Epi-illumination fluorescent light microscopy for the in vivo study of rathepatic microvascular response to cryothermia

To elucidate the hepatic microvascular response to cryothermia, we studied the liver microcirculation of Sprague-Dawley rats after one and two 4-minut e freeze-thaw cycles using intravital fluorescence microscopy. Irrespective of the number of freeze-thaw cycles applied, the nature of hepatic microva scular injury was characterized by complete stasis of sinusoidal blood flow within the central part of the cryolesions and heterogeneous sinusoidal pe rfusion in a critically perfused border zone located at the periphery of th e lesions. Analysis over time (2 hours) revealed a successive shutdown of s inusoidal perfusion within this critically perfused border zone, which was caused by intravascularly lodging cell aggregates, blocking the lumen of in dividual sinusoids. The aggregates consisted of parenchymal cells and cell fragments, but did not include leukocytes or platelets. Strikingly, microva scular perfusion failure was associated with Ito cell disintegration and ma rked dilation of sinusoids (15.6 +/- 0.8 mu m vs. 8.8 +/- 0.8 mu m; P < .05 ). This excludes sinusoidal constriction as the cause of nutritive perfusio n failure, and may indicate dysfunction of Ito cell-regulated vasomotor con trol by cryothermia. However, because circulating cell aggregates were freq uently observed plugging individual microvessels, dilation of sinusoids may just be the result of passive distension caused by outflow blockade. Analy sis of hepatic tissue at 8 weeks after cryothermia did not reveal regenerat ion and microvascular remodeling, but loss of hepatic tissue, which corresp onded well with the tissue area presenting with sinusoidal perfusion failur e during the initial observation period after cryothermia. The fact that th ere was no recovery of sinusoidal perfusion over the initial 2-hour observa tion period, but loss of tissue after 8 weeks, supports the view that cryot hermia induces injury not only by direct low-temperature-mediated action, b ut also through ischemia caused by irreversible deterioration of the microc irculation.