in - stent restenosis ( isr ) is the most common complication associated with coronary stenting and is histologically characterized by occlusive neointima formation . as yet , no adequate treatment modalities are available to treat or prevent development of isr [ 13 ] . the use of drug - eluting stents has significantly reduced the incidence of isr when compared with bare metal stents , but also resulted in increased rates of late stent thrombosis . diabetes mellitus ( dm ) has been associated with increased risk to develop ( in - stent ) restenosis both after percutaneous transluminal coronary angioplasty as well as coronary stenting [ 514 ] . dm is a risk factor for isr development after using both drug - eluting and bare metal stents as revealed by various meta - analyses ( primarily including type 2 diabetic patients ) [ 1517 ] . the beneficial effects on isr of drug - eluting stents over bare metal stents as observed in non - diabetic patients appear to be less clear in diabetic patients . although the exact pathogenetic mechanism underlying increased isr development in diabetic patients is as yet unknown , it at least appears to be due to an exaggerated neointimal response after coronary stent placement as determined by intravascular ultrasound . many studies thus favor for a deleterious effect of dm on the development of isr , although the precise magnitude of this effect is not clear . especially age may act as a confounder as older age is associated with increased restenosis rates independent of dm .   to study the molecular and cellular mechanism(s ) underlying dm - enhanced development of isr however , despite the increasing numbers of diabetic patients worldwide together with coronary artery disease being a significant source of morbidity and mortality in these patients , relevant animal models to study isr are scarce . although the zucker diabetic fatty ( zdf ) rat has been used as a model to study type 2 dm - associated restenosis [ 1921 ] , a reliable model to study the long - term effects of type 1 diabetes on isr development is still lacking . whereas type 1 dm represents only 510% of all diabetics it may be associated with severe coronary artery disease at a relatively young age as demonstrated in a selected population of type 1 diabetics eligible for kidney and/or pancreas transplantation . various rodent models of type 1 dm have been used to study the effects of diabetes on mechanically induced restenosis in mice , rats , or rabbits , in which diabetes is chemically induced using streptozotocin or alloxan and in which contradicting results were obtained . in addition to rodent models , also a streptozotocin - induced diabetes porcine model has been used to study the development of isr . in this model , a high mortality rate ( ~45% ) , a relatively short course of diabetes ( 12 weeks ) , and most likely high costs may actually hamper extensive use of this model . both streptozotocin and alloxan are toxic compounds sharing structural similarities with glucose which explains their selective uptake in cells expressing the glut2 glucose transporter . as pancreatic  - cells have relatively high levels of glut2 , streptozotocin and alloxan are relatively , but not solely , toxic to  - cells . because of the possible toxic side effects of these chemicals and to model more accurately human type 1 ( autoimmune ) dm , the availability of a spontaneous type 1 dm model for the development of ( in - stent ) restenosis is needed . to this end , we here describe the use of inbred diabetes prone biobreeding ( bbdp / wor ) rats as a model to study diabetes - enhanced development of isr . inbred bbdp rats are derived from a canadian colony of outbred wistar rats ( i.e. , the bb wistar rat ) in which diabetes developed spontaneously in the 1970s . bbdp / wor rats develop autoimmune diabetes spontaneously due to severe lymphopenia and preferential lack of immunoregulatory t cells [ 2628 ] . using the rat abdominal aorta stenting model described previously , we tested the hypothesis that long - term suboptimally treated hyperglycaemic bbdp rats develop enhanced isr compared with non - diabetic age - matched control rats . specified pathogen - free diabetes prone bb ( bbdp / wor ) and diabetes - resistant ( bbdr / wor ) rats were bred at the central animal facility of the university medical center groningen , university of groningen , groningen , the netherlands . rats were kept under clean conventional conditions and were fed standard rat chow and acidified water ad libitum . all animals received humane care in compliance with the principles of laboratory animal care ( nih publication no . 85 - 23 , revised 1996 ) and the dutch law on experimental animal care . in the bbdp / wor colony maintained at the university medical center groningen , 8090% of the rats spontaneously develop diabetes from ~70 days of age . rats were considered diabetic when blood glucose levels exceeded 15  mmol / l as measured in peripheral blood obtained by tail vein puncture and by using a glucose sensor ( accu - chek sensor comfort , roche diagnostics nederland b.v . , 9 recent onset diabetic bbdp rats were initially included ( both male and female ) which received a 1/2 ( ~3 - 4  mm length ) linplant sustained release insulin implant ( linshin canada inc , toronto , on , canada ) subcutaneously at diagnosis using a trocar . according to the manufacturer the implants had an estimated insulin release of ~1  u/24  h/(1/2 ) implant for > 40 days . we aimed at maintaining the blood glucose values in insulin - treated diabetic rats between 1520 mmol / l during the entire follow - up period ( up to 10 months of age ) . during follow - up diabetic rats blood glucose levels were measured as described above . when blood glucose levels exceeded 20 mmol / l in the presence of substantial weight loss , rats were reimplanted with an insulin implant as described above . in addition to the insulin - treated diabetic bbdp rats also a control group of age - matched non - diabetic bbdp rats ( initially n = 9 ) was included . diabetes development was prevented by performing thymectomy at the age of 21 days as we described in detail elsewhere . weight and blood glucose levels of thymectomized non - diabetic bbdp rats were measured once every two weeks to obtain basal values . to determine whether long - term suboptimal insulin treatment of diabetic bbdp rats is associated with increased glycated hemoglobin ( hba1c ) levels indicative of poor glycaemic control , we analyzed hba1c levels in a separate cohort of long - term diabetic bbdp rats ( n = 5 ) , as well as in spontaneously protected non - diabetic bbdp rats ( n = 2 ) and non - diabetic bbdr / wor rats ( n = 6 ) . hba1c levels were determined in freshly obtained peripheral blood ( tail vein puncture ) using the bio - rad in2it a1c analyzer ( bio - rad laboratories b.v . , two weeks before stenting renal function of the long - term diabetic and non - diabetic bbdp rats was determined by housing the rats in individual , urine - collecting metabolic cages for 24 hours and by collection of blood plasma . plasma and urine creatinine levels were determined using the enzymatic colorimetric assay crea plus ( roche diagnostics gmbh , mannheim , germany ) . total urinary protein excretion was determined using the roche diagnostics urinary / csf protein assay ( roche diagnostics gmbh , mannheim , germany ) [ 32 , 33 ] . at a median diabetes duration of 28 weeks ( 7 months ) 9 bbdp rats were stented in the abdominal aorta as described previously . as a control , 9 age - matched briefly , under anesthesia ( 2% isoflurane ( abbott , hoofddorp , the netherlands ) , 0.4  l / min o2 and 0.4  l / min n2o ) the abdominal cavity was opened . next , two vascular clips were placed onto the aorta distal to the renal arteries and proximal to the aortic bifurcation . a small incision was then made in the distal abdominal aorta , and the balloon catheter was inserted and inflated to 9  atm pressure to deploy a premounted 2.5  9  mm bestent 2 bare metal stent ( medtronic - bakken research , maastricht , the netherlands ) . after deflation and removal of the balloon , the aortic incision was closed with a 9 - 0 suture . reperfusion was established by removing the clips , and the abdomen was closed with 4 - 0 sutures . starting 5 days before stenting until the end of the experiment , rats were fed chow containing 0.33  mg clopidogrel / gram chow ( plavix , sanofi - aventis , gouda , the netherlands ) in order to prevent platelet aggregation and to mimick the human clinical setting . four weeks after stenting , rats were anesthesized and heparinized systemically with 500  iu i.v . the stented aortas were harvested , fixed in 4% formaldehyde , and embedded in methyl metacrylate for further histological analysis . during stenting , 3 non - diabetic control bbdp rats died because of perforation of the aorta , resulting in an overall mortality of 17% . in addition , 2 stents from long - term diabetic bbdp rats were lost during histological processing . overall , stents from 7 diabetic and 6 non - diabetic bbdp rats were included for histological analysis . to quantify the severity of isr , computerized morphometric analysis was performed on lawson ( elastin ) stained sections obtained from the proximal , middle , and distal parts of each stent . the neointimal area was calculated by measuring the total area within the internal elastic lamina and the remaining lumen using an olympus bx-50f4 microscope equipped with an olympus c-3030 zoom digital camera and olympus dp - soft version 3.0 software ( olympus , tokyo , japan ) . total surface neointima ( in m ) was then calculated by subtracting surface remaining lumen from the total surface area within the internal elastic lamina . the mean vessel injury score was determined as described previously   using a method originally developed by schwartz et al . . vessel injury at every stent strut within a cross - section ( 9 - 10  struts / cross - section ) was determined based on the anatomic vessel structures penetrated by each strut . this value ranged from 0 ( least injury ) to 3 ( most injury ) . for each cross - section 68 cross - sections ( taken from the proximal , middle , and distal parts of the stent ) were analyzed . the total mean injury score was expressed as the mean of the injury scores of all cross - sections analyzed within one stent . differences between two groups were analyzed for statistical significance using an independent samples t - test . all p values were two - tailed , and a p - value < .05 was considered statistically significant . diabetes incidence in the bbdp colony maintained at the university medical center groningen is 8090% . median age of diabetes onset in the bbdp rats ( n = 7 ) that were stented and included for histological analysis was 82 days ( table 1 ) . insulin treatment using insulin - releasing implants resulted in an almost permanent hyperglycaemic state ( blood glucose > 8  mmol / l ) in all diabetic rats . in individual rats blood glucose oscillations were observed that varied between 8 and 27  mmol / l . depending on the blood glucose levels ( > 20  mmol / l or 1520  mmol / l in the presence of substantial weight loss ( > 10% weight loss compared with previous measurement ) ) , rats received an insulin implant . time between reimplantations varied between 5 and 35 days . as non - diabetic age - matched controls , figure 1 shows the blood glucose oscillations observed in an individual diabetic and non - diabetic bbdp rat during a time frame of 140 days during which the diabetic rat received 9 insulin implants . the median age at stenting was 270 and 298 days for the diabetic and non - diabetic bbdp rats , respectively ( not significantly different ) , translating in a median diabetes duration of 198 days ( table 1 ) . during the total follow - up period till stenting mean blood glucose level after diabetes onset in diabetic bbdp rats was 15.0  0.4  mmol / l compared with 5.3  0.1  mmol / l in thymectomized non - diabetic bbdp rats ( figure 2(a ) , p < .001 ) . as increased hba1c levels have been associated with increased cardiovascular risk in human diabetics , we questioned whether long - term diabetes in our bbdp rat model was also associated with increased hba1c levels . to this end , hba1c levels were determined in a separate group of long - term diabetic bbdp rats ( n = 5 ) with a mean diabetes duration of 249  50 days which was not statistically different from the mean diabetes duration in the stented bbdp rats ( 217  7 days ) at sacrifice . for comparison , hba1c levels were determined in spontaneously protected non - diabetic bbdp rats ( n = 2 ) and non - diabetic bbdr / wor rats ( n = 6 ) . as shown in figure 2(b ) , long - term diabetes resulted in significantly increased hba1c levels ( 11.9  0.2  dcct - hba1c% ) compared with age - matched non - diabetic bb rats ( 5.1  0.1  dcct - hba1c% ) . two weeks before stenting part of the long - term diabetic and non - diabetic bbdp rats were housed in metabolic cages for 24 hrs to collect urine . long - term diabetes and the associated permanent hyperglycaemic state resulted in a 6-fold increased ( p < .001 ) urinary volume compared with non - dm bbdp rats ( figure 3(a ) ) . furthermore , total urinary protein excretion ( figure 3(b ) , p < .01 ) and urinary creatinine excretion ( figure 3(c ) , p < .001 ) were significantly increased in long - term dm bbdp rats compared with non - dm bbdp rats . increased urinary creatinine excretion in dm bbdp rats suggests the presence of glomerular hyperfiltration which is commonly observed in diabetics . hyperfiltration was supported by the slightly increased creatinine clearance rates observed in the long - term dm rats ( 1.7  0.3  ml / min in dm versus 1.0  0.2  ml / min in non - dm , not significant ) . despite the presence of proteinuria and hyperfiltration in long - term dm bbdp rats , plasma creatinine levels in these rats were similar to the levels detected in age - matched non - dm bbdp rats indicating preserved renal function in the presence of diabetes ( table 1 ) . histological analysis of pas - stained renal sections did not reveal increased interstitial and glomerular matrix expansion in long - term dm bbdp rats ( not shown ) . in total , 18 rats ( 9 dm and 9 non - dm bbdp rats ) received a bare metal stent ( figure 4(a ) ) in the abdominal aorta . during the stenting procedure , 3 non - dm bbdp rats died because of perforation of the aorta ( mortality rate of 17% ) . age of the dm and non - dm bbdp rats at stenting was similar between both groups ( table 1 , median age , resp . diabetes duration ( median ) in dm bbdp rats at stenting was 198 days ( table 1 ) . stents from 2 dm bbdp rats were lost during histological processing . finally , stents from 7 dm and 6 non - dm bbdp rats were histologically analyzed for the severity of isr . at 28 days poststenting in both non - dm and dm bbdp rats development of isr was detected which was characterized by neointima formation surrounding the stent struts . figures 4(d ) , 4(e ) , 4(f ) , and 4(g ) show representative photomicrographs of isr in , respectively , non - dm and dm bbdp rats . quantitative analysis revealed that long - term dm resulted in a 32% increase in surface neointima compared with non - dm bbdp rats ( figure 4(b ) , p = .02 ) . this increase in neointima formation in dm bbdp rats was not associated with an increased mean injury score ( figure 4(c ) , p = .957 ) . diabetes mellitus ( dm ) is associated with increased risk for the development of in - stent restenosis ( isr ) [ 1517 ] . underlying pathogenetic mechanisms are as yet unknown , and adequate treatment modalities are lacking . in order to increase our insights into the molecular and cellular mechanism(s ) underlying type 1 dm - enhanced development of isr a clinically relevant rodent model might be of great value . yet a model as such is not available . we therefore tested the hypothesis that long - term spontaneously diabetic hyperglycaemic bbdp rats develop enhanced isr and may be used as a suitable model to study the molecular and cellular mechanism(s ) involved in dm - enhanced development of isr . the hyperglycemic syndrome in bbdp rats develops spontaneously due to a disturbed balance in autoreactive and regulatory t cells [ 2628 ] . the bbdp rat has been suggested to represent the best rodent model for human type 1 dm as in this model diabetes manifests during adolescence and involves an autoimmune disorder without the need of exogenous intervention . to achieve suboptimally controlled dm with manifest hyperglycaemia and elevated hba1c levels , recent onset diabetic bbdp rats were treated with insulin using slow release insulin implants . by doing so , we were able to maintain dm bbdp rats in a rather permanent hyperglycaemic state for at least 8 months . the hyperglycaemic state was associated with increased hba1c levels as well as polyuria , polydipsia , proteinuria , and glomerular hyperfiltration . severity of proteinuria was within the range reported by cohen et al . who also determined protein excretion in long - term dm bbdp rats that were treated with daily insulin injections . as older age is associated with increased restenosis rates independent of dm , for our study it was important to include age - matched non - diabetic controls rather than prediabetic young bbdp rats . in order to prevent diabetes development in bbdp rats thymectomy dm and non - dm bbdp rats were followed for 7 months after which they received a bare metal stent implanted in the abdominal aorta . our results clearly demonstrated that long - term dm significantly enhanced the development of isr by 32% compared with non - dm age - matched thymectomized bbdp rats . this enhanced development of isr in diabetic bbdp rats was not associated with increased vascular injury , that is , increased penetration of the stent struts through the internal elastic lamina into the medial layer . the severity of isr was previously shown to be positively correlated with the mean injury score . however , in the same study we demonstrated differences in severity of isr between bare metal and sirolimus - eluting stents , which was independent of the mean injury score . our data suggest that t1 dm enhances the development of isr by factors other than direct vascular injury . the current study was performed as a proof of concept to demonstrate that long - term t1 dm in bbdp rats indeed enhances the development of isr following stenting in the abdominal aorta . although we did not study the underlying mechanism(s ) of enhanced isr in diabetic bbdp rats yet , we suggest that long - term diabetes increases the proliferative and migratory capacity of medial and neointimal smooth muscle cells , thereby facilitating neointima formation [ 36 , 37 ] . in addition , reduced endothelial repair capacity in diabetic bbdp rats might have contributed to enhanced isr , but this needs to be determined . the major advantage of the bbdp model to study the development of isr over other t1 dm rodent models is the fact that diabetes develops due to autoimmune - mediated destruction of pancreatic islets without having the toxic side effects of the use of streptozotocin or alloxan . by using slow release insulin implants we showed that long - term studies are feasible without the need of daily insulin injections . a possible weakness of the model is that bbdp rats are t cell lymphopenic and are , in that respect , not fully immunocompetent . however , despite t cell lymphopenia bbdp rats do develop autoimmune diabetes which is mediated by autoreactive t cells indicating that functional t cells are present in bbdp rats . percutaneous transluminal coronary angioplasty has been shown to induce t cell activation in a small cohort of 10 patients with stable angina . patients that developed restenosis had higher t cell activation levels than patients that did not develop restenosis . however , treatment with the calcineurin inhibitor cyclosporine to prevent t cell activation did not reduce the development of restenosis in rabbits . we therefore assume that the presence of t cell lymphopenia in bbdp rats has not been of major influence on the development of isr . the bbdp rat model for type 1 diabetes is suitable for studies on the long - term effects of hyperglycaemia on the development of isr . , future studies aiming at the identification of the molecular and cellular mechanisms involved as well as on testing the efficacy of novel therapeutic interventions in this model are warranted .
type 1 diabetic patients have increased risk of developing in - stent restenosis following endovascular stenting . 
 underlying pathogenetic mechanisms are not fully understood partly due to the lack of a relevant animal model to study the effect(s ) of long - term autoimmune diabetes on development of in - stent restenosis . 
 we here describe the development of in - stent restenosis in long - term ( ~7 months ) spontaneously diabetic and age - matched , thymectomized , nondiabetic diabetes prone biobreeding ( bbdp ) rats ( n = 6 - 7 in each group ) . 
 diabetes was suboptimally treated with insulin and was characterized by significant hyperglycaemia , polyuria , proteinuria , and increased hba1c levels . 
 stented abdominal aortas were harvested 28 days after stenting . 
 computerized morphometric analysis revealed significantly increased neointima formation in long - term diabetic rats compared with nondiabetic controls . in conclusion , long - term autoimmune diabetes in bbdp rats enhances in - stent restenosis . 
 this model can be used to study the underlying pathogenetic mechanisms of diabetes - enhanced in - stent restenosis as well as to test new therapeutic modalities .