Propidium iodide in vivo: an early marker of neuronal damage in rat hippocampus.
ABSTRACT We have investigated the use of the fluorescent exclusion dye propidium iodide as a marker for acutely degenerating cells in vivo, and report here that combined injection of kainic acid and propidium iodide into the lateral cerebral ventricle results in labelling of CA3 pyramidal cells 1 and 6 h after injection. Alternate sections stained with thionin at these early times revealed little evidence of histologically detectable cell damage.
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ABSTRACT: Neurofibrillary tangles (NFTs) are associated with neuronal loss and correlate with cognitive impairment in Alzheimer disease, but how NFTs relate to neuronal death is not clear. We studied cell death in Tg4510 mice that reversibly express P301L mutant human tau and accumulate NFTs using in vivo multiphoton imaging of neurofibrillary pathology, propidium iodide (PI) incorporation into cells, caspase activation, and DNA labeling. We first observed that in live mice, a minority of neurons were labeled with the caspase probe or with PI fluorescence. These markers of cell stress were localized in the same cells and appeared specifically within NFT-bearing neurons. Contrary to expectations, the PI-stained neurons did not die during a day of observation; the presence of Hoechst-positive nuclei in them on the subsequent day indicated that the NFT-associated membrane disruption, as suggested by PI staining, and caspase activation do not lead to immediate death of neurons in this tauopathy model. This unique combination of in vivo multiphoton imaging with markers of cell death and pathological alteration is a powerful tool for investigating neuronal damage associated with neurofibrillary pathology.Journal of Neuropathology and Experimental Neurology 07/2009; 68(7):757-61. · 4.37 Impact Factor
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ABSTRACT: Subthreshold retinal phototherapy demonstrated clinical efficacy for the treatment of diabetic macular edema without visible signs of retinal damage. To assess the range of cellular responses to sublethal hyperthermia, expression of the gene encoding a 70 kDa heat shock protein (HSP70) was evaluated after laser irradiation using a transgenic reporter mouse. One hundred millisecond, 532 nm laser exposures with 400 μm beam diameter were applied to the retina surrounding the optic nerve in 32 mice. Transcription from the HSP70 promoter was assessed relative to the control eye using a bioluminescence assay at 7 hours after laser application. The retinal pigmented epithelium (RPE) viability threshold was determined with a fluorescence assay. A computational model was developed to estimate temperature and the extent of cell damage. A significant increase in HSP70 transcription was found at exposures over 20 mW, half the threshold power for RPE cell death. Computational modeling estimated peak temperature T = 49°C at HSP70 expression threshold. At RPE viability threshold, T = 57°C. Similar temperatures and damage indices were calculated for clinical subvisible retinal treatment parameters. Beneficial effects of laser therapy have been previously shown to extend beyond those resulting from destruction of tissue. One hundred millisecond laser exposures at approximately half the threshold power of RPE damage induced transcription of HSP70, an indication of cellular response to sublethal thermal stress. A computational model of retinal hyperthermia can guide further optimization of laser parameters for nondamaging phototherapy.Investigative ophthalmology & visual science 11/2010; 52(3):1780-7. · 3.43 Impact Factor
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ABSTRACT: Electronic retinal prostheses represent a potentially effective approach for restoring some degree of sight in blind patients with retinal degeneration. However, levels of safe electrical stimulation and the underlying mechanisms of cellular damage are largely unknown. We measured the threshold of cellular damage as a function of pulse duration, electrode size, and number of pulses to determine the safe range of stimulation. Measurements were performed in-vitro on embryonic chicken retina with saline-filled glass pipettes for stimulation electrodes. Cellular damage was detected using Propidium Iodide fluorescent staining. Electrode size varied from 115μm to 1mm, pulse duration from 6μs to 6ms, and number of pulses from 1 to 7,500. The threshold current density was independent of electrode sizes exceeding 400μm. With smaller electrodes the current density was scaling reciprocal to the square of the pipette diameter, i.e. acting as a point source so that the damage threshold was determined by the total current in this regime. The damage threshold current measured with large electrodes (1mm) scaled with pulse duration as t-0.5, which is characteristic of electroporation. For repeated electrical pulsed exposure on the retina the threshold current density varied between 0.059 A/cm2 at 6ms to 1.3 A/cm2 at 6μs. The dynamic range of safe stimulation, i.e. the ratio of damage threshold to stimulation threshold was found to be duration-dependent, and varied from 10 to 100 at pulse durations varying between 10μs to 10ms. Maximal dynamic range of 100 was observed near 1ms pulse durations.Proceedings of SPIE - The International Society for Optical Engineering 03/2006; · 0.20 Impact Factor