Influence of strain and age differences on the yields of porcine islet isolation: extremely high islet yields from SPF CMS miniature pigs

BACKGROUND: Porcine pancreas is a potential source of material for islet xenotransplantation. However, the difficulty in isolating islets, because of their fragility and the variability of isolation outcome in donor age and breed, represents a major obstacle to porcine islet xenotransplantation. In this study, we compared the islet isolation yield of specific pathogen-free (SPF) Chicago Medical School (CMS) miniature pigs with that of another miniature pig breed and market pigs from a local slaughterhouse. METHODS: Nine adult CMS miniature (ACM) pigs (>12 months), six young CMS miniature (YCM) pigs (6-7 months), four adult Prestige World Genetics (PWG) miniature (APM) pigs (>12 months), and 13 adult market (AM) pigs from a local slaughterhouse were used for islet isolation. RESULTS: The islet yield per gram of pancreas from ACM pigs (9589 +/- 2823 IEQ/g) was significantly higher than that from APM pigs (1752 +/- 874 IEQ/g, P < 0.05), AM pigs (1931 +/- 947 IEQ/g, P < 0.05), or YCM pigs (3460 +/- 1985 IEQ/g, P < 0.05). Isolated islets from ACM pigs were significantly larger than those from AM pigs or YCM pigs. The in vitro and in vivo function of isolated islets showed no difference among experimental groups. The pancreases of ACM pigs contained higher mean islet volume density percentages and larger size of islets than those of AM or APM pigs. CONCLUSIONS: We isolated extremely high yields of well-functioning islets from ACM pigs bred under SPF conditions. SPF CMS miniature pigs should be one of the best porcine islet donors for clinical porcine islet xenotransplantation.     

Biocompatibility of Ricinus communis polymer with addition of calcium carbonate compared to titanium. Experimental study in guinea pigs

Objective

The aim of the present investigation was to determine whether the difference in inflammatory tissue reaction between the Riccinus communis (castor) polymer with calcium carbonate and the titanium implant is statistically significant.

Methods

Thirty-two Cavia porcellus were allocated into four groups of eight animals each. We implanted the two types of materials in the retroperitoneal space of all the animals. They were euthanized at 7, 20, 30 and 40 days after surgery, and an histological study of the samples was conducted.

Results

All implants showed characteristics of chronic inflammation regardless of the material and timepoint of evaluation. There was no statistically significant difference between Pm+CaCO₃ and Ti with regard to the presence of granulation tissue, tissue congestion, histiocytes, lymphocytes, neutrophils, giant cells, and fibrosis (P> 0.05).

Conclusion

The castor oil polymer plus calcium carbonate implant was not statistically different from the titanium implant regarding inflammatory tissue reaction.     

Survival of Microencapsulated Adult Pig Islets in Mice In Spite of an Antibody Response

The aim of this study was to assess the capacity of simple alginate capsules to protect adult pig islets in a model of xenotransplantation. Adult pig islets were microencapsulated in alginate, with either single alginate coats (SAC) or double alginate coats (DAC), and transplanted into the streptozotocin-induced diabetic B6AF1 mice. Normalization of glucose levels was associated with an improvement of the glucose clearance during intravenous glucose tolerance tests. After explantation, all mice became hyperglycemic, demonstrating the efficacy of the encapsulated pig islets. Explanted capsules were mainly free of fibrotic reaction and encapsulated islets were still functional, responding to glucose stimulation with a 10-fold increase in insulin secretion. However, a significant decrease in the insulin content and insulin responses to glucose was observed for encapsulated islets explanted from hyperglycemic mice. An immune response of both IgG and IgM subtypes was detectable after transplantation. Interestingly, there were more newly formed antibodies in the serum of mice transplanted with SAC capsules than in the serum of mice transplanted with DAC capsules. In conclusion, alginate capsules can prolong the survival of adult pig islets transplanted into diabetic mice for up to 190 days, even in the presence of an antibody response.     

Effects of xenon anaesthesia on intestinal oxygenation in acutely instrumented pigs

Background. Xenon is a narcotic gas that might be able to replacevolatile anaesthetics or nitrous oxide due to its favourablepharmacological properties, such as providing haemodynamic stability.Intestinal oxygenation is affected by most volatile anaestheticsas a result of cardiodepressive effects. Reducing oxygenationof the gut might be a factor leading to perioperative organdysfunction. This animal study was designed to assess the effectsof xenon on intestinal oxygenation. Methods. After ethical approval, 24 anaesthetized, acutely instrumentedpigs were randomly assigned to three groups: nine animals receivedxenon anaesthesia with inspiratory concentrations of 0, 20,50 and 65% in addition to their basic i.v. anaesthesia, nineanimals served as a study control group, and five animals wereused to assess model stability. Measurement of systemic andregional haemodynamic and oxygenation parameters was made 30min after changing the xenon concentration. Results. Xenon elicited dose-dependent systemic haemodynamicchanges: heart rate and cardiac output decreased by 30%, whilemean arterial pressure was stable. Superior mesenteric arteryblood flow was lower in the xenon group. Vascular resistanceof the superior mesenteric artery increased. The small intestinaloxygen supply decreased with increasing xenon concentration;the mucosal tissue oxygen partial pressure decreased but didnot reach hypoxic (<5 mm Hg) values. Serosal tissue oxygenpartial pressure was maintained. Conclusions. Xenon, in addition to basic i.v. anaesthesia, eliciteda decrease in cardiac output and maintained mean arterial pressure.Intestinal oxygenation was maintained, although regional macrohaemodynamicperfusion decreased. Xenon does not impair intestinal oxygenationunder physiological conditions.      

Ion-channel currents of smooth muscle cells isolated from the prostate of guinea-pig

OBJECTIVE: To characterize the voltage-activated ion-channel currents in guinea-pig prostate smooth muscle cells (GPSMCs). MATERIALS AND METHODS: GPSMCs were isolated using collagenase, and used in a whole-cell patch clamp study. RESULTS: When GPSMCs were dialysed with a CsCl solution all the outward K+ currents were blocked and the step-like depolarization (holding voltage -70 mV) of the cell membrane evoked inward currents that were completely blocked by nifedipine (1 micromol/L). With KCl solution, step depolarizations showed outward K+ currents composed of fast, transient outward current (Ito) and outward currents that did not inactivate. Ito was resistant to a high concentration of tetraethylammonium (TEA, 5 mmol/L) but was blocked by 4-aminopyridine (5 mmol/L). The half-activation and half-inactivation voltages of Ito were 6 mV and -58 mV, respectively. With low Ca2+ buffer (0.1 mmol/L EGTA) in the solution, there were spontaneous transient outward currents (STOCs) at depolarized membrane voltages (0 mV). STOCs were blocked by TEA (1 mmol/L) or iberiotoxin (10 nmol/L) but were insensitive to apamin (100 nmol/L). CONCLUSION: This voltage-clamp study showed that GPSMCs have l-type Ca2+ channels and more than two types of K+ channels. The voltage- and time-dependent changes of these ion channels and their interactions might be important in forming action potentials and regulating contractility.     

Habib VesOpen消融导管治疗小型猪模拟门静脉癌栓的安全性研究

目的：探索射频消融（RFA）导管治疗门静脉癌栓（PVTT）的安全性。方法将10头实验小型猪分为3组：A组（n=6）直接行正常门静脉RFA治疗;B组（n=2）先行门静脉球囊封堵,然后行门静脉新鲜血栓RFA治疗;C组（n=2）先建立PVTT模型,待门静脉血栓机化后行门静脉RFA治疗。术后1、3、4周作MRI检查,4周后处死动物作病理学检查。结果 A组动物在功率5 W条件下接受RFA治疗0．6～3．6 min,MRI检查及1个月后病理学检查均未发现明确异常。 B组动物RFA后MRI检查提示门静脉区损伤较A组明显,术后1、3、4周内膜MRI检查提示门静脉水肿逐渐减轻;术后1个月病理学检查证实邻近肝组织损伤严重。C组在功率7 W条件下接受RFA治疗1．5 min,术后MRI检查未见消融区明显水肿,病理学检查可见机化的血栓坏死、血管内皮细胞受损。结论采用Habib VesOpen双极RFA导管治疗PVTT,应根据PVTT严重程度选择合适的RFA功率与时间。PVTT较轻时采用大功率、短时间RFA可能较安全,PVTT较重时改用小功率、长时间RFA更为安全可靠。     

In vitro investigation of pig cells for resistance to human antibody‐mediated rejection

Although human complement‐dependent cytotoxicity (CDC) of α1,3‐galactosyltransferase gene‐knockout (GTKO) pig cells is significantly weaker than that of wild‐type (WT) cells, successful xenotransplantation will require pigs with multiple genetic modifications. Sera from healthy humans were tested by (i) flow cytometry for binding of IgM/IgG, and (ii) CDC assay against peripheral blood mononuclear cells and porcine aortic endothelial cells from five types of pig – WT, GTKO, GTKO transgenic for H‐transferase (GTKO/HT), WT transgenic for human complement regulatory protein CD46 (CD46) and GTKO/CD46. There was significantly higher mean IgM/IgG binding to WT and CD46 cells than to GTKO, GTKO/HT, and GTKO/CD46, but no difference between GTKO, GTKO/HT, and GTKO/CD46 cells. There was significantly higher mean CDC to WT than to GTKO, GTKO/HT, CD46, and GTKO/CD46 cells, but no difference between GTKO and GTKO/HT. Lysis of GTKO/CD46 cells was significantly lower than that of GTKO or CD46 cells. CD46 expression provided partial protection against serum from a baboon sensitized to a GTKO pig heart. GTKO/CD46 cells were significantly resistant to lysis by human serum and sensitized baboon serum. In conclusion, the greatest protection from CDC was obtained by the combination of an absence of Gal expression and the presence of CD46 expression, but the expression of HT appeared to offer no advantage over GTKO. Organs from GTKO/CD46 pigs are likely to be significantly less susceptible to CDC.     

Effects of PEEP on oxygenation and respiratory mechanics during one-lung ventilation

Background. One-lung ventilation-related hypoxaemia (OLV-RH)can occur in patients with healthy lungs. In this case, PEEPfrequently improves oxygenation. The aim of this study was todetermine, in a healthy lung model of OLV, whether the increasein PEEP improved oxygenation and whether the mechanisms involvedinclude both inspiratory lung recruitment and an end-expiratorylung volume increase. Since inhaled nitric oxide (iNO) may havea synergistic effect on oxygenation in the case of PEEP-inducedrecruitment, their association was also tested. Methods. Twenty pigs were studied during open-chest, left OLV.Arterial blood gases and haemodynamic variables were measuredat different levels of PEEP (0, 5, 10 and 15 cm H₂O) appliedin random order with or without iNO 4 p.p.m. Pressure–volumecurves were measured at each level of PEEP. Results. PEEP₅ and PEEP₁₀ improved / ratio (P<0.005) and shunt (P<0.005) regardlessof the presence of iNO. PEEP₁₅ improved oxygenation and shuntonly when it was associated with iNO (P<0.001). Whereas PEEP₅,PEEP₁₀ and PEEP₁₅ were associated with a significant increasein end-expiratory volume (P<0.001), only PEEP₅ and PEEP₁₀were associated with continuous lung volume recruitment (P<0.01).Moreover, PEEP₁₅ induced a significant decrease in linear compliance(P<0.001). Conclusions. In a healthy porcine lung model of OLV-RH, moderatePEEP can improve oxygenation. This effect implies both expiratoryand inspiratory pulmonary recruitment. Co-administration of4 p.p.m. iNO was ineffective.     

Nondermal irritating hyperosmotic nanoemulsions reduce treatment times in a contamination model of wound healing

Increased microbial burden within the wound often complicates wound healing and may lead to subsequent infection or delayed healing. Here, we investigate a novel topical for addressing wound contamination that utilizes hyperosmotic saccharides with a cell membrane disrupting emulsion. These hyperosmotic nanoemulsions (HNE) were administered topically in a full‐thickness biopsy model of wound healing. Results show that HNE were well tolerated in noninfected animals with no indications of dermal irritation or acute toxicity. Additionally, HNE was able to reduce bacterial bioburden (Escherichia coli and Enterococcus faecalis) levels by 3 logs within 24 h when wounds were inoculated with 5 × 10⁶ total CFU. These bactericidal values were similar to wounds treated with silver sulfadiazine. Wound closure showed HNE wounds closed in 7.6 ± 0.2 days while SSD and control required 10.2 ± 0.4 and 10.4 ± 0.3 days, respectively. HNE maintained a moist wound environment, were well debrided, and exhibited improved hemostatic response. Further histological examination revealed enhanced granulation tissue as compared to silver sulfadiazine and control cohorts. These results were corroborated with 3D topographical imprints of the wounds at day 14 which qualitatively showed a smoother surface. In contrast, silver sulfadiazine appeared to delay wound closure. Finally, dermal sensitization and irritation studies conducted in guinea pig and rabbits did not reveal any acute dermal side effects from HNE exposure. The cumulative data indicates nonantibiotic‐based HNEs may be a promising topical treatment for the management of contaminated wounds.     

无菌猪生长、血液以及脏器相关指标的测定

目的 建立无菌太湖猪多项生理数据背景资料。方法 本实验对4头25d无菌猪和4头同窝普通级群体,雌雄比3﹕1,测定并比较血液常规、生化、免疫球蛋白和主要脏器发育指标。运用异速生长建模,对主要脏器重量相对体重的异速生长进行了分析。结果（1）无菌猪与普通级猪的12项血常规和8项血生化指标差异存在显著性（P < 0.05）;（2）无菌猪与普通级猪的平均体重、血清中IgM表达量差异达到显著性水平（P < 0.05）,且无菌猪缺少肠系膜淋巴结;（3）无菌猪的心脏、脾脏和胃与体重呈负异速生长（异速生长指数b < 1）,肠道与体重的异速生长指数为1.78,为正异速生长;而普通级猪的心脏、胃和肠道与体重呈负异速生长（b < 1）,肾脏与体重的异速生长指数最大,为1.20,呈正异速生长。结论 微生物学等级差异影响了猪体重、血液及脏器、免疫系统的发育。