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Murine chronic lymph node window for longitudinal intravital lymph node imaging
Meijer, Eelco F J,Jeong, Han-Sin,Pereira, Ethel R,Ruggieri, Thomas A,Blatter, Cedric,Vakoc, Benjamin J,Padera, Timothy P Nature Publishing Group, a division of Macmillan P 2017 Nature protocols Vol.12 No.8
<P>Chronic imaging windows in mice have been developed to allow intravital microscopy of many different organs and have proven to be of paramount importance in advancing our knowledge of normal and disease processes. A model system that allows long-term intravital imaging of lymph nodes would facilitate the study of cell behavior in lymph nodes during the generation of immune responses in a variety of disease settings and during the formation of metastatic lesions in cancer-bearing mice. We describe a chronic lymph node window (CLNW) surgical preparation that allows intravital imaging of the inguinal lymph node in mice. The CLNCLNCLNW is custom-made from titanium and incorporates a standard coverslip. It allows stable longitudinal imaging without the need for serial surgeries while preserving lymph node blood and lymph flow. We also describe how to build and use an imaging stage specifically designed for the CLNW to prevent (large) rotational changes as well as respiratory movement during imaging. The entire procedure takes approximately half an hour per mouse, and subsequently allows for longitudinal intravital imaging of the murine lymph node and surrounding structures for up to 14 d. Small-animal surgery experience is required to successfully carry out the protocol.</P>
Disruption of Striated Preferentially Expressed Gene Locus Leads to Dilated Cardiomyopathy in Mice
Liu, Xiaoli,Ramjiganesh, Tripurasundari,Chen, Yen-Hsu,Chung, Su Wol,Hall, Sean R.,Schissel, Scott L.,Padera Jr, Robert F.,Liao, Ronglih,Ackerman, Kate G.,Kajstura, Jan,Leri, Annarosa,Anversa, Piero,Ye Ovid Technologies Wolters Kluwer -American Heart A 2009 CIRCULATION - Vol.119 No.2
<P>BACKGROUND: The striated preferentially expressed gene (Speg) generates 4 different isoforms through alternative promoter use and tissue-specific splicing. Depending on the cell type, Speg isoforms may serve as markers of striated or smooth muscle differentiation. METHODS AND RESULTS: To elucidate function of Speg gene isoforms, we disrupted the Speg gene locus in mice by replacing common exons 8, 9, and 10 with a lacZ gene. beta-Galactosidase activity was detected in cardiomyocytes of the developing heart starting at day 11.5 days post coitum (dpc). beta-Galactosidase activity in other cell types, including vascular smooth muscle cells, did not begin until 18.5 dpc. In the developing heart, protein expression of only Spegalpha and Spegbeta isoforms was present in cardiomyocytes. Homozygous Speg mutant hearts began to enlarge by 16.5 dpc, and by 18.5 dpc, they demonstrated dilation of right and left atria and ventricles. These cardiac abnormalities in the absence of Speg were associated with a cellular hypertrophic response, myofibril degeneration, and a marked decrease in cardiac function. Moreover, Speg mutant mice exhibited significant neonatal mortality, with increased death occurring by 2 days after birth. CONCLUSIONS: These findings demonstrate that mutation of the Speg locus leads to cardiac dysfunction and a phenotype consistent with a dilated cardiomyopathy.</P>
Investigation of the Lack of Angiogenesis in the Formation of Lymph Node Metastases
Jeong, Han-Sin,Jones, Dennis,Liao, Shan,Wattson, Daniel A.,Cui, Cheryl H.,Duda, Dan G.,Willett, Christopher G.,Jain, Rakesh K.,Padera, Timothy P. U.S. Dept. of Health, Education, and Welfare, Publ 2015 Journal of the National Cancer Institute Vol.107 No.9
<P><B>Background:</B></P><P>To date, antiangiogenic therapy has failed to improve overall survival in cancer patients when used in the adjuvant setting (local-regional disease with no detectable systemic metastasis). The presence of lymph node metastases worsens prognosis, however their reliance on angiogenesis for growth has not been reported.</P><P><B>Methods:</B></P><P>Here, we introduce a novel chronic lymph node window (CLNW) model to facilitate new discoveries in the growth and spread of lymph node metastases. We use the CLNW in multiple models of spontaneous lymphatic metastases in mice to study the vasculature of metastatic lymph nodes (n = 9–12). We further test our results in patient samples (n = 20 colon cancer patients; n = 20 head and neck cancer patients). Finally, we test the ability of antiangiogenic therapy to inhibit metastatic growth in the CLNW. All statistical tests were two-sided.</P><P><B>Results:</B></P><P>Using the CLNW, we reveal the surprising lack of sprouting angiogenesis during metastatic growth, despite the presence of hypoxia in some lesions. Treatment with two different antiangiogenic therapies showed no effect on the growth or vascular density of lymph node metastases (day 10: untreated mean = 1.2%, 95% confidence interval [CI] = 0.7% to 1.7%; control mean = 0.7%, 95% CI = 0.1% to 1.3%; DC101 mean = 0.4%, 95% CI = 0.0% to 3.3%; sunitinib mean = 0.5%, 95% CI = 0.0% to 1.0%, analysis of variance <I>P</I> = .34). We confirmed these findings in clinical specimens, including the lack of reduction in blood vessel density in lymph node metastases in patients treated with bevacizumab (no bevacizumab group mean = 257 vessels/mm<SUP>2</SUP>, 95% CI = 149 to 365 vessels/mm<SUP>2</SUP>; bevacizumab group mean = 327 vessels/mm<SUP>2</SUP>, 95% CI = 140 to 514 vessels/mm<SUP>2</SUP>, <I>P</I> = .78).</P><P><B>Conclusion:</B></P><P>We provide preclinical and clinical evidence that sprouting angiogenesis does not occur during the growth of lymph node metastases, and thus reveals a new mechanism of treatment resistance to antiangiogenic therapy in adjuvant settings. The targets of clinically approved angiogenesis inhibitors are not active during early cancer progression in the lymph node, suggesting that inhibitors of sprouting angiogenesis as a class will not be effective in treating lymph node metastases.</P>