Nitroblue Tetrazolium Chloride Staining Research Articles

Modulating role of ROS in re-establishing desiccation tolerance in germinating seeds of Caragana korshinskii Kom.

In close agreement with visible germination, orthodox seeds lose desiccation tolerance (DT). This trait can be regained under osmotic stress, but the mechanisms are poorly understood. In this study, germinating seeds of Caragana korshinskii Kom. were investigated, focusing on the potential modulating roles of reactive oxygen species (ROS) in the re-establishment of DT. Germinating seeds with 2 mm long radicles can be rendered tolerant to desiccation by incubation in a polyethylene glycol (PEG) solution (-1.7 MPa). Upon PEG incubation, ROS accumulation was detected in the radicles tip by nitroblue tetrazolium chloride staining and further confirmed by confocal microscopy. The PEG-induced re-establishment of DT was repressed when ROS scavengers were added to the PEG solution. Moreover, ROS act downstream of abscisic acid (ABA) to modulate PEG-mediated re-establishment of DT and serve as a new inducer to re-establish DT. Transcriptomic analysis revealed that re-establishment of DT by ROS involves the up-regulation of key genes in the phenylpropanoid-flavonoid pathway, and total flavonoid content and key enzyme activity increased after ROS treatment. Furthermore, DT was repressed by an inhibitor of phenylalanine ammonia lyase. Our data suggest that ROS play a key role in the re-establishment of DT by regulating stress-related genes and the phenylpropanoid-flavonoid pathway.

Selective photothermolysis to target sebaceous glands: Theoretical estimation of parameters and preliminary results using a free electron laser

The success of permanent laser hair removal suggests that selective photothermolysis (SP) of sebaceous glands, another part of hair follicles, may also have merit. About 30% of sebum consists of fats with copious CH(2) bond content. SP was studied in vitro, using free electron laser (FEL) pulses at an infrared CH(2) vibrational absorption wavelength band. Absorption spectra of natural and artificially prepared sebum were measured from 200 to 3,000 nm, to determine wavelengths potentially able to target sebaceous glands. The Jefferson National Accelerator superconducting FEL was used to measure photothermal excitation of aqueous gels, artificial sebum, pig skin, human scalp, and forehead skin (sebaceous sites). In vitro skin samples were exposed to FEL pulses from 1,620 to 1,720 nm, spot diameter 7-9.5 mm with exposure through a cold 4°C sapphire window in contact with the skin. Exposed and control tissue samples were stained using H&E, and nitroblue tetrazolium chloride staining (NBTC) was used to detect thermal denaturation. Natural and artificial sebum both had absorption peaks near 1,210, 1,728, 1,760, 2,306 and 2,346 nm. Laser-induced heating of artificial sebum was approximately twice that of water at 1,710 and 1,720 nm, and about 1.5× higher in human sebaceous glands than in water. Thermal camera imaging showed transient focal heating near sebaceous hair follicles. Histologically, skin samples exposed to ~1,700 nm, ~100-125 milliseconds pulses showed evidence of selective thermal damage to sebaceous glands. Sebaceous glands were positive for NBTC staining, without evidence of selective loss in samples exposed to the laser. Epidermis was undamaged in all samples. SP of sebaceous glands appears to be feasible. Potentially, optical pulses at ~1,720 or ~1,210 nm delivered with large beam diameter and appropriate skin cooling in approximately 0.1 seconds may provide an alternative treatment for acne.

Effects of non‐invasive, 1,210 nm laser exposure on adipose tissue: Results of a human pilot study

Laser radiation (1,210 nm) has been previously shown to be capable of selective photothermolysis of adipose tissue in vitro when applied non-invasively. The objective of this pilot study was to evaluate the in vivo effects of this laser in human subjects. Twenty-four adult subjects were exposed non-invasively on the abdomen to a 1,210 nm laser at fluences of 70, 80, and 90 J/cm(2), with a 10 mm spot size, 5 seconds pre-cooling, and 3 seconds exposure duration delivered with parallel contact cooling. There was an impairment of the skin-cooling device during the study. Exposure and control sites were biopsied at either 1-3 days or 4-7 weeks. Tissue was processed for nitroblue tetrazolium chloride (NBTC) staining, a marker for thermal damage, and hematoxylin and eosin (H&E) staining. Laser exposures were painful, requiring local anesthesia in most subjects, but otherwise well tolerated. At 1-3 days after exposure, there was a fluence-dependent loss of NBTC staining in the fat and dermis. In 2 of 14 subjects (2 of 42 exposure sites) evaluated at 1-3 days after exposure, epidermal damage was noted within a small portion of the test site, likely due to impaired contact cooling. At 4-7 weeks, lipomembranous changes of the fat were seen in 89% of test sites and 33% of control sites. This in vivo study shows histologic evidence of laser-induced damage of fat. With further development, this might become a useful treatment for disorders involving the fat and/or lower dermis.

The effects of pulse energy variations on the dimensions of microscopic thermal treatment zones in nonablative fractional resurfacing

We examined the effects of pulse energy variations on the dimensions of microscopic thermal injury zones (MTZs) created on human skin ex vivo and in vivo using nonablative fractional resurfacing. A Fraxel SR laser system emitting at 1,550 nm provided an array of microscopic spots at variable densities. Pulse energies ranging from 4.5 to 40 mJ were tested on human abdominal skin ex vivo and in vivo. Tissue sections were stained with hematoxylin and eosin (H&E) or nitro blue tetrazolium chloride (NBTC) and MTZ dimensions were determined. Ex vivo and in vivo results were compared. Dosimetry analyses were made for the surface treatment coverage calculation as a function of pulse energy and collagen coagulation based on H&E stain or cell necrotic zone based on NBTC stain. Each MTZ was identified by histological detection of a distinct region of loss of tissue birefringence and hyalinization, representing collagen denaturation and cell necrosis within the irradiated field immediately, 1, 3, and 7 days after treatment. At high pulse energies, the MTZ depth could exceed 1 mm and width approached 200 microm as assessed by H&E. NBTC staining revealed viable interlesional tissue. In general, no statistically significant difference was found between in vivo and ex vivo depth and width measurements. The Fraxel SR laser system delivers pulses across a wide range of density and energy levels. We determined that increases in pulse energy led to increases in MTZ depth and width without compromising the structure or viability of interlesional tissue.

Selective photothermolysis of lipid-rich tissues: A free electron laser study

In theory, infrared vibrational bands could be used for selective photothermolysis of lipid-rich tissues such as fat, sebaceous glands, or atherosclerotic plaques. Absorption spectra of human fat were measured, identifying promising bands near 1,210 and 1,720 nm. Photothermal excitation of porcine fat and dermis were measured with a 3.5-5 microm thermal camera during exposure to the free electron laser (FEL) at Jefferson National Laboratory. Thermal damage to full-thickness samples exposed at approximately 1,210 nm through a cold contact window, was assessed by nitrobluetetrazolium chloride staining in situ and by light microscopy. Photothermal excitation of fat was twice that of dermis, at lipid absorption bands (1,210, 1,720 nm). At 1,210 nm, a subcutaneous fat layer several mm thick was damaged by FEL exposure, without apparent injury to overlying skin. Selective photothermal targeting of fatty tissues is feasible using infrared lipid absorption bands. Potential clinical applications are suggested by this FEL study.

Depth of morphologic skin damage and viability after one, two, and three passes of a high-energy, short-pulse CO 2 laser (Tru-Pulse) in pig skin

Background: CO2 laser energy is absorbed by water, which is present in all tissue. The depth of penetration of CO2 lasers is narrow with minimal reflection, scatter, or transmission. However, thermal damage has limited the usefulness of conventional, continuous-wave CO2 lasers for debridement as demonstrated by wound healing studies. The development of high-energy CO2 lasers, with pulse durations that are less than the thermal relaxation time of tissue, have made vaporization of skin for resurfacing and wound debridement possible because of the decreased risk of thermal damage. Objective: This study was performed to evaluate thermal damage produced by a CO2 laser. Methods: Routine histopathologic examination and nitroblue-tetrazolium chloride (NBTC) staining were used to evaluate the depth of tissue damage and viability in weanling pig skin after one, two, and three passes of the laser. Results: At a pulse energy of 300 mJ, with a pulse duration of 60 μsec, one pass of the laser produced vaporization of the epidermis with minimal thermal damage. Two passes produced areas of denatured collagen with loss of viable cells in the superficial papillary dermis. Three passes extended the damage into the papillary dermis. Conclusion: Hyalinization of collagen appears to correspond well with the level of thermal damage as measured by NBTC staining. Our findings suggest that the energy necessary to vaporize the dermis may be greater than that needed to vaporize epidermis. (J Am Acad Dermatol 1997;37:204-10.)

Losartan attenuates myocardial ischemia-induced ventricular arrythmias and reperfusion injury in spontaneously hypertensive rats.

To assess the efficacy of losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)biphe nyl-4-yl)methyl]imidazole, potassium salt), an angiotensin II receptor antagonist, on acute myocardial ischemia, 36 four-month-old spontaneously hypertensive rats were used. The animals underwent 45 min of left coronary artery occlusion and 1 h of reperfusion and were randomly assigned to control and losartan-treated groups (2, 5, and 10 mg/kg, intravenously). Losartan was administered 15 min before ischemia. Electrocardiograms (lead II) were monitored continuously throughout the experiment. To assess the anti-infarct effect of losartan, the area at risk was determined by methylene blue dye and the infarct size was determined by nitroblue tetrazolium chloride staining. The areas of risk and infarct were measured by computerized planimetry. Results demonstrated that the low and intermediate doses (2 and 5 mg/kg) of losartan significantly decreased the incidence of ventricular fibrillation and mortality during the ischemic period induced by left coronary artery occlusion. However, a significant reduction in infarct size, calculated as a percentage of the area at risk, was noted in all three losartan-treated groups (control: 41.5% +/- 5.2%, losartan, 2 mg/kg: 11.2% +/- 5.8%, 5 mg/kg: 8.5% +/- 2.7% and 10 mg/kg: 13.7% +/- 1.6%). The results suggest that losartan may be useful in the treatment of ventricular arrhythmias induced by acute myocardial infarction and attenuation of reperfusion injury in hypertension.

Accurate histochemical definition of argon-laser-induced tissue necrosis.

We present a histochemical method using the redox dye nitro blue tetrazolium chloride (NBTC) for the evaluation of argon-laser-induced tissue damage. By this method viable cells are stained blue, devitalized cells not. Our findings suggest that the NBTC staining method is more appropriate for the accurate definition of the necrosis and cell viability after laser treatment than routine histology.