Healing of Osteochondral Defects Implanted with Biomimetic Scaffolds of Poly(ε-Caprolactone)/Hydroxyapatite and Glycidyl-Methacrylate-Modified Hyaluronic Acid in a Minipig.

Articular cartilage is a structure lack of vascular distribution. Once the cartilage is injured or diseased, it is unable to regenerate by itself. Surgical treatments do not effectively heal defects in articular cartilage. Tissue engineering is the most potential solution to this problem. In this study, methoxy poly(ethylene glycol)-block-poly(ε-caprolactone) (mPEG-PCL) and hydroxyapatite at a weight ratio of 2:1 were mixed via fused deposition modeling (FDM) layer by layer to form a solid scaffold. The scaffolds were further infiltrated with glycidyl methacrylate hyaluronic acid loading with 10 ng/mL of Transforming Growth Factor-ß1 and photo cross-linked on top of the scaffolds. An in vivo test was performed on the knees of Lanyu miniature pigs for a period of 12 months. The healing process of the osteochondral defects was followed by computer tomography (CT). The defect was fully covered with regenerated tissues in the control pig, while different tissues were grown in the defect of knee of the experimental pig. In the gross anatomy of the cross section, the scaffold remained in the subchondral location, while surface cartilage was regenerated. The cross section of the knees of both the control and experimental pigs were subjected to hematoxylin and eosin staining. The cartilage of the knee in the experimental pig was partially matured, e.g., few chondrocyte cells were enclosed in the lacunae. In the knee of the control pig, the defect was fully grown with fibrocartilage. In another in vivo experiment in a rabbit and a pig, the composite of the TGF-ß1-loaded hydrogel and scaffolds was found to regenerate hyaline cartilage. However, scaffolds that remain in the subchondral lesion potentially delay the healing process. Therefore, the structural design of the scaffold should be reconsidered to match the regeneration process of both cartilage and subchondral bone.

Transcriptome analysis of the effect of Vibrio alginolyticus infection on the innate immunity-related TLR5-mediated induction of cytokines in Epinephelus lanceolatus.

Epinephelus lanceolatus, considered to be an aquaculture fish species of high economic value in East Asia, is one of the largest groupers in the Epinephelus genus. Vibrio alginolyticus is a bacterial species that causes high morbidity in marine fish; infection can cause exophthalmia, ulcers, septicemia, and corneal opaqueness in fish. Epinephelus lanceolatus larvae infected with Vibrio alginolyticus were subjected to transcriptome analysis to study the immune regulation pathway. Grouper larvae were injected with 2.6 × 10(4) CFU/fish in 20 µl of V. alginolyticus and control larvae were injected with TSB; RNA samples were then collected at 4, 6, 8, 10, 12, 16, 24, and 48 h after infection. Extracted RNA was subjected to reverse transcription, and used to examine the immune gene response of E. lanceolatus by Real-time PCR. Samples taken at 6 h were subjected to next-generation sequencing, resulting in a total read value of 28,705,411 and total base number of 2,152,905,850. The unigene number was 100,848, and 5913 unigenes were filtered using FPKM>0.3, 2FC, p < 0.05. Gene Ontology (GO) analysis of the filtered genes revealed a total of 30 GO numbers in the cellular component, and 58 GO numbers for both biological processes and molecular functions. Of the GO group related to immune pathways, 27 unigenes related to biological processes involving the immune response, 31 related to the immune system, 9 related to the inflammatory response, and 43 related to the response to stress were identified. KEGG pathway analysis only detected 1 to 4 genes, and as such, we selected the GO analysis results for further analysis using GeneSpring. This demonstrated that V. alginolyticus probably stimulates TLR5 activity via the bacterial flagellum, through an MyD88-dependent pathway; the resulting production of IL-1ß and IL-8 through the NFκB pathway induces pro-inflammatory and/or chemotactic effects. Alternatively, serum amyloid A may stimulate neutrophils that induce the secretion of MMP9 from infected tissues, resulting in the cleavage and activation of IL-8. IL-8, in turn, would enhance neutrophil chemotaxis. Infection also induced expression of genes encoding C3, C6, C7, C8, and C9, which induce the complement system and form the membrane attack complex to lyse the bacteria membrane. The qPCR results indicated that TLR5 is significantly increased between 10 and 16 h, IL-1ß between 8 and 16 h, IL-8 between 8 and 12 h, and C6 between 4 and 16 h, as compared to levels in the control. One antimicrobial peptide, hepcidin, was also strongly expressed between 4 and 10 h in infected fish. The results indicate that V. alginolyticus infection probably induces an immune response via TLR5-mediated regulation of down-stream cytokine gene expression. A second possibility is that the complement system and hepcidin may be involved in the immune response. These results may be applied by examining the immune effects of feeding E. lanceolatus larvae on a recombinant protein mixture based on the up-regulated genes.

Cell fusion phenomena detected after in utero transplantation of Ds-red-harboring porcine amniotic fluid stem cells into EGFP transgenic mice.

OBJECTIVES: Amniotic fluid stem cells (AFSCs) are derived from the amniotic fluid of the developing fetus and can give rise to diverse differentiated cells of ectoderm, mesoderm, and endoderm lineages. Intrauterine transplantation is an approach used to cure inherited genetic fetal defects during the gestation period of pregnant dams. Certain disease such as osteogenesis imperfecta was successfully treated in affected fetal mice using this method. However, the donor cell destiny remains uncertain. METHODS: The purpose of this study was to investigate the biodistribution and cell fate of Ds-red-harboring porcine AFSCs (Ds-red pAFSCs) after intrauterine transplantation into enhanced green fluorescent protein-harboring fetuses of pregnant mice. Pregnant mice (12.5 days) underwent open laparotomy with intrauterine pAFSC transplantation (5 × 10(4) cells per pup) into fetal peritoneal cavity. RESULTS: Three weeks after birth, the mice were sacrificed. Several samples from different organs were obtained for histological examination and flow cytometric analysis. Ds-red pAFSCs migrated most frequently into the intestines. Furthermore, enhanced green fluorescent protein and red fluorescent protein signals were co-expressed in the intestine and liver cells via immunohistochemistry studies. CONCLUSION: In utero xenotransplantation of pAFSCs fused with recipient intestinal cells instead of differentiating or maintaining the undifferentiated status in the tissue.

Integration of multiple prognostic predictors in a porcine spinal cord injury model: A further step closer to reality.

Introduction: Spinal cord injury (SCI) is a devastating neurological disorder with an enormous impact on individual's life and society. A reliable and reproducible animal model of SCI is crucial to have a deeper understanding of SCI. We have developed a large-animal model of spinal cord compression injury (SCI) with integration of multiple prognostic factors that would have applications in humans. Methods: Fourteen human-like sized pigs underwent compression at T8 by implantation of an inflatable balloon catheter. In addition to basic neurophysiological recording of somatosensory and motor evoked potentials, we introduced spine-to-spine evoked spinal cord potentials (SP-EPs) by direct stimulation and measured them just above and below the affected segment. A novel intraspinal pressure monitoring technique was utilized to measure the actual pressure on the cord. The gait and spinal MRI findings were assessed in each animal postoperatively to quantify the severity of injury. Results: We found a strong negative correlation between the intensity of pressure applied to the spinal cord and the functional outcome (P < 0.0001). SP-EPs showed high sensitivity for real time monitoring of intraoperative cord damage. On MRI, the ratio of the high-intensity area to the cross-sectional of the cord was a good predictor of recovery (P < 0.0001). Conclusion: Our balloon compression SCI model is reliable, predictable, and easy to implement. By integrating SP-EPs, cord pressure, and findings on MRI, we can build a real-time warning and prediction system for early detection of impending or iatrogenic SCI and improve outcomes.

Titanium nanoparticle anchored functionalized MWCNTs for electrochemical detection of ractopamine in porcine samples with ultrahigh sensitivity.

We develop a disposable electrochemical sensor using a titanium nanoparticles (Ti NPs)-anchored functionalized multi-walled carbon nanotube (Ti@f-MWCNTs) composite as electrochemical sensing interface for the detection of ractopamine (RAC). The sensor demonstrated superior electrochemical sensing ability with a broad linear response range (0.01-185 µM) and ultralow detection limit (0.0038 µM). In addition, the stability, repeatability, reproducibility, and anti-interference ability of the Ti@f-MWCNTs sensor were satisfactory. The practicability of the sensor was effectively employed for the determination of RAC in porcine samples including pork, pig urine, and pig serum with substantial recoveries in the range of 92%-99% and a relative standard deviation of less than 5%.

New design to remove leukocytes from platelet-rich plasma (PRP) based on cell dimension rather than density.

Platelet-rich plasma (PRP) can stimulate the proliferation of stem cells and have a positive effect on tissue repair. Although many commercialized PRP preparation kits are already on the market, first-line clinical workers are still not satisfied with most of the PRP kits. The work of commercial PRP kits is based on the density of blood elements. However, the blood elements are very close in density which makes the separation challenging. Therefore, the mentioned commercialized kits are generally contaminated by leucocytes and erythrocyte. In this study, a home-designed PRP device was developed to use a separation membrane with adequate cut-off pore size of 5 µm, 3 µm and 2 µm for the groups of H5M, H3M, and H2M, respectively, to be placed in the middle of the centrifuge tube. The home-designed H2M showed a very promising results regardless of the final volume (1.82 ± 0.09 ml), platelet yield (8.39 ± 0.44%), Red Blood Cells (0%), White Blood Cells (0%), and Relative Concentration of Platelet Increment value (225.09%). Further, it showed a good result in cell viability and cytotoxicity and confirmed a good multilineage potentials. The concentration in PRP prepared by group H2M was relatively stable and far above average. All the fibrin fibers were linked together as bridging strands or strings and turned into an inter-connected porous structure for nutrients transportation and regenerative cell migration. We believe that the home-designed group H2M should have a great potential to develop into the final product to meet the requirements of first-line clinical workers.

Relative efficacy of casein or soya protein combined with palm or safflower-seed oil on hyperuricaemia in rats.

Diets that ameliorate the adverse effects of uric acid (UA) on renal damage deserve attention. The effects of casein or soya protein combined with palm or safflower-seed oil on various serum parameters and renal histology were investigated on hyperuricaemic rats. Male Wistar rats administered with oxonic acid and UA to induce hyperuricaemia were fed with casein or soya protein plus palm- or safflower-seed oil-supplemented diets. Normal rats and hyperuricaemic rats with or without allopurinol treatment (150 mg/l in drinking water) were fed with casein plus maize oil-supplemented diets. After 8 weeks, allopurinol treatment and soya protein plus safflower-seed oil-supplemented diet significantly decreased serum UA in hyperuricaemic rats (one-way ANOVA; P < 0.05). In addition, soya protein and casein attenuated hyperuricaemia-induced decreases in serum albumin and insulin, respectively (two-way ANOVA; P < 0.05). Safflower-seed oil significantly decreased serum TAG and UA, whereas palm oil significantly increased serum cholesterol, TAG, blood urea N and creatinine. However, soya protein significantly decreased renal NO and nitrotyrosine and palm oil significantly decreased renal nitrotyrosine, TNF-alpha and interferon-gamma and increased renal transforming growth factor-beta. Casein with safflower-seed oil significantly attenuated renal tubulointerstitial nephritis, crystals and fibrosis. Comparing casein v. soya protein combined with palm or safflower-seed oil, the results support that casein with safflower-seed oil may be effective in attenuating hyperuricaemia-associated renal damage, while soya protein with safflower-seed oil may be beneficial in lowering serum UA and TAG.

Does pharmacological dose of parenteral arginine have beneficial effect in rats with sub-acute peritonitis?

PURPOSE: Peritonitis is a life-threatening condition that may occur as a sequela of intra-abdominal infection. The management of peritonitis includes surgical intervention, antimicrobial therapy, and nutritional support. Arginine has been reported to have beneficial and adverse effects in subjects with inflammation, which might be related to the dose, time, and route of supplementation and the disease severity. So far, the optimal doses of parenteral arginine are not known. In this study, we investigated dose effects of parenterally supplemented arginine on anabolism and arginine-derived metabolites in sub-acute inflammation. METHODS AND MATERIALS: Male Wistar rats underwent modified cecal puncture procedure for induction of peritonitis were infused with total parenteral nutrition solutions for 7 days, which contained conventional, low, medium, and high doses of arginine, i.e., 1.61, 2.85, 4.08, and 6.54% of calories from arginine. Healthy, orally fed rats were included as references. RESULTS: On day 7, peritonitic rats had significantly decreased body weight, declined serum albumin, and increased serum nitric oxide (NO) and tumor-necrosis factor-alpha compared to references (ANOVA, P < 0.05). There were no dose effects of parenteral arginine on body weight, nitrogen retention, and serum blood urea nitrogen and creatinine in peritonitic rats. In contrast, plasma arginine, proline, and ornithine, and urinary urea nitrogen were significantly increased, whereas serum NO and plasma glutamine were significantly decreased in dose-dependent manners with parenteral arginine. Pharmacological dose of parenteral arginine may increase the synthesis of ornithine, urea, and proline instead of citrulline and NO in peritonitic rats. CONCLUSION: These results suggest that high dose of parenteral arginine may facilitate ureagenesis and proline conversion without causing augmentation of NO production in sub-acute inflammation. Therefore, pharmacological dose of parenteral arginine may not have benefits in anabolism but does not cause adverse effect in rats with sub-acute inflammation.

Evaluation of Chitosan-based Dressings in a Swine Model of Artery-Injury-Related Shock.

Uncontrolled haemorrhage shock is the highest treatment priority for military trauma surgeons. Injuries to the torso area remain the greatest treatment challenge, since external dressings and compression cannot be used here. Bleeding control strategies may thus offer more effective haemostatic management in these cases. Chitosan, a linear polysaccharide derived from chitin, has been considered as an ideal material for bleeding arrest. This study evaluated the potential of chitosan-based dressings relative to commercial gauze to minimise femoral artery haemorrhage in a swine model. Stable haemostasis was achieved in animals treated with chitosan fibre (CF) or chitosan sponge (CS), resulting in stabilisation of mean arterial pressure and a substantially higher survival rate (100% vs. 0% for gauze). Pigs receiving treatment with CF or CS dressings achieved haemostasis within 3.25 ± 1.26 or 2.67 ± 0.58 min, respectively, significantly more rapidly than with commercial gauze (>100 min). Moreover, the survival of animals treated with chitosan-based dressings was dramatically prolonged (>180 min) relative to controls (60.92 ± 0.69 min). In summary, chitosan-based dressings may be suitable first-line treatments for uncontrolled haemorrhage on the battlefield, and require further investigation into their use as alternatives to traditional dressings in prehospital emergency care.

Low levels of haptoglobin and putative amino acid sequence in Taiwanese Lanyu miniature pigs.

Porcine haptoglobin (Hp) is an acute phase protein. Its plasma level increases significantly during inflammation and infection. One of the main functions of Hp is to bind free hemoglobin (Hb) and inhibit its oxidative activity. In the present report, we studied the Hp phenotype of Taiwanese Lanyu miniature pigs (TLY minipigs; n=43) and found their Hp structure to be a homodimer (beta-alpha-alpha-beta) similar to human Hp 1-1. Interestingly, Western blot and high performance liquid chromatographic (HPLC) analysis showed that 25% of the TLY minipigs possessed low or no plasma Hp level (<0.05 mg/ml). The Hp cDNA of these TLY minipigs was then cloned, and the translated amino acid sequence was analyzed. No sequences were found to be deficient; they showed a 99.7% identity with domestic pigs (NP_999165). The mean overall Hp level of the TLY minipigs (0.21 +/- 0.25 mg/ml; n=43) determined by enzyme-linked immunosorbent assay (ELISA) was markedly lower than that of domestic pigs (0.78 +/- 0.45 mg/ml; p<0.001), while 25% of the TLY minipigs had an Hp level that was extremely low (<0.05 mg/ml). In addition, the initial recovery rate (first 40 min) in the circulation of infused fluorescein isothiocyanate (FITC)-Hb was significantly higher in the TLY minipigs with extremely low Hp levels than those with high levels. This data suggests that the low concentration of Hp-Hb complex is responsible for the higher recovery rate of Hb in the circulation. TLY minipigs have been used as an experimental model for cardiovascular diseases; whether they can be used as a model for inflammatory diseases, with Hp as a marker, remains a topic of interest. However, since the Hp level varies significantly among individual TLY minipigs, it is necessary to prescreen the Hp levels of the animals to minimize variation in the experimental baseline. The present study may provide a reference value for future use of the TLY minipig as an animal model for inflammation-associated diseases.

Can mesenchymal stem cells and their conditioned medium assist inflammatory chondrocytes recovery?

Osteoarthritis (OA), one of the most common joint disease, affects more than 80% of the population aged 70 or over. Mesenchymal stem cells (MSCs) show multi-potent differentiation and self-renewal capability, and, after exposure to an inflammatory environment, also exhibit immunosuppressive properties. In this study, we have used a model of lipopolysaccharide (LPS)-stimulated chondrocytes to evaluate MSC anti-inflammatory efficacy. The anti-inflammatory mechanism was tested in two cell-contained culture systems: (i) MSC-chondrocyte indirect contact system and (ii) MSC-chondrocyte direct contact system, and one cytokine-only culture system: MSC-conditioned medium (CM) system. Results showed that MSCs reduced chondrocyte inflammation through both paracrine secretion and cell-to-cell contact. The inflammation-associated, and free-radical-related genes were down-regulated significantly in the direct contact system on 24 h, however, the TNF-α. IL-6 were upregulated and aggrecan, COLII were downregulated on 72 h in direct contact system. Moreover, we found CM produced by MSC possess well therapeutic effect on inflammatory chondorcyte, and the 10-fold concentrated MSC-conditioned medium could down-regulated chondorcyte synthesis inflammation-associated, and free-radical-related genes, such as TNF-α, IL-1ß, IL-6 and iNOS even treated for 72 h. In conclusion, MSC-CM showed great potential for MSC-based therapy for OA.

Severe Burn Injury in a Swine Model for Clinical Dressing Assessment.

Wound healing is a dynamic repair process and is the most complex biological process in human life. In response to burn injury, alterations in biological pathways impair the inflammation response, resulting in delayed wound healing. Impaired wound healing frequently occurs in patients with diabetes leading to unfavorable outcomes such as amputation. Hence, dressings having beneficial effect in promoting burn wound repair are needed. However, studies on burn wound treatment are limited due to lack of proper animal models. Our previous study demonstrated wound-healing performance in rat and swine models using a minimally invasive surgical technique. This study aimed to demonstrate a swine model of severe burn injury that eliminates wound contraction and more closely approximates the human processes of re-epithelialization and new tissue formation. This protocol provides a detailed procedure for creating consistent burn wounds and examining the wound-healing performance under the treatment of an experimental dressing in a swine model. Six burn wounds were created symmetrically on the dorsum, which were covered with a clinical dressing composed of four layers: an inner contact layer of experimental materials, an inner intermediate layer of waterproof film, an outer intermediate layer of gauze, and an outer layer of adhesive plaster. Upon the completion of experiments, wound closure, wound area, and Vancouver Scar Scale score were examined. The samples of skin resected from each animal post-sacrifice were histologically prepared and stained using hematoxylin and eosin staining. Antibacterial activity of each dressing in the context of wound healing was also examined. The application of the clinical dressing to the wounds in swine model mimics the biological processes of human wound healing with respect to the processes of epithelialization, cellular proliferation, and angiogenesis. Therefore, this swine model provides an easy-to-learn, cost-effective, and robust method to assess the effect of clinical dressings in severe burn injury.

Application of amniotic fluid stem cells in repairing sciatic nerve injury in minipigs.

Many studies have demonstrated that combining nerve conduits with neural stem cells or growth factors can repair peripheral nerve injury in rodents. However, nerve damage does occur with longer gaps in human than in rodents, thus findings from rodent studies are difficult to translate to clinical practice. Minipigs have a longer gap that is more closely applicable to the challenge of human nerve grafting in extensive traumatic nerve damage. In this study, human amniotic fluid stem cells (AFSCs) and polylactate nerve conduits were used to repair sciatic nerve injury in minipigs. The AFSCs exhibited the properties of mesenchymal stem cells with a propensity toward neural stem cells. Measurements of compound muscle action potential implied that administration of conduits with AFSCs was beneficial in function recovery in the minipig model compared with conduits alone. The results of diffusion tensor magnetic resonance imaging (DTI) based fiber tractography assay in the minipig model suggest that combining AFSCs with conduits could expedite the repair of sciatic nerve injury. Further, MR-based DTI provides an effective and non-invasive method to visualize the sciatic nerve and to monitor the regeneration progress of injured nerve in a longitudinal study.

Zhi-fuzi, a cardiotonic Chinese herb, a new medical treatment choice for portal hypertension?

Zhi-Fuzi (Radix Aconiti lateralis preparata) is prescribed fairly frequently in Chinese medicine clinical practice for treating the complications of cirrhosis. However, scientific evidence regarding its efficacy and safety has not been available until now; in addition, its treatment efficacy has not yet been evaluated in well-designed clinical trials. Hence, we investigated the hemodynamic effects of Zhi-Fuzi in conscious rats with portal vein ligation (PVL) and the safety in normal rats. Our study included 3 parts: (i) early administration during which the hemodynamic effects of low and high doses of Zhi-Fuzi (0.4 and 0.8 g/kg twice daily) and propranolol (15 and 30 mg/kg twice daily) administered for 14 days after PVL on male Sprague-Dawley rats were evaluated; (ii) late administration during which the other group of PVL rats received 2.4 g/kg of Zhi-Fuzi twice daily from the 15th to 28th postoperative day; hemodynamic effects were measured when the Zhi-Fuzi treatment was finished; and (iii) safety evaluation during which 2 groups of normal rats were administered Zhi-Fuzi (0.4 and 0.8 g/kg twice daily) for 14 days; biochemical and histopathologic studies were completed after hemodynamic measurement. In early administration the portal pressures in rats receiving low and high doses of Zhi-Fuzi, low and high doses of propranolol, and distilled water were 13.81 +/- 0.11, 11.59 +/- 0.07, 17.09 +/- 0.06, 14.52 +/- 0.29, and 20.11 +/- 0.22 mm Hg, respectively. The high dose of Zhi-Fuzi exerted more portal hypotensive effects than propranolol and simultaneously ameliorated the systemic arterial hypotension in PVL rats. The late administration of Zhi-Fuzi also significantly reduced the elevated portal pressure (14.56 +/- 0.19 vs. 19.50 +/- 0.31 mm Hg in control, P < 0.05). There were no adverse effects seen in normal rats receiving Zhi-Fuzi. The results suggest that Zhi-Fuzi is a potential drug for the prophylaxis and treatment of portal hypertension.

Injecting partially digested cartilage fragments into a biphasic scaffold to generate osteochondral composites in a nude mice model.

This study proposed a novel scaffold with heterogeneous morphology that mimics the natural tissue. Its upper part contains a hollow cavity surrounded by a wall of poly(L-lactic-co-glycolic acid) (PLGA) porous membrane for injecting cartilage tissue and cells. An interconnecting porous structure located under the hollow cavity was made of composite materials that combined PLGA and beta-tricalcium phosphate (beta-TCP) to simulate the subchondral bone. Adult pig articular cartilage was cut and sieved into small fragments. The tissue fragments was partially digested by 0.1% collagenase for 0, 2, 4, and 6 h and injected into the hollow cavity of the biphasic scaffold. The biphasic scaffolds were then implanted into the subcutaneous pocket of nude mice for 4 weeks. No tissue bonding or new cartilaginous tissue formation was identified in the cartilage fragment without enzymatic treatment. The cartilage fragments digested with 2 h of collagenase digestion were partially integrated after implantation. The integrative properties of the cartilage fragment depended on the extent of enzymatic digestion. Releasing cells at the tissue surface enhanced confluence and bonding of the cartilage fragment matrix. Complete integration of the cartilage fragments and cartilage remodeling were achieved by digestion of the tissue fragments with 4 h of enzymatic treatment. The neocartilage grew from the upper hollow cavity into the lower PLGA/beta-TCP porous structure, forming an interface similar to that formed between cartilage and subchondral bone. This study combined the osteochondral scaffold and limited cartilage tissues to generate cartilage tissue in vivo intending for repairing full-thickness articular cartilage defects.

On measuring the instantaneous blood pressure in an artery via the tissue control method.

This paper presents a non-invasive approach to measuring the instantaneous arterial blood pressure based upon the tissue control method. According to animal experiments, there exists a pressed location where the mean blood pressure in the artery is equivalent to the mean pressure measured on the skin. At this location, the stiffness measured on the skin is equivalent to the stiffness of the tissue only. By means of the tissue control method, the pulsations of the artery are excluded from tissues. The displacement measured on the skin and the tracking pressure obtained from the controller identify the dynamical impedance of the blood vessel, and then estimate the instantaneous intra-arterial blood pressure. The experimental results show that errors of mean blood pressure, systolic blood pressure and diastolic blood pressure are less than 4%, and indicate that the tissue control method is a feasible way to detect the instantaneous arterial blood pressure.

Propolis extracts exhibit an immunoregulatory activity in an OVA-sensitized airway inflammatory animal model.

Propolis, which has been used widely in folk medicine, has been shown to exhibit various biological activities but its immunoregulatory and anti-inflammatory activities in intact animals have not been well studied. We investigated these activities of propolis using an ovalbumin-induced asthma animal model. Mice were immunized and sensitized by exposure to ovalbumin (OVA) antigen and administered with low- (65 mg/kg body weight) and high-dose (325 mg/kg body weight) propolis water extracts by tube feeding. The serum OVA-specific IgE titer and cytokine profiles in cultured splenocytes and bronchoalveolar lavage fluids (BALF) were analyzed. The number of eosinophils in BALF was counted. Here we demonstrate that propolis extracts can suppress the serum levels of OVA-specific IgE and IgG(1), and airway hyperresponsiveness (AHR) in OVA-sensitized mice. There are no significant differences in the concentration of eotaxin or the number of eosinophils in BALF among the four groups. However, the higher dose of propolis extracts decreases the level of IL-5 in BALF. The splenocytes from mice administered with propolis extracts (low- and high-dose groups) exhibit a strong inhibition of IL-10 secretion and up-regulation of IFN-gamma secretion in splenocytes stimulated with concanavalin A (ConA). In addition, cytokine (IFN-gamma, IL-6, and IL-10) secretion in OVA-stimulated splenocytes from the propolis groups was significantly lower than that in the control group. These results suggest that propolis extracts may be a potential novel therapeutic agent for asthma.

Ethanol treatment induces significant cell death in porcine corneal fibroblasts.

PURPOSE: To explore the effect of ethanol treatment on corneal stromal cells. METHODS: Primary porcine corneal fibroblasts from passages 3 to 5 were treated with ethanol at concentrations of 10%, 15%, 20%, and 50% for 30 seconds. A control group was treated with phosphate-buffered saline (PBS) for 30 seconds. Morphologic changes were documented with phase-contrast microscopy, and the growth curves were examined with a PicoGreen assay. Cellular viability was examined with an ethidium homodimer and calcein-AM stain, whereas cellular apoptosis and/or necrosis were analyzed by a YO-PRO-1 dye/propidium iodide apoptosis assay coupled with flow cytometry and further confirmed with a genomic DNA pattern assay. Cellular toxicity was examined with a lactate dehydrogenase (LDH) assay. RESULTS: Significant cell rounding and detachment from the culture dish were noticed after 20% ethanol treatment of 30 seconds, despite that the cell morphology remained unchanged in the PBS and 10% and 15% ethanol groups. Twenty percent ethanol induced significant cellular toxicity, causing cell death as shown by ethidium homodimer and calcein-AM stain, YO-PRO-1 dye/propidium iodide apoptosis assay, and LDH assay, although 10% and 15% ethanol caused minimal changes to corneal fibroblasts. Cellular death was most significant 6 hours after the 20% ethanol treatment. The genomic DNA pattern revealed intact DNA in the control, 10% ethanol, and 15% ethanol groups at all times, whereas DNA smearing was noticed at 48 hours after the 20% ethanol treatment. However, none of the DNA examined revealed significant DNA laddering patterns of apoptosis. Fifty percent ethanol treatment of 30 seconds resulted in cell fixation and cell death. CONCLUSION: Treatment with 20% ethanol for 30 seconds induced significant porcine corneal fibroblast cell death, whereas 10% and 15% ethanol treatment of 30 seconds caused minimal changes. We propose that, when applied for 30 seconds, 20% ethanol is the threshold level that causes cell death in cultured porcine corneal fibroblasts.

The effects of epithelial viability on stromal keratocyte apoptosis in porcine corneas stored in Optisol-GS.

PURPOSE: To investigate the effect of corneal epithelium on the viability of corneal stromal keratocytes in Optisol-GS. METHODS: After sterilization, corneoscleral buttons were excised and stored in Optisol-GS for various time periods. Group 1 corneas (n = 40) underwent mechanical corneal epithelial debridement before storage while group 2 corneas (n = 40) were stored with intact epithelium. Changes in corneal thickness, keratocyte density, and keratocyte apoptosis were investigated immediately, at 4 hours, and on days 1, 2, 3, 5, 7, and 14 in the preservation medium. The differences between group 1 and 2 corneas were analyzed. RESULTS: Corneal thickness increased significantly in the second week of preservation in both groups, though more substantially in group 1. Significant corneal epithelial apoptosis was noticed in the first week in group 2 corneas. Corneal stromal keratocyte density decreased with prolonged preservation time. DNA laddering was detected by ligation-mediated polymerase chain reaction throughout the experiment periods in both groups, but the increase of keratocyte apoptosis was more significant after 5 days of preservation, especially in group 1. CONCLUSIONS: Stromal keratocytes underwent apoptosis in Optisol-GS. The absence of corneal epithelium during preservation further increased the stromal keratocyte apoptosis.

Original Research: Porcine model for observing changes due to ischemia/reperfusion injury secondary to intra-abdominal endovascular balloon occlusion.

Compared with conventional aortic cross-clamping, endovascular balloon occlusion (EBO) is a valuable strategy in unstable ruptured abdominal aorta aneurysm patients; however, it is unclear how long the balloon may remain safely inflated. Using a porcine model, we evaluated the influence of different EBO time periods on intra-abdominal pressure (IAP) and the association between various pathophysiologic indicators and reperfusion time. Twelve healthy three-month-old domestic piglets were subjected to ischemia/reperfusion injury using EBO within the abdominal aorta. Animals were grouped as A, B, and C based on 30, 60, or 120 min of ischemic time, respectively. Changes in IAP, hemodynamic data, respiratory and renal function, and histology after reperfusion were compared with baseline measurements. All pigs gradually developed intra-abdominal hypertension after ischemia/reperfusion injury. IAP increased significantly after 4 h of reperfusion in all three groups (all P < 0.001) with maximal IAP reaching > 22 mmHg in 10 pigs. However, no significant intergroup differences were found. Cardiac output remained stable, but mixed venous oxygen saturation decreased significantly at 4 h after reperfusion (P < 0.05). The pH decreased significantly at 10 min in all three groups (all P < 0.001). Histological changes in the small intestine, lung, and kidney occurred secondary to aortic ischemia; however, no significant differences were noted between groups (P > 0.05). EBO within the abdominal aorta induced ischemia/reperfusion injury which led to intra-abdominal hypertension, pathological changes within multiple organs, and decreased mixed venous oxygen saturation after only 30 min of abdominal aortic ischemia.