csil impregnated with either vectors showed luciferase activity or bacterial jgalactosidase activity the successful transfection of the high lfts due to methotrexate hypoxic cardiocytes with pgl, a vector for firefly luciferase enzyme � � fig relative light units of luciferase activity of cardiocytes treated with various preparations and controls in csils is shown in fig as relative light units rlus rlus were determined by the use of a luminometer as can be seen, only hypoxic cardiocytes treated with pglcsils showed increased rlus significantly above normal cells high lfts due to methotrexate with treatment with no vectors similarly, normoxic cardiocytes treated with pglcsil, hypoxic cardiocytes and normoxic cardiocytes treated with plain liposomes, or with only vectors, showed high lfts due to methotrexate no significant gene transcription and expression when hypoxic cardiocytes were treated with csil high lfts due to methotrexate with entrapped psvgal vectors, almost all cells in the field of view under light microscopy exhibited bacterial �galactosidase enzyme activity, following reaction with xgal bromo chloroindolylbetadgalactopyranoside, nm mgcl,mm iqfetcn h, mm kfecn in phosphate buffered saline ph high lfts due to methotrexate [fig a] when this mode of gene expression was compared with transfection of high lfts due to methotrexate psvjgal vector with cationic liposomes, cationic liposome transfection according to the manufacturers protocol resulted in transfection of only a few cells per field of view [fig b], in this micrograph, two cells with intense ��galactosidase activity were observed high lfts due to methotrexate quantitation of the number of cells in the field of view that was high lfts due to methotrexate successfully trans fected with psvjgal vector in csil, cationic liposome, iggliposomes, plain high lfts due to methotrexate liposome and vector alone are shown in fig c only csil and cationic high lfts due to methotrexate transfection showed gene expression csiltransfection or csilfection was more than times more efficient high lfts due to methotrexate in transfecting cells than cationic liposomes although the intensity of gene expression was low with csilfection, using the initial vector concentration of xg of vectors in mg lipids in ml, when the vector concentration was increased to � zg, also in mg lipids in ml, the intensity of gene transfection was high lfts due to methotrexate increased [fig d], this study showed that approximately x xg ��� � m t� !