Reduced porcine islet isolation yield in the presence of hyperemic islets

When studying histological characteristics of porcine pancreata in relation to islet isolation, a remarkably high number of hyperemic islets (HIs) was encountered. The abnormalities observed in these HIs ranged from a single dilated vessel to hemorrhages extending into the surrounding exocrine tissue. The aim of the present study was to compare pancreata with and without HI on islet isolation outcomes. This study involved a histological examination of 143 purebred (74 juvenile and 69 adult) and 47 crossbred (only juvenile) porcine pancreata. Islet isolation was performed in 48 purebred adult pigs and in 25 crossbred pigs. Tissue samples were stained with Aldehyde Fuchsine. The presence of HIs was scored semi-quantitatively (HI-, HI+). We observed HIs in 48% of the purebred and in 68% of the crossbred pigs. However, only 3.3±3.1% and 3.1±4.7% of all assessed islets was hyperemic in HI+ pancreata in purebred and crossbred pigs, respectively. In both groups, significantly higher endocrine cell mass was found in the HI+ pancreata (p<0.01). When the higher endocrine cell mass was taken into account, we found significantly lower yields in the HI+ pancreata in both purebred and crossbred pigs (p=0.03 in both groups). The presence of HIs occurs frequently in porcine donor-pancreata and is associated with reduced isolation outcomes.     

Small rat islets are superior to large islets in in vitro function and in transplantation outcomes

Barriers to the use of islet transplantation as a practical treatment for diabetes include the limited number of available donor pancreata. This project was designed to determine whether the size of the islet could influence the success rate of islet transplantations in rats. Islets from adult rats were divided into two groups containing small (diameter <125 microm) or large (diameter >150 microm) islets. An average pancreas yielded three times more small islets than large. Smaller islets were approximately 20% more viable, with large islets containing a scattered pattern of necrotic and apoptotic cells or central core cell death. Small islets in culture consumed twice as much oxygen as large islets when normalized for the same islet equivalents. In static incubation, small islets released three times more insulin under basal conditions than did large islets. During exposure to high glucose conditions, the small islets released four times more insulin than the same islet equivalencies of large islets, and five times more insulin was released by the small islets in response to glucose and depolarization with K+. Most importantly, the small islets were far superior to large islets when transplanted into diabetic animals. When marginal islet equivalencies were used for renal subcapsular transplantation, large islets failed to produce euglycemia in any recipient rats, whereas small islets were successful 80% of the time. The results indicate that small islets are superior to large islets in in vitro testing and for transplantation into the kidney capsule of diabetic rats.     

Improve islet yields and quality when clinical grade pancreata are preserved by the two-layer method

Background: Research grade pancreata preserved by the two-layer method (TLM) yield significantly greater numbers of islets than organs stored with University of Wisconsin solution (UW). The goal of this study was to determine whether this would hold true for pancreata that meet selection criteria for clinical grade organs. Methods: Pancreata were chosen based upon a pre-defined set of criteria used for selecting clinical grade pancreata. Thirteen of these organs were preserved in UW and five pancreata were preserved by the TLM. Islets were isolated and evaluated according to the Edmonton protocol. Results: The average preservation time was significantly longer for organ preserved with TLM (9.5 + 2.0 h) as compared to UW (5.8 + 0.6 h, p = 0.015). The pancreata of TLM group resulted in a significant increase in islet yields (3588 ± 500 vs. 2536 ± 312 IE/g pancreas, p<0.05). Visual scoring of islets indicated that islets were better from TLM group (8.3 ± 0.3 vs. 7.3 ± 0.2), and islet survival rates after culture were higher from organs stored with the TLM (87 ± 17 vs. 55 ± 7.4, p<0.05). Other parameters such as viability, insulin content, and stimulation index were similar between the two groups. All the preparations from the TLM group, but only 54% of preparations from the UW group, qualified for islet transplantation. The two recipients receiving islets from TLM group, daily insulin requirements were reduced and C-peptide levels were increased. Conclusion: Compared to storage with UW, exposure of pancreata to the TLM resulted in greater islet yields and improved quality of islets despite longer preservation period. Consequently, pancreata that meet clinical grade status should be preserved by the TLM prior to islet isolation.     

Calbindin D-27 kDa: preferential localization in non-B islet cells of the rat pancreas

The presence and abundance of calbindin in rat pancreatic islet cells was assessed by immunohistochemistry of either whole islets or purified B and non-B islet cells, as well as by Western blotting of extracts derived from whole islets and purified B and non-B islet cells. Immunohistochemistry of pancreatic sections indicated a higher calbindin content in non-B cells, located at the periphery of the islets, than in the centrally located insulin-producing B cells. Comparable results were obtained in purified islet cells. Likewise, scanning densitometry of the Western blots indicated that, relative to cell volume, the single calbindin band (Mr 27 kDa) was 5-7 times higher in non-B than in B cells. In the splenic lobe of chick pancreas, however, the opposite situation prevailed. Thus, insulin-producing cells clustered in small roundish islets were more intensely labelled after exposure to anti-calbindin serum than non-B islet cells located in large and irregularly shaped islets. Nevertheless, even in the chick pancreas, non-B islet cells contained an appreciable amount of calbindin.     

An immunohistochemical study of the pancreatic endocrine cells of the nude mouse, Balb/c-nu/nu

The regional distribution and frequency of the pancreatic endocrine cells in the nude mouse, Balb/c-nu/nu were studied by immunohistochemical (peroxidase anti-peroxidase; PAP) methods using specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP). The pancreas of the mouse was divided into two lobes, the splenic and duodenal lobes, and each lobe was subdivided into three regions, the pancreatic islets (central and peripheral regions), the exocrine region and the pancreatic duct region (consisting of duct epithelium and surrounding connective tissue--sub-epithelial connective tissue). In the pancreatic islets, most of insulin-immunoreactive (IR) cells were located in the central region, and glucagon-, somatostatin and hPP-IR cells were located in the peripheral region regardless of the lobe. In the splenic part, glucagon-IR cells were also located in the central regions, and more numerous somatostatin-IR cells were detected in the central regions compared to those of the duodenal part. hPP-IR cells were restricted to the peripheral regions in both lobes but more numerous cells were detected in the duodenal portion as compared to those of the splenic portion. In the exocrine parenchyma of the splenic lobe, only insulin-, glucagon- and somatostatin-IR cells were detected.. Here, the insulin- and glucagon-IR cells formed cell clusters, while somatostatin-IR cells were present as solitary cells. In the exocrine region of the duodenal portion, only insulin-, somatostatin- and hPP-IR cells were observed, with the same distributional pattern as that found in the splenic lobe. However, clusters of cells consisting only of hPP-IR cells were distributed in the pancreas parenchyma as small islets. In the pancreatic duct region, only solitary hPP-IR cells were demonstrated in the sub-epithelial connective tissue regions of the splenic portion. In conclusion, some strain-dependent characteristic distributional patterns of pancreatic endocrine cells, especially of the hPP-IR cells, were found in the nude mouse. In addition, somewhat different distributional patterns were found between the two pancreatic lobes.     

Nestin-positive progenitor cells derived from adult human pancreatic islets of Langerhans contain side population (SP) cells defined by expression of the ABCG2 (BCRP1) ATP-binding cassette transporter

The disease diabetes mellitus arises as a consequence of a failure of the beta-cells in the islets of Langerhans of the pancreas to produce insulin in the amounts required to meet the needs of the body. Whole pancreas or islet transplants in patients with severe diabetes effectively restore insulin production. A lack of availability of donor pancreata requires the development of alternative sources of islets such as the ex vivo culture and differentiation of stem/progenitor cells. Earlier we discovered multipotential progenitor cells in islets isolated from adult human pancreata that express the neural stem cell marker nestin: nestin-positive islet-derived progenitor cells (NIPs). Recently it was shown that the exclusion of the Hoechst 33342 dye, which defines the pluripotential side population (SP) of hematopoietic stem cells, is mediated by the ATP-binding cassette transporter, ABCG2. Here we report that the human islet-derived NIPs contain a substantial subpopulation of SP cells that co-express ABCG2, MDR1, and nestin. Thus NIPs may be a potential source of adult pluripotential stem/progenitor cells useful for the production of islet tissue for transplantation into diabetic subjects.     

Porcine islet isolation, cellular composition and secretory response

Porcine islets were isolated by infusion of a warm collagenase solution into whole pancreata followed by static incubation at 37 degrees C for 15 minutes. The pancreata were then chopped into small pieces and the free islets purified by filtration and centrifugation over a ficoll gradient. The insulin:amylase ratio of the islets compared to that in the intact pancreas was determined in 19 pancreata and indicates that the isolated islets were of a high degree of purity. The distribution of insulin, glucagon, somatostatin and pancreatic polypeptide containing cells in pig pancreas sections was compared with that in rat. Porcine islets were much smaller and less well defined than rat islets with infiltration of acinar material even into the islet core. The levels of insulin, glucagon and somatostatin in porcine pancreas and isolated porcine islets were measured using conventional radioimmunoassay techniques. The ratio of these hormones in the pancreas was 105.1:5.8:1 respectively, and in the islets 105.1:0.68:0.087 respectively. Fragmentation of the islets during the isolation may have led to the loss of glucagon and somatostatin-containing cells. Islets cultured overnight and tested with a range of glucose concentrations for one hour did not show a significant stimulation of insulin secretion in the presence of 8.3 mM or 16.7 mM glucose compared to that in 2.8 mM glucose. However freshly isolated islets challenged with 8.3 mM, 13.9 mM and 22.2 mM glucose showed a 1.8 fold, 2.0 fold and 2.3 fold response respectively, over that in 2.8 mM glucose.(ABSTRACT TRUNCATED AT 250 WORDS)     

Adjustment of digestion enzyme composition improves islet isolation outcome from marginal grade human donor pancreata

Despite improvements and recent attempts to standardize techniques to isolate islets from human donor pancreata, there still exists the problem of consistently recovering sufficient quantities of high quality islets. Moreover, achieving consistent recoveries of high numbers of good quality islets becomes even more challenging from marginal grade human donor pancreata with prolonged cold ischemic times. In this study, we investigate whether addition of Pefabloc SC, a serine protease inhibitor, in combination with Pulmozyme, a recombinant human DNase I, to Liberase HI improves islet isolation outcome from marginal grade human donor pancreata (cold ischemic time > 12 h). Twenty-three marginal grade human donor pancreata were randomly digested using four different enzyme preparations: (1) Liberase alone (n = 6), (2) +Pefabloc (n = 7), (3) +Pefabloc/Pulmozyme (n = 5), and (4) +Pulmozyme (n = 5). Overall, there were no significant differences in donor age, body mass index (BMI), pancreas weight, and cold ischemic time. After purification, significantly higher islet yields (3,281 ± 590 IE/g) were obtained with the Pefabloc/Pulmozyme group as compared to the Liberase alone (1,615 ± 305 IE/g) or the Pefabloc group (1,255 ± 261 IE/g) (P < 0.05). Significant improvements in islet viability were also noted from the Pefabloc/Pulmozyme group (87.3 ± 4.4%) as opposed to islets isolated from the Pefabloc group (75.2 ± 3.9%) (P < 0.05). No significant differences in insulin secretory response to glucose stimulation among the four groups were observed, which indicates that the addition of Pefabloc and/or Pulmozyme does not have a detrimental effect on the functionality of islets. It is concluded that the addition of Pefabloc in combination with Pulmozyme to the Liberse HI significantly improves islet isolation outcome and potentially impacts the viability and morphology of the islets obtained from marginal grade human donor pancreata with prolonged cold ischemic times. This study was presented in a poster session at the American Transplant Congress 2005 in Seattle, Washington, USA (May 2005).     

Transcriptional profiling of stress response in cultured porcine islets

Cell-based diabetes therapy may be achieved through xenotransplantation of adult porcine islets, but tissue quality and immunoreactivity barriers need to be overcome. Early identification and exclusion of irreversibly stressed and dying islets may improve transplant outcomes. We used oligonucleotide microarray and quantitative RT-PCR to identify molecular markers of physiological and immunological stress in porcine islets cultured under stress conditions of elevated glucose (16.7 mM), inflammatory cytokine addition (IL-1β, TNF-α, and IFN-γ), or both, for 48 h. Hyperglycemic conditions were associated with increased thioredoxin interacting protein and metabolic process mRNAs, as observed in rodent and primate species. Cytokine treatment increased expression of JAK-STAT pathway components, oxidative stress (transglutaminase 2), and β cell dysfunction genes. Transglutaminase 2 induction is unique to porcine islets. Biomarkers involved in hyperglycemia and islet inflammation may serve as novel targets for improving and monitoring isolated porcine islet function and viability.     

Characterization of integrin expression in islets isolated from hamster, canine, porcine, and human pancreas.

The reasons for the failure of clinical islet transplantation remain obscure. Islet isolation, however, exposes the islet to variety of cellular stresses, including disruption of the cell-matrix relationship, an event associated with apoptosis. The cell-matrix relationship is characterized by an interaction between cell surface integrin receptors and matrix molecules of the surrounding basement membrane (BM). The purpose of this study was to characterize integrin expression and the distribution of the peri-insular BM in human, porcine, canine, and hamster pancreas, and after routine islet isolation. Whereas islets in the porcine pancreas do not have a demonstrable BM, islets in the human, canine, and hamster pancreas have an almost continuous BM with very little direct exocrine to endocrine cell-cell contact. After islet isolation, the BM was destroyed, only to be reestablished during the period of culture. In the pancreas of all four species, integrin alpha3 was expressed only on islet cells, and integrin alpha5 was present on islet cells as well as on acinar, centroacinar, and duct cells. Integrin alphaV was detected only in human and canine pancreas. Integrin beta1 was demonstrated only in the human pancreas. In isolated islets, integrin alpha3, alpha5, and alphaV expression decreased during the culture period and the intensity of the staining was observed to be coincident with the distribution of the BM. In summary, this is the first report of integrin expression in hamster, canine, porcine, and human islets. After islet isolation, the altered islet cell-matrix relationship is reflected both in the decrease in integrin expression and in the destruction of the peri-insular BM. These profound changes will need to be considered as the process of islet isolation for transplantation is refined. (J Histochem Cytochem 47:499-506, 1999)     

Optimizing Conditions for Rat Pancreatic Islets Isolation

Many procedures have been described for rat pancreatic islet isolation. Several factors contribute to the pancreatic islet isolation outcome. One of the main problems in islet isolation procedure is the formation of a viscouse, gellike structure during collagenase digestion which entraps the free islets and decrease islet yield after density gradient purification. This issue has not been addressed in most techniques described for rat islet isolation. We examined effect of various factors to eliminate formation of gellike material and improve the islets yields. Islet isolation was performed on 26 adult male Wistar Albino rats weighing between 280 and 350 g. We have observed that several factors affect pancreatic islet isolation. Optimum Collagenase enzyme concentration, maintaining pH range between 7.7 and 7.9 in digestion solution, incubation temperature at 38±1 °C and addition of Calcium ion decreased the formation of gellike materials and increased islet yield. Addition of Glycerol as a gelatin solvent has also been helpful in the reduction or complete elimination of gellike material. Precise optimization of rat islet isolation procedure is useful to improve the islet yield in islet transplantation studies.     

Pancreatic polypeptide cells of rat pancreas after chronic ethanol feeding

Male Wistar rats, (2 months old) were randomly divided into two groups according to the diet offered (C-control and E-ethanol treated rats). Final body weight was significantly increased but pancreatic weight as a percentage of body weight was decreased in ethanol treated rats. Volume density, number of pancreatic poly peptide (PP)-cells per islet and per micron 2 of islet were significantly increased. PP-cells were abundant and occupied the whole periphery of islets in the splenic part of the pancreas. Those cells showed strong immunopositivity. At the ultrastructural level PP granules had predominantly less electron density. The mean diameter of PP granules was significantly increased and the number of granules of larger diameter was greater in the E group of rats, than in the controls.     

Human Islet Morphology Revisited: Human and Rodent Islets Are Not So Different After All

There has been great interest in understanding how human islets differ from rodent islets. Three major issues about human islet morphology have remained controversial over recent decades: 1) the proportion of the islet made up of β-cells; 2) whether islet cell types have a non-random mantle-core pattern, as seen in rodents, or are randomly scattered throughout the islet; 3) the relation of the different cell types to the blood vessels within the islet, which has implications for intraislet function. We re-examined these issues on immunostained sections of non-diabetic adult human pancreas. The composition of the islets can vary by the analysis method (number vs volume) and by the sampling of islets by size. The majority of adult human islets have clear, non-random clustering of β-cells and blood vessels that penetrate into the β-cell cores. We conclude that although there is far more variability in islet composition both within each human pancreas and among different human pancreas than in rodent pancreas, the islet architecture is not so different between the species. The intrapancreatic variability raises important questions about how islets evolve and function throughout life and how this might relate to the pathogenesis of diabetes.     

Significance of the peri-insular extracellular matrix for islet isolation from the pancreas of rat,dog, pig,and man

Summary The presence and distribution in the peri-insular region of extracellular matrix, and in particular basement membrane, was investigated in a comparative study comprising pancreata of rat, dog, pig, and man. Basement membrane markers, collagen type-IV and laminin, were determined immunohistochemically. Additional information pertaining to the structural relationships between endocrine and exocrine pancreas, in particular cell-to-cell and cell-to-matrix contacts, was obtained by electron microscopy. In pig, very little periinsular capsule is present, and the structural integration of the porcine islet in the exocrine pancreas almost exclusively depends on cell-to-cell adhesion. In the canine pancreas, the islets are almost completely encapsulated with very little direct exocrine-to-endocrine cell-to-cell contact. In rat and man, the situation is intermediate with a tendency towards predominance of cell-to-matrix adhesion. The intra-insular adhesion mechanisms depend largely on cell-to-cell adhesion in all four species. The ultrastructural results suggest that collagenase preparations employed in islet isolation procedures should be of high purity as to preserve the protease-sensitive intra-islet cell-to-cell adhesion. Under these conditions, however, the endocrine-to-exocrine cell-to-cell contacts will be conserved also, resulting in an exocrine-tissue contamination of the islets of Langerhans. Consequently, additional steps for the effective removal of exocrine tissue and the purification of islets are required.     

An immunohistochemical study of the insulin-, glucagon- and somatostatin-immunoreactive cells in the developing pancreas of the chicken embryo

The distributions and relative frequencies of insulin-, glucagon- and somatostatin-immunoreactive cells were studied in dorsal, ventral, third and splenic lobes of developing chicken pancreas during embryonic periods (10 days of incubation to hatching) by immunohistochemical methods. The regions of pancreas were subdivided into three regions: exocrine, light and dark islet. Round, oval and spherical shaped immunoreactive cells were detected in all four lobes. According to developmental stages, the types of lobes and the regions of pancreas showed various distributions and relative frequencies. In the splenic lobes, insulin, glucagon and somatostatin-immunoreactive cells were detected in exocrine, dark islet and light islet from time differentiation of splenic lobes, 13 days of incubation. The insulin- and somatostatin-immunoreactive cells of the third lobes were detected in exocrine and light islets from 10 days of incubation, and in dark islets from 15 and 11 days of incubation respectively. Glucagon-immunoreactive cells were detected in exocrine, dark and light islets from 16, 11 and 19 days of incubation respectively. These immunoreactive cells of the ventral lobes were detected in exocrine and light islets. However, dark islets were not found in this lobe. Insulin-immunoreactive cells were demonstrated from 10 days of incubation in these two regions. Glucagon-immunoreactive cells were detected from 17 days of incubation in exocrine and 16 days of incubation in the light islets. Somatostatin-immunoreactive cells were demonstrated from 11 days of incubation in exocrine and 14 days of incubation in the light islets. In the dorsal lobes, insulin-immunoreactive cells were demonstrated in exocrine, dark and light islets from 12, 14, and 13 days of incubation, respectively. Glucagon- and somatostatin-immunoreactive cells were detected in dark and light islets from 13 and 14 days of incubation, respectively. Glucagon- and somatostatin-immunoreactive cells were demonstrated from 10 and 11 days of incubation in exocrine respectively. Generally, insulin-immunoreactive cells were increased in light islets but decreased in light islets with developmental stages. However, glucagon-immunoreactive cells were decreased in light islets but increased in dark islets. In addition, somatostatin-immunoreactive cells showed the same frequencies in light and dark islets with developmental stages except exocrine which increased with developmental stages.     

An immunohistochemical study of the pancreatic endocrine cells of the ddN mouse

The regional distribution and frequency of the pancreatic endocrine cells in the ddN mouse were studied using specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP). In the pancreatic islets, most of insulin-immunoreactive (IR) cells were located in the central region, and glucagon-, somatostatin and hPP-IR cells were located in the peripheral region regardless of the lobe. In the splenic part, glucagon-IR cells were also located in the central regions, and more numerous somatostatin-IR cells were detected in the central regions as compared with the duo-denal part. hPP-IR cells were restricted to the peripheral regions in both lobes but more numerous cells were detected in the duodenal portion. In the exocrine parenchyma of the splenic lobe, only insulin- and glucagon-IR cells were detected but all four kinds of IR cells were observed in the duodenal portion. In addition, insulin and hPP-IR cells were also demonstrated in the pancreatic duct regions. In conclusion, some strain-dependent characteristic distributional patterns of pancreatic endocrine cells were found in the ddN mouse with somewhat different distributional patterns between the two pancreatic lobes.     

Genetic engineering of a suboptimal islet graft with A20 preserves beta cell mass and function

Transplantation of an excessive number of islets of Langerhans (two to four pancreata per recipient) into patients with type I diabetes is required to restore euglycemia. Hypoxia, nutrient deprivation, local inflammation, and the beta cell inflammatory response (up-regulation of NF-kappaB-dependent genes such as inos) result in beta cell destruction in the early post-transplantation period. Genetic engineering of islets with anti-inflammatory and antiapoptotic genes may prevent beta cell loss and primary nonfunction. We have shown in vitro that A20 inhibits NF-kappaB activation in islets and protects from cytokine- and death receptor-mediated apoptosis. In vivo, protection of newly transplanted islets would reduce the number of islets required for successful transplantation. Transplantation of 500 B6/AF(1) mouse islets into syngeneic, diabetic recipients resulted in a cure rate of 100% within 5 days. Transplantation of 250 islets resulted in a cure rate of only 20%. Transplantation of 250 islets overexpressing A20 resulted in a cure rate of 75% with a mean time to cure of 5.2 days, comparable to that achieved with 500 islets. A20-expressing islets preserve functional beta cell mass and are protected from cell death. These data demonstrate that A20 is an ideal cytoprotective gene therapy candidate for islet transplantation.     

Immunohistochemical and ultrastructural study of adult porcine endocrine pancreas during the different steps of islet isolation

 Treatment of diabetes mellitus by transplantation of isolated pancreatic islets could constitute an alternative to human pancreas allograft. Before transplantation, porcine islets are submitted to a procedure of isolation and purification. The quality of islets through these different steps may be assessed by morphological and functional studies. The aim of this work was the histological characterization of the four main cell types of porcine adult endocrine islets during the different steps of the isolation procedure using immunohistochemistry (IHC) applied in light (LM) and electron microscopy (EM). In fresh pancreas, islets were various sizes and shapes in LM. The number was not found different between the different portions of the pancreas. In IHC, insulin (Ins)-secreting cells accounted for the majority of the islet cells, while glucagon(Glu)-somatostatin (Som)- and polypeptide(PP)-immunoreactive cells, in decreasing number, were found in the mantle around the core of Ins-cells. In EM, B-cells contained polyhedric granules with a dense central core and clear halo. Glu granules were spherical and very dense. D-cells and PP-cells were characterized by numerous granules, rather spherical and of inequal density for Som and more ellipsoidal for PP granules. After purification in Euroficoll, in EM, the four cellular types remained recognizable, but underwent vacuolization, mitochondrial swelling, and enlargment of intercellular spaces. After 3 days of culture on plastic dishes, as on Biopore membranes in a Millicell insert, microvilli appeared and vacuolization increased in EM. At the seventh day of culture, in EM, most of the cells were lysed in contrast to LM where at the same time, the four cell types were clearly identified by IHC but only in collagen matrix. Important discrepancies were noticed between LM and EM. This fact emphasizes the complementarity of morphological and functional studies in assessment of the quality of an islet isolation. Accepted: 11 June 1996     

Impaired glucose-stimulated insulin secretion in the GK rat is associated with abnormalities in islet nitric oxide production

We investigated implications of nitric oxide (NO) derived from islet neuronal constitutive NO synthase (ncNOS) and inducible NOS (iNOS) on insulin secretory mechanisms in the mildly diabetic GK rat. Islets from GK rats and Wistar controls were analysed for ncNOS and iNOS by HPLC, immunoblotting and immunocytochemistry in relation to insulin secretion stimulated by glucose or l-arginine in vitro and in vivo. No obvious difference in ncNOS fluorescence in GK vs control islets was seen but freshly isolated GK islets displayed a marked iNOS expression and activity. After incubation at low glucose GK islets showed an abnormal increase in both iNOS and ncNOS activities. At high glucose the impaired glucose-stimulated insulin release was associated with an increased iNOS expression and activity and NOS inhibition dose-dependently amplified insulin secretion in both GK and control islets. This effect by NOS inhibition was also evident in depolarized islets at low glucose, where forskolin had a further amplifying effect in GK but not in control islets. NOS inhibition increased basal insulin release in perfused GK pancreata and amplified insulin release after glucose stimulation in both GK and control pancreata, almost abrogating the nadir separating first and second phase in controls. A defective insulin response to l-arginine was seen in GK rats in vitro and in vivo, being partially restored by NOS inhibition. The results suggest that increased islet NOS activities might contribute to the defective insulin response to glucose and l-arginine in the GK rat. Excessive iNOS expression and activity might be deleterious for the beta-cells over time.     

Histological observation of islet hemorrhage induced by diagnostic ultrasound with contrast agent in rat pancreas

Contrast enhanced diagnostic ultrasound CEDUS has been shown to induce capillary hemorrhage in heart and kidney. This study characterized the capillary hemorrhage induced in rat pancreas. The pancreata of anesthetized hairless rats were accessed by laparotomy. A 1.5 MHz diagnostic ultrasound probe with 2.3 MPa peak rarefactional pressure amplitude and 1 s intermittent trigger was used to scan the pancreas, located at the focus (3.8 cm), through saline coupling. The probe was swept to expose the entire organ in 5 min during infusion of Definity® contrast agent at 10 µL/kg/min, and this was repeated in a reverse sweep. The entire pancreas was removed, spread flat for fixation and histological slides were prepared from the mid-plane. Slides were scored blind for islet hemorrhage over the entire area of the organ. Intra-islet microlesions were evident and hemorrhage surrounded many islets. The hemorrhage often impacted nearby acini, and expanded into inter-lobular septa. In CEDUS pancreata removed soon after scanning, 76.2±11.8% (n = 6) of islets had evidence of hemorrhage and/or islet microlesions compared to 1.1±2.5% (n = 5) for sham CEDUS (P<0.001). In pancreata removed after 4 hr, fibrin formation was detected by immunohistology in the hemorrhage and intra-islet microlesions. Diagnostic ultrasound with contrast agent induced substantial capillary hemorrhage in rat pancreas, concentrated particularly in the islets.