Assessment of regional bone tissue perfusion in rats using fluorescent microspheres

Abstract

Disturbances in bone blood flow have been shown to have deleterious effects on bone properties yet there remain many unanswered questions about skeletal perfusion in health and disease, partially due to the complexity of measurement methodologies. The goal of this study was use fluorescent microspheres in rats to assess regional bone perfusion by adapting mouse-specific fluorescent microsphere protocol. Ten fifteen-week old Sprague Dawley rats were injected with fluorescent microspheres either via cardiac injection (n = 5) or via tail vein injection (n = 5). Femora and tibiae were harvested and processed to determine tissue fluorescence density (TFD) which is proportional to the number of spheres trapped in the tissue capillaries. Right and left total femoral TFD (2.77 ± 0.38 and 2.70 ± 0.24, respectively) and right and left tibial TFD (1.11 ± 0.26 and 1.08 ± 0.34, respectively) displayed bilateral symmetry in flow when assessed in cardiac injected animals. Partitioning of the bone perfusion into three segments along the length of the bone showed the distal femur and proximal tibia received the greatest amount of perfusion within their respective bones. Tail vein injection resulted in unacceptably low TFD levels in the tibia from 4 of the 5 animals. In conclusion this report demonstrates the viability of cardiac injection of fluorescent microspheres to assess bone tissue perfusion in rats.

Skeletal vascular perfusion is altered in chronic kidney disease

“…To accomplish this goal, we utilized fluorescent microspheres, which lodge in tissue capillaries in direct proportion to the fraction of cardiac output perfusing the tissue. This technique has been shown to allow measurement of organ perfusion as effectively as radioactive microspheres ( Glenny et al, 1993 ), the experimental gold standard ( McCarthy, 2006 ), and has recently been applied to study skeletal perfusion in rats ( Aref et al, 2017 ).…”

Parathyroid suppression therapy normalizes chronic kidney disease-induced elevations in cortical bone vascular perfusion: a pilot study

“…Female Cy/+ animals do not develop notable changes in cortical bone properties and thus were not studied in the current context. Injection of microspheres in the left ventricle to assess perfusion is based on a set of assumptions and has multiple limitations as discussed in our previous work [14]. While these limitations are important to consider from a technical standpoint, we have now consistently shown in several studies that in CKD in the setting of high PTH there is higher cortical perfusion.…”

“…Microsphere injection was performed as we previously described [14]. Briefly, under isoflurane anesthesia, polystyrene red fluorescent (580/605), 15 µm microspheres (FluoSpheres, ThermoFisher) were injected into the apex of the beating left ventricle.…”

“…The animal was euthanized by cardiac dissection 60 seconds after the completion of the injection, allowing for the circulation of spheres. A total of 5.0x106 spheres/kg were injected, a number sufficient to assess perfusion in skeletal tissue [14].…”

“…Samples isolated for tissue perfusion measures were processed as previously described [14]. Briefly, bone samples were placed in a Cal-Ex Decalcifier solution for 7 days.…”