cardiovascular diseases caused by the progression of atherosclerosis are the leading cause of death in the industrialized nations .
atherosclerosis is a focal , inflammatory fibro - proliferative response of the vascular wall to endothelial injury , resulting in the formation of an extended plaque into the lumen of the vessel , affecting the blood flow through coronary arteries . over 75% of myocardial infarctions
since this complication can be fatal , a percutaneous transluminal ( coronary ) angioplasty ( ptca ) with stent implantation became the first - choice therapy in the current medical practice .
the method allows the dilatation of the narrowed coronary artery and thus the restoration of blood flow .
however , the long - term effect of this therapy is limited by an excessive arterial remodeling and restenosis . by employment of stents ,
the ptca became more effective in the treatment of complicated lesions , allowing revascularization after an acute vessel closure .
this method decreases the incidence of in - stent restenosis to less than 10% . beside these benefits , this first - choice therapy for coronary revascularization bears also the life - threatening risks of in - stent thrombosis and restenosis . in - stent
thrombosis is caused by a de - endothelialization of the vessel , followed by a massive adhesion of platelets and fibrin to the injured site .
26% of patients suffer from in - stent thrombosis and 63% die of myocardial infarction .
restenosis refers to the process of wound healing after mechanical injury to the vessel wall , involving neointimal hyperplasia ( migration and proliferation of vascular smooth muscle cells ( vsmc ) , deposition of extracellular matrix ( ecm ) , and remodeling of the vessel .
often , an invasive re - intervention becomes necessary to dilatate severely narrowed atherosclerotic vessels due to in - stent thrombosis and restenosis . to prevent in - stent thrombosis ,
a long - term treatment with anti - thrombotic drugs is necessary . to prevent restenosis , new generation of drug - eluting stents elute anti - proliferative agents such as immunosuppressive drugs ( e.g. sirolimus , everolimus , zotarolimus ) and anti - cancer drugs ( e.g. paclitaxel ) from a polymer coating for several months .
although these drugs decrease the neointima formation and restenosis , they maintain a high risk of in - stent thrombosis by inhibiting the re - endothelialization .
after arterial injury , the maintenance of the endothelial compartment is essential to prevent thrombotic complications . under physiological conditions , the human endothelium shows a small turnover rate . under pathological conditions , however , the endothelial integrity is impaired , so that a rapid recovery by surrounding mature endothelial cells and circulating endothelial progenitor cells ( epcs ) is required .
the study of these complex molecular mechanisms in larger animals or in mouse aortic artery is a very difficult procedure , offering limited data . to test the efficiency of novel stent - coatings to reduce in - stent thrombosis and restenosis new models are imperative .
nitinol represents the ideal platform for stents because of its ' high elasticity , shape - memory effect and good tolerance in patients , being successfully used as bare - metal stents in clinical use .
this alloy made it possible to create a miniaturized stent with an external diameter of 500 m , which can be coated and implanted into the carotid artery of mice .
the development of a miniaturized nitinol stent for mouse carotid artery , allows the study of precise molecular mechanisms induced by stent implantation and offers the possibility to test quickly and efficiently the effects of different drug - coatings to prevent restenosis .
moreover , the existence of different knock - out mice strains represents a huge advantage in clarifying the role of different molecules involved in neointima growth and in - stent thrombosis .
the stent - struts ( fort wayne metals , castlebar , ireland ) were braided and then cut to the desired size at the institute for textile technology and mechanical engineering , rwth aachen university in germany ( figure 1a ) . before implantation , the stents must be transferred into a 2 cm silicon tube , using forceps , and placed 2 mm at one terminal end , referred front end ( figure 1a ) .
the front end should be cut obliquely , to ensure a sharp tip for implantation . before implantation
10 - 12 weeks old male c57bl/6 wild type mice , 25 - 27 g are anesthetized using intraperitoneal injection of 100 mg / kg ketamine and 10 mg / kg xylazine .
proper anesthetization is confirmed prior to surgery by the lack of reflexes and beard movement . to prevent dryness while under anesthesia
after shaving and proper disinfection of the ventral neck area , a small median incision of 1 cm is performed under a stereomicroscope , using scissors . after separating the 2 fatty bodies with sterile curved forceps ,
. 1 cm of the left common carotid artery and the bifurcation should be free prepared .
1 knot using a 5/0 silk thread will be bound around the left common carotid artery , 2 knots using 7/0 silk threads will be bound around the left external carotid artery , and 1 knot using a 7/0 silk thread will be bound around the internal carotid artery ( figure 1b ) . the blood flow is then interrupted by binding the knots on the internal carotid artery and the proximal external carotid artery firmly , as well as by pulling the knot surrounding the common carotid artery . the vessel should be fixed in a way that the common and external carotid artery are in a straight line .
a small incision at the external carotid artery is performed , near the proximal knot , using a vannas scissor .
the silicon tube containing the stent is introduced into the external carotid artery , with the sharp end in front , using a guide - wire .
after the stent reaches the desired position , the silicon tube is pulled back over the guide - wire and allows the shape - memory expansion of the stent ( figure 1b ) .
the distal knot on the external carotid artery is bind tightly to close the site of incision and the knots at the internal and common carotid artery are removed , thereby restoring the blood flow .
the skin incision is closed using 3 - 4 michel suture clips and a michel forcep .
to study the re - endothelialization , an earlier end - time point is necessary ( 3 - 4 days ) .
we observed in our model of stent implantation that 4 weeks after this surgical intervention , especially by the use of specific coatings to biofunctionalize the miniaturized stents , neoangiogenesis occurs in approximately 30% of specimen .
this is a hind for remodeling and regenerative processes , with different mechanisms and representing another pathological problem . to concentrate on neointima formation , in - stent
stenosis and/or analysis of mechanisms underlying these side - effects after stent implantation an end - time point of 3 weeks would be beneficial not to mix up with the regenerative effects induced by the onset of neoangiogenesis . at the end - time point
, the animals are anesthetized using intraperitoneal injection of 100 mg / kg ketamine and 10 mg / kg xylazine .
proper anesthetization is confirmed prior to surgery by the lack of reflexes and beard movement .
after opening the thoracic cavity and pbs washing via intracardial punction , a body - perfusion with 4% paraformaldehyde ( pfa ) solution is performed for 5 min .
the left carotid artery containing the stent is dissected , directly placed in a 4% pfa solution and at least 16 hr later embbeded in plastic .
50 m thick sections are performed from plastic - embedded samples using a diamond band saw . to measure the plaque size ,
giemsa staining is performed . to analyze the rate of re - endothelialization within the stented area of the vessel , immunohistochemistry for von willebrand factor ( vwf )
the stent - struts ( fort wayne metals , castlebar , ireland ) were braided and then cut to the desired size at the institute for textile technology and mechanical engineering , rwth aachen university in germany ( figure 1a ) . before implantation , the stents must be transferred into a 2 cm silicon tube , using forceps , and placed 2 mm at one terminal end , referred front end ( figure 1a ) .
the front end should be cut obliquely , to ensure a sharp tip for implantation . before implantation
10 - 12 weeks old male c57bl/6 wild type mice , 25 - 27 g are anesthetized using intraperitoneal injection of 100 mg / kg ketamine and 10 mg / kg xylazine .
proper anesthetization is confirmed prior to surgery by the lack of reflexes and beard movement . to prevent dryness while under anesthesia
after shaving and proper disinfection of the ventral neck area , a small median incision of 1 cm is performed under a stereomicroscope , using scissors . after separating the 2 fatty bodies with sterile curved forceps , the left common carotid artery can be seen pulsing along with the trachea
. 1 cm of the left common carotid artery and the bifurcation should be free prepared .
1 knot using a 5/0 silk thread will be bound around the left common carotid artery , 2 knots using 7/0 silk threads will be bound around the left external carotid artery , and 1 knot using a 7/0 silk thread will be bound around the internal carotid artery ( figure 1b ) .
the blood flow is then interrupted by binding the knots on the internal carotid artery and the proximal external carotid artery firmly , as well as by pulling the knot surrounding the common carotid artery .
the vessel should be fixed in a way that the common and external carotid artery are in a straight line .
a small incision at the external carotid artery is performed , near the proximal knot , using a vannas scissor .
the silicon tube containing the stent is introduced into the external carotid artery , with the sharp end in front , using a guide - wire .
after the stent reaches the desired position , the silicon tube is pulled back over the guide - wire and allows the shape - memory expansion of the stent ( figure 1b ) .
the distal knot on the external carotid artery is bind tightly to close the site of incision and the knots at the internal and common carotid artery are removed , thereby restoring the blood flow . the skin incision is closed using 3 - 4 michel suture clips and a michel forcep .
to study the re - endothelialization , an earlier end - time point is necessary ( 3 - 4 days ) .
we observed in our model of stent implantation that 4 weeks after this surgical intervention , especially by the use of specific coatings to biofunctionalize the miniaturized stents , neoangiogenesis occurs in approximately 30% of specimen .
this is a hind for remodeling and regenerative processes , with different mechanisms and representing another pathological problem . to concentrate on neointima formation , in - stent
stenosis and/or analysis of mechanisms underlying these side - effects after stent implantation an end - time point of 3 weeks would be beneficial not to mix up with the regenerative effects induced by the onset of neoangiogenesis .
at the end - time point , the animals are anesthetized using intraperitoneal injection of 100 mg / kg ketamine and 10 mg / kg xylazine .
proper anesthetization is confirmed prior to surgery by the lack of reflexes and beard movement .
after opening the thoracic cavity and pbs washing via intracardial punction , a body - perfusion with 4% paraformaldehyde ( pfa ) solution is performed for 5 min .
the left carotid artery containing the stent is dissected , directly placed in a 4% pfa solution and at least 16 hr later embbeded in plastic .
50 m thick sections are performed from plastic - embedded samples using a diamond band saw . to measure the plaque size ,
giemsa staining is performed . to analyze the rate of re - endothelialization within the stented area of the vessel , immunohistochemistry for von willebrand factor ( vwf )
the implantation of a miniaturized nitinol stent into the left carotid artery of mice takes 25 - 30 min and shows a mortality rate of 10% mostly due to the damage of the vessel during the intervention .
a better survival rate is observed in mice having a weight more than 25 g at the time of stent implantation ( mortality rate of 5% ) .
therefore , we chose for the implantation mice with a weight between 25 - 27 g. after surgery , the mice recover from anesthesia within 2 - 5 min and no physical impairments , like e.g. paralysis , are observed .
micro - computer tomography ( micro - ct ) imaging performed one week after stent implantation showed that the stents are not dislocated by blood flow ( figure 1c ) .
unfortunately , analysis of neointima formation in these images is not possible due to the metal - derived artifacts ( figure 1d , 1e ) .
we did n't observe any vessel or endothelial damages of unstented area of the vessel , immediately below the stent , as detectable by histological ( figure 2a ) and by specific staining for endothelium ( figure 2b , anti - mouse cd31 antibody ) .
for a better overview , the section was scanned using a two - photon laser scanning microscopy ( figure 2b , 2c ) . in the stented vessel , a permanent dilatation of 15%
is detected ( ratio stent : artery , 1,15:1 ) by mice with a weight between 25 - 27 g. neointima formation and thrombus - formation can be analyzed by classical histological stainings ( e.g. hematoxilin - eosin , giemsa , movat , toluidin blue , masson - trichrom - goldner , figure 3a , 3b ) .
since the lamina externa and interna are not visible anymore , plaque size was calculated as the difference of the external and the luminal areas ( mean plaque area : 234566  3315 m , mean luminal area : 12036  2662 m ) .
external circumference was also measured ( mean : 1799  14 m ) . for analysis of the cellular composition ,
the sections need to be deplastified and stained with specific markers . for the re - endothelialization
, we used a cy3-conjugated anti - cd31 antibody and for smooth muscle cell proliferation a fitc - conjugated anti - sma antibody ( figure 3c ) .
re - endothelialization was calculated as percentage of cd-31 positive stained to the total luminal surface ( mean : 23.07  3.14 % ) one week after stent implantation .
of course , an unlimited number of specific staining is possible , depending on each laboratories ' experience .
analysis of myosin heavy chain , for a better characterization of smcs , but also analysis of infiltrated cells ( monocytes , lymphocytes ) , or stainings for different inflammatory cytokines can also be performed , depending on the aim of the study .
the blood flow is interrupted by binding the knots on the internal carotid artery and the proximal external carotid artery firmly , as well as by pulling the knot surrounding the common carotid artery .
the silicon tube containing the stent is introduced into the external carotid artery through a small incision at the external carotid artery .
after the stent reaches the desired position , the silicon tube is pulled back over the guide - wire and allows the shape - memory expansion of the stent .
micro - ct images showing the stent position one week after surgical implantation ( b ) . due to the material - derived artifacts ,
unstented area of the vessel is not affected by the surgical procedure , as shown by toluidin blue ( a ) and endothelial - specific cd31 staining ( b , c ) .
analysis of the plaque can be performed by classical histological stainings ( e.g. masson - trichrom - goldner ) ( a ) .
the organized thrombus can be detected by black - stained fibrin depositions inside the neointima , in some cases a complete occlusion of the vessel is observed ( b ) .
re - endothelialization ( cy3 , red ) or smooth muscle cell proliferation ( fitc , green ) was detected by double immunofluorescence staining using specific markers .
we noticed a completed re - endothelialization of the stent struts ( left , double arrow ) compared to an incompleted luminal re - endothelialization ( right , single arrow ) .
to reduce the risk of in - stent thrombosis and restenosis and to sustain the development of new coatings for drug - eluting stents , an easy , simple and accessible method of stent implantation in an animal model is needed .
mice deliver the ideal system to study the complex mechanisms of arterial remodeling after stent implantation and the efficiency of such drugs .
existing models for in - stent restenosis in mouse are difficult , require high surgical skills and imply high risks of complications as bleeding or paralysis .
for example , in the model of the stent- implantation into thoracic aorta of a donor mouse after balloon - dilatation of the vessel and then transplantation of the stented segment into carotid artery of a recipient mouse , the study of the patho - mechanisms is not influenced only by recipient reaction to donor material , but also by the massive damaging of vasa vasorum and adventitia .
implantation of a stainless steel stent directly into abdominal aorta after balloon - dilatation is followed by a high mortality rate ( 35% ) because of hind leg paralysis after thrombosis or bleeding from abdominal aorta on site of arteriotomy .
implantation of a spiral - shaped self - expanding nitinol - stent into abdominal aorta via femoral artery needs high surgical skills , mostly due to blindly directing the stent along the branching from femoral artery to aorta to place the stent at the right position .
this procedure is followed by a high risk of damaging the femoral nerve , therefore paralysis of the hind leg .
compared with these procedures , our model of stent implantation in mouse does not need high surgical skills .
our model offers a simple , easy and efficient method to analyze the effects of different drug - coatings on arterial remodeling , the placing of the stent is made under sight , and there are no risks of damaging nerves or other structures .
the complex molecular mechanisms can be investigated easier in our model of mouse carotid artery stenting , not only by direct accessibility of the vessel , but also due to the existence of different knock - out mice strains .
as one limitation , comparing with the clinical procedure , our model uses healthy mice / arteries and does n't perform stenting on pre - existing plaques ( not in - stent restenosis , but in - stent stenosis ) .
however , due to the massive damage of the vessel wall in both models , the reparatory processes are similar .
unfortunately , due to the metal - derived artifacts , an in vivo monitoring of the neointimal growth is not possible by existing imaging methods as ultrasound or computer - tomography .
another limiting factor is the thin sectioning of metal - based stents , which requires some expertise in the metal processing . using this method
, we were able to show , that neutrophil - instructing biofunctionalized miniaturized nitinol - stents coated with ll-37 reduce in - stent restenosis , providing a novel concept to promote vascular healing after interventional therapy . despite these limitations
, this model seems to be , until now , the most suitable system , thereby money- and time - saving , to investigate new drug - coatings for stents and their effects on the molecular events during arterial remodeling .
moreover , this model can be easily adapted to the hamster , which is more similar to the human , so that every therapeutical hypothesis can be verified before applying to larger animals or human to avoid unpleasant and unexpected effects .
 despite the considerable progress made in the stent development in the last decades , cardiovascular diseases remain the main cause of death in western countries . beside the benefits offered by the development of different drug - eluting stents , the coronary revascularization bears also the life - threatening risks of in - stent thrombosis and restenosis . 
 research on new therapeutic strategies is impaired by the lack of appropriate methods to study stent implantation and restenosis processes . here 
 , we describe a rapid and accessible procedure of stent implantation in mouse carotid artery , which offers the possibility to study in a convenient way the molecular mechanisms of vessel remodeling and the effects of different drug coatings . 