Stratification, specialization, and proliferation of primary keratinocyte cultures. Evidence of a functioning in vitro epidermal cell system

A population of neonatal mouse keratinocytes (epidermal basal cells) was obtained by gentle, short-term trypsin separation of the epidermal and dermal skin compartments and discontinuous Ficoll gradient purification of the resulting epidermal cells. Over 4--6 wk of culture growth at 32--33 degrees C, the primary cultures formed a complete monolayer that exhibited entire culture stratification and upper cell layer shedding. Transmission and scanning electron microscopy demonstrated that the keratinocyte cultures progressed from one to two cell layers through a series of stratification and specialization phenomena to a six to eight cell layer culture containing structures characteristic of epidermal cells and resembling in vivo epidermal development. The temporal development of primary epidermal cell culture specialization was confirmed by use of two histological techniques which differentially stain the specializing upper cell layers of neonatal mouse skin. No detectable dermal fibroblast co-cultivation was demonstrated by use of the leucine aminopeptidase histochemical technique and routine electron microscope surveillance of the cultures. Incorporation of [3H]thymidine ([3H]Tdr) was greater than 85% into DNA and was inhibited by both 20 micron cytosine arabinoside (Ara-C) and low temperature. Autoradiography and 90% inhibition of [3H]Tdr incorporation by 2 mM hydroxyurea indicated that keratinocyte culture DNA synthesis was scheduled (not a repair phenomenon). The primary keratinocytes showed an oscillating pattern of [3H]Tdr incorporation into DNA over the initial 23--25 days of growth. Autoradiography demonstrated that the cultures contained 10--30% proliferative stem cells from days 2-25 of culture. The reproducibility of both the proliferation and specialization patterns of the described primary epidermal cell culture system indicates that these cultures are a useful tool for investigations of functioning epidermal cell homeostatic control mechanisms.     

Collagen fibril diameters increase and fibril densities decrease in skin subjected to repetitive compressive and shear stresses

Understanding microstructural changes that occur in skin subjected to repetitive mechanical stress is crucial towards the development of therapies to enhance skin adaptation and load tolerance in patients at risk of skin breakdown (e.g. prosthesis users, wheelchair users). To determine if collagen fibril diameter, collagen fibril density, dermal thickness, epidermal thickness, basement membrane length, and dermal cell density changed in response to repetitive stress application, skin subjected to moderate cyclic compressive and shear stresses for 1 h/d, 5 d/week, for 4 week was compared with skin from an unstressed contralateral control. The lateral aspects of the hind limbs of 12 Landrace/Yorkshire pigs were used. Skin from under the stressed site and a contralateral control site was processed for electron microscopy and light microscopy analysis. Electron microscopy results demonstrated significant (p<0.01) increases in collagen fibril diameter of 15.9%, 22.4%, and 22.9% for the upper, mid, and lower layers of the dermis, respectively, for the stressed skin compared with the control skin. Collagen fibril density (fibrils/unit cross-sectional area) decreased significantly for stressed vs. control by 19.8%, 29.2%, and 31.8% for the upper, mid, and lower layers, respectively. Light microscopy results demonstrated trends of a decrease in dermal thickness and an increase in cell density for stressed vs. control samples, but the differences were not significant. Differences in epidermal thickness and basement membrane length were not significant. These results demonstrate that quantifiable changes occur in collagen fibril architecture but not in the gross tissue morphology following in vivo cyclic loading of pig skin.     

Modulation of the population density of identifiable epidermal Langerhans cells associated with enhancement or suppression of cutaneous immune reactivity

The epidermis on the backs or ears of DBA/2 mice treated for 7 days with a 20% concentration of monobenzyl ether of hydroquinone (MBEH) had a significantly greater population density of ATPase- and Ia-positive cells compared with control mice treated with diluent. There was no decrease or increase in ATPase- or Ia-positive cells at sites distal from the treated tissue. This increase in population density of Langerhans cells was associated with a significant increase in functional afferent immune reactivity measured by allergic contact hypersensitivity. We also found evidence for enhanced efferent immune reactivity. Animals treated on the ears for 7 days with MBEH were sensitized to DNFB on untreated back. MBEH treated ears with more Ia-positive Langerhans cells demonstrated a threefold greater increase in swelling after the DNFB challenge than the control mice. Results of other studies suggest that the afferent and efferent enhanced immune reactivity produced by MBEH are local effects. We postulated that MBEH produced its effects by activating the oxidation of arachidonic acid (AA) to prostaglandins. To test this, we applied AA to mouse skin. AA has a biphasic effect on epidermal Langerhans cells: in low doses it increases their number; in high amounts it decreases the number of identifiable cells with either the Ia or the ATPase technique. An increased population density of identifiable epidermal Langerhans cells induced with AA was correlated with an increase in afferent and efferent immune reactivity. In contrast, reduction of Langerhans cells with larger amounts of AA suppress the afferent and efferent limb of the immune response. DNFB applied to skin with decreased Langerhans cell density from AA induced a state that mimics immune tolerance. The findings are significant because we report the only method to either increase or decrease the population density of Langerhans cells: and to modulate up or down the afferent or efferent limbs of the cutaneous immune response. Our results also suggest that the Langerhans cell may be involved in the efferent limb of the immune efferent response. These effects may be modulated in part by products of AA metabolism.     

Level of endothelial cell apoptosis required for a significant decrease in microvessel density

Endothelial cell apoptosis plays a critical role in the disruption of blood vessels mediated by natural inhibitors of angiogenesis and by anti-vascular drugs. However, the proportion of endothelial cells required to mediate a significant decrease in microvessel density is unknown. A system based on an inducible caspase (iCaspase-9) offers a unique opportunity to address this question. The dimerizer drug AP20187 induces apoptosis of human dermal microvascular endothelial cells stably transduced with iCaspase-9 (HDMEC-iCaspase-9), but not control cells (HDMEC-LXSN). Here, we generated blood vessels containing several HDMEC-iCaspase-9:HDMEC-LXSN ratios, and developed a mathematical modeling involving a system of differential equations to evaluate experimentally inaccessible ratios. A significant decrease in capillary sprouts was observed when at least 17% of the endothelial cells underwent apoptosis in vitro. Exposure to vascular endothelial growth factor (VEGF(165)) did not prevent apoptosis of HDMEC-iCaspase-9, but increased the apoptotic requirement for sprout disruption. In vivo experiments showed the requirement of at least 22% apoptotic endothelial cells for a significant decrease in microvascular density. The combined use of biological experimentation with mathematical modeling allowed us to conclude that apoptosis of a relatively small proportion of endothelial cells is sufficient to mediate a significant decrease in microvessel density.     

Myocardial capillaries in the fetal and the neonatal rat: a morphometric analysis of the maturing myocardial capillary bed

Developing myocardial capillaries from 16-day-gestation fetus to adult undergo several morphological changes including a thinning of the lateral extensions of the capillary endothelial cells, the formation of a basal lamina, and an increase in the number of plasmalemmal vesicles. A decrease in the extracellular space, an increase in the number of capillaries, and a decrease in the capillary diameter were also observed during the developmental period. In view of these ultrastructural changes, a morphometric analysis was made on the developing myocardial wall to demonstrate specific quantitative changes. The volumes which were occupied by capillary endothelial cells, capillary lumina, extracellular space, and myocardial myocytes within a reference volume of myocardium were measured; and we found that 8% of the reference myocardial volume was occupied by capillary endothelial cells, 85% was occupied by myocardial myocytes, 4% was occupied by capillary lumina, and, except for a significant change in extracellular space at 16 days gestation, 3% was occupied by extracellular space. Each volume ratio was found to be nearly constant throughout the studied period. In contrast to this constancy in the volume ratios, other parameters which were measured demonstrated significant changes during the developmental period studied. These overall changes include a 135% increase in capillary density, a 63% increase in luminal surface area of capillary endothelial cells, a 24% decrease in capillary diameter, a 12% decrease in diffusion distance, and a 35% decrease in the diameter of the erythrocyte population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological and functional changes in the rat heart after X irradiation: strain differences

The hearts of mature male rats of the Wistar and Sprague-Dawley strains were locally irradiated with single doses of 17.5 and 20.0 Gy of X rays, respectively. These two dose levels had previously been shown to result in a comparable latent period between irradiation and the death of rats of these two strains from cardiac failure. Morphological changes in the myocardium and modifications in cardiac function were assessed in the animals at 28, 70, and 100 days after irradiation. The first radiation-induced change which was observed in the myocardium was a rapid decline in capillary density and a loss of alkaline phosphatase activity by the capillary endothelial cells. The capillary density was reduced to approximately 50% of that of unirradiated control values at 28 days and to approximately 40% of the control values between 70 and 100 days after irradiation. The loss of enzyme activity was also detected at 28 days. Examination of histological sections showed an increase by 70 days in the areas with negative enzyme activity up to approximately 70% of the myocardium. The reduction in capillary density and the loss of enzyme activity occurred before any marked pathological changes were seen in the myocardium. The pathological lesions seen in the myocardium at 100 days after irradiation were qualitatively and quantitatively the same in the two strains of rat. Measurements of cardiac output in Sprague-Dawley rats showed a gradual decline in output after irradiation; however, measurements in Wistar rats showed a progressive increase in cardiac output over the same period of time. It was shown by rubidium extraction that there was an increase in the percentage of the total cardiac output distributed to the ventricular muscle of Sprague-Dawley rats, while similar measurements in Wistar rats showed no significant change. In spite of the marked strain differences observed in cardiac output and rubidium extraction, blood perfusion per gram of ventricular muscle was apparently not modified in both strains of rat after irradiation. These findings indicated that the correlation between morphological effects after irradiation and the functional expression of damage is highly complex.     

On the mechanism of plasma membrane turnover in the salt gland of ducklings

Summary This study provides information on the rates of DNA synthesis, sites of DNA synthesis, and DNA content of the avian salt gland during the osmoticstressing (plasma membrane synthesis) and destressing (plasma membrane turnover) cycle, in an effort to better understand the relationship of cell turnover to the initial events in plasma membrane amplification, differentiation, and turnover. The rate of DNA synthesis increases 12–24 h after the onset of osmotic stress, is maximal at about 24 h of osmotic stress, and decreases thereafter in fully stressed and destressed glands. The maximum DNA and protein content, and the maximum protein/DNA ratio are obtained after about 3 days of stress. Autoradiograms show that at 24 h of stress 70–80% of DNA synthesis occurs in connective tissue cells and 20–30% in parenchymal cells, but by 6 days of stress, synthesis occurs about equally in these cell groups. Because destressing is characterized by a large decrease in plasma membrane and in glandular protein, but by little DNA turnover or loss, the loss of plasma membrane is likely due to some type of cell dedifferentiation rather than cell turnover.     

Early radiation-induced endothelial cell loss and blood-spinal cord barrier breakdown in the rat spinal cord

Using a rat spinal cord model, this study was designed to characterize radiation-induced vascular endothelial cell loss and its relationship to early blood-brain barrier disruption in the central nervous system. Adult rats were given a single dose of 0, 2, 8, 19.5, 22, 30 or 50 Gy to the cervical spinal cord. At various times up to 2 weeks after irradiation, the spinal cord was processed for histological and immunohistochemical analysis. Radiation-induced apoptosis was assessed by morphology and TdT-mediated dUTP nick end labeling combined with immunohistochemical markers for endothelial and glial cells. Image analysis was performed to determine endothelial cell and microvessel density using immunohistochemistry with endothelial markers, namely endothelial barrier antigen, glucose transporter isoform 1, laminin and zonula occludens 1. Blood-spinal cord barrier permeability was assessed using immunohistochemistry for albumin and (99m)Tc-diethylenetriamine pentaacetic acid as a vascular tracer. Endothelial cell proliferation was assessed using in vivo BrdU labeling. During the first 24 h after irradiation, apoptotic endothelial cells were observed in the rat spinal cord. The decrease in endothelial cell density at 24 h after irradiation was associated with an increase in albumin immunostaining around microvessels. The decrease in the number of endothelial cells persisted for 7 days and recovery of endothelial density was apparent by day 14. A similar pattern of blood-spinal cord barrier disruption and recovery of permeability was observed over the 2 weeks, and an increase in BrdU-labeled endothelial cells was seen at day 3. These results are consistent with an association between endothelial cell death and acute blood-spinal cord barrier disruption in the rat spinal cord after irradiation.     

Pigment cell mechanism of postembryonic stripe pattern formation in the Japanese four‐lined snake

Postembryonic changes in the dermal and epidermal pigment cell architecture of the striped and nonstriped morph of the Japanese four‐lined snake Elaphe quadrivirgata were examined to reveal stripe pattern formation after hatching. The striped and nonstriped morphs were distinguishable at the hatching, suggesting that the basis of stripe pattern was formed during embryonic development. In the striped morph, the color of stripes changed from red‐brown in juveniles to vivid dark‐brown in adults, and density of dermal melanophore increased much more in the stripe than background dorsal scales with growth. This increase in density of dermal melanophore was accompanied not only by the increased epidermal melanophore density but also by the change in vertical structures of dermal melanophore. By contrast, the density of epidermal and dermal melanophore evenly increased over the dorsal scales in the nonstriped morph. Thus, the increased vividness of the stripe pattern after hatching is achieved through localized increase of melanophore density particularly in the stripe region but not over the whole dorsal scales. J. Morphol. 277:196–203, 2016. © 2015 Wiley Periodicals, Inc.     

The tumor promoter, 12-O-tetradecanoylphorbol-13-acetate accelerates keratinocyte differentiation and stimulates growth of an unidentified cell type in cultured human epidermis

Studies were conducted to determine the effects of the mouse skin tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) on cultured human epidermal cells for comparison with known effects on mouse keratinocytes. In contrast to its effect on mouse cells, TPA did not stimulate human epidermal cell DNA synthesis. TPA stimulated differentiation in human keratinocytes resulting in sloughing of many cells by the 3rd day after exposure. Quantitative assays revealed that 50% of the TPA-exposed population was composed of cornified cells as opposed to 8% in untreated controls. A morphologically distinct cell type (TT cell) emerged after TPA treatment which was triangular in shape, did not stratify, appeared to proliferate rapidly and at most TPA concentrations became the predominant cell type within 1–2 weeks. Cultures composed predominantly of TT cells formed few cornified envelopes, grew well in the absence of TPA and formed colonies at low cell input. In contrast to its effect on keratinocytes, TPA enhanced TT colony formation 3–4-fold and decreased the doubling time of TT cells. Studies were performed to determine the origin of TT cells. Immunofluorescent staining indicated that TT cells lacked the keratinocyte antigens keratin, pemphigus and pemphigoid. Tonofilaments and desmosomes were not seen by electron microscopy. The lack of both melanosomes and standard histochemical DOPA oxidase staining indicated that TT cells were probably not of melanocyte origin. Tests used to identify Langerhans cells were negative. Whereas TT cells, as well as dermal fibroblasts, yielded positive immunofluorescence with antibodies to vimentin, TT cells gave a weak histochemical leucine aminopeptidase reaction, while the reaction of fibroblasts exposed to TPA was strong. Treatment of human dermal fibroblasts with TPA did not yield TT cells. The endothelial cell antigen factor VIII-associated protein was absent by immunofluorescence. These results suggest that the primary effect of TPA on cultured human epidermis is to accelerate terminal differentiation in the keratinocyte population and to stimulate growth of an as yet unidentified cell type.     

Comparison of three methods for human corneal cryopreservation that utilize dimethyl sulfoxide.

We compared endothelial cell survival in human corneas after cryopreservation by three methods that utilize dimethyl sulfoxide. Twenty-eight human cadaver corneas were cryopreserved by one of three methods, stored briefly over liquid nitrogen, thawed, cultured at 37 degrees C for 3 days, and fixed for scanning electron microscopy. Seventeen control corneas underwent identical cryoprotectant immersion and culture protocols but were not frozen. Endothelial photographs taken after 1 and 3 days of culture were analyzed. Endothelial cell losses in cryopreserved corneas by Methods 1, 2, and 3, respectively, were 36, 22, and 10% after 1 day of culture and 57, 36, and 27% after 3 days of culture. Cryopreservation by Method 3 had less cell loss than Methods 1 or 2 (P<0.02) but greater cell loss than the control corneas for Method 3 (P<0.001). No loss of cells occurred in the control corneas for Methods 1 and 3 but substantial cell loss (26%) occurred in the control corneas for Method 2. Polymegethism and pleomorphism of the endothelial cells were seen in the corneas that lost cells. The endothelial cell loss of 10% seen after 1 day of culture in human corneas cryopreserved by Method 3 is similar to the loss that occurs during organ culture storage as currently used clinically and therefore would be acceptable for clinical use. After 3 days of culture, however, the cell loss had increased significantly to 27%. This additional decrease in cell number that occurs in culture may represent latent cryodamage and must be understood and overcome in vivo before the technique can be used clinically.     

Growth factor responses of human arterial endothelial cells in vitro

Summary Human arterial endothelial cells were cultured in vitro for up to 40 cumulative population doublings. Culture conditions similar to those required for long-term propagation of human umbilical vein endothelial cells were employed. These included fibronectin-coated culture vessels, 5 to 20% fetal bovine serum, endothelial cell growth factor, and heparin. Thoracic aorta endothelial cells were larger than iliac artery endothelial cells. Both cell types stained positively for Factor VIII antigen by immunofluorescence. A decrease in confluent density as a function of population doubling level was correlated with the appearance of large, senescent cells in the cultures. Serum growth factors to which the arterial endothelial cells responded included insulin, transferrin, epidermal growth factor, thrombin, and somatomedins. The effect of thrombin did not require the availabilty of the active site of the protease. The effect of the somatomedins was only seen in the presence of heparin. Neither platelet-derived growth factor nor hydrocortisone induced arteiral endothelial cell proliferation. These growth factor responses were also observed on the part of human umbilical vein endothelial cells. This work was supported in part by Public Health Service grants HL01030, HL01734, and AG00599.     

Effect on prostatic growth of 2-difluoromethylornithine, an effective inhibitor of ornithine decarboxylase.

2-Difluoromethylornithine (DFMO), an enzyme-activated irreversible inhibitor of ornithine decarboxylase, causes marked changes in the polyamine metabolism of ventral prostate when given to adult rats in drinking water (20 g/l) for 3 consecutive days. A 90% inhibition of ornithine decarboxylase activity is accompanied by approx. 80% decreases of the concentrations of putrescine and spermidine and by a 36% decrease in spermine. Concomitantly, S-adenosylmethionine decarboxylase activity increases 7-fold and the concentration of decarboxylated S-adenosylmethionine 450-fold. When DFMO is given to immature rats for 12 consecutive days the above described changes are accompanied by a marked reduction in the age-dependent increases of the wet weight and RNA and DNA contents of the ventral prostate. In adult rats DFMO decreases the weight and RNA content of the ventral prostate within 4 days by 32% and 24% respectively and maintains them constant for the next 19 days. After 23 days of treatment, the prostatic weight is 46% of that of control animals of the same age, whereas the weights of other organs are only slightly decreased. Cytological studies carried out at this time show that DFMO reduces the size of both prostatic acini and the epithelial cells lining the acini.     

The effects of ionizing radiation on the pulmonary vasculature of intact rats and isolated pulmonary endothelium

We studied the effects of ionizing radiation on the morphology of the pulmonary circulation using an in vivo rat model and an in vitro pulmonary artery endothelial cell model. Gamma radiation was given as either an acute (30 Gy) or fractionated (5 X 6 Gy) dose to one hemithorax of rats. An acute 30-Gy dose delivered resulted in a 70% decrease in pulmonary arterial perfusion, using technetium-99m microaggregated albumin (99mTc-MAA), in the irradiated lung by 2-3 weeks after irradiation. Pulmonary microradiographs, using a barium sulfate perfusion method, obtained 2-3 weeks after irradiation demonstrated widespread loss of capillary filling and segmentation of the vessels. Histologic examination demonstrated intact capillaries, suggesting that the alterations in pulmonary perfusion were at the precapillary level. Similar abnormalities in lung perfusion and morphology were found after delivery of fractionated doses of radiation, but the onset of the changes was delayed, occurring 4-6 weeks postirradiation. Using cultured pulmonary endothelial cell monolayers, cell sloughing and retraction from the surface substrate were observed within 24 h after in vitro delivery of 30 Gy. Similar findings occurred in monolayers given fractionated doses (5 X 6 Gy) of radiation 2-3 days after the final dose. The in vivo animal and in vitro endothelial cell models offer a useful means of examining the morphologic alterations involved in radiation lung vascular damage.     

Migration of epidermal melanophores to the dermis through the basement membrane during metamorphosis in the frog, Rana japonica

The number of epidermal melanophores of the skin decreases dramatically during metamorphosis in the frog, Rana japonica. This decrease may represent an adaptation for rapid color change, a property which the animal acquires after metamorphosis. We concluded that the decrease was due to the migration of epidermal melanophores to the dermis. Epidermal melanophores and epidermal cells are tightly associated with each other in the young tadpole. The association becomes looser at the metamorphic stage and, occasionally, small breaks in the basement membrane are seen. These breaks may facilitate the migration. The migration was observed exclusively at the metamorphic stage, in spite of careful observation of other stages under the electron microscope. The migration of epidermal melanophores was induced by treatment with thyroxine of cultured skin from tadpoles at stage 15, and this hormone may act directly on epidermal melanophores. Until now, the increase in the number of dermal melanophores during metamorphosis has been explained by the differentiation of dermal melanophores from melanoblasts and by their mitotic division. Our results show that the migration of epidermal melanophores to the dermis may be a factor which accounts for the increase in the number of dermal melanophores.     

Time-dependent effects of removal of the pars distalis of the pituitary gland on toad epidermal cell and tissue kinetic parameters

Abstract. Amphibian moulting and its hormonal control has been extensively studied, but the possible influence of hormones on epidermal proliferation has been less investigated. In the present contribution to studies on the control of epidermal homeostasis, the proliferative pattern of the toad epidermis following ablation of the pars distalis of the pituitary gland is analysed by an investigation of the changes in the epidermal cell number, metaphase index and [³H]thymidine incorporation at various times after the operation. During the first 24 hr after pars distalis ablation, labelling index (LI) increased concurrently with an initial decrease in the metaphase index (MI), followed by an increase. During the same period of time the mean grain count (MGC) and the grain distribution pattern also changed. From 24 hr to 7 days after operation, MI, LI, and MGC were significantly lower than those of controls, but increased to control levels at 14 days after the operation. Phase durations and their possible changes were not measured directly, but data showed that the S-phase duration (Ts) and the mitotic duration (TM) must have changed in relation to each other during the experiment. Assuming that the MGC is a rough estimate of the DNA-synthesis rate, the compatibility of a postulated change in phase duration with the observed MGC was analysed. This analysis revealed that TS and TM could have decreased up to 18 hr after the operation, whereas these phase durations, after 24 hr and during the rest of the experiment, increased compared to those of controls. Even with these possible changes in phase durations, and in the absence of direct assessment of cell division rates, the observed cell kinetic parameters appeared incompatible with an increased rate of proliferation. This was surprising since the efflux of cells from the living, subcorneal epidermis to the stratum corneum was significantly increased after pars distalis ablation, without a concurrent decrease in the stratum corneum recruitment cell pool (SCRP-number of subcorneal epidermal cells per mm surface). Possible reasons for the discrepancy between the expected increase in proliferation following pars distalis ablation, and the failure to demonstrate this, are discussed.     

Effects of fibroblasts and microenvironment on epidermal regeneration and tissue function in long-term skin equivalents

In vitro generated skin models find growing interest as promising tools in basic research and clinical application in regenerative medicine. Here, we present further details of an improved long-term skin equivalent (SE) enabling mechanistic studies on skin reconstruction and epidermal function. Growth conditions of fibroblasts in a 3D scaffold were analysed to optimise the dermal microenvironment by providing an authentic dermal matrix for regular tissue reconstruction and function of cocultured keratinocytes. These SEs demonstrate sustained epidermal viability - over 12 weeks - with regular differentiation as substantiated by in vivo-like patterns of all differentiation products, exemplified here by the cornified envelope components loricrin and repetin. The continuous expression of all major tight junction components in the granular layer, shown here for ZO-1 in coherence with the presence of epidermal barrier lipids, and ultrastructural accumulation of lamellar bodies, collectively indicate proper epidermal barrier structures. Remarkably, cocultured keratinocytes exerted an ongoing proliferation-stimulating effect on fibroblasts colonising the scaffold comparable to a cocktail of fibroblast growth factors. Consequently, precultivation of dermal equivalents (DEs) in basal or growth factor-enriched media had only minor effects on the quality of epidermal regeneration in cocultures. As to the role of fibroblast numbers, complete absence of dermal cells resulted in atrophic epithelia but the effect of cell numbers as low as 5 x 10(4)cells/cm(2) on epidermal tissue quality equalled that of the standard density (2 x 10(5)cells/cm(2)). Surprisingly, precultivation of fibroblasts in the DEs for 7 days (standard) showed no better effect on epidermal tissue reformation as compared to 2 days whereas a precultivation period of 14 days resulted in atrophic epidermal and dermal tissue development. These data demonstrate, (i) the strict dependence of epidermal tissue regeneration on the presence of fibroblasts, (ii) the mutual keratinocyte-fibroblast interactions for cell proliferation and organogenesis, and (iii) the importance of the proper microenvironment for epidermal tissue function and supposedly for establishment of a stem cell niche in vitro.     

The effect of perioperative adriamycin on skin-graft take and adherence in the rat

This two-part study was undertaken to evaluate the effect of perioperative Adriamycin on skin-graft take and adherence in an animal model. Thirty-four male Fisher rats were divided into control and experimental groups. The controls received intravenous saline, and the experimental animals received Adriamycin 6 mg/kg (LD 10) 24 hours preoperatively. Each animal was then grafted with an autogenous split-thickness skin graft on a contiguous dermal and fascial bed. Skin-graft take was judged and measured at 7 and 14 days postoperatively. The average skin-graft take for controls was 6.1 cm2 on both the dermal and fascial beds. This was significantly better than the average skin-graft take sustained by the experimental groups of 4.3 and 3.5 cm2 on the dermal and fascial beds, respectively (p less than 0.01). Another 30 animals were divided into three control and three experimental groups. They were treated with saline and Adriamycin as before, and they underwent a similar surgical procedure. Skin-graft adherence, which was measured at 12, 24, and 48 hours postoperatively, was significantly less in the experimental groups compared with the control groups at all times measured. These data suggest that a single supratherapeutic dose of Adriamycin given preoperatively decreases skin graft take in the experimental model studied and that this decrease may be the result of a concomitant decrease in graft adherence.     

Early inhaled nitric oxide treatment decreases apoptosis of endothelial cells in neonatal rat lungs after vascular endothelial growth factor inhibition

Vascular endothelial growth factor (VEGF) receptor blockade impairs lung growth and decreases nitric oxide (NO) production in neonatal rat lungs. Inhaled NO (iNO) treatment after VEGF inhibition preserves lung growth in infant rats by unknown mechanisms. We hypothesized that neonatal VEGF inhibition disrupts lung growth by causing apoptosis in endothelial cells, which is attenuated by early iNO treatment. Three-day-old rats received SU-5416, an inhibitor of VEGF receptor, or its vehicle and were raised in room air with or without iNO (10 ppm). SU-5416 reduced alveolar counts and lung vessel density by 28% (P < 0.005) and 21% (P < 0.05), respectively, as early as at 7 days of age. SU-5416 increased lung active caspase-3 protein by 60% at 5 days of age (P < 0.05), which subsided by 7 days of age, suggesting a transient increase in lung apoptosis after VEGF blockade. Apoptosis primarily colocalized to lung vascular endothelial cells, and SU-5416 increased endothelial cell apoptotic index by eightfold at 5 days of age (P <0.0001). iNO treatment after SU-5416 prevented the increases in lung active caspase-3 and in endothelial cell apoptotic index. There was no difference in alveolar type 2 cell number between control and SU-5416-treated rats. We conclude that neonatal VEGF receptor inhibition causes transient apoptosis in pulmonary endothelium, which is followed by persistently impaired lung growth. Early iNO treatment after VEGF inhibition reduces endothelial cell apoptosis in neonatal lungs. We speculate that enhancing endothelial cell survival after lung injury may preserve neonatal lung growth in bronchopulmonary dysplasia.     

Markers of cell death in salivary glands of males of the tick Rhipicephalus sanguineus (Latreille, 1806) (Acari, Ixodidae)

The salivary glands of Rhipicephalus sanguineus males at stages: unfed (control), at day seven post-attachment, and at days three and seven post-detachment from the host were examined using methods of enzymatic analysis and cell viability. At these stages of feeding, different staining patterns were observed in the cells of type IV, III, II and I acini, which were affected by degeneration in this sequence. Acid phosphatase reaction was inversely proportional to that of ATPase, while ATPase reaction was proportional to membrane integrity. Salivary gland cells of unfed males exhibited intact nucleus and plasma membrane, suggesting that the acid phosphatase detected may participate in the normal physiology of acini. In males at day seven post-attachment, intact membranes were observed in almost all types of acini, as well as stronger reaction for acid phosphatase, nuclear changes, and decrease in ATPase reaction, changes associated with the degenerative process. At days three and seven post-detachment degeneration progress, being observed loss of membrane integrity, nuclear changes, prominent decrease in ATPase reaction, and an increase in acid phosphatase reaction in the first case and a decreased of it at day seven post-detachment from the host. During cell death, alterations occurred in the following sequence: a) nuclear changes, b) loss of ATPase reaction, c) loss of integrity of the plasma membrane, and d) increase of acid phosphatase. The latter might be associated with the late degradation of cytoplasmic remnants, characterizing the process of cell death in glands of R. sanguineus males as atypical or non-classic apoptosis.