Terminal Deoxynucleotidyl Transferase-mediated Deoxyuridine Triphosphate-biotin Research Articles

Ncx (Enx, Hox11L.1) is required for neuronal cell death in enteric ganglia of mice

Background/Purpose Ncx (Enx, Hox11L.1)–deficient (Ncx-/-) mice develop mega-ileo-ceco-colon with a larger number of neuronal cells in the enteric ganglia. We investigated mechanisms related to this abnormality and directed our attention to the effects on gastrointestinal tract functions. Methods The number of NADPH diaphorase or cuprolinic blue–positive neuronal cells in the enteric ganglia was examined during growth of the mice. Neuronal cell death of enteric ganglia was assayed by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick end labeling. Function of the gastrointestinal tract was determined by measuring excretion time of the barium chloride given into the stomach. Results The number of neuronal cells decreased in control mice older than 2 weeks, and neuronal cell death was evident in the ganglia. However, the number of neuronal cells did not decrease in Ncx-/- mice, and cell death was rare. Excretion time of barium chloride was prolonged in all Ncx-/- mice examined and was improved by the administration of an inhibitor of nitric oxide synthase. Conclusions Ncx participates in cell death of enteric neurons. Motor abnormality of the gastrointestinal tract in Ncx-/- mice may be attributed to the large number of neuronal cells.

Use of Mitochondrial Antioxidant Defenses for Rescue of Cells With a Leber Hereditary Optic Neuropathy–Causing Mutation

To explore a treatment paradigm for Leber hereditary optic neuropathy (LHON), we augmented mitochondrial antioxidant defenses to rescue cells with the G11778A mutation in mitochondrial DNA. Cells homoplasmic for the G11778A mutation in mitochondrial DNA were infected with an adeno-associated viral vector containing the human mitochondrial superoxide dismutase (SOD2) gene. Control cells were infected with an adeno-associated viral (AAV) vector expressing the green fluorescent protein (GFP). Two days later, the high-glucose culture medium was exchanged for a glucose-free medium containing galactose. After 1 or 2 days, cellular production of superoxide was assessed using the fluorescent probe dihydroethidium, and we used TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling) staining to detect apoptotic nuclei. The effect of SOD2 on LHON cell survival was quantitated after 2 or 3 days. Comparisons of AAV-SOD2-infected LHON cells relative to control cells infected with AAV-green fluorescent protein showed increased expression of mitochondrial SOD that attenuated superoxide-induced fluorescence by 26% (P = .003) and suppressed TUNEL-induced fluorescence by 21% (P = .048) after 2 days of growth in galactose medium, when cell survival increased by 25% (P=.05). After 3 days in galactose medium, SOD2 increased LHON survival by 89% (P = .006) relative to controls. Protection against mitochondrial oxidative stress may be useful for treatment of LHON. Gene therapy with antioxidant genes may protect patients with LHON against visual loss.

Histological changes and apoptosis of cartilage layer in human anterior cruciate ligament tibial insertion after rupture

The purpose of this study is to investigate the histological changes and apoptosis of cartilaginous layers in human anterior cruciate ligament (ACL) tibial insertion at different time periods after rupture. By using a core reamer, 35 tibial insertions of ruptured ACLs were obtained during primary ACL reconstructions (number of days after injury: 19-206 days). A histological examination was performed and a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining assay was carried out to detect apoptosis. The average thickness of the cartilage layer, the glycosaminoglycan-stained area and the number of chondrocytes per millimeter decreased with time. The percentage average of TUNEL-positive chondrocytes was 42.0 +/- 16.2. The histological degenerative changes of the cartilage layer in the ruptured ACL tibial insertion progressed with time, especially in the first 2 months. Moreover, chondrocyte apoptosis continued from 19 to 206 days after rupture. The results may help elucidate the etiology of the histological changes of the insertion, and may help in devising optimal treatment protocols for ACL injuries if apoptosis is controlled. Moreover, we consider that using a surviving ligament and minimizing a debridement of ACL remnant during ACL reconstruction may be important for ACL reconstruction to maintain cartilage layers in ACL insertions.

Effects of the Integrin-Linked Kinase Inhibitor QLT0267 on Squamous Cell Carcinoma of the Head and Neck

To study the expression of integrin-linked kinase (ILK) in human squamous cell carcinoma of the head and neck (SCCHN) tumor specimens and cell lines and the efficacy of the novel small molecule QLT0267. Immunohistochemical analysis of 17 SCCHN tumor tissue specimens and 3 normal tongue tissue specimens for ILK expression and in vitro analysis of the effectiveness of QLT0267 on SCCHN cells. Academic medical center. Expression levels of ILK in SCCHN tumor specimens and cell lines and the efficacy of QLT0267 in inhibiting cell growth and inducing apoptosis in SCCHN cell lines. Most SCCHN tumor specimens stained for ILK, whereas none of the 3 normal tongue tissue specimens stained for ILK. Integrin-linked kinase was expressed in all 6 SCCHN cell lines tested. In 4 pairs of normal and SCCHN tumor specimens, ILK expression and activity were higher in most tumor samples tested. A kinase assay showed that QLT0267 inhibited the ILK activity of 2 SCCHN cell lines (TU167 and MDA1986). Modified tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, DNA fragmentation ladder, and TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end()labeling) assays showed that QLT0267 inhibited cell growth and induced apoptosis in these 2 cell lines. A dose-dependent decrease in Akt phosphorylation was observed for these 2 cell lines on treatment with QLT0267. Integrin-linked kinase is overexpressed in SCCHN tumor specimens. Targeting ILK with the small-molecule ILK inhibitor QLT0267 inhibits cell growth and induces apoptosis in SCCHN cell lines by reducing ILK activity and Akt phosphorylation. Integrin-linked kinase may be an attractive target for molecular therapy with which to enhance treatment of SCCHN.

Chondrocyte Apoptosis and Decrease of Glycosaminoglycan in Cranial Cruciate Ligament Insertion after Resection in Rabbits

We examined time-dependent histological changes of the calcified fibrocartilage area in a tibial cranial cruciate ligament (CCL) insertion after ligament resection in rabbits. The animals were divided into two groups: those undergoing CCL substance resection in the right stifle (resected group) and those receiving the same operation without CCL resection in the left stifle (sham operated group). Five animals were euthanized with deep anaesthesia at four time periods (1, 2, 4 and 6 weeks), and Haematoxylin-eosin and Safranin-O stainings and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining were performed. The average percentage of TUNEL-positive chondrocytes and the average thickness of the glycosaminoglycan (GAG)-stained area in the calcified fibrocartilage area were measured. Two and 4 weeks after the surgery, the average percentages of TUNEL-positive chondrocytes in the resected group (23.8 +/- 10.3% and 15.9 +/- 6.7%, respectively) were significantly higher than those in the sham operated group (8.9 +/- 3.8% and 7.4 +/- 1.6%, P<0.05, respectively). Six weeks after the surgery, the average thickness of the GAG-stained area in the resected group (7.7 +/- 13. 5 microm) was significantly smaller than that in the sham operated group (69.4 +/- 39.9 microm, P<0.05). Our results suggest that the average percentage of TUNEL-positive chondrocytes became a peak in 2 weeks and that histological changes occurred in 6 weeks. The chondrocyte apoptosis can induce decrease of GAG-stained area after resection of CCL. Therefore, chondrocyte apoptosis in the calcified cartilage area in the CCL tibial insertion might lead to histological changes.

Ras Activation Modulates Methylglyoxal-Induced Mesangial Cell Apoptosis Through Superoxide Production

Aims. While previous studies have demonstrated that diabetic nephropathy is attributable to glucose-derived dicarbonyl compounds, methylglyoxal (MGO)-inducing apoptosis in renal mesangial cells, the molecular mechanism of upper stream redox signaling modulation, has not been fully elucidated. Methods: Rat mesangial cells pretreated with or without superoxide dismutase, diphenyloniodium, SB203580, and manumycin A were cultured in methylglyoxal stress-induced apoptosis. Signaling protein expression, flow cytometry, and morphological features of apoptotic cell death were assessed. Results: Methylglyoxal decreased cell viability in mesangial cells. Superoxide mediated methylglyoxal-induced caspase 3 cleavage. Pretreatment with diphenyloniodium, SB203580, and manumycin A reduced methylglyoxal augmentation of superoxide synthesis and caspase-3 activation. Methylglyoxal rapidly enhanced Ras activation and progressively increased cytosolic P38 and nuclear c-Jun activation. Scavenging of superoxide by superoxide dismutase or diphenyloniodium, inhibiting P38 by SB203580, and inhibiting Ras with manumycin A successfully reduced the promoting effect of methylglyoxal on P38 and c-Jun phosphorylation (activation). Furthermore, pretreatment with superoxide dismutase, diphenyloniodium, SB203580, and manumycin A significantly attenuated methylglyoxal induction of apoptosis on the basis of Annexin-V assay and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labelling (TUNEL) staining. Conclusions. This study has shown that methylglyoxal increased Ras modulation of superoxide-mediated P38 activation and c-Jun activation, which resulted in increased apoptosis.

Hippocalcin protects hippocampal neurons against excitotoxin damage by enhancing calcium extrusion

Hippocalcin, which is a member of the neuronal calcium-sensor protein family, is highly expressed in hippocampal pyramidal cells. Recently, it was demonstrated that hippocalcin deficit caused an increase in neuronal cell death in the field CA3 of Ammon’s horn (CA3) region of the hippocampus following the systemic injection of kainic acid. Treatment with kainic acid results in seizure-induced cell death in CA3. In the present study, we injected quinolinic acid, which is an N-methyl- d-aspartate receptor agonist, into the hippocampal field CA1 of Ammon’s horn (CA1) region in hippocalcin-knockout (−/−) mice, a procedure which mimics transient ischemia. Although significant pyknotic changes were observed at the injected site in wild-type (+/+) mice 24 h after injection, the area of pyknotic cells extended throughout the hippocampus in −/− mice. The quantification of cell numbers in Nissl-stained sections indicated that the cell damage in −/− mice was more severe than that in +/+ mice. The density of terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick-end labeling-positive cells roughly paralleled that of Nissl-stained pyknotic cells. Primary cultures of hippocampal neurons showed that the number of surviving neurons from −/− mice after 7 days in culture was smaller than the number from +/+ mice. The measurement of intracellular calcium concentrations in single cells revealed that the calcium extrusion from −/− neurons was slower than that from +/+ neurons. The involvement of hippocalcin in the upkeep of calcium extrusion was confirmed using hippocalcin-expressing COS7 cells. These results suggest that hippocalcin plays an important role in calcium extrusion from neurons and, in turn, helps to protect them against calcium-dependent excitotoxin damage in the hippocampus.

Staining Ability and Biocompatibility of Brilliant Blue G

To evaluate the effectiveness and biocompatibility of brilliant blue G (BBG) for capsular visualization for continuous curvilinear capsulorrhexis. The capsular staining ability of BBG was evaluated at graded concentrations of 10.0, 1.0, 0.5, 0.25, 0.1, and 0.01 mg/mL in enucleated pig's eyes. The biocompatibility of BBG was assessed in rat's eyes for 2 months. The eyes were analyzed using light, fluorescence, transmission electron, and scanning electron microscopy. TUNEL (terminal deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling) was used to detect apoptotic cells, and endothelial cell counts were analyzed using scanning electron microscopy. The results were compared using indocyanine green and trypan blue. The BBG improved capsular visualization, and a complete capsulorrhexis could be performed. In the rat model, no apparent toxic effect was observed using biomicroscopy during 2 months. Histologically, BBG showed satisfactory biocompatibility. Apoptotic cell death of the endothelial cells was detected in only the trypan blue group. In contrast to BBG, indocyanine green and trypan blue showed degeneration of corneal endothelial cells using transmission and scanning electron microscopy. The BBG contributed to better capsular visualization and caused no apparent complications to the corneal endothelium.Clinical Relevance The BBG is effective and safe capsular staining for continuous curvilinear capsulorrhexis.

ICSI in Cases of Sperm DNA Damage: Beneficial Effect of Oral Antioxidant Treatment

referral clinic. Patient(s): We collected 303 semen samples from patients undergoing IVF with or without ICSI. Intervention(s): Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay, measurement of fertilization rates, good embryo rates, and pregnancy rates for the IVF/ICSI program. Main Outcome Measure(s): The percentage of sperm with DNA fragmentation, correlated with semen analysis parameters and IVF/ICSI outcomes. Result(s): Sperm DNA fragmentation rates were significantly higher in patients with abnormal sperm parameters than in those with normal sperm parameters. When sperm DNA fragmentation was 10%, fertilization rates were affected. Sperm DNA fragmentation rates were negatively correlated with sperm velocity parameters but did not affect pregnancy outcomes. Conclusion(s): The results indicated that sperm DNA fragmentation affects fertilization rates and sperm motility but might not affect pregnancy rates. Editorial Comment: Since ICSI allows a single immotile sperm, even a dead one, to fertilize an ovum, an assay for sperm DNA integrity would prove useful in counseling couples undergoing the procedure. These investigators assessed sperm DNA integrity as measured by the TUNEL assay, and correlated outcomes in 303 women undergoing in vitro fertilization (86 with ICSI.) DNA fragmentation correlated poorly with bulk seminal parameters, including morphology, supporting the concept that the look of a sperm and its DNA are different things, and drawing into question the ability of a technician to select a good sperm for ICSI based on appearance alone. DNA fragmentation did affect fertilization rates and embryo quality, suggesting a possible prognostic value for sperm TUNEL assay.

Effect of L-Arginine on Pulmonary Artery Smooth Muscle Cell Apoptosis in Rats with Hypoxic Pulmonary Vascular Structural Remodeling

This study investigated the effect of L-arginine (L-Arg) on the apoptosis of pulmonary artery smooth muscle cells (PASMC) in rats with hypoxic pulmonary vascular structural remodeling, and its mechanisms. Seventeen Wistar rats were randomly divided into a control group (n=5), a hypoxia group (n=7), and a hypoxia+L-Arg group (n=5). The morphologic changes of lung tissues were observed under optical microscope. Using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay, the apoptosis of PASMC was examined. Fas expression in PASMC was examined using immunohistochemistry. The results showed that the percentage of muscularized artery in small pulmonary vessels, and the relative medial thickness and relative medial area of the small and median pulmonary muscularized arteries in the hypoxic group were all significantly increased. Pulmonary vascular structural remodeling developed after hypoxia. Apoptotic smooth muscle cells of the small and median pulmonary arteries in the hypoxia group were significantly less than those in the control group. After 14 d of hypoxia, Fas expression by smooth muscle cells of median and small pulmonary arteries was significantly inhibited. L-Arg significantly inhibited hypoxic pulmonary vascular structural remodeling in association with an augmentation of apoptosis of smooth muscle cells as well as Fas expression in PASMC. These results showed that L-Arg could play an important role in attenuating hypoxic pulmonary vascular structural remodeling by upregulating Fas expression in PASMC, thus promoting the apoptosis of PASMC.

Inhibition of vascular endothelial growth factor receptor 2 activity in experimental brain contusions aggravates injury outcome and leads to early increased neuronal and glial degeneration

Angiogenesis following traumatic brain injuries (TBIs) may be of importance for post-traumatic reparative processes and the development of secondary injuries. We have previously shown expression of vascular endothelial growth factor (VEGF), a major regulator of endothelial cell proliferation, angiogenesis and vascular permeability, and VEGF receptors (VEGFR1 and 2) after TBI in rat. In the present work we tried to further elucidate the role of VEGF after TBI by performing specific VEGFR2 activity inhibition. In rats subjected to VEGFR2 blockage we report an increased haemorrhagic area (P < 0.05), early increase in serum levels of neural injury marker neuron-specific enolase (P < 0.05) and glial injury marker S100beta (P < 0.05), and increased numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling- (TUNEL-) and FluoroJade B- (P < 0.05) positive cells, all increases preceding the known VEGF/VEGFR vascular response in brain trauma. An increase in lesion area, as measured by decreased microtubuli-associated protein 2 expression (P < 0.05) and increased glial fibrillary acidic protein reactivity (P < 0.05), could also be demonstrated. In addition, vascular density, as measured by von Willebrandt factor-positive cells, was decreased (P < 0.05). No differences in post-traumatic inflammatory response, as measured by stainings for macrophages, granulocytes and intracellular adhesion molecules, were shown between the groups. Taken together, our findings point towards VEGF/VEGFR2 up-regulation after TBI as being an important endogenous cytoprotective mechanism in TBI. The possible importance of VEGF on the vascular, neuronal and glial compartments of the neurovascular unit after TBI is discussed.

Interleukin-1 beta induction of neuron apoptosis depends on p38 mitogen-activated protein kinase activity after spinal cord injury1

Interleukin-1 beta (IL-1beta) has been implicated as an extracellular signal in the initiation of apoptosis in neurons and oligodendrocytes after spinal cord injury (SCI). To further characterize the apoptotic cascade initiated by IL-1beta after SCI, we examined the expression of IL-1beta, p38 mitogen-activated protein kinase (p38 MAPK) and caspase-3 after SCI, and further investigated whether p38 MAPK was involved in neuron apoptosis induced by IL-1beta. Adult rats were given contusion SCI at the T-10 vertebrae level with a weight-drop impactor (10 g weight dropped 25.0 mm). The expression levels of IL-1beta, p38 MAPK and caspase-3 after SCI were assessed with Western blots, immunohistochemistry staining, and real time reverse transcription polymerase chain reactions (RT-PCR). Neuron apoptosis was assessed with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) method. Increased levels of IL-1beta and p38 MAPK were observed soon after injury, with a peak in expression levels within 6 h of injury. By 24 h after injury, caspase-3 expression was markedly increased in the injured spinal cord. TUNEL-positive cells were first observed in the lesioned area 6 h after SCI. The largest number of TUNEL-positive cells was observed at 24 h post-SCI. Intrathecal injection of the IL-1 receptor antagonist IL-1Ra significantly reduced expression of p38 MAPK and caspase-3, and reduced the number of TUNEL-positive cells. Moreover, intrathecal injection of an inhibitor of p38 MAPK, SB203580, also significantly reduced the expression of caspase-3, and reduced the number of TUNEL-positive cells in the injured spinal cord. The p38MAPK signaling pathway plays an important role in IL-1beta mediated induction of neuron apoptosis following SCI in rats.

P2X7 receptor expression after ischemia in the cerebral cortex of rats.

Large amounts of adenosine 5'-triphosphate (ATP) released from cellular sources under pathological conditions such as ischemia may activate purinoceptors of the P2X and P2Y types. In the present study, the expression of the P2X7 receptor-subtype in the brain cortex of spontaneously hypertensive rats was investigated using a permanent focal cerebral ischemia model. Immunocytochemistry with antibodies raised against the intracellular C-terminus of the P2X7 receptor showed a time-dependent upregulation of labeled cells in the peri-infarct region after right middle cerebral artery occlusion (MCAO) in comparison to controls. Double immunofluorescence visualized with confooal laser scanning microscopy indicated the localization of the P2X7 receptor after ischemia on microglial cells (after 1 and 4 days), on tubulin betaIII-labeled neurons (after 4 and 7 days), and on glial fibrillary acidic protein (GFAP)-positive astrocytes (after 4 days). In the following experiments, changes occurring 4 days after MCAO were investigated in detail. Western blot analysis of the cortical tissue around the area of necrosis indicated an increase in the P2X7 receptor protein. Immunoelectron microscopy revealed the receptor localization on synapses (presynaptically), on dendrites, as well as on the nuclear membrane of neurons (postsynaptically) and glial cells. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling in combination with P2X7 receptor immunocytochemistry indicated a co-expression on the apoptotic cells. Active caspase 3 was especially observed on GFAP-positive astrocytes. In conclusion, the present data demonstrate a postischemic, time-dependent upregulation of the P2X7 receptor-subtype on neurons and glial cells and suggest a role for this receptor in the pathophysiology of cerebral ischemia in vivo.

Innocuity of tetradecyl-dimethyl-benzyl ammonium fluoride on the DNA of human spermatozoa

Tetradecyl-dimethyl-benzyl-ammonium fluoride (TDBAF) is a powerful new spermicide. In cases of failure of local contraception, there is a theoretical risk that a spermatozoon exposed to a sublethal concentration of spermicide might suffer DNA strand breaks, with transmission of damaged DNA to the oocyte. The present study aims to ascertain the innocuity of TDBAF in this regard. Spermatozoa from 32 fertile men were exposed to a sublethal concentration of TDBAF. The degree of DNA fragmentation was measured by modified terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin end labeling (TUNEL) assay, pulsed field gel electrophoresis of purified sperm DNA and radioenzymatic labeling. With the TUNEL assay, a statistically significant increase in sperm labeling was observed after TDBAF treatment compared to the controls. However, pulsed field gel electrophoresis and enzymatic labeling of purified DNA showed no evidence of augmented DNA fragmentation in TDBAF-treated sperm. Under electron microscopy, TDBAF caused sperm chromatin decondensation. In the TUNEL assay, this phenomenon would allow easier access of terminal transferase to its DNA substrate, explaining the apparent increase in DNA fragmentation in the presence of TDBAF. In conclusion, the use of TDBAF as a spermicide does not place progeny at heightened risk of abnormalities should pregnancy occur. In regard to the TUNEL assay, we suggest that pretreatment of spermatozoa with TDBAF promotes a more accurate measurement of sperm DNA fragmentation.

Regional low-flow perfusion improves neurologic outcome compared with deep hypothermic circulatory arrest in neonatal piglets

BackgroundRegional low-flow perfusion is an alternative to deep hypothermic circulatory arrest, but whether regional low-flow perfusion improves neurologic outcome after deep hypothermic circulatory arrest in neonates remains unknown. We tested neurologic recovery after regional low-flow perfusion compared with deep hypothermic circulatory arrest in a neonatal piglet model. MethodsSixteen neonatal piglets underwent cardiopulmonary bypass, were randomized to 90 minutes of deep hypothermic circulatory arrest or regional low-flow perfusion (10 mL · kg−1 · min−1) at 18°C, and survived for 1 week. Standardized neurobehavioral scores were obtained on postoperative days 1, 3, and 7 (0 = no deficit to 90 = brain death). Histopathologic scores were determined on the basis of the percentage of injured and apoptotic neurons in the neocortex and hippocampus by hematoxylin and eosin and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate–biotin nick-end labeling (0 = no injury to 4 = diffuse injury). Differences between groups were tested by using the Wilcoxon rank sum test, and results are listed as medians within a range. ResultsThere were no significant differences between groups during cardiopulmonary bypass. Postoperative neurobehavioral scores were abnormal in 25% (2/8) of the regional low-flow perfusion animals versus 88% (7/8) of controls. Regional low-flow perfusion animals had significantly less neurologic injury compared with controls on postoperative day 1 (0.00 [range, 0-5] vs 12.5 [range, 0-52]; P < .008). There was a trend for less severe injury in the regional low-flow perfusion group (2.0 [range, 1-4] vs 0.0 [range, 0-50]; P = .08) on hematoxylin and eosin. The degree of apoptosis was significantly less in the regional low-flow perfusion group (0.0 [range, 0-1] vs 2.5 [range, 0-4]; P = .03). ConclusionsRegional low-flow perfusion decreases neuronal injury and improves early postoperative neurologic function after deep hypothermic circulatory arrest in neonatal piglets.

Different Susceptibility of Cells of Porcine Skin and Internal Organs to Ultraviolet A - Induced Breaking of Nuclear DNA

There is a continuously growing interest in medical applications of ultraviolet radiation (UV-A and long-wavelength UV-B) especially for laser surgery, phototherapy and photodiagnostics of human internal organs. UV-B and UV-A radiation is potentially mutagenic, however, there has been very little information published to date concerning the significance of possible deleterious action of such photons on cells of internal tissues. The aim of this study is to compare the sensitivities of skin cells to those of internal organs upon exposure to UV-A. To assess this sensitivity we have determined the UV-A dose-dependent frequency of nuclear DNA breaks detected with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) technique. The materials for the study were macroscopic samples of porcine skin, colon and esophagus. The UV-A dose ranged from 0.1 to 1000 mJ/cm2, which is similar to doses received by cells in regions examined with laser-induced fluorescence or by cells surrounding areas subject to a laser ablation. To reduce the influence of DNA repair processes the tissue samples were kept at a low temperature during the irradiation and were deep frozen immediately after completing the irradiation procedure. The cells of the internal organs are much more susceptible to UV-A-induced breaking of DNA than the skin cells. The percentage fractions and the spatial distributions of the damaged cells and the characteristics of the UV-A dose dependence seem to vary by type of internal organ.

Hydroxyl radicals generated in the rat spinal cord at the level produced by impact injury induce cell death by necrosis and apoptosis: protection by a metalloporphyrin

We previously measured the time courses of hydrogen peroxide (H 2O 2), hydroxyl radical ( •OH), and catalytic iron increases following traumatic spinal cord injury (SCI). This study determines whether the SCI-elevated level of •OH causes cell death. OH was generated by administering H 2O 2 and Fe 2+ at the concentrations attained following SCI, each through a separate microdialysis fiber inserted laterally into the gray matter of the cord. The duration of •OH generation mimics the duration of its elevation after SCI. The death of neurons and astrocytes was characterized at 24 h post- •OH exposure and quantitated by counting surviving cells along the fiber track in sections stained with Cresyl Violet, or immunohistochemically stained with anti-neuron-specific enolase (anti-NSE) and anti-glial fibrillary acidic protein (anti-GFAP). DNA fragmentation in neurons was characterized by double staining with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) and anti-NSE. Using a one way ANOVA followed by the Tukey test, we demonstrated that •OH generated in the cord induced significant losses of neurons in both Cresyl Violet ( P<0.001) and anti-NSE-stained sections ( P<0.001), and of astrocytes in GFAP-stained sections ( P=0.001). •OH generated in the cord increased numbers of TUNEL-positive neurons compared with Ringer's solution administered as a control ( P=0.001). Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic and a broad spectrum reactive species scavenger, significantly reduced •OH-induced death of neurons ( P<0.001 in anti-NSE stained sections and P=0.002 in the Cresyl Violet-stained sections) and astrocytes ( P=0.03). It also reduced the numbers of TUNEL-positive neurons ( P=0.01). Electron microscopy confirmed that generated •OH induced neuronal and glial death with characteristic features of both necrosis and apoptosis. We conclude that 1) SCI-elevated •OH is sufficient to induce both necrosis and apoptosis, criteria for identifying an endogenous secondary damaging agent; 2) MnTBAP reduces •OH-induced cell death, perhaps by removing H 2O 2 administered in the tissue, thereby blocking formation of •OH, and also by scavenging downstream reactive species.

Effects of ionizing radiation on brain tissue surrounding arteriovenous malformations: an experimental study in a rat caroticojugular fistula model

Arteriovenous malformation (AVM) may change the cerebral hemodynamics. The purpose of this study was to detect the effects of ionizing radiation (IR) on tissues surrounding AVM in a rat caroticojugular fistula model. Forty rats were divided into four groups. Eight weeks after caroticojugular fistulas and chronic hypoperfusion were created in groups 1 and 2, IR was administered to groups 1 and 3. Group 4 was the control. Brain tissue samples were taken 72 h after irradiation. Comet assay to detect DNA strand breaks (DSB), terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) assay for apoptosis, and free radical measurement were performed. Although the difference between fistula plus irradiation (group 1) and fistula (group 2) was statistically insignificant in terms of DSB and free radical measurement, apoptotic cell count was significantly higher in group 1. Nonetheless, apoptotic cell count corresponded well with both free radicals and DSB in the irradiated group (group 3). Ionizing radiation resulted in significant apoptosis in both groups with or without fistulas. Chronic hypoperfusion might not prevent cerebral damage after IR. Optimal care should be taken with brain tissue around AVM during radiotherapy, regardless of presence or absence of the "steal" phenomenon.

Quantitative tumor apoptosis imaging using technetium-99m-HYNIC annexin V single photon emission computed tomography.

Radiolabeled annexin V may allow for repetitive and selective in vivo identification of apoptotic cell death without the need for invasive biopsy. This study reports on the relationship between quantitative technetium-99m- (99mTc-) 6-hydrazinonicotinic (HYNIC) radiolabeled annexin V tumor uptake, and the number of tumor apoptotic cells derived from histologic analysis. Twenty patients (18 men, two women) suspected of primary (n = 19) or recurrent (n = 1) head and neck carcinoma were included. All patients underwent a spiral computed tomography (CT) scan, 99mTc-HYNIC annexin V tomography, and subsequent surgical resection of the suspected primary or recurrent tumor. Quantitative 99mTc-HYNIC annexin V uptake in tumor lesions divided by the tumor volume, derived from CT, was related to the number of apoptotic cells per tumor high-power field derived from terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end-labeling (TUNEL) assays performed on sectioned tumor slices. Diagnosis was primary head and neck tumor in 18 patients, lymph node involvement of a cancer of unknown primary origin in one patient, and the absence of recurrence in one patient. Mean percentage absolute tumor uptake of the injected dose per cubic centimeter tumor volume derived from tomographic images was 0.0003% (standard deviation [SD], 0.0004%) at 1 hour postinjection (PI) and 0.0001% (SD, 0.0000%) at 5 to 6 hours PI (P =.012). Quantitative 99mTc-HYNIC annexin V tumor uptake correlated well with the number of apoptotic cells if only tumor samples with no or minimal amounts of necrosis were considered. In the absence of necrosis, absolute 99mTc-HYNIC annexin V tumor uptake values correlate well with the number of apoptotic cells derived from TUNEL assays.

Evidence for altered mast cell proliferation and apoptosis in cutaneous mastocytosis.

Mastocytosis presents as a focal or generalized increase of mast cells, particularly in the skin, but also in other organs. Activating mutations of KIT (formerly c-kit), the receptor of the mast cell growth factor stem cell factor (SCF), appear to play a key role in the pathogenesis of sporadic adult onset mastocytosis. However, these mutations are not present in childhood-onset and familial mastocytosis and also fail to explain the heterogeneity of adult-onset disease. Other factors such as prolonged survival of mast cells may therefore participate in causing and modulating the pathological increase of mast cells in mastocytosis. To examine the expression of proliferation and apoptosis markers in the mast cells of cutaneous mastocytosis lesions in order to gain further insight into the pathogenesis of mastocytosis. Lesional cutaneous biopsies from eight infants with solitary mastocytomas, five children with multiple mastocytomas, 11 children with generalized urticaria pigmentosa, 12 adults with urticaria pigmentosa, and skin from seven normal controls were used in this study. Serial sections were stained with toluidine blue to quantify mast cell numbers and with antibodies against the proliferation marker Ki67 protein, the tumour suppressor protein p53, and the inhibitor of cyclins and cyclin-dependent kinases p21WAF1/CIP1, using the alkaline phosphatase antialkaline phosphatase technique. The terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labelling (TUNEL) method was used to assess apoptosis. Cutaneous mast cell counts were significantly increased in all patient sections, particularly in childhood lesions, and similarly, a small but significant increase of proliferation was found in the lesional mast cells of all patients. Enhanced mast cell numbers and proliferation was associated with a significant decrease of TUNEL staining, particularly in mastocytomas. p53 expression was highly variable, with an overall significant increase in all patient skin mast cells, whereas p21 expression was barely observed at all. These findings further support the concept that an imbalance of mast cell proliferation and apoptosis is prevalent in mastocytosis lesions that may account in part for the increased focal mast cell accumulation in this condition.