Quantitative method for measuring adjuvant-induced granuloma angiogenesis in insulin-treated diabetic mice

The angiogenesis of adjuvant-induced pouch granuloma was studied in insulin-treated diabetic mice by a newly established method using carmine dye. The 10% carmine suspension in 5% gelatin solution was infused through the tail vein of mice to be distributed to the end of the capillaries in the granulation tissue without leakage. The carmine dye was extracted from the tissue with 3 N NaOH solution and then measured by spectrophotometry. The content of carmine dye in the granuloma tissue in alloxan diabetic mice was observed to be significantly low during the first week after adjuvant injection when compared with normal mice, indicating poor development of blood vessels in the diabetic state. Diabetes-induced inhibition of the angiogenesis was completely restored by the treatment with insulin in a dose producing no hypoglycemic effect. These results were directly reflected by the formation of granuloma tissue. This method was established to be explicitly useful for measuring the angiogenesis, especially in mouse granuloma tissue.