Two-dimensional electrophoresis and histomorphometry were used to determine if equivalent protein changes occurred within native rabbit corneas and engineered corneal tissue models following in vitro exposure to single pulse, 1540 nm laser light operating at a pulse width of 0.8 milliseconds. Frozen sections of exposed tissues were processed to detect laser-induced protein changes. Isoelectric points, molecular weights and relative densities were used to characterize corneal proteins of interest that were then identified using MALDI-MS peptide fragment analysis. Increasing radiant exposures of corneal tissues were associated with progressively more severe necrosis of the epithelium and stroma in both the native and engineered tissues.

The purpose of this study was to evaluate the laser-tissue interactions of engineered human skin and in-vivo pig skin following exposure to a single 3.8 micron laser light pulse. The goal of the study was to determine if these tissues shared common histologic features following laser exposure that might prove useful in developing in-vitro and in-vivo experimental models to predict the bioeffects of human laser exposure. The minimum exposure required to produce gross morphologic changes following a four microsecond, pulsed skin exposure for both models was determined. Histology was used to compare the cellular responses of the experimental models following laser exposure. Eighteen engineered skin equivalents (in-vitro model), were exposed to 3.8 micron laser light and the tissue responses compared to equivalent exposures made on five Yorkshire pigs (in-vivo model). Representative biopsies of pig skin were taken for histologic evaluation from various body locations immediately, one hour, and 24 hours following exposure. The pattern of epithelial changes seen following in-vitro laser exposure of the engineered human skin and in-vivo exposure of pig skin indicated a common histologic response for this particular combination of laser parameters.

Recent advances in the field of tissue engineering have led to the development of complex three-dimensional tissue constructs. It has become clear, however, that the traditional tools used for studying standard cell cultures are not always adequate for diagnostically studying thick, highly-scattering cultured tissues. Furthermore, many techniques used for studying three-dimensional constructs are invasive or require exogenous fluorophores, which damage the tissue and prevent time-course studies of tissue development. An integrated optical coherence tomography (OCT) and multi-photon microscope (MPM) has been constructed for visualizing 3-D engineered tissues. OCT was used for imaging structure and cell organization, while MPM was used for assessing functional properties of cells. We demonstrate technical developments involved in the construction of this instrument and its use in the non-destructive investigation of cell movement and tissue organization in engineered tissues. Cells labeled with GFP and exogenous fluorescent probes have also been imaged with OCT and confocal microscopy. Studies indicate that an integrated microscope has the potential to be an enabling diagnostic tool for future studies in the growth and organization of engineering tissues and in cell-cell and cell-matrix interactions.

Objective Determine the effect of a 3-dimensional alginate matrix on the growth and differentiation of cells isolated from porcine retinal pigment epithelium (RPE).Procedures Porcine RPE cells were harvested from enucleated eyecups, isolated by differential gravity sedimentation and cultured in either alginate alone (Group 1) or on plastic tissue culture plates followed by alginate (Group 2). Group 1 cells were cultured in alginate to evaluate the efficacy of the matrix as a culture medium. Group 2 cells were initially cultured on plastic to induce dedifferentiation. The cells were then harvested, suspended in alginate beads, and incubated for a second culture period to determine if the induced dedifferentiation was reversible.Results The number of Group 1 cells was significantly greater (Pless than or equal to0.01) at the end of the culture period. The amount of pigment and cell morphology of Group 1 cells at the end of the culture period was similar to that seen at initial cell isolation. The initial culture of Group 2 cells on plastic showed characteristic features of dedifferentiation marked by the loss of pigment and alterations in microscopic appearance. Secondary culture of dedifferentiated Group 2 cells in alginate beads resulted in a return to pigmentation and characteristic morphology for a majority of the cultured cells.Conclusions Porcine RPE cells can be propagated in alginate culture with a significant increase in cell numbers while maintaining normal morphology. Under the conditions described in the present study, the dedifferentiation of porcine RPE induced by standard in vitro culture methods is reversible.

In vitro exposures of explant rabbit corneas to single pulse 1540 nm infrared, laser light operating at a pulse width of 0.8.milliseconds resulted in coagulative necrosis of both the corneal epithelium and stroma. Histomorphometric data correlated with increasing tissue radiant exposures. Histologic alterations in the corneal stroma were typical of matrix remodeling within the beam path and reactive to antibodies against matrix metalloproteinase-2. A two-dimensional electrophoretic analysis, using a mini-gel format, was developed to determine if specific corneal protein changes within tissue sections could be detected. Frozen sections taken through the center of the laser lesion were evaluated for proteomic data using tissue isoelectric focusing in the first dimension and polyacrylamide gel electrophoresis in the second dimension. Histomorphometric data describing the extent of the laser lesions were compared to the isoelectric points, molecular weights and relative densities of individual corneal proteins. Increasing radiant exposures of corneal tissues were associated with characteristic histomorphometric and proteomic changes.

Significant damage to rabbit corneal tissue was produced by a single pulse, in vitro exposure of 1540 m infrared laser light operating in either millisecond or nanosecond pulse widths. Millisecond pulse widths of infrared laser light produced a marked coagulative necrosis of both the corneal epithelium and stroma. We also noted histologic alterations in the stromal matrix within the beam path that we interpreted as matrix remodeling. To test this interpretation, we used an indirect immunohistochemical procedure to detect Matrix Metalloproteinase-2 (MMP-2) activity. Immunohistochemistry revealed that the MMP-2 reaction was mostly limited to the margins of the beam path. In addition, the MMP-2 reaction was less intense than expected given the significant tissue changes observed in the histologic sections. Exposure of rabbit corneal tissue to the nanosecond pulse widths produced a less severe coagulative necrosis of the tissue when compared to the millisecond exposures. However, a markedly stronger immunohistochemical pattern than would have been predicted from the histologic sections was observed, with approximately half of the beam path filled with MMP-2 reaction product. These data suggest an association between infrared laser pulse width and the degree of extracellular matrix remodeling in rabbit corneal tissue.

Proliferation of chondrocytes from nucleus pulposus (NP) and anulus fibrosus (AF) was confirmed in three-dimensional culture using alginate microspheres. Cells isolated from NP and AF were incorporated in microspheres and cultured for 14 days. Round mononuclear cells of 20-25 microm in diameter proliferated and formed aggregates. At day 14, alcian blue positive matrix surrounded the proliferating cells. The cells had cytoplasmic vacuoles stained positively by toluidine blue. On electron microscopy, the cells contained proteoglycan vacuoles and lipid droplets in the cytoplasm and synthesized collagen fibrils and electron dense granules surrounding the cell. These features of the cells were characteristic for chondrocytes. This culture system should be useful to further investigate metabolic activities of intervertebral disk chondrocytes.

Dutch Belted rabbit corneas and corneal equivalent (CE) tissue were exposed to 0.8 millisecond pulses of 1540 nm laser light. We report the single pulse ED50 for Dutch belted rabbits and for in-vitro corneal equivalent tissues. A histological comparison between the two tissues is presented. Remarkable similarities between the two models in both location and extent of damage are noted. We postulate which cellular energy absorption mechanisms are significant at 1540 nm and how this relates to the histopathology presented.

We investigated the effects of phytoestrogen on global myocardial ischemia-reperfusion injury in five groups of female rats. A high-phytoestrogen group (HPE) was ovariectomized (Ovx) and fed a diet containing soybean protein and a high-isoflavone soy extract. Another Ovx group of rats was fed the same diet as the HPE group but treated with the estrogen receptor blocker ICI-182,780 (HPE + ICI). A third group of Ovx rats was fed a diet containing soybean protein alone (low-phytoestrogen content; LPE). A fourth Ovx group was fed a diet free of phytoestrogen (Ovx). The fifth group of rats was sham ovariectomized (sham). Hearts from all rats were subjected to 30 min of global, hypothermic (4 degrees C), cardioplegic ischemia and 120 min of normothermic (37 degrees C) reperfusion with oxygenated Krebs-Henseleit buffer. Compared with either the sham or the HPE group, the Ovx and HPE + ICI groups had significantly decreased first derivative of left ventricular pressure (dP/dt), coronary flow rate (CFR), nitrite production and mitochondrial respiratory function and significantly increased Ca2+ accumulation and myocardial histological and ultrastructural injury. The CFR of the LPE group was significantly different from that of either Ovx or HPE + ICI group but the dP/dt, nitrite production, Ca2+ accumulation, and mitochondrial function were not. Our results indicate that diets containing phytoestrogen extract play a cardioprotective role in global myocardial ischemia-reperfusion in female rats.

Single pulse, 1540 nm laser light with a pulse width of 1 microsecond altered the morphologic appearance of explant rabbit and pig corneas following ex vivo exposure. Using digital images of the post-exposure corneas projected onto a measuring grid, we could accurately locate the relative position of the circular laser lesion in the embedded tissue. This allowed us to section through the lesion with micrometer precision and accurately resolve the inside edge, middle and outside edge of the laser lesion. All tissue sections used for morphometric analysis were taken through the middle of the lesion. Several features of the response to laser exposure may reflect species-specific tissue differences. The rabbit corneal epithelium showed a homogeneous coagulative necrosis with a distinct demarcation between necrotic and normal epithelium. The pig epithelium also showed a distinct demarcation between necrotic and normal epithelium, however, there were several remarkable differences in the tissue response between the two species including coagulative necrosis pattern and nuclear morphology. These changes suggested a different and less severe response of the pig epithelium to the laser light when compared to the rabbit epithelium.

Mechanisms of tissue damage are investigated for skin and cornea exposures from 1540 nm ('eye safe') laser single pulses of 0.8 milli-seconds. New skin model data point out the advantages of using the Yucatan mini-pig versus the Yorkshire pig for in-vivo skin laser exposures. Major advantages found include similarities in thickness and melanin content when compared with human skin. Histology from Yucatan mini-pig skin exposures and the calculation of an initial ED50 threshold indicate that the main photon tissue interaction may not be solely due to water absorption. In-vitro corneal equivalents compared well with in-vivo rabbit cornea exposure under similar laser conditions. In-vivo and in-vitro histology show that initial energy deposition leading to damage occurs intrastromally, while epithelial cells show no direct injury due to laser light absorption.

We investigate the use of in-vitro corneal equivalents as a replacement model for in-vivo rabbit corneas used in laser damage threshold studies. In-vitro corneal equivalents (CE) were exposed to 0.8 millisecond (ms), 1540 nanometer (nm) single laser light pulses ranging from 124 J/cm(2) to 58 J/cm(2). After exposure, CE's were evaluated opthalmoscopcopically, imaged using confocal microscopy and examined histologically to investigate the mechanisms of laser induced damage. Preliminary results indicate that the 50% damage threshold for CE's, ED50, is approximately 70 J/cm(2) with a 0.5 mm diameter spot size. Additional data points are required to determine a statistically significant ED50. Preliminary exposures of in-vivo rabbit corneas using the same laser parameters as the CE's are also reported. Comparisons between the in-vivo and in-vitro models are presented. Histopathological images from both models show remarkable similarities in the location and extent of damage throughout the full thickness of each tissue. Keywords: laser, corneal equivalent, probit analysis, confocal microscopy, rabbits.

Several environmentally and socially important chemicals such as d-limonene and unleaded gasoline have been demonstrated to induce alphas-globulin (a2u) nephropathy in male rats. Substantial progress has been made in characterizing the biological effects of these chemicals on the kidney and in further defining prerequisite events in the pathogenesis of this syndrome. The ot2u increase in the kidney is hypothesized to be the proximal event in the toxicologic and tumorigenic sequelae associated with administration of these xenobiolics over the male rat's lifetime rather than a direct effect of the administered chemical. The administered chemical appears to simply mediate the increase in ot2u concentration in the kidney. To further investigate the properties of a2u, this protein was tested in the pH 6.7 Syrian hamster embryo (SHE) cell transformation assay. The a2u caused morphological transformation in these cells, whereas another protein, bovine serum albumin, did not induce transformation at equimolar concentrations, suggesting a protein-specific phenomenon. Neither rf-limonene nor trimethylpentane (a causarcomponent in unleaded gasoline) induced SHE cell transformation. These results support the hypothesis that a2u increase in proximal convoluted tubules may directly cause renal tumorigenesis in male rats. The SHE cell transformation assay may be a useful tool for mechanistic studies of this syndrome.

OBJECTIVES:To study the effect of flunixin meglumine on short circuit current (Isc) in equine right ventral colon in vitro.SAMPLES:Intestinal mucosa from healthy horses and ponies.PROCEDURE:Isc was measured in mucosa from the right ventral colon mounted in Ussing chambers. In experiment 1, collection and incubation solutions were: control (no additions); flunixin meglumine, 4 micrograms/ml; indomethacin, 10(-6) M; and flunixin meglumine (4 micrograms/ml) with 10(-6) M prostaglandin E2. In experiment 2, incubation conditions were: control [plain Krebs-Ringer bicarbonate [KRB] solution]; flunixin meglumine, 4 micrograms/ml in KRB; chloride-free buffer solution; flunixin meglumine (4 micrograms/ml) in a chloride-free buffer solution; and plain KRB with 10(-6) M prostaglandin E2. In experiment 3, tissue from 3 groups (n = 6 each) of animals: controls, physiologic saline solution given IV at 10 minutes before euthanasia; flunixin meglumine (1.1 mg/kg of body weight, IV) given at 10 minutes before euthanasia; and treatment similar to controls, except that tissues were incubated with 8 micrograms of flunixin meglumine/ml of bathing medium.RESULTS:Flunixin meglumine and indomethacin reduced Isc to approximately a third of control current (P < 0.05), but coincubation with flunixin meglumine and 10(-5) M prostaglandin E2 restored Isc close to the control value. Incubation with 10(-6) M prostaglandin E2 alone did not change Isc. When chloride was substituted with isethionate, flunixin meglumine had no effect on Isc. Flunixin meglumine given before euthanasia or included at a concentration of 8 micrograms/ml in all tissue preparation and incubation solutions reduced Isc (P < 0.05).CONCLUSIONS:Flunixin meglumine given IV or added to bathing solutions decreased Isc in equine right ventral colon by a mechanism that appeared to involve prostaglandin-mediated chloride secretion.CLINICAL RELEVANCE:Our results suggest that flunixin meglumine given IV to horses at recommended doses could alter putative effects of colonic prostaglandins.

The diffusion of phytochemicals in health promoting products is growing, but studies related to their effects on healthy subjects are still lacking despite the large consumption of natural products as nutraceuticals or food supplements. In many cases, research supports the in vitro antioxidant activity of phytochemicals, but the health claims attributed to the final marketed nutraceutical products have dubious scientific foundation. Also, studies focussed on the definition of their biological targets and mechanisms of action can be useful to assess their efficacy and safety.In this study, the effect of oral administration of 80 mg/kg of Curcuma longa Linn. extract to 12 healthy rats over 25 days was evaluated by monitoring the changes of urinary composition. 24-h urine was collected during the animal experiment and the composition was analyzed by 1H NMR and HPLC–MS. The two datasets were studied individually through a metabolomic approach and the multivariate analysis revealed significant differences between the control and the treated group. Curcumin levels were also measured in 24-h urine samples by HPLC–MS. Both the 1H NMR and the HPLC–MS dataset showed that the administration of 80 mg/kg of Curcuma longa extract to healthy animals induces changes in urinary composition. Decreased allantoin urinary levels can be considered a partial demonstration of the in vivo effect of curcumin on oxidative stress in a healthy animal model.

Tissue isoelectric focusing (TIF) was used to establish a reference bovine corneal protein profile and to compare the human, rabbit, and bovine corneal response to in vitro chemical exposure. TIF provided a direct, semiquantitative comparison of tissue-irritant interactions without the potential limitations of standard homogenization and extraction procedures. The in vitro exposure of corneal sections to acetic acid (2%) and ammonium hydroxide (1%) solutions resulted in selective modifications of tissue protein profiles, which were evaluated by scanning densitometry. Comparison of the human and rabbit corneal protein changes associated with in vitro chemical exposure revealed a similar acid/base effect on a common rabbit and human corneal protein band (pI = 8.1). The pI = 8.1 protein band did not occur in the bovine protein profile; however, a similar acid/base effect occurred with a protein band having a pI = 7.2.

Enzyme substrate-based analysis of canine aqueous humor was used to determine plasminogen activator inhibitor (PAI) activity. Commercially available, antibody-based methods for detection of PAIs are not cross-reactive with canine fibrinolytic mediators. Substrate-based enzymatic assays are not dependent upon the cross-reactivity of a primary antibody, but rather on specific enzyme-substrate interactions. The mean value (+/- 1 SD) for PAI concentration in aqueous humor of dogs was 4.2 +/- 1.1 IU/ml (n = 20). The median value for PAI concentration was 4 IU/ml. The distribution of PAI values was positively skewed with a range of values from 2.7 to 6.9 IU/ml. There was no significant difference (p > 0.05) between the mean PAI concentrations determined for male and female dogs.

Topically administered tissue plasminogen activator (tPA) was evaluated for its penetration into aqueous humor of clinically normal dogs. Two concentrations of tPA (5 mg/ml and 10 mg/ml) were evaluated in a single-dose study, and a concentration of 5 mg of tPA/ml was used for a multiple-dose study. The contralateral eye served as a nontreated control. Enzyme substrate analysis of aqueous humor was used to determine tPA activity. The activity of tPA in aqueous humor was significantly (P < or = 0.05) greater in treated eyes of all dogs, compared with that in control eyes. Significant differences in activity of tPA were not detected at different doses in treated eyes.