Testosterone treatment induces erythrocytosis that could potentially affect blood viscosity and cardiovascular risk. dose (5 mg/kg). Our results indicate no rheological difference among normal males females and castrated males at steady-state. Short-term high-dose testosterone increased hematocrit and whole-blood viscosity in both females and castrated males. This effect diminished after long-term treatment in association with increased erythrocyte deformability in the testosterone-treated mice suggesting the presence of adaptive mechanism. Considering that cardiovascular events in human trials are seen early after intervention rheological changes as potential mediator of vascular events warrant further investigation. The use of testosterone as a prescription drug has increased dramatically in recent years. Several studies raised concerns about the cardiovascular risk of testosterone therapy (1 -3). Epidemiologic studies suggested that both very low and very high testosterone levels are linked to cardiovascular and cerebral risk (4 5 but the mechanism remains unclear. Elevated blood viscosity is associated with cardiovascular and cerebral risk especially in the context of therapeutic administration of erythropoiesis-stimulating agents (6 -9). Likewise testosterone administration consistently increases hematocrits in men (10 11 Because erythrocyte mass is a major contributor to blood viscosity testosterone administration might thus increase blood viscosity. However relevant literature is scant and inconclusive (12 -14). On the other hand increased erythrocyte deformability has been reported in erythropoietin-transgenic mice which allows the mice to offset their whole-blood viscosity to a much lower level than that predicted from their exceptionally high hematocrits (15). It is unclear whether men or animals acquire similar adaptations to testosterone-induced erythrocytosis. Here we report the effects of testosterone on blood viscosity and erythrocyte deformability in mice after short-term and long-term interventions. (-)-Huperzine A Materials and Methods Animals For long-term intervention experiments precastrated male sham male and female C57BL/6 mice were purchased from The Jackson Laboratory at 6 weeks of age. For short-term experiments precastrated male and female mice were purchased as retired breeders (7-8 mo). At the end point all mice were about 8-9 months. Mice were randomized to vehicle or testosterone groups with matching levels of hematocrits at baseline. Testosterone propionate was injected sc weekly at 50 mg/kg in 100-μL medium-chain triglyceride oil (Life Enhancement) for either 2 weeks (females and castrated males) or 5 months (females) and (-)-Huperzine A 7 months (castrated males). In response to the reviewers’ suggestion additional female mice were tested with low-dose testosterone propionate (5 mg/kg). All control mice were weekly injected with 100-μL vehicle oil. The number of animals for each experiment is listed in the corresponding figure legend. All mice were housed in the Center for Animal Resources at Harvard Medical School with controlled temperature at 21°C and a 12-hour light 12 dark cycle with free access to water and standard chow. The use of animals was approved by the Institutional Animal Care and Use Committee of Harvard Medical School. Blood analysis Erythrocyte packing difference (EPD) was measured as a marker for erythrocyte sedimentation rate (16). Briefly a microhematocrit tube was filled with tail-vein blood and spun at 100for 30 minutes at room temperature for the apparent hematocrit reading. Then the same tube was spun in CritSpin (Iris Sample Processing Inc) to obtain the standard hematocrit reading. EPD was calculated as the difference between these 2 readings. Complete blood counts were obtained from the Hematology Core at Boston Children’s Hospital (Boston MA). Blood viscosity was measured as described (15). Blood was obtained via cardiac puncture into an EDTA blood collection (-)-Huperzine A tube and analyzed within PTPRC 5-10 minutes using a rotation viscosimeter (DVIII_ Rheometer; Brookfield Engineering Laboratories). Viscosity was recorded at 37°C under different shear rates. Plasma viscosity was analyzed similarly except that samples were prestored at ?80°C. Erythrocyte deformability was measured as the elongation rate (-)-Huperzine A (15). Whole blood (0.3 mL) was mixed with 3 mL of PBS containing 14.4% Dextran (number D6030; USbiological) with a viscosity of 10 mPas close to human peripheral artery blood viscosity at low shear rate (17). For selected experiments a high viscosity Dextran solution was also tested (24 mPas)..