Single-cell microglial transcriptomics during demyelination defines a microglial state required for lytic carcass clearance.

BACKGROUND: Microglia regulate the response to injury and disease in the brain and spinal cord. In white matter diseases microglia may cause demyelination. However, how microglia respond and regulate demyelination is not fully understood. METHODS: To understand how microglia respond during demyelination, we fed mice cuprizone-a potent demyelinating agent-and assessed the dynamics of genetically fate-mapped microglia. We then used single-cell RNA sequencing to identify and track the microglial subpopulations that arise during demyelination. To understand how microglia contribute to the clearance of dead oligodendrocytes, we ablated microglia starting at the peak of cuprizone-induced cell death and used the viability dye acridine orange to monitor apoptotic and lytic cell morphologies after microglial ablation. Lastly, we treated serum-free primary microglial cultures to model distinct aspects of cuprizone-induced demyelination and assessed the response. RESULTS: The cuprizone diet generated a robust microglial response by week 4 of the diet. Single-cell RNA sequencing at this time point revealed the presence of several cuprizone-associated microglia (CAM) clusters. These clusters expressed a transcriptomic signature indicative of cytokine regulation and reactive oxygen species production with altered lysosomal and metabolic changes consistent with ongoing phagocytosis. Using acridine orange to monitor apoptotic and lytic cell death after microglial ablation, we found that microglia preferentially phagocytose lytic carcasses. In culture, microglia exposed to lytic carcasses partially recapitulated the CAM state, suggesting that phagocytosis contributes to this distinct microglial state during cuprizone demyelination. CONCLUSIONS: Microglia serve multiple roles during demyelination, yet their transcriptomic state resembles other neurodegenerative conditions. The phagocytosis of cellular debris is likely a universal cause for a common neurodegenerative microglial state.

Can a less radical surgery using photodynamic therapy with acridine orange be equal to a wide-margin resection?

BACKGROUND: Wide-margin resections are an accepted method for treating soft tissue sarcoma. However, a wide-margin resection sometimes impairs function because of the lack of normal tissue. To preserve the normal tissue surrounding a tumor, we developed a less radical (ie, without a wide margin) surgical procedure using adjunctive photodynamic therapy and acridine orange for treating soft tissue sarcoma. However, whether this less radical surgical approach increases or decreases survival or whether it increases the risk of local recurrence remains uncertain. QUESTIONS/PURPOSES: We determined the survival, local recurrence, and limb function outcomes in patients treated with a less radical approach and adjunctive acridine orange therapy compared with those who underwent a conventional wide-margin resection. METHODS: We treated 170 patients with high-grade soft tissue sarcoma between 1999 and 2009. Fifty-one of these patients underwent acridine orange therapy. The remaining 119 patients underwent a conventional wide-margin resection for limb salvage surgery. We recorded the survival, local recurrence, and functional score (International Society of Limb Salvage [ISOLS]) score) for all the patients. RESULTS: The 10-year overall survival rates in the acridine orange therapy group and the conventional surgery group were 68% and 63%, respectively. The 10-year local recurrence rate was 29% for each group. The 5-year local recurrence rates for Stages II, III, and IV were 8%, 36%, and 40%, respectively, for the acridine orange group and 13%, 27%, and 33%, respectively, for the conventional surgery group. The average ISOLS score was 93% for the acridine orange group and 83% for the conventional therapy group. CONCLUSION: Acridine orange therapy has the potential to preserve limb function without increasing the rate of local recurrence. This therapy may be useful for eliminating tumor cells with minimal damage to the normal tissue in patients with soft tissue sarcoma. LEVEL OF EVIDENCE: Level IV, therapeutic study. See Guidelines for Authors for a complete description of the levels of evidence.

Acridine orange excited by low-dose radiation has a strong cytocidal effect on mouse osteosarcoma.

The study was conducted to clarify the cytocidal effect of combination therapy consisting of administration of acridine orange (AO), which is a photosensitizer, and radiation therapy using in vitro and in vivo mouse osteosarcoma models. The results revealed that AO combined with low-dose X-ray irradiation of about 1-5 Gy had a strong cytocidal effect on the cultured mouse osteosarcoma cells regardless of their chemosensitivity, and that this combination therapy inhibited growth of the in vivo mouse osteosarcoma by induction of tumor necrosis. This effect was inhibited by L-histidine, but not by mannitol. These findings suggested that AO might be excited by X-rays and kill osteosarcoma cells through the release of singlet oxygen, which is toxic to living cells. This mechanism is similar to that of photodynamic therapy with AO.

Genetic toxicological testing of some dyes by the micronucleus test.

Three widely used dyes, acridine orange, blue VRS and fast green FCF were administered to male mice in order to study the induction of gross chromosomal anomalies using the micronucleus test. All 3 compounds were shown to be clastogenic.