Related Experiment Videos
Measurement of tissue viability using intravital microscopy and fluorescent nuclear dyes
R F Potter1, G Peters, M Carson
1A. D. McLachlin Vascular Surgery Research Laboratory, Victoria Hospital, London, Ontario, Canada.
Abstract:
Intravital microscopy used with fluorescent vital stains provides the opportunity to measure the temporal and spatial extent of tissue injury following disease processes. However, this assumes that prolonged exposure to such dyes does not alter microvascular perfusion or cellular viability. To test this hypothesis, the extensor digitorum longus (EDL) muscle in 24 male Wistar rats, anesthetized with sodium pentobarbital (Somnotal, 65 mg/kg, ip), were prepared for microscopy. The EDL was either bathed continuously (n = 6) in Krebs solution containing bisbenzimide (5 micrograms/ml; labels nuclei of all cells) and ethidium bromide (5 micrograms/ml; labels nuclei of injured cells) or had dyes topically applied 1 hr (n = 4) and 4 hr (n = 4) following dissection of the muscle. Noxious stimuli (i.e., hypoxia:FiO2 of 8-10% (n = 3), 95% ethanol (n = 3), and 2 hr ischemia followed by 90 min reperfusion (n = 4) were used to test the ability of ethidium bromide, when used in conjunction with intravital microscopy, to differentiate injured tissue. Video recordings at the surface of the EDL muscle were made every 30 min for 5 hr from which the number of perfused capillaries was counted (NCper). The numbers of bisbenzimide- and ethidium bromide-labeled nuclei were counted at the surface of the muscle and at two to three additional locations within the muscle (to a maximum depth of approximately 120-160 microns). The average NCper (19.05 +/- 1.7) remained constant over 5 hr, while the number of nuclei stained by bisbenzimide increased linearly with time from an initial value of 1218 +/- 125.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Fluorescent vital stains used in intravital microscopy may alter tissue viability. This study found that prolonged dye exposure did not affect microvascular perfusion or cellular viability in rat muscle tissue, validating its use for injury assessment.
Area of Science:
- Physiology
- Microscopy
- Toxicology
Background:
- Intravital microscopy with fluorescent vital stains is crucial for assessing tissue injury.
- Concerns exist regarding potential alterations in microvascular perfusion and cellular viability due to prolonged dye exposure.
Purpose of the Study:
- To evaluate the impact of prolonged exposure to bisbenzimide and ethidium bromide on microvascular perfusion and cellular viability.
- To validate the use of these dyes in assessing tissue injury using intravital microscopy.
Main Methods:
- Wistar rats' extensor digitorum longus (EDL) muscles were prepared for microscopy.
- EDL muscles were continuously bathed or topically exposed to dyes (bisbenzimide and ethidium bromide).
- Noxious stimuli (hypoxia, ethanol, ischemia-reperfusion) were applied to test dye efficacy in differentiating injured tissue.
Main Results:
- The number of perfused capillaries (NCper) remained constant over 5 hours.
- Bisbenzimide-labeled nuclei counts increased linearly with time, indicating no dye-induced toxicity.
- Ethidium bromide effectively differentiated injured tissue when used with intravital microscopy.
Conclusions:
- Prolonged exposure to bisbenzimide and ethidium bromide does not significantly alter microvascular perfusion or cellular viability in rat EDL muscle.
- These vital stains are suitable for use in intravital microscopy to assess the extent of tissue injury.