Quantitative imaging of lymph function

Functional lymphatic imaging was demonstrated in the abdomen and anterior hindlimb of anesthetized, intact Yorkshire swine by using near-infrared (NIR) fluorescence imaging following intradermal administration of 100–200 μl of 32 μM indocyanine green (ICG) and 64 μM hyaluronan NIR imaging conjugate to target the lymph vacular endothelial receptor (LYVE)-1 on the lymph endothelium. NIR fluorescence imaging employed illumination of 780 nm excitation light (∼2 mW/cm2) and collection of 830 nm fluorescence generated from the imaging agents. Our results show the ability to image the immediate trafficking of ICG from the plexus, through the vessels and lymphangions, and to the superficial mammary, subiliac, and middle iliac lymph nodes, which were located as deep as 3 cm beneath the tissue surface. “Packets” of ICG-transited lymph vessels of 2–16 cm length propelled at frequencies of 0.5–3.3 pulses/min and velocities of 0.23–0.75 cm/s. Lymph propulsion was independent of respiration rate. In the case of the hyaluronan imaging agent, lymph propulsion was absent as the dye progressed immediately through the plexus and stained the lymph vessels and nodes. Lymph imaging required 5.0 and 11.9 μg of ICG and hyaluronan conjugate, respectively. Our results suggest that microgram quantities of NIR optical imaging agents and their conjugates have a potential to image lymph function in patients suffering from lymph-related disorders.

Plasma protein concentrations in interstitial fluid from human aortas

The concentration of plasma proteins was examined in interstitial fluid collected from human aortas, obtained at autopsy, from patients from whom a blood sample had been taken for routine analysis shortly before death. The interstitial fluid was absorbed onto small, preweighed pieces of filter paper which were inserted into natural strip planes in the tunica intima and inner tunica media. After equilibration the papers were removed and weighed to measure the amount of interstitial fluid collected, then analysed by quantitative immunoelectrophoresis for three plasma proteins covering a range of molecular masses ( M r ): low density lipoprotein (LDL) with molecular mass 2.4 x 10 6 , α 2 -macroglobulin ( M r 720 000) and serum albumin ( M r 68 000). In interstitial fluid obtained from normal intima of 20 men and women, the mean levels of the proteins were LDL 215%, α 2 -macroglobulin 115% and albumin 54% of the concentration in the patient’s own plasma. Concentrations were not influenced by depth within the intima, or by distance down the aorta. The patients covered the age range 31-96 years; relative concentrations of LDL, α 2 -macroglobulin and albumin increased in parallel by about 10% per decade. Large samples (up to 7 μl) of interstitial fluid were collected from inner media but they contained no measurable LDL; the levels of α 2 -macroglobulin and albumin were respectively 11 and 18% of plasma concentration. Analysis of interstitial fluid from one sample of normal intima obtained at vascular surgery, and from two freshly killed pigs, suggested that results were not invalidated by the use of autopsy material. Direct comparison of whole intimal tissue and interstitial fluid provided no evidence of preferential binding of LDL in tissue. There was no evidence of preferential adsorbtion of LDL by the filter paper. Interstitial fluid of six samples of normal intima from four patients was compared with normal plasma by two-dimensional immunoelectrophoresis. The peaks produced in the antiserum to LDL were identical in mobility, shape and staining properties. Thus the concentration of LDL found in interstitial fluid from normal aortic intima was more than twice the plasma concentration, and the relation between concentration and molecular mass was the inverse of that reported for peripheral interstitial fluid and lymph. Endothelium appears to trap LDL in the intima instead of keeping it out, and this raises fundamental questions about the function of arterial endothelium.