Multiphoton Intravital Microscopy of the Transplanted Mouse Kidney

Abstract

Graft outcomes after kidney transplantation continue to be adversely affected by ischemia-reperfusion injury and rejection. High-resolution, real-time imaging of the transplanted kidney could shed valuable insights into these dynamic processes, but such methodology has not been established. Here we describe a technique for intravital imaging of the transplanted mouse kidney using multiphoton fluorescence microscopy. The technique enabled real-time, high-resolution imaging and quantitation of renal filtration, cell death, leukocyte adhesion and capillary blood flow after transplantation. Using this technique, we found that brief graft ischemia associated with the transplantation procedure led to a rapid decline in renal filtration accompanied by a significant increase in microvascular leakage and renal tubular epithelial cell death within the first 3 h after transplantation. No significant changes in leukocyte adhesion or capillary blood flow were observed during the same time period. This report establishes multiphoton fluorescence microscopy as a sensitive tool for simultaneously studying functional and structural perturbations that occur in the mouse kidney after transplantation and for investigating the migration of leukocytes to the graft.

Non-self recognition by monocytes initiates allograft rejection

“…Surgical procedures. Heterotopic transplantation of primarily vascularized cardiac and kidney grafts was performed as previously described (43,44). Cardiac allograft rejection was defined as cessation of a palpable heartbeat and was confirmed by histological analysis.…”

“…2-photon intravital microscopy. Multiphoton intravital microscopy was performed on transplanted kidneys using an established method (44). An Olympus FluoView FV1000 microscope equipped with a Mai Tai DeepSee femtosecond-pulsed laser (Spectra-Physics) tuned and mode-locked to 915 nm was used for all experiments.…”

Cognate antigen directs CD8+ T cell migration to vascularized transplants

“…To investigate the roles of cognate antigen and Gα i in the transmigration of effector T cells across the graft endothelium, we performed real-time, 2-photon intravital imaging of mouse kidney transplants, a procedure previously established in our laboratory (25). Unlike the flow cytometry approach used in Figure 2, intravital microscopy permitted the direct visualization and enumeration of T cells early after transfer, prior to their proliferation in the graft or secondary lymphoid tissues.…”

“…2-photon intravital microscopy was performed on transplanted kidneys using a previously established method (25). An Olympus FluoView FV1000 microscope with a Mai Tai DeepSee femtosecond-pulsed laser (Spectra-Physics), tuned and mode-locked to either 825 nm or 860 nm, was used for all experiments.…”

“…Splenectomies and heterotopic transplantation of vascularized heart and kidney grafts were performed as previously described (2,25,33). Heart graft rejection, defined as cessation of palpable heartbeat, was confirmed by histological analysis.…”