N. J. M. London

Islet isolation assessment in man and large animals

SummaryRecent progress in islet isolation from the pancreas of large mammals including man, accentuated the need for the development of precise and reproducible techniques to assess islet yield. In this report both quantitative and qualitative criteria for islet isolation assessment were discussed, the main topics being the determination of number, volume, purity, morphologic integrity andin vitro andin vivo function tests of the final islet preparations. It has been recommended that dithizone should be used as a specific stain for immediate detection of islet tissue making it possible to estimate both the total number of islets (dividing them into classes of 50 µ diameter range increments) and the purity of the final preparation. Appropriate morphological assessment should include confirmation of islet identification, assessment of the morphological integrity and of the purity of the islet preparation. The use of fluorometric inclusion and exclusion dyes together have been suggested as a viability assay to simultaneously quantitate the proportion of cells that are intact or damaged. Perifusion of islets with glucose provides a dynamic profile of glucose-mediated insulin release and of the ability of the cells to down regulate insulin secretion after the glycemic challenge is interrupted. Although perifusion data provides a useful guide to islet viability the quantity and kinetics of insulin release do not necessarily predict islet performance after implantation. Therefore, the ultimate test of islet viability is their function after transplantation into a diabetic recipient. For this reason,in vivo models of transplantation of an aliquot of the final islet preparation into diabetic nude (athymic) rodents have been suggested. We hope that these general guidelines will be of assistance to standardize the assessment of islet isolations, making it possible to better interpret and compare procedures from different centers.

Angiogenesis and the atherosclerotic carotid plaque: An association between symptomatology and plaque morphology

Purpose: Symptomatic carotid disease resulting from generation of thromboemboli has been associated with plaque instability and intraplaque hemorrhage. These features of the lesion could be influenced by the fragility and position of neovessels within the plaque. The purpose of this study was to determine whether any association exists between neovessel density, position, morphology, and thromboembolic sequelae. Methods: Carotid endarterectomy samples were collected from 15 asymptomatic patients with greater than 80% stenoses and from 13 highly symptomatic patients who had suffered ipsilateral carotid stenotic events within 1 month of surgery. Both groups were matched for gender, age, risk factors, degree of carotid artery stenosis, and plaque size. Samples were stained with hematoxylin/eosin and van Geison. Immunohistochemistry was performed by using an endothelial specific antibody to CD31. Plaques were assessed for histologic characteristics, and neovessels were counted and characterized by size, site, and shape. Results: There were significantly more neovessels in plaques (P < .00001) and fibrous caps (P < .0001) in symptomatic compared with asymptomatic plaques. Neovessels in symptomatic plaques were larger (P < .004) and more irregular. There was a significant increase in plaque necrosis and rupture in symptomatic plaques. Plaque hemorrhage and rupture were associated with more neovessels within the plaque (P < .017, P < .001) and within the fibrous cap (P < .046, P < .004). Patients with preoperative and intraoperative embolization had significantly more plaque and fibrous cap neovessels (P < .025, P < .001). Conclusion: Symptomatic carotid disease is associated with increased neovascularization within the atherosclerotic plaque and fibrous cap. These vessels are larger and more irregular and may contribute to plaque instability and the onset of thromboembolic sequelae. (J Vasc Surg 1999;30:261-8.)

Beneficial effects of clopidogrel combined with aspirin in reducing cerebral emboli in patients undergoing carotid endarterectomy.

Background—Postoperative thromboembolic stroke affects 2% to 3% of patients undergoing carotid endarterectomy (CEA) and is preceded by 1 to 2 hours of increasing cerebral embolization. Previous work has demonstrated that high rates of postoperative embolization are associated with increased platelet reactivity to adenosine 5′-diphosphate (ADP). Our hypothesis was that preoperative administration of the platelet ADP antagonist clopidogrel could reduce postoperative embolization. Methods and Results—One hundred CEA patients on routine aspirin therapy (150 mg) were randomized to 75 mg clopidogrel (n=46) or placebo (n=54) the night before surgery. Platelet response to ADP was assessed by whole-blood flow cytometry. The number of emboli detected by transcranial Doppler within 3 hours of CEA was independently quantified. Time taken from flow restoration to skin closure was used as an indirect measure of the time to secure hemostasis. In comparison with placebo, clopidogrel produced a small (8.8%) but significant reduction in the platelet response to ADP (P <0.05) while conferring a 10-fold reduction in the relative risk of those patients having >20 emboli in the postoperative period (odds ratio, 10.23; 95% CI, 1.3 to 83.3; P =0.01, Fisher’s exact test). However, in the clopidogrel-treated patients, the time from flow restoration to skin closure (an indirect marker of hemostasis) was significantly increased (P =0.04, Fisher’s exact test), although there was no increase in bleeding complications or blood transfusions. Conclusions—This is the first study to show that a CEA patient’s postoperative thromboembolic potential can be significantly reduced by targeted preoperative antiplatelet therapy without increasing the risk of bleeding complications.

Prevention of postoperative thrombotic stroke after carotid endarterectomy: The role of transcranial Doppler ultrasound

PURPOSE To determine the incidence of particulate embolization after carotid endarterectomy (CEA), the effect of dextran-40 infusion in patients with sustained postoperative embolization, and the impact of transcranial Doppler (TCD) monitoring plus adjuvant dextran therapy on the rate of postoperative carotid thrombosis. METHODS Prospective study in 100 patients who underwent CEA with 6-hour postoperative monitoring using a TCD that was modified to allow automatic, intermittent recording from the ipsilateral middle cerebral artery waveform (10 minute sample every 30 minutes). An incremental dextran-40 infusion was commenced if 25 or more emboli were detected in any 10-minute period. RESULTS Overall, 48% of patients had one or more emboli detected in the postoperative period, particularly in the first 2 hours. However, sustained embolization that required Dextran therapy developed in only five patients. In each case, the rate of embolization rapidly diminished. CONCLUSIONS A small proportion of patients have sustained embolization after CEA, which in previous studies has been shown to be highly predictive of thrombotic stroke. Intervention with dextran reduced and subsequently stopped all the emboli in those in whom it was used and contributed to a 0% perioperative morbidity and mortality rate in this series.

Interpretation of Embolic Phenomena During Carotid Endarterectomy

BACKGROUND AND PURPOSE Air and particulate emboli are a major source of morbidity during carotid endarterectomy (CEA); however, amplitude overload and poor time resolution have restricted the ability of transcranial Doppler ultrasound to differentiate between the two. METHODS We have now overcome these two limitations by (1) rerouting embolic signals away from the audio frequency amplifier to avoid amplitude overload and (2) substituting the Wigner distribution function for the fast Fourier transform to improve time and frequency resolution. Thus, we can now accurately determine embolic duration and embolic velocity, the product of which is the sample volume length (SVL). This measurement represents the physical distance over which an embolic signal can be detected. The underlying hypothesis was that air reflected more ultrasound and would therefore be detected over a greater SVL. RESULTS The median SVL (interquartile range) for 75 in vitro air emboli was 1.97 cm (range, 1.70 to 2.35) compared with 0.27 cm (range, 0.16 to 0.43) for 185 particulate emboli detected during the dissection phase of CEA. Off-line analysis on an additional 560 embolic signals detected during different phases of CEA suggested that 46 of 143 (32%) of emboli immediately after shunt insertion were particulate, as were 19 of 33 (58%) occurring during shunting, 28 of 78 (36%) after restoration of flow in the external carotid artery, 23 of 251 (9%) after restoration of flow in the internal carotid artery, and 55 of 55 (100%) of those emboli detected during the early recovery phase. CONCLUSIONS This development provides objective physical criteria upon which embolus characterization (particulate/air) can be based. This could have major implications for future patient monitoring with respect to modification of surgical technique and pharmacological intervention.

Islet microencapsulation: a review

The original report on the microencapsulation of islets of Langerhans used sodium alginate and poly-l-lysine (PLL) to form the capsules. Although several alternative materials have subsequently been used with varying degrees of success, it is those studies using islets encapsulated in alginate-PLL-alginate which are reviewed in detail in this article. Since the first report of islet microencapsulation, many studies have demonstrated excellent in vitro viability of encapsulated islets. However, transplantation experiments into chemically induced diabetic recipients have yielded varied results, with some studies showing good long-term graft function whilst in others grafts failed due to pericapsular fibrosis. The use of naturally occurring animal models of type 1 (insulin-dependent) diabetes has demonstrated a decline in graft function, suggesting that this presents a more complex problem to be solved than that in chemically induced diabetic recipients. Fibrosis of capsules has been the major problem causing graft failure, and this has been demonstrated to be more severe in spontaneously diabetic models. However, recent advances in alginate purification and attempts to reduce the size of the encapsulated islets are major steps towards encapsulated islet transplants becoming a viable proposition for the treatment of type 1 diabetic patients.

Association Between the Coronary Artery Disease Risk Locus on Chromosome 9p21.3 and Abdominal Aortic Aneurysm

Background—Recent genome-wide studies have shown a significant association of a locus on chromosome 9p21.3 and coronary artery disease. We performed a case-control study to investigate the association between this locus and abdominal aortic aneurysm (AAA). Methods and Results—A total of 1714 patients (899 patients with AAA and 815 controls) were genotyped for the lead single-nucleotide polymorphism, rs1333049, on chromosome 9p21. The frequency of the C (risk) allele of rs1333049 in the control group was 0.471. There was a significant association between the C allele and AAA (odds ratio, 1.22; 95% confidence interval, 1.06 to 1.39; P=0.004). The genotypic-specific odds ratios (compared with the GG genotype) were 1.17 (95% confidence interval, 0.93 to 1.47; P=0.191) for the GC genotype and 1.50 (95% confidence interval, 1.14 to 1.97; P=0.004) for the CC genotype. In logistic regression modeling, the association of the CC genotype with AAA was independent of the presence of clinical coronary artery disease (odds ratio, 1.46; 95% confidence interval, 1.11 to 1.94; P=0.008). Conclusions—Our study shows that the recently identified chromosome 9 variant that increases risk of coronary artery disease is also associated with the presence of AAA. The findings suggest that the effect of this locus on risk of cardiovascular disease extends beyond the coronary circulation.

HUMAN SAPHENOUS VEIN ORGAN CULTURE : A USEFUL MODEL OF INTIMAL HYPERPLASIA?

OBJECTIVES Although cell culture techniques and animal models of intimal hyperplasia have increased our current understanding of the aetiology of vein graft stenosis, the results of such studies have been difficult to relate to the human situation. DESIGN The present study was designed to validate an organ culture of human saphenous vein by comparing the changes occurring in cultured vein with those seen in pathological vein graft stenoses and to identify a suitable marker of cell proliferation. MATERIALS AND METHODS Saphenous vein segments were cultured for 14 days, fixed in formalin and processed for immunohistochemistry. Freshly excised stenoses were fixed and processed similarly. A number of markers of cell proliferation were evaluated in the culture system in order to identify the one best suited to this particular model. RESULTS Marked similarities were observed in the cellular and extracellular matrix composition, and electron microscopy revealed that both the neointima of the cultured vein and the pathological lesion contained an abundance of smooth muscle cells of a secretory phenotype. Bromodeoxyuridine proved to be the most reliable proliferation marker and revealed that early proliferation in the superficial layers of the vein intima gave rise to the formation of neointima. Both proliferation and neointimal thickness were maximal by day 14 in culture. Proliferation declined rapidly thereafter, and the neointima was maintained. CONCLUSIONS The changes occurring in cultured vein and graft stenoses bore many similarities, thereby justifying the use of organ culture as a valuable experimental tool.

Functional studies of rat, porcine, and human pancreatic islets cultured in ten commercially available media

There have been no extensive studies investigating the effect of tissue culture media on the in vitro functional characteristics of rat, porcine and human Islets of Langerhans. We therefore aimed to compare ten commercially available tissue culture media on the basis of their ability to maintain islet viability. Following isolation, islets were cultured free-floating in the ten media (RPMI 1640-11mM glucose (control), RPMI 1640-2.2mM glucose, Dulbeccos MEM, TCM 199, CMRL 1066, Iscoves MEM, Waymouths MEM, Serum-Free medium, Ex-cell 300, Hams F-12) and viability was assessed after 24 hr, 3 days, and 7 days on the basis of macroscopic appearance, cell membrane integrity, and insulin secretion in response to glucose stimulation both by dynamic incubation and by perifusion. Each islet species demonstrated physiological insulin release characteristics in all media--however, it was possible to distinguish between the media by comparing the stimulation indices calculated from the insulin release studies. Significantly higher stimulation indices were produced in Iscoves MEM for rat islets, in Hams F-12 for porcine islets and in CMRL 1066 for human islets. Over the entire culture period a significant deterioration in function was observed in all species cultured in the control media, although this was reversed when islets were cultured in the optimal media. Furthermore, in the case of porcine and human islets a significant improvement in function over the seven-day period was noted in the optimal media. In conclusion, of the commercially available media, the optimal tissue culture medium for rat islets is Iscoves MEM, for porcine islets is Hams F-12, and for human islets is CMRL 1066.

Conservative Management of Type 2 Endoleaks is not Associated with Increased Risk of Aneurysm Rupture

OBJECTIVE Endovascular repair (EVAR) of abdominal aortic aneurysms (AAAs) has led to a reduction in the perioperative mortality when compared with open repair. However, re-intervention for complications, such as endoleak, may be required in up to 20% of the cases. Controversy exists over the management of type 2 endoleaks. This study examined the outcomes of patients with type 2 endoleaks treated conservatively to inform the ongoing management debate. METHODS All patients with a confirmed type 2 endoleak after EVAR for an infrarenal AAA were included in the study. Data regarding device details, endoleak and time point, aneurysm sac growth, intervention and outcome were collected retrospectively from case notes and the vascular research database. RESULTS Forty-one type 2 endoleaks were seen in 369 EVARs performed for infrarenal AAA between March 1994 and June 2006. Twenty-five were isolated type 2 endoleaks and 16 occurred in conjunction with other endoleaks. Of the 25 isolated type 2 endoleaks, 18 (72%) patients demonstrated no increase in sac size, six (24%) patients showed an enlargement of the sac and one patient was lost to follow-up. Only one patient underwent intervention for an isolated type 2 endoleak. After a mean follow-up period of 4 years, approximately half of the patients (48%) remain under observation (with an enlarging or stable sac), whilst the others (48%) have spontaneously sealed. Only five patients under surveillance (20%) have an enlarging sac. There were no ruptured aneurysms or aneurysm-related deaths and no patients required conversion to open repair. CONCLUSIONS In this study, a policy of regular surveillance for type 2 endoleaks was not associated with any adverse events. We therefore advocate the conservative approach for type 2 endoleaks.