the development of artificial bioengineered constructs largely depends on adequate vascularization to guarantee sufficient oxygen supply . the arteriovenous
( av ) loop model is an established method to obtain transplantable bioartificial tissue in vivo .
originally based on the findings of erol and spira   and subsequently modified by murphy et al .   and tanaka et al .  
this model of benign angio - inductive behavior triggers the formation of novel vessel sprouts remarkably without additional extrinsic and angio - inductive factors . in brief , a femoral vein is dissected , harvested , and as an autologous graft , interposed between the contralateral femoral artery and vein .
this results in an arteriovenous fistula , which is then embedded in a fibrin - filled teflon chamber .
a mature microcirculatory system originates mainly from the venous graft , thereby facilitating cultivation , harvesting , and transfer of rudimentary organ - like structures to a secondary defect site , thus minimizing donor site morbidity [ 69 ] .
as vascularization is crucial to promote clinical implementation of bioengineered constructs , tremendous research efforts have been undertaken to further optimize vessel formation within the av loop .
the current understanding of angiogenesis is evolving , as the underlying nature and mediating signaling pathways fundamentally differ in various types of angiogenesis .
watson et al .   demonstrated that vascular flow is essential for hypoxia - driven angiogenesis in embryonic development .
furthermore , we recently pointed out that distinct av associated changes of the hemodynamic load  namely the shear rate  are responsible for triggering the angio - inductive behavior of endothelial cells within the venous graft .
both of these observations fundamentally rely on the presence of angio - inductive endothelial cells and a viable venous graft . however , polykandriotis et al .   and westerband et al
moreover , zdolsek et al .   demonstrated that utterly decellularized and avital vein allografts are continuously able to maintain angio - inductive properties within the av loop . due to the constant progress in reconstructive surgery and due to improved and standardized microsurgical treatment algorithms , the range of medical indications for reconstructive vascularized tissue transfers has increased dramatically during the last decades .
thus , microvascular flaps are exposed to high - dose radiation after defect coverage with increasing frequency . for this reason , the vascular behavior of novel reconstructive approaches , such as intrinsically vascularized av loop - based constructs ,
allows an excellent evaluation of the accompanying angiogenesis , and is of special interest in evaluating the response to radiation .
furthermore , the understanding of the av loop associated angiogenesis is of special translational importance .
there is an ongoing discussion as to whether the interposed venous graft is vitally important for the induction of angiogenesis under conditions of high flow . in order to alter the cellular hemostasis specifically within the grafted vein we exposed the graft intraoperatively to 2 gy of ionizing radiation ( a level of radiation considered a relevant rat dose - rate ) .
we wanted to demonstrate that the graft possessed vital intrinsic factors that were at least partially responsible for triggering flow - mediated angiogenesis in the av loop model , and we generally wanted to investigate the effect of radiation in the developing av loop microvasculature . beside three - dimensional micro - ct
, a previously described observer - independent automatic software algorithm was used to assess vessel number , density , and area by analyzing two - dimensional histological cross sections in order to characterize the differences of angiogenesis in detail .
all operations were performed by the same investigator ( jmc ) using a surgical microscope ( magnification 16 , opmi ifc , carl zeiss , oberkochen , germany ) .
2 to 4 month old male lewis rats ( n=14 ) with an average body mass of 340 g were obtained from charles river laboratories ( sulzfeld , germany ) .
all experiments were approved by the institutional animal care and use committee of the regierungsprsidium mittelfranken ( az 54 - 2532.1 - 34/09 ) and in accordance with the german animal welfare act .
for induction of anesthesia , the rats received 5% isoflurane ( baxter , vienna , austria ) inhalation . before surgery
, they received buprenorphin for analgesia , ( 0.3 mg / kg body weight , temgesic , essex chemie ag , luzern , switzerland ) , as well as heparin anticoagulation ( 80 iu / kg liquemin , ratiopharm ulm , germany ) .
the right femoral vascular bundle was exposed by a mid - ventral incision . the femoral vein was dissected and a 20 mm long venous graft was harvested .
the grafts either underwent irradiation or were directly interposed as a loop ( figure 1 ) between the contralateral femoral artery and vein using 11/0 nylon sutures ( ethilon , ethicon , norderstedt , germany ) after similar incubation in sodium heparin solution ( sham treatment , 10,000 ie / l ) .
the av loop was embedded in the isolation chamber , which was filled with 800 l of fibrin sealant ( tissucol , baxter ) composed of fibrinogen ( 10 mg / ml ) , thrombin ( 2 iu / ml ) and aprotinin ( 1,500 kie / ml ) as described previously .
the chamber was sutured subcutaneously onto the underlying adductor fascia with 6 - 0 polypropylene ( prolene 6/0 , ethicon , norderstedt , germany ) . for wound closure ,
interrupted vertical mattress sutures with vicryl 4 - 0 ( ethicon , norderstedt , germany ) were used .
all rats received buprenorphin and heparin ( 80 iu / kg liquemin , ratiopharm ulm , germany ) postoperatively .
the rats were kept at a 12-hour dark / light cycle in the animal facility of the university of erlangen medical centre with free access to standard chow ( sniff ) and water . at the end of the experiment ,
the rats were sacrificed under deep anesthesia ( 5% isoflurane ) by exsanguination , and subsequent reperfusion with india ink or microfil ( flowtech , ma , usa ) . the autologous venous graft was harvested and rinsed in sodium heparin solution ( 10,000 ie / l ) .
a dose of 2 gy ionizing radiation was applied to the vein graft within 30 seconds using an isovolt titan x - ray machine with a 120 kv power supply ( ge sensing & inspection technologies , ahrensburg , germany ) .
after 15 days , an abdominal midline incision was performed and newly formed vessels along the main vessel axis were detected by cannulating the distal descending aorta using a 24-gauge catheter .
the descending vascular system was cleansed with 100 ml of prewarmed ( 37c ) isotonic salt solution containing heparin ( 100 ie / ml ) and subsequently perfused with 30 ml of prewarmed ( 37c ) india ink solution [ 50% v / v india ink ( rohrer & klinger ) ] in 5% gelatin and 4% mannitol in order to detect additional vessels around the constructed av loop .
micro - ct analysis required the reperfusion of the circulatory system with 20 ml of warmed yellow microfil ( mv-122 ) containing 5% of mv curing agent ( flowtech inc . , carver , usa ) .
incubation at 4c for 24 hours allowed the gelatin and mannitol to solidify before continuing with downstream applications . for histological analysis
then 5 m histological sections in standardized planes perpendicular to the longitudinal av loop axis were produced using a microtome ( leica microsystems , wetzlar , germany ) .
hematoxylin and eosin ( h&e ) staining was performed using an autostainer ( st5010 , leica microsystems ) according to standard protocol .
isolectin b4 ( sigma l 2140 , sigma - aldrich , hamburg , germany ) staining was used to detect luminal endothelial cells by means of immunohistochemistry as described elsewhere .
histological cross sections were visualized using an inversed microscopy ( olympus ix81 , 10 magnification , olympus corporation , hamburg , germany ) and analyzed using an automatic two - dimensional quantification algorithm established previously by our group for superior morphometric quantification .
high - resolution micro - ct scans were acquired on a cone - beam micro - ct scanner at the institute of medical physics , university erlangen , germany ( forbild scanner ) .
further technical specifications and specialized algorithms for optimization and enhancement of images obtained were described in detail elsewhere . for statistical analysis and figures , originpro 2015 ( b9.2.214 , originlab corp . ,
for analysis of more than two groups , analysis of variance ( one - way anova ) was used .
all operations were performed by the same investigator ( jmc ) using a surgical microscope ( magnification 16 , opmi ifc , carl zeiss , oberkochen , germany ) .
2 to 4 month old male lewis rats ( n=14 ) with an average body mass of 340 g were obtained from charles river laboratories ( sulzfeld , germany ) .
all experiments were approved by the institutional animal care and use committee of the regierungsprsidium mittelfranken ( az 54 - 2532.1 - 34/09 ) and in accordance with the german animal welfare act .
for induction of anesthesia , the rats received 5% isoflurane ( baxter , vienna , austria ) inhalation . before surgery
, they received buprenorphin for analgesia , ( 0.3 mg / kg body weight , temgesic , essex chemie ag , luzern , switzerland ) , as well as heparin anticoagulation ( 80 iu / kg liquemin , ratiopharm ulm , germany ) .
the right femoral vascular bundle was exposed by a mid - ventral incision . the femoral vein was dissected and a 20 mm long venous graft was harvested .
the grafts either underwent irradiation or were directly interposed as a loop ( figure 1 ) between the contralateral femoral artery and vein using 11/0 nylon sutures ( ethilon , ethicon , norderstedt , germany ) after similar incubation in sodium heparin solution ( sham treatment , 10,000 ie / l ) .
the av loop was embedded in the isolation chamber , which was filled with 800 l of fibrin sealant ( tissucol , baxter ) composed of fibrinogen ( 10 mg / ml ) , thrombin ( 2 iu / ml ) and aprotinin ( 1,500 kie / ml ) as described previously .
the chamber was sutured subcutaneously onto the underlying adductor fascia with 6 - 0 polypropylene ( prolene 6/0 , ethicon , norderstedt , germany ) . for wound closure ,
interrupted vertical mattress sutures with vicryl 4 - 0 ( ethicon , norderstedt , germany ) were used .
all rats received buprenorphin and heparin ( 80 iu / kg liquemin , ratiopharm ulm , germany ) postoperatively .
the rats were kept at a 12-hour dark / light cycle in the animal facility of the university of erlangen medical centre with free access to standard chow ( sniff ) and water . at the end of the experiment ,
the rats were sacrificed under deep anesthesia ( 5% isoflurane ) by exsanguination , and subsequent reperfusion with india ink or microfil ( flowtech , ma , usa ) .
the autologous venous graft was harvested and rinsed in sodium heparin solution ( 10,000 ie / l ) .
a dose of 2 gy ionizing radiation was applied to the vein graft within 30 seconds using an isovolt titan x - ray machine with a 120 kv power supply ( ge sensing & inspection technologies , ahrensburg , germany ) .
after 15 days , an abdominal midline incision was performed and newly formed vessels along the main vessel axis were detected by cannulating the distal descending aorta using a 24-gauge catheter .
the descending vascular system was cleansed with 100 ml of prewarmed ( 37c ) isotonic salt solution containing heparin ( 100 ie / ml ) and subsequently perfused with 30 ml of prewarmed ( 37c ) india ink solution [ 50% v / v india ink ( rohrer & klinger ) ] in 5% gelatin and 4% mannitol in order to detect additional vessels around the constructed av loop .
micro - ct analysis required the reperfusion of the circulatory system with 20 ml of warmed yellow microfil ( mv-122 ) containing 5% of mv curing agent ( flowtech inc . , carver , usa ) .
incubation at 4c for 24 hours allowed the gelatin and mannitol to solidify before continuing with downstream applications . for histological analysis , the chambers with the av loop were explanted .
formalin - fixed , paraffin - embedded tissue sections were obtained using standard protocols . then 5 m histological sections in standardized planes perpendicular to the longitudinal av loop axis were produced using a microtome ( leica microsystems , wetzlar , germany ) .
hematoxylin and eosin ( h&e ) staining was performed using an autostainer ( st5010 , leica microsystems ) according to standard protocol .
isolectin b4 ( sigma l 2140 , sigma - aldrich , hamburg , germany ) staining was used to detect luminal endothelial cells by means of immunohistochemistry as described elsewhere .
histological cross sections were visualized using an inversed microscopy ( olympus ix81 , 10 magnification , olympus corporation , hamburg , germany ) and analyzed using an automatic two - dimensional quantification algorithm established previously by our group for superior morphometric quantification .
high - resolution micro - ct scans were acquired on a cone - beam micro - ct scanner at the institute of medical physics , university erlangen , germany ( forbild scanner ) .
further technical specifications and specialized algorithms for optimization and enhancement of images obtained were described in detail elsewhere .
for statistical analysis and figures , originpro 2015 ( b9.2.214 , originlab corp . , northampton , ma , usa ) was used .
all data are presented as mean sem and compared using unpaired t - test . for analysis of more than two groups , analysis of variance ( one - way anova )
all rats survived the experiments and tolerated the anesthesia and surgical procedures well ( n=14 ) .
one rat in the control group had a significant swelling in the groin in the first week after surgery most likely due to seroma formation .
after explantation of the chamber , the peripheral tissue was examined by macroscopic assessment . the matrix embedding the av loop within the chambers had a milky translucent appearance at implantation .
stained main vessel axis was visible in all rats indicating a successful reperfusion and patency of the av loop at the time of reperfusion .
fifteen days after the loop implantation , angiogenesis was visualized by three - dimensional micro - ct analysis in two rats from each group . in line with previous results , control loops showed homogenous vessel sprouting that originated predominantly from the lumen of the graft ( figure 2a ) . local graft irradiation considerably impaired neovessel formation along the graft ( figure 2b ) .
lumina of the microcirculatory system along the graft appeared darkly stained in h&e and lectin stained cross sections of india ink - or microfil - perfused av loop constructs .
the microcirculatory system along irradiated grafts was predominantly aligned in close proximity to the main vessel axis . when a non - irradiated control graft was used , however , newly formed vessels extended further peripherally from the main vessel axis ( figure 3 ) .
the degree of vascular sprouts was assessed by an automatic observer - independent quantification algorithm , as described earlier , and yielded a total amount of 739111 ( n=7 ) vessels per cross section .
no significant morphometrical differences were found between the inflow ( 43183 , n=7 ) and the outflow tract ( 30843 , n=7 ) of non - irradiated control grafts ( figure 4a ) .
the amount of vessels of the graft inflow ( 17458 , n=7 ) was not statistically different from the graft outflow ( 13738 n=7 ) .
mean vessel area ( figure 4b ) assessed by cross section analysis within constructs where control grafts were used amounted to 238,92463,126 m and , similar to the vessel number , there were no statistical significant differences among control grafts with an area of 151,73051,975 m ( n=7 ) at inflow segments and 84,33722,964 m ( n=7 ) at outflow segments .
irradiated grafts behaved correspondingly with an area of 21,9986,850 m , n=7 ( graft inflow ) and 21,1405,897 m , n=7 ( graft outflow ) respectively .
although luminal sprouting was strongly impaired , the formation of new vessels from the luminal graft was not thoroughly abolished .
single - dose irradiation of 2 gy reduced the mean number of vessel sprouts to 31173 ( n=7 ) and was significantly different from the control ( figure 4a ) .
irradiation reduced mean vessel area to 43,13710,224 m ( n=7 ) compared to 238,92463,126 m ( n=7 ) in corresponding control grafts ( figure 4b ) .
the mean number of vessels and area of irradiated grafts were significantly lower when compared to non - irradiated control grafts ( figure 4a and 4b ) . while not statistical significant , mean vessel diameter after graft irradiation ( 141.8 m , n=7 ) tended to be lower compared to control grafts ( 192.6 m , n=7 , figure 5 ) .
densities of neovessels were differently distributed when comparing av loop constructs with control and irradiated grafts .
vessel formation aroused primarily within a peripheral radius of 401600 m when normalized to the main vessel axis .
in contrast , mean vessel diameter began to differ at radii greater than 400 m , suggesting that radiation may have negative effects on peripheral maturation of newly formed vessels ( figure 6b ) .
all rats survived the experiments and tolerated the anesthesia and surgical procedures well ( n=14 ) .
one rat in the control group had a significant swelling in the groin in the first week after surgery most likely due to seroma formation .
after explantation of the chamber , the peripheral tissue was examined by macroscopic assessment . the matrix embedding the av loop within the chambers had a milky translucent appearance at implantation .
stained main vessel axis was visible in all rats indicating a successful reperfusion and patency of the av loop at the time of reperfusion .
fifteen days after the loop implantation , angiogenesis was visualized by three - dimensional micro - ct analysis in two rats from each group . in line with previous results , control loops showed homogenous vessel sprouting that originated predominantly from the lumen of the graft ( figure 2a ) . local graft irradiation considerably impaired neovessel formation along the graft ( figure 2b ) .
lumina of the microcirculatory system along the graft appeared darkly stained in h&e and lectin stained cross sections of india ink - or microfil - perfused av loop constructs .
the microcirculatory system along irradiated grafts was predominantly aligned in close proximity to the main vessel axis .
when a non - irradiated control graft was used , however , newly formed vessels extended further peripherally from the main vessel axis ( figure 3 ) .
the degree of vascular sprouts was assessed by an automatic observer - independent quantification algorithm , as described earlier , and yielded a total amount of 739111 ( n=7 ) vessels per cross section .
no significant morphometrical differences were found between the inflow ( 43183 , n=7 ) and the outflow tract ( 30843 , n=7 ) of non - irradiated control grafts ( figure 4a ) .
the amount of vessels of the graft inflow ( 17458 , n=7 ) was not statistically different from the graft outflow ( 13738 n=7 ) .
mean vessel area ( figure 4b ) assessed by cross section analysis within constructs where control grafts were used amounted to 238,92463,126 m and , similar to the vessel number , there were no statistical significant differences among control grafts with an area of 151,73051,975 m ( n=7 ) at inflow segments and 84,33722,964 m ( n=7 ) at outflow segments .
irradiated grafts behaved correspondingly with an area of 21,9986,850 m , n=7 ( graft inflow ) and 21,1405,897 m , n=7 ( graft outflow ) respectively .
although luminal sprouting was strongly impaired , the formation of new vessels from the luminal graft was not thoroughly abolished . single - dose irradiation of 2 gy reduced the mean number of vessel sprouts to 31173 ( n=7 ) and was significantly different from the control ( figure 4a ) .
irradiation reduced mean vessel area to 43,13710,224 m ( n=7 ) compared to 238,92463,126 m ( n=7 ) in corresponding control grafts ( figure 4b ) .
the mean number of vessels and area of irradiated grafts were significantly lower when compared to non - irradiated control grafts ( figure 4a and 4b ) . while not statistical significant , mean vessel diameter after graft irradiation ( 141.8 m , n=7 ) tended to be lower compared to control grafts ( 192.6 m , n=7 , figure 5 ) .
densities of neovessels were differently distributed when comparing av loop constructs with control and irradiated grafts .
vessel formation aroused primarily within a peripheral radius of 401600 m when normalized to the main vessel axis .
in contrast , mean vessel diameter began to differ at radii greater than 400 m , suggesting that radiation may have negative effects on peripheral maturation of newly formed vessels ( figure 6b ) .
to the best of our knowledge , this is the first study investigating the effects of ionizing radiation within an av loop - based neovascular system .
grafts were exposed intraoperatively to a single dose of 2 gy ionizing radiation prior to loop construction in order to elucidate whether they provide angio - inductive features under conditions of high flow that are mandatory for av loop associated angiogenesis . as assessed by three - dimensional micro - ct analysis and two - dimensional quantification of mean vessel number and area
, we demonstrated that vessel sprouting was significantly impaired when irradiated vein grafts were used for av loop construction . further dissecting this angio - inductive phenomenon histologically , we provided evidence that the emerging microcirculatory system was homogeneously distributed along the main vessel axis
additionally , we illustrated that radiation compromises neovessel stabilization and maturation as mean vessel diameter primarily decreased in more peripheral regions of constructs containing irradiated vein grafts .
changes in mechanical load following vein grafting into an arteriovenous shunt flow have been shown to foster angio - inductive properties in the graft endothelium .
however , it has also been demonstrated that vascular grafts are exposed to a significant amount of endothelial denudation .
notably , even decelluarized and avital vascular allografts seem sufficient to provide angiogenesis in the av loop .
thus , it has been proposed that the venous graft merely serves as a connecting prosthesis providing sufficient length to ensure a tension - free av anastomosis .
contrary to this believe , we repeatedly observed luminal sprouts emerging from the grafted vein .
observed by means of scanning electron microscopy ( sem ) of corrosion casts , that luminal sprouting was present in the vein and the grafted vein but not in the arterial segment between day 10 and 14 after av loop creation .
we previously pointed out that , in contrast to pulsatile flow , increased vascular shear rate induced specific cellular responses linked to angio - inductive behavior .
notably , intercellular gap junction protein connexin ( cx ) 43 is specifically upregulated in vein graft endothelium , and it has been suggested that it is vital for angiogenic signaling and vessel growth .
thus , we believe that the interposed graft transduces mechanosensitive stimuli after exposure to high flow .
the detrimental effects of ionizing radiation have long been recognized and are widely used in adjuvant and neoadjuvant treatment options worldwide .
hydroxyl radicals ( oh ) alter cellular lipid hemostasis , membrane transport , and constitute significant genotoxic stressors on vascular cells .
double - strand dna breaks ultimately activate proto - oncogenes and propel malignant cell transformation .
the molecular mechanisms behind radiation - induced genetic alterations are for the time being largely unidentified but may , among others factors , be linked to dysfunctional expression and nuclear translocation of cytoplasmic transcription factors . within blood vessels , endothelial cells are the most vulnerable to irradiation .
radiation - induced vascular injury correlates histologically with desquamated endothelial cells , which leads to small vessel obstruction and subsequent tissue ischemia .
it has been previously demonstrated that the acute effect of radiation - induced vascular injury depends on vessel diameter .
severity of adventitial fibrosis and medial hyalinization are most distinctively recognized in vessels < 500 m . in the presented study
, we showed that single - dose graft irradiation with 2 gy impaired the development of a functional microcirculatory system likely due to dysfunctional graft - specific and angio - inductive properties .
the clinical management of head and neck tumors and extended extremity sarcomas continue to challenge reconstructive surgeons worldwide ; av loop - based tissue transfer may provide an essential adjunct for vascularized tissue transfer after oncologic resection .
as the majority of these patients regularly require adjuvant radiotherapy , the effect of radiation on av loop vascularization is of great clinical importance .
although strongly decreased , vessel sprouting was not fully abolished after irradiation in our study .
hence , vascular shear rate might constitute a fairly sturdy and vital determinant for loop - associated neoangiogenesis even after isolated and high - dose radiation of the mediating vein . nevertheless , we only examined short - term effects of radiation on av loop associated angiogenesis .
as vascular tissue mainly consists of slowly proliferating cells , single - dose local graft irradiation may impede vascular hemostasis of bioengineered constructs primarily in the long term .
further analysis of radiation - mediated ramifications of the av loop model may contribute to safe clinical implementation and adequate patient selection .
the isolated and homogeneous chamber environment , as well as the established quantification algorithms , render the av loop model eligible for compartment - specific characterization of newly developing vascular networks .
as various malignant microenvironments induce different dynamic and angio - inductive behavior , the av loop model may conceivably be a useful tool for further in vivo characterization of anti - angiogenic treatment options .
dynamic and molecular interactions of various cancer stem cells with a defined vascular supply , as well as their intrinsic susceptibility to radiotherapy and other novel and locally restricted treatment options , may conveniently and separately be studied within the av loop chamber . in the presented study , we investigated the effects of radiation in a benign angiogenesis rat model .
these findings provide a useful tool for further molecular characterization of tumor growth ; especially as various tumor cells differently interact with vascular structures thereby altering radiation sensitivity .
taken together , we provided evidence that the venous graft constitutes a vital part of the av loop model and is essential to initiate vascular luminal sprouts .
it remains speculative whether reduced graft viability or cellular alterations are responsible , as irradiation induces a plethora of vascular and metabolic changes within the vascular tissue .
although not completely impaired , graft - specific irradiation negatively influences av loop associated vascularization .
radiation compromises neovessel stabilization and maturation primarily in peripheral regions of av loop - based constructs .
these results add to the current understanding of av loop - based neovascularization and suggest clinical implications for patients relying on combined av loop - based tissue transfer and adjuvant radiotherapy .
 backgroundthe arteriovenous ( av ) loop model enables axial vascularization to gain a functional microcirculatory system in tissue engineering constructs in vivo . these constructs might replace surgical flaps for the treatment of complex wounds in the future . 
 today , free flaps are often exposed to high - dose radiation after defect coverage , according to guideline - oriented treatment plans . 
 vascular response of av loop - based constructs has not been evaluated after radiation , although it is of particular importance . 
 it is further unclear whether the interposed venous av loop graft is crucial for the induction of angiogenesis.material/methodswe exposed the grafted vein to a single radiation dose of 2 gy prior to loop construction to alter intrinsic and angio - inductive properties specifically within the graft . 
 vessel loops were embedded in a fibrin - filled chamber for 15 days and radiation - induced effects on flow - mediated vascularization were assessed by micro - ct and two - dimensional histological analysis.resultsvessel amount was significantly impaired when an irradiated vein graft was used for av loop construction . however , vessel growth and differentiation were still present . 
 in contrast to vessel density , which was homogeneously diminished in constructs containing irradiated veins , vessel diameter was primarily decreased in the more peripheral regions.conclusionsvascular luminal sprouts were significantly diminished in irradiated venous grafts , suggesting that the interposing vein constitutes a vital part of the av loop model and is essential to initiate flow - mediate angiogenesis . 
 these results add to the current understanding of av loop - based neovascularization and suggest clinical implications for patients requiring combined av loop - based tissue transfer and adjuvant radiotherapy . 
Background
Material and Methods
Experimental design and surgical procedure
Irradiation of the interpositional venous graft
Histological and micro-CT analysis
Statistical analysis
Results
Surgery, patency, and macroscopic appearance upon explantation
Non-quantitative assessment of the effect of irradiation on loop angiogenesis by means of micro-CT and histological cross sections
Automatic morphometric quantification of the AV loop vascularization in histological cross sections
Local graft irradiation and neovessel formation in the AV loop
Discussion
Conclusions