Volume 71, Issue 4, Pages 584-593 (April 2017) Ex Vivo Model of Human Penile Transplantation and Rejection: Implications for Erectile Tissue Physiology  Nikolai A. Sopko, Hotaka Matsui, Denver M. Lough, Devin Miller, Kelly Harris, Max Kates, Xiaopu Liu, Kevin Billups, Richard Redett, Arthur L. Burnett, Gerald Brandacher, Trinity J. Bivalacqua  European Urology  Volume 71, Issue 4, Pages 584-593 (April 2017) DOI: 10.1016/j.eururo.2016.07.006 Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 1 Schematic of human cavernous tissue mixed lymphocyte reaction cultured for 48h in media. (a–e) During (a) penile prosthesis surgery, (c) “donor” cavernous tissue and (d) donor peripheral blood mononuclear cells (PBMCs) are taken from patients; (b) from a healthy volunteer, (e) “recipient” PBMCs are taken. These materials are combined in various combinations to model human cavernous tissue transplant. (f) Cavernous tissue (pieces measuring approximately 1×2cm) is cultured in media as a nontransplant control reaction (Media). (g) Cavernous tissue and autologous PBMCs are cultured as an autotransplant control reaction (Auto). (h) Cavernous tissue from the donor is cultured with allogenic PBMCs from the recipient to model tissue rejection (Allo). (i–k) Representative proliferation plots of 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE)–labeled PBMCs. Proliferation is assessed by CFSE signal loss in daughter cells, which have decreased dye content due to mitosis, seen as the left-shifted peaks, with less CFSE intensity as the parent peak. (i) PBMCs in media without penile tissue demonstrated the baseline proliferative rate of PBMCs in culture. (j) Donor PBMCs cultured with autologous penile tissue demonstrated a proliferation rate similar to PBMCs cultured in media (i), suggestive of no rejection reaction occurring. (k) Recipient PBMCs cultured with donor penile tissue demonstrated increased proliferation indicative of activation due to tissue rejection. European Urology 2017 71, 584-593DOI: (10.1016/j.eururo.2016.07.006) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 2 Rejection impairs nerve-mediated cavernous tissue contraction and relaxation induced by electrical field stimulation. Tissue myography was performed on penile tissues following incubation for 48h in either autologous or allogenic mixed lymphocyte reaction (Allo). A) Allo samples demonstrated impaired nerve-mediated smooth muscle contraction. B) Allo tissues demonstrated impaired nerve-mediated smooth muscle relaxation induced by EFS following maximal precontraction with phenylephrine. These data suggest that rejection impairs both the cavernous nerves and the smooth muscle tissue of the penis, resulting in impaired tissue function, including erectile dysfunction. n=4 per group. Allo=allogenic; Auto=autologous; EFS=electrical field stimulation; PE=phenylephrine. European Urology 2017 71, 584-593DOI: (10.1016/j.eururo.2016.07.006) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 3 An additional experimental group (a) composed of an allotransplant (Allo) mixed lymphocyte reaction (MLR) treated with 1μM cyclosporine A (CsA) was added to investigate the effects of immunosuppression treatment on penile rejection and function. (b) Heat map of an 84-gene polymerase chain reaction human transplant array of peripheral blood mononuclear cells (PBMCs) removed from cavernous tissue MLR after 48h of culture. Expression was normalized to the autotransplant group. Allotransplant PBMCs demonstrated generalized increased expression of transplant-associated genes. PBMCs treated with 1μM CsA demonstrated decreased transplant-associated gene expression compared with Allo. (c) Bar graphs of gene expression of transplant-associated genes grouped by function demonstrated rejection occurring in the Allo group that was mitigated by CsA treatment. n=3 per group. Allo=allotransplant; Auto=autotransplant; CsA=cyclosporine A; PBMC=peripheral blood mononuclear cell. European Urology 2017 71, 584-593DOI: (10.1016/j.eururo.2016.07.006) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 4 Representative histology of human cavernous tissue cultured for 48h in control (media) and mixed lymphocyte reaction (MLR) conditions. (a–d) Representative hematoxylin and eosin and (e–h) Masson's trichrome staining of tissue groups demonstrating smooth muscle and connective tissue content. (i–l) Giemsa stain of inflammatory cells with outlined high-power fields (m–p) demonstrating a qualitative increase in the presence of inflammatory cells in the allograft MLR (Allo) compared with media, autologous MLR, and 1-μM cyclosporine A–treated Allo. (q–t) Terminal deoxynucleotidyl transferase dUTP nick-end labeling stain demonstrating a qualitative increase in apoptotic nuclei in Allo tissues. Bar represents 100μM. Allo=allograft mixed lymphocyte reaction; Auto=autologous mixed lymphocyte reaction; CsA=1-μM cyclosporine A–treated allograft mixed lymphocyte reaction; H&E=hematoxylin and eosin; TUNEL=terminal deoxynucleotidyl transferase dUTP nick-end labeling. European Urology 2017 71, 584-593DOI: (10.1016/j.eururo.2016.07.006) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 5 Live laser confocal microscopy of penile tissues cultured in media and mixed lymphocyte reaction (MLR) with autologous peripheral blood mononuclear cells (PBMC; Auto), allogenic PBMCs (Allo), and allogenic PBMCs treated with 1μM cyclosporine A (CsA) for 48h. SNARF-1 was used to identify living cavernous tissues, and activated caspase-3/7 was used to identify cells undergoing apoptosis. Cavernous tissues (a–l) demonstrated (g–i) increased apoptosis in the setting of rejection (Allo) compared with (a–c) media and (d–f) Auto. (j–l) Allo-associated apoptosis was prevented by CsA treatment. Cavernous nerves were found in Auto, Allo, and CsA penile tissues. Similar to cavernous tissues, (p–r) nerve tissue demonstrated increased apoptosis in the setting of rejection (Allo) compared to (m–o) Auto, which was prevented by (s–u) CsA treatment. Bar represents 100 uM. Allo=allograft mixed lymphocyte reaction; Auto=autologous mixed lymphocyte reaction; CsA=1-μM cyclosporine A–treated allograft mixed lymphocyte reaction. European Urology 2017 71, 584-593DOI: (10.1016/j.eururo.2016.07.006) Copyright © 2016 European Association of Urology Terms and Conditions

Fig. 6 Assessment of immunosuppression treatment on cavernous tissue function physiology by myography. (a) Allograft mixed lymphocyte reaction (MLR; Allo) and Allo tissues treated with 1μM cyclosporine A (CsA) were cultured for 48h. Electrical field stimulation (EFS)–induced nerve-mediated smooth muscle contraction was not different between treated and untreated tissues. (b) EFS-induced nerve-mediated relaxation following maximal precontraction with phenylephrine demonstrated that CsA-treated tissues had impaired relaxation compared with rejecting Allo tissues, despite prevention of rejection. Given these surprising findings and concern that CsA may have direct smooth muscle relaxation impairment, cavernous tissues without peripheral blood mononuclear cells (ie, non-MLR culture) were incubated in media, 1μM cyclosporine A (Cyclo A), or 20nM FK506 for 24h. (c) No difference in EFS-induced tissue contraction was seen between groups. (d) Cyclo A tissues demonstrated impaired contraction compared with media and FK506-treated tissues. These data suggest that cyclosporine A impairs nerve-mediated cavernous tissue relaxation, whereas FK506 does not, demonstrating the importance of immunosuppression regimens in the setting of penile transplantation. n=4 per group. Allo=allograft mixed lymphocyte reaction; CsA=1-μM cyclosporine A–treated allograft mixed lymphocyte reaction; Cyclo A=1-μM cyclosporine A; EFS=electrical field stimulation; PE=phenylephrine. European Urology 2017 71, 584-593DOI: (10.1016/j.eururo.2016.07.006) Copyright © 2016 European Association of Urology Terms and Conditions